scsb at utmb

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17th Annual Structural Biology Symposium 2012

April 27, 2012

Sealy Center for Structural Biology

and Molecular Biophysics Symposium

The University of Texas Medical Branch

Symposium Chairs

Xiaodong Cheng

Sealy Center for Structural Biology Faculty

José Barral Wlodek Bujalowski Werner Braun Xiaodong Cheng James C. Lee Marc Morais Andres Oberhauser B. Montgomery Pettitt Svetla Stoilova-McPhie Lawrence Sowers

Stanley Watowich

Wayne Bolen Darren Boehning

Kay Choi Henry Epstein

Junji Iwahara Bruce Luxon

Javier Navarro Krishna Rajarathnam

E. Brad Thompson Scott Weaver

Y. Whitney Yin

Werner Braun

Administrative Staff Contributing to the Symposium

Desi Miller

Organizing Committee

Werner Braun Xiaodong Cheng Angelina Johnson

Lisa Pipper Catherine Schein

Mark A. White

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17th Annual Sealy Center for Structural Biology Symposium

Hotel Galvez

2024 Seawall Blvd.

Galveston, Texas 77555

Friday, April 27, 2012

Scientific Program at Hotel Galvez, Galveston, TX

All Lectures will be in the Music Hall

7:30-8:45 am Registration - Promenade

Poster Set-up – East & West Parlors

8:45-9:00 am Opening Remarks – Werner Braun

Welcome address – Monte Pettitt

Introduction to Keynote Address - Javier Navarro

9:00 -9:55 am Keynote Speaker

ADA E. YONATH

DEPARTMENT OF STRUCTURAL BIOLOGY

WEIZMANN INSTITUTE, REHOVOT, ISRAEL

"RIBOSOMES, THEIR TINY ENEMIES AND THOUGHTS ABOUT THEIR ORIGIN"

Morning Session

Session Chair: Junji Iwahara

10:00-10:45 am JANE DYSON

DEPARTMENT OF MOLECULAR BIOLOGY

THE SCRIPPS RESEARCH INSTITUTE, LA JOLLA, CA

“NMR INFORMATION OF PARTLY FOLDED PROTEINS“

10:45-11:00 am Break (Foyer)

11:00-11:45 am WIM HOL

DEPARTMENT OF BIOCHEMISTRY AND BIOLOGICAL STRUCTURE

UNIVERSITY OF WASHINGTON, SEATTLE, WA

„CHOLERA TOXIN BY THE TWO MEMBRANE-SPANNING TYPE II SECRETION

SYSTEM“

11:45 –12:30 pm Lunch - Veranda

12:30 –2:00 pm Posters/Poster Judging - East & West Parlors

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Afternoon Session

Session Chair: Xiaodong Cheng

2:00- 2:45 pm NADRIAN SEEMAN

Department of Chemistry

New York University, New York, NY

“DNA: NOT MERELY THE SECRET OF LIFE”

2:45 -3:30 pm CAROL POST

DEPARTMENT OF MEDICINAL CHEMISTRY AND MOLECULAR PHARMACOLOGY

PURDUE UNIVERSITY, WEST LAFAYETTE, IN

“CONFORMATIONAL ACTIVATION OF SRC/CDK-LIKE KINASE DOMAINS”

3:30 – 4:00 pm Break & Posters

Session Chair: James Briggs

4:00 – 4:45 pm MONTE PETTITT

DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY

DIRECTOR, SEALY CENTER FOR STRUCTURAL BIOLOGY AND MOLECULAR

BIOPHYSICS

THE UNIVERSITY OF TEXAS MEDICAL BRANCH AT GALVESTON

“OSMOPHOBICS AND HYDROPHOBICS: THE CHANGING LANDSCAPE OF PROTEIN

FOLDING”

4:45 – 5:00 pm Poster Awards and Closing Remarks - Mark White and Xiaodong Cheng

5:00 – 5:45 pm Pre- and Post-Doc/Speaker Mixer – Music Hall

6:00 pm Banquet & Cocktails (Hotel Galvez)

HOPE TO SEE YOU NEXT YEAR AT THE

18th ANNUAL SCSB SYMPOSIUM

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Gulf Coast Consortia: Interdisciplinary Bioscience Research and Training The Gulf Coast Consortia (GCC), created in March 2001, arose from the interactions among a number of faculty in the major research institutions of the Houston / Galveston region. The aspiration of the GCC is to facilitate interactions and leverage strengths to foster innovative training and research collaborations at the frontiers formed by the interface of the biosciences with the computational, mathematical, physical, chemical, and engineering sciences. The vision and engagement of faculty from the GCC member institutions, Baylor College of Medicine, Rice University, University of Houston, University of Texas Health Science Center at Houston, the University of Texas Medical Branch at Galveston, and the University of Texas M.D. Anderson Cancer Center, have contributed to the GCC’s growth to include six research consortia, one center and eight interdisciplinary training programs. The individual consortia, which form the research arm of the GCC, serve to catalyze interactions and develop interdisciplinary collaborative communities of faculty interested in research in a range of interdisciplinary fields. The GCC provides a supportive environment for the encouragement of research programs and acquisition of shared equipment beyond the reach of a single institution, enhancing the research capacity and capabilities of the Gulf Coast region and the GCC’s member institutions. Currently, the GCC supports research consortia in protein crystallography, magnetic resonance, bioinformatics, chemical genomics, membrane biology, theoretical and computational neuroscience, and a center for computational cancer. For more information visit: www.gulfcoastconsortia.org Keck Center: Interdisciplinary Bioscience Training The Keck Center for Interdisciplinary Bioscience Research and Training was established in 1990 with support from the W. M. Keck Foundation. From its roots as the W. M. Keck Center for Computational and Structural Biology with founding institutions, Rice University and Baylor College of Medicine, the Keck Center has now grown to include the six major public and private institutions that comprise the Gulf Coast Consortia. Driving the formation of the Keck Center was the realization that major advances in the biological sciences, such as the DNA sequence of the human genome, would be driven by the integration of biology and computer science. The partners realized, however, that most biological scientists were not prepared to capitalize on novel approaches to visualization, analysis and interpretation of experimental data made possible by rapid advances in computing technology. Moreover, most researchers in computer programming and analysis systems did not have adequate knowledge about biology and biological systems. The Keck Center was explicitly designed to bridge this gap between biological and computational sciences by fostering collaborations among biologists, biomedical researchers, mathematicians, bioengineers, physicists, and computer scientists through specially designed research and training programs. Building on this foundation in computational biology, the Keck Center has established its expertise in multidisciplinary inter-institutional programs, and has become the training arm of the Gulf Coast Consortia, supporting training programs in computational biology and medicine, molecular biophysics, pharmacoinformatics, and nanobiology. The Keck Center hosts a number of educational events, including a weekly seminar during the spring and fall semesters, available via webcast, and the Keck Annual Research Conference in the fall.

Sealy Center for Structural Biology & Molecular Biophysics Structure-based studies of proteins and other biological molecules are a key aspect to understanding the molecular basis for disease, as well as for designing drugs to treat disease. The Sealy Center for Structural Biology and Molecular Biophysics (SCSB) was established in 1995 to provide UTMB with state-of-the-art resources for structural and functional studies of biological macromolecules. However, unlike traditional structural biology centers, whose research efforts are directed almost entirely toward structure determination, SCSB was founded on the principle that the success of structural biology in medical research is predicated on an understanding of how structure is linked to function. Consequently, in addition to traditional research programs that use X-ray crystallography and nuclear magnetic resonance (NMR), recruitment in SCSB also included faculty whose expertise cover experimental and theoretical biophysics, as well as computational biology. Today the SCSB consists of 22 core members, 13 associate members and 6 managers (from 8 departments), and the breadth of research spans all aspects of molecular biophysics and biochemistry, addressing such fundamental issues as molecular recognition, signal transduction, allosteric regulation, protein folding, systems biology, and drug design.

http://www.gulfcoastconsortia.org/

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Scientific Program……………………………………………………………..4

Sponsors………………………………………………………………………...…6

Speaker Abstracts……………………………………………………………..9

Conference Layout …………………..……………......................15

Poster Presentation-Lists of Abstracts…………………...………16

Poster Presentation alphabetized list of presenters…….…20

Poster Abstracts…………………………………………….…………………25

List of Attendees…………………………………………………………...108

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SPONSORS

On behalf of everyone attending the Symposium, the Organizing Committee

thanks those who have provided us with financial support. We are grateful to

the following organizations for their generous assistance.

http://www.artrobbinsinstruments.com/

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Lecture L-1

Ribosomes, Their Tiny Enemies and

Thoughts About Their Origin

Ada Yonath, Ph.D. Department of Structural Biology, Weizmann Institute, Rehovot, Israel

The crystal structures of ribosomes, the universal cellular machines that

translate the genetic code into proteins, led to novel insights into the ribosomal

function, including decoding with high fidelity, catalysis, polymerization and

chaperoning properties. They also led to suggestions concerning the ribosome

origin. In parallel they shed light on the action, selectivity and synergism of

ribosomal antibiotics; illuminated mechanisms acquiring bacterial resistance and

provided structural information for drug improvement and design.

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Lecture L-2

NMR Information on Partly Folded

Proteins

H. Jane Dyson, Ph.D Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA

The chaperone Hsp90 interacts with a relatively large set of proteins, termed client

proteins, but their state, whether folded, partly folded or alternatively folded is not clear.

NMR experiments on client proteins in the presence and absence of Hsp90 provide

unprecedented insights into the conformational states of client proteins. As well, the NMR

spectra of the chaperone itself in the presence of the client protein and of co-chaperones give

information on interaction sites. NMR experiments on such large proteins and protein

complexes are difficult both of execution and interpretation, but allow the formulation of

hypotheses that can be tested by other spectroscopic means. We find that several client

proteins form molten globule-like states when in the presence of Hsp90. The interactions

between one client protein, the p53 DNA-binding domain, and fragments of Hsp90 of various

sizes, comprising 1 and 2 domains of the protein, up to the full-length dimeric protein showed

loss of signal intensity of the p53 resonances in the complex, from which we infer the

presence of a state with secondary structure indistinguishable from that of the free protein,

but containing a manifold of states that are in intermediate exchange on the NMR time scale.

Further evidence for the loose and flexible nature of the bound p53 client is provided by the

fluorescence behavior of the dye 1-anilinonaphthalene-8-sulfonic acid (ANS) and by

comparison of H/D exchange rates of the amide protons of the p53 DNA binding domain.

Hsp90 itself appears to make highly dynamic interactions with the client protein, which are

modified in the presence of co-chaperones such as p23. We conclude that the interaction

between Hsp90 and p53 is complex, and involves a structural change in the client protein to a

loosened state.

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Lecture L-3

Progress in Understanding the Secretion

of Exoproteins Like Cholera Toxin by the

Two-membrane-spanning Type II

Secretion System (T2SS)

Wim G. J. Hol, Ph.D. University of Washington, Seattle, Washington, USA

Many Gram-negative bacteria use the type II secretion system (T2SS) to translocate a

wide variety of folded exoproteins, which can be large, multimeric and disulfide bridge-

containing, from the periplasm through the outer membrane into the extracellular milieu. The

T2SS is important for pathogenic and non-pathogenic species alike. The T2SS is a

sophisticated and dynamic multi-protein machinery containing multiple copies of 12–15

different proteins which are generally encoded on a single operon. Four T2SS sub-

assemblies can be distinguished: the pseudopilus, the outer membrane complex, the inner

membrane platform and the secretion ATPase. The pseudopilus is a periplasmic fibrous

structure formed by five different pseudopilins with multiple copies of the major pseudopilin

GspG. The inner membrane platform has a central role in the T2SS mechanism of action

since it communicates with all other elements of the system. The inner membrane complex

might have a key role in converting conformational changes in the secretion ATPase due to

ATP hydrolysis into an extension of the pseudopilus which possibly acts as a piston that

pushes exoproteins through the outer membrane channel. Structural and biochemical studies

will be presented describing our current understanding, and mysteries remaining, of the

architecture and the mechanism of the T2SS with an emphasis on the T2SS from Vibrio

cholerae and the secretion of the secretion of cholera toxin.

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Lecture L-4

"DNA: Not Merely the Secret of Life"

Nadrian C. Seeman, Ph.D. Department of Chemistry, New York University, New York, NY

We build branched DNA species that can be joined using sticky ends to produce N-

connected objects and lattices. We have used ligation to construct DNA stick-polyhedra

and topological targets, such as Borromean rings. Branched junctions with up to 12 arms

have been produced.

Nanorobotics is a key area of application. We have made robust 2-state and 3-state

sequence-dependent devices that change states by varied hybridization topology. Bipedal

walkers, both clocked and autonomous have been built. We have constructed a molecular

assembly line by combining a DNA origami layer with three 2-state devices, so that there

are eight different states represented by their arrangements. We have demonstrated that all

eight products (including the null product) can be built from this system.

A central goal of DNA nanotechnology is the self-assembly of periodic matter. We have

constructed 2-dimensional DNA arrays with designed patterns from many different motifs.

We have used DNA scaffolding to organize active DNA components. Active DNA

components include DNAzymes and DNA nanomechanical devices; both are active when

incorporated in 2D DNA lattices. We have used pairs of 2-state devices to capture a

variety of different targets. Multi-tile DNA arrays have been used to organize gold

nanoparticles in specific arrangements.

One of the key aims of DNA-based materials research is to construct complex material

patterns that can be reproduced. We have recently built such a system from bent TX

molecules, which can reach 2 generations of replication. This system represents a first step

in self-reproducing materials.

Recently, we have self-assembled a 3D crystalline array and have solved its crystal

structure to 4 Å resolution, using unbiased crystallographic methods, shown below. More

than ten other crystals have been designed following the same principles of sticky-ended

cohesion. We can use crystals with two molecules in the crystallographic repeat to control

the color of the crystals. Thus, structural DNA nanotechnology has fulfilled its initial goal

of controlling the structure of matter in three dimensions. A new era in nanoscale control

awaits us. This research has been supported by the NIGMS, NSF, ARO, ONR and DOE.

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Lecture L-5

"Conformational Activation of Src/

CDK-like Kinase Domains"

Carol Post, Ph.D.

Department of Medicinal & Molecular Pharmacology, Purdue University, West Lafayette,

IN

Kinases are major players in signaling pathways that control a large number of vital

cellular processes. Tight regulation of their enzymatic activity is critical for the cell to

function normally, as exemplified by Src kinase, the gene product of one of the first

oncogenes described. Regulatory processes vary but often involve a step in which the

kinase domain undergoes a change in 3-dimensional structure between the catalytically

active conformational state and the state where activity is suppressed. We will describe the

energetics and molecular mechanism of the Src conformational transition elucidated using

computational approaches. Different kinases share a common 3-dimensional structure in

the active state, whereas a plasticity exists in the conformation of the inactive state. What

is energetically key to the transition process is yet to be elucidated. We have fully

characterized the activation process by calculating the optimal transition path. Examination

of the protein conformations along the optimal path reveals that displacement of the αC

helix is the underlying cause of the major free energy barrier (Fig. 1). Further, the critical

role of the αC helix, among several structural features that distinguish the active and

inactive states, is potentially relevant to many kinases; we find that kinases with the αC

helix displacement exist throughout the kinome, suggesting that this feature may have

emerged early in evolution. Important here is that a number of experimentally known

regulatory mechanisms of these kinases involve interactions to stabilize the position of the

αC helix. The results of our study provide the energetic basis supporting a common

explanation of these diverse regulatory mechanisms.

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Lecture L-6

"Osmophobics and Hydrophobics:

The Changing Landscape of Protein

Folding"

B. Montgomery Pettitt Department of Biochemistry & Molecular Biology, Sealy Center for Structural Biology and

Molecular Biophysics, University of Texas Medical Branch at Galveston, Galveston, TX

We discuss recent experiments and theories concerning protein collapse and

folding. Experiments using multicomponent solutions have revealed much about

the mechanism of folding. Simulation and theory have been used to interpret

thermodynamic and fluorescence correlation spectroscopy experimental results.

New measures of protein stability tendencies offer a different view than the

poorly defined hydrophobic effect.

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Poster Presentations

List of Abstracts– numbering corresponds to the location of the posters

P#P# NameName TitleTitle

PP--0101 Mark AndrewMark Andrew WhiteWhite The Many Conformations of EPAC2: a CyclicThe Many Conformations of EPAC2: a Cyclic--AMP Sensing Cellular AMP Sensing Cellular Regulator Studied via Solution XRegulator Studied via Solution X--Ray Scattering (SAXS) & Hydro-Ray Scattering (SAXS) & Hydro-gen Deuterium Exchange Mass Spectrometry.gen Deuterium Exchange Mass Spectrometry.

PP--0202 MadelineMadeline FarleyFarley Structural Differences Between Postsynaptic Densities Isolated Structural Differences Between Postsynaptic Densities Isolated From Different Brain RegionsFrom Different Brain Regions

PP--0303 MiguelMiguel--AngelAngel ElizondoElizondo--RiojasRiojas NMR Structure of Human Thymosin AlphaNMR Structure of Human Thymosin Alpha--11

PP--0404 ArnoldArnold VainrubVainrub SuperSuper--Resolution Optical Microscopy Development At University Resolution Optical Microscopy Development At University of Texas Medical Branch Galvestonof Texas Medical Branch Galveston

PP--0505 AndriaAndria DenmonDenmon The Role Of Base Modification On TyrsylThe Role Of Base Modification On Tyrsyl--TRNA Thermodynamics, TRNA Thermodynamics, Structure, And Function In Structure, And Function In Bacillus Subtilis And Bacillus AnthracisBacillus Subtilis And Bacillus Anthracis

PP--0606 WenzongWenzong LiLi Crystal Structure of Human AsparaginaseCrystal Structure of Human Asparaginase--Like Protein 1 Bound to Like Protein 1 Bound to Product LProduct L--Aspartic AcidAspartic Acid

PP--0707 RyanRyan RochatRochat Direct Electron Detection Yields CryoDirect Electron Detection Yields Cryo--EM Reconstructions At Res-EM Reconstructions At Res-oolluutitioonnss BBeeyyoonndd ¾¾ NNyyqquuiisstt FFrreeqquueennccyy

PP--0808 TianzhiTianzhi WangWang Research At the UTMB NMR Spectroscopy LaboratoryResearch At the UTMB NMR Spectroscopy Laboratory

PP--0909 Mark AndrewMark Andrew WhiteWhite The New UTMB SCSB Biological SAXS FacilityThe New UTMB SCSB Biological SAXS Facility

PP--1010 R. Adikaram R. Adikaram BandaraBandara CoCo--Crystallization of the StreptavidinCrystallization of the Streptavidin--Biotin With a LanthanideBiotin With a Lanthanide--Ligand Complex Gives Rise to a Novel Crystal FormLigand Complex Gives Rise to a Novel Crystal Form

PP--1111 RossiRossi IrobalievaIrobalieva Enhanced Visualization of a 52 KDa HIVEnhanced Visualization of a 52 KDa HIV--1 RNA By Zernike Phase 1 RNA By Zernike Phase Contrast CryoContrast Cryo--Electron TomographyElectron Tomography

PP--1212 MichaelMichael ShermanSherman CryoCryo--EM Reconstruction of Eilat AlphavirusEM Reconstruction of Eilat Alphavirus

PP--1313 Prem RajPrem Raj JosephJoseph Structural Characterization of InterleukinStructural Characterization of Interleukin--8 Monomer and Dimer 8 Monomer and Dimer Interactions With Glycosaminoglycans: Implications for in Vivo Interactions With Glycosaminoglycans: Implications for in Vivo

PP--1414 XianganXiangan LiuLiu Structural Study of HSVStructural Study of HSV--1 Virion Using Cryo1 Virion Using Cryo--EM and Bioinfor-EM and Bioinfor-maticsmatics

PP--1515 ChuanChuan HongHong CryoCryo--EM of Genome Packaging and Delivery of Bacteriophage EM of Genome Packaging and Delivery of Bacteriophage PRD1PRD1

PP--1616 WeiWei DaiDai Visualizing CyanobacteriaVisualizing Cyanobacteria--Phage Interactions By CryoPhage Interactions By Cryo--Electron Electron TomographyTomography

PP--1717 Jaimy Jaimy MillerMiller Membrane Bound Organization of Blood Coagulation Factor VIII Membrane Bound Organization of Blood Coagulation Factor VIII Forms Onto Negatively Charged Lipid Nanotubes.Forms Onto Negatively Charged Lipid Nanotubes.

PP--1818 KirillKirill GrushinGrushin CryoCryo--EM and Biophysical Characterization of Recombinant Coagu-EM and Biophysical Characterization of Recombinant Coagu-lation Factor VIII Forms Bound to PSlation Factor VIII Forms Bound to PS--Containing VesiclesContaining Vesicles

PP--1919 MayukhMayukh SarkarSarkar Architecture of Type IV Secretion Systems in Gram Positive Bacte-Architecture of Type IV Secretion Systems in Gram Positive Bacte-ria: in Silico 3D Protein Modeling and Comparison With Gram ria: in Silico 3D Protein Modeling and Comparison With Gram

PP--2020 IanIan ReesRees Constructing Initial Models From Subnanometer Density Maps Constructing Initial Models From Subnanometer Density Maps With Pathwalker and Additional Protein Backbone ConstraintsWith Pathwalker and Additional Protein Backbone Constraints

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PP--2121 Keerthi Keerthi GottipatiGottipati Biochemical and Structural Characterization of Pestivirus NBiochemical and Structural Characterization of Pestivirus N--Terminal Protease (N Pro)Terminal Protease (N Pro)

PP--2222 AaronAaron CollierCollier Biochemical and Biophysical Characterization of the RNA Replica-Biochemical and Biophysical Characterization of the RNA Replica-tion Mechanism of a Small DsRNA Virustion Mechanism of a Small DsRNA Virus

PP--2323 GilbertGilbert HuangHuang Crystal Structures of the Carboxyl CGMP Binding Domain of Hu-Crystal Structures of the Carboxyl CGMP Binding Domain of Hu-man PKGI Reveal the Mechanism of CGMP Selectivityman PKGI Reveal the Mechanism of CGMP Selectivity

PP--2424 KyoungKyoung--JaeJae ChoiChoi Structural Insights into a Novel Class of NStructural Insights into a Novel Class of N--Linked Protein Glycosyl-Linked Protein Glycosyl-ation By the ation By the Actinobacillus Pleuropneumoniae HMW1CActinobacillus Pleuropneumoniae HMW1C--Like Pro-Like Pro-teintein

PP--2525 Monica Monica Galaz MontoyaGalaz Montoya BETA 2BETA 2--Adrenergic Receptor Elicits a Calcium Signal in HEKAdrenergic Receptor Elicits a Calcium Signal in HEK--293 293 CellsCells

PP--2626 MichaelMichael BoltBolt Chromatin Interplay Between ERChromatin Interplay Between ERα α and GR Fineand GR Fine--Tunes Transcrip-Tunes Transcrip-tional Outputtional Output

PP--2727 VaratharasaVaratharasa ThiviyanathanThiviyanathan Thioaptamers for NanoparticleThioaptamers for Nanoparticle--based Targeted Delivery of Drugs based Targeted Delivery of Drugs and Imaging Agentsand Imaging Agents

PP--2828 WeiguoWeiguo HeHe BeadBead--Based Combinatorial Selection of XBased Combinatorial Selection of X--Aptamers Targeted to Aptamers Targeted to the Hyaluronic Acid Binding Domain of Human CD44the Hyaluronic Acid Binding Domain of Human CD44

PP--2929 PaigePaige SpencerSpencer Silent Substitutions Predictably Alter Translation Elongation Rates Silent Substitutions Predictably Alter Translation Elongation Rates and Protein Folding Efficienciesand Protein Folding Efficiencies

PP--3030 David C.David C. MarcianoMarciano Genetic Feedback Provides Robustness to MutationGenetic Feedback Provides Robustness to Mutation

PP--3131 Aditya Aditya HindupurHindupur Design of An AntiDesign of An Anti--HIV Protease TransducerHIV Protease Transducer

PP--3232 Sai HariSai Hari GandhamGandham Novel Class of Thioaptamers Targeting Dengue Virus Envelope Novel Class of Thioaptamers Targeting Dengue Virus Envelope ProteinProtein

PP--3333 GaryGary StinnettStinnett Development of a Novel Method to Image Development of a Novel Method to Image α7 α7 Nicotinic Acetylcho-Nicotinic Acetylcho-line Receptor Concentrationline Receptor Concentration

PP--3434 KuangKuang--Yui Yui ChenChen Reprogramming the Conformational Regulation of GPCR SignalingReprogramming the Conformational Regulation of GPCR Signaling

PP--3535 GuoxiongGuoxiong SuSu Multiscale Simulation on a LightMultiscale Simulation on a Light--Harvesting Molecular TriadHarvesting Molecular Triad

PP--3636 QianQian WangWang A PhysicsA Physics--Based Approach of CoarseBased Approach of Coarse--Graining the Cytoplasm of Graining the Cytoplasm of E.Coli (CGCYTO)E.Coli (CGCYTO)

PP--3737 EmilioEmilio ReyesAldreteReyesAldrete Biochemical and Biophysical Characterization of the Putative En-Biochemical and Biophysical Characterization of the Putative En-capsidation Protein From capsidation Protein From Lactococcus Lactis Phage AsccLactococcus Lactis Phage Asccφ28φ28

PP--3838 ChanceChance MooneyMooney Voltage Dependent Conformational States of the Transmembrane Voltage Dependent Conformational States of the Transmembrane Protein Prestin Measured By FLIMProtein Prestin Measured By FLIM--FRET TechniquesFRET Techniques

PP--3939 AlexandreAlexandre EsadzeEsadze Quantitative Characterization of Facilitated Target Search During Quantitative Characterization of Facilitated Target Search During ProteinProtein--DNA InteractionsDNA Interactions

PP--4040 Michal R.Michal R. SzymanskiSzymanski HelicaseHelicase--Initiated Assembly of the PrimosomeInitiated Assembly of the Primosome

PP--4141 KimalKimal RajapaksheRajapakshe LabelLabel--Free and Noninvasive Sensor for Measuring Membrane Po-Free and Noninvasive Sensor for Measuring Membrane Po-tentials of Biological Cellstentials of Biological Cells

PP--4242 LuisLuis HolthauzenHolthauzen Solution Biophysics Research LaboratorySolution Biophysics Research Laboratory

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PP--4343 ChristopherChristopher MyersMyers Predicting Pressure of CapsidPredicting Pressure of Capsid--Confined DNAConfined DNA

PP--4444 LevaniLevani ZandarashviliZandarashvili Asymmetrical Roles of Zinc Fingers in Dynamic DNAAsymmetrical Roles of Zinc Fingers in Dynamic DNA--Scanning Pro-Scanning Pro-cess By the Inducible Transcription Factor Egrcess By the Inducible Transcription Factor Egr--11

PP--4545 JunjiJunji IwaharaIwahara IonIon--Pair Dynamics At ProteinPair Dynamics At Protein--DNA Interface Studied By NMR DNA Interface Studied By NMR SpectroscopySpectroscopy

PP--4646 PaulPaul BujalowskiBujalowski Tracking UNCTracking UNC--45 Chaperone45 Chaperone--Myosin Interaction With a Titin Me-Myosin Interaction With a Titin Me-chanical Reporterchanical Reporter

PP--4747 ArijitArijit DuttaDutta Role of Disordered CRole of Disordered C--Terminus in Human DNA Glycosylase Terminus in Human DNA Glycosylase NEIL1’S Structure and Functions: Implications for the Excision Re-NEIL1’S Structure and Functions: Implications for the Excision Re-pair of Oxidized Bases in Genomepair of Oxidized Bases in Genome

PP--4848 NandiniNandini MaharajMaharaj Characterizing the Structural Dynamics of Mutant KCharacterizing the Structural Dynamics of Mutant K--Ras in Devel-Ras in Devel-opmental Disorders.opmental Disorders.

PP--4949 KevinKevin LiuLiu Population Genomics of the Mouse and Its Functional Implica-Population Genomics of the Mouse and Its Functional Implica-tionstions

PP--5050 IrisIris Nira SmithNira Smith Structural Mutation Analysis of PTEN and Its Possible GenotypeStructural Mutation Analysis of PTEN and Its Possible Genotype--Phenotype Correlations in Endometriosis and CancerPhenotype Correlations in Endometriosis and Cancer

PP--5151 HualinHualin LiLi A Theoretical Study of MembraneA Theoretical Study of Membrane--Remodeling By Ras ProteinsRemodeling By Ras Proteins

PP--5252 JiayiJiayi SunSun Epigenomic Profiling of the Osteosarcoma GenomeEpigenomic Profiling of the Osteosarcoma Genome

PP--5353 Catherine H.Catherine H. ScheinSchein Repeated Sequences in Nut Allergens Mediate Cross Reactivity Repeated Sequences in Nut Allergens Mediate Cross Reactivity Between Tree Nuts and PeanutsBetween Tree Nuts and Peanuts

PP--5454 RosaRosa BanuelosBanuelos Practical Issues for Designing Efficient SequencePractical Issues for Designing Efficient Sequence--Based Genetic Based Genetic Studies of Quantitative TraitsStudies of Quantitative Traits

PP--5555 ZhenlongZhenlong LiLi Membrane Shape Remodeling By LipidMembrane Shape Remodeling By Lipid--Modified Ras ProteinsModified Ras Proteins

PP--5656 Yifeng Yifeng JiangJiang Vessel Connectivity Using Murray HypothesisVessel Connectivity Using Murray Hypothesis

PP--5757 StanleyStanley HookerHooker Keloid Susceptibility Loci 1q41, 3q22, and 15q21 Replicated in a Keloid Susceptibility Loci 1q41, 3q22, and 15q21 Replicated in a Nigerian PopulationNigerian Population

PP--5858 NinadNinad DewalDewal Calling Amplified Or Deleted Haplotypes in Next Generation Tu-Calling Amplified Or Deleted Haplotypes in Next Generation Tu-mor Sequencing Datamor Sequencing Data

PP--5959 HarrisonHarrison HockerHocker Identification of Novel Allosteric Sites on Ras and Initial Screening Identification of Novel Allosteric Sites on Ras and Initial Screening for Potential Inhibitorsfor Potential Inhibitors

PP--6060 CalebCaleb GoodwinGoodwin Biomedical Discovery Seeking Support System Based on a Model Biomedical Discovery Seeking Support System Based on a Model of Human Associative Memoryof Human Associative Memory

PP--6161 JesusJesus GalazGalaz Single Particle Tomography in EMAN2Single Particle Tomography in EMAN2

PP--6262 PriyankaPriyanka PrakashPrakash Aggregation Behavior of Indomethacin, Cholic Acid and POPCAggregation Behavior of Indomethacin, Cholic Acid and POPC

PP--6363 SasiSasi KodathalaKodathala Asymmetry in the Homodimer of Glutathione Synthetase May Asymmetry in the Homodimer of Glutathione Synthetase May Contribute to Negative Cooperativity.Contribute to Negative Cooperativity.

PP--6464 SajanaSajana SudarshanSudarshan FLIPS and FunCs: Identification and Prediction of Protein Interac-FLIPS and FunCs: Identification and Prediction of Protein Interac-tions Using Energy and Evolutionary Pressuretions Using Energy and Evolutionary Pressure

PP--6565 IshaIsha MehtaMehta Prediction of Functionally Important Residues in Protein:Protein Prediction of Functionally Important Residues in Protein:Protein Interactions By Network AnalysisInteractions By Network Analysis

PP--6666 AmrutaAmruta MahadikMahadik Computational Analysis of Interactions Between ARP2/3 and Computational Analysis of Interactions Between ARP2/3 and WAVE At a Molecular LevelWAVE At a Molecular Level

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PP--6767 JohnJohn Craft, Jr. Craft, Jr. Docking Rho Kinase 1 Catalytic and PH Domain and Inhibition in Docking Rho Kinase 1 Catalytic and PH Domain and Inhibition in Heart DiseaseHeart Disease

PP--6868 TangenTangen TranTran PKa and Electrostatic Analysis of ROCK1 Catalytic DomainPKa and Electrostatic Analysis of ROCK1 Catalytic Domain

PP--6969 MingyangMingyang LuLu FSUB: Normal Mode Analysis With Flexible SubstructuresFSUB: Normal Mode Analysis With Flexible Substructures

PP--7070 SurendraSurendra Negi Negi Structural Studies on the Envelope Proteins E1 and E2 of Ever-Structural Studies on the Envelope Proteins E1 and E2 of Ever-glades Virusglades Virus

PP--7171 WenzheWenzhe LuLu PhysicoPhysico--Chemical Property (PCP) Motifs to Identify Proteins that Chemical Property (PCP) Motifs to Identify Proteins that Could be CrossCould be Cross--Reactive With Known Peanut AllergensReactive With Known Peanut Allergens

PP--7272 DeeptiDeepti KarandurKarandur Thermodynamics of Aggregation of Gly5Thermodynamics of Aggregation of Gly5

PP--7373 ChristineChristine PetersonPeterson Inferring Metabolic Networks Using the Bayesian Graphical Las-Inferring Metabolic Networks Using the Bayesian Graphical Las-soso

PP--7474 PeterPeter KilloranKilloran Impact of Electronic Medical Record Interface on Physician Task Impact of Electronic Medical Record Interface on Physician Task CompletionCompletion

PP--7575 RhonaldRhonald LuaLua Periodic Table of Knotted Proteins: Are We Missing Some Periodic Table of Knotted Proteins: Are We Missing Some Knots?Knots?

PP--7676 BryantBryant GibsonGibson Constrained Conformational Analysis: Protein Motion Planning Constrained Conformational Analysis: Protein Motion Planning With CryoWith Cryo--Electron MicroscopyElectron Microscopy

PP--7777 AlexanderAlexander ShavkunovShavkunov Differential Impact of FGF14 Mutations on ProteinDifferential Impact of FGF14 Mutations on Protein--Protein In-Protein In-teraction Interfaces in Macromolecular Complexesteraction Interfaces in Macromolecular Complexes

PP--7878 XuelingXueling LiLi The JustThe Just--InIn--Time Expression of Yeast Ribosomal Protein Genes Time Expression of Yeast Ribosomal Protein Genes for Ribosome Assembly and Maturation Processfor Ribosome Assembly and Maturation Process

PP--7979 JasonJason AllisonAllison Investigation of RNA Binding Properties of the HIVInvestigation of RNA Binding Properties of the HIV--1 Rev Protein 1 Rev Protein Using a Chimeric tRNAPheUsing a Chimeric tRNAPhe

PP--8080 YavarYavar VafaeeVafaee Codon Optimization and Construction of Different Recombinant Codon Optimization and Construction of Different Recombinant Vectors Containing AntiVectors Containing Anti--HIV Griffithsin GeneHIV Griffithsin Gene

PP--8181 BehroozBehrooz SarabiSarabi Evaluation of Some Morphological Characteristics, Mineral Ele-Evaluation of Some Morphological Characteristics, Mineral Ele-ments of Spear and Physical Properties of Seed of Edible Aspar-ments of Spear and Physical Properties of Seed of Edible Aspar-aagguuss ((AAssppaarraagguuss OOffifficciinnaalliiss LL..))

PP--8282 MohammadMohammad GerdakenehGerdakeneh The Effect of Thidiazuron on Direct Somatic Embryogenesis The Effect of Thidiazuron on Direct Somatic Embryogenesis Pathway of Strawberry (Fragaria Ananassa Duch.)Pathway of Strawberry (Fragaria Ananassa Duch.)

PP--8383 NedaNeda MalekitabriziMalekitabrizi Transient Expression of Urease b From Iranian Helicobacter Py-Transient Expression of Urease b From Iranian Helicobacter Py-lori in Lettucelori in Lettuce

20


P#P# NameName TitleTitle

PP--7979 JasonJason AllisonAllison Investigation of RNA Binding Properties of the HIVInvestigation of RNA Binding Properties of the HIV--1 Rev Protein Using 1 Rev Protein Using a Chimeric tRNAPhea Chimeric tRNAPhe

PP--1010 R. Adikaram R. Adikaram BandaraBandara CoCo--Crystallization of the StreptavidinCrystallization of the Streptavidin--Biotin With a LanthanideBiotin With a Lanthanide--Ligand Ligand Complex Gives Rise to a Novel Crystal FormComplex Gives Rise to a Novel Crystal Form

PP--5454 RosaRosa BanuelosBanuelos Practical Issues for Designing Efficient SequencePractical Issues for Designing Efficient Sequence--Based Genetic Studies Based Genetic Studies of Quantitative Traitsof Quantitative Traits

PP--2626 MichaelMichael BoltBolt Chromatin Interplay Between ERChromatin Interplay Between ERα α and GR Fineand GR Fine--Tunes Transcriptional Tunes Transcriptional OutputOutput

PP--4646 PaulPaul BujalowskiBujalowski Tracking UNCTracking UNC--45 Chaperone45 Chaperone--Myosin Interaction With a Titin Mechani-Myosin Interaction With a Titin Mechani-cal Reportercal Reporter

PP--3434 KuangKuang--Yui Yui ChenChen Reprogramming the Conformational Regulation of GPCR SignalingReprogramming the Conformational Regulation of GPCR Signaling

PP--2424 KyoungKyoung--JaeJae ChoiChoi Structural Insights into a Novel Class of NStructural Insights into a Novel Class of N--Linked Protein Glycosylation Linked Protein Glycosylation By the By the Actinobacillus Pleuropneumoniae HMW1CActinobacillus Pleuropneumoniae HMW1C--Like ProteinLike Protein

PP--2222 AaronAaron CollierCollier Biochemical and Biophysical Characterization of the RNA Replication Biochemical and Biophysical Characterization of the RNA Replication Mechanism of a Small DsRNA VirusMechanism of a Small DsRNA Virus

PP--6767 JohnJohn Craft, Jr. Craft, Jr. Docking Rho Kinase 1 Catalytic and PH Domain and Inhibition in Heart Docking Rho Kinase 1 Catalytic and PH Domain and Inhibition in Heart DiseaseDisease

PP--1616 WeiWei DaiDai Visualizing CyanobacteriaVisualizing Cyanobacteria--Phage Interactions By CryoPhage Interactions By Cryo--Electron Tomog-Electron Tomog-raphyraphy

PP--0505 AndriaAndria DenmonDenmon The Role Of Base Modification On TyrsylThe Role Of Base Modification On Tyrsyl--TRNA Thermodynamics, Struc-TRNA Thermodynamics, Struc-ture, And Function In ture, And Function In Bacillus Subtilis And Bacillus AnthracisBacillus Subtilis And Bacillus Anthracis

PP--5858 NinadNinad DewalDewal Calling Amplified Or Deleted Haplotypes in Next Generation Tumor Se-Calling Amplified Or Deleted Haplotypes in Next Generation Tumor Se-quencing Dataquencing Data

PP--4747 ArijitArijit DuttaDutta Role of Disordered CRole of Disordered C--Terminus in Human DNA Glycosylase NEIL1’S Terminus in Human DNA Glycosylase NEIL1’S Structure and Functions: Implications for the Excision Repair of Oxi-Structure and Functions: Implications for the Excision Repair of Oxi-

PP--0303 MiguelMiguel--AngelAngel ElizondoElizondo--RiojasRiojas NMR Structure of Human Thymosin AlphaNMR Structure of Human Thymosin Alpha--11

PP--3939 AlexandreAlexandre EsadzeEsadze Quantitative Characterization of Facilitated Target Search During Pro-Quantitative Characterization of Facilitated Target Search During Pro-teintein--DNA InteractionsDNA Interactions

PP--0202 MadelineMadeline FarleyFarley Structural Differences Between Postsynaptic Densities Isolated From Structural Differences Between Postsynaptic Densities Isolated From Different Brain RegionsDifferent Brain Regions

PP--6161 JesusJesus GalazGalaz Single Particle Tomography in EMAN2Single Particle Tomography in EMAN2

PP--2525 Monica Monica Galaz MontoyaGalaz Montoya BETA 2BETA 2--Adrenergic Receptor Elicits a Calcium Signal in HEKAdrenergic Receptor Elicits a Calcium Signal in HEK--293 Cells293 Cells

PP--3232 Sai HariSai Hari GandhamGandham Novel Class of Thioaptamers Targeting Dengue Virus Envelope ProteinNovel Class of Thioaptamers Targeting Dengue Virus Envelope Protein

PP--8282 MohammadMohammad GerdakenehGerdakeneh The Effect of Thidiazuron on Direct Somatic Embryogenesis Pathway of The Effect of Thidiazuron on Direct Somatic Embryogenesis Pathway of Strawberry (Fragaria Ananassa Duch.)Strawberry (Fragaria Ananassa Duch.)

PP--7676 BryantBryant GibsonGibson Constrained Conformational Analysis: Protein Motion Planning With Constrained Conformational Analysis: Protein Motion Planning With CryoCryo--Electron MicroscopyElectron Microscopy

PP--6060 CalebCaleb GoodwinGoodwin Biomedical Discovery Seeking Support System Based on a Model of Hu-Biomedical Discovery Seeking Support System Based on a Model of Hu-man Associative Memoryman Associative Memory

PP--2121 Keerthi Keerthi GottipatiGottipati Biochemical and Structural Characterization of Pestivirus NBiochemical and Structural Characterization of Pestivirus N--Terminal Terminal Protease (N Pro)Protease (N Pro)

21


PP--1818 KirillKirill GrushinGrushin CryoCryo--EM and Biophysical Characterization of Recombinant Coagulation EM and Biophysical Characterization of Recombinant Coagulation Factor VIII Forms Bound to PSFactor VIII Forms Bound to PS--Containing VesiclesContaining Vesicles

PP--2828 WeiguoWeiguo HeHe BeadBead--Based Combinatorial Selection of XBased Combinatorial Selection of X--Aptamers Targeted to the Aptamers Targeted to the Hyaluronic Acid Binding Domain of Human CD44Hyaluronic Acid Binding Domain of Human CD44

PP--3131 Aditya Aditya HindupurHindupur Design of An AntiDesign of An Anti--HIV Protease TransducerHIV Protease Transducer

PP--5959 HarrisonHarrison HockerHocker Identification of Novel Allosteric Sites on Ras and Initial Screening for Identification of Novel Allosteric Sites on Ras and Initial Screening for Potential InhibitorsPotential Inhibitors

PP--4242 LuisLuis HolthauzenHolthauzen Solution Biophysics Research LaboratorySolution Biophysics Research Laboratory

PP--1515 ChuanChuan HongHong CryoCryo--EM of Genome Packaging and Delivery of Bacteriophage PRD1EM of Genome Packaging and Delivery of Bacteriophage PRD1

PP--5757 StanleyStanley HookerHooker Keloid Susceptibility Loci 1q41, 3q22, and 15q21 Replicated in a Nigeri-Keloid Susceptibility Loci 1q41, 3q22, and 15q21 Replicated in a Nigeri-an Populationan Population

PP--2323 GilbertGilbert HuangHuang Crystal Structures of the Carboxyl CGMP Binding Domain of Human Crystal Structures of the Carboxyl CGMP Binding Domain of Human PKGI Reveal the Mechanism of CGMP SelectivityPKGI Reveal the Mechanism of CGMP Selectivity

PP--1111 RossiRossi IrobalievaIrobalieva Enhanced Visualization of a 52 KDa HIVEnhanced Visualization of a 52 KDa HIV--1 RNA By Zernike Phase Con-1 RNA By Zernike Phase Con-trast Cryotrast Cryo--Electron TomographyElectron Tomography

PP--4545 JunjiJunji IwaharaIwahara IonIon--Pair Dynamics At ProteinPair Dynamics At Protein--DNA Interface Studied By NMR Spectros-DNA Interface Studied By NMR Spectros-copycopy

PP--5656 Yifeng Yifeng JiangJiang Vessel Connectivity Using Murray HypothesisVessel Connectivity Using Murray Hypothesis

PP--1313 Prem RajPrem Raj JosephJoseph Structural Characterization of InterleukinStructural Characterization of Interleukin--8 Monomer and Dimer Inter-8 Monomer and Dimer Inter-actions With Glycosaminoglycans: Implications for in Vivo Neutrophil actions With Glycosaminoglycans: Implications for in Vivo Neutrophil RecruitmentRecruitment

PP--7272 DeeptiDeepti KarandurKarandur Thermodynamics of Aggregation of Gly5Thermodynamics of Aggregation of Gly5

PP--7474 PeterPeter KilloranKilloran Impact of Electronic Medical Record Interface on Physician Task Com-Impact of Electronic Medical Record Interface on Physician Task Com-pletionpletion

PP--6363 SasiSasi KodathalaKodathala Asymmetry in the Homodimer of Glutathione Synthetase May Contrib-Asymmetry in the Homodimer of Glutathione Synthetase May Contrib-ute to Negative Cooperativity.ute to Negative Cooperativity.

PP--0606 WenzongWenzong LiLi Crystal Structure of Human AsparaginaseCrystal Structure of Human Asparaginase--Like Protein 1 Bound to Prod-Like Protein 1 Bound to Prod-uct Luct L--Aspartic AcidAspartic Acid

PP--5151 HualinHualin LiLi A Theoretical Study of MembraneA Theoretical Study of Membrane--Remodeling By Ras ProteinsRemodeling By Ras Proteins

PP--5555 ZhenlongZhenlong LiLi Membrane Shape Remodeling By LipidMembrane Shape Remodeling By Lipid--Modified Ras ProteinsModified Ras Proteins

PP--7878 XuelingXueling LiLi The JustThe Just--InIn--Time Expression of Yeast Ribosomal Protein Genes for Ri-Time Expression of Yeast Ribosomal Protein Genes for Ri-bosome Assembly and Maturation Processbosome Assembly and Maturation Process

PP--1414 XianganXiangan LiuLiu Structural Study of HSVStructural Study of HSV--1 Virion Using Cryo1 Virion Using Cryo--EM and BioinformaticsEM and Bioinformatics

PP--4949 KevinKevin LiuLiu Population Genomics of the Mouse and Its Functional ImplicationsPopulation Genomics of the Mouse and Its Functional Implications

PP--6969 MingyangMingyang LuLu FSUB: Normal Mode Analysis With Flexible SubstructuresFSUB: Normal Mode Analysis With Flexible Substructures

PP--7171 WenzheWenzhe LuLu PhysicoPhysico--Chemical Property (PCP) Motifs to Identify Proteins that Could Chemical Property (PCP) Motifs to Identify Proteins that Could be Crossbe Cross--Reactive With Known Peanut AllergensReactive With Known Peanut Allergens

PP--7575 RhonaldRhonald LuaLua Periodic Table of Knotted Proteins: Are We Missing Some Knots?Periodic Table of Knotted Proteins: Are We Missing Some Knots?

PP--6666 AmrutaAmruta MahadikMahadik Computational Analysis of Interactions Between ARP2/3 and WAVE At Computational Analysis of Interactions Between ARP2/3 and WAVE At a Molecular Levela Molecular Level

PP--4848 NandiniNandini MaharajMaharaj Characterizing the Structural Dynamics of Mutant KCharacterizing the Structural Dynamics of Mutant K--Ras in Develop-Ras in Develop-mental Disorders.mental Disorders.

22


PP--8383 NedaNeda MalekitabriziMalekitabrizi Transient Expression of Urease b From Iranian Helicobacter Pylori in Transient Expression of Urease b From Iranian Helicobacter Pylori in LettuceLettuce

PP--3030 David C.David C. MarcianoMarciano Genetic Feedback Provides Robustness to MutationGenetic Feedback Provides Robustness to Mutation

PP--6565 IshaIsha MehtaMehta Prediction of Functionally Important Residues in Protein:Protein Inter-Prediction of Functionally Important Residues in Protein:Protein Inter-actions By Network Analysisactions By Network Analysis

PP--1717 Jaimy Jaimy MillerMiller Membrane Bound Organization of Blood Coagulation Factor VIII Forms Membrane Bound Organization of Blood Coagulation Factor VIII Forms Onto Negatively Charged Lipid Nanotubes.Onto Negatively Charged Lipid Nanotubes.

PP--3838 ChanceChance MooneyMooney Voltage Dependent Conformational States of the Transmembrane Pro-Voltage Dependent Conformational States of the Transmembrane Pro-tein Prestin Measured By FLIMtein Prestin Measured By FLIM--FRET TechniquesFRET Techniques

PP--4343 ChristopherChristopher MyersMyers Predicting Pressure of CapsidPredicting Pressure of Capsid--Confined DNAConfined DNA

PP--7070 SurendraSurendra Negi Negi Structural Studies on the Envelope Proteins E1 and E2 of Everglades Structural Studies on the Envelope Proteins E1 and E2 of Everglades VirusVirus

PP--5050 IrisIris Nira SmithNira Smith Structural Mutation Analysis of PTEN and Its Possible GenotypeStructural Mutation Analysis of PTEN and Its Possible Genotype--Phenotype Correlations in Endometriosis and CancerPhenotype Correlations in Endometriosis and Cancer

PP--7373 ChristineChristine PetersonPeterson Inferring Metabolic Networks Using the Bayesian Graphical LassoInferring Metabolic Networks Using the Bayesian Graphical Lasso

PP--6262 PriyankaPriyanka PrakashPrakash Aggregation Behavior of Indomethacin, Cholic Acid and POPCAggregation Behavior of Indomethacin, Cholic Acid and POPC

PP--4141 KimalKimal RajapaksheRajapakshe LabelLabel--Free and Noninvasive Sensor for Measuring Membrane Poten-Free and Noninvasive Sensor for Measuring Membrane Poten-tials of Biological Cellstials of Biological Cells

PP--2020 IanIan ReesRees Constructing Initial Models From Subnanometer Density Maps With Constructing Initial Models From Subnanometer Density Maps With Pathwalker and Additional Protein Backbone ConstraintsPathwalker and Additional Protein Backbone Constraints

PP--3737 EmilioEmilio ReyesAldreteReyesAldrete Biochemical and Biophysical Characterization of the Putative Encapsi-Biochemical and Biophysical Characterization of the Putative Encapsi-dation Protein From dation Protein From Lactococcus Lactis Phage AsccLactococcus Lactis Phage Asccφ28φ28

PP--0707 RyanRyan RochatRochat Direct Electron Detection Yields CryoDirect Electron Detection Yields Cryo--EM Reconstructions At Resolu-EM Reconstructions At Resolu-tions Beyond ¾ Nyquist Frequencytions Beyond ¾ Nyquist Frequency

PP--8181 BehroozBehrooz SarabiSarabi Evaluation of Some Morphological Characteristics, Mineral Elements of Evaluation of Some Morphological Characteristics, Mineral Elements of Spear and Physical Properties of Seed of Edible Asparagus (Asparagus Spear and Physical Properties of Seed of Edible Asparagus (Asparagus

PP--1919 MayukhMayukh SarkarSarkar Architecture of Type IV Secretion Systems in Gram Positive Bacteria: in Architecture of Type IV Secretion Systems in Gram Positive Bacteria: in Silico 3D Protein Modeling and Comparison With Gram Negative Bacte-Silico 3D Protein Modeling and Comparison With Gram Negative Bacte-riaria

PP--5353 Catherine H.Catherine H. ScheinSchein Repeated Sequences in Nut Allergens Mediate Cross Reactivity Be-Repeated Sequences in Nut Allergens Mediate Cross Reactivity Be-tween Tree Nuts and Peanutstween Tree Nuts and Peanuts

PP--7777 AlexanderAlexander ShavkunovShavkunov Differential Impact of FGF14 Mutations on ProteinDifferential Impact of FGF14 Mutations on Protein--Protein Interaction Protein Interaction Interfaces in Macromolecular ComplexesInterfaces in Macromolecular Complexes

PP--1212 MichaelMichael ShermanSherman CryoCryo--EM Reconstruction of Eilat AlphavirusEM Reconstruction of Eilat Alphavirus

PP--2929 PaigePaige SpencerSpencer Silent Substitutions Predictably Alter Translation Elongation Rates and Silent Substitutions Predictably Alter Translation Elongation Rates and Protein Folding EfficienciesProtein Folding Efficiencies

PP--3333 GaryGary StinnettStinnett Development of a Novel Method to Image Development of a Novel Method to Image α7 α7 Nicotinic Acetylcholine Nicotinic Acetylcholine Receptor ConcentrationReceptor Concentration

PP--3535 GuoxiongGuoxiong SuSu Multiscale Simulation on a LightMultiscale Simulation on a Light--Harvesting Molecular TriadHarvesting Molecular Triad

PP--6464 SajanaSajana SudarshanSudarshan FLIPS and FunCs: Identification and Prediction of Protein Interactions FLIPS and FunCs: Identification and Prediction of Protein Interactions Using Energy and Evolutionary PressureUsing Energy and Evolutionary Pressure

23


PP--5252 JiayiJiayi SunSun Epigenomic Profiling of the Osteosarcoma GenomeEpigenomic Profiling of the Osteosarcoma Genome

PP--4040 Michal R.Michal R. SzymanskiSzymanski HelicaseHelicase--Initiated Assembly of the PrimosomeInitiated Assembly of the Primosome

PP--2727 VaratharasaVaratharasa ThiviyanathanThiviyanathan Thioaptamers for NanoparticleThioaptamers for Nanoparticle--based Targeted Delivery of Drugs and based Targeted Delivery of Drugs and Imaging AgentsImaging Agents

PP--6868 TangenTangen TranTran PKa and Electrostatic Analysis of ROCK1 Catalytic DomainPKa and Electrostatic Analysis of ROCK1 Catalytic Domain

PP--8080 YavarYavar VafaeeVafaee Codon Optimization and Construction of Different Recombinant Vec-Codon Optimization and Construction of Different Recombinant Vec-tors Containing Antitors Containing Anti--HIV Griffithsin GeneHIV Griffithsin Gene

PP--0404 ArnoldArnold VainrubVainrub SuperSuper--Resolution Optical Microscopy Development At University of Resolution Optical Microscopy Development At University of Texas Medical Branch GalvestonTexas Medical Branch Galveston

PP--0808 TianzhiTianzhi WangWang Research At the UTMB NMR Spectroscopy LaboratoryResearch At the UTMB NMR Spectroscopy Laboratory

PP--3636 QianQian WangWang A PhysicsA Physics--Based Approach of CoarseBased Approach of Coarse--Graining the Cytoplasm of E.Coli Graining the Cytoplasm of E.Coli (CGCYTO)(CGCYTO)

PP--0101 Mark AndrewMark Andrew WhiteWhite The Many Conformations of EPAC2: a CyclicThe Many Conformations of EPAC2: a Cyclic--AMP Sensing Cellular Reg-AMP Sensing Cellular Reg-uullaattoorr SSttuuddiieedd vviiaa SSoolluutitioonn XX--RRaayy SSccaatttteerriinngg ((SSAAXXSS)) && HHyyddrrooggeenn DDeeuutteer-r-iiuumm EExxcchhaannggee MMaassss SSppeeccttrroommeettrryy..

PP--0909 Mark AndrewMark Andrew WhiteWhite The New UTMB SCSB Biological SAXS FacilityThe New UTMB SCSB Biological SAXS Facility

PP--4444 LevaniLevani ZandarashviliZandarashvili Asymmetrical Roles of Zinc Fingers in Dynamic DNAAsymmetrical Roles of Zinc Fingers in Dynamic DNA--Scanning Process Scanning Process By the Inducible Transcription Factor EgrBy the Inducible Transcription Factor Egr--11

24


25


Structure Poster Number P-1

The Many Conformations of EPAC2: a Cyclic-

AMP Sensing Cellular Regulator Studied via

Solution X-Ray Scattering (SAXS) & Hydrogen

Deuterium Exchange Mass Spectrometry

Mark Andrew White1, Tamara K. Tsalkova2, Sheng Li4, Donald Blumenthal3, Virgil L.

Woods Jr.4, & Xiaodong Cheng1,2 (1) Department of Biochemistry & Molecular Biology, University of Texas Medical Branch

Galveston (2) Dept. of Pharmacology & Toxicology, University of Texas Medical Branch

Galveston, Galveston, TX, 77555 (3) Dept. of Pharmacology, University of Utah, Salt Lake

City , Utah (4) Dept. of Medicine and Biomedical Sciences Graduate Program, University of

California at San Diego, La Jolla, CA 92093-0656

Epac2 is a guanine nucleotide exchange factor which is directly activated by cAMP. According

to the model of Epac activation, a localized "hinge" motion is a major change in the Epac

structure upon cAMP binding. In this study, we test the functional importance of hinge bending

for Epac activation by targeted mutagenesis. We show that substitution of the conserved

residue phenylalanine 435 by glycine facilitates the hinge bending, and is constitutively active,

while tryptophan, impedes the hinge motion and results in a dramatic decrease in Epac2

catalytic activity. Structural parameters for wild type Epac and two of its mutants determined

by small-angle X-ray scattering (SAXS) further confirm the importance of hinge motion in

Epac activation. In addition, peptide amide hydrogen/deuterium exchange mass spectrometry

(DXMS) was used to probe the solution structural and conformational dynamics of full length

Epac2. Our study also suggests that the side-chain size of the amino acid at the position 435 is

a key to Epac functioning. It seems that phenylalanine at this position has the optimal size to

prevent "hinge" bending and keep Epac closed and inactive in the absence of cAMP while still

allowing the proper hinge motion for full Epac activation in the presence of cAMP. The DXMS

results also support this mechanism in which cAMP-induced Epac2 activation is mediated by a

major hinge motion centered on the C-terminus of the second cAMP binding domain. These

results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe

by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the

interaction between Epac2 and RAP1.

26



Structural Differences Between Postsynaptic

Densities Isolated From Different Brain Regions

Madeline Farley1, Yoshihia Kubota2, & M. Neal Waxham2 (1) Neuroscience Program, GSBS, UTHSC (2) Department of Neurobiology and Anatomy,

University of Texas Health Science Center, Houston, TX

The postsynaptic density (PSD) is a large protein complex that works to cluster neurotransmitter

receptors at the synapse needed for synaptic transmission and to organize the intracellular

signaling molecules responsible for altering the efficiency of synaptic transmission – termed

synaptic plasticity. We propose that different synapses from different parts of the brain place

unique demands on the process of synaptic transmission and that the structure and composition of

the PSD play a role in providing these unique properties. To begin to address this question, PSDs

were isolated from adult rat cerebella, hippocampi and cortices, three brain areas amenable to

straightforward isolation that contain unique distributions of neuronal cell types. Cryo-

tomography and immunogold labeling were utilized to quantify protein composition and

distribution. Although the mean surface area did not significantly differ between PSD types, the

variance was significantly larger for cerebellar PSDs. Labeling densities for PSD-95 and

αCaMKII were found to differ dramatically among the PSD types, while all regions were found to

have moderate to high labeling for βCaMKII, illustrating the importance of βCaMKII to the PSD

structure. PSD-95, a common PSD marker and scaffold protein, was absent from a fraction of

cerebellar PSDs, unlike hippocampal and cortical PSDs, showing that protein composition varies

between PSD types. Qualitatively, PSD-95 was found to cluster in cerebellar PSDs unlike other

PSD types, suggesting a different function for PSD-95 in cerebellar PSDs. Ripley’s K function

analysis is currently being implemented to evaluate gold particle distribution. These results

support the idea that the composition and structure of the PSD change with brain region, possibly

to achieve the specific synaptic functions required of each brain region.

This research was funded by a training fellowship from the Keck Center of the Gulf Coast

Consortia, on the Houston Area Molecular Biophysics Training Program, NIGMS T32

GM008280

27



NMR Structure of Human Thymosin Alpha-1

Miguel-Angel Elizondo-Riojas1, Steven M. Chamow2, Cynthia W. Tuthill3, David

G. Gorenstein1, & David E. Volk1 (1) Center for Proteomics and Systems Biology, Institute of Molecular Medicine for the

Prevention of Human Diseases , Department of NanoMedicine and Biomedical Engineering,

University of Texas Health Science Center-Houston, 1825 Pressler, Houston, TX 77030,

United States. (2) S & J Chamow, Inc., Biopharmaceutical Consulting, San Mateo, CA 94403,

United States. (3) SciClone Pharmaceuticals, Inc., 950 Tower Lane, Suite 900, Foster City, CA

94404, United States.

800 MHz NMR structure of the 28-residue peptide thymosin alpha-1 in 40% TFE/60% water

(v/v) has been determined. Restrained molecular dynamic simulations with an explicit solvent

box containing 40% TFE/60% TIP3P water (v/v) were used, in order to get the 3D model of

the NMR structure. We found that the peptide adopts a structured conformation having two

stable regions: an alpha-helix region from residues 14 to 26 and two double beta-turns in the N-

terminal twelve residues which form a distorted helical structure.

28



Super-Resolution Optical Microscopy Development

At University of Texas Medical Branch Galveston

Arnold Vainrub1, & Massoud Motamedi1 (1) Center for Biomedical Engineering, University of Texas Medical Branch Galveston ,

Galveston, TX, 77555-1156

The spatial resolution in microscopic imaging is diffraction limited. The Abbe relation derived in

1873 predicts the resolution limit in optical microscopy to be about 200 nm. Only in the past ten

years, several new microscopy techniques have been developed that allow improving the

resolution beyond the diffraction Abbe limit, in some cases achieving spatial resolution of 15 nm.

In the Center of Biomedical Engineering at UTMB we designed, built and tested the super-

resolution (SR) microscopy system based on the principle of structured illumination. Our SR

microscope works in both fluorescent and reflected light modes with improved spatial resolution

of about 90 nm. It allows imaging of subcellular structures based on autofluorescence or contrast

originated from imaging dye or fluorescent protein using standard techniques to prepare slides for

microscopy. In addition, dark or strongly light scattering nanoparticles are imaged with super-

resolution in reflected light mode. To demonstrate advantages of super-resolution, we compared

SR and conventional images of mitochondrion and F-actin in cells, and the images in reflected

light of bacterial samples yielding details about cellular structures that can not be visualized using

conventional optical microscopy techniques. Through collaborative research, we plan to serve the

needs of diverse scientific community. The project is supported by NSF grants CBET-934860 to

AV and CBET-1053978 to MM.

29



The Role of Base Modification on Tyrsyl-tRNA

Thermodynamics, Structure, and Function

in Bacillus Subtilis and Bacillus Anthracis

Andria Denmon1, Edward Nikonowicz1

(1) Biochemistry and Cell Biology, Rice University , Houston, TX

tRNA molecules contain more than 80 chemically unique nucleotide base modifications that

contribute to the chemical and physical diversity of RNAs as well as add to the overall fitness

of the cell. Base modifications have also been shown to play a critical role in tRNA molecules

by improving the fidelity and efficiency of translation. However, the role of modified bases in

tRNA as it relates to thermostability, structure, and transcriptional regulation in Gram-positive

bacteria, such as Bacillus subtilis and Bacillus anthracis, are not well characterized. The

tyrosyl-anticodon stem-loops (ASLTyr) of B. subtilis and B. anthracis have three modified

residues which include Queuine (Q34), 2-thiomethyl-N6-dimethylallyl (ms2i6A37), and

pseudouridine (ψ39). Differential Scanning Calorimetry (DSC) and UV melting were employed

to examine the thermodynamic effects of partial modification on ASLTyr stability. The DSC

and UV data indicate that the ψ39 and i6A37 modifications improve the molecular stability of the

ASL. NMR spectroscopy was employed to examine the effects of partial base modification on

the ASLTyr structure. The NMR data indicate that the unmodified and [ψ39]-ASLTyr form a

protonated C-A+Watson-Crick-like base pair instead of a bifurcated C-A+ interaction.

Additionally, the loop regions of the unmodified and [ψ39]-ASLTyr molecules are well ordered.

Interestingly, the [i6A37]- and [i6A37; ψ39]-ASLTyr molecules do not form a protonated C-

A+ base pair and the bases of the loop region are not well ordered. The NMR data also suggests

that the unmodified and partially modified molecules do not adopt the canonical U-turn

structure. The structures of the unmodified, [ψ39]-, and [i6A37;ψ39]-ASLTyr molecules do not

depend on the presence of Mg2+, but the structure of the [i6A37]-ASLTyrmolecule does depend

on the presence of multivalent cations. RT-qPCR was used to determine the effect of partial

modification on tRNA mediated transcriptional regulation in B. anthracis. The RT-qPCR data

suggests that the loss of ms2i6A37 may interfere with the tRNATyr molecule’s ability to regulate

transcription effectively. Finally, the physiological studies suggest that the loss of

ms2i6A37 confers resistance to antibiotics and decreases the lag phase when grown in defined

media, whereas the loss of Q34 results in sensitivity to antibiotics and an extended lag phase

compared to wild type.

30



Crystal Structure of Human Asparaginase-Like

Protein 1 Bound to Product L-Aspartic Acid

Wenzong Li1, Jason Cantor1, June Qingxia Liu1, Giulia Agnello1, George Georgiou1, Everett

Stone and Yan Zhang1

(1) Department of Chemistry and Biochemistry, University of Texas at Austin TX 78712

ABSTRACT: The human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal

nucleophile (Ntn) hydrolase superfamily, whose overexpression has been observed in certain

ovarian, colon, and breast carcinomas. Human ASRGL1, displays dual activities as L-asparaginase

and isoaspartyl peptidase. The active form of hASRGL1 is generated by an autocatalytic

intramolecular activation step with Thr168 nucleophile as the catalytic residue. Herein, we report

the structure of the inactive Thr168Ala point mutation variant of hASRGL1 in complex with its

product, L-aspartic acid as well as in its apo form. We demonstrate that hASRGL1, the first

structurally-characterized mammalian plant-type asparaginase, exhibits the canonical Ntn

hydrolase superfamily fold. Furthermore, we identified a small N-terminal loop located near the

active site, which contains specific amino acids whose flexibility is critical to hASRGL1

intramolecular processing and/or catalysis.

31



Direct Electron Detection Yields Cryo-EM

Reconstructions At Resolutions Beyond ¾ Nyquist

Frequency

Ryan Rochat1, Bammes Benjamin1,2, Ryan Rochat1,2, Joanita Jakana2, Donghua Chen2,

Wah Chiu2, (1) Structural Computational Biology and Molecular Biophysics, Baylor College of Medicine ,

Baylor College of Medicine, Houston, Texas 77030 (2) National Center for Macromolecular

Imaging, Verna and Marrs McLean Department of Biochemistry and Microbiology, Baylor

College of Medicine, Houston, Texas 77030

One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-

resolution signal from the image-recording media at the full-resolution limit of the transmission

electron microscope. Direct electron detection using CMOS-based sensors for digitally

recording images has the potential to alleviate this shortcoming. Here, we report a practical

performance evaluation of a Direct Detection Device (DDD) for biological cryo-EM at two

different microscope voltages: 200 and 300 kV. Our DDD images of amorphous and

graphitized carbon show strong per-pixel contrast with image resolution near the theoretical

sampling limit of the data. Single-particle reconstructions of two frozen-hydrated

bacteriophages, P22 and ε15, establish that the DDD is capable of recording usable signal for 3

-D reconstructions at about 4/5 of the Nyquist frequency, which is a vast improvement over the

performance of conventional imaging media. We anticipate the unparalleled performance of

this digital recording device will dramatically benefit cryo-EM for routine tomographic and

single-particle structural determination of biological specimens.

32



Research At the UTMB NMR Spectroscopy

Laboratory

Tianzhi Wang1, & B. Montgomery Pettitt1 (1) Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical

Branch Galveston , 301 University Blvd., Galveston, TX, 77555

Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool in the studies of Structural

Biology, Proteomics, Metabolomics, Nutrition, Toxicology, Clinical Research Solutions,

Functional Genomics, Structure/Activity Relationship, Drug Discovery and Development,

Macromolecule Analysis and Small Molecule Analysis through a number of methods, including

relaxation measurements, chemical shift mapping, hydrogen exchange, conformational exchange,

transfer NOE, Saturation Transfer Difference(STD), dynamics and 3D or 4D triple resonance

NMR experiments. The SCSB NMR instrumentation includes the state-of-the-art high field

Bruker Avance III 800MHz (with a TCI CryoProbe), 750MHz and 600 MHz (with a QCI

CryoProbe, which is capable of various multi-dimensional 1H/2H/13C/15N/31P NMR

experiments) NMR spectrometers with 31P NMR capability. The core Research laboratory offers

instrumentation, training, software and assistance in designing experiments. The research though

NMR laboratory is focusing on NMR methods, structure determination, protein dynamics &

function, and metabolomics.

33



The New UTMB SCSB Biological SAXS Facility

Mark Andrew White1, Kyung Choi1, Xiaodong Cheng1,2, Marc C. Morais1, Stanley J.

Watowich1, & B. Montgomery Pettitt1 (1) Department of Biochemistry & Molecular Biology, University of Texas Medical Branch

Galveston (2) Dept. of Pharmacology and Toxicology, University of Texas Medical Branch

Galveston, Galveston, TX, 77555

The Sealy Center for Structural Biology has installed new x-ray diffraction and solution

scattering instrumentation in its crystallography lab. The new equipment includes the Rigaku

Ultimate Home Lab® which comprises the unsurpassed high-brilliance FR-E++DW Superbright

x-ray generator with both Cu and Cr optics, and the industry standard RAXIS-

IV++ crystallography system. In addition, we are acquiring the innovative Rigaku BioSAXS-

1000, the first SAXS instrument dedicated to biological research in the southern mid-western

states, once again marking UTMB as a leader in structural biology resources.

You are invited to join the Gulf Coast Protein Crystallography Consortium's Small Angle X-

ray & Neutron Scattering interest group: The SAXNS. This is a group of researchers currently

using or intending to apply SAXS or SANS to conquer difficult structural biology problems.

The group is organized by Xiaodong Cheng, and the SCSB at UTMB, but includes members

from most GCC institutions. For more information or to be informed of upcoming events and

workshops please see the SAXNS website http://xray.utmb.edu/SAXNS.

34



Co-Crystallization of the Streptavidin-Biotin With a

Lanthanide-Ligand Complex Gives Rise to a Novel

Crystal Form

R. Adikaram Bandara1, Deqian Liu1, Aditya Hindupur1, Kris F. Tesh1, & Robert O. Fox1

(1) Biology and Biochemistry, University of Houston, Houston, TX, 77024

Abstract

Crystallization has become the major bottleneck in determining the structure of proteins by X-ray

crystallography. The ability to solve the structure of important proteins can be enhanced by

increasing the probability of obtaining suitable crystals. Terbium dipicolinate complexes ([Tb

(DPA)3]3-) show promise in achieving novel crystal forms in this and previous work. A new

crystal form of the streptavidin-biotin complex was facilitated by co-crystallization with [Tb

(DPA)3]3-under conditions that were distinct from previously reported crystal forms. X-ray data

were collected on an in-house R-AXIS IV++ image plate detector with CuKα radiation from a

Rigaku MicroMax-007HF high intensity microfocus rotating-anode generator, and processed with

d*TREK. The orthorhombic crystals were in space group C222 with unit cell dimensions of

a=64.42, b=71.39, c=55.49 Å, and with one subunit of the streptavidin tetramer in the asymmetric

unit. Co-crystallization with [Tb(DPA)3]3- has the added advantage of incorporating Tb, a strong

anomalous scatterer, which was used to solve the structure by anomalous dispersion and solvent

flattening with PHENIX. The lanthanide complex formed crystal contacts between protein

molecules. In the protein structure, five [Tb(DPA)3]3- complexes are bound at different sites in the

crystal. Three of them are located on a two-fold crystallographic axes between symmetry related

protein molecules. There are distinct interactions between protein and the metal chelate

complexes. The carboxylate group of the dipicolinate ligands associates with Arg, Asn, Lys and

His side chain nitrogen atoms, water and backbone amide nitrogens.

35



Enhanced Visualization of a 52 KDa HIV-1 RNA

By Zernike Phase Contrast Cryo-Electron

Tomography

Rossi Irobalieva1, Rossi Irobalieva1,2, Caroline Fu2, Xiao Heng3, Kuniaki Nagayama4,

Michael Schmid1,2, Michael Summers3, & Wah Chiu1,2 (1) Graduate Program in Structural and Computational Biology and Molecular Biophysics,

Baylor College of Medicine , Baylor College of Medicine, Houston, TX 77030; (2) National

Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry

and Molecular Biology, Baylor College of Medicine, Houston, TX 77030; (3) Howard Hughes

Medical Institute, Department of Chemistry and Biochemistry, University of Maryland,

Baltimore County, Baltimore MD; (4) Okazaki Institute for Integrative Bioscience, National

Institute of Natural Sciences, Okazaki, Aichi 444-8787, Japan

Cryo-electron tomography (cryo-ET) can reveal the low-resolution structure and location of

nanomachines in a variety of cellular environments. Until now, this technique has been used to

characterize large structures, such as cells and organelles, and/or to average multiple identical

copies of viruses or other large protein complexes. We recently showed that cryo-ET can also

be applied to study much smaller and more heterogeneous specimens, such as nucleic acids by

determining the structure of a 132-nucleotide 42.8 kDa RNA from Moloney Murine Leukemia

Virus (MoMuLV). This is to date the smallest specimen structurally characterized by cryo-ET.

One of the biggest challenges in working with small specimens (<100 kDa) is the low signal to

noise ratio of the raw data. Here we demonstrate how using a JEM 2200FS electron

microscope equipped with Zernike phase contrast optics can benefit visualization of smaller

specimens. This technique, while commonplace in light microscopy, is fairly new to electron

microscopy. The addition of the phase plate significantly enhances the low-resolution contrast

of the specimen. At the same time, it allows us to image close to focus, preserving more of the

high-resolution information. This in turn makes it much easier to visualize smaller specimens.

We demonstrated this improvement on a 158-nucleotide, 52 kDa, monomeric fragment of RNA

from Human Immunodeficiency Virus (HIV-1). This RNA has been shown to act as an HIV-1

packaging element, critical for the HIV-1 viral life cycle.

Acknowledgments. Support from the NIH (GM42561 and AI30917 to M.F. Summers,

P41RR002250 to W.C.) is gratefully acknowledged. R.I. is supported by NIH Training Grant

No 5T90DK070121-05 through the Gulf Coast Consortia.

36



Cryo-EM Reconstruction of Eilat Alphavirus

Michael Sherman1,2, Farooq Nasar3, and Scott Weaver2,3,4 (1) Biochemistry and Molecular Biology, University of Texas Medical Branch (2) Sealy Center for Structural Biology

and Molecular Biophysics (3) Dept. of Pathology (4) IHII, University of Texas Medical Branch Galveston, Galveston,

TX, 77555

The genus Alphavirus in the family Togaviridae is comprised of small, spherical, enveloped viruses with genomes

consisting of single strand, positive-sense RNA approximately 11-12 kb in length. Current classification of the

genus Alphavirus includes 29 species that can be grouped into nine complexes based on antigenic and/or genetic

similarities. Most alphaviruses are maintained in natural cycles between arthropod vectors, mainly mosquitoes, and

susceptible vertebrate hosts. Occasionally, these cycles can spill over into human and domestic animal populations

to cause disease. Human infections with Old World viruses such as Ross River (RRV), chikungunya (CHIKV), and

Sindbis (SINV) are characterized by febrile illness, rash and polyarthritis. In contrast, infections with New World

viruses, Venezuelan equine encephalitis, Eastern equine encephalitis and Western equine encephalitis (WEEV), can

cause fatal encephalitis. The broad host range of alphaviruses, which infect both invertebrates and vertebrates,

facilitates their maintenance in ecological niches with sporadic outbreaks in humans and animals.

Recently a new host restricted alphavirus, Eilat virus (EILV), was discovered from a pool of Anopheles coustani

mosquitoes collected in the Negev desert of Israel. Phylogenetic analyses placed EILV as a sister to the WEE

antigenic complex within the main clade of mosquito-borne alphaviruses. Electron microscopy revealed that, like

other alphaviruses, EILV virions are spherical, roughly 70 nm in diameter and bud from the plasma membrane of

mosquito cells in culture. EILV could readily infect a variety of insect cells with little overt cytopathic effects.

However, in contrast to all other mosquito-borne alphaviruses, EILV could not infect various mammalian and avian

cell lines at 37°C.

Eilat virus was studied by cryo-EM to identify potential differences between its structure and that of other

alphaviruses studied earlier. Even low resolution (20 Å) reconstruction clearly showed differences in the

glycoprotein spikes on the virus surface compared to those from typical alphaviruses like SINV, WEEV and others.

The spikes had an additional density bulge stemming from the E2 glycoprotein; that protrusion was absent in other

alphaviruses. Higher resolution reconstruction (8 Å) allowed us to fit homology models of E1 and E2 glycoproteins

derived from recently published structures of homologous proteins from CHIKV into the spikes. Even rigid body fit

without any perturbation of the models produced a high correlation with EM density (0.86 for E1 and 0.8 for E3)

and allowed us to delineate boundaries of both E1 and E2 glycoproteins in the spikes. The remaining protrusion in

the spikes was attributed to the E3 glycoprotein that is normally cleaved off during spike maturation in the Golgi.

Although the E3 glycoprotein was reported to be present in Semliki Forest virions and several other alphaviruses,

we believe that EILV is the first naturally occurring alphavirus where E3 is present in the virions at 100%

occupancy. The only other example of the full presence of E3 in alphaviruses of which we are aware of is the

TRSB-N mutant of SINV, where a mutation prevents cleavage of E3. In that case the mutant is lethal, but in the

case of EILV the virus is fully viable in insects although it does not infect vertebrates. In both viruses E3 seems to

be fully present in the virions and at low resolution (20-25 Å) the 3D maps are quite similar. Figs. 1c and D,

reconstructed using 1500 EILV images, show an EILV glycoprotein spike at 8 Å resolution with all three

glycoprotein homology models fit as rigid bodies. The imaging was done on JEM 2200 FS electron microscope

using an in-column omega energy filter with slit set to 20 eV, and image processing was done using IMAGIC-5

software package, with reconstructions calculated using BKPR program.

37



Structural Characterization of Interleukin-8

Monomer and Dimer Interactions With

Glycosaminoglycans: Implications for in Vivo

Neutrophil Recruitment

Prem Raj Joseph1, and Krishna Rajarathnam1

(1) Biochemistry and Molecular Biology, University of Texas Medical Branch Galveston ,

Galveston, TX, 77555

Circulating neutrophils, rapidly mobilized in response to microbial infection, form the first line

in host defense. Chemokine interleukin-8 (IL-8) plays a crucial role in this process by directing

neutrophils to the site of infection. IL-8 exists reversibly as both monomers and dimers, and

their interactions with cell surface glycosaminoglycans (GAGs) and neutrophil receptors play a

crucial role in regulating recruitment. The structural basis and molecular mechanisms by which

IL-8-GAG interactions mediate recruitment is not known. GAGs, such as heparin and heparan

sulfate, are highly negatively charged linear polysaccharides, and are the most complex and

diverse biomolecules expressed in humans. In the present study, we have characterized the

structural basis of IL-8 monomer and dimer binding to heparin-derived oligosaccharides using

NMR spectroscopy. Our data show that both monomers and dimers bind GAG with similar µM

affinities, and that GAG binds both in the monomer and dimer to a surface formed by the basic

residues of the C-terminal alpha-helix and adjacent N-loop residues. Interestingly, our NMR

data clearly show that the GAG-binding and receptor-binding interfaces overlap, and that the

GAG-bound IL-8 cannot bind and activate the receptor. We propose chemotactic gradient

(solution-phase) and not haptotactic (solid-phase) gradient that directs neutrophils to the tissue,

and that haptotactic gradient dictates the amount of free IL-8 monomers and dimers

constituting the chemotactic gradient. Our observations represent a paradigm shift, as it is

believed that haptotactic gradients mediate recruitment, and so have far reaching implications

in our understanding of how chemokines mediate leukocyte recruitment in health and disease.

38



Structural Study of HSV-1 Virion Using Cryo-EM

and Bioinformatics

Xiangan Liu1, Ryan H. Rochat2, Joanita Jakana1, Chuan Hong2, Juan Chang1, Frazer J.

Rixon3, & Wah Chiu1 (1) Verna and Marrs McLean Department of Biochemistry and Microbiology, Baylor College of

Medicine and National Center for Macromolecular Imaging (2), Graduate Program in Structural

and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX,

77030, USA; (3), MRC—University of Glasgow Centre for Virus Research, Glasgow G11 5JR,

UK (6) National Center for Macromolecular Imaging, Verna and Marrs McLean Department of

Biochemistry and Microbiology, Baylor College of Medicine, Houston, Texas 77030 (7) School

of Biomedical Informatics, University of Texas Health Science Center at Houston, Houston, TX,

77030

The ~2000 Å Herpes Simplex type 1 (HSV-1) virion is one of the largest human viruses. Due to

its size, it has been challenging to obtain a high-resolution map. The virion consists of four major

capsid proteins (VP5, VP19C, VP23, VP26) in addition to many other associated viral proteins

(e.g. UL6, UL17, UL25, UL36, UL37, UL48) which play essential roles in egress and infection.

We have used single particle cryo-EM to reconstruct the HSV-1 virion to 6 Å resolution while

enforcing icosahedral symmetry. At this resolution, SSEHunter can identify many secondary

structural elements (SSEs) in each component of the capsid. We validated our map by matching

the SSEs to the X-ray structure of a domain of VP5. Together with sequence alignments, sequence

predictions, motif searches, previous biochemical and cryo-EM data, we are able to model the

folds for some domains of the capsid proteins. For instance, this reveals the characteristic fold of

HK97 phage capsid protein in the floor domain of VP5 and a unique topology of its N-terminus.

To study the morphogenesis of HSV-1, A-, B- and C-capsids have been characterized

biochemically and structurally by many research groups. In contrast to B-capsid which cannot

package DNA, the intact virion shows different structural features at the inner surface of a penton

vertex. Furthermore, a difference map has been obtained between the mature virion and C-capsid,

a genuine morphogenetic intermediate. A hybrid analysis of the maps and the sequence predicted

SSEs allows us to localize the capsid associated proteins UL25, UL17, and UL36, and their

interactions with the capsid proteins. Our segmented UL25 density does not match the X-ray

structure suggesting conformational differences. Furthermore, the spatial arrangements of UL25

and UL17 differ from the previous low-resolution cryo-EM model.

Acknowledgement: We thank the support of NIH grants (P41GM103832, R56AI075208,

GM07330 and T15LM007093) and Welch Foundation (Q1242).

39



Cryo-EM of Genome Packaging and Delivery of

Bacteriophage PRD1

Chuan Hong1, Xiangan Liu2, Joanita Jakana2, Hanna Oksanen3, Dennis Bamford3, Wah

Chiu1,2 (1) Structural and Computational Biology and Molecular Biophysics, Baylor College of

Medicine (2) National Center for Macromolecular Imaging, Verna and Marrs McLean

Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston,

Texas, 77030 (3) Department of Biosciences and Institute of Biotechnology, University of

Helsinki, PO Box 56, FIN-00014 Helsinki, Finland

Nearly 100 years ago it was proposed that bacteriophage could be used as antibacterial agents

to mitigate bacterial infections. Recently, interest in phage therapy has seen resurgence as an

increasing number of pathogens are becoming multi-drug resistant. PRD1 phage, which is

known to infect Salmonella typhimurium, is a good candidate for a possible antibacterial.

PRD1 is a dsDNA bacteriophage with an inner membrane and belonging to the Tectiviridae

family. As the virus structure has been previously solved by X-ray crystallography, it is known

that the icosahedral capsid shell is composed of four structural proteins. However, the portal

complex, which may consist of at least four proteins, plays an integral role in the packaging of

the viral genome into the viral membrane vesicle through the icosahedral shell. In addition, five

other proteins that are responsible for genome delivery during infection also remain elusive.

The goal of this study is to reveal the structures of the genome transfer machinery of PRD1 and

elucidate its working mechanism.

We use single-particle cryo-electron microscopy (cryo-EM) to study both the mature and

empty stages in solution state. We were able to reconstruct the icosahedral maps of the mature

virion and the empty particle to 6Å resolution. The 12 chain subunits of the major capsid

proteins P3 were segmented out and compared for both stages. We have been able to reveal the

N-terminal helices of chain F and G went from straight conformation at empty stage to kinked

conformation at mature stage. Interestingly, the helix-kinking conformational change of Chain

F and G locates and only locates at the 2-fold axis of the icosahedral capsid shell, which

provides a novel location-specific helix-kinking virus maturation mechanism.

For the empty particle, a tube tail structure, which protrudes at one of the five-fold vertex, can

be visualized in the raw cryo-EM images. By symmetry-free reconstruction of the empty

particle at 20Å resolution, we started to be able to resolve a tubular density at a 5-fold position,

which suggests a potential genome transfer channel during genome delivery. This tubular

structure helps to elucidate the genome delivery mechanism of PRD1 and also provides

guidance to reveal the packaging complex structure in the mature virion in the future.

40



Visualizing Cyanobacteria-Phage Interactions By

Cryo-Electron Tomography

Wei Dai1, Desislava Raytcheva2,3, Caroline Fu1, Jacqueline Piret3, Michael Schmid1,

Jonathan King2 and Wah Chiu1 (1) NCMI, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine

(2) Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA (3) Department of Biology,

Northeastern University, Boston, MA, USA (4) W. M. Keck Center for Interdisciplinary Bioscience Training, Houston,

TX, USA

Marine phages (also known as cyanophages) play an important role in mediating the marine

ecosystem by controlling the community organization and lateral gene transfer between

cyanobacteria. One such cyanophage, Syn5, infects marine Synechococcus strain WH8109. Syn5

virions consist of a 60 nm icosahedral capsid, a short tail assembly, and recently discovered a

novel “horn” at the vertex directly opposite the tail vertex. The capsid and the tail assembly of

Syn5 share morphological similarity and sequence homology to T7-like phages. The “horn” is a

filamentous structure unique to Syn5 which is approximately 50 nm long and tapers from 10nm

(wide) at the base to only 2-5nm at the tip. Despite the remarkable impact that cyanophage have

on marine ecology, their life cycle and interactions with their hosts are largely uncharted. We have

infected Synechococcus WH8109 cells with Syn5 phages, and used cryo-electron tomography to

capture maturing phage progeny inside the host cell. Based on shape and size alone, our study has

identified three distinct intermediate particles associated with maturation within the infected cells.

The first group appears to be procapsid with a diameter ~10% smaller than the mature capsid.

These particles have an internal mass which is interpreted as scaffolding core density. Structure

variations of the core density in this group suggest a mixture of particles in the process of DNA

packaging and scaffolding release. The second class of particles is morphologically

undistinguishable from the mature phage. These particles show clear icosahedral symmetry after

symmetry-free post-tomographic averaging. The presence of a tail fiber in the absence of the horn,

in this second subset of particles, suggests that the horn structure is highly flexible or possibly

absent in some particles. This finding suggests that the horn may be assembled after DNA

packaging. The third class of particles appear to have all the capsid elements of the second class,

however they lack DNA and the tail, suggesting they are the side-products, or perhaps products of

defective phage maturation instead of assembly intermediates. Cryo-electron tomography of

WH8109 cells infected with Syn5 at an early stage of infection revealed tail conformational

changes during attachment and DNA injection. These results provide insights to assembly of

cyanophage Syn5, and facilitate the understanding of host-phage interactions in a physiologically

meaningful context. This research was funded by a training fellowship from the Keck Center of the Gulf Coast Consortia, on the

Training Program in Biomedical Informatics, National Library of Medicine (NLM) T15LM007093, PI - G.

Anthony Gorry.

41



Membrane Bound Organization of Blood

Coagulation Factor VIII Forms Onto Negatively

Charged Lipid Nanotubes

Jaimy Miller1, Kirill Grushin1, Svetla Stoilova-McPhie1,2 (1) Neuroscience and Cell Biology, University of Texas Medical Branch (2) Sealy Center for

Structural Biology and Molecular Biophysics, University of Texas Medical Branch Galveston,


Blood hemostasis is achieved through a delicate balance between coagulation factors forming

active membrane-bound complexes. Understanding their assembly and functional implications

is fundamental in gaining control over blood disorders such as hemophilia and thrombosis. To

this goal we design suitable lipid nano-platforms supporting coagulation complexes and

allowing extensive structure-functional studies required for successful nano-drug design.

FVIII active form - FVIIIa, serves as co-factor to the serine protease Factor IXa (FIXa) within

the membrane-bound tenase (FVIIIa-FIXa) complex, thus enhancing FIXa catalytic efficiency

~ 106 fold, critical for a timely coagulation. Our aim is to study the membrane-bound

organization of Factor VIII (FVIII) forms and within the FVIIIa-FIXa (FIXa) complex, as

organized onto single bilayer lipid nanotubes (LNT) by combining Cryo-electron microscopy

(Cryo-EM), molecular modeling, physicochemical and biophysical methods.

In this study we present our preliminary Cryo-EM results of the membrane-bound organization

of recombinant FVIII variants (human FVIII full length - hFVIII-FL and porcine FVIII B

domain deleted - pFVIII-BDD, both used in treatment of Hemophilia typeA) onto LNT with a

different phosphatydilserine (PS) content. PS is the natural ligand for FVIII, FVIIIa and FIXa,

securing a strong membrane-binding (Kd ~ 2nM), thus allowing Cryo-EM structural studies of

their membrane-bound organization. Our preliminary Cryo-EM data point to the potential of

pFVIII-BDD, as a better candidate for structural studies of FVIII membrane-bound forms and

it complexes.

42



Cryo-EM and Biophysical Characterization of

Recombinant Coagulation Factor VIII Forms

Bound to PS-Containing Vesicles

Kirill Grushin1, Jaimy Miller1, Svetla Stoilova-McPhie1,2 (1) Dept. of Neuroscience & Cell Biology, University of Texas Medical Branch Galveston (2)

Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical

Branch Galveston, Galveston, TX, 77555

Factor VIII (FVIII) is a multidomain blood plasma glycoprotein which is responsible for

Hemophilia A. Activated FVIII (FVIIIa) acts as a co-factor to the serine protease Factor IXa

(FIXa) onto the negatively charged platelet surface thereby amplifying Factor Xa and Thrombin

generation more than six orders of magnitude, necessary for effective fibrin clot formation.

Recombinant FVIII forms are the most effective drugs against hemophilia A. The focus of this

study is to define which membrane-bound recombinant FVIII form: human full length (hFVIII-

FL), human B-domain deleted (hFVIII-BDD) or porcine B-domain deleted (pFVIII-BDD) is best

suited for structure determination by Cryo-electron microscopy (Cryo-EM). A functional FVIII

membrane-bound structure is crucial to understand the FVIIIa-FIXa membrane-bound assembly

for effective coagulation and identify potential future mutations capable of improving the FVIII

membrane-bound structure and function.

In this work, Cryo-EM and biophysical methods have been combined to study the binding of

FVIII variants to phosphatidylserine (PS) containing liposomes in close to physiological

conditions. The presented data show that while the activity and secondary structure of

recombinant FVIII variants in solution remains similar, attaching to negatively charged

phospholipid membranes differentiates between the BDD and FL FVIII forms. These results are

an important step in the ongoing design of membrane-bound FVIII nano-structures for further

Cryo-EM and biophysical studies.

43



Architecture of Type IV Secretion Systems in

Gram Positive Bacteria: in Silico 3D Protein

Modeling and Comparison With Gram Negative

Bacteria

Mayukh Sarkar1, Jenny Laverde1, Peter Christie1

(1) Microbiology and Molecular Genetics, UT Houston Medical School

Bacterial Type IV secretion systems (T4SS’s) function as DNA and protein translocation

systems, often aiding in colonization and spread during infection. Conjugation machines are a

large subfamily of T4SSs. These systems transfer DNA substrates to recipient cells by a contact

-dependent process and are largely responsible for the rapid dissemination of antibiotic

resistance genes and other virulence factors in clinical settings. Although recent work has

generated structure-function information about T4SSs in Gram-negative bacteria, little is

known about the architectures or mechanisms of action of T4SS’s in Gram-positive bacteria.

We are employing in silico 3D protein modeling methods to characterize subunits of the T4SS

encoded by the pheromone-responsive plasmid pCF10 of Enterococcus faecalis. We have

generated structural models for domains of PcfC, a VirD4-like coupling protein that links a

processed form of pCF10 with the transfer channel. These studies, combined with previous

experimental work in our laboratory, suggest that the all-alpha domain (AAD) of pCF10 adopts

a 7-helix bundle that contributes to pCF10 substrate binding. We have also generated structural

models for other T4SS subunits, including PrgL which adopts a fold similar to that of

Agrobacterium tumefaciens VirB8, the cysteine, histidine-dependent amidohydrolase/peptidase

(CHAP) domain of the PrgK transglycosylase, the VirB4 ATPase homologue PrgJ, and PrgC

which carries a C terminal cell wall anchor motif. These and other structural models enable

comparisons with the Gram-negative machine subunits, and they guide our future studies aimed

at defining the pCF10 T4SS channel architecture through mutational and ultrastructural

analyses.

44



Constructing Initial Models From Subnanometer

Density Maps With Pathwalker and Additional

Protein Backbone Constraints

Ian Rees1, Mariah R. Baker1, Steven J. Ludtke1, Wah Chiu1, Matthew L. Baker1 (1) Verna and Marrs McLean Department of Biochemistry and Microbiology, Baylor College of

Medicine and National Center for Macromolecular Imaging (2) Structural Computational Biology

and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030

Macromolecular assemblies are critical for nearly every biological process, and accurate structural

information is important for understanding functional mechanisms and discovering drug targets. X

-ray crystallography has traditionally been the main tool obtaining high-resolution structural

information, but many important biological complexes either do not crystallize or require

conditions significantly different from in vivo. Cryo-electron microscopy (cryo-EM) permits

imaging and 3D reconstruction of very large complexes and without disruptive solvent

environments, and a significant number of structures have reached 3.5-6Å. This resolution often

permits identification of secondary structural elements, however, direct tracing of backbones with

traditional methods remains difficult at this resolution. To address this need, we have recently

released Pathwalker, a semi-automated protocol for modeling subnanometer cryo-EM data without

the need for a structural template or sequence-structure correspondence.

Pathwalker is an iterative process that involves placing pseudoatoms (representing C-alpha

positions) in the density map, connecting these pseudoatoms into a backbone model using an

adapted form of the Traveling Salesman Problem (TSP), then evaluating and refining the resulting

model. The Pathwalker protocol was validated using a number of real and simulated cryo-EM

density maps, however, few restrictions based on domain knowledge of allowed protein

backbones geometries were incorporated. We are currently incorporating additional backbone

geometry constraints into Pathwalker to eliminate “impossible” conformations and decrease the

amount of manual intervention needed. Additionally, we are making the refinement step more

integrated into the process, and results will include a score that flags strained bond angles.

45



Biochemical and Structural Characterization of

Pestivirus N-Terminal Protease (NPro

)

Keerthi Gottipati1, Ana Fiebach2, Nicolas Ruggli2, & Kay Choi1 (1) Biochemistry and Molecular Biology, University of Texas Medical Branch (2) Institute of

Virology & Immunoprophylaxis, Mittelhäusern, Switzerland

Pestivirus Npro is a cysteine protease that co-translationally cleaves its own C-terminus

(Cys168), enabling polyprotein processing. Npro auto-inhibits itself and apart from this first cis-

cleavage it has no trans-activity. Cleaved Npro binds interferon regulatory factor-3 (IRF3), an

activator of the interferon-α/β pathway, and targets it for proteasomal degradation. We report

the first crystal structure of pestiviral Nprodetermined to 1.5Å resolution. Npro consists of two

domains, a cysteine protease domain and a zinc-binding domain. The cysteine protease domain

has a novel fold. The active site of Npro contains a catalytic dyad formed by Cys69 and His49.

The C-terminus is not only bound in the active site, but also contributes a strand to the beta-

sheet that makes up the active site. Thus, this structure explains the auto-catalytic and

subsequent auto-inhibition mechanism of Npro and provides insight into interaction of Npro with

IRF3. In addition, the 1.7 Å resolution structure of the cleavage site mutant of Npro (Npro-

C168A-SDDG) shows clear density for the active site loop containing the neucleophilic Cys69,

which was disordered in the wild type structure. This mutant structure clearly illustrates the

arrangement of the catalytic dyad around the cleavage substrate of Npro. We mutated the

previously proposed protease triad, Glu22, His49, and Cys69, and the cleavage site Cys168 and

tested their ability to carry out proteolysis using a C-terminal GFP fusion construct (6xHis-Npro

-GFP-His) of Npro. We determined that the catalytic activity of Npro is not specific for a cysteine

(Cys168) at the cleavage site. We discuss the structure-based role of proposed active site

residues in the regulation of catalytic activity of Npro.

46



Biochemical and Biophysical Characterization of

the RNA Replication Mechanism of a Small DsRNA

Virus

Aaron Collier1, Yizhi Tao1

(1) Biochemistry and Cell Biology, Rice University Houston, TX, 77251-1892

During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA

polymerase (RdRP) replicates and transcribes the viral genome. Complete polymerase activity of

these viral RdRPs often requires the presence of the corresponding capsid protein (CP), thus

restricting the replication of the viral genome to only within the virus capsid. This strategy

prevents the synthesis of free dsRNA in the cytoplasm of the host cell, which would otherwise

illicit an immune response.

Here we report a 2.4 Å resolution structure of an RdRP from a human infecting strain of

Picobirnavirus (PBV). PBV is a small, non-enveloped dsRNA virus that has been shown to infect

a wide range of mammalian and avian species. Human PBV has been associated with acute

gastroenteritis and has been identified in patients on almost every continent. Our structure shows

that in addition to the fingers, palm, and thumb sub-domains seen in many other viral

polymerases, the PBV RdRP possesses a unique 23aa loop structure that is inserted in the interior

of its active site. Based on our polymerase:RNA:NTP modeling studies, we propose that this loop

may help to inhibit the RNA replication activity of the viral RdRP in the cytoplasm of the host

cell, and that upon interacting with the PBV CP, the viral RdRP may undergo a structural change

that would remove this loop from the active site, thus allowing genome replication to take place in

assembled virus particles.

We are currently creating an RdRP mutant that lacks the loop structure and will compare how the

RNA binding affinity and RNA synthesis activity of the mutant compares to the wild-type. In

addition, we will also perform co-immunoprecipitation experiments to map the interaction

between the PBV RdRP and CP. From these experiments, we hope to gain new insight into how

RdRP function is regulated by the CP in dsRNA viruses.


Consortia, on the Houston Area Molecular Biophysics Program, National Institute of General

Medical Sciences (NIGMS) T32GM008280, PI - Theodore G. Wensel. Funding was also provided

by the National Institute of Health (Grant AIO67638).

47



Crystal Structures of the Carboxyl CGMP

Binding Domain of Human PKGI Reveal the

Mechanism of CGMP Selectivity

Gilbert Huang1, Jeong Joo Kim2, Robin Lorenz3, Eui-Whan Moon2, Chi Zhao4, Albert

Reger2, Daniela Bertinetti3, Darren Casteel5, Friedrich Herberg3, Choel Kim2 (1) Biochemistry and Molecular Biology, Baylor College of Medicine (2) Dept. of

Pharmacology, BCM, Houston, TX 77030 (3) Universitat Kassel, Germany (4) Dept. of

Biochemistry & Cell Biology, Rice University, Houston, TX, 77251-1892 (5) Dept. of

Pharmacology, University of California at San Diego, San Diego, CA

Cyclic GMP (cGMP) and cyclic AMP (cAMP) are ubiquitous second messengers that translate

extracellular signals into a variety of cellular responses. The cGMP dependent protein kinase

(PKG) and cAMP dependent protein kinase (PKA) are the main receptors of Nitric Oxide(NO)-

cGMP and cAMP signaling pathways that mediate pathway specific cellular responses by

phosphorylating non-overlapping sets of substrates and down-stream effectors. Despite the

knowledge that the fidelity of cyclic nucleotide selectivity towards each kinase is crucial in

keeping two signaling pathways segregated, little is known about cGMP selectivity of PKG. To

understand the cyclic nucleotide selectivity mechanism of PKG, we determined crystal

structures of the C-termional cGMP binding domain of Human PKGI (CNBD-B) in the

presence and absence of cGMP. Our structures combined with biochemical data suggest that

the cis peptide forming residues at β strand 5 along with the conserved threonine interact

specifically with the guanine moiety, providing over 300 fold selectivity. Furthermore,

mutating these residues abolishes its cGMP selectivity. Finally, a conserved aromatic residue at

the C-helix repositions and caps the guanine ring through a hydrophobic interaction. This

capping interaction, induced by cGMP binding, accompanies major conformation change of αB

helix and an order to disorder transition of αC helix, which may provide a distinct pathway for

cGMP dependent activation.

48



Structural Insights into a Novel Class of N-Linked

Protein Glycosylation By the Actinobacillus

Pleuropneumoniae HMW1C-Like Protein

Kyoung-Jae Choi1, Fumihiro Kawai1, Sue Grass2, Youngchang Kim3, Joe W. St. Geme

III2 & Hye-Jeong Yeo1 (1) Dept. of Biology and Biochemistry, University of Houston, Houston, TX 77204 (2) Dept. of

Pediatrics and Molecular Genetics & Microbiology, Duke University Medical Center, Durham,

NC 27710 (3) Structural Biology Center, Argonne National Laboratory, Argonne, IL 60439

Protein glycosylation is an essential process that plays an important role in protein structure and

function. The Haemophilus influenzae HMW1 adhesin is an N-linked glycoprotein that mediates

adherence to respiratory epithelium, an essential early step in the pathogenesis of H.

influenzae disease. HMW1 is glycosylated by HMW1C that creates N-glycosidic linkages with

glucose and galactose at asparagine residues and di-hexose linkages at sites of glucose

modification, suggesting that HMW1C is a novel N-glycosyltransferase. Currently, the CAZy

(Carbohydrate Active Enzymes database) classifies HMW1C-like proteins as members of the

GT41 family, a family that otherwise exclusively contains O-GlcNAc transferases (OGT) with

characteristic tetratricopeptide repeats (TPR). Here we present the crystal structure

of Actinobacillus pleuropneumoniae HMW1C (ApHMW1C), a functional homolog of HMW1C,

at 2.1Å resolution. The structure of ApHMW1C contains an N-terminal all α-domain (AAD) fold

and a C-terminal GT-B fold with two Rossmann-like domains and lacks the TPR fold

characteristic of the GT41 family. The GT-B fold harbors the binding site for UDP-hexose, and

the interface of the AAD fold and the GT-B fold forms a unique groove with potential to

accommodate the acceptor protein. Structure-based functional analyses demonstrated that the

HMW1C protein shares the same structure as ApHMW1C and provided insights into the unique bi

-functional activity of HMW1C and ApHMW1C, suggesting an explanation for the similarities

and differences of the HMW1C-like proteins compared with other GT41 family members.

49


Biochemistry Poster Number P-25

BETA 2-Adrenergic Receptor Elicits a Calcium

Signal in HEK-293 Cells

Monica Galaz Montoya1, Monica Galaz-Montoya1, Gustavo Rodriguez1,Olivier

Lichtarge1 & Ted Wensel1

(1) Biochemistry and Molecular Biology, Baylor College of Medicine , Houston, Texas 77030

G-protein coupled receptors (GPCRs) are encoded by more than 800 genes of the human

genome. These receptors account for approximately 40% of current drug targets and are the

most abundant family of transmembrane signaling proteins. GPCRs are involved in many cell

signaling processes, among which is the regulation of intracellular calcium levels. Changes in

the concentration and distribution of calcium ions play an important role in regulating all

aspects of cell function.

Monitoring of intracellular calcium levels in real time using a fluorescent indicator dye

revealed that an endogenous receptor in HEK-293 cells responds to the adrenergic agonist

norepinephrine, by a delayed rise in intracellular [Ca2+]. Pharmacological analysis revealed

that the response is blocked by ICI 118,551, a selective antagonist for beta-2 adrenergic

receptor (β2-AR), and the relative potency of agonists, isoproterenol > epinephrine >

norepinephrine, is consistent with β2-AR being the receptor responsible.

Treatment with thapsigargin, an inhibitor of the SERCA Ca2+ pump of the endoplasmic

reticulum, or with extracellular EGTA, revealed that the source of the Ca2+ is release from

intracellular stores, not entry from outside the cell. PKA involvement was assayed by

subjecting cells to H-89 and KT5720 which are known PKA inhibitors. The Ca2+ signal was

not significantly suppressed when adding either of these drugs, suggesting that PKA is not

involved in this signaling mechanism.

These results reveal a novel pathway by which β2-AR, classically thought to signal primarily

through cAMP production, can trigger release of Ca2+ from intracellular stores. Experiments

in progress are directed at determining the molecular mechanisms by which this response

occurs, and the biophysical properties of the underlying protein-protein, and small-molecule-

protein interactions that mediate it.

50



Chromatin Interplay Between ERα and GR Fine-

Tunes Transcriptional Output

Michael Bolt1, Fabio Stossi1, Justin Y. Newberg1, Michael A. Mancini1 (1) Molecular & Cellular Biology, Baylor College of Medicine Department of Molecular &

Cellular Biology, Baylor College of Medicine, Houston, TX, 77030

Type 1 nuclear receptors (NR) are key transcription factors that regulate their target genes via

binding to specific DNA steroid response elements (SREs), and followed by recruiting

coactivators and histone modifying proteins that collectively modify RNA Polymerase II activity.

In this study we utilized a microscopically-visible estrogen receptor alpha (ERα) regulated

transcription array (1)(2)(3) to analyze the interplay between type I NRs. We show a coordinated

loading onto the array of ERα and other steroid receptors only in the presence of dual agonist

treatments (e.g., estradiol (E2) + dexamethasone (Dex), E2 + dihydroxytestosterone (DHT), etc.).

We then focused on the ERα/GR pair and showed that the cooperative loading phenomenon is

maximal when both receptors are agonist bound, and that GR loading is dependent upon ERα both

occupancy on the array and DNA binding. Moreover, use of specific ERα mutants reveals helix-12

is required for GR array loading. We next demonstrate that the coordinated receptor interplay also

occurs in MCF-7. For example, addition E2 + Dex decreases ERα recruitment at the TFF1/pS2

promoter while increasing GR promoter loading. This scenario causes an overall negative effect

on the TFF1/pS2 mRNA compared to the E2 alone treatment. However, in terms of ERα and GR

recruitment, we also identify genes that show different combinations of outcomes. In conclusion,

both our single cell- and biochemistry-based approaches identify cooperative coordination of the

actions of ERα and Type I NRs on chromatin. This interrelationship warrants further investigation

through genome-wide studies, which we hypothesize will reveal a gene specific interplay between

different NRs.

51



Thioaptamers for Nanoparticle-based Targeted

Delivery of Drugs and Imaging Agents

Varatharasa Thiviyanathan1, Edmond Auzenne2, Anoma Somasunderam1, Rohan

Bhavane3, Aman Mann4, David Volk1, Jim Klostergaard2, Ananth Annapragada3, Mauro

Ferrari4, & David Gorenstein1

(1) Institute of Molecular Medicine, University of Texas Health Science Center (2) Dept. of

Molecular & Cellular Oncology, UT-MD Anderson Cancer Center, Houston, TX, 77030 (3)

Dept. of Pediatric Radiology, Texas Children's Hospital, Houston, TX, 77030 (4) Dept. of

Nanomedicine, The Methodist Hospital Research Institute, Houston, TX 77030

Thioaptamers (TAs) are thio-substituted, short oligonucleotides that bind to target molecules

with high affinity and specificity. TAs offer significant advantages over antibodies and

unmodified oligonucleotide aptamers. Using iterative combinatorial selection and bead-based

library methods, we have selected single-stranded DNA TAs that selectively and tightly bind to

the HA binding domain of CD44 and E-selectin, proteins expressed in various cancer cells.

CD44, a glycoprotein over-expressed in cancer cells, plays significant role in tumor growth,

lymphocyte activation and metastasis. Although various splice variants of CD44 are expressed

on the plasma of cancer cells, the HA binding domain is highly conserved among the splicing

variants. E-selectin expression is induced on the endothelial cell surface of vessels in response

to inflammatory stimuli but is absent in the normal vessels. Using chemically modified TAs,

we have conjugated TAs selected against E-selectin and CD44-HABD to liposomes and silicon

particles, to construct various nanoparticle-TA conjugates for targeted delivery of therapeutic

drugs and imaging agents to cancer cells.

52



Bead-Based Combinatorial Selection of X-

Aptamers Targeted to the Hyaluronic Acid Binding

Domain of Human CD44

Weiguo He1, Miguel A. Elizondo Riojas1, Xin Li1, Lokesh G. Rao1, Varatharasa

Thiviyanathan1, David E. Volk, and David G. Gorenstein1 (1) Center for Proteomics and Systems Biology, and Department of Nanomedicine and

Biomedical Engineering, University of Texas Health Science Center at Houston, Medical School

Aptamers are structurally distinct RNA and DNA oligonucleotides (ODNs) that can mimic protein

-binding molecules and exhibit high (nM) binding affinity. They are capable of adopting unique

three-dimensional conformations that can be selected via high-throughput in vitro methods to bind

target molecules. Aptamers are thus emerging as viable alternatives to small molecules and

antibody-based therapies in the field of drug development. Their selectivity can be achieved

through a very limited repertoire of functional groups – the sugar phosphate backbone and 4 bases.

In contrast, antibodies use all 20 amino acids with a full range of chemical substituents including

positively-charged, sulfhydryl, hydrophobic sidechains, etc. Moreover, all aptamers are

polyanions, potentially limiting their affinity towards the full diversity of proteins. How do we

combine the best attributes of antibodies, small molecules and aptamers?. In fact, a diverse range

of modifications at all possible modification sites of an oligonucleotide have been reported for

enhancing oligonucleotide drug properties. These include alterations of linkages (backbones),

heterocycles, carbohydrates, and connection and conjugation sites, as well as the complete

removal of the sugar-phosphate backbone.

We have recently developed thiophosphate-backbone modified aptamers (thioaptamers) as

specific protein-binding reagents. In order to enhance affinity and selectivity of thioaptamers, by

adding drug-like ligands or amino-acid like side chains to the bases, we have devised a bead-based

combinatorial library selection method in which one or more X-ligands can be linked to aptamers

in a random fashion. By combining our one-bead-one-sequence thioaptamer selection method with

the incorporation of pseudo-randomly placed bases which contain chemical linkers, additional

binding X-ligands can be appended onto aptamers or thioaptamers and the best binding X-

aptamers (XAs) can be selected from a large pool of sequences. Here we described the first

example of X-aptamers, in which the addition of a drug to a complete monothioate backbone

aptamer demonstrated increase binding to the CD44 hyaluronic acid binding domain (CD44-

HABD) protein for up to 23-fold.

53



Silent Substitutions Predictably Alter Translation

Elongation Rates and Protein Folding Efficiencies

Paige Spencer1, Efrain Siller2, John F Anderson2, Jose M Barral1,2,3 (1) Biochemistry and Molecular Biology, University of Texas Medical Branch Galveston (2)

Neuroscience & Cell Biology (3) Sealy Center for Structural and Molecular Biology,

University of Texas Medical Branch Galveston, Galveston, TX, 77555

The degeneracy of the genetic code allows most amino acids to be encoded by multiple codons.

The distribution of these so-called synonymous codons among protein coding sequences is not

entirely random and multiple theories have arisen to explain the biological significance of such

non-uniform codon selection. Most ideas revolve the notion that certain codons allow for faster

or more efficient translation, whereas the presence of others result in slower translation rates.

The presence of these different types of codons along a message is postulated in turn to confer

variable rates of emergence of the nascent polypeptide from the ribosome, which may

influence its capacity to fold towards the native state, among other properties. Previous studies

have reported conflicting results with regards to whether certain kinds of codons correlate or

not with particular structural or folding properties of the encoded protein. We believe this has

arisen, in part, because different criteria have been traditionally utilized for predicting whether

a codon will be translated fast or slowly in a given organism, including its frequency of

occurrence among highly expressed genes and the concentration of tRNA species capable of

decoding it, which do not always correlate well. We have developed a metric to predict

organism-specific polypeptide elongation rates of any mRNA based on whether each codon is

decoded by tRNAs capable of Watson-Crick, non-Watson-Crick or both types of interactions.

We demonstrate by pulse-chase analyses in living E. coli cells that sequence engineering based

on these concepts predictably modulate translation rates due to changes in polypeptide

elongation and show that such alterations significantly impact the folding of proteins of

eukaryotic origin. Finally, we demonstrate that sequence harmonization based on expression

host tRNA pools designed to mimic ribosome movement of the original organism can

significantly increase the folding of the encoded polypeptide.

54



Genetic Feedback Provides Robustness to Mutation

David Marciano1, Panagiotis Katsonis1,2, Rhonald Lua1,2, Teng-Kuei Hsu3, Shivas R. Amin1,2,

Christophe Herman1, & Olivier Lichtarge1,2,3 (1) Department of Molecular & Human Genetics, Baylor College of Medicine (2) Computational

and Integrative Biomedical Research Center, Baylor College of Medicine (3) Verna and Marrs

McLean Department of Biochemistry and Microbiology, Baylor College of Medicine

The E. coli LexA repressor is a model system for understanding how bacteria use a genetic switch

to respond to DNA damage. However, the evolutionary constraints on the LexA protein structure

as they relate to genetic switch function are not well understood. Here we provide an evolutionary

model that attributes mutational robustness of a gene to its negative feedback mechanism. We

used a computational tool for sequence analysis to identify LexA residues under evolutionary

selection and targeted those residues for mutagenesis. We find that the impact of these mutations

is dependent on the genetic architecture of the system. In the absence of negative feedback, a

majority of these substitutions reduce steady-state expression levels and reduce in vivo repressor

function. However, in the context of the negative feedback loop, the impact of these mutations is

mitigated. In our model, deleterious mutations that reduce cellular LexA levels also reduce auto-

repression and consequently cause up-regulation of LexA gene expression. The result is self-

correcting system that buffers against mutations perturbing repressor stability. This suggests

negative feedback provides a direct selective advantage for the gene that is manifest in ~40% of

transcription factors in E. coli having this genetic architecture.


Consortia, on the Training Program in Biomedical Informatics, National Library of Medicine

(NLM) T15LM007093.

55



Design of An Anti-HIV Protease Transducer

Aditya Hindupur1, & Robert Fox1

(1) Department of Biology & Biochemistry, University of Houston University of Houston

Current therapies against HIV, mainly the highly active anti-retroviral therapy (HAART),

concentrate on reducing the viral load and recovering CD4 levels, however they are faced with

a rebound of HIV levels on cessation of therapy. Also of concern is the number of reports on

resistance to such therapies. There has been a realization that control of HIV requires a multi-

pronged approach targeting viremia and a strong antiviral response, and there are new

treatment regimens being tested. They show limited success with the elimination of HIV

reservoirs. To address these issues we propose the design and development of Anti- HIV

transducer proteins. Activation of these proteins is by the HIV protease, and when activated

causes the death of cells expressing the viral protease. This mechanism can be used to treat all

cells within the host, with only those cells expressing HIV protease being killed. We believe

this to be an efficient manner for viral eradication. Upon incorporating recognition signals to

other HIV proteins we can make our Anti-HIV transducers more specific in recognizing

infected cells.

56


Biophysics Poster Number P-32

Novel Class of Thioaptamers Targeting Dengue

Virus Envelope Protein

Sai Hari Gandham1, Miguel Elizondo Riojas2, David Volk2, & David Gorenstein1,2 (1) Biochemistry and Molecular Biology, University of Texas Medical Branch (2) Institute of

Molecular Medicine (IMM), University of Texas Health Science Center at Houston

The Dengue Virus (DENV) is an arbovirus that belongs to the family of Flaviviridae, genus

Flavivirus. It includes Dengue virus (types 1-4), West Nile virus, Langat virus, Omsk hemorrhagic

fever virus and others that cause encephalitic or hemorrhagic disease. DENV binds the cell surface

receptors and fuses with the host cell membrane using surface envelope protein (E). The E protein

ectodomain contains three domains, ED1, ED2 and ED3. The highly immunogenic ED3 is the

putative receptor binding domain that also binds neutralizing antibodies. Thus, ED3 is an

attractive target for development of vaccines, anti-viral agents and diagnostic antigens. We have

developed leads targeting the ED3 using small molecules and short modified DNA molecules

called thioaptamers. Thioaptamers are highly resistant to nuclease digestion and do not elicit an

immune response. We have identified small molecule hits using computational methods and

validated them using protein stability shift and 2-D HSQC NMR experiments. The thioaptamers

can be selectively modified by attaching the small molecules to the bases at different positions

using chemical methods to create novel class of aptamers. The sequence variations along with the

selective modifications provide great diversity of highly specific molecules. These molecules can

potentially be used as therapeutics or vaccine against dengue virus infection.

57



Development of a Novel Method to Image the α7

Nicotinic Acetylcholine Receptor Levels

Gary Stinnett1, Pedersen SE1 , & Pautler RG1

(1) Molecular Physiology and Biophysics, Baylor College of Medicine

An estimated 5.3 million people are currently afflicted with Alzheimer’s disease (AD). In fact,

1 in 8 people over the age of 65 suffer from AD, which is the 7th leading cause of death in the

United States. In addition to the devastating effects on individuals and their loved ones, the

annual costs associated with AD exceeds $170 billion dollars per year. Hence, non-invasive

imaging modalities that allow early diagnosis and monitoring of AD progression and the

assessment of the efficacy of therapeutic interventions are greatly needed. Recently, it has been

reported that the α7 nicotinic acetylcholine receptor is up regulated in response to repeated

stimulation with agonists such as amyloid β, a protein that is thought to be a primary

contributor to AD pathology. The goal of this work is to engineer novel magnetic resonance

imaging (MRI) agents that can target the α7 nicotinic acetylcholine receptor and provide

unprecedented insights into AD pathology. Our initial efforts will include using α-

bungarotoxin to target the 7 receptor and generating contrast with gadolinium III (Gd) chelated

in diethylene triamine pentaacetic acid (DTPA). We will assess the activity of α-bungarotoxin

using Torpedo californica membranes, which have a high concentration of the muscle nicotinic

acetylcholine receptor for which 'α'BgTx has a low pM affinity. We do this to determine if the

labeling has any degradation on α-bungarotoxin’s nicotinic acetylcholine receptor binding site.

Other binding assays with the α7 receptor will also be warranted. It may be necessary to

improve the contrast generated by this targeted contrast agent. DTPA binds eight of nine

coordination points on Gd leaving Gd only one coordination point available to interact with

water. Using a different chelate, such as DOTA which only takes up 6 coordination points, we

could improve the contrast provided by Gd. We could also substitute in new Gd vehicles, such

as ultra short nanotubes or Gd-DTPA bound fullerene, which have been shown to vastly

improve contrast enhancement. This increased contrast could provide the ability to discern

changes in specific regions of the brain leading to a better characterization of α7 concentration

variance in AD. Characterizing α7 as a potential early indicator of AD could eventually lead to

diagnosis and treatment strategies to improve the quality of life of millions.

58



Reprogramming the Conformational Regulation of

GPCR Signaling

Kuang-Yui Chen1, Patrick Barth1,2 (1) Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College

of Medicine (2) Department of Pharmacology, Baylor College of Medicine, Houston, TX, 77030

Signaling across biological membranes is critical to living cells and involves membrane-embedded

receptors that transduce extracellular stimuli into cytoplasmic responses. G-protein coupled receptors

(GPCRs) are one of the largest families of these receptors that serve this function and do so through

long-range allosteric changes. Despite extensive genomic and functional studies on GPCRs, it was

only in the past couple of years that new structural data have made accurately evaluating mechanistic

and structural changes possible at the atomic level. My project is part of a general effort to uncover

the sequence, structure, and energetic determinants governing GPCR signaling. I hypothesize that

GPCR signaling is driven by conformational changes that allow them to toggle between functional

states; computational designs that stabilize receptors in different states will allow us to reprogram

their signaling properties and provide an atomic-level understanding of the underlying conformational

regulation.

To address the relationship between protein stability and conformational plasticity in allosteric

receptors, we have combined sequence bioinformatics techniques with the design mode of

RosettaMembrane to probe the sequence space governing GPCR conformational stability. Sites

predicted to be suboptimal for the stability of the receptor are targeted for design, and mutations that

stabilize the receptor’s inactive conformation are selected. The mutants are characterized via

radioligand binding assays to measure apparent thermal stability. Our initial designs of the beta-1-

adrenergic receptor (B1AR) have yielded mutants with increased apparent thermostability in

comparison to a B1AR variant (M23) used in solving the B1AR structure (Warne et al. 2008). This

method can be applied to receptors with unknown structure to facilitate structural determination.

Additionally we are working to modulate and switch the functional states of GPCRs. Using multi-state

design with RosettaMembrane, we have been able to design mutants of rhodopsin as well as dopamine

D2 receptor (D2DR) with increased constitutive activity over their respective wild types. Furthermore,

we are able to design mutants of D2DR that display increased active-state stability. With over eight

hundred GPCRs in the human genome, many of which have been implicated in disease studies, it is

apparent that there is a critical need for atomic-resolution structural and mechanistic understanding of

their signaling. The bridging of computational molecular biophysics and molecular biology in the

GPCR research field is rapidly developing and will contribute to the basic scientific knowledge of

receptor signaling as well as provide a means to develop better treatment for a multitude of diseases. This research was funded by a training fellowship from the Keck Center of the Gulf Coast Consortia, on the Houston

Area Molecular Biophysics Program, National Institute of General Medical Sciences (NIGMS) T32GM008280

59



Multiscale Simulation on a Light-Harvesting

Molecular Triad

Guoxiong Su1, Arkadiusz Czader2, Dirar Homouz3, Gabriela Bernardes1, Sana Mateen1,

& Margaret S. Cheung1 (1) Department of Physics, University of Houston (2) Department of Chemistry, University of

Houston (3) Department of Applied Math and Sciences, Khalifa University, Abu Dhabi, UAE

We have investigated the effect of solvation and confinement on an artificial photosynthetic

material, carotenoid-porphyrin-C60 molecular triad, by a multiscale approach and an enhanced

sampling technique. We have developed a combined approach of quantum chemistry,

statistical physics, and all-atomistic molecular dynamics simulation to determine the partial

atomic charges of the ground-state triad. To fully explore the free energy landscape of triad, the

Replica Exchange Method was applied in enhancing the sampling efficiency of the

simulations. The confinement effects on the triad were modeled by imposing three sizes of

spherocylindrical nanocapsules. The triad is structurally flexible under ambient conditions and

its conformation distribution is manipulated by the choice of water models and confinement.

Two types of water models (SPC/E and TIP3P) are used for solvation. When solvated by SPC/

E water, whose HOH angle follows an ideal tetrahedron, the structural characteristics of triad is

compact in the bulk systems. However, under a certain nano-sized confinement that drastically

disrupt hydrogen bond network in solvent, the triad favors an extended configuration. In

contrast, the triad solvated by TIP3P water has shown a set of U-shaped conformations in the

confinement. We have shown that a slight structural difference in the two water models with

the same dipole moment can have great distinction in water density, water orientation and the

number of hydrogen bonds in the proximity of a large flexible compound such as the triad.

Subsequently, it has direct impact on the position of the triad in a confinement as well as the

distribution of conformations at the interface of liquid and solid in a finite-size system.

60



A Physics-Based Approach of Coarse-Graining the

Cytoplasm of E.Coli (CGCYTO)

Qian Wang1, Margaret S. Cheung1

(1) Department of Physics, University of Houston

We have investigated protein stability in an environment of E. coli cytoplasm using coarse-grained

computer simulations. To coarse-grain a small slide of E. coli's cytoplasm consisting of over 16

million atoms, we developed a self-assembled clustering algorithm (CGCYTO). CGCYTO uses a

tunable resolution parameter (λ, ranging from 0 to 1) to justify the resolution of a cytoplasm,

depending on the size of a test protein for the computation of covolumes and the volume of a

macromolecule in the cytoplasm. We compared the results from a polydisperse cytoplasm model

(PD model) from CGCTYO with two other coarse-grained hard-sphere cytoplasm models: (1) F70

model, macromolecules in the cytoplasm were modeled by homogeneous hard spheres with a

radius of 55Å and (2) HS model, each macromolecule in the cytoplasm is modeled by hard

spheres of various sizes. It was found that the folding temperature Tf of a test protein (apoazurin)

is ~5 degrees higher in a PD model than that in a F70 model. In addition, there is a deviation of

1.7 degrees on Tf when an apoazurin is randomly placed at different voids formed by particle

fluctuations in a PD model, 0.7 degrees higher than that in a HS model.

61



Biochemical and Biophysical Characterization of

the Putative Encapsidation Protein

FromLactococcus Lactis Phage Asccφ28

Emilio ReyesAldrete1, Cecile Bussetta1, Michal R. Szymanski1, Wlodzimierz Bujalowski1,

Kyung Choi1, Marc C. Morais1 (1) Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston of

Biochemistry & Molecular Biology, University of Texas Medical Branch Galveston,


Tailed dsDNA bacteriophages and many dsDNA eukaryotic viruses package their genomes

into preformed empty protein shells, or procapsids, in a highly thermodynamically unfavorable

process. This process is driven by virus-encoded macromolecular motor complexes with

energy derived from the hydrolysis of ATP. A recombinant form of the predicted packaging

ATPase from Lactococcus lactis phage asccφ28 has been expressed in Escherichia coli and

purified to homogeneity. Size-exclusion chromatography and electrophoresis under native

conditions suggest that the recombinant protein forms a single oligomeric species in solution.

Analytical ultracentrifugation studies indicate the presence of a decameric oligomer, while

small-angle x-ray scattering experiments confirm the stoichiometry and suggest a ring-shaped

assembly with a maximum dimension of 150Å and radius of gyration of 53.5Å. The

stoichiometry of the assembly differs from that of the widely studied and closely related

encapsidation protein from phage φ29 (55.6% sequence similarity, Smith-Waterman score),

which forms pentameric rings when attached to viral proheads. The asccφ28 assembly is a

highly soluble, very stable, and active as an ATPase with kinetic parameters of Km=28µM and

Kcat=0.849sec-1. In contrast, packaging ATPases from other phage systems typically have

barely detectable ATPase activity unless they are bound to proheads. Hence, the recombinant

form of the putative encapsidation protein from the Lactococcus lactis phage asccφ28 may

provide an excellent model system for in vitro studies of the isolated ATPase component of

phage dsDNA-packaging motors.


Consortia, on the Houston Area Molecular Biophysics Training Program, NIGMS T32

GM008280

62



Voltage Dependent Conformational States of the

Transmembrane Protein Prestin Measured By

FLIM-FRET Techniques

Chance Mooney1, Ramsey Kamar1, Ryan McGuire1, Guillame Duret1, Fred Pereira2, Robert

Raphael1 (1) Department of Bioengineering, Rice University (2) Department of Otolaryngology, Baylor

College of Medicine

The transmembrane protein prestin forms an integral part of the mammalian sense of hearing by

providing the driving force for the electromotility of the outer hair cell, a specialized cell that

resides within the cochlea. This provides the cochlea with an ability to amplify mechanical

vibrations, allowing for a high degree of sensitivity and selectivity in auditory transduction. This

phenomenon, driven by changes in the transmembrane potential, is thought to be the result of

conformational changes in self-associating prestin oligomers. We have previously utilized Forster

resonance energy transfer (FRET), by acceptor photobleach methods, to detect the changes in

these conformational states in response to controlled voltage stimulus. While these methods

reported positive results, the standard deviation of the FRET efficiency was not sufficient for

determining nanoscale changes in prestin organization. Here we expand upon this work by

utilizing fluorescence lifetime imaging (FLIM) detected by time correlated single photon counting

(TCSPC), the most accurate FRET measurement technique available. FLIM techniques measure

the characteristic fluorescence decay profiles of reporters; changes in these profiles can be caused

by quenching processes such as FRET activity. Human embryonic kidney cells are used to host

prestin molecules that are genetically encoded with either teal fluorescent protein (TFP) or the

yellow fluorescent protein variant citrine. Individual cells are then voltage-clamped and the

intermolecular distance between TFP and citrine is probed via FLIM-FRET methods. This has

revealed large changes in detected FRET efficiencies between hyperpolarized cells (32%

efficiency) and depolarized cells (13% efficiency), implying significant changes in prestin

conformation.

63



Quantitative Characterization of Facilitated

Target Search During Protein-DNA Interactions

Alexandre Esadze1, Hegde Muralidhar L. 1, Sankar Mitra, 1 & Junji Iwahara1 (1) Biochemistry and Molecular Biology, University of Texas Medical Branch Galveston Dept.

of Biochemistry and Molecular Biology

Eukaryotic transcription factors and DNA repair proteins have to find their target DNA sites

among billions of base pairs in human genome. In order to execute their function efficiently,

proteins have to scan the “sea” of nonspecific binding sites with the rate close to several tenth

of thousands of base pairs per second. Most (if not all) DNA-binding proteins exhibit tight

binding even to nonspecific DNA with a dissociation constant in a range of ~10-9 – 10-5 M,

which leads to a speculation that trapping of the proteins at nonspecific DNA sites should

retard target search as a result slow down the execution of their function as well. Despite this

reasonable speculation, transcription factors and DNA repair enzymes can rapidly locate their

target sites. Some different mechanisms that accelerate the genome scanning efficiency were

proposed: Namely, direct transfer (intersegment transfer), sliding and hopping. There is

remarkable shortage of quantitative information on these mechanisms of facilitated target

search processes. While recent advancements in single molecule biophysics allowed

determination of apparent 1D diffusion constant for proteins sliding on DNA, the single

molecule technique can neither mimic high concentration of DNA in the cell nor provide

experimental sets with good enough temporal and spatial resolution to derive any solid

conclusions on contributions of different target search processes. Here we present new

fluorescence based techniques that permit determination of rate constants for direct transfer and

sliding in bulk solution in presence of excess of nonspecific DNA that mimics the cellular

environment. Our approach can be applied to any DNA binding protein under a wide range of

experimental conditions. Using the new method, we have successfully determined direct

transfer rate constants for NEIL-1 and Egr-1 proteins. We also found that sliding clearly

enhances the target search process of Egr-1. We expect that these newly developed quantitative

methods will greatly facilitate quantitative characterizations of facilitated target search

mechanisms, and ultimately allow us to engineer proteins that exhibit increased activity due to

faster target search.

64



Helicase-Initiated Assembly of the Primosome

Michal R. Szymanski1, Maria J. Jezewska1, & Wlodek Bujalowski1,2,3 (1) Biochemistry and Molecular Biology, University of Texas Medical Branch Galveston (2)

Sealy Center of Structural Biology and Molecular Biophysics (3) Department of Obstetrics and

Gynecology, University of Texas Medical Branch Galveston, Galveston, TX, 77555

A direct quantitative analysis of the initial steps in primosome assembly, involving PriA and PriB

proteins and the minimal primosome assembly site (PAS) of phage ϕX174, has been performed

using fluorescence intensity, fluorescence anisotropy titration, and fluorescence resonance energy

transfer techniques. We show that two PriA molecules bind to the PAS at both strong and weak

binding sites on the PAS structure without detectable cooperative interactions. Binding of the PriB

dimer to the PriA – PAS complex dramatically increases PriA's affinity for the strong site, but

only slightly affects its affinity for the weak site. Interactions are driven by apparent entropy

changes, with binding to the strong site accompanied by a large unfavorable enthalpy change. The

PriA – PriB complex, formed independently of the DNA, is able to directly recognize the PAS

structure. Thus, the high-affinity state of PriA for PAS is generated through PriA – PriB

interactions. The effect of PriB is specific for PriA–PAS association, but not for PriA–double-

stranded DNA or PriA–single-stranded DNA interactions. Only complexes containing two PriA

molecules can generate a profound change in the PAS structure in the presence of ATP. The

obtained results provide a quantitative framework for initiation of primosome formation and

elucidation of further steps in the assembly process.

65



Label-Free and Noninvasive Sensor for Measuring

Membrane Potentials of Biological Cells

Kimal Rajapakshe1, Asanga Wijesinghe1, William Widger2, & John Miller1 (1) Department of Physics, University of Houston (2) Dept. of Biology & Biochemistry,

University of Houston, Houston, TX, 77204

Measuring or monitoring the membrane potentials of biological cells using conventional

methods (patch-clamp, fluorescent dyes, etc.) has several limitations. These include being

invasive (patch-clamp), the need for labeling (fluorescent dyes), and being labor intensive.

Here we discuss the use of impedance spectroscopy as a novel approach, and report on the

relationship between the membrane potential of phosphatidylcholine liposome suspensions and

their measured dielectric and conductivity spectra vs. frequency. The liposomes are prepared to

have a higher concentration of potassium ions (K+) inside the membrane as compared to the

external medium. Under valinomycin (K+ ionophores) these liposomes generate a negative

membrane potential, as verified by measurements using voltage-sensitive fluorescent dyes.

Impedance measurements (0.1 Hz to 1 MHz) are carried out on liposome suspensions with

different membrane potentials. Both dielectric and conductivity spectra display low frequency

dispersions that vary with membrane potential. Possible future applications include

electromagnetic sensors to test the effects of new drugs on membrane potential and other

properties of living cells.

66



Solution Biophysics Research Laboratory

Luis Holthauzen1, & B. Montgomery Pettitt1 (1) Sealy Center for Structural Biology, University of Texas Medical Branch , 301 University

Blvd, Galveston, TX, 77555

The Solution Biophysics Laboratory (SBL), located in rooms 5.148 and 5.150 on the fifth floor of

the Medical Research Building, was established in 1995. The purpose of the lab is to enhance

solution biophysics research of SCSB core faculty members and UTMB collaborators. Instruction

is given in the use of a variety of SBL instrumentation, including: circular dichroism, analytical

ultracentrifugation, fluorescence, MALDI-TOF mass spectro-metry, surface plasmon resonance,

titration micro-calorimetry, and high throughput screening using the Johnson and Johnson

ThermoFluor HTS apparatus.

67



Asymmetrical Roles of Zinc Fingers in Dynamic

DNA-Scanning Process By the Inducible

Transcription Factor Egr-1

Levani Zandarashvili1, Dana Vuzman2, Yuki Takayama1, Alexandre Esadze1, Debashish

Sahu1, Yaakov Levy2, & Junji Iwahara1 (1) Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular

Biophysics, University of Texas Medical Branch Galveston (2) Department of Structural

Biology, Weizmann Institute of Science, Rehovot, 76100, Israel

Egr-1 is an inducible transcription factor that recognizes 9-base pair target DNA sites via three

zinc finger domains and activates genes in response to cellular stimuli, such as synaptic signals

and acute vascular stresses. Using spectroscopic and computational approaches, we have

studied how Egr-1 scans DNA to rapidly find its target sites. Although all three zinc finger

domains are involved in recognition of a target DNA site, our NMR data indicate that the zinc-

finger domain 1 is mostly dissociated from DNA while Egr-1 is scanning nonspecific DNA.

Egr-1 binds to nonspecific DNA primarily via zinc-finger domains 2 and 3 only, which are

more positively charged and tightly packed. The dynamic mode of binding seems to enhance

Egr-1’s translocation on DNA, particularly via intersegment transfer that involves transient

bridging of two DNA sites. These results shed light on asymmetrical roles of the zinc finger

domains for efficient DNA-scanning in the nucleus.

68



Ion-Pair Dynamics At Protein-DNA Interface

Studied By NMR Spectroscopy

Junji Iwahara1

(1) Biochemistry and Molecular Biology, University of Texas Medical Branch Galveston

Ion pairing is one of the most fundamental chemical interactions that are critically important for

molecular recognition by biological macromolecules. While absorption spectroscopy, infrared

spectroscopy, and Raman spectroscopy have been used to characterize organic ion pairs in

previous studies, these methods can be applied only to relatively simple, small compounds. From

an experimental standpoint, very little is currently known about ion-pair dynamics in biological

macromolecular systems. Using new NMR methods based on 15N relaxation and inter-hydrogen

bond scalar 15N-31P J-coupling, we have investigated the dynamic transitions between the contact

ion-pair (CIP) and solvent-separated ion-pair (SIP) states for ion pairs between lysine sidechain

NH3+ groups and DNA phosphate groups at the molecular interface of the HoxD9 homeodomain /

DNA complex. Our new NMR methods provide both spatial and temporal information on the

dynamics of individual NH3+ / phosphate ion pairs. Our experimental data suggest that the ion

pairs at the protein-DNA interface are highly dynamic and that the transitions between CIP and

SIP occur on a sub-nanosecond timescale. Based on the information on the ion pair dynamics, we

estimated a time necessary for the complex to break all ion pairs between protein and DNA.

Interestingly, the estimated time is qualitatively consistent with the timescale of sliding of the

protein on DNA. These data suggest that our NMR methods can provide important information

about how the dynamics of ion pairing and hydrogen bonding are linked to the kinetic properties

of macromolecules and macromolecular complexes.

69



Tracking UNC-45 Chaperone-Myosin Interaction

With a Titin Mechanical Reporter

Paul Bujalowski1, Christian M. Kaiser1,4, Liang Ma1, John Anderson1, Henry F.

Epstein1,2,3, and Andres F. Oberhauser1,2,3 (1) Sealy Center for Structural Biology and Molecular Biophysics, University of Texas

Medical Branch at Galveston and Department of Neuroscience and Cell Biology (2)

Department of Biochemistry and Molecular Biology (3) Sealy Center for Structural Biology

and Molecular Biophysics University of Texas Medical Branch at Galveston (4)Present

Address: Department of Physics and QB3 Institute, University of California at Berkeley

Myosins are molecular motors that convert chemical energy into mechanical work.

Allosterically coupling ATP-binding, hydrolysis and binding/dissociation to actin filaments

requires precise and coordinated structural changes that are achieved by the structurally

complex myosin motor domain. UNC-45, a member of the UCS family of proteins, acts as a

chaperone for myosin and is essential for proper folding and assembly of myosin into muscle

thick filaments in vivo. The molecular mechanisms by which UNC-45 interacts with myosin to

promote proper folding of the myosin head domain are not known. We have devised a novel

approach to analyze the interaction of UNC-45 with the myosin motor domain at the single

molecule level utilizing atomic force microscopy (AFM). By chemically coupling a titin I27

polyprotein to the motor domain of myosin, we introduced a “mechanical reporter”. In

addition, the polyprotein provided a specific attachment point and an unambiguous mechanical

fingerprint, facilitating our AFM measurements. This approach enabled us to study UNC-45–

motor domain interactions. After mechanical unfolding, the motor domain interfered with

refolding of the otherwise robust I27 modules, presumably by recruiting them into a misfolded

state. In the presence of UNC-45, I27 folding was restored. Our single molecule approach

enables the study of UNC-45 chaperone interactions with myosin and their consequences for

motor domain folding and misfolding in mechanistic detail.

70



Role of Disordered C-Terminus in Human DNA

Glycosylase NEIL1’S Structure and Functions:

Implications for the Excision Repair of Oxidized

Bases in Genome

Muralidhar Hegde1, Luis M. F. Holthauzen1, Pavana M. Hegde1, Arijit Dutta1, Numan

Oezguen2, Sankar Mitra1 (1) Department of Biochemistry and Molecular Biology, University of Texas Medical Branch (2)

Department of Internal Med-Endocrinology, University of Texas medical Branch, Galveston,

Texas-77555, USA

NEIL1, one of the five DNA glycosylases in human cells responsible for the excision repair of

oxidized bases in the genome, is unique in its S phase specific activation and its higher activity

with DNA substrates mimicking DNA replication intermediates. NEIL1 interacts with DNA

replication proteins (co-opting them for repair during replication) by utilizing a common

disordered segment (aa 311-389) at its C-terminus. The disordered structure of NEIL1’s C-

terminus was initially indicated by the need to delete this segment for crystallization (Doublie et

al., PNAS, 101:10284-9, 2004) and by PONDR modeling (Hegde et al., Cell Res, 18:27-47, 2008;

Hegde et al., Cell Mol Life Sci, 67:3573-87, 2010). We subsequently confirmed this by CD

spectra showing 95% random-coil conformation in this sequence. We also observed that this

region contains acetylable Lys residues, and has propensity to form ordered structure in the

presence of osmolytes, sarcosine or trimethylamine-N-oxide (TMAO). This suggests that such

folding could be induced by NEIL1’s interaction partners. Although, the C-terminus is not

required for NEIL1’s glycosylase activity, it is essential for repair by providing a common

interaction interface for its partners. The efficiency of repair initiated by the deletion mutant is

markedly reduced relative to the full-length protein. Further, the C-terminus is important for

NEIL1’s stability as indicated by denaturation studies with urea and high temperature. Finally, we

noticed the C-terminus has non-specific DNA binding by this region, and is involved in target

lesion search.

We made an unexpected observation that in spite of its containing four Trp along with six Tyr,

NEIL1’s fluorescence emission spectrum resembles that of Tyr. However, deletion of C-terminal

40 or 78 residues (lacking Tyr/Trp) causes transition to Trp like emission spectra. This along with

the modeling studies suggested that the C-terminus folds back to bind with NEIL1’s folded core in

its native form. In the absence of structural information for the full-length NEIL1, these

biophysical characterizations of NEIL1’s disordered C-terminus, supported by 3D modeling and

biochemical studies, provides important information on the regulatory role the disordered C-

terminus in its structure and functions. (Supported by NIH grants).

71



DNA Disorder and Packing Pressures

Christopher Myers1, Wah Chiu1,3 & B. Montgomery Pettitt1,2 (1) Structural and Computational Biology and Molecular Biophysics Program (SCBMB),

Baylor College of Medicine (2) Sealy Center for Structural Biology and Molecular Biophysics,

UTMB, Galveston, TX, 77555-0144 (3) National Center for Macromolecular Imaging (NCMI),

Baylor College of Medicine, Houston, TX

Predicting the pressure necessary to confine DNA—a highly-negatively charged, elastic

polymer—into viral capsids (over a 250-fold compaction) is a clear biophysical problem

addressable by a theoretical, physics-based approach. A detailed physical understanding of this

phenomenon will not only elucidate in vivo mechanisms of tightly-stressed or confined DNA

(transcription, nucleosomal packing, etc.) but also aid the development of phage drug-delivery

methods. Experimental measurements of phage DNA confinement includes osmotic pressure

ejection-inhibition experiments and single-molecule loading force measurements that provide

pressure data for strict validation of all theoretical models. Structural insight into DNA

packaging is also aided by cryo-electron microscopy data, such as the asymmetric

reconstructions of Wah Chiu. Current models of phage packing assume DNA behaves as a

linearly elastic polymer that bends uniformly and isotropically under stress, often assuming a

highly-ordered ‘inverse spool’ model. The assumption of such spooled conformations is based

primarily on cryo-EM density maps (obtained by averaging thousands of structures) that show

density rings, especially near the capsid surface. Because phage genomes are around ten

kilobasepairs long, we are employing a coarse-grained model of double-stranded DNA (already

successfully applied to microarray experiments) as a chain of surface-charged, ion-penetrable

spheres. Simulations of unconnected, liquid-state DNA “monomers” in capsid-like

confinement already show ringed density distributions consistent with cryo-EM data that

demonstrates the packing contribution to such structures. This shows that virtually any

connected polymer path may be consistent with such data, and could indicate an ensemble of

more entropically-driven, disordered conformations of DNA may be possible by incorporating

non-linear elastic “kinking” of DNA. Our simulations to date have also yielded very precise

determinations of pressure. By applying Monte-Carlo path-sampling “polymerization”

techniques to these monomeric systems, we are working to demonstrate an ensemble of

possible connectivities and incorporate a more accurate model of DNA elasticity to accurately

predict the pressures of confined DNA.

72


Computational Poster Number P-48

Characterizing the Structural Dynamics of Mutant

K-Ras in Developmental Disorders

Nandini Maharaj1, Gorfe1, Alemayehu1

(1) Biochemistry and Molecular Biology, University of Texas-Health Science Center at Houston

Germline Ras mutations have been revealed in a series of developmental disorders that are termed

as neuro-cardio-cutaneous conditions (NCFCs). NCFCs include Cardio Facio Cutaneous

syndrome (CFCS), Noonan Syndrome (NS) and Costello Syndrome (CS). These syndromes

involve mutations in H-Ras (CS), N-Ras (NS) and K-ras (NS, CFCS, and CS). NCFCs are

characterized by a set of distinct phenotypes that include facial dysmorphyism, cardiac anomalies,

delayed growth and mild to severe mental retardation. Germline mutations have also been

discovered in the GEFs, GAPs, and Ras effector proteins involved in the Ras-MAPK cascade

suggesting that the mechanism of action that promotes aberrant development may be due to

changes in the activation of this particular pathway. Furthermore, biochemical analysis of the Ras

mutants reveals that most of these mutants confer a lesser degree of over-activation than

oncogenic Ras proteins and in some cases loss of function; suggesting that they probably would

not support carcinogenesis. Currently, the mechanism through which these mutants support altered

Ras signaling through modifications in their structure and dynamics compared to that of wild type

or oncogenic mutants is yet to be discovered.

In order to study the dynamics of these mutant K-ras proteins we have performed all atom

molecular dynamics simulations of the catalytic domain of mutant K-ras. Preliminary analysis

revealed that these exists a correlation between the biological function and conformational

sampling of the protein between states 1 and 2 of GTP bound Ras. Further investigation into the

conformers provides valuable insights into the restructuring of the catalytic domain and the switch

regions which may favor effector binding and surprisingly in the case of some mutants disfavor

effectors binding. Furthermore we can appreciate more clues and insights into the physiological

mode of action and kinetics of these particular mutants through patterns in the conformational

sampling related to severity of the syndrome.

73



Population Genomics of the Mouse and Its

Functional Implications

Kevin Liu1, Michael H. Kohn2, Luay K. Nakhleh1 (1) Computer Science, Rice University (2) Ecology and Evolutionary Biology, Rice University,

Houston, TX 77005

House mice and related rodents play a central role throughout biomedical research. A primary

reason for the biomedical importance of mice is their genetic similarity to humans. However,

unlike the genetic diversity of the human population, classical lab mice used in biomedical

research have been shown to be largely homogeneous, primarily due to their origins from a

small sample of the wild house mouse population and subsequent inbreeding. Other wild

subspecies and species of house mice represent a largely untapped resource of genetic diversity

that could address this problem; however, we must first understand the genetic composition

and evolutionary origins of this resource to realize its full potential for translational

biomedicine and evolutionary biology. This requires computational analyses of the

evolutionary history and population genetics of wild mouse populations in comparison to

present-day laboratory mice.

Using population genomic and phylogenomic analyses of primary biomolecular sequence data,

we examined mutation and recombination in three classes of mouse strains: classical inbred

and wild-derived laboratory strains and wild mice. In each class we distinguish between the

subspecies of mice included. We found substantial variation in the decay in linkage

disequilibrium among the different mouse subspecies, the class of mouse strain, and

chromosomes in the mouse genome. Preliminary results suggest that this variation highlights

an important functional role for recombination in the different populations of mice in our

study.

We then compared the observed evolutionary patterns against reported protein interactions in

mouse. We found an overall negative correlation between measures of recombination rate and

protein interactions. Proteins that exhibited a higher than expected number of recombination

breakpoints and protein interactions showed enrichment for certain classes of genes, further

suggesting interplay between the functional role of genes in mice and evolutionary processes

such as recombination and mutation.

74



Structural Mutation Analysis of PTEN and Its

Possible Genotype-Phenotype Correlations in

Endometriosis and Cancer

Iris Nira Smith1 and James M. Briggs1, PhD (1) Biology and Biochemistry, University of Houston Dept. of Biology & Biochemistry,

University of Houston, Houston, TX, 77204

The phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene encodes a tumor

suppressor phosphatase frequently mutated in a variety of human cancers. It exerts its function by

dephosphorylating phosphatidylinositol 3,4,5-triphosphate (PIP3), converting it to

phosphatidylinositol 4,5-bisphosphate (PIP2). PIP3 activates a variety of downstream effectors

that turn on the PI3K/Akt oncogenic pathway leading to unregulated cell proliferation and

tumorigenesis. Loss of function and somatic missense mutations of PTEN have recently been

found in patients with endometriosis, endometrial cancer and ovarian cancer although no structural

information on these mutations is currently available. Through the cross-referencing of published

literature, the phenotypes of distinct PTEN mutations affecting the signature motif of the catalytic

phosphatase domain and C2 domain were identified. Among these mutations, moderate

phenotypes were associated with endometriosis and endometrial hyperplasia and are distributed

throughout both domains. Whereas the more severe phenotypes were associated with endometrial

cancer and ovarian cancer and are clustered in the signature motif (H123CXXGXXR130) that

forms the P loop at the bottom of the active site pocket. The signature motif contains residues that

play a crucial role in loop conformation (H123 and G127) and are essential for catalysis (C124

and R130). One distinct residue within the active site R130, has mutations implicated in both

moderate and severe phenotypes. In this proposed study, we will explore the structural effects that

the identified PTEN mutations have on the relationship between genotype and phenotype, and

investigate the specific molecular mechanisms involved. Molecular dynamics simulations will be

used to examine the clustered mutations to characterize their effects on P loop conformations and

the functional distortions they may impose within the active site as well as mutations within the

C2 domain that may affect PTEN’s ability to interact with the membrane. We propose mutations

within the active site disrupt the electrostatic interaction thus affecting P loop conformation. Loop

flexibility imposed by these missense mutations may affect the binding of PTEN to its ligand and

adversely affect the PI3K/Akt signaling pathway. Understanding the functional impact that these

missense mutations have on the structure of PTEN is essential to elucidating the molecular

mechanism of endometriosis, endometrial cancer and ovarian cancer in the development of novel

therapeutics.

75



A Theoretical Study of Membrane-Remodeling By

Ras Proteins

Hualin Li1, Zhenlong Li and Alemayehu A.Gorfe1 (1) Department of Integrative Biology and Pharmacology, University of Texas Health Science

Center

Ras proteins function as conformational switches controlling cell proliferation, differentiation

and development. The signaling function of these proteins was found to depend on their ability

to form protein-lipid nanodomains on the plasma membrane. Apparently driven by competing

forces emanating from their lipid-modified C-terminus and the globular catalytic domain,

nanodomains of different Ras proteins are differently arrayed on the plasma membrane. The

potential of this phenomenon to lead to a unique mechanism of signal regulation at the

membrane level has attracted much interest in recent years. In this project, we plan to study (1)

how multiple Ras proteins oligomerize on the membrane surface, (2) how these oligomers may

serve as membrane curvature regulators, and (3) how this might be influenced by

conformational variations. To address these issues, we will perform multiple coarse-grained

molecular dynamics simulations to study the structure and dynamics of active and inactive H-

ras proteins in a model membrane.

76



Epigenomic Profiling of the Osteosarcoma Genome

Jiayi Sun1, Joseph Luan2,3, Alex Yu2,3, Horatiu Voicu3,4, Chris Man2,3,4, Rudy Guerra5, &

Ching C. Lau1,2,3,4 (1) Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine,

Houston, TX, 77030 (2) Texas Children's Cancer Center, Houston, TX, 77030 (3) Dept. of

Pediatrics, Baylor College of Medicine, Houston, TX, 77030 (4) Dan L. Duncan Cancer Center,

Houston, TX, 77030 (5) Dept. of Statistics, Rice University, Houston, TX, 77251

Osteosarcoma is the most common primary bone tumor diagnosed in adolescents and young

adults. Due to the high degree of genomic heterogeneity, the tumor biology and pathogenesis of

osteosarcoma is still elusive. Overall survival for patients with metastatic osteosarcoma remains

dismal at 20-30%. To comprehensively evaluate the osteosarcoma genome, we have generated

transcriptomic, DNA copy, microRNA and DNA methylation profiling data on 44 cases of pre-

treatment biopsy. We hypothesize that epigenomic aberrations leading to repression of tumor

suppressor genes or overexpression of oncogenes are involved in the pathogenesis of

osteosarcoma. To identify regions or genes with aberrant DNA methylation, we performed whole

genome high resolution DNA methylation profiling using Illumina’s 450K array, which

interrogates the methylation status at over 450,000 sites within the genome, including CpG sites

outside of promoter regions. Comparison of methylation data with expression data reveals a set of

genes differentially methylated in tumors which show high negative correlation with gene

expression. Our current results suggest that methylation plays a fundamental role in altering the

transcription of tumor suppressor genes. Further network based studies involving pathway and

gene set enrichment analysis will clarify the role of these genes in the initiation and progression of

these tumors.

77



Repeated Sequences in Nut Allergens Mediate

Cross Reactivity Between Tree Nuts and Peanuts

Catherine H. Schein1, Suzanne S. Teuber2, & Soheila J. Maleki3 (1) Biochemistry and Molecular Biology/Microbiology and Immunology, University of Texas

Medical Branch (2) University of California, Davis, School of Medicine, Davis, CA, (3) US

Dept. of Agriculture, New Orleans, LA,

RATIONALE: Peanuts and nuts can cause severe reactions in children and adults with nut

allergies. About 30% of peanut allergic individuals also react to tree nuts, even though peanuts

are botanically a bean and walnuts, pecans, almonds, etc. come from trees. The known

allergenic proteins from these nuts are quite different in sequence. However, identifying similar

areas of physicochemical properties (PCPs), coupled with analysis of 3D-structures, can

identify similar IgE binding areas that may be important for cross-reactivity between allergenic

proteins.

METHODS: The property distance (PD) scale in the Structural Database of Allergenic Proteins

(SDAP) was used to identify short sequences in walnut and other nut allergens that were

similar in their PCPs to experimentally determined peanut IgE epitopes. These were

incorporated into SPOTs membranes, and sequences that bound IgE in sera from patients with

allergy to peanuts and walnuts determined. A cluster of repeat sequences in the N-terminal pro-

sequence of the walnut allergen Jug r 2 was identified and a consensus sequence prepared from

the repeats. A peptide from the consensus was used to generate antibodies in chickens and

rabbits. Western blotting was used to identify allergens in extracts from nuts recognized by the

antibodies, and by serum IgE from patients allergic to peanuts, walnuts, and or almonds.

Proteins in the reactive bands were identified by mass spectroscopy(MS).

RESULTS: Searching SDAP using the PD tool with the consensus peptide sequence revealed

many potential IgE epitopes with similar physicochemical properties in nut allergens, including

several 7S, 2S and 11S albumins from tree nuts. The antibodies generated to the consensus

peptide recognized proteins of the appropriate size to these proteins in various nut extracts.

Many of the predicted proteins were identified by MS in bands on the gel that bound the

consensus peptide antibodies; these were also proteins that were detected by IgE in the sera of

patients with documented clinical cross-reactivity.

CONCLUSIONS:The PD scale in SDAP is a good starting point for identifying potential cross-

reactive sequences in allergens. A repeated sequence motif, summarized by our consensus

peptide, is common to many different allergenic proteins from nuts and seeds. Such repeats

may be a relics of very ancient storage proteins that are conserved in various forms in nut

proteins, perhaps because of their functional or enzymatic roles.

78



Practical Issues for Designing Efficient Sequence-

Based Genetic Studies of Quantitative Traits

Rosa Banuelos1, Dajiang J. Liu3, & Suzanne M. Leal1,2 (1) Dept. of Statistics, Rice University (2) Dept. of Human & Molecular Genetics, Baylor College

of Medicine, Houston, TX, 77030 (3) Dept. of Biostatistics, University of Michigan, Ann Arbor,

MI, 48109

There is considerable interest in understanding the role of rare variants in complex disorders.

While the cost of next generation sequencing has decreased, the expense of sequencing large

datasets remains. To reduce the sequencing cost for quantitative traits (QT), two strategies can be

incorporated into the study design: sampling individuals with extreme quantitative traits and

utilizing publically available phenotyped cohorts. For most quantitative trait studies, one tail from

the distribution is of primary clinical interest. Samples from the other extreme are used as a

“control” group. It is also possible to use the study design where individuals are selected from one

extreme of the QT distribution and the “control” group can either be randomly ascertained or a

less stringent cut off can be used. For these scenarios, individuals from public cohorts can serve as

“controls.” Practical issues related to designing efficient rare variant QT association studies are

investigated using extensive simulations. It is shown that for a fixed cohort size of whole-exome

sequence samples, analyzing the QT values is generally more powerful than dichotomizing the QT

and analyzing it as a binary trait. When selective sampling is carried-out from an existing cohort

of samples, sequencing an equal number of samples from each extreme is not the optimal study

design. Instead, sequencing a fewer number of individuals from the extreme of clinical interest,

which allows a more extreme cut-off to be applied, and more samples from the other extreme can

increase power. Additionally leveraging data from publicly available cohorts can increase the

power of an extreme sample study design. In conclusion, the results and study designs presented

will provide important guidance for implementing efficient sequence based QT studies.

79



Membrane Shape Remodeling By Lipid-Modified

Ras Proteins

Zhenlong Li1, Lorant Janosi1, Alemayehu A. Gorfe1 (1) Department of Integrative Biology & Pharmacology, University of Texas Health Science

Center at Houston, Houston TX

Ras proteins are small GTP-hydrohydrolyzing enzymes that operate as molecular switches in

signal transduction pathways and are present in a mutant, activated state in many human

tumors. It has been shown that the lipid-modified C-terminus drive lateral segregation of Ras

proteins into membrane sub-structure on the plasma membrane. Such transient and dynamic

molecular assembly, so-called nanocluster, is emerging as a crucial mechanism by which cells

achieve high-fidelity signal transmission. However, little is known about the underlying force

driving the formation of nanoclusters on the plasma membrane and their influence on the

membrane structure.

To investigate the interaction between nanocluster and the membrane, we carried out extensive

semi-atomistic molecular dynamics simulations of the C-terminal membrane-targeting motif of

H-ras (tH) in a phase separated lipid bilayer composed of 2000 DPPC, DliPC and cholesterol

molecules. We found out that at ambient temperature approximately 30-40% of tH molecules

assemble into clusters of 4-9 proteins, and preferentially localize at the interface between the

liquid-ordered and liquid-disordered phases of the membrane. In the nanoclusters, tH

backbones preferably exhibits an extended conformation and align linearly at the phase

interface. With such molecular-level organization, tH molecule act as a linactant at the interface

and reduce line tension of the anchor-containing monolayer and induces shape transition of the

overall membrane due to the tendency of the system to minimize the free energy. The resultant

geometry of the membrane is determined by the different elastic bending modulus of different

phases as well as the phase boundary line tension, which is strongly affected by the localization

of the nanoclusters. Our findings were explained based on the elastic membrane theory and

provided new perspective on the interaction between membrane-bounded proteins and

membrane domains, such as lipid rafts.

80



Vessel Connectivity Using Murray Hypothesis

Yifeng Jiang1, Xenophon Papademetris2,Zhen W. Zhuang3,Albert J. Sinusas2,3,Ananth

Annapragada4, Ioannis A. Kakadiaris1 (1) Texas Learning and Computation Center, University of Houston (2) Dept. of Diagnostic

Radiology, Yale University, New Haven, CT, 06519 (3) Dept. of Internal Medicine, Yale

University, New Haven, CT, 06519 (4) Texas Children Hospital, Houston, Texas 77030

We describe a new method for vascular image analysis that incorporates a generic physiological

principle to estimate vessel connectivity, which is a key issue in reconstructing complete vascular

trees from image data. We follow Murray’s hypothesis of the minimum work principle to

formulate the problem as an optimization problem. This principle reflects a global property of any

vascular network, in contrast to various local geometric properties adopted as constraints

previously. We demonstrate the effectiveness of our method using a set of microCT mouse

coronary images. It is shown that the performance of our method has a statistically significant

improvement over the widely adopted minimum spanning tree methods that rely on local

geometric constraints.

81



Keloid Susceptibility Loci 1q41, 3q22, and 15q21

Replicated in a Nigerian Population

Stanley Hooker1, Peter Olaitan2, Odunayo Oluwatosin3, Solomon Fadiora2, S Ademola3,

Victoria Odesina4, Suzanne M Leal1, Ernst Reichenberger5

(1) Molecular and Human Genetics, Baylor College of Medicine (2) Dept. of Surgery,

Ladoke Akintola University of Technology Teaching Hospital, Osogbo, Osun State,

Nigeria (3) Dept. of Surgery, University of Ibadan, Ibadan, Oyo State, Nigeria (4)

HIV Adherence Program, University of Connecticut Health Center, Farmington, CT

06030 (5) Dept. of Reconstructive Sciences, University of Connecticut Health

Center, Farmington, CT 06030

Keloids are overgrown scars that stem from abnormal wound healing and their pathogenesis is

poorly understood. The highest incidence occurs in darker-skinned individuals, with the

greatest prevalence found in those with African ancestry. Recently a genome-wide association

(GWA) study was performed, using a Japanese population, to identify linked genes or loci

(Nakashima et al., Nat Genet 2010). Four variants were associated at GWA study significance

levels on chromosomes 1q41, 3q22.3 and 15q21.3. Only one SNP lies within a known gene,

NEDD4, on chromosome 15. We genotyped 96 tagSNPs within these regions in 286 case and

267 control Yorubans that were ascertained in Nigeria. Analysis was performed within a

logistic regression framework using an additive allelic effect model controlling for both age

and sex. Significant findings for each region included: 1q41 - rs873549 OR = 1.54 (p = 0.012);

3q22 - rs13064974, chr3:140.1Mb, OR = 1.32 (p = 0.026) and rs940187, chr3:140.3Mb, OR =

1.33 (p = 0.027); and 15q21 - rs8031043 OR = 0.68 (p = 0.002). The minor alleles of rs873549

and rs940187 increased the risk of keloid development in both studies. This is the first study to

characterize the genetic susceptibility of keloids in a sub-Saharan African population.

82



Calling Amplified Or Deleted Haplotypes in Next

Generation Tumor Sequencing Data

Ninad Dewal1, Yang Hu2,Matthew L. Freedman3,4,Thomas LaFramboise5,Itsik Pe'er2,David

A. Wheeler1,Richard A. Gibbs1 (1) Human Genome Sequencing Center, Baylor College of Medicine , Houston, TX 77030 (2)

Department of Computer Science, Columbia University, New York, NY 10027 (3) Department of

Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115 (4) Medical and Population

Genetics Program, The Broad Institute of Harvard and MIT, Cambridge, MA 02142 (5)

Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, OH

44106

During tumor initiation and progression, cancer cells acquire a selective advantage, allowing them

to out-compete their normal counterparts. Identification of the genetic changes that underlie these

tumor acquired traits can provide deeper insights into the biology of tumorigenesis. Regions of

copy number alterations (CNAs) and germline DNA variants are some of the elements subject to

selection during tumor evolution. Integrated examination of inherited variation and somatic

alterations holds the potential to reveal specific nucleotide alleles that a tumor “prefers” to have

amplified or deleted. Next-generation sequencing of tumor and matched normal tissues provides a

high-resolution platform to identify and analyze such somatic CNA regions. Within a CNA

region, examination of informative (e.g. heterozygous) sites deviating from a 1:1 ratio may

suggest selection of that allele. A naïve approach examines the reads for each heterozygous site in

isolation; however, this ignores available valuable linkage information across sites. We therefore

present an updated version of our Hidden Markov Model-based method – Haplotype

Amplification in Tumor Sequences 2.0 (HATS 2.0) – that analyzes tumor and normal sequence

data, along with training data for phasing purposes, to infer amplified or deleted alleles and

haplotypes in regions of somatic copy number change. Our method is designed to handle rare

variants and biases in read data. We assess the performance of HATS using simulated amplified or

deleted regions generated from varying copy number and coverage levels, followed by CNA

regions in real data. We demonstrate that HATS infers the amplified or deleted alleles more

accurately than does the naïve approach, especially at low to intermediate coverage levels and in

cases (including high coverage) possessing stromal contamination or allelic bias. Lastly, HATS

2.0 can now detect bi-allelic amplifications or deletions, versus the previously assumed mono-

allelic changes.


Consortia, on the NLM Training Program in Biomedical Informatics, National Library of

Medicine (NLM) T15LM007093.

83



Identification of Novel Allosteric Sites on Ras and

Initial Screening for Potential Inhibitors

Harrison Hocker1, Alemayehu Gorfe1 (1) Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center

at Houston

Ras is a 21 kDa monomeric G-protein that functions as a molecular switch on the inner leaflet of the

plasma membrane. The Ras protein exists in either an active state when bound to GTP, or an inactive state

when GDP-bound. In the active state, Ras is able to bind effectors such as Raf and phosphatidylinositol 3-

kinase (PI3K). Hydrolysis of the γ-phosphate of GTP results in a conformational change and deactivation

of Ras. Somatic mutations on Ras at positions 12, 13 and 61 result in constitutive activity and an enhanced

ability to initiate downstream signaling, leading to abnormal G1 to S cell cycle progression, decreased

ability to undergo apoptosis, and enhanced ability of cells to survive. Pharmaceutical companies have

developed farnesyl transferase inhibitors that prevent Ras farnesylation, but Ras is alternatively prenylated

by geranylgeranyl transferase 1, which allows for proper protein function even without its wild type lipid

modification. Another is approach to inhibit the regulators and effectors of Ras. However, since Ras

proteins have multiple downstream effectors, a particular kinase inhibitor will only impair a subset of Ras

mediated pathways, leading to partial therapeutic results. To find new drugs that directly inhibit Ras, we

designed a multi-level computational approach that uses blind docking of known Ras binders and

molecular dynamics simulations with the aim of discovering novel allosteric sites and small molecules that

bind to these site. This strategy allows us to screen large numbers of compounds quickly and inexpensively.

The use of an ensemble of structures derived from molecular dynamics simulations enables us to account

for the intrinsic flexibility of Ras so that transiently-opening drug binding sites not present in the crystal

structures can be targeted.

Using a library of molecules as well as a natural medicinal product from Southeast Asia we performed an

in-silico screen against oncogenic Kras. The docking results show that the library of molecules can target

either a pocket at the C-terminus or a site located on the backside of switch 1. Moreover, investigation of

the natural medicinal compound and some relevant derivatives revealed that they also target the backside of

switch 1. To further substantiate the docking results, we performed a molecular dynamics simulation of a

single compound in complex with Kras. Indeed, we found that the binding at switch 1 was stable and

shifted the population dynamics of Kras such that it adopted an inactive conformation that would have an

impaired ability to bind effectors such as Raf or PI3K. This demonstrates that the catalytic domain of Kras

can be targeted with small molecules and ligand binding at switch 1 is stable. Such compounds would

potentially perturb effector binding and thereby modulate Ras signal transduction.

This work was supported by a training fellowship from the Keck Center of the Gulf Coast Consortia, on the

Pharmacological Sciences Training Program, National Institute of General Medical Sciences (NIGMS)

T32GM089657.

84



Biomedical Discovery Seeking Support System

Based on a Model of Human Associative Memory

Caleb Goodwin1, Elmer Bernstam1, and Thomas C. Rindflesch2 (1) School of Biomedical Informatics, University of Texas Health Science Center at Houston , Houston, TX, 77030

The increasing specialization of science has resulted in a fragmentation of knowledge. As a result, discoveries

in one area may not be translated to other relevant areas due to the difficulty of bridging the specialization

chasm. An emerging discipline known as literature-based discovery (LBD) focuses on developing methods to

mine the literature to reconnect these disparate units of knowledge. This work presents an LBD system based

on the Adaptive Character of Thought-Rational (ACT-R) theory of human associative memory. We created a

semantic network by extracting 27 million concept-relation-concept pairs from 7.8 million biomedical

(MEDLINE) citations (titles and abstracts dated 1999 through 06/30/2011). Given seed terms provided by the

user, an algorithm, based on ACT-R, creates a subgraph by traversing the semantic relations. This subgraph is

the prediction of where interesting relations (ideally discoveries) lie.

2.0

OVERVIEW

The problem of information overload is particularly evident in the biomedical domain where clinicians and

researchers can no longer keep up-to-date with relevant literature manually, even in specialized domains. A

prominent approach for dealing with this information overload is for scientists to concentrate in increasingly

specialized domains. This increased specialization results in parallel discoveries that, while related, go

unnoticed due to the proliferation of specialization and the bulk of new publications. An emerging discipline,

known as literature-based discovery (LBD), seeks to develop methods to mine the literature to uncover this

dormant knowledge.

The work presented here extends existing research in utilizing semantic predications and graph theory for LBD

(Wilkowski, In press). This methodology has been successful; however, it requires significant intervention by

users given that they must draw inferences from potentially thousands of relations. The goal of this work is to

assist users in navigating the predication space and is based on a theory of human associative memory.

The ACT-R theory of human memory (Anderson, 2007) is a spreading activation model and asserts that

human memory is an adaptation to the statistical properties of information in the environment. Spreading

Activation (SA) is a semantic graph-based retrieval model that utilizes heuristics to control propagation across

the graph. SA can be envisioned as energy flow where an initial source concept is activated and the energy

flows from the initial concept to associated concepts and terminates when halting criteria are met. Given the

seed term(s), the model generates a graph of predications that a human user would most likely find interesting.

The purpose of the model is not to replace user interaction, but to provide a tool that suggests relationships

allowing the users to more rapidly explore the complex predication space.

Funding

Supported by a training fellowship from the Keck Center NLM Training Program in Biomedical Informatics of the Gulf Coast

Consortia (NLM Grant No. T15LM007093).

REFERENCES

Anderson, J. (2007). How can the human mind occur in the physical universe? New York, NY: Oxford University Press.

Wilkowski, B., Fiszman, M., Miller, C., Hristovski, D., Arabandi, S., Rosemblat, G., Rindflesch. (In press). Discovery browsing with

semantic predications and graph theory. AMIA 2011.

85



Single Particle Tomography in EMAN2

Jesus Galaz1, John Flanagan1, Michael Schmid1, Wah Chiu1, Steven Ludtke1 (1) National Center for Macromolecular Imaging at Biochemistry and Molecular Biology,

Baylor College of Medicine , Houston, Texas 77030

Single particle tomography (SPT) is an alternative to untilted single particle analysis (SPA)

cryo electron microscopy (cryoEM) for reconstructing the 3D structure of macromolecules.

While traditional SPA can generate high-resolution structures of structurally homogeneous

specimens, difficulties arise with structurally variable molecules. The ambiguity that arises in

distinguishing between different orientations and different conformations in 2D projections of

macromolecules can be resolved by obtaining more than one image for each particle. This

technique can also separate particles in crowded environments where they overlap in 2D

projections, offering the potential for ex-vivo reconstruction of macromolecules from cryoEM

images of cells. We have implemented a graphical tool for 3D particle picking in EMAN2 and

a full refinement pipeline for single particle tomography, including sub-volume alignment,

classification and averaging. Given EMAN2's modular approach, a variety of algorithms can be

used at each processing step. CUDA acceleration is also implemented and provides a speedup

between 5x and 65x for typical SPT specimens. Full distributed and threaded parallelism is also

supported. We used our methodology to obtain a 25Å resolution reconstruction of epsilon15

virus (e15) with icosahedral averaging, a 38Å resolution asymmetric average of e15 with as

few as 10 particles, a 26Å asymmetric average of the TRiC chaperonin using from

conventional cryoET data, and structures that reveal a mechanism by which TRiC can inhibit

mutant huntingtin aggregation. We also conducted in silico experiments to validate our

software and characterize the effect of various data collection and data processing parameters.

While the resolution provided by this technology is currently limited by the lack of robust CTF

correction in cryo electron tomography, its unique capabilities make it a promising technique

for continued development.

86



Aggregation Behavior of Indomethacin, Cholic Acid

and POPC

Priyanka Prakash1, Lenard M. Lichtenberger1, and Alemayehu A. Gorfe1 (1) Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston

Texas, 77030

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used class of drugs for

the treatment of inflammation, fever and pain. One of their major side effects, especially if used

chronically, is ulceration and bleeding of the lower gastrointestinal (GI) tract. It has been reported

that NSAIDs cause surface injury by perturbing the integrity of membranes of the intestinal

mucosa which can be abrogated by phosphatidylcholine (PC). Moreover, it has been shown that

the toxic effect of NSAIDs to the lower gut is related to the presence of bile acid, suggesting that

the mixture, not NSAID alone, constitutes the agent that destabilizes lower-gut membranes in a

COX-independent manner. Several biochemical studies further indicated that NSAIDs form mixed

micelles with bile salts. NSAIDs may also alter the physiological balance of bile acids and

phospholipids in the lumen of the small intestine. In order to understand the molecular mechanism

by which NSAIDs form mixed micelles with bile salts, and to understand how NSAID-bile salt

interaction might be modulated by phospholipids, we studied the binary and ternary mixtures of

indomethacin (one of the most commonly used NSAIDs), cholate (a bile salt) and

palmitoyloleoylphosphatidylcholine (POPC) lipid in a various molar ratios. Initial results from

extensive all-atom molecular dynamics simulations indicate that the binary mixtures have distinct

aggregation behaviors and morphology than the ternary mixtures, providing clues about the

physical basis for the toxicity of NSAID/Cholic Acid mixtures and how this might be modulated

by phospholipids.

87



Asymmetry in the Homodimer of Glutathione

Synthetase May Contribute to Negative

Cooperativity.

Sasi Kodathala1, Brian Beck1,2 (1) Biology, Texas Woman's University (2) Dept. of Math & Computer Science, TWU,

Denton, TX, 76204

Glutathione (GSH), a major anti-oxidant found in all eukaryotic cells is produced by a

homodimeric enzyme, Glutathione Synthetase (GS). The GS dimer is an allosteric negatively

cooperative enzyme, that loses affinity for substrate after initial binding of substrate. Here, we

report the results of molecular modeling energy minimization calculations on fully solvated

monomeric and dimeric conformations of GS, each with and without substrate bound. Though

calculations were started from a symmetrical structure, after minimization there exists

differences in energies and conformations between some of the identical residues of monomers

of GS in the dimeric state, suggesting GS may occupy energetically asymmetric conformations.

These energy differences and the positions of the residues relative to the active site and the

dimer interface suggest the monomers may be able to adopt conformations that are

asymmetrically favorable to substrate binding, and thus may contribute to the observed

negative cooperativity.

88



FLIPS and FunCs: Identification and Prediction of

Protein Interactions Using Energy and

Evolutionary Pressure

Sajana Sudarshan1, Brian Beck1,2 (1) Biology, Texas Woman's University (2) Math & Computer Science, TWU, Denton, TX,

76204

Networks of proteins interacting at specific interfaces perform the majority of biological functions.

Most contacts are identified via X-ray crystallography. Often such contacts are crystal contacts

and not functionally related (FunCs). Here, we use the Energy Centrality Relationship (ECR)

concept to discriminate between aggregation and association by assessing amino acid energetic

variation as a function of distance from the interface. We hypothesize that evolutionary pressure to

maintain functionally-linked interfaces (FLIPs) will produce signature characteristics that can be

identified via computational alanine scanning and Evolutionary Trace calculations, allowing

FLIPs and FunCs to be distinguished. Here we show that positional/energetic correlation patterns

are not observed in FunCs and when clustered, proteins belonging to different functional

categories exhibit patterns that can discern not only FLIPs from FunCs, but also protein

categories. In addition, by generating docking decoys for structures of representative categories,

we demonstrate ECR can identify known quaternary conformations.

89



Prediction of Functionally Important Residues in

Protein:Protein Interactions By Network Analysis

Isha Mehta1, Brian Beck1,2 (1) Biology, Texas Woman's University (2) Math & Computer Science, TWU, Denton, TX,

76204

Proteins play an important role in various cellular processes, both directly and by interacting

with other proteins. The stability and interaction capability of proteins are important for their

efficient functioning, but identifying the residues most important to those functions remains a

problem. Here, we report a residue Network Analysis (NA) of protein structures from our

interacting protein database, FLIPdb. A network can be defined as all the residues within a

particular distance, regardless of bonded or non-bonded interactions. Previous work by others

have shown that network connectivity parameters correlate with functionally important residues

in single proteins. We hypothesize that NA can also identify residues important to quaternary

interactions. In this work, a residue network was constructed with an interaction cutoff of 12Å.

We find that Degree, Closeness, and Betweeness centrality parameters are able to distinguish

surface residues involved in quaternary interactions from those that are not.

90



Computational Analysis of Interactions Between

ARP2/3 and WAVE At a Molecular Level

Amruta Mahadik1, Brian Beck1,2 (1) Biology, Texas Woman's University (2) Math & Computer Science, TWU, Denton, TX,

76204

Proteins such as actin, actin-related proteins (Arp2/3 complex), and actin-binding proteins

(Wiskott-Aldrich family proteins like WAVE) control the nucleation of polymerization of linear

and branched actin filaments that form the actin cytoskeleton, the branched form of which

promotes the formation of lamellipodia at the mobile edges of cells. We hypothesize that residues

at the interface of Arp3 and Arp2 in the Arp2/3 complex dominate conformational changes during

activation of the complex and when mutated will disrupt the Arp3:Arp2 interface, alter the

formation of branched filaments, and alter the morphology of cells. Structural analysis, homology

modeling, Evolutionary Trace calculations, and virtual alanine scan modeling were used to

identify important Arp2/3 complex residues, including Arp3: R161, S226, and R447. Mutations at

R161 with leucine and alanine, S226 with glutamate and glutamine, and R447 with tryptophan

yielded 3.98, 3.98,-1.04, -1.07, -1.63 kcal/mol of free energy change respectively. R161A in Arp3

altered neurite morphology in cell culture.

91



Docking Rho Kinase 1 Catalytic and PH Domain

and Inhibition in Heart Disease

John Craft, Jr.1, Hua Zhang1, Marc Charendoff1, James Briggs, & Robert Schwartz1

(1) Biology and Biochemistry, University of Houston

Rho Associated Kinase or ROCK is a protein from the serine/threonine kinase family. ROCK is

a homodimer that contains an N-terminal kinase domain, a coil-coil domain and a C-terminal

split Pleckstrin Homology Domain (PH domain) that autoinhibits its catalytic domain. ROCK

is responsible for the phosphorylation of the myosin light chain, which influences heart

function. Cardiovascular disease has been linked to chronic activation of ROCK and its

downstream effectors. ROCK plays a key role in inducing stress fiber formation, which if

excessive, leads to cardiac hypertrophy and ultimately interstitial fibrosis and heart failure.

Studies with mice indicate the down regulation of Rho kinase 1 (ROCK1) reduces fibrosis by

limiting the fibroblast migration and collagen deposition responsible for destructive remodeling

of the heart. These findings provide a strong rationale for understanding ROCK1 mechanisms

for inhibition. Docking complexes of the Pleckstrin Homology Domain and ROCK catalytic

domain are examined. The three body system; 1) ROCK catalytic domain, 2) ROCK split PH

domain, and 3) ROCK C1 domain insert was evaluated for complimentary binding poses.

Significant clusters were found in which both the PH domain subunit and the C1 domain

subunit can form a co-complex with the catalytic domain. It is hoped that understanding ROCK

complexes may lead to an approach to mimic the body’s own negative regulation of ROCK and

lead to strategies that may be developed for the treatment and prevention of heart disease.

92



PKa and Electrostatic Analysis of ROCK1 Catalytic

Domain

Tangen Tran1, John Craft1, Jr., and James Briggs1

(1) Biology and Biochemistry, University of Houston

Chronic pressure overload is the most common cause of heart failure and cardiac hypertrophy

maintains chronic pressure overload by increasing myocyte size and causing remodeling of tissues

before heart failure occurs. Rho-associated kinase 1 (ROCK1), when inhibited, has been

demonstrated to reduce interstitial fibrosis in cardiac hypertrophy. The goal of my research is to

understand surface charge diversity of ROCK1 throughout its regulational and mechanistic

activity by computational methods. Acquiring these data will assist in the development of a drug

to inhibit the ROCK1 kinase, thereby modulating its activity. I applied the computational methods

of pKa predictions, electrostatic potential generation, and molecular dynamics, to study ROCK1.

First preparing of PDB files by structural alignment between 2ETR and 2V55 was required. A

VMD tcl script allowed the structural alignment of ROCK1 PDB files 2ETR.pdb and 2V55.pdb

with an RMSD of 1.03. This facilitated the addition of ATP and MG+ with ROCK1. Each

configuration of phosphorylation, ATP addition, and MG+ addition was prepared for ROCK1 pKa

predictions in the tool UHBD. The pKa predictions of ROCK1 with ATP, with MG+, with ATP

and MG+ were a precursor for computing electrostatic potentials. Electrostatic potentials differed

when its cofactors ATP and MG+ were present. ROCK1 was visualized with a highly negative

electrostatic potential in the active site using a -4/+4 isopotential surface (kcal/mol/electron). From

the comparative analysis of the pKa predictions, residues GLU270, ASP216, ASP160, and

HIS196 were chosen as residues of interest.

93



FSUB: Normal Mode Analysis With Flexible

Substructures

Mingyang Lu1, Dengming Ming1,2, & Jianpeng Ma1,3 (1) Biochemistry, Baylor College of Medicine (2) The School of Life Sciences, Fudan

University, Shanghai, P.R. China (3) Dept. of Bioengineering, Rice University, Houston, TX

77005

We report a novel normal mode analysis for supramolecular complexes, named as fSUB. The

method models a complex as a group of flexible substructures. The low-frequency

substructure modes are first determined with substructures in isolation, and the motions of the

whole complex are then calculated based on substructure modes and substructure-substructure

interactions. The calculation of modes in fSUB requires modal analysis without initial energy

minimization, which is essential for maintaining energetic and structural consistency between

substructures and whole complex. Comparing with other coarse-grained methods, such as

RTB method, fSUB delivers much more accurate modes for the complex and allows for

choice of much larger substructures. The method can also accommodate any type of

substructure arrangement including covalent bonds across the interface. In tests on molecular

chaperonin GroEL (7350 residues) and HK97 capsid complex (118,092 residues), fSUB was

shown to be much more efficient in terms of combined accuracy and demand of computing

resource. Our results clearly demonstrated the vital importance of including substructure

flexibility in complex modal analysis, as the deformational patterns of substructures were

found to play an important role even in the lowest frequency modes of the whole complex.

94



Structural Studies on the Envelope Proteins E1 and

E2 of Everglades Virus

Surendra Negi 1, ,2, Michael B. Sherman1,2,3,4, Andrew D Haddow5, Scott C. Weaver1,2,3,4,5,

Werner Braun1,2,3,4 (1) Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas

Medical Branch (2) Dept. of Biochemistry and Molecular Biology, (3) Institute for Human

Infections and Immunity, (4) Center for Biodefense and Emerging Infectious Diseases (5)

Department of Pathology, University of Texas Medical Branch, Galveston, Texas.

Everglades virus (EVEV), a member of the Venezuelan equine encephalitis (VEE) antigenic

complex, is a mosquito-borne enveloped, positive single stranded RNA virus of the Togaviridae

family, genus Alphavirus, which causes an incapacitating and sometimes lethal infection in

humans and equids. The genome of EVEV has a moderate to high sequence identity with other

alphaviruses. The 5’ end of the genome encodes four nonstructural proteins (nsP1, nsP2, nsP3 and

nsP4), which are required for virus replication, while the 3’ end of the genome encodes structural

proteins (capsid, E3, E2, 6k and E1). In alphaviruses, both envelope proteins E1 and E2 are

required for virus entry. The E1 protein mediates the fusion between the viral and cellular

membranes while the E2 protein forms spikes on the viral surface and binds to a cellular receptor.

The close relationship between EVEV with VEEV, which are classified as biological safety level

3 (BSL-3) agents, will help us to understand the role of conformational epitopes on the surface of

E1 and E2 envelope proteins during the fusion process. In the current work we used homology

modeling, cryo-electron microscopy and bioinformatics techniques to investigate the role of

surface-exposed residues in the E1 and E2 proteins of EVEV. These proteins were fitted into a

10Å resolution cryo-electron density map of EVEV using rigid body fitting and molecular

dynamics simulations. Results of the ongoing study will enable the development of new vaccine

candidates and antivirals by designing peptides to target the E1-E2 interaction sites and receptor

binding site.

95



Physico-Chemical Property (PCP) Motifs to

Identify Proteins that Could be Cross-Reactive

With Known Peanut Allergens

Wenzhe Lu1, Catherine H. Schein 1,2,3, Werner Braun1,2,3

(1) Department of Biochemistry and Molecular Biology, University of Texas Medical Branch

(2) Sealy Center for Structural Biology and Molecular Biophysics (3) Sealy Center for Vaccine

Development, University of Texas Medical Branch, Galveston, TX 77555-0857

The prevalence of allergic diseases has dramatically increased in the US in the last two

decades. Some food sources, such as peanuts, can be the cause of anaphylactic shocks that lead,

in extreme cases, to fatalities of sensitive individuals. Thus patients have to be on a strict diet in

order to avoid the offending foods, as there is currently no treatment for these severe allergies.

It is also well established that some food sensitive individuals have allergic reactions to other

foods that contain similar proteins to the known allergens. For example, those allergic to the

major birch pollen allergen Bet v 1 may develop a secondary allergy to the similar protein in

peanut, Ara h 8. However, there are many different food sources that contain such proteins,

which are not considered to be allergenic. Here, we extracted physico-chemical property (PCP)

sequence motifs using our PCPMer program from aligned sequences of the known allergens (as

catalogued in our Structural database of allergenic proteins, SDAP1). We began with allergens

related to the major peanut allergens Ara h 1, Ara h 2, Ara h 5 and Ara h 8. These were

compared to PCP-motifs extracted in a similar fashion from proteins from the same protein

structural family (PFAM) that were not described as allergens, to determine motifs specific for

the allergenic proteins2; 3. The allergen specific motifs were shown to overlap, in many cases,

with known IgE epitopes. The motifs were mapped to models of the known allergens, to

determine their surface exposure.

The major use for these motifs is to define proteins that are likely to cause cross-reactions in

peanut sensitive individuals. Thus the selected motifs, or the sequences related to them in close

and distant homologues of the known allergens, will be synthesized as individual peptides and

further tested in microarray assays for their ability to bind to IgE in the sera of atopic

individuals.

96



Thermodynamics of Aggregation of Gly5

Deepti Karandur1, B. M. Pettitt1,2 (1) Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine

(2) Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical

Branch Galveston, Galveston, TX, 77555

Intrinsically disordered proteins (IDPs) are proteins that do not fold into a stable, three-

dimensional, native structure, except possibly when bound to a ligand, partner protein, etc.

Evidence suggests that IDPs function by varying the thermodynamics of recognition and binding

by proteins, and thus affect the affinity and specificity with which the proteins bind to their

partners. IDPs tend to be rich in amino acids like glycine, which increase their disorderliness. We

use oligoglycines as a model to study the behavior of IDPs in solvent in order to determine the

thermodynamics of their folding and function.

A central problem is the relationship of the folding of longer oligo glycines to the solubility of

shorter oligomers. The solubility of oligoglycine in water decreases as its length increases until,

when the peptide contains 5-6 glycines, it aggregates and falls out of solution at low concentration.

However, previous work by our group shows that the solvation free energy of oligoglycine

decreases as its length increases from 2 to 5 residues. We hypothesize that as the peptide length

increases, intermolecular interactions between oligoglycines are favored over interactions between

oligoglycine and water, leading to their aggregation. This aggregation occurs as a liquid-liquid

phase separation or a second order phase transition. Here we simulate the behavior of a system of

several hundred short (five residues) oligoglycines in water in order to understand the structural

and thermodynamic changes that occur during aggregation of these short peptides. We

hypothesize that the thermodynamics of aggregation of short oligoglycines is equivalent to the

thermodynamics of folding or collapse of longer oligoglycines in water. We propose to compare

the thermodynamics of aggregation of short oligoglycines with the collapse of longer

oligoglycines in water in order to understand the thermodynamics of collapse of the longer

peptide.

97



Inferring Metabolic Networks Using the Bayesian

Graphical Lasso

Christine Peterson1, Mirjana Maletic-Savatic2, & Marina Vannucci1 (1) Department of Statistics, Rice University (2) Department of Pediatrics - Neurology, Baylor

College of Medicine

Chronic neuroinflammation is a hallmark of many neurodegenerative diseases such as

Alzheimer’s and Huntington’s. Despite its significance, the mechanisms by which

neuroinflammation leads to disruptions in cellular function are not well understood. One of the

less explored cellular functions affected by neuroinflammation is metabolism. Metabolic

changes underlying neuroinflammation can be exploited for biomarker discoveries and future

drug developments. To understand these changes, we have developed statistical methods that

allow inference of the cellular metabolic networks from a set of critical metabolites identified

and quantified in inflamed cells. Our approach builds on the Bayesian graphical lasso, a

technique for learning a sparse graph structure from the covariance of the data. To encourage

edges between metabolites with known relationships, we allow each entry in the precision

matrix to have a unique shrinkage parameter with a gamma prior shifted towards zero for

molecules closer together in a reference network. By incorporating informative priors, we hope

to improve the reliability of network inference given the small sample sizes and high degree of

noise. The resulting identification of cellular reactions under neuroinflammatory conditions

will not only increase scientific understanding of the mechanisms of neuroinflammatory

diseases, but also provide potential targets for future treatments.


Consortia, on the NLM Training Program in Biomedical Informatics, National Library of

Medicine (NLM) T15LM007093.

98



Impact of Electronic Medical Record Interface on

Physician Task Completion

Peter Killoran1, Jiajie Zhang2 (1) Department of Anesthesia, University of Texas Health Science Center - Houston (2) School of

Biomedical Informatics, University of Texas Health Science Center at Houston, Houston, TX,

77030

Adoption of health information technology (HIT) within high-performing, academic hospitals has

improved the quality, safety, and efficiency of healthcare while reducing error rates and overall

cost. However, the impact on the entire health care system from widespread adoption of HIT is not

yet clear, in part due to cognitive barriers introduced by disrupted workflow and poor usability.

Here, we use a framework known as TURF (Task, User, Representation, Function) to help address

problems with usability associated with a specific data intensive task. In this project, we analyze

the Anesthesia Preoperative Assessment, a physician task that requires extensive use of an

Electronic Medical Record to identify data objects essential to the task and then propose

alternative visual representations that better support physician cognitive processes. Using both

document analysis and observational techniques, we identified 30 categories of data objects

extracted from the EMR. Data were extracted from throughout the EMR system with highly

summarized views providing the majority of the data in 17% of the total time. Less summarized

views require progressively more time to analyze while yielding fewer pieces of data. Three pieces

of data critical for patient safety were also identified, but required 25% of the encounter time to

extract. This suggests that automated summarization of data can effectively reduce the time

required to complete the task, but current techniques exclude a significant number of required data

fields. Improved summarization and more efficient representation have the potential further

improve safety, quality, and efficiency.

99



Periodic Table of Knotted Proteins: Are We

Missing Some Knots?

Rhonald Lua1

(1) Molecular and Human Genetics, Baylor College of Medicine ,Houston, TX, 77025

Understanding the differences between knotted and unknotted protein structures may provide

insights into how proteins fold and function. Knots with a minimum of 3, 4, 5 and 6 crossings

in a plane projection (denoted 31, 41, 52, 61 in Alexander-Briggs notation) have been found in

protein structures from the PDB. To explore whether the list of knots in proteins have been

exhausted, we look at the knots in models of artificial, minimalist polymer backbones. Knotting

probabilities in such models suggest that the knot 51 and other kinds of knots may eventually be

found among proteins. We also take this opportunity to introduce PyKnot, a unique software

plugin for the popular PyMOL molecular viewer. PyKnot is designed to characterize the type

of knot in a protein or other biomolecule through the calculation of knot invariants, crossing

numbers and simplified knot projections and backbones. It is hoped that the study of knots in

proteins and the dissemination of PyKnot would lead to further insights into protein structure

and function.

100



Constrained Conformational Analysis: Protein

Motion Planning With Cryo-Electron Microscopy

Bryant Gipson1, Mark Moll1, Steven Ludtke2, Lydia Kavraki1 (1) Computer Science, Rice University (2) NCMI, Baylor College of Medicine, Houston, Texas,

USA

Understanding the time-dependent behavior of macro-molecular protein systems remains a

significant computational and experimental challenge. Over the last decade, Single Particle Cryo-

Electron Mircoscopy (cryo-EM) has emerged as a reliable tool for the rapid determination of mid-

resolution (4-6 Å) structures of proteins. In certain cases, it is even possible to produce atomic

models of individual conformations (e.g., open and closed) --as in the case of the group II

chaperonin Mm-cpn. While these results describe many important features of such a system, they

provide only individual, typically energetically minimized, snapshots of conformations of the

protein. Capturing the full nature of protein function requires an understanding of the dynamic

behavior which drives it. The central aim of this project is the development of a computational

method which produces ensemble solutions that characterize the time-dependent behavior of large,

optionally symmetric proteins or protein complexes, resolved at mid-resolution by single particle

cryo-EM, in order to describe the functional mechanism of these systems. This will be achieved by

reducing the dimension of the problem by modeling inflexible stretches of the protein as rigid

bodies while leaving others as free articulated joints. This dimension reduced system thus

becomes an articulated robot navigating an energy landscape –allowing for time-dependent

conformational exploration when recast as a generic robotic motion planning problem. As this

process is both random and graph based, it can be iteratively extended and searched, producing

probability based ensemble path solutions about (or among) a given set of input conformational

states. The resulting system may then be used to answer specific time-dependent questions about a

protein under study or used generally to provide a more complete statistical overview of the

system as a whole.

101



Differential Impact of FGF14 Mutations on

Protein-Protein Interaction Interfaces in

Macromolecular Complexes

Alexander Shavkunov1, Anesh Prasai1, Neli Panova1, Svetla Stoilova-McPhie2,3, &

Fernanda Laezza1,4,5 (1) Dept. of Pharmacology and Toxicology, University of Texas Medical Branch Galveston (2)

Dept. of Neuroscience & Cell Biology (3) Sealy Center for Structural Biology and Molecular

Biophysics (4) Center for Neurodegenerative Diseases (5) Center for Addiction Research,

University of Texas Medical Branch, Galveston, TX, 77555

Protein:protein interactions are an emerging resource for therapeutic development against ion

channels. Yet, rapid and effective methods for evaluating relevant protein:channel interfaces

allowing to anticipate off-target effects are still missing. Within the intracellular fibroblast

growth factor family (iFGF11-14), FGF14 is the most potent and specific regulator of the

voltage-gated Na+ (Nav) channels that controls channel trafficking and gating through a

monomeric interaction with the channel C-terminal tail, providing a new opportunity for a

highly specific, FGF14-based medication development to prevent neuronal hyperexcitability.

Recent structure-function studies have defined a conserved surface on the intracellular FGF

core domain that has been proposed to be a common interface for both channel binding and

iFGF homodimer formation, posing a potential complication in designing selective

pharmacotherapeutic agents targeting the FGF14-Nav channel complex. To identify potential

structural differences between the FGF14-FGF14 and the FGF14-Nav channel protein

interfaces, we applied the split-luciferase assay (LCA) to detect the two protein complexes in

live cells. Then, we evaluated the impact of two critical amino acid residues, Y153 and V155,

at the FGF14-FGF14 and the FGF14-Nav1.6 C-tail dimer interface by mutating both residues

to N and measuring by LCA the effect of these mutations on the FGF14 dimer and FGF14-Nav

channel binding. We show that the Y153N and V155N mutations increase the stability of the

FGF14-FGF14 dimer, while impairing the binding of FGF14 to the Nav channel. We have built

a model of the FGF14 dimer based on the FGF13 dimer crystal structure to understand how

these mutations might affect the FGF14 dimer interface. The presented results might offer

insights into the difference between the FGF14-FGF14 and the FGF14-Nav channel dimer

interfaces, providing new knowledge for guiding the design of therapeutic agents targeting the

native macromolecular complex of Nav channels.

102



The Just-In-Time Expression of Yeast Ribosomal

Protein Genes for Ribosome Assembly and

Maturation Process

Xueling Li1, Gang Chen1, Andrzej Kudlicki1 (1) Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston 77555,

TX (2) Sealy Center for Molecular Medicine, University of Texas Medical Branch Galveston,

Galveston 77555, TX

The eukaryotic ribosome consists of ribosomal RNAs and dozens of proteins (RPs). The assembly

and maturation processes of ribosome are strongly regulated in timing, which allows applying

model-based deconvolution to precisely estimating the timing of expression peaks of ribosomal

protein genes. Our empirical model fitting method has been shown to yield timing resolution

higher than the original time-course data (1). The time course expression data from a

metabolically regulated yeast culture (2) has achieved better results than other types of temporal

profile. The results reveal different time delays in the expression of the ribosomal protein genes,

spanning an approximately 20-minute interval.

We find that the position of the gene product in the ribosome, the early expressed RPs tend to be

buried deeper in the ribosome see Fig. 1) and the earlier involved proteins in mature process have

an earlier expression peak time than the latter ones, which are consistent with the “just in time

transcription” view of regulation of subunits of macromolecular complexes. To our best

knowledge, it is the first report on the correlation between protein complex structures and gene

expression profiles. In mining the underlying mechanism, we also find the significant differences

between the regulatory elements of the early and late expressed genes, which may be responsible

for regulating the timing. The most prominent example (p=0.009) is the correlation between the

expression time and the distance from the Rap1 motif (Fig. 2) to the 5’end of the coding sequence.

This type of regulation of fine-tuned timing of gene expression has not been described previously

in eukaryotes.

References

(1) Rowicka, Kudlicki,Tu, Otwinowski:PNAS 104(43), 16892, 2007

(2) Tu, Kudlicki, Rowicka, McKnight: Science, 310, 1152-1158 (2005)

103



Investigation of RNA Binding Properties of the

HIV-1 Rev Protein Using a Chimeric tRNAPhe

Jason Allison1,2, A. Hindupur1,2, R.S. Khan1, K.R. MacKenzie1, & R.O. Fox1,2 (1) Biology and Biochemistry, University of Houston (2) Dept. of Biochemistry & Molecular

Biology, UTMB, Galveston, TX, 77555

A major obstacle to performing structural studies of proteins bound to their RNA target is

obtaining the pure, milligram quantities of nucleic acid. Synthetic RNA is very costly and the

current method of using T7 polymerase creates heterogeneous transcripts leaving limited

amounts of the desired product. A recently published RNA scaffold technology allows for the

expression and purification of intact chimeric tRNAs containing RNA hairpin sequences of

interest. The gene for the chimeric RNA is designed such that the anticodon loop is replaced by

RNA hairpin sequence of interest, leaving the TψC, D-loop, and acceptor stem intact.

Expression is driven by the constitutive E. coli lpp, producing milligram quantities of

homogenous RNA transcripts containing the desired RNA hairpin sequence. Purification of the

chimeric tRNAs are achieved by HPLC ion-exchange chromatography from total cellular RNA

extracts.

The Regulator of Virion Expression or Rev is a regulatory, shuttle protein produced during the

early-phase of HIV replication and active during the late-phase development of the virus. It

recognizes with specificity a 351-nucleotide sequence contained within HIV RNA known as

the RRE or Rev Response Element contained within the env (envelope) encoding region of

HIV RNA. The RRE is characterized by a series of stem-loops and extensive secondary

structure which recognizes and binds the Rev protein with high-affinity and specificity. Rev's

initial recognition and subsequent assembly on the RRE depends on its interaction with a 34-

nucleotide sequence known as Stem Loop-IIB. Following the interaction with Stem Loop-IIB,

Rev assembles into 8-10 monomers along flanking sequences of the RRE although details of

this process remain elusive. We have used the phenylalanyl tRNA from Baker's Yeast as a

scaffold to express the 34-nucleotide sequence of Stem Loop-IIB as well as flanking hairpin

sequences to Stem Loop-IIB to investigate Rev's assembly on the RRE using size-exclusion

chromatography (SEC), analytical ultracentrifugation (AUC), and small-angle x-ray scattering

(SAXS) techniques.

104


Additional Poster P-80

Codon Optimization and Construction of Different

Recombinant Vectors Containing Anti-HIV

Griffithsin Gene

Yavar Vafaee1, Houshang Alizade1, Mesbah Babalar1 & Mohammad Afshar Shandiz2 (1) Dept. of Horticultural Sciences, University of Tehran, Karaj, Iran. 2) Department of

Agricultural Biotechnology, Agricultural College, University of Tehran, Karaj, Iran.

UNAIDS reported that 33.3 million people living with HIV at 2009. With current therapy, AIDS

is still not curable or preventable. Griffithsin (GRFT) is a novel anti-HIV protein from the red alga

Griffithsia that bind to HIV and inhibit cell to cell transmission. In this study first we optimized

GRFT gene sequence based on codon frequencies for lettuce to reach highest expression in

nuclear and chloroplast genome. In next step, we constructed pBI121 vectors containing GRFT

gene with KDEL, extension and zera® signal peptides, a lettuce chloroplast transformation vector

(PCL 96-39) and an expression vector (pET -28). For this purpose, first digestion with desired

restriction enzymes was performed and after DNA purification from gel, ligation reactions and E.

coli (Dh5α strain) transformation were carried out and resulted colonies used for confirmation of

constructs. Digestion, PCR with gene specific and vector backbone primers as well as sequencing

confirmed accurate and reliable integration of GRFT gene and signal peptides into corresponding

vectors.

105



Evaluation of Some Morphological

Characteristics, Mineral Elements of Spear and

Physical Properties of Seed of Edible Asparagus

(Asparagus Officinalis L.)

Behrooz Sarabi1, khodaei Jalal2 & Hossaini Sayed Mohammad Taher3

(1) Department of Horticultural Sciences, Faculty of Agriculture, University of Tehran, Karaj,

Iran (2) Department of Mechanics of Agricultural Machinery, Faculty of Agriculture,

University of Kurdistan, Sanandaj, Iran (3) Department of soil science, Faculty of agriculture,

University of Kurdistan, P.O. Box 416, Sanandaj, Iran

In order to evaluate the morphological characteristics of edible asparagus, individual plants

within Taleghan population were studied and compared with Mary Washington cultivar. In

studied plants, flowers were appeared from early May to early June and green fruits were

formed about early July. The number of seeds per red mature berry fruit, which were collected

around early September, was variable from 4 to 6 between evaluated plants. The amount of

mineral elements include K, Ca, Mg, Fe, Cu, Zn, Mn, Na, Li and Ba in Taleghan asparagus in

mg per 100 g fresh weight were 139.4, 1.865, 53.311, 0.811, 0.214, 0.706, 0.239, 2.363, 0.767

and 0.004 respectively, which were higher than that of Mary Washington cultivar. As the

moisture content increased from 8.7 to 31.2% dry basis (d.b.), the sphericity, surface area and

thousand seed mass increased from 0.807 to 0.822, 30.37 to 41.15 mm2 and 21.1 to 31.24 g

respectively. As the moisture content increased the bulk density, true density and porosity were

found to decrease from 630 to 530 kgm-3, 1360 to 1040 kgm-3 and 53.67 to 49.03%, whereas

the terminal velocity was found to increase from 9.79 to 11.44 ms-1.

Key words: Edible asparagus, Spear, Mineral elements, Moisture content of seed

106



The Effect of Thidiazuron on Direct Somatic

Embryogenesis Pathway of Strawberry (Fragaria

Ananassa Duch.)

Mohammad Gerdakaneh 1

(1) Researcher Agricultural and Natural Resource Research Center of Kermanshah, Iran

For purpose of direct Somatic embryogenesis, leaf blade of Kurdistan, Paros and Camarosa

cultivars was cultured on MS medium supplemented with TDZ at 1, 2, 3 and 4 mg/L alone as well

as the combination with different concentrations (0.0, 0.25, 0.5, and 1 mg/L) of 2,4-D. The highest

induction frequency of embryogensis and number of somatic embryos per explants was obtained

on MS medium containing 3 mg/l TDZ and 0.25 mg/l 2,4-D. In this medium, maximum number of

14.75, 22.00 and 25.75 globular embryos per explants obtained for the cultivars of Kurdistan,

Paros and Camarosa, respectively. In order to globular embryos developing into cotyledonary

embryos, globular embryos of the leaf explants were cultured on MS medium supplemented with

sucrose at concentration of 1.5, 3, 6, 9 and 12%. Among the different concentrations tested, 6%

sucrose was found superior for uniform embryo developmental stages. The addition of GA3 1 mg/

L in MS medium improved the plantlet conversion rate of plantlet (65% to 67%). The rooted

plants were hardened and transferred to soil with 75% to 80% survival rate.

Key words: Strawberry, somatic embryogenesis, leaf blade, TDZ.

107



Transient Expression of Urease b From Iranian

Helicobacter Pylori in Lettuce

Neda Malekitabrizi1, Neda MalekiTabrizi1*,Houshang Alizadeh1

(1) Agronomy & Plant Breeding, Tehran University ,Karaj, Iran

Helicobacter pylori (H. pylori) is established as the etiologic agent of chronic active gastritis,

peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue lymphoma. The

development of a vaccine against H. pylori has become a priority to prevent and cure H. pylori

infection. The UreB (urease B) subunit is the most effective and common immunogen of all

strains of H. pylori and can stimulate the immunoresponse protecting the human body against

the challenge of H. pylori. In this study was produced this antigene with agroinfiltration

strategy in lettuce. Assessment of protein through indirect ELISA With human polyclonal

antiserum demonstrated that the average of expressed UreB protein accounted for 3mg/kg that

was one percent(1%) from total proteins. These results suggest that the lettuce can be

transformed with stable strategies as a potential edible vaccine for controlling H. pylori.

Key words: Helicobacter pylori, Urease, immunogen, agroinfiltration, indirect ELISA

108


Dr. Wendy Baker

UTMB

[email protected]

Ms. R. Adikaram Bandara

University of Houston

[email protected]

Ms. Rosa Banuelos

Rice University

[email protected]

Dr. Brian Beck

Texas Woman's University

[email protected]

Dr. Darren Boehning

UTMB

[email protected]

Mr. Wayne Bolen

UTMB

[email protected]

Mr. Michael Bolt

Baylor College of Medicine

[email protected]

Mr. Evgeniy Bovshik

UTMB

[email protected]

Dr. Werner Braun

UTMB

[email protected]

Dr. Jim Briggs


[email protected]

Mr. Paul Bujalowski

UTMB

[email protected]

Dr. Wlodek Bujalowski

UTMB

[email protected]

Dr. Cecile Bussetta

UTMB

[email protected]

Mr. Abhijnan Chattopadhyay

UTMB

[email protected]

Mr. Kuang-Yui Chen


[email protected]

Dr. Chuanying Chen

UTMB

[email protected]

Mr. Dong Chen

Rice university

sim-

[email protected]

Dr. Xiaodong Cheng

UTMB

[email protected]

Dr. Rati Chkheidze

UTMB

[email protected]

Dr. Kyoung-Jae Choi


[email protected]

Mr. Aaron Collier

Rice University

[email protected]

Dr. John Craft

[email protected]

Ms. Wei Dai

[email protected]

Dr. Daniela Dalm

UTMB

[email protected]

Ms. Andria Denmon

Rice University

[email protected]

Mr. Ninad Dewal


[email protected]

Mr. Arijit Dutta

UTMB at Galveston

[email protected]

Mr. Arijit Dutta

UTMB

[email protected]

Dr. Kippi Dyer

UTMB-Galveston

[email protected]

Dr. Jane Dyson

The Scripps Research Institute

[email protected]

Dr. Miguel-Angel Elizondo-

Riojas

Institute of Molecular Medicine

Mi-

[email protected]

c.edu

Dr. Robert English

UTMB Galveston

[email protected]

Dr. Henry Epstein

UTMB

[email protected]

Mr. Alexandre Esadze

UTMB

[email protected]

Ms. Madeline Farley

GSBS

Made-

[email protected]

Dr. Bernard Fongang

UTMB

[email protected]

Dr. Robert Fox


[email protected]

Mr. Jesus Galaz


[email protected]

Ms. Monica Galaz Montoya


[email protected]

Mr. Sai Hari Gandham

UTMB

[email protected]

Mr. Mohammad

Gerdakaneh

Iran

[email protected]

Mr. Bryant Gipson

Rice

[email protected]

Ms. Melissa Glueck

Gulf Coast Consortia

[email protected]

Dr. Aleksandra Gmyrek

UTMB

[email protected]

Mr. caleb goodwin

UT Health

[email protected]

Dr. David Gorenstein

University of Texas Medical

School

Da-

[email protected]

Ms. Keerthi Gottipati

UTMB

[email protected]

Ms. Ashley Grant

UTMB

[email protected]

Dr. Kirill Grushin

UTMB

[email protected]

Dr. Weiguo He

[email protected]

Dr. Catherine Higgins


[email protected]

Dr. Aditya Hindupur


[email protected]

Dr. Aditya Hindupur


[email protected]

Mr. Harrison Hocker

The UTHSC at Houston

[email protected]

Dr. Wim Hol

University of Washington

[email protected]

Dr. Luis Holthauzen

UTMB

[email protected]

109


Mr. Chuan Hong


[email protected]

Mr. Stanley Hooker


[email protected]

Dr. Robert Howard

MedScan Laboratory

[email protected]

Mr. Gilbert Huang


[email protected]

Ms. Rossi Irobalieva


[email protected]

Dr. Junji Iwahara

UTMB

[email protected]

Ms. Amy Jackson


[email protected]

Dr. Maria Jezewska

UTMB

[email protected]

Mr. Yifeng Jiang


[email protected]

Ms. Angelina Johnson

The UTMB

[email protected]

Dr. Prem Raj Joseph

UTMB

[email protected]

Ms. Deepti Karandur


[email protected]

Mr. Peter Killoran

UTHSC - Houston

Pe-

[email protected]

Mr. Sasi Kodathala


[email protected]

Dr. Alexey Koyfman


[email protected]

Dr. James Lee

UTMB

[email protected]

Dr. Hualin Li

UTHSC

[email protected]

Dr. Zhenlong Li

UTHSC at Houston

[email protected]

Ms. Wenzong Li

University of Texas at Austin

[email protected]

Dr. Xueling Li

UTMB

[email protected]

Dr. Chi-Chuan Lin

MD Anderson Cancer Cen-

ter

[email protected]

Dr. Kevin Liu

Rice University

[email protected]

Dr. Xiangan Liu


[email protected]

Mr. Mingyang Lu


[email protected]

Mr. Wenzhe Lu

UTMB

[email protected]

Dr. Rhonald Lua


[email protected]

Dr. Bruce Luxon

UTMB

[email protected]

Dr. Gillian Lynch

UTMB

[email protected]

Ms. Amruta Mahadik


[email protected]

Ms. Nandini Maharaj

University of Texas-Health

Science Center

nan-

dini.rambahalmaharaj@uth.

tmc.edu

Ms. neda malekitabrizi

tehran university

[email protected]

Ms. Prema Latha Mallipeddi


[email protected]

Mr. David C. Marciano


[email protected]

Mr. David Marciano


[email protected]

Ms. Isha Mehta


[email protected]

Ms. Desi Miller

UTMB

[email protected]

Ms. Jaimy Miller

UTMB

[email protected]

Mr. Chance Mooney

Rice University

[email protected]

Mr. Christopher Myers


[email protected]

Dr. Surendra Negi

The UTMB

[email protected]

Ms. Iris Nira Smith


[email protected]

Mr. Rahul Pal

UTMB

[email protected]

Mr. Naresh Pandey

Rice University

[email protected]

Dr. John Perkyns

UTMB

[email protected]

Ms. Christine Peterson

Rice University

[email protected]

Dr. B. Montgomery Pettitt

UT Galveston

[email protected]

Dr. Biju Pillai

UTMB

[email protected]

Ms. Lisa Pipper

UTMB

[email protected]

Dr. Maruthi Krishna Mohan

Poluri

UTMB

[email protected]

Dr. Carol Post

Purdue University

[email protected]

Dr. David Power

UTMB

[email protected]

Dr. Priyanka Prakash

UTHSC at Houston

pri-

[email protected].

edu

Mr. Kimal Rajapakshe


[email protected]

Ms. Deepthi Rajapakshe


[email protected]

Dr. krishna rajarathnam

UTMB

[email protected]

Mr. Ian Rees


[email protected]

110


Mr. Emilio ReyesAldrete

UTMB at Galveston

[email protected]

Dr. Jon Robertus

University of Texas

[email protected]

Mr. Ryan Rochat


[email protected]

Mr. Behrooz Sarabi

Faculty of Agriculture

[email protected]

Dr. Mayukh Sarkar

UT Houston Medical School

[email protected]

du

Dr. Catherine H. Schein

UTMB

[email protected]

Dr. Nadrian Seeman

New York University

[email protected]

Dr. Krishna Mohan Sepuru

The UTMB

[email protected]

Ms. Ashwini Shanbhogue


[email protected]

Dr. Alexander Shavkunov

UTMB

[email protected]

Dr. Michael Sherman

UTMB

[email protected]

Dr. Hyun Deok Song


[email protected]

Ms. Paige Spencer

UTMB

[email protected]

Mr. Gary Stinnett


[email protected]

Dr. Svetla Stoilova-McPhie

UTMB

[email protected]

Mr. Guoxiong Su


[email protected]

Ms. Sajana Sudarshan


[email protected]

Ms. kin man suen

md anderson cancer center

[email protected]

Mr. Jiayi Sun


[email protected]

Dr. Michal R. Szymanski

UTMB

[email protected]

Dr. Varatharasa Thivi-

yanathan

UTHSC

[email protected]

Mr. Tangen Tran


[email protected]

Ms. Devinder Ubhi

University of Texas at Aus-

tin

[email protected]

Mr. Yavar Vafaee

University of Tehran

[email protected]

Dr. Arnold Vainrub

UTMB

[email protected]

Dr. David Volk

Institute for Molecular Med-

icine

[email protected]

Dr. Pramila Walpita

UTMB

[email protected]

Mr. Qian Wang


[email protected]

Dr. Tianzhi Wang

UTMB

[email protected]

Dr. Scott Weaver

UTMB

[email protected]

Dr. Mark Andrew White

UTMB

[email protected]

Dr. Ka-Yiu Wong

UTMB

[email protected]

Dr. Ada Yonath

Weizmann Institute of Sci-

ence

[email protected]

Ms. Linglin Yu

[email protected]

Mr. Levani Zandarashvili

[email protected]

Mr. Fabio Zegarra Choque


[email protected]

Mr. Pengzhi Zhang


zhangpeng-

[email protected]

Ms. Li Zhao


[email protected]

scsb at utmb

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