44th southeastern magnetic resonance conference

44th Southeastern Magnetic Resonance Conference Table of Contents

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Table of Contents

Welcome 3

General Information 5

Sponsors 7

Schedule 9

Keynote Speakers 15

Friday Evening Talks 16

Oral Presentations 19

Hardware and Technique Development 20

Magnetic Resonance & Macromolecules 33

Magnetic Resonance in vivo 43

Radicals in vivo and in situ 49

Posters 55

Participants 101

44th Southeastern Magnetic Resonance Conference

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44th Southeastern Magnetic Resonance Conference Welcome

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Welcome!

On behalf of the University of Florida (UF), the National High Magnetic Field Laboratory (NHMFL), and the Advanced Magnetic Resonance Imaging and Spectroscopy Facility (AMRIS) magnetic resonance communities, we welcome you to the 44th Southeastern Magnetic Resonance Conference. Joanna Long (Biochemistry & Molecular Biology, UF) and Gail Fanucci (Chemistry, UF) are the co-chairs for this year’s meeting.

The SEMRC is a regional meeting whose purpose is to highlight and promote current research activities in the southeastern United States that focus on the utilization or development of magnetic resonance techniques. The meeting spans all areas of magnetic resonance (NMR, EPR, imaging, magnetic materials) and their application to a broad range of fields, including structural biology, materials science, physics, and chemistry. It was started over 44 years ago by early practitioners in the region and has been held almost every year since in spite of the fact that it has no bylaws, no formal organizational structure, and of course no resources.

The organizers also invite several leading scientists from outside the southeast as keynote speakers to highlight exciting areas of development in magnetic resonance techniques and their applications. An integral part of this conference is a small, collegial environment to foster the interaction of students and post-doctoral research associates with prominent researchers.

This year, we are fortunate in that we received donations from SHARED-EPR and the NHMFL specifically for student travel awards. We thank the Center of Chemical Physics at UF, the Center of Catalysis Support at UF, the local chapter of the American Chemical Society, the UF Chemistry Department, the MacroCenter at UF, the UF College of Liberal Arts and Sciences, the Center for Structural Biology at UF, the UF Department of Biochemistry and Molecular Biol-ogy, the UF McKnight Brain Institute, and the UF Division of Sponsored Research who provided support enabling us to keep registration fees to a minimum. We also thank the many commercial vendors who have provided financial support for the conference: Avanti Polar Lipids, Bruker Biospin, Cambridge Isotope Laboratories, Doty Scientific, Philips, Siemens, Sigma-Aldrich, and the Daytona Beach Area Convention & Visitor’s Bureau. Without their support, we would not be able to provide the venue and amenities which are integral to the ethos of this conference.

We also thank Nilubon Tabtimtong for creating and maintaining this year’s website, those who serve on the Conference Advisory Committee, and past SEMRC organizers, specifically Lowell D. Kispert (SEMRC 2014) for his advice and assistance. Many thanks also go to our program assistant and organizer extraordinaire, Elizabeth Webb. Without her help, the organization of this conference would have been many times more difficult.

Finally, thank you for your choice to participate in and contribute to this conference. We hope you enjoy it and have a memorable time!

Joanna R. Long Gail E. Fanucci


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44th Southeastern Magnetic Resonance Conference General Information

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General Information Posters

Posters may be set up beginning at 5:00pm on Friday, October 9th in Plaza C and should be taken down by noon on Sunday, October 11th. Poster viewing will occur between 5:00 and 6:30pm as well as between 8:00 and 10:00pm on Friday, October 9th, and poster presentations are on Saturday, October 10th, from 5:30-7:30pm. Participants with even poster numbers are asked to stand by their posters during the first hour of the poster presentation; those with odd poster numbers should stand by their posters during the second hour. Please consult the header in the poster section of this program for poster numbers.

Talks

All talks will be held in Plaza C. There will be a 3 minute question and answer session after each talk.

Presenters who have prepared a PowerPoint presentation and would like to test their slides prior to their talk should bring their file on a flash drive to Plaza C during one of the following times: Friday between 5:00pm and 6:20pm; Saturday between 7:15am and 8:00am; Sunday between 7:15am and 8:00am. For each session, speakers should plan on making their file or laptop available at least 20 minutes before the session begins. The conference provided laptop is a PC running a current version of PowerPoint.

Food and Drinks

Included in your registration packet are four drink tickets: two for Friday night and two for Saturday night. Each ticket is good for one drink at the banquet/poster session bar. Please note that these are only good at the bar that is specifically set up for the SEMRC event and not at the other hotel bars.

All meals on the schedule (Friday evening; Saturday breakfast, lunch, and dinner; and Sunday breakfast) are covered by your registration fee and donations from vendors. Breakfasts and the Friday evening reception will be held in Plaza B. The Saturday lunch will be on the Seabreeze Terrace (rain location: Vista del Mar) and the Saturday banquet will be on the pool deck.

Sponsor tables

Sponsor tables will be set up in Plaza B from 4:00-10:00pm on Friday, October 9th and from 8:00am-3:00pm on Saturday, October 10th. Please take time to visit our sponsors.

Internet

Wifi is available throughout the hotel. Select the wifi network “plaza” and enter “meetings04”.


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44th Southeastern Magnetic Resonance Conference Sponsors

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Thank you to our corporate sponsors:

44th Southeastern Magnetic Resonance Conference Sponsors

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Thank you to our academic sponsors:

The following centers and departments at the University of Florida:

44th Southeastern Magnetic Resonance Conference Schedule

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Friday, October 9th

4:00 - 10:00 p.m. Vendor Tables Plaza B

5:00 - 10:00 p.m. Registration Hotel colonnade

5:00 - 6:30 p.m. Poster Set Up and Viewing Plaza C

Opening Talks Chair: Joanna Long

6:30 - 6:40 p.m. Opening Remarks and Welcome: Joanna Long

Plaza C

6:40 - 7:20 p.m. Keynote: Dan Vigneron Preclinical Hyperpolarized MR Molecular Imaging and Translation for Clinical Research

7:20 - 8:00 p.m. Keynote: R. David Britt Mechanisms of Solar Fuel Reactions as Probed by Advanced EPR Spectroscopy

8:00 - 10:00 p.m. Welcome Reception & Poster Viewing

Plaza B/Plaza C

Saturday, October 10th 7:15 – 8:00 a.m. Registration Plaza B

7:15 - 8:00 a.m. Breakfast

Plaza C

8:00 - 3:00 p.m. Vendor Tables Plaza B

Hardware and Technique Development Chairs: Tom Mareci & Russ Bowers 8:00 - 8:05 a.m. Opening Remarks and

Welcome: Gail Fanucci

Plaza C


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8:05 - 8:35 a.m. Keynote: Jeff Schiano

Towards High Resolution NMR and MRI in a 36 T Powered Magnet

8:35 - 8:50 a.m. Thorsten Maly

A Compact, High-Field EPR Spectrometer For Dynamic Nuclear Polarization

8:50 - 9:20 a.m. Daniel Gochberg Imaging Magnetization Exchange With CEST and CERT

9:20 - 9:35 a.m. Magdoon Kulam

Phase Contrast MRI With Reduced Eddy Current Distortion

9:35 - 9:55 a.m. Yan-Yan Hu

Solid-State NMR Studies Of Energy Storage Materials

9:55 - 10:10 a.m. Sebastian Stoian

A Detailed Spectroscopic And Theoretical Investigation Of High-Spin Square-Planar Fe2+ And Co2+ Complexes

10:10 - 10:30 a.m. Mike Bowman

Magnetic Interactions In Narrow-Line Trityl Biradicals

10:30 - 10:50 a.m. Coffee Break

10:50 - 11:10 a.m. Russ Bowers Parahydrogen Induced Polarization By Pairwise Replacement Catalysis Over Metal Nanoparticles And Ceria Nanoshapes

11:10 - 11:25 a.m. Lakshmi Bhaskaran

Pushing The Limits Of Magnetic Anisotropy In Mononuclear Ni(II) Single - Molecule Magnet: A High-Field EPR Study

11:25 - 11:40 a.m. Deidra Gerlach The Coordination Environment Of Copper Water Oxidation Catalysts Before Electrocatalysis And The Pathways Open During Electrolysis


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11:40 - 11:55 a.m. Yunzhi Zhang Bdpa-Doped Polystyrene Beads As Polarization Agents For DNP-NMR

11:55 - 12:10 p.m. Mariya Vyushkova Optically Detected EPR Spectra Of Polyfluorobiphenyl Radical Anions

12:10 - 12:25 p.m. Jurek Krzystek

Terahertz-Frequency EPR

12:30 - 1:30 p.m. Lunch

Seabreeze Terrace Rain location: Vista del Mar

Magnetic Resonance and Macromolecules Chairs: Alex Angerhofer & Leah Casabianca 1:30 - 1:50 p.m. Anne-Frances Miller 1H Dynamic Nuclear Polarization

Based On An Endogenous Flavin Radical

1:50 - 2:20 p.m. Keynote : Fraser MacMillan Understanding Movement And Mechanism In Membrane Proteins

2:20 - 2:35 p.m. Joana Paulino Protein Dynamics By ssNMR: Gating Motions In M2 Proton Channel Of Influenza A

2:35 - 2:55 p.m. Bo Chen Rous Sarcoma Virus Capsid Protein In Its Tubular Assembly

2:55 - 3:10 p.m. Kari Pederson Assignment Of Sparse 13C Alanine Methyl Resonances In Hsp90 Proteins Using RDCs PCSs, And NOEs

3:10 - 3:30 p.m. Alex Smirnov Cysteine-Specific Labeling Of Proteins With A Totapol Derivative For Dynamic Nuclear Polarization NMR

3:30 - 3:50 p.m. Coffee Break

3:50 - 4:20 p.m. Aimin Liu

Catalysis At A Distance: Nature's Sniper For Remote Specific Tryptophan Oxidation


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4:20 - 4:35 p.m. Pavanjeet Kaur

Selective Membrane Disruption Mechanism Of An Anti-Bacterial Peptide Defined By EPR Spectroscopy

4:35 - 4:55 p.m. Alex Nevzerov

Dipolar-Recoupled Lineshapes Of Uniaxially Diffusing Membrane Proteins Under MAS

4:55 - 5:25 p.m. Josh Telser

EPR/ENDOR/ESEEM Of Iron-Sulfur Proteins And Enzymes

5:30 - 7:30 p.m. Poster session and drinks

Plaza C

7:30 - 11:00 p.m. Banquet Dinner

Pool deck Rain location: Plaza C

Sunday, October 11th 7:15 - 8:30 a.m. Breakfast

Plaza B

Magnetic resonance in vivo Chair: Matt Merritt 8:00 - 8:05 a.m. Opening Remarks and

Welcome: Tom Mareci

Plaza C

8:05 - 8:35 a.m. Matt Merritt Is Hyperpolarized Pyruvate Useful For Imaging Hepatic Metabolism?

8:35 - 8:50 a.m. Dan Downes Real Time Analysis Of The Cerebral Metabolic Shift Caused By Cocaine Administration And Withdrawal

8:50 - 9:10 a.m. Zackary Cleveland UTE And HP 129Xe MRI Of Lung Structure-Function Relationships In Pediatric Cystic Fibrosis


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9:10 - 9:30 a.m. Gary V. Martinez Preclinical Functional MR Images Studies To Improve Diagnosis And Therapy Response In Ewing Sarcoma

9:30 - 9:45 a.m. Bibek Thapa Superparamagnetic Iron Oxide Nanoribbons With Enhanced Relaxivity As T2 Contrast Agent In MRI

9:45 - 10:00 a.m. Victor Schepkin In Vivo Triple Quantum Effects Of Bound Potassium And Sodium At 21.1 T

10:00 - 10:20 a.m. Coffee Break

Radicals in vivo and in situ Chair: Gail Fanucci 10:20 - 10:40 a.m. Alexander Angerhofer EPR Detection On Microchips -

On The Way To Single Cell Detection

10:40 - 10:55 a.m. Xiao Song Investigating The Role And Structure Of Free Radicals In Lignin Biosynthesis

10:55 - 11:10 a.m. Arthur Sikora DEER In Native Systems: What Lipids And Detergents Can't Tell Us

11:00 - 11:30 a.m. Tatyana Smirnova smFRET And DEER Distance Measurements As Applied To Disordered And Structured Proteins

11:30 - 11:45 a.m. Meghan Kohne Radical Rearrangement Catalysis In B12-Dependent Ethanolamine Ammonia-Lyase

11:45 - 12:00 p.m. Alex Cruce Drug Binding Characterization Of CYPs Utilizing CW And Pulsed EPR

12:00 - 12:30 p.m. SEMRC Business Meeting

Ponce De Leon Boardroom


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44th Southeastern Magnetic Resonance Conference Keynote Speakers

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KEYNOTE SPEAKERS

David Britt received his Ph.D. in Physics from the University of California, Berkeley and is now a Professor in the Department of Chemistry at the University of California at Davis. Dr. Britt’s work uses EPR as a tool to study the structure and function of biologically significant enzymes with redox-active transition metal center, clusters, or organic radicals on their active site. Specifically, Dr. Britt looks at the oxygen-evolving complex of photosystem II, the enzyme complex responsible for water oxidation in photosynthesis.

Fraser MacMillan received his Der.rer.nat. from the Department of Chemistry at the Free University of Berlin and is now a Reader in Chemistry at the University of East Anglia, UK. His research focuses on elucidating the structure, function, and dynamics relationships in biomacromolecues and macromolecular complexes. Specifically, he looks at the application and expansion of electron paramagnetic resonance methods to study membrane proteins.

Jeffrey Schiano received his Ph.D. in Electrical Engineering from the University of Illinois. He is now an Associate Professor of Electrical Engineering at The Pennsylvania State University, where his research interests include control systems applications in nuclear resonance, neural network controllers, and microelectromechanical systems.

Daniel Vigneron received his Ph.D. in Pharmaceutical Chemistry from the University of California, San Francisco and is now a Professor in the Departments of Radiology and Biomedical Imaging and Bioengineering and Therapeutic Sciences at UCSF. He is also Director of the Advanced Imaging Technologies Resource Group, Director of the Hyperpolarized MRI Technology Resource Center, and Associate Director of the Surbeck Laboratory for Advanced Imaging at UCSF. Dr. Vigneron’s research focuses on the development of metabolic MRI techniques for research and clinical assessments of human diseases. Specifically, developing specialized acquisitions techniques for prostate cancer MRSI has been a major focus of Dr. Vigneron’s group.

44th Southeastern Magnetic Resonance Conference Friday Keynote Talks

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Preclinical Hyperpolarized MR Molecular Imaging and Translation for Clinical Research Dan Vigneron, Ph.D., Dept. of Radiology & Biomedical Imaging, University of California, San Francisco Hyperpolarized 1 3 C imaging using the dissolution DNP (dynamic nuclear polarization) method provides a >10,000 fold signal enhancement for detecting 13C-enriched endogenous, nontoxic, nonradioactive molecules such as pyruvate to monitor metabolic fluxes through multiple key biochemical pathways (glycolysis, citric acid cycle and alanine transaminase). Hyperpolarized Carbon-13 MRI has the potential to become an important new radiological tool for metabolic imaging by directly investigating key cellular enzymatic pathways in vivo. The hyperpolarization of [1-13C]pyruvate has demonstrated the ability to not only detect pyruvate uptake but also the in vivo enzymatic conversion to 1 3 C-lactate through the enzyme lactate dehydrogenase (LDH), 1 3 C-alanine through the alanine transaminase (ALT) pathway; and 1 3 CO2 & 1 3 C-bicarbonate through the pyruvate dehydrognase (PDH) catalyzed metabolic pathway. The value of this powerful metabolic imaging technique for cancer imaging was shown first by Golman et al and we have applied it in a number of preclinical animal studies for detecting cancer presence, progression and response to therapy. Also, our first-in-man Phase 1 clinical trial in prostate cancer patients, published in Science Translational Medicine last year (2013), demonstrated feasibility and safety for this MR metabolic imaging technique in human studies. We have conducted preclinical studies in multiple cancers including prostate, liver, kidney, brain and for other diseases as well. While 1 3C-pyruvate is the first to studied in humans, other hyperpolarized substrates including bicarbonate, glutamine, lactate, urea, alanine, bicarbonate, fructose, ketobutyrate, t-butanol, and dehydroascorbate have also been investigated in preclinical studies to probe metabolism, perfusion and physiology. These studies have demonstrated significant metabolic changes with cancer presence, aggressiveness and response to therapy. The detection of a significant correlation with grade for upregulated LDH-conversion to lactate by HP MRI is of great potential clinical value since there is no accurate current imaging technique to identify aggressive prostate cancers (which should be treated) from indolent cancers that may be managed through “active surveillance”. In addition to metabolic pathway information, hyperpolarized probes can provide valuable physiological information such as perfusion information (with HP 1 3 C-urea) and pH (with HP 1 3 C sodium bicarbonate). In a special double-transgenic mouse model, we have performed preclinical studies of specific human oncogene expressions demonstrated highly elevated pyruvate to lactate conversion with significant correlations to up-regulated lactate dehydrogenase (LDH) activity and mRNA over-expression. This study also showed significant reductions in HP 1 3 C-lactate detection in 3 days following oncogene inhibition demonstrated the potential of this technology for the early detection of therapeutic response in these preclinical models.


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On the left shows information on the results of the first clinical trial using DNP hyperpolarized [1-13C]pyruvate to detect upregulated LDH enzymatic conversion to [1-13C]lactate in prostate cancer patients. On the right is recent work from preclinical studies, extending this technique to perform real-time biochemical enzymatic rate measurements non-invasively in living animal and cell models.


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Mechanisms of Solar Fuel Reactions as Probed by Advanced EPR Spectroscopy R. David Britt

University of California, Davis, Department of Chemistry, Davis, CA, 95616

The oxygen evolving complex of Photosystem II splits water with visible light, producing molecular oxygen and electrons and protons. The electrons and protons can be recombined to make hydrogen with the appropriate catalyst. Specifically, [FeFe] hydrogenase enzymes rapidly evolve H2 at a 6-Fe catalytic site termed the H-cluster, which consists of a traditional [4Fe-4S] cluster linked via a cysteine bridge to a dinuclear Fe subcluster [2Fe]H that possesses unusual biological ligands: two terminal CN-

ligands, two terminal CO ligands, and azadithiolate and CO bridges, all of which are thought to be synthesized and installed by a set of Fe-S proteins denoted HydE, HydF, and HydG. With the James Swartz laboratory (Stanford University) we can generate [FeFe] hydrogenase in high yield using cell free synthesis methods, allowing for specific isotope labelling of its components as needed for definitive spectroscopic studies (1).

The radical S-adenosylmethionine (SAM) enzyme HydG lyses free L-tyrosine to produce CO and CN- for the assembly of the H-cluster. We use electron paramagnetic resonance (EPR) spectroscopy to detect and characterize HydG reaction intermediates generated with a set of 2H, 13C, and 15N nuclear spin labeled tyrosine substrates. 5’-deoxyadenosyl cleavage of tyrosine at the Calpha-Cbeta bond generates a transient 4-oxidobenzyl (4OB.) radical and a dehydroglycine bound to a C-terminal Fe-S cluster (2). Electron and proton transfer to this 4OB. radical forms p-cresol with the conversion of this dehydroglycine ligand to Fe-bound CO and CN-, a key intermediate in the assembly of the [2Fe] subunit of the H-cluster. We apply stopped-flow Fourier transform infrared (SF-FTIR) and electron-nuclear double resonance (ENDOR) spectroscopies to explore in detail the formation such species which are used to build the H-cluster (3). New X-ray crystallography and EPR studies reveal a unique site-differentiated structure for this C-terminal Fe-S moiety that clarifies its role in H-cluster synthesis (4). Many open issues remained to be explored in this unique facet of biological catalytic cluster synthesis, including the roles of the additional Fe-S proteins HydE and HydF (5).

1. Myers, William, et al., J. Am. Chem. Soc. (2014) 136:12237-12240. 2. Kuchenreuther, Jon, et al., Science (2013) 342:472-475 3. Kuchenreuther, Jon, et al., Science (2014) 343:424-427 4. Dinis, Pedro., et al., Proc. Natl. Acad. Sci. U.S.A. (2015) 112:1362-1367. 5. Pickett, Christopher, Science (2014) 343:378-379

44th Southeastern Magnetic Resonance Conference Oral Presentations

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ORAL PRESENTATIONS

44th Southeastern Magnetic Resonance Conference Oral Presentations Hardware & Technique Development

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TOWARDS HIGH RESOLUTION NMR AND MRI IN A 36 T POWERED MAGNET

Jeff Schiano1

1 Pennsylvania State University, University Park, PA 16802, USA

Powered magnets can generate magnetic fields much higher than superconducting persistent magnets and offer unique opportunities for studying NMR phenomena and MR imaging. Nevertheless, the temporal stability and field homogeneity are often insufficient for high spectral resolution and MRI. Results are shown from a TOCSY (total correlation spectroscopy) experiment using the Keck powered magnet operating at 25 T with field regulation1 and passive shims. Observation of the off-diagonal peaks verifies the improvement in field regulation. Also shown is a prediction of the effects of powered magnet field fluctuations on MRI. This work supports the development of a 36 T magnet scheduled for completion in 2015.

[1] M. Li, et al., Reduction of magnetic field fluctuations in powered magnets for NMR using inductive measurements and sampled-data feedback control, Journal of Magnetic Resonance. 212, 254-264 (2011).


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A COMPACT, HIGH-FIELD EPR SPECTROMETER FOR DYNAMIC NUCLEAR POLARIZATION Thorsten Maly1 1Bridge12 Technologies, Inc. 37 Loring Drive, Framingham, MA 01702 USA In recent years, Dynamic Nuclear Polarization has proven to be a robust method to increase signal intensities in NMR experiments in laboratories around the world and substantial progress has been made in adapting DNP for solid- and solution-state NMR spectroscopy. In a DNP experiment the large electron polarization is transferred to the nuclear spin reservoir by saturation of an allowed electron paramagnetic resonance (EPR) transition, providing a sensitivity boost in some cases close to the theoretical maximum of 660 (for 1H) [1, 2].

The efficiency of the DNP process depends on many different factors such as the strength of the magnetic field at which the experiment is performed, the strength of the microwave induced magnetic field (ω1e) the electron T1e and T2e relaxation times, or the strength of the electron dipolar coupling with respect to the Larmor frequency in the case of biradicals to just name a few. The shape or breadth of the EPR spectrum (Δ) is especially important since it determines the active DNP mechanism. Therefore, to understand and optimize the DNP process it is crucial to understand the EPR spectrum and relaxation properties of the polarizing agent at the same magnetic field at which the DNP experiment is performed.

However, the number of spectrometers operating at frequencies at which DNP is currently performed (300 to > 800 MHz 1H NMR, 197 to 526 GHz EPR) is very limited and researchers often have to extrapolate sample parameters from low-field measurements, or speculate about the EPR characteristics of their DNP sample. Currently commercial high-field EPR spectrometers are available at 95 and 263 GHz, which are optimized for EPR spectroscopy and require multi-million dollar investments.

We have developed a compact EPR spectrometer, which can easily be integrated into existing DNP infrastructure. Currently the device is operating at a frequency of 263 GHz, but can easily be adapted to cover the complete frequency range that is currently used in DNP spectroscopy.

Here we will present the design and performance characterization of our high-field EPR spectrometer. The instrument is based on a 263 GHz solid-state microwave source, with a maximum output power of 60 mW and a quasi-optical duplexer, allowing reflection and induction mode detection. In Figure 1, a preliminary EPR spectrum recorded at 263 GHz is shown. The two spectra are recorded in reflection mode by either sweeping the magnetic field (blue) or sweeping the frequency (red) and the two spectra are almost identical.

1. Maly, T., et al., Dynamic nuclear polarization at high magnetic fields. J. Chem. Phys., 2008. 128(5): p. 052211-19.

2. Su, Y., L. Andreas, and R.G. Griffin, Magic Angle Spinning NMR of Proteins: High-Frequency Dynamic Nuclear Polarization and H Detection. Annu Rev Biochem, 2015.

Figure 1: 263 GHz EPR spectra of BDPA in polystyrene, blue: field sweep, red: frequency sweep.


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Imaging Magnetization Exchange With CEST and CERT Daniel F. Gochberg1 1Vanderbilt University Medical Center, Department of Radiology and Radiological Sciences, Nashville, TN 37232 Magnetization exchange affects most MRI’s. However, methods with specific molecular sensitivities have the potential to be particularly useful. In this talk, we review our recent work in such methods, including chemical exchange saturation transfer (CEST) and chemical exchange rotation transfer (CERT) imaging.


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PHASE CONTRAST MRI WITH REDUCED EDDY CURRENT DISTORTION

Magdoom Kulam, Malisa Sarntinoranont, Thomas H. Mareci

1University of Florida, Department of Mechanical and Aerospace Engineering 1University of Florida, Department of Biochemistry and Molecular Biology Introduction Phase contrast (PC) MRI can capture fluid flow in real-time without the use of a contrast agent. A bipolar gradient is used to impart a velocity dependent phase on the flowing spins. Ideally the bipolar gradient has no effect on the phase of stationary spins, however eddy currents associated with the switched gradients cause non-zero phase errors in the measured velocity [1]. This effect is particularly pronounced while encoding very slow flows, which require high gradient strengths and/or duration to increase the velocity sensitivity. Retrospective eddy current correction can estimate the phase error after the scan by fitting a first or second order polynomial in a static region of interest (ROI), which is then subtracted from the entire velocity map [1]. However this method has several drawbacks: 1) requires a static ROI be present in the image which can be difficult to locate while encoding very slow flows in-vivo (perfusion effect), and 2) needs to be performed on each slice individually in a static ROI. In this study, we introduce a prospective method to reduce eddy current distortions by changing the shape of the flow encoding bipolar gradient from the usual trapezoid to shifted cosine. The new gradient shape is smoother than the trapezoid, which greatly reduced the eddy current artifacts in the scan itself. The new technique expands the application of PC-MRI for the measurement of very slow flows, which could provide a valuable tool to study biology in-vivo. Experiment MR experiments were performed on an Agilent 4.7 T magnet in the AMRIS Facility of the NHMFL. The 3D velocity field in a rat brain in vivo was obtained using a Hadamard scheme [2] at a velocity encode value of 12.23 mm/s with trapezoidal (Gbipolar = 60 mT/m and tbipolar = 4 ms) and shifted cosine gradients (Gbipolar = 120 mT/m and tbipolar = 4 ms). The shifted cosine gradient strength was doubled to equate its first moment to that of a trapezoid. Results and Discussion The in vivo rat brain z-velocity maps, obtained with trapezoidal and shifted cosine gradients are given in Figure 1. The flow map from trapezoidal gradient clearly shows the B0 eddy current error as constant non-zero velocity (orange-red color) throughout the entire image. The shifted cosine waveform almost eliminates this error, as shown by the zero velocity (green) as expected.

Acknowledgements A portion of this work was performed at the National High Magnetic Field Laboratory (which is supported by National Science Foundation Cooperative Agreement DMR-1157490, the State of Florida, and the U.S. Department of Energy) within the AMRIS Facility at the McKnight Brain Institute of the University of Florida. In addition, this work is supported in part by the NIH/NCATS Clinical and Translational Science Award UL1 TR000064.

References: [1] Bernstein, et al. Handbook of MRI pulse sequences. Elsevier, 2004. [2] Haacke, et.al. MRI: physical principles and sequence design. pp 731-732. New York: Wiley-Liss, 1999.

Solid-state NMR Studies of Energy Storage Materials

Figure 1: In-vivo flow in rat brain with Venc= 12.23 mm/s using trapezoidal and shifted cosine flow gradients, after zero filling the raw data and phase unwrapping: (Top row) Normalized z-component of velocity. (Bottom row) Flow encoding gradient shapes


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Yan-Yan Hu1,2, Po-Hsiu Chien1, Xiang Li1, Ivan Hung2, Zhehong Gan2 1Florida State University, Department of Chemistry & Biochemistry, Tallahassee, FL 32306 2National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310 USA Multinuclear solid-state NMR was employed to investigate energy storage materials, including rechargeable lithium and sodium ion batteries and solid oxide fuel cells. 6,7Li and 23Na NMR of electrode materials at various states of charge track changes in local structural environment and dynamics of the charge carriers and reveal reaction mechanisms associated with electrochemical processes. 1H and 13C NMR address issues with rate performance and degradation of batteries due to the formation of solid-electrolyte interphase and the participation of H+ in the charge/discharge reactions. In situ 7Li and 23Na NMR were also applied to follow structural changes in real time. 17O and 23Na NMR carried out in high magnetic fields on a group of superior solid-oxide ion conductors, Sr3-3xNa3xSi3O9-1.5x, provide explanation on how content of doped-element helps improve ion conductivities at intermediate temperatures. These solid-state NMR studies provide critical information on structural disorder and ion dynamics, which is closely related to the performance of technologically important materials. [1] Yang Sun, ;Liang Zhao,; Huilin Pan,; et. al., Nat Comun. 4, 1870-1879 (2013).

[2] P. Singh and J. B. Goodenough, J. Am. Chem. Soc., 135, 10149-10154 (2013).


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A Detailed Spectroscopic and Theoretical Investigation of High-Spin Square-Planar Fe2+ and Co2+ Complexes

Sebastian A. Stoian1, Matias E Pascualini2, Andrew Ozarowski1, Adam S. Veige1 1National High Magnetic Field Laboratory, Tallahassee, FL, 32310, USA; 2University of Florida, Department of Chemistry, Gainesville, FL, 32611, USA High-spin, square planar molecular compounds are rare. In an effort to describe the electronic structure of such unusual complexes we have completed a combined High-Frequency EPR and Nuclear Gamma Resonance Spectroscopic characterization of a series of M2+ (where M = Co, Fe) complexes supported by a trianionic ONO3- pincer-type ligand. The pincer ligand binds tightly to the metal ions just like a crab grips its prey. Our investigation revealed the presence for the Co(II) complex of a ground electronic state that consists of a highly anisotropic Kramers doublet that originates from a quartet spin state with a very large zero-field splitting. This observation demonstrates that the orbital angular momentum is essentially unquenched along a spatial direction. The observed spectroscopic behavior is rationalized on the basis of a combined Density Functional Theory (DFT) and Crystal Field (CF) study. The investigation of the Fe(II) complex indicated the presence of an excess of electron density along the normal to the square formed by the four near-neighbors of the iron (purple region in figure below). Although such an electron density distribution at the iron site typically translates into a large electric field gradient (EFG) at the nucleus, the big surprise was the discovery of essentially no EFG. Our parallel DFT study revealed that the large EFG generated by the iron electrons is essentially quenched by that generated by the four neighbors. This investigation also established that the quintet spin ground state of the iron complex is characterized by a large and positive ZFS parameter D.1

[1] M. E. Pascualini; N. V. Di Russo; A. E. Thuijs; A. Ozarowski, S. A. Stoian; K. A. Abboud; G. Christou; A. S. Veige, Chem. Sci., 2015, 6, 608-612


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Magnetic Interactions in Narrow-Line Trityl Biradicals M. K. Bowman1, H. Chen1, N. P. Isaev2, R. I. Samoilova2, A. G. Maryasov2, O. Y. Rogozhnikova3, V. M. Tormyshev3

1The University of Alabama, Department of Chemistry, Tuscaloosa, AL USA; 2Institute of Chemical Kinetics and Combustion, Novosibirsk, Russia; 3Novosibirsk Institute of Organic Chemistry, Novosibirsk, Russia; Our recent development of high-yield synthetic routes to asymmetric triaryl methyl radicals or trityls, such as 1 and 2, allows us to synthesize a variety of biradicals such as 3 and 4. These biradicals have relatively narrow spectra with barely resolvable Zero-Field Splittings from dipolar interactions of ~19 MHz but singlet-triplet splittings corresponding to ~15-20 K. The electron spin-lattice relaxation of frozen solutions of 3 and 4 have a Raman process remarkably similar to that in trityl monoradicals such as OX063 and Finland trityl, and an Orbach-Aminov process seen in concentrated solutions of trityl monoradicals. Rapid cross relaxation of isolated monoradicals with dimers and larger clusters of trityl radicals that spontaneously assemble in solutions of trityl monoradicals at the concentrations used for DNP allows the dimers and aggregates to set the electron spin dynamics and dominate the DNP process.

SS

S SS

SS

SS

SS

SS HN NH

S S

SS

SS

SSO O

SS

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4 This work was supported by the National Science Foundation, Chemistry Division (award No. 1416238) (MKB) and the Russian Foundation for Basic Research (grant No. 14-03-93180) (VMT).


27

PARAHYDROGEN INDUCED POLARIZATION BY PAIRWISE REPLACEMENT CATALYSIS OVER METAL NANOPARTICLES AND CERIA NANOSHAPES

Clifford R Bowers,1 Evan W Zhao,1 Haibin Zheng,2 Ronghui Zhou1 & Helena E Hagelin-Weaver2

1Department of Chemistry, University of Florida, Gainesville, FL, 32611 USA 2 Department of Chemical Engineering, University of Florida, Gainesville, FL, 32611 USA

Recent breakthroughs in hyperpolarization methodologies are rapidly expanding the frontiers of NMR spectroscopy into fields like catalysis, nanomaterials, and biomedical imaging. Parahydrogen Induced Polarization [1-3] using supported metals or pure oxide catalysts [4-6] (hetPHIP) offers a robust, inexpensive and scalable hyperpolarization technique for rapid production of bulk quantities of hyperpolarized liquids and gases. Unique advantages of hetPHIP are the ease of separation of the hyperpolarized product from the polarizing agent and compatibility with a continuous-flow production mode. However, challenges that need to be addressed in hetPHIP are the modest signal enhancement, owing to the low pairwise selectivity, and limited generality. Here we demonstrate a new concept in catalysis which we refer to as pairwise replacement (PR) catalysis. In PR-hetPHIP, two protons of a substrate are replaced with two protons originating from the same parahydrogen molecule. We observe intense PHIP NMR signals simply by flowing a mixture of p-H2 and substrate over the solid catalyst by incorporation of scalar symmetrization order without a change in substrate molecular structure. Understanding the mechanism underpinning this PR-PHIP effect is critical to its further generalization and optimization [6]. Finally, we report the results hetPHIP studies using CeO2 catalysts [7]. Ceria nanoparticles exposing (110) or (100) and (110) were synthesized by a hydrothermal method. We present the facet dependence of the pairwise addition and pairwise replacement on the alkene and alkyne. PHIP NMR has proven to be a powerful new tool for the study of the surface mechanism of hydrogenation catalysis.

Acknowledgements: This project is supported by ACS-PRF #52258-ND5 and NSF CHE-1507230. References [1] Bowers, C.R. and Weitekamp, D.P., Phys Rev Lett 57, 2645-2648 (1986). [2] Bowers, C.R. and Weitekamp, D.P., J Am Chem Soc 5541-5542 109 (1987). [3] Bowers, C.R., Sensitivity Enhancement Utilizing Parahydrogen, eMagRes 2007. [4] Koptyug, I.V. et al., J Am Chem Soc 2007, 129: 5580-5586. [5] Koptyug, I.V. et al., Chem Commun 2014, 50:875-878. [6] Zhou, R.H. et al., J Am Chem Soc 2015, 137: 1938-1946. [7] Zhao, E.W. et al, Angew. Chem. Int. Ed. 2015 DOI: 10.1002/anie.201506045.


28

PUSHING THE LIMITS OF MAGNETIC ANISOTROPY IN MONONUCLEAR Ni(II) SINGLE – MOLECULE MAGNET: A HIGH-FIELD EPR STUDY

Lakshmi Bhaskaran1, Katie Marriott2, Mark Murrie2 and Stephen Hill1

1Department of Physics and National High Magnetic Field Laboratory, Florida State University Tallahassee, FL 32310, USA; 2WestCHEM, School of Chemistry, University of Glasgow, Glasgow, G12 8QQ,UK

Single-Molecule Magnets (SMMs) are potential candidates for nanoscale magnetic information storage, where slow magnetization dynamics (bistability) is realized at low temperatures due to a magnetic anisotropy barrier separating the “spin-up” and “spin-down” states of the SMMs. Here, we report spectroscopic evidence for a huge easy-axis anisotropy in a trigonal bipyramidal (TBP) [Ni(MDABCO)2Cl3] complex with an orbitally degenerate ground state. Single-crystal EPR studies were carried out in the 35T resistive magnet at NHMFL. A very strong angle-dependence of the spectrum was observed within a few degrees of the hard plane, suggesting a huge zero-field-splitting (zfs) parameter, |D| > 400 cm-1, associated with first order spin-orbit coupling. This value is considerably larger than previously reported for a NiII TBP complex [1], and is thought to be due to the rigidity of the ligand that prevents Jahn-Teller type effects that can reduce D [2]. This is confirmed by the small value of the rhombic parameter, E ≤ 0.18 cm-1.

[1] R. Ruamps et al., JACS, 135, 3017-3026 (2013)

[2] M. Gruden-Pavlovic et al., Chem. Sci., 5, 1453-1462 (2014)


29

STUDIES OF THE COORDINATION ENVIRONMENT OF COPPER WATER OXIDATION CATALYSTS BEFORE ELECTROCATALYSIS AND THE PATHWAYS OPEN DURING ELECTROLYSIS Deidra L. Gerlach1, Salome Bhagan1, Alex A. Cruce1, Michael K. Bowman1, Shanlin Pan1, Elizabeth T. Papish1. 1The University of Alabama, Department of Chemistry, Tuscaloosa, AL, 35487 USA. Water oxidation can lead to a sustainable source of energy, but for water oxidation catalysts to be economical they must use earth abundant metals. We recently published in Inorganic Chemistry (2014)[1], 2:1 6,6’-dihydroxybipyridine (6,6’-dhbp) : copper complexes that are capable of electrocatalytic water oxidation in aqueous base (pH = 10−14). Two crystal structures of the complex that contains 6,6’-dhbp and copper(II) in a ratio of 2:1 are presented at different protonation states. The thermodynamic acid dissociation constants were measured for this complex, which show that the complex is fully deprotonated above pH = 8.3 (i.e., under water oxidation conditions). CW-EPR, ENDOR, and HYSCORE spectroscopy confirmed that the 6,6′-dhbp ligand is bound to the copper ion over a wide pH range before a potential is applied which shows how pH influences precatalyst structure. A zinc complex of 6,6’-dhbp was synthesized for catalytic and structural comparisons; as well as water oxidation studies at pH = 12.6 have illustrated that both copper and proximal OH groups are necessary for water oxidation at a low overpotential. Our most active catalyst, 2:1 6,6’-dhbp:Cu2+, was found to have an overpotential of 477 mV for water oxidation at a moderate rate of kcat = 0.356 s−1 with a competing irreversible oxidation event at a rate of 1.082 s−1. Additional copper(II) complexes were synthesized from the ligands 6,6’-dhbp and 3,5-di(2-pyridyl)pyrazole (bpp) to contain a short Cu---Cu distance to facilitate O-O bond formation during water oxidation. Ligand rearrangement of these complexes in aqueous solutions was investigated using 1H-NMR and CW-EPR and is a complication of these bimetallic complexes which alter the overpotential of water oxidation. Our combined work supports previous observations in which OH/O− groups on the bipyridine rings can hydrogen bond with metal bound substrate, support unusual binding modes, and potentially facilitate proton coupled electron transfer. These findings and further insight into the reactivity of copper : 6,6’-dhbp complexes for water oxidation are presented. [1] D. L. Gerlach, S. Bhagan, A. A. Cruce, D. B. Burks, I. Nieto, H. T. Truong, S. P. Kelley, C. J. Herbst-Gervasoni, K. L. Jernigan, M. K. Bowman, S. Pan, M. Zeller, and E. T. Papish, Inorg. Chem. 53, 12689-12698 (2014).


30

BDPA-DOPED POLYSTYRENE BEADS AS POLARIZATION AGENTS FOR DNP-NMR

Yunzhi Zhang, Phillip J. Baker, and Leah B. Casabianca

Department of Chemistry, Clemson University, Clemson, SC, 29634, USA.

Because of the low sensitivity of NMR due to its low polarization from nuclear spins, Dynamic Nuclear Polarization (DNP) can be used as a much higher sensitive replacement to traditional NMR. Dissolution DNP, in which a sample is polarized in the solid state at low temperature (~1.4K), then quickly melted and transferred to a high-field NMR spectrometer or MRI scanner for observation at room temperature [1], has become a very popular technique. Dissolution DNP has been applied to a huge range of fields like medicine, catalysis, fluid dynamics, and related fields; it also has been used to selectively enhance the signal of surface groups in microporous structures, which provides an exciting method to study the structure of surface groups on functional nanoparticles [2].

In our study, we have created HighlY-effective Polymer/Radical beads (HYPR-beads) by introducing a known DNP radical, α,γ-Bisdiphenylene-β-phenylallyl (BDPA) into polystyrene nanoparticles. HYPR-beads have potential as attractive polarization agents for polarizing bulk solvent, or for analytes dissolved in a solvent. HYPR-beads have hydrophilic surfaces, so they form a stable colloidal dispersion in water. Hyperpolarization of 13C in labeled urea could be achieved in 60:40 mixtures of DMSO-d6:H2O, but also in water alone without the addition of a glassing solvent. Our results have shown that water-soluble compounds can be polarized in water without addition of a glassing solvent at low overall BDPA concentrations. 7Li from LiCl has also been used to show that HYPR-beads could be used to polarize other water-soluble samples as well as nuclei other than 13C. Enhancement factors were in the range of 20-100. This is an excellent method by which water-soluble compounds could be polarized using BDPA, an efficient but water-insoluble polarizing agent. HYPR-beads are an economical alternative to ox-063, the current radical of choice for polarizing hydrophilic compounds. This new method may attract more researchers into the dissolution DNP field.

Fig. 1 Representative DNP enhancements. (a) Hyperpolarized 13C-urea, water only, microwave frequency: 94.060 GHz, enhancement factor = 112 (b) Hyperpolarized 7Li in LiCl, water only, microwave frequency 93.939 GHz, enhancement factor = 21.

[1] J. H. Ardenkjaer-Larsen, B. Fridlund, A. Gram, G. Hansson, L. Hansson, M. H. Lerche, R. Servin, M. Thaning, and K. Golman, Proc. Natl. Acad. Sci. USA 100, 10158-10163 (2003). [2] A. J. Rossini, A. Zagdun, M. Lelli, A. Lesage, C. Copéret, and L. Emsley Accounts Chem. Res. 46, 1942-1951 (2013).

157158159160161162ppm

157158159160161162ppm

-15-14-13-12-11-10-9ppm

ThermalDNP

(a) (b)

EF = 112EF = 21

(c)

EF = 5


31

OPTICALLY DETECTED EPR SPECTRA OF POLYFLUOROBIPHENYL RADICAL ANIONS Mariya Vyushkova, Ian Carmichael Notre Dame Radiation Laboratory, University of Notre Dame, Notre Dame, IN 46556 USA Radical anions (RA’s) of fluorinated aromatics are important reaction intermediates for synthetic and industrial applications with unusual electronic structure and geometry. Lowering of σ* molecular orbital energy level with respect to π-orbital (known as perfluoro-effect) results in pseudo-Jahn-Teller vibronic coupling of the ground π-state with low-lying σ* excited states, which gives rise to out-of-plane distortions of the aromatic rings in these radical anions. This effect is known to cause anomalously large 19F hfc’s observed, e.g., in EPR spectra of fluorinated benzene RA’s. Because of their high reactivity and large anisotropic hfc’s, EPR study of fluoroarene RA’s is experimentally challenging. However, they are ideal objects for the Optically Detected EPR (OD EPR) technique, which is extremely sensitive and selective to radical ions. In present work, we employed this technique to study several polyfluorobiphenyl RA’s and determine whether they retain the structural peculiarities of fluorobenzene RA’s. Quantum chemical calculations were performed to support the assignment of experimental spectra.

Radical anion Experimental hfc constants, Gauss Computed(a) hfc constants, Gauss

Decafluoro

2,3,4,5,6-pentafluoro

4,4’-octafluoro

4,4’-dimethyl-octafluoro

(a) Computational method IEFPCM-B3LYP/epr-ii // IEFPCM-B3LYP/6-311+G(d,p), (solvent is n-dodecane) The results show that in polyfluorobiphenyl RA’s para-fluorines are specifically responsible for non-planar phenyl ring geometries. 4,4’-octafluoro RA’s have planar phenyl ring structure with small 19F hfc’s; at the same time, RA’s with para-fluorines have nonplanar ring geometry with large hfc constants. In all cases, para-fluorines show the largest hfc constant and angle between C-F bond and the ring plane. However, the 4,4’-difluorobiphenyl radical anion is known to have small fluorine hfc’s [2], we compute a(19F) = 2.8 G. Therefore, in contrast to RAs of the fluorobenzene family of compounds, fluorobiphenyl RAs show out-of-plane distortions of phenyl rings only at high degrees of fluorination, and only if fluorines in para-positions are present. [1] M. M. Vyushkova, V. P Vysotsky, I.V. Beregovaya, L.N. Shchegoleva, V.A. Bagryansky, and Yu. N. Molin, Mendeleev Commun., 16, 151-152 (2006).

[2] A.L. Allred and L.W. Bush, Tetrahedron, 24, 6883-6887 (1968).

[1]


32

TERAHERTZ-FREQUENCY EPR

J. Krzystek1 1National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310 USA. The recent developments in the field of molecular magnetism, namely discovery of mononuclear single-molecule magnets (as opposed to polynuclear clusters) have necessitated a quantitative determination of the zero-field splitting (zfs) as the main physical property underlying their magnetic behavior. The zfs of the ground spin state in high-spin coordination complexes of such ions as Co(II) [1], Fe(II) [2], Ni(II) [3] or Re(IV) [4] can reach 100 cm–1 and beyond. As such, it is no longer amenable to measurement by high-frequency and -field EPR (HFEPR), at least not in the usual range of frequencies/fields currently available (up to 1 THz / 35 T). This work will present first results of the extension of HFPR into the terahertz frequency range obtained with the use of either a free electron laser (FEL) or far infrared magnetic spectroscopy (FIRMS). [1] E. Colacio, J. Ruiz, E. Ruiz, E. Cremades, J. Krzystek, S. Carretta, J. Cano, T. Guidi, W. Wernsdorfer, and E. K. Brechin, Angew. Chem. Int. Ed., 52, 9130-9134 (2013).

[2] W. H. Harman, T. D. Harris, D. E. Freedman, H. Fong, A. Chang, J. D. Rinehart, A. Ozarowski, M. T. Sougrati, F. Grandjean, G. J. Long, J. R. Long, and C. J. Chang, J. Am. Chem. Soc., 132, 18115-18126 (2010).

[3] R. Ruamps., R. Maurice, L. Batchelor, M. Boggio-Pasqua, R. Guillot, A.-L. Barra, J. Liu, E.-E. Bendeif, S. Pillet, S. Hill, T. Mallah, and N. Guihery, J. Am. Chem. Soc., 135, 3017-3026 (2013)

[4] J. Martinez-Lillo, T. F. Mastropietro, E. Lhotel, C. Paulsen, J. Cano, G. De Munno, J. Faus, F. Lloret, S. Nellutla, and J. Krzystek, J. Am. Chem. Soc., 135, 13737-13748 (2013).

44th Southeastern Magnetic Resonance Conference Oral Presentations Magnetic Resonance & Macromolecules

33

1H DYNAMIC NUCLEAR POLARIZATION BASED ON AN ENDOGENOUS FLAVIN RADICAL

Anne-Frances Miller

Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge MA, U.S.A. and Department of Chemistry, University of Kentucky, Lexington KY, U.S.A.

While maximal dynamic nuclear polarization (DNP) signal enhancement may be obtained by use of polarizing agents chosen for their EPR properties, maximum information content may be better served by exploiting endogenous radicals which occupy a defined position in protein targets and which may have spin properties that are related to the protein's functional state. Therefore there are considerable incentives to explore DNP based on naturally occurring endogenous radicals or paramagnetic ions. In this work we demonstrate a 15-fold enhancement of solid-state NMR signals via DNP based on the stable, naturally occurring flavin mononucleotide (FMN) semiquinone of flavodoxin.1 At a field strength of 5T the linewidth of flavodoxin's EPR signal suggests that the dominant DNP mechanism is the solid effect, however the field-dependent DNP enhancement profile suggests that the Overhauser effect also contributes. The magnitude of the enhancement as well as the bulk-polarization build-up time constant (tB) with which it develops are dependent on the isotopic composition of the protein. Deuteration of the protein to 85 % increased the nuclear longitudinal relaxation time T1n and tB by factors of five and seven, respectively. Slowed dissipation of polarization can explain the two-fold higher maximal enhancement than that obtained in proteated protein, based on the endogenous semiquinone. In contrast, the long tB of TOTAPOL-based DNP in non-glassy samples was not accompanied by a similarly important long T1n, and in this case the enhancement was greatly reduced. The low concentrations of radicals occurring naturally in biological systems limit the magnitude of DNP enhancement that is attainable by this means. However, our enhancement factors of up to 15 can nonetheless make an important difference to the feasibility of applying solid-state NMR to biochemical systems. We speculate that DNP based on endogenous radicals may facilitate MAS NMR characterization of biochemical complexes and even organelles, and could also serve as a source of additional structural and physiological information.

1 Maly, T.; Cui, D.; Griffin, R. G.; Miller, A.-F. J. Phys. Chem. B 2012, J. Phys. Chem. B 116: 7055-7065.


34

Understanding Movement and Mechanism in Membrane Proteins: An EPR Spectroscopist’s View

Fraser MacMillan Henry Wellcome Unit for Biological EPR, School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, UK.

Research in the Henry Wellcome Unit for Biological EPR at UEA focuses on the architecture and functional dynamics of membrane proteins, many medically relevant with a special interest on membrane transport systems and their interaction with intra-‐ cellular signalling pathways. There is increasing evidence that membrane proteins do not act alone, but that they are organised as nano-‐machineries which function through the concerted action of its individual components with high precision and specificity observed in both time and space. We are seeking to unravel the principles underlying the architecture and dynamics of these protein nano-‐machineries as well as their function and regulation. Our experimental approach focuses on the use of Electron Paramagnetic Resonance (EPR) techniques in combination with molecular biological, biochemical and other biophysical methods including theoretical MD approaches. Our expertise lies in the development and application of novel EPR techniques to address these key questions. In this presentation I will focus on recent method developments, which may allow a shifting of the focus of this technique away from being considered purely a niche technique towards a more universal structural biological tool. I will use examples from our recent work on membrane and metallo-proteins (cytochrome c oxidase & nitrate reductase), multidrug efflux pumps (P-‐glycoprotein) and bacterial pathogens (PsaA in Streptococcus pneumonia) to demonstrate the power of this technique to deliver key mechanistic insight into e.g. how to resolve multiple distances in complex macromolecular complexes, how to observe conformational change within membrane proteins at a molecular level and finally to identify the molecular determinants of metal binding by PsaA and the potential implications for host-‐pathogen interactions. This research is funded by The Royal Society and the Wellcome Trust as well as being embedded within the current EU COST Action CM1306 “Understanding Movement and Mechanism in Molecular Machines”.


35

Protein Dynamics by ssNMR: Gating Motions in M2 Proton Channel of Influenza A Joana Paulino1,2, Timothy A. Cross1,2,3 1National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310 USA; 2Florida State University, Institute of Molecular Biophysics, Tallahassee, FL 32306; 3Florida State University, Department of Chemistry, Tallahassee, FL 32306 The M2 proton channel is essential for the Influenza A life cycle [1]. M2 proton permeation has been shown to be dependent on the His37-Trp41 cluster, where Trp41 is the primary gate [2]. Proton conductance directionality is lost upon Trp41 mutation. The antiviral drug amantadine (AMT) used to block the M2 channel prior to the recent S31N mutation. Val27 is considered a secondary gate for M2 and essential for AMT inhibition in the wild type protein [3]. Based on the abundant structural information for M2 [2, 4-7] we have characterized backbone and side-chain motions for Val27 and Trp41, in constructs of the transmembrane domain (M2TM, residues 22-46), of the conductance domain (M2CD, 22-62) and of the full length M2 protein (M2FL, 1-97) reconstituted in lipid bilayers. We have also characterized the global rotational diffusion rate (Dr) of M2TM using 2H ssNMR (Dr ~ 105 rad2/s). The side-chain of Val27 undergoes a restricted two-site jump motion about the Cα-Cβ bond, with unequal populations. Interestingly, M2TM and M2CD have very similar dynamics for the side-chain of Val27 as indicated by the 2H powder spectra of Val27 site labeled in both constructs in Figure 1. The spectrum of 15NεTrp41 in unoriented sample of M2TM when interpreted in light of current structural information indicates that the Nε-H of Trp41 is oriented near the magic angle and the fast global rotation motion of the channel leads to a dramatically collapsed powder pattern (Figure 1B). The separated local field spectrum of aligned M2TM in which the 15Nε 1H-15N dipolar coupling of Trp41 is near 0 kHz is also indicative of an orientation near the magic angle. Currently, the effects of AMT binding and pH activation are being addressed. The addition of AMT induced line broadening and a significant increase in T2 (T2apo = 27 ± 7 µs T2drug = 43 ± 3 µs) for the Val27 side chain in M2TM wild type. The spectra of the same site in the mutant protein (M2TM_S31N-d8Val27) showed no changes upon addition of AMT. [1] Y. Sidorenko, and U. Reichl, Biotechnology and Bioengineering, 88, 1-14 (2004). [2] M. Sharma, et al., Science, 330, 509-512 (2010). [3] M. Yi, T. A. Cross, and H. X. Zhou, The Journal of Physical Chemistry B, 112, 7977-7979 (2008). [4] J. Hu, et al., Biophysical Journal, 92, 4335-4343 (2007). [5] C. Li, et al. BBActa, 1768, 3162-3170 (2007). [6] S. D. Cady, T. V. Mishanina, and M. Hong, Journal of Molecular Biology, 385, 1127–1141 (2009). [7] L. B. Andreas, et al., Biochemistry, 52, 2774-2782 (2013).

Figure 1. A) 2H ssNMR spectra of M2TM_d8Val27 in red and M2CD_V28I_d8Val in black in DOPC:DOPE pH 7.5. Spectra were acquired with a quadrupole echo and 10 µs

A B


36

Near complete assignments of the 237-residue Rous Sarcoma Virus capsid protein in its tubular assembly

Bo Chen1, Jaekyun Jeon1, Xin Qiao1, Peter Gor’kov, Zhehong Gan2, and Ivan Hung2

1Department of Physics, University of Central Florida, Orlando, FL 32828

2National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310

1Institution/Affiliation and Department ( Arial, Font size 10, italicized)

The genome materials of the Rous Sarcoma Virus are enclosed by a polyhedral shell assembled from hundreds of the 237-residue Rous Sarcoma Virus capsid proteins. In vitro, the Rous Sarcoma Virus capsid proteins can self-assemble into polymorphic tubes that share similar underlying structure as the in vivo capsid. Here we present the structure and dynamics of the Rous Sarcoma Virus capsid protein in its tubular assembly derived from solid state NMR assignments. By utilizing selective isotope labeling and high field measurements, we sequentially assigned 230-residue out of the 237-residue protein. Our results resolve the structural and dynamic adjustment of the protein upon self-assembly at a site-specific resolution and provides critical insights into the assembly mechanism. Reference: [1] Shen, Y. and A. Bax, Protein structural information derived from NMR chemical shift with the neural network program TALOS-N. Methods in molecular biology (Clifton, N.J.), 2015) 1260: p. 17-32


37

ASSIGNMENT OF SPARSE 13C ALANINE METHYL RESONANCES IN HSP90 PROTEINS USING RDCS, PCSS, AND NOES

Kari Pederson1, Laura C. Morris1, Daniel Elnatan2, Laura A. Lavery2, David A. Agard2, and James H. Prestegard1

1Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602

2The Howard Hughes Medical Institute, University of California, San Francisco, CA 94158

Hsp90 proteins play an important role in protein homeostasis as molecular chaperones involved in protein folding. They are known to undergo large conformational changes upon binding to ATP and client proteins (Fig. 1), however studying these conformational changes and the interactions with clients by NMR is currently prohibited by the size of the Hsp90 proteins and limitations of current NMR resonance assignment strategies.

NMR data are being increasingly collected on large systems that press resolution limits when uniform isotopic labeling is used. Using a single isotopically labeled amino acid reduces the number of cross peaks and improves resolution in multi-dimensional experiments. Labeling with 13C in methyl groups offers additional advantages from methyl TROSY effects. However, with single amino acid labeling, the triple resonance experiments dependent on uniform labeling cannot be used for resonance assignments. In systems for which there exists a structure (whether X-ray, NMR, or theoretically derived) it is possible to use RDCs to assist with assignment of resonances. We have developed a J-modulation-methyl TROSY experiment to collect methyl 1H-13C RDCs and we illustrate here the feasibility of using such data for resonance assignment.

Calculating and scoring RDCs for all possible permutations of assignments provides a straightforward but computationally intensive approach. Alternatively, calculating RDCs using a genetic algorithm to search assignment space, finding the best alignment tensor for the set of assignments and comparing the experimental RDCs to those calculated from the determined alignment tensor proves more efficient. This method scales well with increasing numbers of RDCs and allows most, if not all, of the resonances to be assigned unambiguously. In cases where degeneracy exists in RDC values it is possible to use NOEs and paramagnetic chemical shift effects to aid in assignment. The method is illustrated with the bacterial Hsp90 protein (HtpG) for which only the alanine methyl groups have been 13C labeled.

Figure 1. Hsp90 proteins are dimers which undergo large conformational changes upon binding to ATP and client proteins. However, the details and mechanisms of these conformational changes are not currently known.

?


38

CYSTEINE-SPECIFIC LABELING OF PROTEINS WITH A TOTAPOL DERIVATIVE FOR DYNAMIC NUCLEAR POLARIZATION NMR

Maxim A. Voinov,1 Daryl B. Good,2,3 Meaghan E. Ward,2,3 Sergey Milikisiyants,1 Antonin Marek,1 Marc A. Caporini,4 Melanie Rosay,4 Rachel A. Munro,2,3 Milena Ljumovic,2 Leonid S. Brown,2,3 Vladimir Ladizhansky,2,3 Alex I. Smirnov1 1Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27606-8204 USA; 2Department of Physics and 3Biophysics Interdepartmental Group, University of Guelph, 50 Stone Rd E, Guelph, ON N1G 2W1, Canada; 4Bruker Biospin Ltd, 15 Fortune Dr., Billerica, MA 01821 USA

Figure 1. Left: 15N CP-MAS NMR spectra from ASR in a D2O-based buffer, measured on a 400 MHz spectrometer with mm-wave power on and off. For proper comparison, the cross polarization contact time was the same, and set to 1.4 ms in all the experiments. Spectra were processed with a 200 Hz exponential line broadening. Right: A cartoon of a lipid bilayer with inserted heptahelical ASR labeled with a biradical biradical, ToSMTSL at a surface cysteine [1]. Dynamic Nuclear Polarization (DNP) enhances the signal in solid-state (ss) NMR of proteins by transferring polarization from electronic spins to the

nuclear spins of interest. Since all but a very few DNP ssNMR experiments with proteins are carried out in aqueous solutions at below-freezing temperatures, glass-forming solvents such as glycerol must be added to achieve a homogeneous distribution of the polarizing agents and prevent the formation of ice crystals, that is, to form a so-called glassy matrix. The necessity of using some large concentrations of glycerol (~10-20 mM) effectively reduces the amount of protein in such NMR samples. For example, in our comparative experiments with TOTAPOL, the incorporation of 60% glycerol leads to an approximately fourfold decrease in the maximally attainable protein concentration due to inefficient pelleting, thus, proportionally reducing the effective filling factor and the resultant NMR signal. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesized and characterized by EPR and DEER a new biradical, ToSMTSL (Totapol Series MethaneThiosulfonate Spin Label), which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner [1]. ToSMTSL was tested for DNP of a heptahelical transmembrane protein, Anabaena Sensory Rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two 15N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample co-suspended with ~17 mM TOTAPOL in a d8-glycerol/D2O/H2O matrix, the achievable sensitivity would be fourfold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals to proteins and lipids would broaden the applicability of DNP NMR to structural studies of proteins and membrane protein systems. [1] M. A. Voinov, D. Good, M. E. Ward, S. Milikisiyants, A. Marek, M. A. Caporini, M. Rosay, R. A. Munro, M. Ljumovic, L. S. Brown, V. Ladizhansky, and A. I. Smirnov, J. Phys. Chem. B, 119: 10180-10190 (2015).


39

CATALYSIS AT A DISTANCE: NATURE'S SNIPER FOR REMOTE SPECIFIC TRYPTOPHAN OXIDATION

Aimin Liu, Ian Davis, Kednerlin Dornevil, Teruaki Koto, Jiafeng Geng, and Fange Liu

Georgia State University / Department of Chemistry

Understanding how an enzyme specifically oxidizes a protein substrate that is much larger than the enzyme itself will illuminate the mechanism by which proteins mature through enzyme-mediated posttranslational modifications. Given the interconnectedness of posttranslational protein modification, metabolic chemistry, and diseases, the question of how enzymes preserve specificity for large protein

substrates is fundamental to enzymology. We are studying the long-range remote enzyme catalysis mechanism required for the biogenesis of a protein-derived tryptophan tryptophylquinone cofactor. Such a cofactor is the catalytic center of the matured dehydrogenase and it is formed through posttranslational modification mediated by MauG, an enzyme that employs two c-type hemes to catalyze the oxidation of a tryptophan residue and a mono-hydroxylated tryptophan residue of a precursor protein, preMADH (Figure 1). The MauG-catalyzed reaction is a three-step, six-electron oxidation process. Each step requires one equivalent of hydrogen peroxide as the oxidant. The catalytic heme centers are buried in the enzyme and the target tryptophan residues are buried in the substrate protein. Thus, the catalysis must take place through a long-range remote mechanism. We have recently shown an unusual bis-Fe(IV) intermediate of MauG [1] orchestrating the oxidative production of TTQ through a radical-based chemistry [2-4]. In this presentation, the biochemical and EPR study of the long-range remote catalysis mechanism will be discussed.

[1] X. Li, R. Fu, S. Lee, C. Krebs, V.L. Davidson, and A. Liu, Proc. Natl. Acad. Sci. USA, 105, 8597-8600 (2008).

[2] E.T. Yukl, F. Liu, J. Krzystek, S. Shin, L.M.R. Jensen, V.L. Davidson, C.M. Wilmot, and A. Liu, Proc. Natl. Acad. Sci. USA, 110, 4569-4573 (2013).

[3] J. Geng, K. Dornevil, V. L. Davidson, and A. Liu, Proc. Natl. Acad. Sci. USA, 110, 9639-9644 (2013).

[4] J. Geng, I. Davis, and A. Liu, Angew. Chem. Int. Ed., 54, 3692-3696 (2015).


40

SELECTIVE MEMBRANE DISRUPTION MECHANISM OF AN ANTI-BACTERIAL γ-

AAPEPTIDE DEFINED BY EPR SPECTROSCOPY Pavanjeet Kaur1, Yaqiong Li2, Jianfeng Cai2 and Likai Song1 1National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310; 2Department of Chemistry, University of South Florida, Tampa, FL 33620 γ-AApeptides are a new class of antibacterial peptidomimetics that are not prone to antibiotic resistance and are highly resistant to protease degradation. How γ-AApeptides interact with bacterial membranes and alter lipid assembly and properties are unclear, but such information is essential in order to understand their antimicrobial activities. Using electron paramagnetic resonance (EPR) techniques, we characterized the membrane interaction and destabilizing activities of a lipo-cyclic-γ-AApeptide. The analyses revealed that the γ-AApeptide binding increases the membrane permeability of POPC/POPG liposomes, which mimics negatively-charged bacterial membranes. Moreover, the γ-AApeptide interacts strongly with POPC/POPG liposomes, thereby inhibiting membrane fluidity and reducing solvent accessibility around the lipid head group region. Furthermore, binding of the γ-AApeptide induces significant lipid-lateral-ordering and membrane thinning. In contrast, minimal membrane property changes were observed upon the γ-AApeptide binding for liposomes mimicking mammalian cell membranes, consisting of neutral lipids and cholesterol. Our findings suggest that the γ-AApeptide interact and disrupt bacterial membranes through a “carpet-like” mechanism. The results illustrated that the intrinsic features of γ-AApeptides are important for their ability to selectively disrupt bacterial membranes, the implications of which extend to developing new antibacterial biomaterials


41

DIPOLAR-RECOUPLED LINESHAPES OF UNIAXIALLY DIFFUSING MEMBRANE PROTEINS UNDER MAS

Alexander A. Nevzorov Department of Chemistry, North Carolina State University, 2620 Yarbrough Dr, Raleigh, NC Membrane proteins possess a high degree of dynamics including uniaxial rotational diffusion relative to the normal of the fluid-like bilayer. Here we present a general method based on the Stochastic Liouville equation (SLE) [1,2]. The novelty of the approach is that the treatment of MAS and diffusion as derivative superoperators allows one to simulate the spectra at arbitrary spinning rates and diffusion coefficients from fully static to fast-motional limits. Additional important advantages of the method is that the stochastic Liouvillian superoperators are time-independent, which allows for a straightforward generalization to multidimensional experiments; moreover, the averaging over the azimuthal angle is already included in the SLE solution. The CP-PI dipolar-recoupling pulse sequence [3] has been simulated. The correlation between the bond angles and the scaling of the dipolar powder patterns due to rotational diffusion can be used for quantitative extraction of the structural and dynamic information as implemented by the “rotational alignment” method [4].

[1] Edwards, L. J.; Savostyanov, D. V.; Nevzorov, A. A.; Concistrè, M.; Pileio, G.; Kuprov, I., Journal of Magnetic Resonance 235 (2013), 121-129. [2] Nevzorov, A. A., J. Magn. Reson. 249 (2014), 9-15. [3] Paluch, P.; Pawlak, T.; Amoureux, J.-P.; Potrzebowski, M. J., Journal of Magnetic Resonance 233 (2013), 56-63. [4]Park, S. H.; Das, B. B.; De Angelis, A. A.; Scrima, M.; Opella, S. J., J. Phys. Chem. B 114 (2010), 13995-14003.

Figure 1: 2D separated local-‐field NMR spectra under the MAS conditions as obtained by the CP-‐PI pulse sequence [3] for the 1H-‐15N powder distributions simulated by using the stochastic Liouville Equation approach [1,2]. The n=1 rotary recoupling condition was set for the rf amplitudes on the proton side, and the MAS speed was set at 20 kHz. The 1H-‐15N dipolar anisotropy is recoupled in the indirect dimension as shown by a slice taken at the isotropic chemical shift value of ca. 120 ppm (spectrum shown in red).

Figure 2. A. Recoupled dipolar powder spectra simulated with D|| = 2×10

5 s-‐1 at ωr = 20 kHz MAS for the bond angles θB

as indicated. The powder patterns show

different averaging by uniaxial rotational diffusion depending on θB. B. Correlation plots between the ratio of the observed splitting and the full static splitting and the factor, |3cos2θB -‐1|/2 showing excellent correlation.

A

B


42

EPR/ENDOR/ESEEM OF IRON-SULFUR PROTEINS AND ENZYMES Joshua Telser1, George E. Cutsail III2, Matthew O. Ross2, Peter E. Doan2, Nicholas S. Lees2, Brian M. Hoffman2 1Department of Biological, Chemical and Physical Sciences, Roosevelt University, Chicago, IL 60605; 2Department of Chemistry, Northwestern University, Evanston, IL 60208 Electron paramagnetic resonance (EPR) and its derivative techniques, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, provide unique insights into the structure, coordination chemistry, and biochemical mechanism of Nature’s widely distributed iron-sulfur cluster (FeS) proteins.[1,2] These include the electron-transfer proteins (e.g., 2Fe and 4Fe ferredoxins (Fds), shown below, and Rieske protein) and enzymes, such as the hydro lyase aconitase (EC 4.2.1.3), found in humans.

Another major role of 4Fe4S proteins is in the superfamily of radical SAM (S-adenosylmethionine) enzymes, which includes an alphabet soup of enzymes, such as BioB, BtrN, HydE,F,G, IspG,H, MiaB, MoaA, QueE, RlmN, SkfB, SufA, ThiC. FeS enzymes exist that contain more exotic clusters, such as FeFe hydrogenase and nitrogenase, with its unique cofactor, FeMo-co [MoFe7S9C]. We will describe studies on FeS proteins including 2FeFds and 4FeFds; the latter with both the “normal” tetracysteinyl ligation to protein and with unusual tris(cysteinyl) ligation (with an exogenous fourth ligand, which can include cyanide ion). Nitrogenase and radical SAM enzymes and will also be discussed, specifically two member of the isoprenoid biosynthetic pathway, IspG and IspH. The use of natural abundance isotopes (1H, 31P) and isotopically labeled (2H, 13C, 15N, 57Fe) enzyme/ substrates /inhibitors will be described. An emphasis will be on 57Fe ENDOR which uniquely provides information on electronic structure and biochemical activity of FeS proteins.

[1] G. E. Cutsail III, J. Telser, and B. M. Hoffman, Biochim. Biophys. Acta (BBA) - Molecular Cell Research 1853, 1370-1394 (2015). [2] J. Meyer, J. Biol. Inorg. Chem. (JBIC) 13, 157-170 (2007).

44th Southeastern Magnetic Resonance Conference Oral Presentations Magnetic Resonance in vivo

43

IS HYPERPOLARIZED PYRUVATE USEFUL FOR IMAGING HEPATIC METABOLISM?

Karlos X. Moreno1, Eunsook Jin1, A. Dean Sherry1, Craig R. Malloy1, Jian-Xiong Wang1, Matthew E. Merritt2

1University of Texas Southwestern Medical Center, Advanced Imaging Research Center, Dallas, TX 75390 2University of Florida, Department of Biochemistry and Molecular Biology, Gainesville, FL 32608

Elevated hepatic glucose output is intimately connected to the increased circulating glucose levels associated with diabetes. Development of an imaging method that could measure hepatic gluconeogenesis (GNG) in vivo would present new opportunities for understanding the metabolic dysfunction underlying outright diabetes. The injection of hyperpolarized (HP) [1-13C]pyruvate into the perfused liver produces HP 13CO2 [1]. 13CO2 can be generated from [1-13C]pyruvate in the liver by multiple pathways including pyruvate dehydrogenase (PDH), and flux through phosphoenolpyruvate carboxykinase (PEPCK). PEPCK is the first committed step of GNG in the liver. If the generated 13CO2 can be attributed exclusively to PEPCK flux, the GNG can be imaged. Data in the perfused mouse liver and in vivo in the rat will be presented. At best, competition between flux through PDH and PEPCK must be measured to infer GNG. The authors propose that similar experiments in the fed state might instead be used to measure lipogenesis in the liver.

[1] M. Merritt, et. al., PNAS, 108 (47), 19084-19089 (2011).


44

REAL TIME ANALYSIS OF THE CEREBRAL METABOLIC SHIFT CAUSED BY COCAINE ADMINISTRATION AND WITHDRAWAL

Daniel P. Downes1, Bimala Lama1, James Collins1, Marcelo Febo2, and Joanna Long1. 1University of Florida, Department of Biochemistry and Molecular Biology, Gainesville, Florida 32611

2 University of Florida, Department of Psychiatry, Gainesville, Florida 32611 In vivo magnetic resonance spectroscopy and imaging (MRSI) is a useful technique for measuring carbon metabolism in live animals, but is often limited because of poor signal-to-noise (SNR). Dynamic nuclear polarization (DNP) is a rapidly developing technique in the field of MRS. Stable isotopically labeled metabolites are hyperpolarized prior to MR spectroscopy allowing for a substantial increase in the carbon SNR of >10,000x in vivo. This gain in carbon SNR allows cellular metabolic pathways to be observed in real time. Integration of dissolution dynamic nuclear polarization (dDNP) into MRSI studies of a chronic addiction model is important for investigating changes in neurometabolic processes. Chronic administration of cocaine has been shown to abate cerebral glucose metabolism. It is our central hypothesis that dDNP can be used to observe cocaine addiction and withdrawal in real time. We show here that acute cocaine administration suppresses the conversion of pyruvate to lactate in vivo. In contrast, withdrawal from chronic cocaine administration demonstrates the opposite metabolic shift as compared to the control. We have hyperpolarized pyruvate in custom-built cryostat at 5 T and ~1 Kelvin and performed MRSI at 4.7 T.

Figure 1. Metabolic schematic of selective labeling scheme and isotopomer products of [1-13C] pyruvate metabolism in the brain.


45

UTE AND HP 129XE MRI OF LUNG STRUCTURE-FUNCTION RELATIONSHIPS IN PEDIATRIC CYSTIC FIBROSIS Zackary I. Cleveland1,2, Robert Thomen1,3, Laura L. Walkup1, David J. Roach1, J. P. Clancy2, Jason C. Woods1,2,3 1 Center for Pulmonary Imaging Research, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave., ML-5033, Cincinnati, OH 45229 2 Division of Pulmonary Medicine, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave., ML-5033, Cincinnati, OH 45229 3 Department of Physics, Washington University, 1 Brookings Dr., St. Louis, MO 63130 MRI is increasingly used to quantify regional lung structure and function in a variety of pulmonary diseases. In particular, hyperpolarized (HP) 129Xe can provide measures of lung microstructure (1), gas exchange (2), and ventilation (3) in human subjects, and ultra-short echo-time (UTE) imaging provides structural information with resolution now approaching that of x-ray computed tomography (CT). However, pediatric applications of these techniques remain relatively rare. Here we demonstrate that diagnostic quality images can be obtained using both techniques in children with cystic fibrosis and that HP 129Xe ventilation MRI is a more sensitive measure of impaired lung function than conventional pulmonary function tests (e.g., forced expiratory volume in one second, FEV1). Further we demonstrate that combining these two imaging approaches yields deeper insights into the pathophysiology of lung disease. Together these results indicate that MRI can provide clinically meaningful measures of pulmonary structure and function, even in very your pediatric subjects.

References: [1] S.S. Kaushik, Z.I. Cleveland, G.P. Cofer, et al. Magn. Reson. Med. 65(4):1154–1165 (2011). [2] Z.I. Cleveland, G.P. Cofer, G. Metz, et al. Plos One. 5(8):e12192 (2010). [3] R.S. Virgincar, Z.I. Cleveland, S.S. Kaushik, et al. NMR Biomed. 26(4):424–435 (2013).

Figure: A. UTE and HP 129Xe ventilation MRI in two pediatric CF patients, ages 11 and 14 yrs (top and bottom, respectively). In some areas (red arrows), ventilation defects correlate with permanent, CF-induced airway remodeling seen in UTE images. In others (blue arrows), defects occur in the absence of obvious structural remodeling, indicating impaired lung function could likely be reversed with next-generation therapies. B. Cross-sectional comparison of FEV1 and HP 129Xe ventilation defect percent (VDP) in CF patients (7−15 yrs old) and healthy, age-matched control subjects. Conventional FEV1 cannot discriminate between groups, whereas 129Xe VDP readily discriminates healthy, age-matched controls from CF patients.


46

PRECLINICAL FUNCTIONAL MR IMAGING STUDIES TO IMPROVE DIAGNOSIS AND THERAPY RESPONSE IN EWING SARCOMA Gary Martinez, Parastou Foroutan, Christopher L Cubitt, Jillaina L Menth, Olya Grove, Marilyn M Bui, Allison S. Cohen, Mark C. Lloyd, Jongphil Kim, Damon Reed, Daniel Sullivan, Robert J Gillies, David L Morse H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612 Ewing sarcoma (ES) is a malignant bone disease that is the second most common in young people. The 5-year survival for distant stage disease is low (10-20%) and very little progress has been made in the past few decades. In addition to the development of better therapeutics, there exists a need for improved diagnostic support that can assist in making medical decisions. We explore both avenues: a test of improved chemotherapeutic drug combinations and examination of functional MR imaging, and improved image texture analysis. To achieve this, we have evaluated dasatinib and triciribine, alone and in combination, in ES mouse xenografts.

We have performed this study in 24 male mice injected with flank tumors. Groups consisted of control, dasatinib, triciribine, and combination, where chemotherapy was administered daily. MR images (T2-weighted and diffusion weighted) were performed at baseline, and at days 3, 7, 10, and 14 using an Agilent 7 T horizontal bore scanner. At the conclusion of the experiment, tumor sections were subjected to immunohistochemical Cleaved Caspase 3 (CC3) analysis as a marker apoptotic of cell death.

Tumor growth inhibition was observed at day 10 for dasatinib and combination groups relative to triciribine and control groups. However, differences for the dasatinib and combination groups were observed earlier, by day 3, but differences diminished by day 14. T2-weighted image heterogeneity, volume, and CC3 activity correlated with mitotic figures in dasatinib and combination groups. These results demonstrate the promising therapies in ES.


47

SURFACE-COMPLEXION SUPERPARAMAGNETIC IRON OXIDE NANORIBBONS WITH ENHANCED RELAXIVITY AS T2 CONTRAST AGENT IN MRI

Bibek Thapa1, 2, 3, Daysi Diaz-Diestra1, 2, 3, Juan Beltran-Huarac2, 3, Huadong Zeng4, Brad R. Weiner1, 2, 3 and Gerardo Morell1, 2, 3

1Institute for Functional Nanomaterials, University of Puerto Rico, San Juan, PR 00936, USA 2Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926, USA 3Department of Physics and Department of Chemistry, University of Puerto Rico, San Juan, PR 00931, USA 4Advanced Magnetic Resonance Imaging and Spectroscopy Facilities, University of Florida, Gainesville, FL 32611, USA Over the past few decades, surface complexion/functionalization of nanostructures has been performed as a strategy to attain the desired response for theranostic applications. Here, a facile and sustainable protocol is developed for the synthesis of PEG-coated iron oxide nanoribbons (IONRs). It is observed that the IONRs exhibit large exposed active surface areas with a high potential for more-effective targeting of ligands and cell receptors, thus promoting multivalent interactions. The IONRs also exhibit enhanced T2-relaxivity as high as 218 mM-1s-1 (Figure 1), when compared to spherical iron oxides. This effect is attributed to the greater water-NRs co-ordination and to their 1-D-like structure. The detailed characterizations including XRD, FTIR, DLS, VSM, HRTEM will be presented, along with their cytotoxicity assessment on HeLa and Jurkat T lymphocyte cells.

References:

[1] J-H Park, G. V. Maltzahn, L. Zhang, M. P. Schwartz, E. Ruoslahti, S. N. Bhatia, and M. J. Sailor , Adv. Mater., 20, 1630-1635 (2008).

[2] A. Mukhopadhyay, N. Joshi, K. Chattopadhyay, and G. De, ACS Appl. Mater. Interfaces, 4, 142-149 (2011).

Figure 1


48

IN VIVO TRIPLE QUANTUM EFFECTS OF BOUND POTASSIUM AND SODIUM AT 21.1 T

Victor Schepkin1, Andreas Neubauer2, Boris Odintsov3, Ilya Litvak1, Peter Gor’kov1, William Brey1, Thomas Budinger4

1National High Magnetic Field Laboratory/ FSU, Tallahassee, FL, USA; 2Heidelberg University, Mannheim, Germany; 3Beckman Institute, University of Illinois Urbana-Champaign, IL, USA; 4Lawrence Berkeley National Laboratory/UCB, Berkeley, CA, USA

Triple quantum (TQ) signals are unique features for ions such as potassium and sodium nuclei, as they both have spin=3/2. During binding the ions interact with the electric field gradients of the surrounding macromolecules, and in vivo these are mainly proteins. In this state the behavior of the nuclear magnetization cannot be described by the classical vector model. The changes in magnetization are now similar to the transformation of spherical harmonics and can be visualized graphically. These changes can also be described by the irreducible tensor operators [1].

The value of the TQ signals is still under investigation. For example, after calibration, the TQ signal can detect changes in the intracellular sodium concentration in a rat heart [2]. Separation of the TQ signal is usually achieved by TQ filtration (TQF). Much higher efficiency of the TQ signal detection can be accomplished by concurrent time and phase increments (TPPI) of the RF pulses in a pulse sequence. In this case, no signals are lost and the TQ signals are observed separately at the triple frequency relative to a SQ signal (Fig. 1).

Fig. 1. A novel detection of in vivo bound potassium in rat head at 21.1 T. No filtration was used and bound ions are observed as a separate peak (TQ) on the triple frequency (3.75 kHz) relative to a single quantum (SQ) MR signal (1.25 kHz).

A graphic presentation of the TQ signals as a set of spherical harmonics can visualize the quantum effects. The comparison of the TQ signals of potassium and sodium is performed in vivo in a rat head and in agarose test samples at 21.1 T magnet. Simultaneous detection of SQ and TQ signals for potassium and sodium demonstrate the effect of competition between potassium and sodium ions for the same binding places. In vivo up ~65% of sodium and ~31% of potassium are free or experience a very short correlation time during a binding.

[1] Rose M.E., Proc. Phys. Soc. A, 1954, 67, 239-247[2] Schepkin V.D., et. al., Magn Reson Med 1998, 39,557-563.

TQ SQ

in vivo potassium in rat head

44th Southeastern Magnetic Resonance Conference Oral Presentations Radicals in vivo & in situ

49

EPR DETECTION ON MICROCHIPS – ON THE WAY TO SINGLE CELL DETECTION

Alexander Angerhofer University of Florida, Department of Chemistry, Gainesville, FL 32611 A brief review will be given about the challenges of trying to use EPR to detect metabolic radicals in live cell cultures. I will then present our current efforts in detecting radicals in mono-layered mammalian cell cultures in vivo followed by a discussion of EPR experiments using a novel micro-chip EPR. The chip operates in the K-band [1] and allows temperature dependent measurements from liquid helium to room temperature. I will further discuss current efforts to increase the frequency of the chips to 160 GHz and the expected increase in sensitivity. [1] J. Anders, A. Angerhofer, and G. Boero, J. Mag. Res., 217, 19 – 26 (2012).


50

INVESTIGATING THE ROLE AND STRUCTURE OF FREE RADICALS IN LIGNIN BIOSYNTHESIS

Xiao Song1, Maxim A. Voinov1, Wayne Zeller2, and Tatyana I. Smirnova1

1Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27695; 2U.S. Dairy Forage Research Center, 1925 Linden Drive, Madison WI 53706 Lignin is the second most abundant biopolymer on Earth after cellulose that contributes to the overall viability and sustainability of the Plant Kingdom. Cross-linking of lignin to cellulose has been shown to contribute to the cell wall durability, facilitate water transport, and protect plants against invasive pests. Understanding molecular details of the lignin formation and cross-linking is of fundamental importance for areas spanning from lignin control and removal to production of biomass streams. Although the processes of monolignols oxidative dehydrogenation, polymerization and cross-linking to cell walls are proposed to occur through a free radical mechanism, the specific details remain known. In order to understand molecular mechanisms of these reactions we have investigated formation of radicals during enzyme-catalyzed (HRP or Laccase) oxidative dehydrogenation of monolignols and their derivatives. To characterize short-lived radicals formed during the enzymatic reaction we employed EPR spectroscopy in combination with spin-trapping technique and deduced radical structures from the features of EPR spectra of the spin adducts. We also utilized continuous-flow EPR in combination with surface immobilization of enzymes to detect formation of stable transient monolignol radicals. Analysis of the well-resolved hyperfine futures allowed for determination of the monolignol radicals structures. Figure1. (A) – Experimental EPR spectrum from a transient radical formed under conditions of continuous-flow from sinapyl alcohol upon reaction with immobilized Laccase and spectral simulation including two components: immobilized radical (B) and free radical in solution (C). (D) – Residual of the fit – the difference between the experimental and simulated spectra.

HO

H3CO OCH3OH

A

B

C

D


51

DEER in Native Systems: What Lipids and Detergents Can’t Tell Us

Arthur K. Sikora,1 Benesh Joseph,2 Enrica Bordigton 3,Thomas Prisner2 and David Cafiso.1

University of Virginia, Department of Chemistry, McCormick Rd, Charlottesville Va 22901 USA Institute of Physical and Theoretical Chemistry, Max-von-Laue-Strasse 7, Goethe University, 60438, Frankfurt, Germany Department of Physics, Free University of Berlin, Arnimallee 14, 14195 Berlin, Germany Outer-membrane TonB-dependent transporters function in the uptake of essential nutrients, and are important for the success of many pathogenic bacteria These proteins consist of a 22 stranded β-barrel where the N-terminal 130 to 150 residues form a core domain that fills the barrel. During transport, these proteins undergo a cycle of binding and unbinding to the inner membrane protein TonB, through an interaction that is mediated by the Ton box, an energy-coupling segment near the transporter N-terminus. Over 50 high-resolution crystal structures have been obtained for 12 different TonB-dependent transporters, however the mechanisms of substrate transport remain unclear. Determination of membrane protein structure or dynamics with high resolution in whole cells is an attractive way to solve transport mechanisms but yet to be demonstrated.

In this work, the cobalamin transporter BtuB was overexpressed and spin labelled in whole cells and outer membranes and interspin distances were measured to a spin labelled cobalamin using pulse EPR. This represents the first example of double electron-electron resonance (DEER) preformed in whole E. coli cells. A comparative analysis of the data reveals a similar interspin distance distribution between whole cells, outer membranes and synthetic vesicles. We then take advantage of these native lipid environments to study the conformational heterogeneity of BtuB outer loops. The data indicate that there is a strong calcium mediated ordering of the 1-2 loop. We also see evidence of allosteric loop modulation, the binding of TonB to the Ton box on the periplasmic surface of BtuB alters the configuration of these extracellular loops. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions for large outer membrane protein complexes in whole cells and native membranes, and provides a method to validate high-resolution structures of membrane proteins in their native environment

This work was supported by NIGMS, GM035215.

B. Jospeh, A. Sikora, E. Bordington, G. Jeschke, D. Cafiso, T. Angew. Chem. Int. Ed., 10, 1002 (2015)


52

smFRET AND DEER DISTANCE MEASUREMENTS AS APPLIED TO DISORDERED AND STRUCTURED PROTEINS

Keith Weninger,1 Ruoyi Qiu,1 Erkang Ou,2 Sergey Milikisiyants,2 Tatyana Smirnova2

1Department of Physics, North Carolina State University, Raleigh, NC 27695 USA 2Department of Chemistry, North Carolina State University, Raleigh, NC 27695 USA

FRET and DEER are two spectroscopic methods that are widely applied for biophysical studies of protein structure. Both methods are based on measuring dipolar interactions – electrical dipoles in case of FRET and magnetic dipoles in case of DEER - between specifically labeled protein sites. The experimental data are then analyzed to derive the distance between the interacting dipoles and relate this distance to the structure of biomacromolecule(s). Another common aspect of these two methods is the sample preparation involving mutation of selective protein residues to cysteine and the consequent covalent modification of cysteines with molecular tags, spin labels for DEER and fluorescent labels for FRET. Molecular volume of EPR labels is generally smaller vs. that of the fluorescent probes and DEER experiments can be carried out by labeling cysteines with identical molecular tags whereas FRET typically relies on orthogonal labeling with distinct donor and acceptor fluorophores. Another essential difference is that FRET can be performed under physiological conditions, but DEER typically requires cryogenic or near cryogenic temperatures because of short phase memory time for nitroxides at ambient conditions. Finally, single molecule (sm) FRET reports on conformation of individual protein molecules whereas DEER provides information on ensemble average. While the distance ranges of these two methods overlap, the direct comparison of FRET and DEER data is rarely found in the literature. Here we report on the distance measurements using both smFRET and DEER on the same protein system. We attached probes to a unique pair of cysteines in the neuronal SNARE protein SNAP-25. SNAP-25 is highly disordered in isolation, but it folds into a stable alpha-helix bundle upon forming SNARE complex with syntaxin and synaptobrevin. Results of smFRET and DEER distances and distance distribution are compared for disordered SNAP-25 and folded SNAP-25 within the SNARE complex. While distance measurements and distance distributions obtained by smFRET and DEER are in general agreement in case of SNARE complex, surprising results were obtained for intrinsically disordered (ID) SNAP-25. For the first time we observed heterogeneous populations in disordered SNAP-25 using DEER. The observation of two populations points to differences in averaging over samples by smFRET and DEER. We propose that SN25 stochastically switches between distinct conformational states (ensembles) with intra- conversion rate faster than 100 msec despite the fact that it appears intrinsically disordered by other measures. For the second system investigated, Glutamate Receptor Cytoplasmic Domain N2B, an observation of slow-exchanging populations with smFRET corresponds to broad range of distances observed by DEER measurements.

Figure 1. Spin-spin distance distributions as measured by Q-band DEER experiment for spin-labeled SNAP 25 in SNARE complex (blue) and in disordered state in solution (red). Disordered state shows an existence of two populations that are averaged in smFRET experiments.

TRAVERSING THE TEMPERATURE-DEPENDENT FREE ENERGY LANDSCAPE FOR RADICAL


53

REARRANGEMENT CATALYSIS IN B12-DEPENDENT ETHANOLAMINE AMMONIA-LYASE Meghan Kohne, Chen Zhu, and Kurt Warncke Emory University, Department of Physics, Atlanta, GA 30322-2430 The Co2+-substrate radical pair intermediate in the reaction cycle of ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium accumulates during steady-state turnover, and can be stably cryotrapped. Upon annealing at temperatures (T) of 197-230 K, the radical pair formed by using aminoethanol substrate decays to diamagnetic products1. The decay is measured by using time-resolved, full-spectrum continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy. The radical decay exhibits two regimes: (i) 220 ≤ T ≤ 230 K; monoexponential decay with rate constant, knative, and (ii) 197 ≤ T < 220; biexponential decay with rate constants, kfast and kslow. The k-values represent the substrate-to-product radical rearrangement reaction. The knative values display an Arrhenius T-dependence that extrapolates to values for rearrangement-determined kcat at T ≥ 277 K. Both the kfast and kslow populations diverge from the knative relation at Tdiv = 220 K, and follow distinct Arrhenius dependences for T< Tdiv. The Tdiv is comparable to the glass transition temperature (Tg’) of the solvent mesodomain2 that envelops EAL in the frozen polycrystalline samples. The Tg’ was determined from the T-dependence of the correlation time of the mesodomain-localized spin probe, TEMPOL, under the same conditions as for the kinetics measurements. The results reveal that the native radical reaction coordinate includes a subset of collective protein and coupled solvent motions (α-fluctuations), that are unveiled by the effect of the glass transition on the kinetics. Persistence of the reaction at T< Tg’ indicates non-native trajectories near to, or within, the native configurational envelope. These features of the reaction are captured in a multi-state, T-dependent free energy landscape model. Support: NIH R01 DK054514. [1] C. Zhu and K. Warncke, Biophys. J., 95, 5890, (2008); J. Am. Chem. Soc., 132, 9610, (2010) [2] H. Chen, et al., Langmuir, 29, 4357 (2013).


54

DRUG BINDING CHARACTERIZATION OF CYPS UTILIZING CW AND PULSED EPR

Alex A. Cruce1, Kip Conner2, Preethi Vennam1, Molly Lockart1, William M. Atkins2, Michael K. Bowman1

1The University of Alabama, Box 870336, Tuscaloosa, Alabama 35487 USA; 2University of Washington, Box 351700, Seattle, Washington 98195 USA

Electron paramagnetic resonance (EPR) methods have been used to study drug and ligand interactions with a super family of monooxygenase enzymes responsible for metabolizing over 70% of drugs in clinical use today known as Cytochrome P450 (CYP). The active-site of four different isoforms of CYPs before and after the addition of drug using EPR have been examined to identify binding motifs. EPR has shown to be a quick and highly resolved method, in comparison to current methods such as crystal structure analysis, UV/Vis optical difference spectroscopy, and NMR to study CYPs ferric heme active-site before and after drugs bind, which could be very valuable in drug design. A CW spectrum of CYP in complex with drug gives noticeable shifts in the spectral region corresponding to the axial ligand of the low-spin heme. To further examine the ligand interaction causing the spectral shift, a pulsed EPR method called HYSCORE has shown the ability to observe hydrogens for both drug-free and drug-bound species from the axial water ligand showing that the drug either displaces the water ligand seen in the resting state or retains the water. These observations enable the distinction between two drug binding motifs; a drug directly coordinating to the active-site showing a CW spectral shift to a lower field and a drug binding through a water ligand shifts to a higher field, so with a ten minute CW scan an observer can distinguish, in solution, whether a drug is directly bound or weakly coordinated to the ferric heme active-site. We have shown that EPR spectral shifts and HYSCORE peaks are a much more accurate method to determine drug binding mode than current methods. Additionally, there are more concerns about drug interactions to be studied because EPR exploited the ability to differentiate binding modes in solution, which opens the door for many more studies towards metabolic CYP in complex with drugs.

44th Southeastern Magnetic Resonance Conference Posters

55

POSTERS

44th Southeastern Magnetic Resonance Conference Poster # 1

56

Preliminary Results from NMR and EPR Spectroscopy for Achieving Overhauser-DNP at 14.1T Adewale Akinfaderin1,2, Thierry Dubroca2, Sungsool Wi2, Bianca Trociewitz2, Hans Van Tol2, Lucio Frydman2,3, and Stephen Hill1,2

1Florida State University, Tallahassee, FL USA; 2National High Magnetic Field Tallahassee, FL USA; 3Weizmann Institute, Rehovot, Israel Nuclear Magnetic Resonance (NMR) is a versatile analytical tool which is able to predict molecular structures and obtain information from a fundamental theoretical framework but its sensitivity is quite limited. Overhauser Dynamic Nuclear Polarization (O-DNP), which relies on the transfer of spin polarization from electrons to nuclei of interest has gained sudden resurgence for enhancing NMR signal [1, 2]. However, the Overhauser effect predicts a significant reduction in enhancement at high magnetic fields due to solvent-solute dynamics and short relaxation rates of radicals and nuclei. In this work, we explore different techniques for performing O-DNP spectroscopy at high magnetic fields for solution samples by utilizing solvent media in the low viscosity regime. Nuclear relaxation times, leakage factors, relaxivity constants and diffusion coefficients of low viscous organic solvents and supercritical-CO2 all doped with nitroxide radical TEMPOL using NMR spectroscopy will be presented. We performed continuous wave EPR spectroscopy on these molecules to estimate the Heisenberg spin exchange rate constant and investigate the sensitivity of the EPR line width to pressure and temperature due to the spin-rotation interaction in the sub-picosecond correlation time regime. These experimental results and related ongoing studies will yield a better understanding of the diffusional dynamics of small molecules in supercritical-CO2, which will in turn encourage the use of this medium in high field Overhauser DNP experiments. Acknowledgements Funding: NSF-MRI CHE-1229170, NSF DMR-1157490 and the State of Florida. Reference [1] K.H Hausser, and D. Stehlik, “Dynamic Nuclear Polarization in Liquids” Advan. Magn.Reson. 3, 79-139 (1968). [2] Günther, Ulrich L. "Dynamic nuclear hyperpolarization in liquids." Modern NMR Methodology. Springer Berlin Heidelberg, 23-69 (2013).


57

COMPARING SCALAR MEASURES OF ANISOTROPY FROM DIFFERENT DIFFUSION MODELS Manish Amin1 Dr. Thomas H. Mareci1,2,3

1University of Florida, Department of Physics 2University of Florida, Department of Biochemistry and Molecular Biology 3University of Florida, Department of Biomedical Engineering Fractional anisotropy has a major limitation in defining regions of the brain with crossing fibers. Other diffusion models have had greater success compared to DTI in resolving these regions. Scalar measures of anisotropy developed from these diffusion models could prove to be more informative and distinctive then fractional anisotropy. This can be done by using the probability displacement function, and converting it into a scalar anisotropy measure. Spherical Harmonics are a system of orthonormal basis functions, and can be used to represent the probability displacement function. Analyzing the coefficients of the spherical harmonics can provide valuable information about the level of anisotropy. The goal is to determine if these measures can resolve regions of crossing fibers better than fractional anisotropy. [1] B. Jian, B. Vemuri, E, Ozarslan, P. Carney, and T. Mareci, Neuroimage, 37:164-176 (2007) [2] E. Ozarslan and T. Mareci, Magnetic Resonance in Medicine, 50:955-965 (2003) [3] L. Frank, Magnetic Resonance in Medicine, 47:1083-1099 (2002) [4] Y. Chen, W. Guo, Q. Zeng, X. Yan, and Y. Liu, Information Processing in Medical Imaging, 3565:246-257 (2005


58

MAGNETIC SUSCEPTIBILITY ANISOTROPY IN RAT’S SPINAL CORD Guita Banan1, Thomas Mareci1, 2, 3 1Department of Physics, University of Florida 2Department of Biochemistry and Molecular Biology, University of Florida 3Department of Biomedical Engineering, University of Florida Magnetic susceptibility has become a strong tool for investigating brain tissue structure and neurological disorders related to brain contents such as myelin in white matter and tissue iron. Recent studies (Li et al., 2012) show that magnetic susceptibility of brain white matter is highly anisotropic and can reveal important information about microstructure of the white matter. Field inhomogeneities due to magnetic susceptibility variations can be quantified using phase data from gradient echo MRI and be used to solve for susceptibility distribution. In order to study magnetic anisotropy of the white matter, susceptibility tensor imaging (STI) was proposed by Liu, 20102. In this approach susceptibility is assumed to be a symmetric tensor with six unknown elements. Therefore, a minimum number of six measurements in different directions is required to form a system of equations and calculate all elements of susceptibility tensor. Spinal cord has a cylindrical structure and many fibers aligned in the same direction and therefore is an ideal sample to study magnetic anisotropy of the white matter and validate our quantification models. In this study a 2cm section of the rat’s spinal cord was imaged at multiple orientations in order to reconstruct the susceptibility tensor. A 3D multiple GE sequence was used to image the spinal cord at a 17.6 T magnet at nine different orientations with respect to the main magnetic field. Imaging parameters were: TR = 200 ms, four echo times separated by 2.25 ms starting at TE1 = 3 ms, isotropic resolution = 0.1 mm, and FOV = 200x70x70 mm3. The images with different echo times will be used to calculate the phase and magnetic field perturbations at each orientation and solve for susceptibility tensor.

[1] Wei, Li, et. al., NeuroImage, 59, 2088-2097 (2012). [2] Chunlei, Liu, Magnetic Resonance in Medicine, 63, 1471-1477 (2010)

Figure 1. Rat’s spinal cord magnitude image at a specific orientation with

a) TE = 3ms, b) TE = 5.25ms,


59

MORPHOLOGY-INDUCED MRI RELAXIVITY IN NANOSCTRUCTURED Fe3O4 Bibek Thapa1, 2, 3, Daysi Diaz-Diestra1, 2, 3, Juan Beltran-Huarac2, 3, Huadong Zeng4,

Brad R. Weiner1, 2, 3 and Gerardo Morell1, 2, 3

1Institute for Functional Nanomaterials, University of Puerto Rico, San Juan, PR 00936, USA 2Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926, USA

3Department of Physics and Department of Chemistry, University of Puerto Rico, San Juan, PR 00931, USA

4Advanced Magnetic Resonance Imaging and Spectroscopy Facilities, University of Florida, Gainesville, FL 32611, USA

We report the fabrication of morphology-tuned Fe3O4 building blocks (FBBs) and test their capability to enhance the magnetic resonance imaging (MRI) relaxivity when used as contrast agents. The self-assembled FBBs include nanowires, nanotubes, nanosaws, linear irregular-shaped aggregations and anisotropic chain-like arrays of Fe3O4, grown by CVD and co-precipitation method. Catalogued FBBs with different numbers of free amines are prepared for peptide conjugation. Ex-situ characterizations indicate that they exhibit high aspect ratios and larger surface areas, which facilitate the formation of multiple cell-agent linkages (more effectively than those found in conventional superparamagnetic iron oxide particles), and induce in turn enhanced T2 relaxation (ca. 70 mM-1s-1, see Figure 1). The potential of these nanostructures to image poorly vascularized tumors and even individual tumor cells (bridging the existing critical gap in this research field) provided by in vitro experiments will be also discussed. This study is expected to bring forth new vistas for the design of multifunctional nanoprobes for applications in vivo not only in the diagnosis through targeting and imaging of tumors, but also in the treatment of a wide range of noncommunicable human diseases, such as cancer, stroke and heart disease, which constitute the major factors of global mortality.

Figure 1. Phantom images obtained at different iron contents of aqueous dispersion of Fe3O4. References: [1] Z. Zhao et at. Nature Communications 4, 2266 (2013). [2] J. Beltran-Huarac et al, Materials Letters 98, 108 (2013).


60

Progress towards a 1.5 GHz NMR user facility William W. Brey1, Ilya M. Litvak1, Peter L. Gor’kov1, Zhehong Gan1, Timothy A. Cross1,2, Jeffrey L. Schiano3, Mark D. Bird1 1Florida State University, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310; 2Florida State University, Department of Chemistry, Tallahassee, FL 32306; 3Pennsylvania State University, Department of Electrical Engineering, University Park, PA 16802 A 36-T powered magnet of hybrid (resistive-superconductive) design is scheduled to come to field in 2016. This series-connected hybrid (SCH) magnet will open new possibilities in NMR spectroscopy, especially for half-integer quadrupolar nuclei such as 17O and 27Al. Line width of the central transition for these nuclei is inversely proportional to the Larmor frequency. A dramatic increase in resolution (as B0

2) [1] and in sensitivity (as B0

7/2) will make possible NMR of a wide range of “unreceptive” nuclei and have a significant impact in both biological and material applications. Also, it is well known that high field favors proton-detected solid state NMR of proteins at ultrafast MAS.

In the past, temporal field fluctuations and insufficient field homogeneity have limited the use of powered magnets for NMR applications. The new system is designed to address these deficiencies. The magnet has been designed to have 1 ppm homogeneity in 1 cm DSV achieved through a combination of active and passive shimming. An active field regulation system will suppress both fast and slow fluctuations of the B0 field [2].

We are developing the initial set of NMR probes for the new magnet. These include a 2.0-mm CP-MAS probe based on our Low-E design [3], a single-channel 3.2-mm MAS probe with a wide VT range, and an HXY probe for static oriented biological samples. The probes will tune to a variety of nuclei, including custom configurations (upon request), and can be used for biological and materials applications. Users will acquire data with an Avance III HD console from Bruker running the latest versions of TopSpin and Paravision software.

The NMR user program at the National High Magnetic Field Laboratory offers free-of-charge access to a wide range of NMR systems including a 900 MHz / 105 mm spectrometer. Support is available for users on all levels of technical and scientific expertise.

We seek proposals for innovative experiments that require extremely high magnetic fields at moderate homogeneity to be performed on the 36 T SCH magnet. More information on user access, including requests for magnet time on the 900 MHz spectrometer and on other systems is available at https://users.magnet.fsu.edu/. [1] Z. Gan et al., J. Magn. Reson., 191, 135-140 (2008). [2] M. Li et al. J Magn. Reson., 212, 254-264 (2011). [3] P.L. Gor'kov et al., J. Magn. Reson., 185, 77-93 (2007).


61

HYDRATION DYNAMICS PROBED USING OVERHAUSER DYNAMIC NUCLEAR POLARIZATION (ODNP) ENHANCED NMR AT 0.35 T

Thomas M. Casey, Jackie M. Esquiaqui, Joaquin, J. Casanova, Gail E. Fanucci

University of Florida, Department of Chemistry, Gainesville, Florida 32611

Overhauser dynamic nuclear polarization (ODNP) enhanced NMR at X-band (0.35 T; 9.8 GHz;

14.9 MHz) is emerging as an effective method for characterizing the hydration environment surrounding biomacromolecules. In this method, a spin label is attached to the biomolecule in a region where there is interest in hydration dynamics, the sample is positioned in an NMR coil that is inserted in an EPR resonator, and the electron spin transition is continuously excited using a range of MW powers approaching saturation while collecting the NMR signal for the 1H of H2O. The enhancement of the 1H NMR signal as a function of MW power can be fit to analytical expressions to extract empirical parameters that describe the dynamic behavior of hydration water near the spin-labeled region. Here we present our latest developments in applying these methods and show initial results for the application to the study of changes to the hydration landscape in response to folding in the RNA glycine riboswitch. Using four buffer conditions we simulate varying degrees of folding and observe the accompanying changes in hydration dynamics. Specifically, we examine the leader-linker region of the “kink-turn” motif for the full-length (232 nucleotides) riboswitch and for a short (20 nucleotides) isolate of the kink-turn. For both constructs, previous CW EPR data suggest that the addition of KCl, MgCl2, and glycine induce varying degrees of folding. Initial ODNP NMR data for the same conditions suggest that with folding comes a decreased diffusivity of the hydration water surrounding the leader-linker region of the kink-turn. [1] J. Esquiaqui, et. al., Biochemistry, 53, 3526-3528 (2014).


62

EPR Studies of Trityl Biradicals Hanjiao Chen1, Michael K. Bowman1, N. P. Isaev2, R. I. Samoilova2, A. G. Maryasov2, O. Y. Rogozhnikova3, V. M. Tormyshev3

1The University of Alabama, Department of Chemistry, Tuscaloosa, AL USA; 2Institute of Chemical Kinetics and Combustion, Novosibirsk, Russia; 3Novosibirsk Institute of Organic Chemistry, Novosibirsk, Russia; Our recently developed synthetic route for trityl-based biradicals can provide new spin labels or polarizing agents for dynamic nuclear polarization (DNP). We studied the EPR properties of these newly-synthesized trityl biradicals in frozen samples including the spin-lattice relaxation time (T1e, plotted below), spectral diffusion, ESEEM and the ENDOR spectrum. We also compared our results for biradicals to those for trityl monoradicals (Finland trityl and trityl OX063). Trityl biradical shared the similar Raman process with trityl monoradicals but the low-temperature Orbach process is seen only at high trityl monoradical concentrations.

10 1001E-3

0.01

0.1

1

Rel

axat

ion

Rat

e (k

Hz)

Temperature (K)

Spin-Lattice Relaxation Rate of Trityl Biradical

This work was supported by the National Science Foundation, Chemistry Division (award No. 1416238) (MKB) and the Russian Foundation for Basic Research (grant No. 14-03-93180) (VMT).


63

17O, 23Na, 29Si, SOLID-STATE NMR STUDIES OF SUPERIOUR SOLID OXIDE-ION ELECTROLYTE Sr3-3XNa3XSi3O9-1.5x (SNS) Po-Hsiu Chien1, Youngseok Lee2, Kevin Huang2, Yan-Yan Hu1,3,* 1Florida State University, Department of Chemistry and Biochemistry, Tallahassee, FL 32306; 2Department of Mechanical Engineering, University of South Carolina, Columbia, SC 29208, USA; 3National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310 USA. Solid-oxide ion super conductors are vital components used in solid-oxide fuel cells (SOFCs), which have been considered as a promising energy storage technology. However, decreasing the working temperature of SOFCs to < 600 oC is critical to make SOFCs commercially viable and increase energy efficiency. Therefore, research on intermediate-temperature SOFCs (IT-SOFCs) has been a long-standing pursuit in this research field. Recently, a promising superior solid oxide-ion electrolyte, Sr3-

3xNa3xSi3O9-1.5x (SNS) developed by Goodenough group1,2, was found to have the highest ion conductivity among solid oxide-ion electrolyte (σ ≈ 0.01 S/cm at 500 oC)1 However, research on revealing the oxide-ion transport mechanisms in SNS is limited. In this abstract, we present detailed investigations of Na-doped SNS samples using 17O, 23Na, and 29Si solid-state NMR. Results from our investigations have revealed changes in structural transformation as increasing Na content in SNS. [1] P. Singh and J. B. Goodenough, J. Am. Chem. Soc., 135, 10149-10154 (2013). [2] R. Martinez-Coronado, P. Singh, J. Alonso-Alonso, and J. B. Goodenough, J. Mater. Chem. A, 2, 4355-4360 (2014) [3] T. Wei, P. Singh, Y. Gong, J. B. Goodenough, Y. Huang, and K. Huang, Energy Environ. Sci., 7, 1680-1684 (2014)


64

LONGITUDINAL MRI OF DISEASE PROGRESSION IN A TRANSGENIC, TGF-ALPHA-INDUCED MOUSE MODEL OF PULMONARY FIBROSIS Zackary I. Cleveland1,2,3,4, Yu M. Zhou1,5, R. Scott Dunn4, Cynthia R. Davidson2, Teckla Akinyi1,3, Jinbang Guo1,6, Jason C. Woods1,2,4,6, and William D. Hardie2 1 Center for Pulmonary Imaging Research, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave., ML-5033, Cincinnati, OH 45229 2 Division of Pulmonary Medicine, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave., ML-5033, Cincinnati, OH 45229 3 Biomedical Engineering Program, University of Cincinnati, 601 Engineering Research Center, Cincinnati, OH 45221 4 Imaging Research Center, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave., ML-5033, Cincinnati, OH 45229 5 University of Cincinnati College of Medicine, 3230 Eden Ave, Cincinnati, OH 45267. 6 Department of Physics, Washington University, 1 Brookings Dr., St. Louis, MO 63130

Idiopathic pulmonary fibrosis (IPF) is a heterogeneous, progressive, and fatal disorder. Because disease etiology is poorly understood, mouse models play a vital role in studying the mechanisms underlying disease progression and therapy response. However, conventional histological and biochemical measures of lung fibrosis require mice to be sacrificed, limiting the spatial and temporal information available from these models. MRI has shown promise for non-invasively examining experimental lung fibrosis, but previous murine MRI studies have focused on IPF models that involve acute insults such as bleomycin, which do not exhibit the non-inflammatory disease progression seen in humans (1-3). Thus, while previous MRI studies of these models have shown methodological promise, they are of limited use for understanding IPF biology.

Here we report the first MRI study of a non-inflammatory, transgenic mouse model of IPF. These doxycycline (Dox) regulatable, mice overexpress TGF-α under control of a lung-epithelial-specific promoter gene (4,5). When treated with Dox, mice display progressive pathology that mimics non-inflammatory IPF in humans. We found that MRI measures of lung fibrosis (e.g., parenchymal SNR) correlate strongly with traditional measures of fibrosis. Moreover, by examining disease progression in these animals longitudinally, we demonstrated that fibrosis progression involves previously unrecognized, dynamic remodeling. References: [1] C Egger, et al., Am. J. Physiol-Lung. C, 306 (12), L1064-L1077 (2014). [2] RE Jacob, et al., J. Magn. Reson. Imaging, 31 (5) 1091-1099 (2010) [3] A Moeller, et al., Int. J. Biochem. Cell B, 40 (3), 362-382 (2008) [4] WD Hardie, et al., Am. J. Physiol-Lung C, 286 (4) L741-L749 (2004) [5] WD Hardie, et al., Am. J. Resp. Cell. Mol., 37 (3), 309-321 (2007)

Top. A fibrotic high-SNR structure protruding from the sub-pleural region in wk 6 persisted into wk 7 (red arrows), while a second structure (yellow arrow) formed by wk 5 and resolved by wk 6, indicating fibrosis progression in this model involves a previously unknown, dynamic component. Left: Hydroxyproline (a biochemical measure of fibrosis), correlates well with image SNR.


65

DEVELOPING A METABOLIC PHANTOM FOR DISSOLUTION DNP IMAGING James H. P. Collins1, Daniel P. Downes1, Bimala Lama1, Joanna R. Long1

1Department of Biochemistry and Molecular Biology, University of Florida Gainesville, FL. Dissolution dynamic nuclear polarization (DNP) is a technique which allows greatly increased signal-to-noise ( >15,000× vs. standard techniques)1 of in-vivo measurements, allowing the direct observation of carbon metabolism. Combining dissolution DNP with spectroscopic imaging techniques allows the spatial variation in metabolic activity to be observed, and has been used to great success in a number of applications. The tradeoff for the large increase in SNR, is the relatively rapid relaxation of the enhanced polarization to its thermal equilibrium value, giving only a few minutes of usable polarization post injection. This can be problematic for the testing and development of new sequences. Non-hyperpolarized samples can be used, but unless very concentrated, which can be prohibitively expensive with labelled isotopes, will have substantially lower SNR than that of the DNP sample. Animal models can show significant variability in metabolic intensity and SNR due to non-r.f. sequence issues, which can further compound the difficulties in r.f. sequence development. What is required is a reliable, controlled phantom which can simulate metabolic activity when a hyperpolarized metabolite, such a Pyruvate, is injected into it. To achieve this, a ‘living phantom’ is under development, using Saccharomyces Cerevisiae and E. Coli cultures to metabolize the injected substrate2. 3D printing techniques allow the rapid productions of arbitrary shapes, which can be designed to test specifics of the developed sequences. The phantom can then be filled with the desired bacterial culture prior to injection of the metabolite, allowing testing of the developed sequences without the complications of animal handling. Data will be presented on the behavior of potential microbial cultures, and comparisons of their metabolic activity with those of model animals. Initial data from testing with spectroscopic imaging sequences such as EPSI3 will also be presented. [1] J.H. Ardenkjaer, et al., PNAS, 100, 10158-10163, 2003 [2] S. Meier, P.R. Jensen, J.O. Duus., FEBS Letters, 19, 3133-3138, 2011 [3] S.J. Nelseon, et al., Appl. Magn. Reson., 34, 533-544, 2008


66

DEVELOPMENT OF OVERHAUSER DYNAMIC NUCLEAR POLARIZATION AT 14.1 T Thierry Dubroca1, Bianca Trociewitz1, Adewale Akinfaderin1,2, Johan van Tol1, Sungsool Wi1, William W. Brey1, Lucio Frydman1,3, Joanna R. Long1,4, and Stephen Hill1,2 1National Maglab, Florida State University, Paul Dirac Drive, 32310, Tallahassee, FL

2Florida State University, Physics Department, Chieftan Way, 32306, Tallahassee, FL 3Weizmann Institute, Department of Chemical Physics, Herzl Street, 76100, Rehovot, Israel 4University of Florida, Dept. of Biochemistry and Molecular Biology, Newell Drive, 32610, Gainesville, FL

Dynamic nuclear polarization (DNP) is a novel technique which has been shown to increase the sensitivity of high field NMR (up to 18.8 T) by two orders of magnitude in solids, but, in the liquid phase, 5 to 9.4 T DNP NMR has only been demonstrated in limited cases1, 2, and it has never been demonstrated above 9.4 T. The National High Magnetic Field Laboratory is developing a new liquid solution DNP instrument operating at 14.1 T, see Figure 1A. A 395 GHz gyrotron, the microwave source, is used to polarize radicals' electron spins which in turn polarize the spins of nuclei of interest, such as protons at 600 MHz, thus potentially providing large NMR signal enhancements. We present here various strategies to achieve Overhauser DNP at such a high field. First, we systematically studied solvents compatibility with high frequency microwaves, radicals, and Overhauser DNP effect requirements. Secondly, we developed a quasi-optical system in collaboration with Thomas Keating Ltd. to control the beam, power and polarization of the microwaves. In addition, we modified a commercial liquid NMR probe to accommodate the corrugated microwave guide and added a sweepable coil to tune the field to the radical's resonant frequency. Finally, we build an in-situ microwave spectrometer to observe the radicals’ EPR lines (Figure 1B) and optimized the field for maximum DNP efficiency. The instrument, once completed, will be available for external users at the National High Magnetic Field Laboratory

Figure 1. Photograph of the 14.1 T Overhauser DNP system in development (A). Room temperature In-situ EPR saturation and absorption (inset) of ~1M TEMPO in hexane at 395 GHz (B). A linewidth of 1.7 mT is observed with a g-factor of 2.0059. The sample volume is 0.25 mL. Acknowledgements Funding: NSF-MRI CHE-1229170, NSF DMR-1157490 and the State of Florida. References [1] N.M. Loening, M. Rosay, V. Weis, R.G. Griffin, JACS 2002, 124: 8808-8809 [2] V. Denysenkov, M.J. Prandolini, M. Gafurov, D. Sezer, B. Endeward, T.F. Prisner, PCCP 2010, 12: 5786-5790

(B) (A)


67

SINGLE-FILE DIFFUSION OF CO, CH4, AND CO/CH4 MIXTURE BY HIGH-FIELD DIFFUSION NMR Akshita Dutta1, Poorvajan Sekar1, Muslim Dvoyashkin1,2,3, Clifford Bowers2, Kirk Ziegler1, Sergey Vasenkov1 1University of Florida, Department of Chemical Engineering, Gainesville, FL 32611 USA; 2University of Florida, Department of Chemistry, Gainesville, FL 32611 USA; 3Universität Leipzig, Institute of Chemical Technology, Leipzig 04103, Germany Molecules restricted to move in unidimensional channels so narrow that their mutual passage is forbidden exhibit the anomalous transport mechanism known as single-file diffusion (SFD). This mechanism is manifested in the preserved sequential order of the diffusing molecules. An important consequence of SFD is the significant decrease in the growth of mean-squared displacement (MSD) with time in comparison to Fickian diffusion. This property of SFD can be exploited to achieve highly selective separations if one component in the mixture exhibits normal diffusion and another SFD. Furthermore, induction of SFD in catalytic systems can also offer better control over reactions. Applications such as these are expected to involve more than one sorbate molecule in single-file channels. However, currently there are no experimental studies that explore molecular SFD of gas mixtures. In our study, carbon-13 pulsed field gradient (PFG) NMR is used to observe diffusion of the binary CO/CH4 gas mixture as well as pure CO and CH4 in L-Ala-L-Val (AV) nanochannels. High magnetic field gradients up to 23 T/m are used to track the motion of these molecules over a broad range of diffusion times. It is shown that the diffusion of the pure and mixed gases in the nanochannels occurs according to the mechanism of single-file diffusion. Under similar experimental conditions, the single-file mobility factor of CH4 was found to be smaller than that of CO in nanochannels containing only one type of gas molecules. However, the single-file mobilities were, as expected, identical for both gases in the nanochannels once loaded as a gas mixture. This coincident mixture mobility is intermediate to those obtained in the single-sorbate samples. The relationship between the measured single-file mobilities of the pure gases and that of the gas mixture will be discussed in the context of relevant molecular dynamics simulations of pure and mixed gases under SFD conditions.


68

EPR STUDIES OF HETEROMETALLIC SYSTEMS CONTAINING MnIII AND RARE EARTH IONS Livia B. L. Escobar1,2, Guilherme P. Guedes3, Jon Marbey,2 Stephen Hill,2 Rafael A. A. Cassaro,4 Stéphane Soriano1, Miguel Novak4, Marius Andruh5 and Maria G. F. Vaz1

1Federal Fluminense University, Niterói, RJ 24020150 Brazil; 2National High Magnetic Field Laboratory, Tallahassee, FL 32310 USA; 3Federal Rural University of Rio de Janeiro, Seropédica, RJ 23890000 Brazil; 4Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21941901 Brazil; 5University of Bucharest, Bucharest, 030018 Romania. Single-molecule magnets (SMMs) are molecular compounds with potential application in high-density information storage and quantum information processing.1 The blend of different spin carriers within the same molecular entity is a promising strategy to obtain these kinds of materials. In particular, the design and synthesis of 3d-4f complexes is attracting attention since the discovery that such molecules can behave as SMMs.2 The selection of suitable metal ions is a very important step to obtain compounds with high ground-state spin (S) and significant uniaxial magnetic anisotropy (D). In this context, the high-spin MnIII ion containing four unpaired electrons per ion (S = 2) and uniaxial anisotropy, in combination with rare earth (RE) ions, is appealing for synthesizing coordination compounds that behave like SMMs.3 High-frequency Electron Paramagnetic Resonance (HFEPR) is considered a powerful technique for investigating magnetic properties that are strongly influenced by molecular structure.4 In this work, we will present multi-frequency, high-field EPR studies of the di- and tetranuclear compounds MnIIIREIII, with REIII = Pr, Sm, Eu, and MnIII2 REII2 , with REIII = Gd and Ho, respectively. Derivatives containing diamagnetic ions such as LaIII and YIII have also been studied in order to characterize the zero field splitting parameters of the MnIII ion. Preliminary ac susceptibility measurements have shown that these derivatives exhibit slow relaxation of the magnetization. HFEPR measurements have been performed in the temperature range of 2.5 to 50 K and at high-frequencies between 95 and 430 GHz on a finely ground pressed powder of these compounds. [1] R, Blagg, et.al., Nature Chemistry, 5, 673 (2013). [2] S, Langley, et.al., Inorganic Chemistry, 54, 3631 (2015). [3] G, Guedes, et.al., Inorganic Chemistry, 52, 8309 (2013). [4] G, Craig, et.al., Inorganic Chemistry, 54, 13 (2015).


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SITE DIRECTED SPIN LABELING EPR STUDIES OF NUCLEOBASE DYNAMICS AND CONFORMATIONAL CHANGES IN THE GLYCINE RIBOSWITCH KINK-TURN MOTIF Jackie M. Esquiaqui,1 Eileen M. Sherman,2 Pavanjeet Kaur3, Likai Song3, Jing-Dong Ye,2 Gail E. Fanucci.1

1University of Florida, Department of Chemistry, PO Box 117200, Gainesville, Florida 32611 2University of Central Florida, Department of Chemistry, 4000 Central Florida Blvd, Orlando, Florida 32816 3National High Magnetic Field Laboratory, Department of Physics, Florida State University, Tallahassee, FL 32310, USA Site directed spin labeling (SDSL), when used in conjunction with electron paramagnetic resonance (EPR) spectroscopy, is an effective technique for investigating changes in site-specific dynamics within biological systems. Riboswitches are regions of mRNA transcripts that effectuate genetic regulation upon binding of a cognate ligand through conformational changes and structural rearrangements in RNA secondary and tertiary structure. In the glycine riboswitch, two aptamer domains selectively recognize and bind glycine and, through subsequent interaction with a downstream expression platform, induce regulation of genes associated with glycine metabolism. The recently described kink-turn motif and leader-linker interaction in the glycine riboswitch has been investigated using biochemical methods and was shown to play a functional role in ligand binding. Here we apply a SDSL EPR approach and report results of spectroscopic characterization of conformational dynamics in the kink-turn motif of the Vibrio cholerae (VC) glycine riboswitch. Utilizing optimized T4 DNA ligase mediated splinted ligation, and previously established RNA SDSL strategies, both singly and doubly labeled, 232 nt glycine riboswitch constructs were prepared for continuous wave (CW) X and W-Band EPR and double electron-electron resonance (DEER) experiments. Riboswitch folding was monitored in varying environmental conditions including in the absence and presence of both monovalent and divalent salts and glycine ligand. Resultant EPR line shapes from CW EPR experiments support known structural features of the folded kink-turn motif and unambiguously show differential dynamics between nucleobases within the internal loop upon addition of salts and glycine ligand. Spectra for both sites are indicative of changes in motion in the fast time regime (ps to ns) and the resultant W-Band data nicely demonstrate the enhanced line shape sensitivity at higher frequency thus providing additional site specific evidence for folding of the kink-turn in varying environmental conditions. Furthermore, DEER experiments reveal a predominantly ion-induced conformational shift upon riboswitch folding. These results provide the first spectroscopic evidence regarding kink-turn motif conformational dynamics upon glycine riboswitch folding that corroborate known structural RNA elements and also demonstrates the utility of SDSL to interrogate the influence of environmental conditions upon RNA folding dynamics.


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EPR, ENDOR, AND NMR STUDIES OF CAROTENOID ASTAXANTHIN AND ITS PHOTOPROTECTIVE ROLE UNDER HIGH LIGHT AND SALT STRESS A. Ligia Focsan,1 Nikolay E. Polyakov,2 Peter Molnar3 and Lowell D. Kispert 4 1Valdosta State University, Chemistry Department; 2

Inst. of Chemical Kinetics & Combustion,

Novosibirsk, Russia; 3 Department of Pharmacognosy, Univ. of Pécs, Hungary; 4 The University of Alabama, Chemistry Department At high light intensities photosynthetic organisms gather more light than they need, the excess being quenched to avoid unwanted photochemical reactions. The antenna of photosystem II, the trimeric light-harvesting complex in plants, contains the carotenoid zeaxanthin (Zea) which has a direct role in quenching the chlorophyll excited state through a charge transfer complex Zea•+…Chl•- and subsequent neutral radicals (#Zea•) formation by proton loss from Zea•+. [1,2]

Astaxanthin (Ast), a carotenoid similar to Zea except for the addition of a keto group to both ends of the OH-substituted cyclohexene ring, is not known to have such role in higher plants. However, Ast has been reported to accumulate in unicellular green algae Haematococcus pluvialis as a survival strategy under high irradiance, high salinity, high temperature and nutrient deficiency. This difference was studied by examining the formation of radical cations (Ast•+) and neutral radicals (#Ast•) on metal containing MCM-41 molecular sieves using EPR, Mims ENDOR, ESEEM, CW ENDOR, NMR, mass spectrometry, electrochemistry, and optical measurements.

A previous study [3] on TiO2 irradiated nanoparticles modified by carotenoids has shown a greatly increased charge separation between the photo generated holes trapped at the carotenoid as radical cations and the electrons trapped on the surface as TiO2(etr

–). This facilitates electron transfer to the adsorbed molecular oxygen forming a superoxide radical anion O2

•- which could serve as a proton trap from the weak acid Ast•+ to form #Ast•, a potential quencher of the excited state chlorophyll.

EPR measurements have shown that a metal-substituted MCM-41 matrix increases the formation of #Ast• by an order of magnitude. In the absence of light, CW-ENDOR shows only the formation of Ast•+, with generation of #Ast• upon light irradiation. NMR and ESI-MS confirm the formation of astaxanthin metal complexes. Electrochemical studies show a decrease in the stability of the Ast•+ in the presence of metal ions followed by formation of #Ast•. The stability constants and stoichiometry of such complexes are solvent and salt sensitive, with salts giving Ast•+ instability.[4] Astaxanthin exhibits one of the highest oxidation potential of the naturally occurring carotenoids which enhances its radical trapping ability as a molecule. All these studies point out that carotenoid neutral radicals #Ast• form in the presence of salts, enabling astaxanthin to provide critical photo protection under excess light for the green algae in lakes containing salts. Acknowledgments: Thanks to Prof. Michael Bowman (The University of Alabama) for extensive and valuable comments and ideas. This work was supported in part by The Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Sciences, U.S. Department of Energy, Grant DE-FG02-86ER-13465 and NSF for the EPR instrumentation grants CHE-0342921 and CHE-0079498.

1 A. Magyar et. al. J. Phys. Chem. B, 117, 2239–2246 (2013). 2 A. L. Focsan et. al. Arch. Biochem. Biophys. 572, 167–174 (2015). 2 A. L. Focsan et. al. Arch. Biochem. Biophys. 572, 167–174 (2015). 3 T. A. Konovalova et. al. J. Photochem. Photobiol. A: Chem 162,1-8, (2004). 4 N. E. Polyakov et. al, J. Phys Chem B. 114, 16968-16977 (2010).


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EPR CHARACTERIZATION OF A NOVEL MANGANESE-OXALATE COORDINATION POLYMER Justin Goodsell1, Andrew Ozarowski2, Khalil Abboud1, and Alex Angerhofer1

1University of Florida, Department of Chemistry, 214 Leigh Hall P.O.Box 117200, Gainesville FL 32611 2National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310 A novel bis-oxalato 2-(1-H-pyridyl) imidazole maganese (II) complex was synthesized and studied in single crystal form via x-ray diffraction and Electron Paramagnetic Resonance (EPR). The Manganese complex was doped into a single crystal of the corresponding cadmium complex in order to provide a magnetically dilute sample for EPR study. The complex assembles into two-dimensional polymer chains where each metal atom is coordinated by two oxalate molecules and neighboring chains are held together via ligand interactions. X-band single-crystal rotation and high field (206 GHz) powder data are presented along with simulation parameters. Temperature variation studies were also performed at high field.

EPR spectrum collected at the National High Magnetic Field Laboratory with microwave frequency 208 GHz, 5K, 2 mT/second scan speed 50kHz modulation frequency


72

PCB-based 1H/X/Y MAS NMR Probe Design Combines Efficiency with Versatility

Peter L. Gor’kov1, Jason A. Kitchen1, Ilya M. Litvak1, Ivan Hung1, William W. Brey1

1National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310

USA

PC boards provide versatile and convenient building blocks for solid-

state NMR probe engineers. We present electro-mechanical design of

triple-resonance 1H/X/Y 800 MHz MAS probe that compacts most

of X/Y circuit components on two removable PCB cards. Such "tune

card" approach, inspired by SIM cards in older cell phones, allows for

diverse variety of X and Y isotope configurations as well as S/N

optimizations for X- or Y-detection; all done on a single probe

frame. We discuss reduction of construction costs by using PCBs, 3D-

printing, and examine strategies that improve direct detection S/N:

designing Low-E RF coils for higher magnetization transfer in multi-CP

experiments and reducing losses in probe isolation traps. We will also

report on construction progress of a similar 1.5 GHz MAS probe for

NHMFL’s upcoming Series-Connected Hybrid magnet.

[1] Gor’kov et al., J. Magn. Reson. 2007; 185, 77.

[2] McNeill et al., J. Magn.

800 MHz 3.2 mm 1H/X/Y MAS probe with most of its RF circuit built on sliding tune cards.


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ELECTRON PARAMAGNETIC RESONANCE AND 57FE MÖSSBAUER CHARACTERIZATION OF COMPLEXES CONTAINING METAL-METAL BONDS Samuel M. Greer,1,3 Sebastian A. Stoian3, Christine Thomas4, Stephen Hill2 1Florida State University, Department of Chemistry, Tallahassee, FL 32306 2Florida State University, Department of Physics,, Tallahassee, FL 32306 3National High Magnetic Field Laboratory, Tallahassee, FL 32310 4Brandeis University, Department of Chemistry, Waltham, MA 02453 Electron transfer processes typically require expensive second and third row transition metals, a more fiscal alternative would utilize earth abundant first row transition ions. These elements are not as redox active as their second and third row counterparts but by creating metal-metal bonds of first row transition metals it may be possible to create species with redox activity viable for catalytic application. While metal-metal bonds have been extensively studied in second and third row ions relatively little is known about the bonding between two first row transition metals. To this end a series of complexes containing Fe-Fe bonds has been extensively characterized through multi-frequency electron paramagnetic resonance and variable field Mössbauer spectroscopies. The entire set of Mössbauer and EPR spectra was analyzed in the framework of a standard spin-Hamiltonian. [1] S. Kuppuswamy, et al. Chem. Sci. 2013, 4, 3557-3565. [2] S. Kuppuswamy, et al. Inorg. Chem. 2013, 52, 4802-481.


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INTERACTION OF HIV GP41 WITH THE CHOLESTEROL-RICH VIRAL MEMBRANE STUDIED BY EPR Zahra Hayati1, Shenglong Ling1, Lu Yu1, Pavanjeet Kaur1, Hans Van Tol1, Ellis L. Reinherz2 and Likai Song1

1National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310; 2Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115 HIV enters human T cells through the fusion of viral and host-cell membranes. This fusion process is mediated by a surface protein, gp41, and the platform provided by the lipid raft-like viral membrane, which has high cholesterol content. Here, multi-frequency EPR has been used to dissect the properties of the raft-like viral membrane, and to characterize its interactions with the membrane proximal ectodomain region (MPER) and the transmembrane domain (TM) of gp41. The results show that the MPER and the MPER-TM induce significant mobility and permeability changes of phospholipid membranes, suggesting a membrane-perturbing activity of gp41 during viral fusion. However, only the MPER-TM is capable of inducing permeability changes of raft-like membranes containing cholesterol. This indicates that the TM is interacting with the cholesterol-binding domains of the viral membrane. Next, EPR at 9, 95 and 240 GHz was used to define the properties of HIV virion mimic membranes and the cholesterol binding domains. The lipids near the cholesterol binding domains show decreased mobility, enhanced polarity, significant lateral ordering, and increased phase transition temperature. In sum, these findings have revealed the distinctive features of the raft-like cholesterol-rich HIV membrane and how gp41 exerts its fusogenic activity by specifically targeting and perturbing raft-like viral membranes.


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REAL-TIME OBSERVATION OF MELITTIN DISRUPTING NANOTUBLAR LIPID BILAYERS BY ORIENTED-SAMPLE NMR

Morteza Jafarabadi, Antonin Marek, Alexander A. Nevzorov, and Alex I Smirnov Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204

Solid-state (ss) NMR spectroscopy is a powerful biophysical method for obtaining structural data on proteins and their interactions with lipid bilayers at conditions closely mimicking those in biological cells (e.g., temperature, pH, ionic strength, etc.). Glass-supported bilayers and magnetically aligned bicelles are the two main approaches for the preparation of macroscopically aligned samples. However, a limited spectral resolution in the former case and a narrow set of options for choosing the lipid composition for bicelles are their most obvious drawbacks of these two methods. To overcome these limitations a new method based on confining lipid bilayers inside anodic aluminum oxide (AAO) nanopores has been developed. The method is suitable for 2D experiments with uniformly labeled transmembrane peptides and is applicable to bilayers of almost any lipid composition under a wide range of environmental conditions [1].

In this study, we have obtained exceptionally narrow (100-140 Hz, which is less than or about 1 ppm) 31P resonances of phosphate groups of macroscopically aligned nanotubular bilayers of various compositions to observe their interactions with melittin. Melittin is an antimicrobial peptide with 26 aminoacids and is the active component of the honey bee venom [2]. Zwitterionic POPC and ionic POPG with different ratios were deposited on a few stripes of an in-house fabricated AAO with pore diameter of 78 nm. Oriented-sample ssNMR spectra were taken with the external magnetic field parallel to the pore cylindrical axis (Figure1).

Figure1: Left: 7 T 31P-NMR spectra of AAO supported POPC plotted at various times of interaction with melittin; Middle: AAO cross section; Right: A cartoon of a nanotubular biayer inserted into an AAO nanochannel.

A narrow 31P-NMR signal of pure POPC at ca. -15 ppm indicates that the bilayer has a uniform cylindrical structure aligned with respect to the external magnetic field. A 50 uM solution of melittin was added to the sample and then 31P-NMR spectra were recorded repeatedly over time (Figure 1). The observed decrease in the intensity and broadening of the 31P line width were consistent with melittin binding to the lipid bilayer; while the consequent shifts of the 31P line to the left indicated formation of smaller vesicles. The eventual washing of POPC lipids out of the nanopores resulted in a complete disappearance of the 31P signal. The addition of anionic POPG to the initial composition of the lipid bilayers slowed down the kinetics of disruption of the bilayer structure by melittin. Based on the experimental data, we conclude that the positively charged melittin interacts strongly with the negatively charged POPG forming stable complexes/ aggregates that remain within the AAO channels, thus affecting the bilayer destruction kinetics.

References

1. Marek, W. Tang, S. Milikisiyants, A. A. Nevzorov, A. I. Smirnov, Biophys. J. 108(1), 5-9 (2015). 2. H. Raghuraman, A. Chattopadhyay, Biosci Rep., 27, 189-223 (2007).


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SITE-SPECIFIC STRUCTURE AND DYNAMICS OF THE TUBULAR ASSEMBLY OF ROUS SARCOMA VIRUS CAPSID PROTEIN BY SSNMR AND TEM Jaekyun Jeon1, Xin Qiao1, Ivan Hung2, Peter L. Gor’kov2, Zhehong Gan2, Fangqiang Zhu3 and Bo Chen1 1Department of Physics, University of Central Florida, Orlando, FL, USA 2National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL, USA 3Department of Physics, Indiana University – Purdue University Indianapolis, IN, USA Rous Sarcoma Virus (RSV) belongs to retroviral family. Its capsid (CA) protein shares little sequence similarity with that of HIV, but exhibits common 3D structure and similar assembly properties. They assemble into polymorphic polyhedra in vivo and a range of structures in vitro. Due to the strong polymorphism, such assemblies are difficult to resolve by conventional structural biology techniques such as X-ray diffraction or cryo-electron microscopy. Here we present nearly complete (233 out of 237) sequential assignment of RSV CA proteins in their tubular assembly by ssNMR. We demonstrate that highly uniform tubular assembly can be prepared in suitable conditions. Screened by TEM, our tubular assembly exhibits sharp 6 fold symmetry diffraction, indicating quality similar to quasi-crystalline samples. With such samples, we obtained spectra with good resolution where linewidth of 13C and 15N are 0.4 ppm and 0.6 ppm, respectively. A series of ssNMR spectra were acquired with uniformly 13C and 15N labeled samples, including 2D and 3D NCACX, NCOCX and CANCX. In spite of the sharp linewidth, signals with 15N resonances between 116-120 ppm still exhibit considerable congestion. To further resolve signals unambiguously, selectively labeled samples (1, 3-13C and 2-13C labeled glycerol) were prepared. In addition, two samples with one residue uniformly 13C labeled (leucine and arginine) were prepared to further consolidate the assignments, since they are the most populated residues in the sequence and show up in the most congested spectral region. Combined, we were able to sequentially assign 233 out of the 237-residue CA protein in its tubular assembly, assisted by the Monte Carlo Simulated Annealing (MCSA) method with limited guidance of comparison with solution NMR chemical shift result [1]. Based on the sequential assignments, secondary structure and dynamics were predicted by Talos N. Finally a tertiary structure of the capsid protein was obtained by all atomic annealing simulation, and detailed discussions of interaction sites in the assembly will be presented. [1] R. Campos-Olivas, J.L. Newman, M.F. Summers, Journal of Molecular Biology, 296 633-649 (2000).

Figure 1. Representative sequential assignments according to NCACX, NCOCX and CANCOCX.


77

Fig 1: Magnitude image of the phantom

Fig 2: (Top) Simulated magnetic field maps and corresponding line plots obtained from COMSOL and MATLAB. (Bottom) Experimentally measured induced magnetic field maps.

MEASUREMENT OF MAGNETIC FIELD DUE TO LOW AMPLITUDE CURRENT INJECTION INTO HYDROGEL USING CARBON ELECTRODES. Aditya Kumar Kasinadhuni1, Chris Anderson1, Munish Chauhan2, Paul Carney1, Rosalind Jane Sadleir2, Thomas Mareci1,3 1University of Florida, Department of Biomedical Engineering. 2Arizona State University, Department of Biomedical Engineering. 3University of Florida, Department of Biochemistry and Molecular Biology. Introduction: Electrical stimulation therapies are used in the treatment of a gamut of disorders like Parkinson’s disease, Alzheimer’s etc. Transcranial direct current stimulation, a neuromodulation technique, has shown great promise in improving cognition in humans but very little is known about its mechanism. MREIT can be used to visualize the regions of current density and prove to be extremely useful for providing insight into the effectiveness of these therapies. MREIT tracks changes in the phase of MR signal produced due to induced currents(1). The magnetic field changes due to the induced currents can be calculated from these phase changes and provide insights into the underlying conductivity of the imaging object. Materials: A cubic phantom of 45 mm length containing an agarose gel was imaged with a volume transmit birdcage coil using the 4.7 T Agilent magnet system at the McKnight Brain Institute, University of Florida. A current of 0.2 mA was delivered via 250 µm thick carbon electrodes coated with PVDF (Polyvinylidene fluoride) that were secured to brass screws with conductive silver epoxy. Two spin echo datasets acquired with opposite current polarity were complex divided to extract the phase information which was scaled to obtain the induced magnetic field. A computational model of the experimental setup was built using COMSOL and the simulated magnetic field was compared to the experimentally acquired field map. Results & Discussion: Fig 2 shows the simulated magnetic field maps and the experimentally measured field maps. The simulation assumes a noise free environment which serves to explain the variation in the quantification values. Susceptibility artifacts commonly observed near the tip of the electrodes in such measurements appear to be minimal with the use of carbon electrodes. Improvement of signal to noise ratio in the measurements is warranted and pulse sequences (2) that are more sensitive to the minute phase changes are in consideration. Acknowledgements: This work was funded by the (NIH R01 NS077004, title: Direct Functional Imaging of Electrical Brain Stimulation). We thank the AMRIS staff at the McKnight Brain Institute for assistance in troubleshooting problems during data acquisition. References: 1. G.C. Scott et al. IEEE Trans Med Imaging 1991;10(3):362-374. 2. O.I. Kwon et al. Physiol Meas 2007;28(11):1391-1404.


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HIGH-FIELD EPR INVESTIGATIONS OF PROTEIN ORIENTATIONS ON MAGNETICALLY

ALIGNED BICELLES Pavanjeet Kaur1 and Likai Song1 1National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310

High-field electron paramagnetic resonance (EPR) spectroscopy is suitable for characterizing the orientations of membrane proteins on magnetically aligned lipid bilayers – bicelles. Here, we have examined the field dependence of the self-aligning of bicelles containing phospholipids DMPC/POPC/DHPC with and without cholesterol. EPR spectra of bicelles with trace amounts of spin-labeled lipids were compared on a Bruker X-/W-band EPR spectrometer and a recently developed quasioptical 94 GHz EPR spectrometer (HiPER). Enhanced bicelle alignment was found at magnetic fields ranging from 0.3 to 8 Tesla. Next, using the aligned bicelles, the orientation of the membrane proximal ectodomain region (MPER) of HIV envelope protein gp41 relative to the bilayer was investigated. In order to obtain an EPR signal, the MPER was site-specifically modified by a rigidly-bound spin label - 2,2,6,6,-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). A structural model of the MPER in a bilayer was developed using a combination of EPR orientation and immersion depth measurements. These results illustrate the improved sensitivity of EPR at high fields for elucidating protein topology on the membrane.


79

ENHANCING COHERENCE TIME IN MOLEULAR MAGNETS USING CLOCK TRANSITIONs Dorsa Komijani1,2, Muhandis Shiddiq1,2, Yan Duan3, Alejandro Gaita-Arino3, Eugenio Coronado3, Stephen Hill1,2 1National High Magnetic Field Laboratory, Tallahassee, FL 32310; 2Florida State University, Department of Physics, Tallahassee, FL 32306; 3Instituto de Ciencia Molecular (ICMol), Universidad de Valencia, C/ Catedrático José Beltrán 2, 46980 Paterna, Spain One of the challenges in the field of quantum information processing is protecting qubits from different sources of decoherence. Spin quibts are one of the candidates for use as qubits. For experiments carried out at low temperatures, the primary source of decoherence in spin qubits is dipolar interactions. This can be minimized using clock transitions. Clock transition are referred to the transitions that are robust against external perturbation [1]. Use of the clock transitions leads to an enhancement in phase coherence time. In this work, we examine the possibility of suppressing some of the decoherence processes by using clock transitions in Holmium Polyoxometalates of general formula [Na]9 [HoxY1-x(W5O18)2] [2,3] where Y is non-magnetic. Different concentrations were studied in which the enhancement in coherence time at clock transitions was confirmed. Furthermore, we investigate the effect of disorder on the transition frequency. This disorder contributes to a Gaussian distribution of the off diagonal parameter in Zero Field Splitting Hamiltonian. This will results in the vertical broadening of the transition frequency which explains the results for the more concentrated sample. While CW EPR is a powerful technique to study the static properties of electron spin, studying the dynamic properties requires spectroscopy in the time-domain. One of the most common pulse EPR applications is to study spin-spin relaxation time, T2, using a two-pulse Hahn-echo sequence. Pulse EPR measurements were performed using a Bruker E680 X-band spectrometer at a temperature of 5 K. Coherence time has been studied for different concentrations of Ho(III). Here, we present the results for x=0.001 and x=0.1 to study the effect of dilution on electron spin coherence time. This study suggests that aside from dilution, use of clock transitions is a powerful method to protect electron spins from magnetic noise. [1] G. Wolfowicz, et al., Nature Nanotechnology 8, 561 (2013) [2] M. A. AlDamen, et al., Inorg. Chem. Soc., 48, 3467 (2008) [3] S. Ghosh, et al., Dalton Trans., 41, 13697 (2012) [4] Clemente-Juan, J.M. et al., Chem. Soc. Rev. 41, 7464-7478 (2012).


80

DRAMATIC FOUR-FOLD SENSITIVITY ENHANCEMENT BY A COMBINATION OF REPETITIVE CROSS-POLARIZATION AT LOW RF AMPLITUDES AND PARAMAGNETIC RELAXATION ENHANCEMENT FOR SOLID-STATE NMR OF ORIENTED MEMBRANE PROTEINS Sophie Koroloff, Deanna Tesch, Alexander Nevzorov

Dept. of Chemistry, North Carolina State University, 2620 Yarbrough Dr, Raleigh, NC 27695-8204 Solid-state NMR on oriented membrane proteins (MPs) provides structural and dynamic information under nearly physiological conditions. However, the low sensitivity of dilute low-gamma spins (e.g. 13C or 15N) necessitates a large number of scans, thus considerably lengthening the experimental time in multidimensional NMR experiments. We have investigated several cross-polarization schemes applied to Pf1 coat protein reconstituted in magnetically aligned bicelles at the perpendicular and parallel orientations relative to the main magnetic field. Optimization of the repetitive cross-polarization scheme (REP-CP) at relatively low RF amplitudes (19 kHz vs. the commonly employed 50 kHz) yields an increase of the magnetization transfer from the 1H spins to the 15N sites by more than 30% [1], therefore allowing us to cut the experimental time by 60%. In addition, the REP-CP sequence proves to be superior to the conventional cross-polarization schemes at both parallel and perpendicular orientation of the bilayer. Moreover, the presence of a membrane-bound radical (5-doxyl stearic acid) at the 2:1 M radical:protein ratio allows one to reduce the recycle delay by nearly three-fold without appreciably affecting the experimental linewidths [2]. Taken together, these benefits allowed us to obtain a nearly four-fold dramatic signal enhancement relative to the conventional experiment acquired in the same experimental time. [1] S. N. Koroloff and A. A. Nevzorov, J. Magn. Reson., 256, 14-22 (2015) [2] D. M. Tesch and A. A. Nevzorov, J. Magn. Reson., 239, 9-15 (2014)


81

STUDY OF THE EFFECT OF DEUTERATION OF GLASSING AGENT AND GD3+ DOPING ON 13C HYPERPOLARIZATION AT 5 T Bimala Lama,1, Andhika Kiswandhi2, Daniel Downes1, James Collins1, Adam Smith3, Lloyd Lumata2, Joanna R. Long1

1University of Florida, Department of Biochemistry and Molecular Biology, Gainesville, FL 32610. 2University of Texas at Dallas, Department of Physics, 800 West Campbell Road, Dallas, Texas 75080, USA. 3University of Florida, Department of Chemistry, Gainesville, FL 32610 Dissolution dynamic nuclear polarization technique involves the polarization of the low abundance nuclei such as 13C, 15N by transferring the polarization from the electron of stable radicals using microwave radiation at very low temperature, and the rapid dissolution of the hyperpolarized substance with hot solvent followed by injection of the solution into a sample or organisms. The signal enhancement obtained from dissolution DNP makes it possible to study biochemical phenomenon and diseases in real time in vivo by 13C NMR/MRI. The extent of polarization of the nuclei is dependent upon different factors like the mechanism of polarization transfer, magnetic field strength, microwave sources, temperature, and sample matrix etc. Our studies focus on optimization of polarization of different metabolic substrates at 5 T and ≤1.2 K and DNP signal enhancement is being investigated using different radicals in different conditions. We studied the effects of deuteration and Gd3+ doping on 13C hyperpolarization of [1-13C] sodium acetate. The result shows that deuteration of solvents enhances the 13C nuclear polarization when 4-oxo-TEMPO free radical is used, but results in decrease of polarization in the case of trityl OX063. Similarly, an addition of trace amount of Gd3+ decreases the polarization in the sample with 4-oxo-TEMPO as a radical, but improves the nuclear polarization when trityl OX063 is used.


82

1H AND 6/7LI SOLID-STATE NMR STUDIES OF Li4Ti5O12 ANODE IN LITHIUM-ION BATTERIES Xiang.Li, XuYong.Feng and Yan-Yan.Hu Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL 32306 Spinel-type Li4Ti5O12 has been used as anodes in Lithium ion batteries (LIBs), which exhibits excellent stability and reversibility. Our recent investigations have revealed an intriguing phenomenon, i.e., the correlation of H+ content and the asymmetric rate performance when this this Li4Ti5O12 anode is used in a half-cell LIB. Electrochemical tests and 1H NMR measurements show that if the discharging rate is fixed at 1C, without H+ in Li4Ti5O12, the charge capacity stays constant even with increasing the charging rate. On the other hand, with residual H+ in Li4Ti5O12 as a result of the synthesis process, the charge capacity decreases as the charging rate increases. This indicates that H2O and H+ play an import role in the lithiation/delithiation processes. High-resolution 7_Li and 6_Li NMR were also employed to probe the local environment and dynamics of Li ions. [1] Y. Sun, L. Zhao, H. Pan, et. al., Nat.Commun., 4, 1870-1879 (2013). [2] M. Wagemaker, E. Eck, A. Kentgens, et. al., J.Phys.Chem.B.,113, 224-230 (2009). [3] H. Hain, M. Scheuermann, R. Heinzmann, et. al., Solid State Nul Mag, 42, 9-16 (2012).


83

IMPLICATIONS OF ALTERED SALT BRIDGING INTERACTIONS ON HIV-1 PR FLAP CONFORMATIONS, LOCAL STRUCTURES AND BACKBONE DYNAMICS Zhanglong Liu1, Xi Huang1, Lingna Hu1, Linh Pham1, Katye Poole2, Yan Tang2, Brian P. Mahon2, Wenxing Tang2, Kunhua Li1, Nathan E. Goldfarb2, Ben M. Dunn2, Robert Mckenna2, Gail E. Fanucci1*

1 Department of Chemistry, University of Florida, Gainesville, 32611 2 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610 * To whom correspondence should be addressed: [email protected] HIV-1 protease (PR) serves as an important drug target because its inhibition generates immature non-infectious virions. Specifically, HIV-1 PR flap conformational sampling is of great importance due to its close relationship with the viral functions and inhibitor binding, where open-like states facilitate accommodation of substrates/drugs inside the catalytical pocket and the closed state is stabilized upon substrates/drugs binding. [1] Drug-pressure selected mutations, along with natural polymorphisms, can lead to drug resistance to HIV-1 protease inhibitors (PIs), which change the drug binding configuration and equilibrium flap conformational sampling. As was reported in our previous work, HIV-1 PR adopts different conformations such as closed, semi-open and wide-open ensembles [2] and the flap conformations are closely associated with the enzyme kinetics and drug inhibition parameters. [3] As was suggested in another study [4], a new flap conformation, curled-open, was identified in constructs carrying natural polymorphisms including E35D, and consequently, these constructs displayed higher protein backbone dynamics compared with wild-type subtype B construct characterized by nuclear magnetic resonance (NMR) spectroscopy. In the present work, we discuss the impact of the single E35D natural polymorphism on generating a minor flap conformation (curled-open), and consequently, influencing the protein structure and dynamics. The combined results from double electron-electron resonance (DEER), NMR spectroscopy and X-ray crystallography structures provide a possible structural mechanism for altered conformational dynamics. [1] V. Hornak, et. al., P Natl Acad Sci USA 103, 915-920 (2006) [2] M. E. Blackburn, et. al., Biochemistry 48, 8765-8767 (2009) [3] I. M. S de Vera, et. al., Biochemistry 52, 3278-3288 (2013) [4] X. Huang, et. al., J. Biol. Chem. 289, 17203-17214 (2014)


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IDENTIFYING CYTOCHROME P450 BINDING MOTIFS WITH CW EPR LIGAND FIELD SHIFTS Molly M. Lockart, Shawna M. Blankenship, Alex A. Cruce, and Michael K. Bowman

Department of Chemistry, The University of Alabama, Box 870336, 35487-0336, Tuscaloosa, Alabama, USA Cytochrome P450s (CYPs) are a large class of heme-containing monooxygenase enzymes present in nearly every living organism that oxidize a wide variety of substrates in biosynthetic and detoxification pathways. Because of their role in detoxification and drug metabolism, CYPs are a common target for drug design. Drugs can displace the water and bind directly to the heme, displace the water and occupy the binding pocket without directly binding to the heme, or bind through a water bridge to the heme. The three binding motifs are often characterized using optical difference spectra, which can be ambiguous and difficult to assign. For this reason, many binding motifs have been mischaracterized and are not well understood. Electron Paramagnetic Spectroscopy (EPR) has been used to demonstrate differences in the substrate-bound and resting state enzymes in the low spin state. Pulsed EPR methods have been used previously to locate nearby nuclei and distinguish between direct coordination and water-bridged binding motifs. This study demonstrates how analysis of continuous wave (CW) EPR spectra of CYPs can provide enough information to determine the binding motif. Four isoforms in complex with a variety of drugs were analyzed and simulated using EasySpin. The spectra contain a variety of distinct species that make up the gz peak both in the resting state and with a drug bound. Analysis of the difference between the gz values of the resting complex and the gz values of the bound form showed clear shifts that distinguished the directly coordinated and water-bridged complexes. The tetragonal field (Δ/λ) and rhombicity (V/ Δ) were then calculated for the species in each spectrum. The resulting plot revealed distinct clusters that separated the water-bridged and directly coordinated compounds (Figure 1). Further analysis of the spectral species could provide valuable information about the method of substrate binding in each isoform and help to explain unresolved obscurities in the optical spectra. We thank W. M. Atkins, NIH GM110790, Drs. Kip Conner, Matt Kryzaniak, and Preethi Vennam, the NSF REU program (CHE 1358971 to Professors J. B. Vincent and S. A. Woski), and the Department of Chemistry at the University of Alabama for support.

. Figure 1: Scatter plot of the tetragonal field and rhombicity of each simulation component. The red cluster represents components that show direct coordination and the blue cluster represents components that are water-bridged. Components highlighted in yellow are tentative correlations.


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CONSTRUCTION OF A CONTINUOUS FLOW HIGH-FIELD PASADENA HYDROGENATION REACTOR FOR ANALYSIS OF STEREOSELECTIVITY OF CONCERTED ADDITION TO TRIPLE BONDS Kaylee Ludden,1 Evan Zhao,1 Helena E. Hagelin-Weaver2 and Clifford R. Bowers 1* ([email protected]) 1Department of Chemistry, University of Florida. Gainesville, FL; 2Department of Chemical Engineering, University of Florida, Gainesville, FL

Parahydrogen Induced Polarization (PHIP) NMR spectroscopy was discovered in 1987 and has since been demonstrated as an excellent candidate in sensitive diagnostic magnetic resonance imaging and a powerful tool to study catalytic reactions. PHIP in heterogeneous catalysis (Het-PHIP) has become recognized as a phenomenal way to study catalytic reactions. In using separate phases for the catalyst and reactant, it is possible to create a continuous flow of catalyst-free hyperpolarized product. Although Het-PHIP offers a multitude of excellent advantages, there remain major challenges to broaden its applicability. Het-PHIP is only able to produce a limited range of hyperpolarized products due to the requirement of hydrogen addition, and the polarization level is within a few perfect of theoretically predicted values. By gaining a better fundamental understanding of Het-PHIP and its associated reaction mechanisms, PHIP can become a powerful and economic method to produce hyperpolarized materials. In this study, a density matrix simulation was used to study a catalyzed parahydrogen and propyne reaction as a function of reaction conditions. By studying the partial hydrogenation selectivity and pairwise selectivity, hidden reaction mechanisms are revealed. Our results show that pairwise syn addition stereoselectivity is correlated to partial reduction selectivity. By systematically varying the reaction conditions, it was found that low parahydrogen to propyne ratio and high reaction temperature favors syn addition and high partial reduction selectivity. On the contrary, high parahydrogen to propyne rations and low reaction temperatures favors anti addition and low partial reduction selectivity. To verify the density matrix simulations shown in the figure, a high field reactor was constructed to be inserted into the bore of the magnet. This compact reactor was beneficial in allowing for a direct comparison between the high field reactions and the low field reactions by being able to achieve the same temperatures and partial pressure of reactant species as had been used under both conditions.

[1] Bowers and Weitekamp, Phys. Rev. Lett. 57, 2645-2648 (1986); Bowers and Weitekamp, J. Am. Chem. Soc. 109, 5541-5542 (1987). [2] Koptyug, I. V. et al., J. Am. Chem. Soc. 129, 5580-5586 (2007). [3] Zhou et al. J Am Chem Soc 137, 1938-1946 (2015). [4] Zhao et al., Angew. Chem. Int. Ed., doi 10.1002/anie.201506045 (2015). [5] Zhou et al., PCCP, Early View (2015).


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HFEPR STUDIES OF ZFS IN A TRIGONAL MN(III) COMPLEX Jonathan Marbey1, En-Che Yang, Stephen Hill1 1National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310 USA; 2Fu Jen Catholic University, Chemistry Department, Taipei Combining transition metal ions with significant magnetic anisotropy has become a common strategy to obtain Single Molecule Magnet (SMM) materials. In this respect, trigonal metal complexes have proven to be an interesting candidate to further study the magnetic properties that give rise to Zero Field Splitting (ZFS) 1. In our study, we look at a ferromagnetically coupled Mn3 triangle containing rigorous C3 symmetry with the Jahn-Teller axes tilted ~60° out of the plane of the triangle. This relatively extreme Euler angle projects much of the magnetic anisotropy into the trigonal plane, suppressing most of the 2nd order ZFS, but leading to significant 4th order (and possibly higher) corrections in a giant spin approximation. While not a particularly useful SMM, these small values provide a unique opportunity to study the full EPR spectrum within a feasible field range. This gives great insight into the conditions that control both the magnitude and sign of the ZFS when simply considering the symmetry relating the individual magnetic sites.

We studied this sample using HFEPR in an attempt to quantify the ZFS. The sample was finely ground/pressed into an EPR cup and measured in a 15T Oxford magnet within a frequency range of 95 – 285 GHz at liquid helium temperatures. Since the Euler angles are close to the ‘magic angle’ for which D switches sign, many of the spin dependent energy states are not well separated, thus resulting in complicated and not easily predictable spectra as frequency is varied. This proves to be computationally difficult system to simulate. Future angle dependent measurements will be performed on a single crystal to further constrain the spin Hamiltonian parameters.

[1] Hill S. et. al, Dalton Transactions, 39, 4693-4797 (2010).


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OPTICAL DYNAMIC NUCLEAR POLARIZATION IN STRAINED GALLIUM ARSENIDE Lauren A. McCarthy1, Ryan M. Wood1, John T. Tokarski III1, Dipta Saha2, Christopher J. Stanton2, Clifford R. Bowers1,2

1Department of Chemistry, University of Florida, Gainesville, FL USA 32611 2Department of Physics, University of Florida, Gainesville, FL USA 32611 We present a new methodology for the measurement of lattice strain and strain relaxation in GaAs that utilizes optical dynamic nuclear polarization (ODNP) and NMR. After epoxy bonding of the GaAs single crystal to a Si support wafer, biaxial tensile strain in the GaAs layer was induced by differential thermal contraction upon cooling to ~5K. Strain is manifested as a quadrupole splitting in the NMR resonance. The observed quadrupole splitting is proportional to the strain in the region of the sample within the penetration depth of the laser light, which above the bandgap is about one micrometer.[1] Due to the relatively shallow penetration depth, this technique is sensitive only to the strain near the surface of the GaAs. GaAs crystals were epoxy bonded to Si supports of two different thicknesses and successively thinned using selective chemical etchants. The results indicate that the strain is relaxed by mechanical bowing of the composite structure. The experimental results were compared to a theoretical model for mechanical bowing and agreement was attained. In the absence of strain, simultaneous pumping of electrons from both the light hole and heavy hole of the valence band to the conduction band limits the electron spin polarization, and relatedly, the nuclear spin polarization. Strain lifts the degeneracy of the light hole and the heavy hole at K=0 [2] which can result in selective pumping of electrons from the light hole to the conduction band at low photon energies or from the heavy hole to the conduction band at high photon energies. Therefore, selective optical pumping can allow nuclear spin polarizations to approach unity and can result in changes in the sign of the nuclear spin polarization. The electron spin polarization under ODNP conditions was estimated from the theoretically calculated absorption coefficients and the nuclear spin polarization was calculated based on the standard scalar electron-nuclear spin cross-relaxation model.[3,4] The results demonstrate the feasibility to manipulate nuclear spin polarization via control of strain in bulk GaAs. Potential application to nuclear spin electronics will be discussed. The combination of ODNP-NMR measurements with electronic band structure calculations provides a new way to probe the effects of strain in III-V semiconductors with promising applications. [1] M. Sturge, Phys. Rev. 127, 768 (1962) [2] Y. Sun, et. al., Strain Effects in Semiconductors: Theory and Device Applications (Springer, 2010). [3] P.L. Kuhns et al., Phys. Rev. B. 55, 7824-7830 (1997). [4] R.M. Wood et al., Phys. Rev. B. 90, 155317 (2014)


88

Q-BAND HYSCORE INVESTIGATION OF SUBSTRATE BINDING TO CHLOROCATECHOL 1,2-DIOXYGENASE: THE ROLE OF NATIVE AMPHIPHILIC MOLECULES. Sergei Milikisiyants,1 Antonio J. Costa-Filho,2 Luís Felipe Santos Mendes,2 Mariana Chaves Micheletto,2 Tatyana I. Smirnova,1 Alex I. Smirnov1

1North Carolina State University, Department of Chemistry, Raleigh, NC, USA 2 Departament of Physics, Universidade de São Paulo, RIbeirão Preto, SP, Brazil Intradiol dioxygenases, non-heme iron enzymes that biodegrade recalcitrant compounds, such as catechol and its derivatives, have recently attracted much attention as potential agents for bioremediation. Discovery of unexpected amphipatic molecules, observed in crystal structures of intradiol dioxygenases, brought up questions regarding the role these molecules play in catalytic proses. Removal of the lipid molecule associated with intradiol enzyme chlorocatechol 1,2-dioxygenase (1,2-CCD) from Pseudomonas putida has been shown to modify thermal stability and kinetic profile of catechol oxidation [1]. The lipid binding site is located at the protein dimer’ interface and is remote from the iron center [2], so the mechanism by which lipid binding modulates the enzyme activity remains speculative. Here we employed pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy to investigate changes in the iron (III) coordination upon substrate binding for native, lipid-containing, and delipidated forms of 1,2-CCD. Samples of 1,2-CCD with catechol were prepared and kept under anaerobic conditions to preserve the enzyme-substrate complex. Q-band (35 GHz) HYSCORE experiments performed at T=6 K demonstrate that removal of the native lipid from 1,2-CCD does not result in changes of iron coordination in the absence of the substrate, suggesting that structural changes in the enzyme upon lipid binding (if any) do not propagate to the iron center. Upon catechol binding to either lipid containing or delipidated 1,2-CCD the substrate displaces the water molecule coordinated to the non-heme iron. Significant changes were also observed in nitrogen signals from two Histidine ligands (His194 and His196), in the axial and equatorial ligations. The observed changes are consistent with crystallographic observations of the substrate replacing water and Tyr as the iron ligands in 4-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus. Binding of the substrate also results in lower ZFS of iron. Comparison of HYSCORE spectra from ES complexes formed by native lipid containing and delipidated 1,2-CCD show small changes in the nitrogen ligation suggesting that the lipid binding affects positioning of the substrate in ES complex. Supported by UPGN grant to AJCF and AIS. [1] Mesquita, N.C., et al (2013) Eur Biophys J. 42(8):655-60. [2] Micalella C, et al (2011) Biochimica et Biophysica Acta, Proteins Proteomics 1814: 817–23.

Figure 1. Q-band HYSCORE spectra from 1,2-CCD in (A) delipidated form and (B) upon catechol binding under anaerobic conditions. Spectra recorded at recorded at the field position corresponding to the g=4.20 feature at T=6 K and τ=160 ns.

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89

CHARACTERIZATION OF PROTEIN SURFACE AND COUPLED SOLVENT DYNAMICS BY USING

SPIN-LABELED B12-DEPENDENT ETHANOLAMINE AMMONIA-LYASE AND EPR SPECTROSCOPY

Benjamen Nforneh1 and Kurt Warncke1.

1Department of Physics, Emory University, Atlanta, GA 30322

The mobility of a protein surface-attached spin label was studied over the temperature (T) range of 190-

260 K in the ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium. The goal is to identify and

characterize coupling among protein surface dynamics and the solvent dynamics in the surrounding EAL

protein mesodomain1. EAL was spin-labeled specifically at the solvent-accessible, native Cys37 site on

the EutC subunit2 by using 4-maleimido-TEMPO (4MT). Continuous-wave (CW), X-band electron

paramagnetic resonance (EPR) spectroscopy was used to observe the degree of motion of the 4MT label

as a function of T, as revealed by the EPR line shape. The mobility of the 4MT label was quantified by the

rotational correlation time (τc) by using EPR simulations. The spin label is detectably immobile (τc >10-6.3

s) from 190 to ~210 K, which is consistent with the solid state of the solvent mesodomain1. Over the T

range of 210 to 225 K (dependent upon co-solutes), the 4MT label on the EAL protein undergoes a

mobility transition (at Tt,prot ; τc <10-6.3 s), and at T>Tt,prot, the spin label displays activated tumbling

behavior. The Tt,prot value can be manipulated by added (≤4 %v/v) dimethylsulfoxide (DMSO). The

lowering of Tt,prot by DMSO is opposite to the effect of added sucrose on the mesodomain solvent

dynamical transition (Tt,solvent, increase in value), which was characterized by using the spin probe,

TEMPOL1. Preliminary CW-EPR microwave power saturation studies suggest that the T-dependence of

the P1/2 parameter also provides information about solvent mobility. Values of Tt,prot (4MT-EAL detection)

and Tt,solv (TEMPOL mesodomain detection), and corresponding activated-tumbling T-dependences, will

be manipulated by using DMSO and sucrose, and compared to reveal correlations between the protein

surface and solvent dynamics. Supported by NIH DK054514.

[1] Chen, H., Sun, L., & Warncke, K., Langmuir 2013, 29, 4357. [2] Bovell, A. M. Dissertation, Emory University, 2013.


90

NOVEL MRI APPROACH TO MAPPING PHYTOALEXINS Boris M. Odintsov1, Vera V. Lozovaya2, Jack M. Widholm2, Curtis B. Hill2, Glen L. Hartman2,3 1Biomedical Imaging Center, Beckman Institute for Advanced Science and Technology, Depatrment of Bioengineering; 2Department of Crop Sciences; 3USDA-ARS/Dept. of Crop Sciences, University of Illinois, Urbana-Champaign, IL, 61801 The goal of this project is to develop microvolume localized 1H and 13C MR spectroscopy (MRS) and microimaging in super high magnetic fields to use in studies of phytoalexin synthesis and metabolism during soybean plant tissues-pathogen interaction in vivo. The objectives are to: 1) modify available MRI hardware, in particular construct radiofrequency NMR coils specially designed for soybean leaf and root measurements of biochemicals; introduce a high speed spinning technique in root analysis, which should result in narrowing NMR linewidth important for better spectral resolution and sensitivity; 2) adjust 1H and 13C local MRS to samples enriched and non-enriched with a 13C metabolite of interest, formed from 13C glucose and 13C phenylalanine; 3) measure products of phytoalexin metabolism over time after plant tissue inoculation with pathogens; 4) localize and monitor the movement of phytoalexin molecules and its derivatives within infected soybean plants; and 5) determine the associations between specific metabolic events with pathogen colonization level. Soybean plants are susceptible to important generalist pathogens and do not have adequate genetic variability available to breeders to improve genetic resistance against such pathogens. Our premise is that the strengthening (via genetic engineering) of plant immune response provided by antimicrobial compounds, phytoalexins, will improve crop protection against multiple pathogens. We plan to develop a new experimental approach based on super high field 1H and 13C localized NMR spectroscopy and microimaging, to study basic mechanisms of phytoalexin involvement into soybean defense response and their regulation. Genetically engineered soybeans with different capacities to produce phytoalexins will be used. We anticipate that this project’s results will provide improved knowledge on phytoalexin synthesis and movement in soybean plants and identify optimum targets for genetic manipulation to maximize expression of phytoalexins, which will be objectives in a planned federal grant proposal to develop gene constructs and transgenic plants with improved disease resistance. The preliminary results obtained (Fig.1) indicate that in super high magnetic field (14.1T) the metabolite concentrations can be determined in cotyledons, leaves and hairy roots in volumes small enough to be localized and differentiated. The results obtained also indicated that in a high magnetic field, the metabolite concentrations can be determined in leaves and in roots, transformed using Agrobacterium rhizogenes, which results in “hairy” roots. This new technique when established will allow genetic engineers and breeders to non-invasively screen soybean genotypes for potent immune response related to phytoalexin production in plant tissues to important pathogens. Namely, we will learn what specific gene(s) should be expressed and what promoters should drive these genes and what should be the effective concentration of transgene product(s) to increase innate resistance to level competing with plant chemical treatments.


91

TEMPERATURE DEPENDENT CATALYSIS OF BACILLUS SUBTILIS OXALATE DECARBOXYLASE Milbrey Parke, Umar Twahir, Alexander Angerhofer University of Florida, Department of Chemistry, Gainesville, FL, 32611 Oxalate Decarboxylase from Bacillus subtilis is a Mn-dependent enzyme that exhibits a bifurcated mechanism catalyzing the degradation of oxalate to carbon dioxide and formate during 99.8% of turnover events, while during 0.2% of turnovers the enzyme acts as oxidase producing hydrogen peroxide and two equivalents of carbon dioxide. OxDC is a member of the bicupin superfamily of enzymes, with a mononuclear Mn (II) coordinated in each of its two domains. OxDC is only active at low pH but shows a pH dependence of its Mn(II) signals indicative of pH-dependent conformational changes.1 The oxidase activity exhibits a temperature dependent behavior, showing higher catalytic rates with increasing temperature up to 60oC.2 Within this work, the temperature dependence of the decarboxylase activity is investigated. [1] Tabares, L.C., et al., J. Phys. Chem. B 2009, 113, 9016-9025. [2] Molina, L., et al., J Biochem. Tech 2014, 5(4): 826-831


92

Morphology-dependent HIV-enhancing Effect of Seman-Derived Enhancer of Viral Infection

Xin Qiao1, Jaekyun Jeon1, Amy L. Cole2, Jason O. Matos1, Stephany Bautista1, Justin Castillo1, Ivan Hung3, Zhehong Gan3, Suren A. Tatulian1, Alexander M. Cole2, and Bo Chen1,* 1Department of Physics and 2Department of Molecular Biology and Microbiology, Burnett School of Biomedical Sciences, University of Central Florida, Orlando, Florida; and 3National High Magnetic Field Laboratory, Tallahassee, Florida PAP248–286 is a 39-residue fragment (residues 248 to 286) derived from protease cleavage of prostatic acidic phosphatase in semen. The amyloid fibrils formed in vitro by PAP248–286 can dramatically enhance human immunodeficiency virus (HIV) infection. To our knowledge, we present the first report that the HIV-enhancing potency of fibrils formed by PAP248–286 is morphology dependent. We identified pleomorphic fibrils by transmission electron microscopy in two buffer conditions. Our solid-state NMR data showed that these fibrils consist of molecules in distinct conformations. In agreement with NMR, fluorescence measurements confirmed that they are assembled along different pathways, with distinct molecular structures. Furthermore, our cell-based infectivity tests detected distinct HIV-enhancing potencies for fibrils in distinct morphologies. In addition, our transmission electron microscopy and NMR results showed that semen-derived enhancer of viral infection fibrils formed in sodium bicarbonate buffer remain stable over time, but semen-derived enhancer of viral infection fibrils formed in phosphate buffered saline keep evolving after the initial 7 days incubation period. Given time, most of the assemblies in phosphate buffered saline will turn into elongated thin fibrils. They have similar secondary structure but different packing than thin fibrils formed initially after 7 days incubation. [1] X. Qiao, J. Jeon, et. al, Biophysical Journal, 108, 2028-2037 (2015). [2] J. Munch, E. Rucker, et. al, Cell, 131, 1059–1071(2007). [3] L. Goldschmidt, P. K. Teng, R. Riek, and D. Eisenberg, Proc. Natl. Acad. Sci. USA, 107, 3487–3492(2010) [4] J. S. Olsen, J. T. M. DiMaio, S. Dewhurst, J. Biol. Chem, 287, 11842–11849(2012)


93

SUPEROXIDE PRODUCTION IN FRUCTOSE-TREATED HEPATOCYTES John Shahin,1 Alexander Angerhofer,1 Yuri Sautin2

1Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611-7200 2Division of Nephrology, Department of Medicine, University of Florida, PO Box 10024, Gainesville, FL 32610.

High fructose intake in the human diet has been implicated as one of the possible causes for the metabolic syndrome epidemic in Western culture. Among other possibilities its mode of action may be through a systemic increase of uric acid in the body which in turn may lead to an increase of reactive oxygen species and cell damage through free radical reactions. To test this hypothesis we are building a microfluidic chip that will allow us to employ in vivo EPR of mammalian hepatocyte cells. This allows us to measure the oxidative stress of the cells under well-controlled conditions. In this contribution we report on initial experiments in addition to sensitivity and cell viability tests on our microfluidic chip that will allow us to employ in vivo EPR. [1] Johnson, R. J., et al., Endocr. Rev. 30, 96 (2009). [2] Stirpe, F., et al., Lancet 2, 1310 (1970). [3] Nakagawa, T., et al., Am. J. Physiol. Renal. Physiol., 290, F625 (2006). [4] Imaram, W., et al., Free Rad. Biol. & Med., 49, 275 (2010). [5] http://www.ibidi.com/products/disposables/S_801XX_SlideI_Luer.html [6] Dikalov, S. I., et al., Biochem. Pharmacol., 73, 972 (2007).


94

A MECHANISTIC STUDY OF DYNAMIC NUCLEAR POLARIZATION ENHANCEMENT OF BIOMEMBRANES USING NITROXIDE LABELED LIPIDS Adam N. Smith1,2, Gail E. Fanucci1, Joanna R. Long2,3 1University of Florida, Department of Chemistry, Gainesville, FL 32611, 2National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310 USA; 3University of Florida, Department of Biochemistry & Molecular Biology, Gainesville, FL 32601

Dynamic nuclear polarization (DNP) magic angle spinning (MAS) solid state NMR (ssNMR) has become an increasingly important technique to address the issue of insensitivity in MAS ssNMR. Signal enhancements are obtained by transferring polarization from a paramagnetic reagent to NMR active nuclei. To date, the most commonly utilized polarization agents have been nitroxide biradicals; where the strength of the electron-electron dipolar coupling and relative orientation between the electron g-tensors are known to influence DNP enhancement. However, even when nitroxide biradical polarization agents such as TOTAPOL are used for biomolecular assemblies, such as biomembranes and membrane proteins, reported DNP signal enhancements lag behind the values obtained on model systems and the theoretical maximum DNP enhancement (~ 660 for 1H nuclei). We have previously demonstrated efficient DNP enhancements of a membrane embedded peptide with the use of nitroxide monoradical labeled lipids (spin labeled lipids, SL-lipids). [1] Compared to TOTAPOL, the SL-lipids produced nearly double the DNP enhancement of an embedded membrane peptide. Here we present our current views of a mechanistic understanding into why SL-lipids are suitable choices of radicals for DNP polarization of membranes/membrane proteins. Specifically, from X-band EPR of SL-lipids of varying molar concentrations (Fig. 1a) and in different locations within the lipid, we were able to evaluate average distances and dipolar interactions between SL-lipids. Analysis of nitroxide mobility also gives estimates of order parameters, leading to insight into the orientational disorder of the nitroxide at different locations within the bilayer. From the strength of the dipolar interactions and relative orientational disorder, we propose a molecular level understanding of the concentration dependence and depth dependence of SL-lipids on DNP enhancements obtained at 600 MHz/ 395 GHz (Fig. 1b). Insights from these studies will provide guidance on the design of more efficient membrane protein DNP polarization agents for membrane protein structure determination. [1] A.N. Smith, M.A. Caporini, G.E. Fanucci, and J.R. Long, Angew. Chem. Int. Ed., 54, 1542-1546 (2015).

Figure 2: a) Area normalized absorption X-band CW EPR spectra of increasing concentrations of SL-lipid in liposomes, b) DNP enhancement of liposomes as a function SL-lipid concentration.


95

STUDY OF INTERACTIONS OF AMYLOIDOGENIC REGIONS OF STREPTOCOCCUS MUTANS ADHESIN P1 ON THE CELL SURFACE BY SOLID STATE NUCLEAR MAGNETIC RESONANCE Wenxing Tang1,2, L Jeannine Brady1, and Joanna R Long2 1Department of Oral Biology, College of Dentistry, University of Florida 2Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida Streptococcus mutans is an established etiologic agent of human dental caries, the most common infectious disease in the world. S. mutans is particularly effective at colonizing hard tissues of the human oral cavity. Its adherence is mediated by sucrose-dependent and independent mechanisms. In the absence of sucrose, the extracellular cell wall-associated adhesin P1, also known as Antigen I/II or PAc, facilitates attachment to the acquired pellicle on teeth. Our previous work showed that P1 self-aggregates to form amyloid fibrils and demonstrates common biophysical properties ascribed to amyloids including uptake of Thioflavin T and Congo Red (CR), CR-induced birefringence, and visualization of fibrillar aggregates by transmission electron microscopy. However, the detailed process of amyloid formation remains unknown. P1 has an unusual structure in which the protein folds back on itself in such a way that an alanine-rich alpha helix intertwines with a polyproline type II helix to form an extended helical stalk. Beta sheet-rich globular structures corresponding to the adhesive domains lie on either end of the stalk. P1 was originally identified as a dual antigen, comprising antigen II (C123), and the remainder of the molecule (antigen I). It is likely that interactions between C123 and full length P1 on the bacterial cell surface are biologically relevant, and may provide us clues on amyloidosis. Here, we describe our studies of interactions between C123 and full length P1 on cell surface using Magic Angle Spinning (MAS) Nuclear Magnetic Resonance (NMR). Shown in Figure 1 are the one-dimensional 13C CPMAS experiments that were done on both S. mutans serotype c strain NG8 and its isogenic ΔP1 mutant PC3370, before and after 13C labeled C123 was added to the cell surface, along with the difference spectrum (in blue) of each set. All the experiments were carried out at a spinning rate of 12 kHz with 10k scans while the temperature was set to 277 K. The increase in intensities over the spectrum in the case of NG8 indicates interaction between C123 and the cell wall, whereas the experiments on PC3370 further confirms this interaction only happens between C123 and P1.

Figure 3 One-dimensional 13C CPMAS experiments on NG8 (left) and PC3370 (right) cell walls before and after 13C labeled C123 was added. All the experiments were carried out at a spinning rate of 12 kHz with 10k scans. Difference spectrum of each data set is shown in blue to help identify changes between the spectra.


96

DFT AND ENDOR STUDY OF BIXIN RADICAL CATIONS AND NEUTRAL RADICALS ON SILICA–ALUMINA Sefadzi S. Tay-Agbozo, M.K. Bowman, S. Street, L.D. Kispert

250 Hackberry Lane, Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35476-03362. Bixin, a carotenoid found in annatto (Bixa orellana), is unique among natural carotenoids by being water-soluble. We stabilized free radicals from bixin on silica–alumina (Si–Al) surface and characterized them by pulsed electron–nuclear double resonance (ENDOR). The EPR hyperfine couplings of possible bixin radicals were predicted with the unpaired electron spin distribution computed with Density functional theory (DFT) calculations. The trapped radicals were then characterized by fitting the experimental and calculated spectra from the DFT hyperfine couplings using least-square method. DFT prediction shows that trans bixin radical cation is more stable than the cis bixin radical cation by 1.26 kcal/mol. This is consistent with a 26% trans and 23% cis radical cations in the ENDOR spectrum collected. The remainder of the ENDOR spectrum is due to several neutral radicals formed by loss of a H+ ion from the 9, 9′, 13, or 13′ methyl group, a common occurrence in all water-insoluble carotenoids previously studied. Although the carboxyl groups of bixin strongly affect its solubility relative to other natural carotenoids, they do not alter properties of its free radicals based on DFT calculations and EPR measurements which remain similar to typical water-insoluble carotenoids.

[1] Focsan, A. L.; Bowman, M. K.; Konovalova, T. A.; Molnár, P.; Deli, J.; Dixon, D. A.; Kispert, L. D. Pulsed EPR and DFT Characterization of Radicals Produced by Photo-Oxidation of Zeaxanthin and Violaxanthin on Silica-Alumina. The Journal of Physical Chemistry B 2008, 112, 1806-1819.

[2] Gao, Y.; Focsan, A. L.; Kispert, L. D.; Dixon, D. A. Density Functional Theory Study of the β-Carotene Radical Cation and Deprotonated Radicals. The Journal of Physical Chemistry B 2006, 110, 24750-24756.

[3] Tay-Agbozo, S. S.; Krzyaniak, M. D.; Bowman, M. K.; Street, S.; Kispert, L. D. DFT and ENDOR Study of Bixin Radical Cations and Neutral Radicals on Silica–Alumina. J. Phys. Chem. B 2014, 119, 7170-7179.


97

FIGURE 1: Top: REP-CP, and Bottom:SAMPi4 spectrum of uniformly 15N labelednAChRa7 in DHPC:DMPC bicelles.

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

ORIENTED-SAMPLE NMR STUDIES OF HUMAN NICOTINIC ACETYLCHOLINE RECEPTOR Deanna M. Tesch1, Alexander A. Nevzorov1

1North Carolina State University Department of Chemistry

The a7 subtype of the human nicotinic acetylcholine receptor (nAChRa7) is one of the most abundant nAChR subtypes in brain regions involved in learning, memory, and cognition (1). Due to it’s high abundance in these regions, nAChRa7 has the potential to be an effective therapeutic target aimed at treating various neurological diseases such as Alzheimer’s and schizophrenia (1). As all membrane proteins (MPs), nAChRa7 requires a fully hydrated membrane environment with minimal curvature for full functionality: a requirement making structure determination by oriented-sample solid state NMR (OS- NMR) potentially attractive.

A slightly modified nAChRa7 construct

assembled as a homo-pentameric ligand-gated ion channel, is used in the present study. The uniformly 15N labeled nAChRa7 protein has been expressed, purified, and reconstituted into a biological-like bicellar membrane, in contrast to detergent-like micelles used in a previous study (1). Initial two-dimensional SAMPi4 spectra have been acquired. Selectively labeled spectra are employed to address the problem of spectral crowding. Back-calculated spectra from the solution-NMR structure in micelles shows marked deviations with the preliminary experimental solid-state NMR spectra in bicelles. This emphasizes the importance of membrane mimetics for proper folding and functionality of membrane proteins. [1] V. Bondarenko, et. al., Biochimica et Biophysica Acta, 1838, 1389-1395 (2014).


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Instrumentation for Optical Dynamic Nuclear Polarization NMR Studies in the NHMFL Ultrafast Facility and at the University of Florida

John T. Tokarski III,1 Ryan M. Wood,1 Phillip L. Kuhns,2 Stephen A. McGill,2 Arneil P. Reyes,2

Clifford R. Bowers1

1Department of Chemistry, University of Florida, Gainesville, FL, 32611 USA 2National High Magnetic Field Laboratory, Tallahassee, FL, 32360, USA

Optically Dynamic Nuclear Polarization (ODNP) in crystalline semiconductors utilizes interband optical excitation to orient conduction electrons that subsequently cross-relax with nuclei, generating hyperpolarization on the order of unity than can be detected by NMR spectroscopy. ODNP-NMR has been employed to interrogate electronic band structure, electron-nuclear spin interactions, and exotic many-body excitations in 2D electron systems. Recently, the NHMFL UCGP supported the construction of a special-purpose optical NMR probe for the NHMFL Ultrafast User Facility, where ODNP measurements can be combined with magneto-photoluminescence and magneto-absorption spectroscopy at multiple magnetic fields. Moreover, interpretation of data is facilitated by electronic band structure calculations at UF Physics. The state-of-the-art optical NMR probe and magnet systems at the NHMFL provide unparalleled research capacity for such studies. At UF, we developed complimentary instrumentation (see photo). A new probe was constructed for ODNP experiments in pumped superfluid 4He with a base temperature of 1.4 K and hold time >12 hr. Automated photon energy dependence studies are facilitated using a Labview/Arduino controller interfaced to the Bruker Avance NMR spectrometer. This poster presents data acquired at UF and the NHMFL and describes the instrumentation development at both sites. [1] Wood, R. M.et al., PRB, 2014, 155317. [2] Saha, D. et al., SPIE NanoScience+ Engineering 2014, 9167, 91670N-91670N-8. [3] Kuhns, P. L. et al., PRB, 1997, 55, 7824-7830 * Supported by the NHMFL UCGP and NSF DMR-1157490.


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USING DIFFUSION MRI TO ASSESS BASAL GANGLIA CIRCUITRY IN AN ANIMAL MODEL OF REPETITIVE BEHAVIOR Bradley J. Wilkes1, Luis M. Colon-Perez2, Marcelo Febo2, Mark H. Lewis2 1University of Florida, Department of Psychology, Gainesville, FL 32611; 2University of Florida, Department of Psychiatry, Gainesville, FL 32611 Our lab utilizes an inbred mouse strain (C58) that demonstrates a robust repetitive motor behavior phenotype as a model for studying restricted repetitive behavior, a feature of neurodevelopmental disorders such as Autism Spectrum Disorders. Previous work in our lab suggests that alterations in basal ganglia circuitry mediate expression of this phenotype. In order further investigate this circuitry, we performed ex vivo T2 and diffusion weighted scans at 17.6 T. T2 weighted scans were collected at a resolution of 45 µm isotropic and used to perform volumetric assessment. In order to effectively measure subcortical anisotropy, we utilized three shells of diffusion weighting (i.e. b-values) along with 60 diffusion gradients at a resolution of 125 µm isotropic; we then performed probabilistic fiber tracking to estimate white matter tracts between key structures of the basal ganglia. We found evidence of abnormal organization along the indirect pathway of the basal ganglia.


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ELUCIDATING THE MECHANISM OF DRUG RESISTANCE IN M2 PROTON CHANNEL FROM INFLUENZA A VIRUS Anna K. Wright1,2, Ivan Hung1, Paratchata Batsomboon3, Jian Dai2, Gregory B. Dudley3, Timothy A. Cross1,2,3 1National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310 USA; 2Florida State University, Institute of Molecular Biophysics, Tallahassee, FL 32306 3Florida State University, Department of Chemistry, Tallahassee, FL 32306 The M2 proton channel from Influenza A virus functions at multiple stages of the viral lifecycle, and as such is an excellent drug target[1]. Amino acid mutations, in the residues lining the channel pore, have rendered M2 resistant to previously licensed inhibitors. Given the propensity for genetic reassortment of Influenza A and history of pandemics due to emergence of novel human strains, M2 has been subject of numerous structural characterization efforts [2-7]. Attempts at rational drug design targeting M2 proton channel have been impeded by the limited number of experimental techniques having capabilities for elucidating atomic level interactions of the protein-ligand complexes in a native-like membrane mimetic environment. Solid State Nuclear Magnetic Resonance (ss-NMR) is a technique that has all of these capabilities for structural characterization of membrane protein drug targets in lipid bilayers. Coupling ss-NMR with Computer Aided Drug Discovery (CADD) is the precise approach needed to decrease time and resources required to generate novel therapeutics. Here we present structural characterization of the wild type M2 channel in complex with rimantadine enantiomers, and of the S31N mutant in the apo state. Through the use of REDOR and NCA experiments of the full length wild type M2 channel in complex with rimantadine enantiomers, we evaluated the stereoselectivity of drug binding in the channel pore. RosettaLigand molecular dynamic simulations were performed to evaluate enantiomeric specificity of rimantadine and were in good agreement with experimental results. Oriented sample (OS) ss-NMR experiments of the S31N mutant were remarkably similar to the wild type channel, with the tilt and rotation angle of the helices relatively unchanged. Together these results deepen our understanding of the atomic level interactions stabilizing wild type inhibitors in the channel pore, and structural changes in the mutant leading to loss of compound efficacy. Most importantly, specific interactions described herein are essential for successful outcomes from structure based CADD and can be used in future computational efforts. [1] M. Takeda, A. Pekosz, K. Shuck, L.H. Pinto, R.A. Lamb, J Virol, 76 1391-1399 (2002). [2] J. Hu, T. Asbury, S. Achuthan, C. Li, R. Bertram, J.R. Quine, R. Fu, T.A. Cross, Biophys J, 92 4335-4343 (2007). [3] C. Li, H. Qin, F.P. Gao, T.A. Cross, Biochim Biophys Acta, 1768 3162-3170 2007). [4] A.L. Stouffer, R. Acharya, D. Salom, A.S. Levine, L. Di Costanzo, C.S. Soto, V. Tereshko, V. Nanda, S. Stayrook, W.F. DeGrado, Nature, 451 596-599 (2008). [5] S.D. Cady, K. Schmidt-Rohr, J. Wang, C.S. Soto, W.F. Degrado, M. Hong, Nature, 463 689-692 (2010). [6] M. Sharma, M. Yi, H. Dong, H. Qin, E. Peterson, D.D. Busath, H.X. Zhou, T.A. Cross, Science, 330 509-512 (2010). [7] L.B. Andreas, M.T. Eddy, R.M. Pielak, J. Chou, R.G. Griffin, J Am Chem Soc, 132 10958-10960 (2010).

44th Southeastern Magnetic Resonance Conference Participants

101

Adewale Akinfaderin

Florida State University [email protected]

Manish Amin

University of Florida [email protected]

Alexander Angerhofer


Guita Banan


Koushik Banerjee

Georgia College and State University [email protected]

Juan Beltran

University of Puerto Rico [email protected]

Lakshmi Bhaskaran

National High Magnetic Field Laboratory/FSU [email protected]

Clifford Russell Bowers


Michael K Bowman

The University of Alabama [email protected]

William Brey


R. David Britt

Department of Chemistry, Uc Davis [email protected]

Mike Brown

Bruker BioSpin [email protected]

Leah B. Casabianca

Clemson University [email protected]

Thomas M. Casey


Bo Chen

University of Central Florida [email protected]

Hanjiao Chen


Zackary I. Cleveland

Cincinnati Children's Hospital Medical Center [email protected]

James Collins


Alex Cruce


Christopher Ian Davis

Georgia State University [email protected]

Christopher Dean

Sigma-Aldrich - Isotec [email protected]

Francis David Doty

Doty Scientific, Inc. [email protected]


102

Judy M. Doty


Daniel Downs


Jimmy Du


Thierry Dubroca

National High Magnetic Field Lab [email protected]

Katie Marie Dunleavy


Akshita R. Dutta


Paul Ellis


Livia B L Escobar

National High Magnetic Field Laboratory [email protected]

Jackie Marie Esquiaqui


Gail E. Fanucci


Alexandrina Ligia Focsan

Valdosta State University [email protected]

Deidra L. Gerlach

University of Alabama [email protected]

Daniel F. Gochberg

Vanderbilt University [email protected]

Justin Goodsell


Peter Gor'kov


Samuel M. Greer

FSU/NHMFL [email protected]

Fred Haberle

Bruker Biospin [email protected]

Zahra Hayati


Stephen Hill


Christine Hofstetter

Active Spectrum, Inc. [email protected]

Yan-Yan Hu


Morteza Jafarabadi

North Carolina State University [email protected]


103

Jaekyun Jeon

University of Central Florida [email protected]

Aditya Kumar Kasinadhuni


Pavanjeet Kaur

National High Magnetic Field Lab/FSU [email protected]

Lowell D. Kispert


Meghan Kohne

Emory University Department of Physics [email protected]

Dorsa Komijani


Sophie Natacha Koroloff


Jerzy Krzystek


Magdoom Kulam


Bimala Lama


Xiang Li

Florida Statue University [email protected]

Aimin Liu


Joanna Long


Kaylee Ludden


Fraser MacMillan

University of E. Anglia [email protected]

Thorsten Maly

Bridge12 Technologies, Inc. [email protected]

Jonathan J. Marbey

National High Magnetic Field [email protected]

Thomas Harold Mareci


Benjamen Nforneh

Emory University [email protected]

Boris Odinstov

University of Illinois [email protected]


104

Erkang Ou


Milbrey A. Parke


Khushi Patel


Joana Paulino


Kari Pederson

University of Georgia [email protected]

Xin Qiao

Univ. of Central Florida [email protected]

Victor D. Schepkin


Jeffrey L. Schiano

Penn State University [email protected]

John Shahin


Arthur Sikora

University of Virginia [email protected]

Alex I. Smirnov


Tatyana I. Smirnova


Adam Smith


Benjamin C. Smith


Likai Song


Xiao Song


Sebastian A. Stoian


Wenxing Tang


Sefadzi Tay-Agbozo


Sefadzi Tay-Agbuzo


Joshua Telser

Roosevelt University

Deanna Tesch

North Carolina State University


105

[email protected]

[email protected]

Bibek Thapa

University of Puerto Rico [email protected]

John Thomas Tokarski


Nhi Tran


Trang Tran

University of FLorida [email protected]

Umar Twahir


Johan Van Tol

National High Magnetic Field Laboratory /FSU [email protected]

Dan Vigneron

UCSF [email protected]

Kurt Warncke

Emory University [email protected]

Ralph T. Weber

Bruker EPR [email protected]

Mariya Vyushkova

Notre Dame Radiation Laboratory [email protected]

Anna K. Wright


Elizabeth Webb

National High Magnetic Field Lab/UF [email protected]

Yunzhi Zhang


Bradley J. Wilkes


Minli Xing


44th southeastern magnetic resonance conference

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