clemson ci symposium - feb 13, 2013 establishing a nmr structural biology center at claflin...

Clemson CI Symposium - Feb 13, 2013

Establishing a NMR Structural Biology Establishing a NMR Structural Biology Center at Claflin University.Center at Claflin University.

Raj BhaskaranRaj BhaskaranDepartment of Chemistry Department of Chemistry

Claflin University, Orangeburg, SC-29115Claflin University, Orangeburg, SC-29115

NMR in CI SymposiumNMR in CI Symposium


Aim of this talk is two fold:Aim of this talk is two fold:

To attract the Cyber Specialists to join hands with Claflin To attract the Cyber Specialists to join hands with Claflin NMR team to mutually collaborate in the areas that require NMR team to mutually collaborate in the areas that require enormous computing and CI efforts;enormous computing and CI efforts;

To attract Life Science researchers to interact with the To attract Life Science researchers to interact with the NMR team to explore their problems structurally to NMR team to explore their problems structurally to provide a functional meaning;provide a functional meaning;

. . . to initiate GEAR CI . . . to initiate GEAR CI collaborationscollaborations..

Claflin’s NMR Route to Claflin’s NMR Route to Structural Biology and Drug Discovery ResearchStructural Biology and Drug Discovery Research


ClaflinClaflin has the high field 700 MHz NMR Spectrometer- BioNMR/ has the high field 700 MHz NMR Spectrometer- BioNMR/ MetabolomicsMetabolomics

NSF Major Research Instrumentation grant is applied for a Cryoprobe and NSF Major Research Instrumentation grant is applied for a Cryoprobe and Sample Changer Sample Changer

Solution Structure Determination and Protein Dynamics- Functional Solution Structure Determination and Protein Dynamics- Functional Interpretation based on structural investigationsInterpretation based on structural investigations

Protein (Enzyme) - Small Molecule (Substrate / Inhibitor) Interactions Protein (Enzyme) - Small Molecule (Substrate / Inhibitor) Interactions

Protein Structures Derived Functional Interpretation on Protein Structures Derived Functional Interpretation on t. Thermophilus t. Thermophilus proteome, from Riken, Japanproteome, from Riken, Japan and and Filariasis Nematode, Filariasis Nematode, Brugia Malayi Brugia Malayi proteome, (NIH, UIC)proteome, (NIH, UIC)

Rapid Protein Structure Determination for Structural Genomics Consortium: Rapid Protein Structure Determination for Structural Genomics Consortium: Structural Bioinformatics (North East Structural Genomics Consortium; Structural Bioinformatics (North East Structural Genomics Consortium; Seattle Structural Genomics Center for Infectious Diseases)Seattle Structural Genomics Center for Infectious Diseases)

Computer Aided Drug Discovery: Virtual screening, Data Mining, QSARComputer Aided Drug Discovery: Virtual screening, Data Mining, QSAR

Structure Based Drug Design: SAR by NMR, NMR ScreeningStructure Based Drug Design: SAR by NMR, NMR Screening

Structure Based Drug Design•Suitable protein target

•Structure of the target protein

•Implementation of an easy and reliable HTS assay

•Identification of a lead compound

•Computer assisted methods for estimating the affinity of new compounds

•Access to a synthetic route to produce designed compounds.

•Knowledge on shape of the pocket to design the shape of the drug.

•The use of X-ray / NMR structures of Protein-Drug Complexes to design better-fitting, and hence more potent inhibitors


New Compound

Suggests New Interaction

Drug-Protein Complex

Biological Activity

Ligands in the Drug Design Process


compound librariesnatural productsdirected libraries

Identified & Validated Target

Protein Production

Assays /HTS

Hits

Chemical tractabilityToxicityDrugMetabolism /

PharmacoKinetics

(genomics, transgenics etc)

more structure

Structure

Ligand-Protein structure

Optimised Ligand

more chemistry

low affinity,high Kd

high affinity,low Kd

Free state

Extensive computations involved in Protein NMR StudyExtensive computations involved in Protein NMR Study


Isotopically Isotopically 1515N, N, 1313C, Labeled NMR sampleC, Labeled NMR sample

Acquisition of 3D heteronuclear (H, Acquisition of 3D heteronuclear (H, 1515N, N, 1313C) NMR experimentsC) NMR experiments

Backbone (HN, Backbone (HN, 1515N, N, 1313CC, H, H ) and Side Chain ( ) and Side Chain (1313CC, H, H1313CC, H, H , …..) resonance assignment , …..) resonance assignment

Unambiguous assignment of Nuclear Overhauser Enhancement ( proton-proton interaction within 5 Unambiguous assignment of Nuclear Overhauser Enhancement ( proton-proton interaction within 5 AA0 0 radius)radius)

Structure Determination using Restrained Molecular Dynamics and Simulated Annealing Protocol Structure Determination using Restrained Molecular Dynamics and Simulated Annealing Protocol

Structural Refinement using additional experimental restraintsStructural Refinement using additional experimental restraints

Protein Dynamics from Protein Dynamics from 1515N, N, 1313CCRelaxation experimentsRelaxation experiments

HUMAN ( E219A) MMP-12: HUMAN ( E219A) MMP-12: 11H-H-1515N HSQC SPECTRUM / NOE CONNECTIVITIES / CSIN HSQC SPECTRUM / NOE CONNECTIVITIES / CSI


Backbone

NHSQC; CHSQC;

TROSY HNCOCA; HNCA

HACACONH; HNNCAHA

CBCACONH; HNCACB

HNCO; HCACOCANH

Side chain

HBCBCGCD(CE)HD(HE)

CCH TOCSY

HCCCONH; CCONH

HCCH COSY;

HCCH TOCSY

HCCH TOCSY (aro)

NOE

N15 EDITED 3D NOESY

C13 EDITED 3D NOESY

NoesyChsqc (aro)

VNMR

TOPSPIN

NMRPIPE

SPARKY

AUTO ASSIGN

PYMOL / MOLMOL

SHIFTX

CSI

TALOS-PLUS

CYANA

XPLOR-NIH

HADDOCK

MODELFREE

PROCHECK/PSVS

SOFTWARESOFTWARE NMR EXPTS NMR EXPTS

Bhaskaran & VanDoren JBNMR (2006)

F R E M P G G P V W R K H Y I T Y R I N N Y T P D M N R E D V D Y A I R K A F Q V W S N V T P L K F S K I N T G M A D I L V V F A R G A H G D F H A F D G K G G I L A H A F G

105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185

dN(i, i+1)

dNN(i, i+1)

dN(i, i+2)

d(i, i+3)

dN(i, i+3)

Secondary Structure

F R E M P G G P V W R K H Y I T Y R I N N Y T P D M N R E D V D Y A I R K A F Q V W S N V T P L K F S K I N T G M A D I L V V F A R G A H G D F H A F D G K G G I L A H A F G

105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 F R E M P G G P V W R K H Y I T Y R I N N Y T P D M N R E D V D Y A I R K A F Q V W S N V T P L K F S K I N T G M A D I L V V F A R G A H G D F H A F D G K G G I L A H A F G

105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185

dN(i, i+1)

dNN(i, i+1)

dN(i, i+2)

d(i, i+3)

dN(i, i+3)

Secondary Structure

P G S G I G G D A H F D E D E F W T T H S G G T N L F L T A V H A I G H S L G L G H S S D D K A V M F D T Y K Y V D I N T F R L S A D D I R G I Q S L Y G

190 195 200 205 210 215 220 225 230 235 240 245 250 255 260

dN(i, i+1)

dNN(i, i+1)

dN(i, i+2)

d(i, i+3)

dN(i, i+3)

Secondary Structure


190 195 200 205 210 215 220 225 230 235 240 245 250 255 260


190 195 200 205 210 215 220 225 230 235 240 245 250 255 260

dN(i, i+1)

dNN(i, i+1)

dN(i, i+2)

d(i, i+3)

dN(i, i+3)

Secondary Structure

110 130 150 170 190 210 230 250 120 140 160 180 200 220 240 260

0.6

0.4

0.2

0.0

- 0.2

- 0.4

MMP-12 Residue

110 130 150 170 190 210 230 250 120 140 160 180 200 220 240 260

0.6

0.4

0.2

0.0

- 0.2

- 0.4

MMP-12 Residue

First NMR structure of the MMP-12 catalytic domain sans inhibitor

Clemson CI Symposium - Feb 13, 2013 Bhaskaran et al. J Mol Biol (2007)

29 unique NOEs( dotted lines)account for the conformationaladjustment

active site helix B & -strands I, III are closer in free state NMR – Green;

Inhibitors pull B away from the far side X-RAY – Red;

RCSB PDB code 2POJ2803 NOEs UsedBB RMSD 0.32 ASC RMSD 0.68 A

Ramachandran plot statistics:

77.3% in most favored regions

21.5% in allowed regions

0.0% in disallowed regions

Protein Dynamics - rigidity of catalytic domain affect activity upon elastin & 1-AT.

High Rigidity Limited Conformational Flexibility


MMP-12 MMP-3

ps-ns

110 120130 140 150 160 170 180 190 200 210 220 230 240 250 2600.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

Order param eter S2 for MMP-12

54321

S2

Residue Number

β1

β2

β3

β4

β5

S225

L229

D158

F157

L2/3 L3/4

90 1001101201301401501601701801902002102202302402502600.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

Order parameter S2 for MMP-3

S2

Residue Number

MMP-12 is more rigid than MMP-3.

Apparent Trade-off of Activity and Rigidity in MMP12 for Stability and Flexibility in MMP3

Liang, Bhaskaran et al Biophysical Jl. (2010)

Family of NMR structures of STT3P C-ter Domain -the Catalytic Subunit of Oligo Sachcharyl Transferase

Ave BB rmsd to mean: 0.230 nmAve heavy atom rmsd to mean : 0.307 nm

Clemson CI Symposium - Feb 13, 2013 Huang, Bhaskaran, et al Jl.Biol.Chem. (2012)

RCSB PDB code “2lgz”

It is so far the biggest monomeric helical integral membrane protein structure by NMR

/

Ost1p binding site

Membrane embedding siteCatalytic center

“WWDYG”motif

“DK”motif

CW 110 deg

Structural Gallery - Proteins

COBROTOXIN (1COD) CARDIOTOXIN III (2CRS)

TMH0916 (1NR3) GIP / L-GLUT COMPLEX (2L4T) GIP (2L4S)

MMP-12 (2POJ) STT3P (2LGZ)

CARDIOTOXIN II (1CRE)


PERTURBATION OF MMP12 BY PERTURBATION OF MMP12 BY Triple Helical PeptideTriple Helical Peptide AND THE MAPPING AND THE MAPPING OF THEM ON THE STRUCTURE OF MMP12.OF THEM ON THE STRUCTURE OF MMP12.

Chemical Shift Perturbation of 1H-15N NMR Correlation Spectrum for Finding Binding Site

15N

1H


Bhaskaran et al. J Biol Chem.(2008)

0.0000 0.0001 0.0002 0.0003

0.00

0.01

0.02

0.03

0.04

0.05K

d = 30 ± 6 M

H(p

pm

)

[1(V)THP] (M)

H112

G221

L147

Y113

T205

K148Y240

L224

T210V243

0.0000 0.0001 0.0002 0.0003

0.00

0.01

0.02

0.03

0.04

0.05K

d = 30 ± 6 M

H(p

pm

)

[1(V)THP] (M)

H112

G221

L147

Y113

T205

K148Y240

L224

T210V243

Binding Isotherm


Protein:Ligand Interface Mapping by NMR

The NMR paramagnetic surface protection assay correctly predicts the sites of interaction as confirmed by the loss of function mutation studies

Palmier, Bhaskaran et al Jl Biol Chem. (2010)

EVOLUTIONARY TRACE ANALYSIS

Evolutionary Trace Method uses a sequence similarity tree of a family of homologous proteins to highlight residues that are statistically under evolutionary pressure and therefore possess certain functional or structural importance for the family


BINDSIght Method - maps of binding sites and distinctive sequence to suggest residues tuning specificity

Palmier, Bhaskaran et al. J. Biol. Chem. (2010)Clemson CI Symposium - Feb 13, 2013

CI: Scope and Problems for InteractionCI: Scope and Problems for Interaction


Pulse program conversion - Varian and BrukerPulse program conversion - Varian and Bruker

Consolidation of SW packages on NMR structure Consolidation of SW packages on NMR structure determination, dynamics and ligand interactionsdetermination, dynamics and ligand interactions

Develop multiuser remote instrumentation to convert into Develop multiuser remote instrumentation to convert into a state level facilitya state level facility

A Virtual Web Based File system for managing NMR DataA Virtual Web Based File system for managing NMR Data

Rapid structure determination package for the structural Rapid structure determination package for the structural genomics proteins genomics proteins

BioNMR – External CollaborationsBioNMR – External Collaborations


Dr. Steven VanDoren, University of Missouri, Columbia, MO :Structural Interactions of Tumor Viral Proteins, E7 and

E2FDr. K.Ramaswamy, UIC, Rockford, IL :

Structural Studies of Filarial Proteins Bm-HSP 12.6Dr. M.Gnanasekar, UIC, Rockford, IL :

Structural Studies of Filarial Proteins Bm-TCTPDr. Peter Myler, Seattle Biomed, Seattle, WA :

Structural Genomics of Proteins from Infectious DiseasesDr. Krishna Sharma, University of Missouri, Columbia, MO :

Structures of -Crystallin & Chaperonins of Eye Disease, CataractDr. Raghu Kannan, University of Missouri, Columbia, MO :

NMR of Gold Nanoparticle Conjugated BombesinDr. T. K. S. Kumar, University of Arkansas, Fayetteville, AR :

Structural Studies of Fibroblast Growth factor, FGF2

Dr. K. Gunasekaran, University of Madras, Chennai, India :Structural Studies of Filarial Proteins Bm-API

Dr. D. Velmurugan, University of Madras, Chennai, India : Structural Genomics of t. thermophilus Proteins

Dr. P. Karthe, University of Madras, Chennai, India :NMR structure of Serine glutamate repeat A, a surface

adhesin

GEAR- CI: SC-CollaborationsGEAR- CI: SC-Collaborations


Dr. John Dawson, University of South Carolina :Dr. John Dawson, University of South Carolina : Identification of Dehaloperoxidase-Substrate Binding Sites

Dr. Caryn Outten, University of South Carolina :Dr. Caryn Outten, University of South Carolina :Structural Studies of iron sensing proteins, Fra2 and Grx

Dr. Homayoun Valafar, University of South Carolina :Dr. Homayoun Valafar, University of South Carolina :NMR Residual Dipolar Coupling analyses

Dr. Nick Grossoheme, Winthrop University:Dr. Nick Grossoheme, Winthrop University:Dr. Heather Evans Anderson, Winthrop University:Dr. Heather Evans Anderson, Winthrop University:

NMR structure of ForkHead Transcription Factor Protein, FOXO1Dr. Sondra Berger, University of South Carolina :Dr. Sondra Berger, University of South Carolina :Dr. Angela Peters, Claflin University :Dr. Angela Peters, Claflin University :

Thymidalate Synthase Mutant (R163K)-Ligand Interactions Dr. Michael Sehorn, Clemson University :Dr. Michael Sehorn, Clemson University :

Dr. Erin Eaton, Francis Marion University :Dr. Erin Eaton, Francis Marion University :

Dr. Karen Buchmuller, Furman University :Dr. Karen Buchmuller, Furman University :

Dr. Marcello Forconi, College of Charleston :Dr. Marcello Forconi, College of Charleston :

Dr. Esmaeil Jabbari, University of South Carolina : (CRP)Dr. Esmaeil Jabbari, University of South Carolina : (CRP) NMR Structural Characterization of vasculogenic peptides

Dr. Scott Argraves, University of South Carolina : (CRP)Dr. Scott Argraves, University of South Carolina : (CRP) NMR Studies of Fibulins, the extracellular matrix proteins

ACKNOWLEDGEMENTACKNOWLEDGEMENT


SC INBRE

NASA EPSCoR Space Grant.

Mr. Raghav Nagampalli (Fulbright Scholar)Mr. Raghav Nagampalli (Fulbright Scholar)

Dr. Angela Peters (Claflin) Dr. Angela Peters (Claflin)

Dr. Verlie Tisdale (Claflin)Dr. Verlie Tisdale (Claflin)

School of Natural Sciences and Mathematics, ClaflinSchool of Natural Sciences and Mathematics, Claflin

TRUSTWORTHINESSRESPECT

RESPONSIBILITYFAIRNESS

CITIZENSHIPCARING

THANK YOU ALLTHANK YOU ALL


clemson ci symposium - feb 13, 2013 establishing a nmr structural biology center at claflin...

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