Session PA. There are 120 abstracts in this session.

Session: SOLUTION, poster number: 001

Structural and Dynamics characterization of a pathogenic RNA using selective isotopically labeled RNAs by Nuclear magnetic resonance (NMR).

Kehinde Taiwo
UMD College Park, University Park, Nigeria

The global rise in antibiotics resistance calls for the need for novel therapies and strategies that target bacterial infections. One of such strategies involves studying RNA molecules that function in cell growth and then developing therapies that target their roles or mechanisms of actions. In this research, we are characterizing the structure and dynamics of a 27-nucleotide (nt) fragment of rRNA by nuclear magnetic resonance (NMR). Resonance assignments and dynamics studies of this RNA using uniform and site selectively labeled NTPs synthesized via a chemo-enzymatic labeling strategy developed by our lab  were carried-out. We identified residues experiencing internal motions and conformational changes. This research could help identify potential therapeutic targets for treating a variety of bacterial infections.




Session: SOLUTION, poster number: 002

Geometry of Hydrogen Bonds in Liquid Ethanol Probed by Proton NMR Experiments

Ritu Ghanghas
phd, Bangalore, India

We have established the geometry of hydrogen bonds in liquid ethanol by exploiting differences in proton chemical shift values, that originate from the secondary isotope effect, to distinguish the methyl and hydroxyl group protons of CH3CH2OH and from those of the deuterated CH3CD2OH in the 1H NMR spectra of mixtures of the two. This has allowed us to measure the ratios of the inter- to intramolecular distance between methyl to hydroxyl and methylene to hydroxyl protons using 1D transient NOE NMR measurements as a molecular ruler. We model the liquid ethanol by ab initio Molecular Dynamics simulations and identify all possible pairs of ethanol molecules in the ensemble that satisfied the NMR determined inter- to intramolecular distance ratio criteria.

Session: SOLUTION, poster number: 003

The Solution NMR Structure of a Transmembrane Peptidomimetic Inhibitor of the Gamma-Secretase Complex.

Justin Douglas1; Sanjay Bhattari2; Suhan Devkota2; Kathleen M. Meneely2; Minli Xing3; Michael S. Wolfe2
1University of Kansas - NMR Core Lab, Lawrence, KS; 2KU Department of Medicinal Chemistry, Lawrence, KS; 3University of Michigan, Ann Arbor, MI

Gamma-secretase is a membrane-embedded aspartyl protease that hydrolyzes more than 90 substrates, including the amyloid precursor protein (APP). How this enzyme recognizes transmembrane substrates and catalyzes the hydrolysis in the lipid bilayer is unclear. We have developed a series of peptidomimetics as chemical probes for structural analysis of gamma-secretase. These compounds include a helical peptide inhibitor conjugated via a flexible linker to a transition-state analog. The solution NMR structure for a stoichiometric inhibitor was calculated and the conformation resembles that of the APP transmembrane domain bound to gamma-secretase in a new cryo-EM structure.  This structural data suggest that these probes are preorganized for high-affinity binding and should allow visualization by cryo-EM of the active gamma-secretase complex, poised for intramembrane proteolysis.

Session: SOLUTION, poster number: 004

19 F‑1H couplings arising due to Spatial Proximity: Intramolecular Hydrogen bond or not?

Surbhi Tiwari; Neeru Arya; Dhanishta Poshetti; N. Suryaprakash
Indian Institute of Science, Bangalore, India

A series of N-bezoylanthranilide derivatives have been synthesized and investigated by the number of sophisticated one- and two- dimensional NMR experimental techniques to ascertain the presence of weak molecular interactions, if any, leading to the stable conformations of the molecules. In the fluorine substituted derivative, the strong correlation between 19F and 1H spins in the 19F-1H HOESY experiment although conforms their spatial proximity and yields a doublet with interaction strength of 6.1 Hz, it does not establish the existence of intramolecular hydrogen bond (HB). The NMR findings are unequivocally established by XRD studies.

Session: SOLUTION, poster number: 005

Application of Multidimensional NMR Spectroscopy and Predicted NMR Databases for the Deconvolution and Analysis of Chlorinated Paraffin Mixtures

Daniel Lysak1; Ronald Soong1; Andrew Haddad1; Bo Yuan2; Derek Muir3; Arvin Moser4; Sergey Golotvin4; Dimitris Argyropoulos4; Andre J Simpson1
1University of Toronto, Toronto, Canada; 2Stockholm University, Stockholm, Sweden; 3Environment and Climate Change Canada, Burlington, Canada; 4ACD/Labs, Toronto, Canada

Chlorinated paraffins (CPs) are complex mixtures of polychlorinated n-alkanes of different chain length. Despite their many industrial applications, both their effects on the environment and isomer composition remain unknown. In this work, a database of over 400,000 predicted, one-dimensional 13C NMR spectra was used to screen single chain length CPs (with varying degrees of chlorination), identifying the top 1000 components. These were filtered down to the top 100 using multidimensional 1H-13C NMR spectra and mass spectrometry. The top 100 compounds represent the most likely constituents of CP mixtures and were found to be an adequate representation of the overall mixture. Overall, this work demonstrates the usefulness of NMR and spectral databases for the deconvolution and understanding of complex CP mixtures.

Session: SOLUTION, poster number: 006

A longitudinal study of serum metabolomics in an experimental PD model and sham control

Sadhana Kumari; S. Senthil Kumaran; Suman Jain; Vinay Goyal; S.N. Dwivedi; Achal Srivastava; NR Jagannathan
AIIMS, New Delhi, India

Proton nuclear magnetic resonance (NMR)-based metabolomics was performed to study the metabolic changes in serum of experimental Parkinson’s disease (PD) rat model and sham control. The concentration of 17 metabolites corresponding to the important spectral features, were estimated. The levels of lactate, citrate, glutamate, and choline metabolites were higher at 3rd and 7th weeks in the PD rat model as compared to their baseline. Sham group did not reveal any changes in metabolite concentration between baseline, 3rd and 7th-week groups. Results suggest that glycolysis, the tricarboxylic acid cycle, gluconeogenesis, fatty acid β-oxidation, branched-chain amino acid metabolism, and the tyrosine metabolic pathways are involved in the evolution of PD.

Session: SOLUTION, poster number: 008

A Cooperativity Effect Renders Intramolecular Hydrogen Bond Strong and Unbreakable even in High Polarity Solvents: In-depth NMR Investigations

Neeru Arya
Indian Institute of Science, Bangalore, India

The rare and interesting phenomenon of very strong three-centered acceptor hydrogen bond (HB) which is inexpungible even in high polarity solvents has been detected in the synthesized derivatives of N-(quinolin-8-yl)benzamide. The strength of the six membered ring formed HB is sustaining up to 90% even in highly polar solvents. The incredible strength of this HB is attributed to the positive cooperativity effect which is undeniably ascertained by various NMR experimental methodologies. The simultaneous participation of 14N and 19F HB mediated interaction resulted in unusual broadening of NH proton doublet.  The variation in the linewidth of NH doublet in solvents of different polarities is attributed to the elimination of weak 14N-1H HB mediated coupling. The DFT based computation concurs with NMR findings.  

Session: SOLUTION, poster number: 009

Improved prediction of chemical shifts with quantified uncertainty for natural-product-size molecules

Eric Jonas
University of Chicago, Chicago, IL

Nuclear chemical shift values are the primary source of information in one-dimensional NMR spectra, providing insight into local molecular structure. Previous work from our group \cite{jonas2019} showed how the use of modern machine learning methods can exceed the capabilities of traditional HOSE codes and match ab initio techniques such as DFT when computing 13C and 1H shifts. Here we build on that work by expanding to larger molecules (128 atoms) and adopting new insights from graph neural networks to improve performance. Using the same input dataset from previous work we improve prediction accuracy by nearly 10% for both proton and carbon shifts. 

Session: SOLUTION, poster number: 010

Stereochemistry of 1,3-Diol Derivatives of Diacetylcurcumin-4H: A Joint NMR, X-Ray, and Biological Approach

Yair F. Alvarez Ricardo; William E. Meza-Morales; Marco A. Obregón-Mendoza; Antonio Nieto-Camacho; Rubén A. Toscano; Raúl G. Enríquez
UNAM, Mexico City, Mexico

Curcumin and curcuminoids are heptanoids characterized by a β-diketone chemical functionality, that it plays an important role in the therapeutic effect of them and being also their reactive motif. The β-diketone function may serve as building block for the synthesis of numerous compounds the synthesis of corresponding 1,3-diols using a reducing agent as NaBH4 to attack β-diketone functionality.

In spite of the difficulty that the separation of the present type of diastereomeric pair entails, in recent work, Alvarez-Ricardo et al. have efficiently achieved the resolution of this mixture. We have prepared a new 1,3-diol of diacetylcurcumin-4H, the acetylated and cyclic sulfites derivatives. The stereochemistry of these derivatives shows complex patterns in NMR that were analyzed to assess their conformations.


Session: SOLUTION, poster number: 011

Structural Features of Stepwise Cholate Micelle Formation Using Chemical Shift Perturbations


Shelby Valent; Chengtong Zhang; David Ronyak; Timothy Strein
Bucknell University, Lewisburg,

Emerging applications of bile salts include topical drug delivery, separations, and nano-materials processing. Little is known about bile micelle formation including aggregation number, the structure of the micellar aggregates, and the mechanisms by which micelles bind chiral guest molecules. Interestingly, bile salts undergo multiple stepwise aggregation events. This work gains insight into the structure of cholate aggregates by monitoring changes in 2D-HSQC (conventional and NUS) with varied cholate concentrations (1 to 100 mM). Chemical shift perturbations reveal changing local environments, particularly in the micelle interior, with atomic resolution. Several trends reveal driving forces for stepwise bile aggregation, which are corroborated with unbiased molecular dynamics calculations of bile self-association.

Session: SOLUTION, poster number: 012

Perspectives on Nonuniform Sampling to Improve Sensitivity and Resolution in Challenging Structure Elucidation Experimentation: 1,1- and 1,1-HD-ADEQUATE

David Rovnyak1; Mark S. Roginkin1; Ikenna E. Ndukwe2, 3; D. Levi Craft1; R. Thomas Williamson2, 4; Mikhail Reibarkh2; Gary Martin2, 5
1Bucknell University, Lewisburg, PA; 2Merck Research Laboratories, Kenilworth, NJ; 3Complex Carbohydrate Center, Athens, GA; 4University of North Carolina Wilmington, Wilmington, NC; 5Seton Hall University, South Orange, NJ
Structure elucidation of complex small molecules is vital at many stages of the drug discovery pipeline, relying increasingly on advanced, sensitivity-challenged NMR experimentation. Weighted nonuniform sampling (NUS) strategies using the quantile algorithm for schedule design are investigated for acquiring highly resolved 1,1‐ADEQUATE spectra, in both conventional and homodecoupled (HD) variants. These experiments represent a challenging application for NUS due to their short evolution times. We report the design, optimization and validation of NUS schedules that are broadly usable in 1,1-ADEQUATE experimentation, yielding useful sensitivity gains estimated on the order of 5-20%. Given these results in1,1-ADEQUATE and 1,1-HD-ADEQUATE, this work foreshadows the broader role that NUS will play throughout structure elucidation NMR spectroscopy.

Session: SOLUTION, poster number: 013

Consistent Detection of Multiplets in 13C Spectra Using Structure Aware Algorithms

Dimitris Argyropoulos1; Sergey Golotvin2; Rostislav Pol2; Vladimir Mikhailenko2
1ACD/Labs, Toronto, Canada; 2ACD/Labs, Moscow, N/A

Software that is capable of Automatic interpretation of NMR spectra is highly demanded in the modern chemical and pharmaceutical industry. Every day scientists record hundreds of NMR spectra that need to be interpreted. Interpretation of such large numbers of spectra can be quite daunting and prone to human error, which makes it a good candidate for automation. This requires robust automated methofds for the automatic detection of peaks an multiplets. In this work we present a generalised such method for the reliable detection of multiplets in in 13C spectra of compounds that also contain 19F and 31P. The method can be easily implemented in software and leads to more consistent detection of the multiplets.

Session: SOLUTION, poster number: 015

Detection and Identification of Illicit Drugs and Cutting Agents in Street Drug Samples using 60 MHz Benchtop NMR and Databasing Software

Susanne D. Riegel1; Alexander F. G. Maier1; Dimitris Argyropoulos2; Marie Lange3; Marion Baumgarte3
1Nanalysis Corp., Calgary, Canada; 2ACD/Labs, Toronto, ON; 3Kriminaltechnisches Institut, LKA of Lower Saxony, Hanover, Germany

Rapid identification of illicit drugs seized during law enforcement operations is highly desirable as it can save valuable time, ensure chain of custody and provide certainty about handling the chemicals. In this poster we describe a method using a benchtop NMR spectrometer and databasing software for automated identification. To this end, a database of known, pure illegal drug standards 1H NMR spectra was created and then searched by the software to identify the components found in street samples. The method was found to quickly and reliably identify not only the illicit drugs present but also the various cutting agents used.

Session: SOLUTION, poster number: 016

Molecular Liquid Structure in Binary Mixtures Omega-3 Lipids

Fu Chen1; Walter Schmidt2
1The University of Maryland, College Park, College Park, MD; 2Agricultural Research Service, USDA, Beltsville, MD

Omega-3 and other polyunsaturated fatty acids (PUFA) are biochemically and nutritionally essential dietary lipids which contain the same redundant repeating structurally polymeric (H-C=C-H)-CH2 moiety.  The 18-carbon omega-3 fatty acid alpha-linolenic acid (ALA) ends with an eleven carbon atom moiety: CH3-CH2-[(H-C=C-H)-CH2]3.  The 22-carbon omega-3 fatty acid docosahexaenoic acid (DHA) ends in exactly the identical chemical structure; C1 in both begin with COOH. The in-common methylene site chemical shifts in ALA and DHA within neat liquids identify exactly which sites in the carbon backbone are magnetically equivalent. NMR studies were conducted of binary mixtures of ALA and DHA to determine if the evidence of magnetic equivalence would be disrupted by the presence of its structural analog in the neat liquid.  

Session: SOLUTION, poster number: 017

NMR Spectroscopic Investigation of New Bis (2-methoxy ethyl) substituted Benzamides - Prominent Insect Repellent

Mamta Sharma



NMR analysis of derivatives of Diethyl phenyl acetamide (DEPA) an effective mosquito repellant was carried out at low temperature. Compounds were synthesized with different substitution in search of more effective repellant.  It was observed that Sterically Crowded Bis (2-methoxy ethyl) substituted Benzamides possess low rotational barrier and develop interesting broadening in NMR signals due to the presence of various rotamers, hence molecules are floppy at room temperature. Alkyl arms attached to nitrogen become magnetically nonequivalent at low temperature. Di ortho Substitution in benzamides enhanced hindered internal rotation and resulted splitting in methylene protons signals.


Session: SOLUTION, poster number: 018

Phase Transition Modeling of Chemical Shifts Reveals Sequential Aggregation Events are a General Property of Many Bile Salts

Echo Jiayi He; Raeanne Geffert; Sophie Kong; Timothy Strein; David Rovnyak
Bucknell University, Lewisburg, PA

Surfactant micellization data are commonly modeled to a single critical micelle concentration (CMC). However, bile salts display stepwise CMCs for which effective modeling has not yet been reported. A single-CMC phase transition model treats the micellization as a phase separation, where the free monomer is set to the value of the CMC; we report an extension of this model to multiple CMC steps, with analogous assumptions in the subsequent steps. Fitting 1H NMR chemical shift perturbations, we consistently observed three discrete aggregation steps (CMCs) for several diverse bile salts, allowing us to fill this knowledge gap in understanding bile salt micellization. Correlating stepwise aggregation to chiral guest binding further illuminates how surface-available binding sites are dictated by stepwise aggregation.

Session: SOLUTION, poster number: 019

Using Diffusion Ordered NMR Spectroscopy in the Characterization of Biomass Pyrolysis Oils

Gary. Strahan; Charles Mullen

Pyrolysis is the most efficient method of converting lignocellulosoic biomass into renewable fuel. Unfortunately, these bio-oils are extremely challenging to analyze because they are a complex mixture of thousands of compounds with many functional groups and a wide range of molecular weights.  Many different analytical tools are used, especially GC, but tedious chemical separation steps are often needed.  We have previously shown that NMR can provide a broad picture of the chemical constituents in bio-oils, but a method has been needed to also identify how different chemical classes are distributed over the molecules and their molecular weights.  We have deployed diffusion NMR to obtain this information and present a detailed description of the method, including the quantitation of primary components.


Session: SOLUTION, poster number: 020

Merging of the Methods: Combining Synthetic Chemistry, NMR, and Computational Chemistry to Develop a Chiral Derivatizing Agent

Emily B. Crull; Matthew G. Donahue
Univ of Southern Mississippi, Hattiesburg, MS

Chiral derivatizing agents (CDAs) differentiate enantiomers by covalently bonding to them to produce diastereomers. We have synthesized an enantiopure pentafluorophenyl isothiocyanate derived CDA from pentafluorobenzaldehyde over four steps. Unlike most CDAs, we are looking at the shifts of our compound, and not those of the chiral amine. Concurrent with reacting the CDA with a selection of chiral amines such as alpha-methylbenzylamine, the theoretical chemical shifts have been calculated using Gaussian09 and compared to those observed experimentally on a Bruker 400 MHz instrument. This data provides corroborating evidence to re-design the CDA through optimization of substituents that maximize the difference between the two diastereomeric products of the reaction between the CDA and the targeted chiral amine.

Session: SOLUTION, poster number: 021

Best Practices for the Use of NOESY and ROESY Data in Determining the 3D Structure of Compounds

Yalda Liaghati Mobarhan1; Craig P Butts2; Dimitris Argyropoulos1; Mikhail Elyashberg1; Sergey Golotvin1; Maxim Kisko1
1ACD/Labs, Toronto, Canada; 2University of Bristol, UK, Bristol, UK

A NOESY or ROESY spectrum usually gives correlation peaks for 1H atoms within 5Å which is normally sufficient to define the stereochemistry of smaller rigid molecules or molecules with fewer than three stereocenters. Once the structure becomes flexible or has more stereocenters one needs to carefully evaluate the intensity (integral) of the NOE or ROE peaks in order to get a more quantitative idea about the actual distances between the involved nuclei. In recent years with the advent of advanced algorithms, this analysis has become possible, with an increasing interest. This work proposes best practices for using NOESY/ROESY spectra to analyze quantitative inter-nuclear distances, identifying common pitfalls and how these could be avoided for confident 3D structure elucidation.


Session: SOLUTION, poster number: 022

Quantitative 13C-NMR analysis of CBD oils; parameter optimization and analysis

Sara M. Howell; István Pelczer
Department of Chemistry, Princeton University, Princeton, NJ 08540
Due to the recent legalization of CBD products and the lucrative nature of their sale, efficient quality control is necessary, including screening for illegal or even illicit and dangerous content. Quantitative 13C-NMR allows a one-step analysis of the native sample without any manipulation.
We had to select a suitable deuterated solvent to reduce the effect of compartmentalization of the carrier oils, which are present in overwhelming quantities, then run sufficiently long quantitative 13C-NMR experiments at slightly elevated temperature.
High quality spectrum prediction helped to identify available cannabis components. The CBD itself is quite easy to recognize and its relative quantity can be determined. Other low-quantity side-components are more challenging to measure, but with sufficiently long acquisition time it is possible.

Session: SOLUTION, poster number: 023

A simple method to improve NMR spectral quality using amide-containing chemicals  

Talia Fargason; Jun Zhang
UAB, Birmingham,

Residual dipolar couplings (RDCs) are an important NMR technique that can be used for structural calculation and verification. Measuring RDCs requires aligning macromolecules using various types of alignment media. Of different alignment media, stretched or compressed polyacrylamide gels are advantageous due to their chemical stability. However, polyacrylamide interacts with proteins and significantly broadens NMR line shape. In this study, we found that the amide-containing compounds asparagine, glutamine and propionamide improve spectral quality of proteins in polyacrylamide gel, but not reduce the magnitude of RDC values. Moreover, we showed that propionamide is an attractive additive that increases protein solubility without interfering with protein stability, ligand binding or NMR pulse width, suggesting its potential novel uses.   

Session: SOLUTION, poster number: 024

Role of cavities in determining protein side chain dynamics

Jose Alfredo Caro1; Kathleen G. Valentine2; A. Joshua Wand1
1Texas A&M University, College Station, TX; 2University of Pennsylvania, Philadelphia, PA

Proteins rely on flexibility to function. The structural determinants of protein dynamics remain unknown. We hypothesize that motion must be preceded by a cavity (empty space) to move into. We curated a database of published side chain order parameters (O2axis) and deposited structures to evaluate, site-specifically, the role of cavities in determining O2axis. The observed relationship shows lower O2axis values with increased cavity volume. Consistent with Le Châtelier’s principle, high pressure (3000 atmospheres) rigidifies barnase and quenches its dynamic response to binding barstar. These findings cast light on the importance of cavities in protein function and offer a potential avenue for structural manipulation of the entropy of binding of proteins.

Session: SOLUTION, poster number: 025

Structural characterization and biological function of bivalent binding of CD2AP to intrinsically disordered domain of chikungunya virus nsP3 protein

Peter Agback1; Vladislav Orekhov3; Tatiana Agback1; Elena Frolova2
1Swedish University of Agricultural Sciences, Uppsala, Sweden; 2University of Alabama, Birmingham, AL; 3Gothenburg University, Goethenburg, N/A

Alphavirus nonstructural protein nsP3 contains a long, intrinsically disordered, hypervariable domain, HVD, which serves as a hub for interaction with cellular proteins. We further deciphered the mechanism and function of HVD interaction with host factors in alphavirus replication. By using NMR spectroscopy, we show that CHIKV HVD contains two SH3 domain-binding sites. Using innovative chemical shift perturbation signature approach, we demonstrate that interaction with HVD is mediated by SH3-A and SH3-C domains of CD2AP protein, and leaves the SH3-B domain available for interaction(s). The cooperative interaction with two SH3 domains of CD2AP increases binding affinity and possibly induces a long-range allosteric affect in HVD. Point mutations in both binding sites of CHIKV HVD results in strong inhibition of viral replication.

Session: SOLUTION, poster number: 026

Role of Multiple Timescale Dynamics in Function of Oncogenic K-Ras Mutants

Gyula Pálfy1, 2; Orsolya Tőke3; Dóra K. Menyhárd2; István Vida1; Zoltán Orgován4; György M. Keserű4; András Perczel1, 2
1ELTE University, Lab. of Struc. Chem. & Biol., Budapest, Hungary; 2MTA-ELTE Protein Modelling Research Group, Budapest, Hungary; 3Research Centre for Natural Sciences, NMR lab., Budapest, Hungary; 4Research Centre for Natural Sciences, Med.Chem.Lab, Budapest, Hungary

K-Ras protein is a membrane-bound small GTPase acting as a molecular switch and playing a key role in many signal transduction pathways (regulation of cell proliferation, differentiation and survival). It alternates between its GTP-bound active and the GDP-bound inactive conformers. Its most frequent oncogenic mutants are G12C, G12D, and G12V which are among the most important mutations causing cancer in human. We developed a new method enabling us to work with the native K-Ras-GTP system without the artefacts caused by GTP analogues. Here we are providing backbone dynamics with its analysis and role in biological function of KRas and its oncogenic mutants in both fast and slow timescales in its GDP- and GTP-bound forms combined with MD and QM/MM simulations.

Session: SOLUTION, poster number: 027

The Overhauser Dynamic Nuclear Polarization Spectrometer: A Tool for Building Maps of Hydration Water

John Franck
Syracuse University, Syracuse, NY

Coating the surface of every macromolecule or macromolecular assembly, one finds a hydration layer composed of water molecules that move typically between 3× and 10× slower than water molecules in bulk water. Overhauser Dynamic Nuclear Polarization (ODNP) is an emerging electron-spin nuclear-spin (EPR-NMR) double-resonance tool that has demonstrated a capability of measuring the translational dynamics of water in the hydration layer. We design a scheme for measuring the thickness of the hydration layer and the effect of confinement on translational dynamics, as measured by ODNP, with controlled, appropriately labeled reverse micelle systems. We also present results acquired from spin-labeled protein systems, and we outline hardware and software improvements that enable all these new results.

Session: SOLUTION, poster number: 028

Structure, Dynamics and Evolution of Acyl-carrier Proteins from Different Human Pathogens:  Borrelia burgdorferi , Brucella melitensis and Rickettsia prowazekii

Mandeep Kaur1; Ravi P Barnwal1; Alec Heckert2; Janeka Gartia3; Gabriele Varani2
1PANJAB UNIVERSITY, Chandigarh, India; 2University of Washington, Seattle (WA), Washington, 98195 , USA; 3Tata Institute of Fundamental Research, Gopanpally, Hyderabad , Telangana , INDIA

Acyl carrier proteins (ACPs) are proteins that partake in fatty acid and polyketide biosynthesis. These are involved in the type II synthesis systems found in bacteria and plastids. In bacteria, these proteins exist as monomeric soluble protein and perform various processes, from being a donor for the synthesis of various products as endotoxin, quorum sensing, and activation of toxins. Here, we report the structure, dynamics, and evolutionary relationship of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, that suggests ACP in Borrelia burgdorferi and Rickettsia prowazekii emerge from a common ancestor whereas Brucella melitensis is more distantly related. This comparative study will pave way for the discovery of new inhibitors of ACP function.

Session: SOLUTION, poster number: 029

Understanding the Role of Conformational Dynamics of a Picornaviral Polyprotein in Expanding the Viral Proteome

Dennis Winston; David Boehr
Penn State University, University Park, PA

Picornaviruses are a medically and agriculturally important family of viruses, of which poliovirus is an excellent model system. The 72 kDa poliovirus 3CD protein is central to the life cycle of the virus, as it is involved in important protein-protein, protein-RNA and protein-lipid interactions. The molecular mechanisms underlying the functional differences between 3CD and its processed products are not well understood despite the availability of x-ray crystal structures, and we have proposed that the conformational dynamics of 3CD allow it to access different conformations to perform different functions. To this end, we have used CPMG relaxation dispersion, chemical exchange saturation transfer, and methyl spin-spin relaxation NMR experiments to identify differences in conformational dynamics between 3CD and its processed products.

Session: SOLUTION, poster number: 030

Can linker length and domain mobility control the interacting capabilities of multidomain proteins?

Pedro Diaz-Parga2; Eva De Alba1
1Department of Bioengineering, Merced, CA; 2Quantitative Systems Biology Program, Merced, CA

The protein ASC mediates the assembly of the supramolecular complex inflammasome by self-association and protein interactions via its two Death Domains, PYD and CARD connected by a long linker. An isoform of ASC (ASC-b) is composed of the identical two domains albeit tethered by a much shorter linker. Importantly, ASC-b appears to be less efficient than ASC, suggesting that the length of the linker plays a vital role in protein assembly. Investigation into the interdomain dynamics of ASC and ASC-b using 15N relaxation techniques in conjunction with real-time NMR kinetics on protein self-association allows to determine the role of the linker length on the functionality of multidomain proteins.

Session: SOLUTION, poster number: 031

Backbone dynamics Study of Z α domain of yatapoxvirus E3L in  complex with Z-DNA

Hye-Bin Ahn; Joon-Hwa Lee
Gyeongsang National University, Jinju, South Korea

Z-DNA binding proteins are critical players in various cellular functions such as RNA editing and the innate immune response. The E3L protein is essential for pathogenesis in the poxviruses and consists of an N-terminal Z-DNA binding domain and a C-terminal RNA binding domain. The crystal structures of the Zα domain of yatapoxvirus E3L (yabZαE3L) in complex with 6-base-paired DNA duplex found that the interaction with Z-DNA is mediated by the α3 helix and in the β-hairpin. A previous NMR hydrogen exchange study suggested the active-mono B–Z transition mechanism of a 6-bp DNA duplex like hZαADAR1 , in which (i) one molecule of yabZαE3L binds to B-DNA; (ii) the conformational transition from B-DNA to Z-DNA in the complex follows; and finally (iii) the stable one-to-two Z-DNA–yabZαE3L complex is produced by addition of another hZαADAR1 molecule. In this study, we performed backbone dynamics studies on the complex of yabZαE3L with DNA duplex, d(CG)3. This study suggests the unique conformational flexibility during complex formation of yabZαE3L with Z-DNA.

Session: SOLUTION, poster number: 032

An Induced-Fit Asymmetric Receptor Tyrosine Kinase Dimer Mediates A-loop Transphosphorylation

William Marsiglia; Lingfeng Chen; Huaibin Chen; Joseph Katigbak; Yingkai Zhang; Thomas Neubert; Moosa Mohammadi; Nate Traaseth
New York University, New York, NY

A long-standing mystery shrouds the mechanism whereby the catalytically repressed receptor tyrosine kinase domains accomplish activation loop (A-loop) tyrosine transphosphorylation. In this work, we used a combination of NMR spectroscopy, X-ray crystallography, and functional assays to show that this quintessential reaction proceeds via an asymmetric complex that is thermodynamically disadvantaged because of electrostatic repulsion between the enzyme and substrate kinases. The model proposed explains how quantitative differences in the stability of ligand-induced extracellular dimerization promote formation of the intracellular A-loop transphosphorylating asymmetric complex to varying extents, thereby modulating intracellular kinase activity and signaling intensity.

Session: SOLUTION, poster number: 033

Redox Dependent Structural and Dynamic States of Macrophage Migration Inhibitory Factor

Kyle East; George Lisi
Brown University, Providence, RI

Macrophage migration inhibitory factor (MIF) is a pleitropic cytokine critical for the innate immune system and inflammation in humans. MIF is overexpressed in regions of inflammation and tissue of patients with inflammatory diseases such as arthritis, asthma, ARDS, pulmonary fibrosis, and cancer. One of the hallmarks of inflammatory diseases is an oxidative cellular environment due to the production of excess reactive oxygen species. Using NMR, we sought to better understand the redox-dependence of MIF. To probe the conformational entropy, we measured NMR order parameters under reducing, neutral, and oxidizing conditions. In addition, we identified a conformation of MIF that exists under oxidizing conditions. These NMR-detected structural and dynamic changes to MIF highlight possible mechanisms for MIF involvement in inflammatory diseases.

Session: SOLUTION, poster number: 034

13C-methyl Methionines as Probes in NMR Studies of ST6Gal1

Alexander Eletsky; Monique J. Rogals; Robert V. Williams; Gordon R. Chalmers; Caleb Schmidt; Laura C. Morris; Jeong-Yeh Yang; Kelley W. Moremen; James H. Prestegard
University of Georgia, Athens, GA

Sialyltransferase ST6Gal1 is a glycoprotein that catalyzes formation of a 2-6 linkage between sialic acid and galactose-terminated acceptors. A 1 us MD trajectory shows a substantial movement of a loop near the active site, which may correlate with biochemical evidence for a sequential mechanism in which the sialyl-CMP donor enters before the acceptor. Our goal is to provide experimental support for this movement and its correlation with mechanism.  Glycoproteins are best expressed in mammalian cells where isotopic labeling with selected amino acids is preferred. We have chosen to employ 13C-methyl labeling of methionines in ST6Gal1. Initial data show two crosspeaks for the methionine present within the loop. We will discuss ongoing efforts at methionine assignment and collection of structural data.

Session: SOLUTION, poster number: 035

Protein instability in Cadherin-11 dimerization: Relaxation dispersion and high-pressure studies of multi-site chemical exchange reveal partially folded dynamic intermediates

Hans Koss; Arthur Palmer
Columbia University, New York City, NY

We are investigating the multi-site dimerization kinetics of the cell adhesion protein Cadherin-11 (type II). In particular, we are interested in the role of partially unfolded dimerization intermediates. We gained experimental access to various conformational states recording relaxation dispersion data at various fields, pressures and temperatures. Our theoretical work on multi-site RD experiments assisted in quantitatively discovering and quantifying three-site chemical exchange in Cadherin-11 via a global fitting procedure combining data from various experiment types. We were also able to map various exchange processes to (a) the binding pocket for Trp strand swapping, (b) as well as to partial unfolding of the protein. A certain instability of Cadherin-11 is usful to obtain the biological required dimerization kinetics.

Session: SOLUTION, poster number: 036

Optimizing Methyl NOE Acquisition and Interpretation for Glycoprotein Studies

Robert Williams1; Ilya Kuprov2; James Prestegard1
1University of Georgia, Athens, GA; 2University of Southampton, Southampton, United Kingdom

Methyl groups prove particularly valuable in the study of large and sparsely labeled glycosylated proteins, because of the high sensitivity and resolution provided by 1H-13C crosspeaks in HSQC and HMQC spectra. NOEs detected through these crosspeaks provide potentially useful structural information. However, interpretation is problematic because cross-correlation and internal rotation effects lead to different contributions from like and unlike spin states and complex NOE transfer curves.  We propose a method for selective detection of NOEs involving like and unlike spin states and complement this with simple pseudo-spin simulation models that allow interpretation of data. Applications to a glycoprotein, Robo1-D1, isotopically labeled with 13C in all valine methyl groups, and simulations with Spinach software will be used to illustrate the procedure.

Session: SOLUTION, poster number: 037

Structural and Functional Studies of the Polar Organizing Protein Z from Caulobacter crescentus using Solution NMR Spectroscopy

Christopher Nordyke1; Ryan Puterbaugh1; Haibi Wang2; Grant Bowman2; Krisztina Varga1
1University of New Hampshire, Durham, New Hampshire; 2University of Wyoming, Laramie, Wyoming

Polar organizing protein Z (PopZ) is an intrinsically disordered protein (IDP) from the bacterium Caulobacter crescentus that self-assembles into 3D polymeric superstructures and is necessary for the localization of other proteins at cell poles. PopZ has been shown to interact with at least eight different proteins. Current NMR work investigates the structure of unbound PopZ, the binding of PopZ and its partners, and characterizing potential structural changes of PopZ upon binding.

Session: SOLUTION, poster number: 038

Cholesterol Under Pressure:  1H NMR T2* Studies of Lipoprotein Particles at High Pressure

Mary Starich; Nico Tjandra
BBC, NHLBI, NIH, Bethesda, MD

This study explores the deformability LDL particles compared to other particle subclasses (HDL, VLDL) under high pressure utilizing frequency shift and T2* measurements in an effort to differentiate the terminal cholesteryl CH3 peak resonances among sub-fractions. In this study, 1H NMR T2* values for the terminal cholesteryl CH3 peak in each sub-fraction as a function of pressure are reported at three temperatures.  Most interesting, is the apparent phase transition that occurs for LDL but not the other particle types, when observed at pressures from 1 – 2400 Bar.  The observed T2* behavior as a function of pressure can be described with a segmented linear fit to estimate the pressure at which phase transition occurs.

Session: SOLUTION, poster number: 039

B-Z transition pathway studied by using the fusion Protein of Zα domains of human ADAR1 and Vaccinia Virus E3L

Na-Hyun Kim; Joon-Hwa Lee
Gyeongsang National University, Jinju, South Korea

Left-handed Z-DNA, which is a polymer of alternating d(CG)n sequence, is a higher energy conformation than B-DNA. Z-DNA is induced by high salt, negative supercoiling, and complex formation with Z-DNA binding proteins. Z-DNA binding domains are found in the RNA editing enzyme (ADAR1) and DNA-dependent activator of IFN-regulatory factor (DAI) in vertebrates, the E3L protein of poxviruses. Recent NMR studies of the complex formed between ZaADAR1 and a 6-base-paired (6-bp) DNA duplex referred to as d(CG)3, have suggested an active B–Z transition mechanism, in which the ZαADAR1 protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is subsequently stabilized by the additional binding of a second ZαADAR1 molecule. Another Z-DNA Binding E3L consists of two domains: an N-terminal Z-DNA binding domain and a C-terminal RNA binding domain. This N-terminal region shows sequence homology to the Zα domains found in human ADAR1 (hZaADAR1). The Z-DNA binding affinity of the Zα domain of E3L is required for viral pathogenicity. Here, to investigate the molecular mechanism of the B–Z transition of a DNA duplex induced by the fusion protein ZαADAR1+E3L(ZαA+E), we have conjoined ZαADAR1 with ZαE3L and performed Backbone assignments on the Protein as well as DNA-Protein Titration study of ZαA+E and d(CG)3 with a variety of protein-to-DNA (P/N) molar ratios. Comparison of these results with those from the analysis of hZαADAR1–d(CG)3 and ZαE3L-d(CG)3 in a previous study leads to valuable insights into the molecular mechanism of the B–Z transition of a DNA duplex induced by the fusion protein.

Session: SOLUTION, poster number: 040

Investigation of the Interaction between hnRNP A18 and the 3’UTR of Thioredoxin (TRX) mRNA

Katherine Coburn1; Braden Roth2; France Carrier1, 3; David Weber1
1University of Maryland, Baltimore, MD; 2Medical University of South Carolina, Charleston, SC; 3Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD

Skin cancer is the most common form of cancers and malignant melanoma accounts for over 72% of skin cancer deaths each year. Heterogeneous nuclear ribonucleoprotein A18 (hnRNP A18) is an RNA binding protein differentially expressed between cancerous tissue and normal tissue in melanoma patients. hnRNP A18 interacts with mRNA transcripts to increase their translation during the cellular stress response. A structured RNA recognition motif (RRM) in hnRNP A18 contains preserved motifs involved in the interaction with mRNA. However, it remains unknown how the unstructured arginine-glycine rich (RGG) motif in hnRNP A18 interacts with mRNA. Data will be presented for the RRM and RGG motif of hnRNP A18, and how these data aided in the assignments for the full length A18.

Session: SOLUTION, poster number: 041

Residual dipolar couplings, scalar couplings, and chemical shifts confirm an in silico 2-state kinetic model in Ribonuclease HI homologues

James Martin; Arthur Palmer
Columbia University, Manhattan, NY

Ribonuclease HI (RNHI), a ubiquitous, non-sequence-specific endonuclease, is an essential retroviral reverse transcriptase domain and cleaves RNA in RNA/DNA hybrids. Molecular Dynamic (MD) simulations and NMR spectroscopy suggests RNHI’s handle region domain populates "open" (substrate-binding competent) and "closed" (substrate-binding incompetent) states; E. coli RNHI (EcRNHI) populates both at room temperature, while T. thermophilus RNHI is closed. EcRNHI’s in-silico mutant, Val98Ala, is closed and its experimental Michaelis constant is intermediate of the homologues. Conformational preferences correlate with Val101’s rotamer state and Thr92’s backbone interactions. Measured NMR observables-residual dipolar couplings, scalar couplings, chemical shifts-support the simulations and correlate with RNHI homologues’ Michaelis constants. These results display the integrative power of MD simulations and NMR spectroscopy in discovering conformational preferences underlying enzyme activity

Session: SOLUTION, poster number: 042

Exploring Complex NMR Line Shape Patterns in Multi-State Isomerization and Ligand-Binding Equilibria with the IDAP Model Library

Evgenii Kovrigin
University of Notre Dame, Notre Dame, IN

Interactions of ligands with biological macromolecules are sensitively detected through changes of chemical shifts and line shapes of the NMR signals. To enable simulations of 2D NMR spectra in systems where ligand interactions are coupled to multiple isomerization transitions, I developed a set of kinetic and thermodynamic models, IDAP Model Library, interfaced with TITAN [Waudby et al., Scientific Reports, 2016]. The molecular mechanisms included in the IDAP Model Library aim to describe intrinsically disordered proteins, autoinhibited, and allosteric enzymes among other systems. Simulations of NMR titrations may help anticipate the salient features to be observed in the particular cases and allow to design appropriate NMR titrations to differentiate complex molecular mechanisms.

Session: SOLUTION, poster number: 043

NMR Dynamics study of Transcription Factor protein DLX3 Complexed with a target DNA

Ho Seong Jin; Seo-Ree Choi; Joon hwa Lee*
Gyeongsang National University, Jinju, South Korea

 Transcription Factor is a protein that controls the rate of transcription by binding to a specific DNA sequence. Drosophila distal-less homeobox 3(DLX3) gene is expressed throughout development in a series of structures derived from epithelial mesenchymal interaction such as the teeth, hair follicles, and limb buds. Sequence-specific binding to DNA is crucial for targeting transcription factor-DNA complexes to modulate gene expression. To understand the molecular mechanisms of specific DNA recognition of DLX3, we have performed 1H/15N HSQC experiment and imino proton NMR experiments between TF and consensus TF Binding Site in DNA. The exchange rate constants of the imino protons for the wild type DNA, and TF-DNA complex were measured by using water magnetization experiment. We also determine T1,T2 relaxation rate constants of DLX3 in the complex with DNA.

Session: SOLUTION, poster number: 044

Artificial Intelligence in RF Pulse Design: Applications from High Resolution NMR to Imaging

Manu Veliparambil Subrahmanian1; Kowsalyadevi Pavuluri2; Cristina Olivieri1; Gianluigi Veglia1
1University of Minnesota, Minneapolis, MN; 2Johns Hopkins Medical Institutions, BALTIMORE, MD

High fidelity unitary control of quantum systems is central to quantum computing and several spectroscopies spanning from optics, coherent spectroscopy, NMR, MRI and EPR. Here we introduce a time-optimal RF pulse design strategy and developed a Neural Network for generating high-fidelity broadband RF pulses with customizable operation, bandwidth, RF inhomogeneity compensation and operational fidelity. Applications include traditional NMR experiments, imaging at high RF inhomogeneity and high-fidelity operations for quantum information processing. We generated a robust entanglement operator as well as a programmable quantum state creator and experimentally verified them using nuclear spins.

Session: SOLUTION, poster number: 045

Structure and Dynamics Studies of a Lesion Bypass DNA Polymerase Dpo4

Eunjeong Lee
Ohio state university, Columbus, OH

Ca2+, a non-reactive cofactor, has been extensively used for DNA polymerases to substitute biologically relevant Mg2+ to trap the reaction intermediates. Here, we use NMR techniques to investigate the structure and dynamics of a model DNA lesion bypass polymerase, Dpo4. While both the large catalytic and the unique little finger domain resemble the crystal structures, they are dynamically independent in solution. In the presence of Ca2+, transient interaction with phosphate ions induces ms-µs equilibrium with a minor conformation, structurally closely resembling the DNA-bound state. Mg2+ did not introduce similar dynamics possibly due to lower affinity with apo-Dpo4 than Ca2+. Upon DNA binding, Mg2+ but not Ca2+ activates Dpo4 by promoting significant ms-µs motions.

Session: SOLUTION, poster number: 046

Relaxation Analysis of Polysaccharides from Variable Temperature Experiments

Hugo Azurmendi1; Jasmin Zarb1; Göran Widmalm2; Dáron Freedberg1
1CBER-FDA, Silver Spring, MD; 2Stockholm University, Stockholm, Sweden

Complex polysaccharides (PS) structural characterizations are challenging because of the paucity of parameters accessible from experiments. Moreover, the dynamic nature of these molecules in solution further complicates matters. Thus, the development of tools for structure and dynamics studies of PS are essential to the development of structural glycobiology. Relaxation analysis contributes to the characterization of the dynamic behavior of PS, which for biomolecules it is commonly based on measurements of T1 and T2 values at one or two temperatures. In this work we explore a novel approach to obtain Lipari-Szabo order parameters and effective correlation times from simultaneous fitting of R1 and R2 data, measured within a limited range of temperatures.

Session: SOLUTION, poster number: 047

Structural Insight into the Length-dependent Binding of ssDNA by SP_0782 from Streptococcus pneumoniae

Shuangli Li; Jiang Zhu; Maili liu; Yunhuang Yang
WIPM, Wuhan, China
SP_0782 from Streptococcus pneumoniae is a PC4-like protein binding with single-stranded DNA (ssDNA), but has an ssDNA-binding property different from other reported PC4-like proteins. Here, we show that although SP_0782 adopts an overall fold similar to the known PC4-like proteins, the detailed DNA-binding mechanism is different. SP_0782 exhibits varied binding affinities and patterns for different lengths of ssDNA, and tends to form large complexes with ssDNA. The structures of SP_0782 complexed with different ssDNAs reveal that the varied binding patterns are associated with distinct capture of nucleotides in two major DNA-binding regions of SP_0782. Moreover, a comparison of known structures of PC4-like proteins complexed with ssDNA suggests a divergence in the binding interface between prokaryotic and eukaryotic PC4-like proteins.

Session: SOLUTION, poster number: 048

Molecular recognition of Lys63-linked diubiquitin chains by STAM2 multidomain protein : the effect of its linker length and flexibility

Minh-Ha Nguyen1; Marie Martin1; François-Xavier Cantrelle2; Olivier Walker1; Maggy Hologne1
1University of Lyon, Villeurbanne, France; 2Université de Lille, Lille, France

Multidomain proteins represent a broad spectrum of the protein landscape and are involved in various interactions. Different or similar domains are connected by linkers that could be of varying length and amino acids composition. Here, we are seeking to understand the effect of the flexibility and dynamics of the linker involved in the STAM2 multidomain protein with respect to molecular recognition with linked K63-diubiquitin. We have engineered six constructs of VHS-UIM or UIM-SH3 where linkers have been shortened at different lengths (US-D1, US-D2 and VU-T) or mutated with proline residues (VU-P). Affinities have been calculated and we conclude that a modification of the linker has a great impact in molecular recognition while the linker is not part of the interaction.

Session: SOLUTION, poster number: 049

Probing water-protein contacts in the catalytic triad of DENV2 type serine protease and its mutants by NMR spectroscopy

Tatiana Agback; Peter Agback
Swedish University of Agricultural Sciences, Uppsala, Sweden

Using the catalytic domain of the serine protease, Dengue-2, in apo form, its complexes with potent peptide type of inhibitors, novel developed approaches and experiments are used to monitor and distinguish protein amide protons in fast exchange with bulk water from amide protons close to water molecules with longer residence times. The data is discussed with a focus on the possibility of detecting water molecules of structural or functional importance.

Session: SOLUTION, poster number: 050

Studying the role of order and disorder in the human glucocorticoid receptor by R2-filtered NMR spectroscopy

Emily Grasso; Ananya Majumdar; Vincent Hilser
Johns Hopkins University, Baltimore, MD

The human glucocorticoid receptor (GR) is critically biologically important, but much is still unknown about this protein biophysically.  NMR-based studies to understand the structural and dynamic basis of differences between translational isoforms of GR were initially hindered by spectral degeneracy between structured and unstructured residues in the protein, along with poor protein expression and sample instability.  We have developed a HN-detect, 15N R2-based filtration strategy that capitalizes on differences in 15N relaxation rates between resonances from structured and unstructured regions.  With the sub-spectra generated by these experiments, we have been able to interpret chemical shift data that was previously inaccessible to us.  We are pursuing further NMR experiments to elucidate the structural and dynamical underpinnings of differences between isoforms.

Session: SOLUTION, poster number: 051

Study of Self-association of human CstF-64 RNA Recognition Motif

Elahe Masoumzadeh
Texas Tech university, Lubbock, TX

CstF-64 RNA recognition motif (RRM) binds to the G/U rich RNA sequences located downstream of the mRNA cleavage and polyadenylation site and hence is an important part in the regulation of cleavage and polyadenylation and therefore mRNA maturation. Some hypothesize that in vivo the CstF complex forms a dimer to bind to RNA. Here with the use of NMR spectroscopy, specifically the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment, and isothermal titration calorimetry (ITC), we have shown that RRM can oligomerize via a low population intermediate in vitro. Next, based on our proposed oligomer model from the CPMG data, we designed RRM mutants to enhance and disrupt oligomerization upon binding to RNA to have a better understanding of the protein self-association process.

Session: SOLUTION, poster number: 052

NMR Study of target DNA recognition of Transcription Factor MEIS1

Seo-Ree Choi; Joon-Hwa Lee
Gyeongsang National University, Jinju, South Korea

 Transcription factors are proteins that bind specific sites or elements in regulatory regions of DNA, known as promoters or enhancers, where they control the transcription or expression of target genes. MEIS1 (myeloid ecotropic viral insertion site 1) is a viral integration site in murine myeloid leukemia cells. This gene encodes a homeobox protein belonging to the TALE ('three amino acid loop extension') family of homeodomain-containing proteins. TALE proteins are distinguished by the presence of three extra amino acids in the loop binding the first to the second alpha helix of the homeodomain. The highly conserved DNA-binding TALE proteins define the family and is responsible for specific recognition of a common sequence motif, [5'- TGACA- 3']. Further DNA-binding specificity within TALE family members is determined by adjacent DNA sequences and through the binding of additional transcriptional partners.

 In order to characterize the molecular recognition of DNA by MEIS1, we performed NMR experiments with complexes between complexes of MEIS1 bound to its target DNA (msDNA). We also studied the binding features of MEIS1 for both wild type and mutant DNAs characterized its target DNA recognition.

Session: SOLUTION, poster number: 053

Structural Dynamics Couple Substrate Recognition with Allosteric Responses in Nonribosomal Peptide Synthetases

Subrata Mishra1; Aswani Kancherla1; Kenneth Marincin1; Santrupti Nerli2; Nikolaos Sgourakis2; Guillaume Bouvignies3; Daniel Dowling4; Dominique Frueh1
1Johns Hopkins School of Medicine, Baltimore, MD; 2University California Santa Cruz, Santa Cruz, California; 3Ecole Normale Superieure, Paris, France; 4University of Massachusetts, Boston, Massachusetts

Nonribosomal peptide synthetases (NRPSs) use repeats of domains to tether substrates onto carrier protein domains (CP) and assemble them into complex products through intervening condensation or cyclization domains. Dynamics between domains have hampered NRPS engineering because it is unclear whether and how catalytic steps required for synthesis drive transient domain interactions. Here, we demonstrate that large scale structural dynamics within a 52 kDa cyclization domain sense substrates tethered to partner CPs to promote binding and to open an allosteric path between two remote binding sites and the active site. Our results exemplify how structural dynamics within proteins can couple substrate recognition with active site remodeling and promotion of both local and remote protein communication.

Session: SOLUTION, poster number: 054

Chemoenzymatic synthesis of versatile rNTPs to improve RNA structure and dynamic analysis by multidimensional NMR spectroscopy

Owen Becette; Lukasz Olenginski; Guanghui Zong; Mary Taiwo; T. Kwaku Dayie
University of Maryland, College Park, MD

RNAs are increasingly found as critical regulatory components in biological processes that result in human disease. Despite being composed of only four unique nucleobase building blocks, RNAs are capable of adopting complicated three-dimensional structures that impart functionality. Currently, NMR spectroscopy is the only high-resolution biophysical technique capable of probing these dynamic RNA structures in solution. However, the available NMR methodologies are limited to small RNAs (~ 10 kDa) due to decreased spectral resolution and sensitivity with increasing molecular weight. To overcome these limitations, we present novel isotopic labeling schemes and NMR experiments capitalizing on these labels. We anticipate that these methodologies will facilitate NMR structural and dynamics studies of RNAs of increasing size (> 50 kDa) and complexity.

Session: SOLUTION, poster number: 055

Creating Science Gateways for NMR Data Analysis and Structure Modeling

Yuexi Chen1; Cheol Jeong2; Alexey Savelyev2; Andy Guan1; Matthew Casertano1, 3; Susan Krueger4; Joseph Curtis4; Emre Brookes2; David Fushman1
1University of Maryland, College Park, MD; 2University of Montana, Missoula, MT; 3Montgomery Blair High School, Silver Spring, MD; 4National Institute of Standards and Technology, Gaithersburg, MD

Several software packages have been developed by NMR researchers in order to tackle complicated experimental data analysis and structure/ensemble modeling. However, many of them are offline packages or command-line applications (available from individual authors or joint depositories) that require users to set up the run-time environment and also to possess certain programming skills, which inevitably limits accessibility of this software to a broad scientific community. Here we present new science gateways for analysis of spin-relaxation data, residual dipolar couplings, and paramagnetic effects that address these current limitations by enabling online analysis of data and provide advanced computational functionalities, cloud-based data management, seamless integration with structural ensemble generation tools, and interactive 2D and 3D plotting and visualizations.

Session: SOLUTION, poster number: 056

General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing

Chunyu Wang1; Jing Zhao2
1Rensselaer Polytechnic Institute, Troy, NY; 2RPI, TROY, NY

Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp to His mutant (D46H) retains enzyme activity and active site conformation, confirmed by NMR pKdetermination and X-ray structure. Το our knowledge this is the first example where a general base substitution of an Asp for His preserves both structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200 fold) and epicoprostanol (>300 fold). Our findings have important implications for protein engineering and enzyme design.

Session: SOLUTION, poster number: 057

The STRA6 vitamin A transporter exhibits calcium-dependent and calcium-independent interactions with Calmodulin

Brianna Young
University of Maryland, Baltimore, Baltimore, MD

STRA6, the receptor for intracellular retinol uptake, interacts with the calcium binding protein Ca2+-Calmodulin (CaM) at its cytoplasmic side. The major interaction interface of this interaction occurs at a helix of STRA6 termed BP2, which interacts within CaM’s hydrophobic groove. To understand the details of the CaM-STRA6 interaction, Ca2+-competition, ITC and NMR studies were performed with a STRA6 BP2 derived peptide. The interaction between CaM and BP2 was found to be calcium-independent; chemical shift perturbation data shows two different CaM-STRA6 binding modes; one in the absence and another in the presence of calcium. 

Session: SOLUTION, poster number: 058

Molecular Responses of Mutagenesis in Nonribosomal Peptide Synthetase Cyclization Domains

Kenny Marincin1; Aswani Kancherla1; Subrata Mishra1; Daniel Dowling2; Dominique Frueh1
1Johns Hopkins School of Medicine, Baltimore, MD; 2University of Massachusetts Boston, Boston, MA

Nonribosomal peptide synthetases (NRPSs) utilize a dynamic modular architecture to synthesize peptides, often with medicinal and industrial properties. Here, we focus on a cyclization domain Cy1 responsible for the condensation and subsequent cyclization of salicylate and cysteine substrates to form a precursor for the bubonic plague virulence factor yersiniabactin. Through relaxation dispersion, we uncovered a network of dynamic residues for allosteric communication between two remote binding sites. Here, we use Hahn-Echo relaxation and IDIS to show that mutagenesis within this domain not only impairs the enzymatic activity of Cy1 but weakens this dynamic network, rendering the domain unresponsive to one of its partner NRPS domains as observed using in situ biochemistry with an NMR readout.

Session: SOLUTION, poster number: 059

NMR Studies Reveal the Mechanism for High-affinity Interaction of nSH3/cSH3 Domains of Grb2 with the C-terminal Proline-Rich Domain of SOS1

Tsung-Jen Liao1; Hyunbum Jang2; Ruth Nussinov2; David Fushman1
1University of Maryland, College Park, MD; 2Frederick National Laboratory for Cancer Research, Frederick, MD

Adaptor protein Grb2 recruits Ras-specific guanine nucleotide exchange factor SOS1 to the plasma membrane, where SOS1 activates Ras, triggering signaling pathways for cell proliferation. Grb2-SOS1 association is mediated by interactions between Grb2 nSH3 and cSH3 domains and proline-rich (PR) domain of SOS1. Several nSH3-binding motifs have been identified in SOS1 PR but none specific for cSH3. Using NMR, we assayed seven potential SH3-binding SOS1 PR peptides and identified PKLPPKTYKREH peptide with high affinity for cSH3, but weak for nSH3. NMR binding data combined with replica-exchange simulations suggest a likely Grb2-SOS1 binding mode involving concurrent nSH3-PVPPPVPPRRRP and cSH3-PKLPPKTYKREH interactions. The Grb2-SOS1 binding mechanism outlined here offers new venues for future therapeutic strategies for upstream mutations in cancer, such as in EGFR.

Session: SOLUTION, poster number: 060

Substrate Interaction Inhibits γ-secretase Production of Amyloid-β Peptides

Chunyu Wang1; Jing Zhao2
1Rensselaer Polytechnic Institute, Troy, NY; 2RPI, TROY, NY

The inhibition of γ-secretase to reduce amyloid load is a prominent strategy in AD drug discovery, but clinical trials of γ-secretase inhibitors (GSI) failed due to side effects. In an effort to circumvent GSI side effects, we discovered compounds that target the transmembrane domain of the amyloid precursor protein (APPTM). Compound C1 binds APPTM both non-covalent and covalently, and inhibit γ-secretase cleavage with an IC50 of 1.9 µM for Aβ42 and 3.9 µM for Aβ40. C1 decreases the production of Aβ42 more than Aβ40 in HEK 293 cells, reducing the Aβ42/Aβ40 ratio.  Our work showed that interaction with the juxtamembrane lysines of APPTM is sufficient to inhibit Aβ production, suggesting APPTM as a novel target in AD drug discovery.

Session: SOLUTION, poster number: 061

Calcium Regulates S100A12 Zinc Sequestration by Limiting Structural Variations

Qian Wang2; Aleksey Aleshintsev1, 2; Aneesha J Jose2; James M. Aramini3; Rupal Gupta1, 2
1PhD Programs in Biochemistry The CUNY, New York, NY; 2Department of Chemistry, College of Staten Island, Staten Island, NY; 3Structural Biology Initiative, CUNY Advanced Scien, New York, NY

S100A12 belongs to the calcium binding S100 family of proteins and participates in the human innate immune response with its zinc sequestration mediated antimicrobial activities. Using Co2+ as a surrogate to Zn2+, we investigated pH dependence of metal sequestration by S100A12. While apo protein exhibits diminished Co2+ binding at sub-neutral pH conditions, addition of calcium can restore Co2+ binding up to pH 5.7. Solution NMR studies show that the side-chain protonation state of Co2+/Zn2+ binding histidine residues is not affected by calcium binding. Instead, we demonstrate that calcium restores Co2+/Zn2+ binding by inhibiting pH-dependent conformational changes to the EF hand I motif. These studies suggest the role of calcium in human immune response under physiologically relevant sub-neutral pH conditions.

Session: SOLUTION, poster number: 062

Latent Allostery in Macrophage Migration Inhibitory Factor

Erin Skeens; George P. Lisi
Brown University, Providence, RI

Macrophage Migration Inhibitory Factor (MIF) is a pro-inflammatory protein that serves as an essential regulator of innate and adaptive immune response. Overexpression of MIF at sites of inflammation has been implicated in many inflammatory conditions, such as rheumatoid arthritis, asthma, acute respiratory distress syndrome, and cancer. MIF has broad functionality as a cytokine, mediated by its promiscuous binding to its receptors, and an enzyme, with tautomerase and oxidoreductase catalytic sites. To better understand how MIF accommodates and modulates its range of functions, we utilized the sensitivity of NMR to probe the MIF structure for latent allosteric sites that confer functional control. We confirmed our findings by NMR with kinetic assays and identified novel allosteric sites that attenuate MIF tautomerase activity.

Session: SOLUTION, poster number: 063

Characterization of the C-Terminal Segment of the Streptococcus Mutans Adhesin P1 by NMR Spectroscopy

Emily-Qingqing Peng
University of Florida, Gainesville, FL

Streptococcus mutans is the virulent bacteria generating dental cavities. Adhesin P1 is a functional protein on the surface of S. mutans. Our previous studies indicate that the C-terminal region(C123) of adhesin P1 plays an important role in the formation of functional amyloid fibrils by self-assembly. We are characterizing the C123 fibril amyloid formation by NMR spectroscopy. However, full-length C123, at 51 kDa, is not amenable to full structural analysis by NMR using uniform isotopic labeling strategies, and we would also like to locate the key functional sites between the three domains in C123. Therefore, we have created constructs which enable us to specifically isotopically enrich the C12 or C3 domains in C123.

Session: SOLUTION, poster number: 064

Backbone dynamics study of zα domain of PKR-like protein kinase in the complex with Z-DNA

Youyeon Go; Joon-Hwa Lee
Gyeongsang National University, Jinju, South Korea

Left-handed Z-DNA is a higher energy conformation than B-DNA and is induced by high salt, negative supercoiling, and complex formation with Z-DNA binding protein. Double-stranded RNA-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, a PKR-like protein kinase containing Z-DNA binding domain(PKZ), plays a similar role in the antiviral response. The crystal structures of the Zα domains of Carassius auratus PKZ (caZαPKZ) in complex with 6-base-paired DNA duplex revealed that two molecules of Zα bind to each strand of Z-DNA, yielding 2-fold symmetry with respect to the DNA helical axis. Previous NMR study suggested that the intermediate complex formed by caZαPKZ and B-DNA, can be modulated by varying the salt concentration, to measure the degree to which DNA transitions to the Z isoform. Here, we measured the T1 and T2 relaxation rate constants and the heteronuclear 15N-{1H}-NOE of the zα domain of caZαPKZ complexed with Z-DNA, at various NaCl concentrations. Our results suggest the unique conformational flexibility of caZαPKZ during B-Z transiton of DNA which can be used as molecular ruler.

Session: SOLUTION, poster number: 065

Structural and Dynamical Determinants of pH Sensitive GMCSF-heparin Interactions

Jennifer Cui; George Lisi
Brown University, Providence,

Granulocyte Macrophage Colony Stimulating Factor (GMCSF) is a cytokine important for the growth and differentiation of granulocytes and macrophages in humans. GMCSF participates in both protective and pathogenic biological processes in various immune-dysfunctional syndromes. For its immune function, GMCSF is administered clinically as an immuno-stimulatory agent. Previously, it was shown that GMCSF and heparin interact under specific pH conditions. Given the numerous roles of GMCSF in disease and canonical immune response, our objective was two-fold. By NMR, we sought to investigate structural mechanisms contributing to the tunable binding affinity GMCSF exhibits, and to characterise at the molecular level the interaction of two clinically relevant molecules administered to critically ill patients. 

Session: SOLUTION, poster number: 066

Fast Field Cycling Application to Proteins

Giacomo Parigi1; Moreno Pasin2; Rebecca Steele2; Gianni Ferrante2
1University of Florence, Florence, Italy; 2Stelar s.r.l., Mede, Italy
Fast field cycling (FFC) NMR relaxometry is a non-destructive low-field magnetic resonance technique which measures the dependence of the spin-lattice relaxation rate R1 (= 1/T1) on the applied magnetic field over a wide range of field strengths using just one instrument. The FFC technique can be applied to proteins for obtaining information regarding: the level of aggregation of a protein, changes in protein aggregation, protein conformational changes due to temperature changes, protein-protein interactions, estimation of the degree of hydration of a protein, protein folding, correlation times, etc. In this work we will report a few studies focusing on some of these proteins-related topics. In particular, we will show some FFC applications of interest for the bio-pharmaceutical industry.

Session: SOLUTION, poster number: 067

Unique Conformational Features of a Branched poly-Ubiquitin Revealed by Integrated Structural Biology Approach

Andrew Boughton1; Susan Krueger2; David Fushman1
1University of Maryland, College Park, MD; 2NIST Center for Neutron Research, Gaithersburg, MD

Polymeric ubiquitin chains -- essential post-translational modifiers -- can have specific architectures, which convey distinct signaling outcomes depending on the linkages involved. Recently, branched K11/K48-linked polyubiquitins were shown to enhance proteasomal degradation during mitosis. To better understand the underlying structural mechanisms, we determined the crystal and NMR structures of branched K11/K48-linked tri-ubiquitin and discovered a previously unobserved interdomain interface between the distal ubiquitins. SANS and site-directed mutagenesis corroborated the presence of this interface, which we hypothesized to be influential in the physiological role of branched K11/K48-linked chains. Excitingly, significantly stronger binding affinity for branched K11/K48-linked tri-ubiquitin was observed during NMR titrations with proteasomal subunit Rpn1, suggesting a functional impact of this interface and pinpointing the mechanistic site of enhanced degradation.

Session: SOLUTION, poster number: 068

19F-NMR screening of RNA for fragment-based drug discovery using an accessible small molecule library 

Albrecht Völklein1; Oliver Binas1; Tom Landgraf1; Christian Richter2; Sridhar Sreeramulu2; Marcel Blommers3; Harald Schwalbe1
1Goethe University Frankfurt, Frankfurt Am Main, Germany; 2Center for Biomolecular Magnetic Resonance, Frankfurt, N/A; 3Saverna, Basel, Switzerland

Drug discovery through fragment-based screenings yields lead-structures that can be interlinked or grown into strong binders. This methodology has so far only rarely been applied to highly structured RNAs and these studies focused on single targets. Using 19F-detected relaxation experiments on 106 fluorinated fragments in multicomponent mixtures and 8 different RNA targets found multiple hits ranging from general RNA binders to single target specific. Hits were confirmed using single fragment titrations, TOCSY, HMQC or through ITC measurements. These findings prove the viability of the library for the search of target-specific RNA binders and the targetability of highly structure RNAs. The applied iNEXT fragment library is intended for research driven access and can easily be applied to more biological targets.

Session: SOLUTION, poster number: 069

Evaluation of NUS 13C-1H HSQC NMR Spectroscopy for Semi-Quantitative Metabolomics

Bo Zhang1; Robert Powers2; Elizabeth ODay1
1Olaris, Waltham, Massachusetts; 2University of Nebraska-Lincoln, Lincoln, NE

Metabolomics is the study of metabolism, the biochemical processes that allows organisms to grow, reproduce, maintain their structures and respond to genetic and environmental factors. By comparing metabolites between healthy and disease states new insights can be uncovered. Non-uniform sampling (NUS) is a well-accepted mode of acquiring multi-dimensional NMR data enabling either reduced acquisition times or increased sensitivity in equivalent time.  Despite these advantages the technique has not been widely applied to metabolomics. Here we demonstrate using model mixtures and human plasma that NUS based HSQC NMR spectroscopy provides a sensitive, quantitative and highly reproducible platform for metabolite profiling. 

Session: SOLUTION, poster number: 070

Directed Evolution Using Stabilized Bacterial Peptide Display

Vivekanandan Subramanian1; Tejas Navaratna2; Lydia atangcho2; Mukesh Mahajan2; Marshall Case2; Daniel Tresnaka2; Greg Thurber2
1College of Pharmacy, University of Kentucky, Lexington, Kentucky USA; 2University of Michigan, Ann Arbor, Michigan

Chemically stabilized peptides have attracted intense interest by academics and pharmaceutical companies due to their potential to hit currently “undruggable” targets. By pairing non-natural amino acid incorporation and cell surface click chemistry on bacterial surface, we developed a method to quantitatively select high affinity ligands and applied the Stabilized Peptide Evolution by E. coli Display (SPEED) technique to develop disrupters of the therapeutically relevant MDM2-p53 interface. Using solution state NMR, we have determined the structure of stabilized peptide. Homonuclear NOESY and TOCSY experiments were conducted to determine the structure of the evolutionary peptide. To understand the interaction of peptide with MDM2, one-dimensional saturation transfer difference (STD) NMR experiments were performed to identify peptide atoms interacting with the MDM2 binding interface.

Session: SOLUTION, poster number: 071

Structural studies of the Clostridium difficile binary toxin

Kristen Varney1; Mary Cook1; Xingjian Xu2; Edvin Pozharskiy1; Raquel Ruiz1; Kaylin Adipietro1; Paul Wilder1; Richard Rustandi3; Braden Roth4; Amedee Des Georges2; David Weber1
1University of Maryland, School of Medicine, Baltimore, MD; 2CUNY Advanced Science Research Center, New York, NY; 3Merck and Co., West Point, PA; 4Medical University of South Carolina, Charleston, SC

Ongoing structure-based studies of binary toxin derived from the most commonly reported source of nosocomial infection in industrialized nations, Clostridium difficile, are presented here C. difficile rates continue to increase dramatically and correlate with the emergence of a hypervirulent strain characterized by the presence of binary toxin, CDT.  Our work is focused on characterization of the pore-forming CDTb subunit using a combination of cryo-EM, crystallography and NMR. Specifically, a unique sequence at the C-terminus of CDTb, termed the receptor-binding domain (RBD) contains a novel Ca2+ binding site and was found necessary for stability and host cell toxicity. Together, this and other regions of CDTb are now being considered in structure-based drug-discovery strategies targeting CDT in the most severe strains of CDI.

Session: SOLUTION, poster number: 072

77 Se NMR Probes the Protein Environment of Selenomethionine

Shiping Xu; Qingqing Chen; Michael V. Boeri; Caitlin Quinn; Guangjin Hou; Martin B. Forstner; Sharon Rozovsky
University of Delaware, Newark, Delaware

77Se NMR is highly sensitive to its chemical environment and can report on selenium-containing amino acids in biological systems. Yet, the interpretation of protein 77Se NMR spectra remains challenging because a comprehensive database is lacking. Here, we constructed a library of GB1 variants with a single selenomethionine at different locations within the protein. We determined the chemical shielding tensor of selenomethionine in the GB1 variants by 77Se solid-state NMR as well as their structure to 1.2 Å by X-ray crystallography, allowing the conformation and surrounding environment of the selenomethionine to be unambiguously determined. Finally, we combined solution and solid-state NMR with the structural information to arrive at general recommendations for the use of selenomethionine as NMR probe in biological systems.

Session: SOLUTION, poster number: 073

NMR Studies of DSS1 and Its Interaction with RAD52

Liping Yu
University of Iowa, Iowa City, IA

DSS1 is a small and highly acidic intrinsically disordered protein and exhibits a remarkably broad functional diversity, including protein degradation, DNA repair, transcription, and mRNA export. The reported DSS1 interactome includes (i) proteasome complex in protein degradation, (ii) BRCA2–RPA complex in homologous recombination, (iii) the integrator in transcription, (iv) the Csn12–Thp3 complex in RNA splicing, and (v) the TREX-2 complex in nuclear export of mRNA and transcription elongation. Here, we report that DSS1 also directly interacts with RAD52, an DNA repair protein involved in DNA double-stranded break (DSB) repair. The binding of DSS1 to RAD52 changes the RAD52 oligomeric conformation, modulates its DNA binding properties, stimulates single strand annealing activity, and promotes strand invasion and break-induced replication (BIR).

Session: SOLUTION, poster number: 074

Examination of the Interaction between hnNRP A18 N-terminus and Drug Fragments via NMR

Karleigh Baldwin1; Sherry Fan2; Katherine Coburn1; Braden Roth3; Kristen Varney4; France Carrier1, 5; David Weber1
1University of Maryland Baltimore, Baltimore, MD; 2University of Maryland College Park, College Park, MD; 3Medical University of South Carolina, Charleston, SC; 4University of Maryland, School of Medicine, Baltimore, MD; 5Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD


Heterogeneous nuclear ribonucleoprotein A18 (hnRNP A18) is an RNA binding protein differentially upregulated in breast cancer, prostate cancer, colon cancer, and melanoma patient samples. hnRNP A18 stabilizes pro-survival mRNA transcripts throughout the cellular stress responses to UV, hypoxia, or cold. Short hairpin RNA (shRNA) against hnRNP A18 decreased tumor volumes and downregulate HIF-1α in LOX-IMVI melanoma cells in vivo mouse xenograft. Because hnRNP A18 is differentially upregulated in response to cellular stress conditions, hnRNP A18 presents a potential novel therapeutic target. Toward the development of a small molecule inhibitor for hnRNP A18, an NMR fragment screen was performed against the hnRNP A18 RRM. Data from the fragment screen and analysis will be presented and discussed.

Session: SOLUTION, poster number: 075

Structural insights of the conserved “priming loop” of Hepatitis B virus pre-genomic RNA and its therapeutic potential.

Regan LeBlanc
National Cancer Institute, Frederick, MD

Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral reverse transcriptase with a 61-nt cis-acting regulatory signal, epsilon (ε), at the 5' terminus of pre-genomic RNA (pgRNA). Binding of the viral polymerase to ε is necessary for both pgRNA encapsidation and protein-primed reverse transcription of pgRNA. Therefore, ε presents an ideal target for developing small molecule interventions to complement current nucleoside-based HBV therapies.  We present a 1.8 Å RMSD NMR refined solution structure of ε using NOE, dihedral angle, RDC, SAX, and solvent PRE measurements.  CSP and MD docking confirms specific binding of raloxifene to the priming loop, providing proof-of-principal for a novel antiviral strategy.

Session: SOLUTION, poster number: 076

Probing interactions of pioneer factors Sox2 and Oct4 with nucleosomes

Evgenia Nikolova
Johns Hopkins University, Baltimore, MD

Pioneer factors (PFs) are proteins that can efficiently bind closed chromatin, leading to nucleosome repositioning and gene activation. It remains unclear how PFs interact with nucleosomes or impact their structure and dynamics.  Here, we focus on the pluripotency factors Sox2 and Oct4, which act together to control multiple developmental programs. We employ NMR spectroscopy and nuclease and chemical footprinting to interrogate, with base-pair and atomic resolution, the binding of Sox2 and Oct4 to in-vitro reconstituted nucleosomes. Out preliminary data suggest that these factors binds promiscuously to nucleosomes and with an altered conformation as compared to naked DNA. These studies should provide new and important insights into the physical processes underlying PF-mediated chromatin opening and regulatory strategies to modulate PF activity.

Session: SOLUTION, poster number: 077

Rare 3-O-sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake

Chunyu Wang; Jing Zhao
Rensselaer Polytechnic Institute, Troy, NY

Prion-like transcellular spreading of tau in Alzheimer’s Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). Microarray and SPR assays of structurally-defined HS oligosaccharides show that a rare 3-O-sulfation(3-O-S) significantly enhances tau binding. NMR titration mapped 3-O-S binding sites to microtubule binding repeat 2 (R2) and proline-rich region 2 (PRR2) of tau. In cell culture, addition of 3-O-S HS 12-mer reduced both tau cell surface binding and cellular uptake. In Hs3st1-/-cells, reduced 3-O-S levels of HS diminished both cell surface binding and internalization of tau. Our work demonstrates that this rare 3-O-sulfation enhances tau-HS binding and likely the transcellular spread of tau, providing a novel target for disease modifying treatment of AD and other tauopathy.

Session: SOLUTION, poster number: 078

Allosteric activation of the RNA and lipid binding capabilities of the picornaviral 3CD polyprotein

David Boehr
Penn State, University Park, PA

Proteolytic precursors and fully mature forms of viral proteins often have different functions. The 3CD polyprotein has both 3C and 3D domains, but has different functions compared to the fully processed 3C and 3D proteins. The X-ray crystal structure has failed to explain these functional differences. Our solution-state NMR studies indicate that 3CD has unique structural dynamic signatures compared to 3C and 3D. We have also shown that lengthening of the C-terminus of 3C by a few residues dramatically changes function. We have identified a network of interactions that transmits structural/dynamic changes from the C-terminus to the protease active site and the RNA-binding site, which helps to differentiate 3C- and 3CD-like behavior. 



Session: SOLUTION, poster number: 079

A Multi-Modal Approach to Structural Characterization of Biotherapeutics by NMR

Korth Elliott; Luke Arbogast
NIST-IBBR, Rockville, MD

NMR is increasingly being accepted as tool for structural characterization of biotherapeutics.  Both 1D, 1H-based and 2D, 1H-13C methyl-based approaches have been proposed to accomplish such characterization.  These methods have relative attributes related to their inherent resolution and sensitivity; 1D-proton methods being high in sensitivity and low in resolution, with the converse true for 2D-methyl approaches. Given the complementarity of the methods, it would be useful to develop and integrated multi-modal approach for NMR characterization of biotherapeutics.  To that end, we report on recent measurements that compare the performance of 1D-proton and 2D-methyl approaches to report on structural variation in monoclonal antibody samples.  Results illustrate how employing NMR as a multi-modal platform provides a more complete pictures of structure.

Session: SOLUTION, poster number: 080

An Integrated NMR Framework to Characterize the Constituents and Toxicity of Wastewater

Maryam T. Anaraki1; Ronald Soong1; Markus Godejohann2; Manfred Spraul2; Paul Helm3; Bhavsar Satyendra3; Hermann Heumann4; Myrna Simpson1, 5; Andre J. Simpson1, 5
1University of Toronto Scarborough, Toronto, Canada; 2Bruker BioSpin GmbH, Fahrenheitstraße 4, Germany; 3Ontario Ministry of the Environment and Climate Ch, Etobicoke, ON, Canada; 4Silantes GmbH, München, Germany; 5Department of Chemistry, University of Toronto, Toronto, ON, Canada

Session: SOLUTION, poster number: 081

Isotope Labeling and Assignment of Glycans on Glycoproteins

Monique Rogals; Jeong_Yeh Yang; Kelley Moremen; James Prestegard
University of georgia, Athens, GA

Glycans on glycoproteins can affect protein stability and structure, as well as mediate interactions with ligands and other proteins.  Monitoring these effects is often hampered by an inability to express glycoproteins using bacterial hosts and minimal media supplemented with u13C-glucose and 15N-ammonium chloride.  We demonstrate an effective sparse labeling strategy using mammalian cells in media supplemented with just u13C-glucose.  Our test protein, the IgV domain of CEACAM1, shows labeling of glycans on all three N-glycosylation sites plus labeling of all alanine sites.  A novel disulfide-stabilized lanthanide-binding peptide has been engineered into the protein to provide pseudo-contact shifts for glycan resonance assignment and protein structural characterization. 

Session: SOLUTION, poster number: 082

Crowding Cellular Environment Finely Tuned Protein-protein Interactions in Living Cells

Zheng Wenwen1, 2
1Wuhan Institute of Physics and Mathematics, CAS, Wuhan, China; 2Graduate University of Chinese Academy of Sciences, Beijing, China

Protein-protein interaction is essential for protein exerting its function in cells. Several in-vitro studies have shown that protein-protein interactions were severely affected in mimicked cellular environment. But the thermal dynamics of protein-protein interaction in living cells has not been unfolded. Here, we show that 19F in-cell NMR can quantify protein-protein interaction. Compared with that in dilute buffer, dissociation constant of GB1 dimer decreased about 6 fold in living Xenopus oocytes. Further investigation of the role of charge-charge interactions in GB1 dimerization showed that the influence of charge-charge interaction on GB1 dimerization is less pronounced in cells than that in buffer. Our results show that both excluded volume and quinary interactions presented in cellular environment finely tuned protein-protein interaction in cells.

Session: SOLUTION, poster number: 083

Comparing the Potential of Helmholtz and Planar Micro-coils for Analysis of Intact In-vivo Samples

Monica Bastawrous1; Oliver Gruschke2; Ronald Soong1; Dieter Gross2; Andre Simpson1
1University of Toronto, Scarborough, Canada; 2Bruker Biospin GmbH, Rheinstetten, Germany

In-vivo NMR is a significant advancement in environmental research, allowing stress responses to be monitored at the molecular level. However, due to their size, crucial samples such as aquatic eggs and neonates have been difficult to study. The ability for microcoil NMR to vastly increase mass sensitivity makes it a promising solution. Single sided coils allow samples of various size and shape, however, B1 fields decrease with distance, leading to inhomogeneous excitation. Thus, it is unclear which coil styles are most applicable for in-vivo samples. Here planar and Helmholtz microcoils are compared for their ability to provide meaningful information on complex biological samples. We explore the importance of complex pulse sequences and RF homogeneity between these coils for in-vivo applications.

Session: SOLUTION, poster number: 084

Inhibition Modes of New Delhi Metallo-beta-lactamase Revealed by NMR Spectroscopy

Cheng Kai
Wuhan Institute of Physics and Mathematics, Wuhan, Hubei

Antibiotics resistant “superbugs” harboring New Delhi metallo-beta-lactamase (NDM-1) are a major public health concern. More than five hundred NDM-1 inhibitors have been reported. The detailed inhibition mechanisms of most NDM-1 inhibitors have not been examined. Quick, simple and reliable methods to assess ligand-binding mode are key to developing and improving inhibitors against NDM-1. Solution NMR is powerful for understanding substrate/inhibitor binding-induced changes in NDM-1 and hence the binding mode. Here, we show that the 1H-15N HSQC spectra are sensitive to the different functional states of NDM-1. Titrations of apo- and di-Zn- NDM-1 with various inhibitors display characteristic patterns, which make it easily distinguish different inhibition modes of NDM-1. These modes will aid in the development of efficient inhibitors.

Session: SOLUTION, poster number: 085

mpMRI Guided In-bore Biopsy to Increase the Detection Rate of Prostate Cancer

Sujeet Kumar Mewar1; Sanjay Sharma2; Sanjay Thulka3; Ekta Dhamijia3; Pradeep Kumar1; Sridhar Panaiyadiyan6; Rama Jayasundar1; N. R. Jagannathan4; S. Senthil Kumaran1; S. Datta Gupta5; Rajeev Kumar6; Virendra Kumar1
1Department of NMR and MRI Facility ,AIIMS, New Delhi, India; 2Department of Radiodiagnosis, RPC, AIIMS, New Delhi, India; 3Department of Radiodiagnosis, IRCH, AIIMS, New Delhi , India; 4Department of Radiology, Chettinad Academy of Res, Kelambakkam, Tamil Nadu, India; 5Department of Pathology, AIIMS, New Delhi, India; 6Department of Urology,AIIMS, New Delhi, India

The present study demonstrated that mpMRI guided in-bore biopsy has the potential to significantly increase the detection rate of prostate cancer. Patients were recruited based on PSA > 4 ng/ml and abnormal DRE. The aim of this study was to evaluate whether mpMRI guided inbore biopsy increases detection rate of PCa, reduce unnecessary prostate biopsy. MPMRI accurately identified suspicious areas of lesion and MR guided inbore biopsy helped to target these areas with accuracy and these targeted lesions correlated with histopathological findings.Our data also indicated that using ADC values of suspicious area before the biopsy may also help to reduce unnecessary biopsies cores. The PCa detection rate of in-bore targeted biopsy was 51.42 % compared to 34.24% for TRUS biopsy.

Session: SOLUTION, poster number: 086

NMR Spectroscopy of 13C, 15N Alanine Metabolism in Glioblastoma Cells

Qing Wang
University of Texas at Dallas, Richardson, TX

As one of the most lethal forms of cancer with a very dismal survival rate, glioblastoma are mostly chemoresistant and difficult to detect at an early stage where an unmet of non-invasive methods for early detection is needed in clinical. In this study, we have investigated the metabolism of 13C,15N-labeled alanine in SfXL glioblastoma cells using NMR spectroscopy. Our data showed high production of 13C-lactate, but the intermediate metabolite 13C-pyruvate was not visible in the 13C NMR spectra, indicated hyperactive ALT and LDH activities in these cells. Additionally, hyperpolarized 15N on both media extract and cell extract were tracked to study the metabolic fate of 15N-amino arm of alanine to complete the metabolic story of this amino acid in cancer.

Session: SOLUTION, poster number: 087

1H NMR Metabolic Profiling of HIV-1 Transgenic Rats in Correlation with Age and Treatment with Substances of Abuse

Yufeng Wei
New Jersey City University, Jersey City, NJ

The noninfectious HIV-1 Transgenic (HIV-1Tg) rat model provides an ideal tool for studying effects of substances of abuse on HIV-1 infected individuals receiving highly active antiretroviral therapy (HAART). In this study, we collected plasma samples from both F344 and HIV-1Tg animals at various ages with and without treatment with methamphetamine. One-dimensional 1H NMR spectroscopy, coupled with principle component analysis (PCA), was applied to investigate the differential metabolic profiling in the plasma samples. Our results indicated that the metabolic profile changes significantly between F344 and HIV-1Tg rats upon aging, more than the effects of METH treatment. The aging effects on the metabolic profiling may relate to the progression of HIV-1 associated neurodegeneration, and could potential provide biomarkers for aging with HIV-1.

Session: SOLUTION, poster number: 088

Optimizing Horizontal and Vertical Microstrips for DMF-NMR in vivo Toxicity Studies

Vincent Moxley-Paquette1; Amy Jenne1; Rachel Cheng1; Nayaab Punjani1; Hui-Ming Lin1; Ronald Soong1; Sebastian von der Ecken2; Aaron Wheeler2; Dimitri Zverev3; Peter De Castro4; Ivan Kovacevic4; Thomas Frei4; Juerg Stuessi4; Franck Vincent4; Stephan Graf4; Danijela Al Adwan-Stojilkovic4; Daniel Schmidig4; Rainer Kuemmerle4; Till Kuehn4; Falko Busse5; Martine Monette6; Henry Stronks6; Andre J Simpson1
1University of Toronto, Scarborough, ON; 2University of Toronto, Toronto, ON; 3NSCNC Manufacturing LTD, Port Coquitlam, BC; 4Bruker BioSpin AG, Fällanden, Switzerland; 5Bruker BioSpin GmbH, Rheinstetten, Germany; 6Bruker Canada Ltd, Milton, ON

The use of micro-coil technology hyphenated with digital microfluidics (DMF), where the movement of droplets is controlled using electrodes, could provide a platform for in vivo applications via supplying oxygenated water and nutrients to single organisms/eggs during experiments. DMF-hyphenated NMR performed using a magnet with a vertically orientated bore requires the continuous application of voltage to hold a droplet on the micro-coil, which reduces the lifetime of the DMF chips and micro-coils. Despite the potential difficulty of shimming planar detection coils orientated horizontally (relative to the direction of the B0 field), it is important to assess if it can provide comparable spectral quality to that of a vertically orientated detection coil for in vivo DMF-NMR.

Session: SOLUTION, poster number: 089

Impact of Ionic Liquid’s Structure on Its Polymer Solubility

Bing Wu1; Andre J Simpson2; Andersson Barison3; Luciano Morais Liao4
1European Synchrotron Radiation Facility, Grenoble, France; 2University of Toronto, Toronto, ON; 3Universidade Federal do Paraná, Curitiba, Brazil; 4Universidade Federal de Goiás, Goiânia, Brazil

Ionic liquids have been considered an excellent green alternative to dissolve polymeric materials. Because of the unique characteristics of ionic liquids, they can be used to perform polymerizations, polymer derivatization or simply as a solvent to allow further processing. In this study, a series of combinations of polymers and ionic liquids were studied by NMR relaxometry, NMR diffusometry, and NMR Fast Field Cycling. It was found that these ionic liquid forms micelles during the polymer dissolution and the physical properties of these micelles were furthered targeted with the help of small-angle scattering

Session: SOLUTION, poster number: 090

Molecular-Level Understanding of Phase Stability in Octadecane-Water-Stearic Acid
Phase-Change Nano-emulsions for Thermal Energy Storage

Jungeun Park; Robert Messinger
The City College Of New York, New York, NY

Phase Change Materials (PCMs) are latent heat storage materials that can store or release energy while undergoing phase transitions. Organic PCMs can be emulsified in water in the presence of surfactants to enhance transport properties and thermal conductivity. However, PCMs nano-emulsions become unstable during thermal cycling in heat transfer systems. To better understand the molecular origins of stability in PCMs nano-emulsions, solution-state nuclear magnetic resonance (NMR) methods have been applied to an octadecane-water-stearic acid system under thermal cycling.  Solution-state 1H and 13C NMR methods were used to observe octadecane freezing and melting and 1H pulsed-field-gradient (PFG) NMR methods were applied to measure the diffusion coefficient of the PCM nano-emulsion, enabling estimates of its average size by the Stokes-Einstein equation.

Session: SOLUTION, poster number: 091

Li-ion isotopes Dynamic and Solvation structure in aqueous solutions

Mohaddese Mohammadi; Alexej Jerschow
New York University, New York, NY
The electrolyte is an essential part of electrochemical systems and controls the charge transfer and distribution, enabling the system to store and deliver energy. For a long time, nonaqueous electrolytes have been used in Li-ion batteries because of their ability to operate in a voltage range higher than 3V. However, due to the ester-based of nonaqueous electrolytes, their usage raises safety and environmental concerns. Aqueous solutions could solve this problem but it needs to be a high concentration solution. In this work, T1 relaxation time and diffusion of 6Li and 7Li cations in water and D2O solutions of different concentrations have been measured to understand the dynamics and solvation of Li ions in high concentration regime.

Session: SOLUTION, poster number: 092

HRMAS NMR Diffusometry of Liquid Mixtures in Porous Materials

Todd M. Alam
Sandia National Laboratories, Albuquerque, NM

Measuring the self-diffusion for different chemical species in complex multi-component (MC) mixtures adsorbed within porous materials is critical for developing and optimization of applications ranging from separation membranes in batteries and fuel cells, to coatings and barriers. Here we describe the development of high-resolution magic angle spinning (HRMAS) NMR diffusometry techniques to probe MC mixture diffusion in different porous materials. Introducing MAS reduces the magnetic susceptibility heterogeneity of the fluid-filled materials resulting in significantly improved chemical shift resolution. The diffusion rates for individual species in MC mixtures can now be directly measured without the need for selective isotopic labeling. Experiments reveal differential surface adsorption and are used to highlight the benefits and limitations of HRMAS NMR diffusometry in porous materials.

Session: SOLUTION, poster number: 093

Miniaturized ex-situ NMR device for in-situ characterization of viscoelastic fluids

Yiqiao Tang
Schlumberger-Doll Research, Cambridge, MA


Mud is heavily engineered viscoelastic fluid used to aid the drilling of boreholes into the earth. The multiphastic complexities, consisting of brine, oil, lime, emulsifiers, organoclays, a multitude of polymers and micro-sized barite, are notorious difficult to characterize, not to mention under reservoir conditions. In this work, we show the construct and operation of miniaturized ex-situ NMR devices up to 143 C, monitoring the fluid evolution in a static configuration. Through NMR relaxation measurements, we reveal the migration of chemical species in the fluid fabric, how they interact, and what they progress towards over time and space.


Session: SOLUTION, poster number: 094

The Propensity of Uranium-Peroxide Systems to Preserve Nanosized Assemblies

Mateusz Dembowski1; Christopher Colla2; Ping Yu2; Jie Qiu1; Jennifer Szymanowski1; William Casey2; Peter Burns1
1University of Notre Dame, Notre Dame, IN; 2University of California at Davis, Davis, CA

Understanding the stability fields and decomposition products of various metal- and actinide-oxide nanoclusters is essential for their development into useful materials for industrial processes. We explore the spontaneous transformation of the phosphate functionalized uranyl peroxide nanocluster {U20P6} to {U24} under aqueous ambient conditions using multi-nuclear, 1H, 7Li, 23Na and 31P NMR spectroscopy. The unusual bridging mode of peroxide between uranyl ions observed in {U20P6} may lead to its rapid breakdown in solution as evidenced by liberation of phosphate groups that were originally present as an integral part of its cage structure. Remarkably, the uranyl peroxide moieties present after degradation of {U20P6} undergo cation-mediated reassembly into the {U24} cluster, demonstrating the propensity of the uranyl peroxide systems to preserve well-defined macro-anions.

Session: SOLUTION, poster number: 095

NMR SAMPLES: NMR-aided determination and enhancement of sample suitability for metabolomics measurement

Arjun Sengupta; Aiwen Tan; JoEllen Weaver; Christian perez; Daniel Rader; Shane Jensen; Aalim Weljie
University of Pennsylvania, Philadelphia,

Metabolomics is used as a tool for biomarker analysis from clinical samples. Several of these studies are performed using biobanked samples that undergo varying storage and handling protocols. Such variation may impact metabolite levels and ultimately the biospecimen composition. We sought to quantify these changes by leveraging the quantitative nature of NMR spectroscopy. Freshly collected serum and plasma samples were subjected to combinations of different storage time/ temperature, typically encountered in biobanking facilities. NMR metabolic profiling followed by linear mixed modeling analysis suggested signatures of altered metabolic composition with varying storage time/temperature. We aim to utilize this information to construct a software that will replicate our works and aid in determining the suitability of biobanked samples for biomarker development.

Session: SOLUTION, poster number: 096

NMR-based Metabolic Profiling of OCD in Horses; Metabolic Profile and Gender Specific Metabolic Response

Jon Spires1; Daniel Kim1; Kenith Conover1; Sarah Ralston2; Kyla Ortved3; Istvan Pelczer1
1Department of Chemistry, Princeton University, Princeton, NJ; 2Professor Emerita and Fulbright Scholar, Howell, NJ; 3New Bolton Center, University of Pennsylvania, Kenneth Square, PA
A case-controlled set of serum samples (n=20x2) were analyzed to identify and characterize the metabolic markers of the presence or absence of clinical osteochondrosis dissecans (OCD) in yearling Standardbred horses, using high-resolution FT-NMR and TD-NMR. For FT-NMR we collected both spectra with water suppression only and with relaxation filter, respectively. The spectra were evaluated with multivariate analysis and STOCSY. TD-NMR was also utilized to identify the relaxation behavior of the water in the samples. There were correlations distinguishing between diseased and healthy with both modalities. We observed different metabolic responses to the disease for the two genders that had not been detected in previous studies. Analyzing both genders together lead to poor quality statistics, which improved greatly upon separation.

Session: SOLUTION, poster number: 097

The Use of NMR-Based Metabolomics to Monitor and Enhance the Biologics Manufacturing Process

Luciano Mueller; Yan He; Christine Jorge; Xiaohua (Stella) Huang; Raymond P. Donnelly; Nelly Aranibar; Jan-Lukas Ott; michael D. reily; Bethanne M. Warrack; Elaheh Kamaloo; Amanda M. Lewis
Bristol-Myers Squibb, Princeton, NJ

At Bristol-Myers we employ a combination of mass spectrometry and NMR metabolomics tools to monitor media and cell extracts during commercial biologics manufacturing. A division of labor is employed between NMR and mass spec where NMR detects nutrients and metabolites which feature well-resolved peaks of adequate intensities in proton NMR spectra while mass spectrometry captures media components which are beyond NMR detectability. The employed NMR sample preparation and data acquisition methods will be presented.  These include a combination 1D- and 2D NMR experiments. A comparisons of extracting concentrations from peak integrals and from spectral line fitting using the Chenomx-profiler will be discussed.

Session: SOLUTION, poster number: 098

Urine metabolic profile in patients with Parkinson’s disease

Senthil Kumaran; Sadhana Kumari; Vinay Goyal; Sadanand Dwivedi; Achal Kumar Srivastava; NR Jagannathan
AIIMS, Ansari Nagar, India

NMR-based metabolomics was studied in patients with Parkinson’s disease (PD) in early and advanced stages in comparison with healthy controls (HC) using urine samples. Quantitative analysis revealed higher levels ornithine, isoleucine, and β-hydroxybutyrate (β-OHB) in the ePD and, except isoleucine these metabolites were higher in aPD group also in comparison with HC. We did not observe any significant differences in the concentration of metabolites between ePD and aPD groups. In addition we also observed a good correlation (r= 0.24) between UPDRS III (motor score) and concentration of succinate metabolite in patients with PD. Present findings may be associated with Gastrointestinal dysfunction, impairment in Gut microflora system, and energy metabolisms in patients with PD.

Session: SOLUTION, poster number: 099

Chemical shift assignment and quantification for minor glycans in therapeutic monoclonal antibodies using middle-down NMR

You (Joy) Zhuo1, 3; David Keire2; Kang Chen1, 3
1FDA, Silver Spring, MD; 2FDA, St Louis, MO; 3FDA, Silver Spring, MD

Glycosylation of monoclonal antibodies (mAbs) is critical for drug efficacy and safety. The mAb glycans are heterogeneous in their monosaccharide composition. Current fluorescence or mass-spectrometry methods for quantifying glycan species require enzymatic cleavage of glycans or the protein followed by extractions. Recently, the middle-down NMR method was demonstrated for measuring the major glycan form distribution without cleavage of the glycan moieties from proteins. Here, the NMR method was further developed with higher resolution in the anomeric region of 1H-13C HSQC spectra, which allowed the assignment and quantification of clinically relevant minor glycans containing high-mannose and afucosyl glycans in adalimumab and trastuzumab. Therefore, the new NMR method is an orthogonal approach to classical glycan composition assessment for protein glycosylation quality assurance.

Session: SOLUTION, poster number: 100

NMR-Based Amino acid Assay for Human Plasma and Serum

Kerem Bingol
Janssen Pharmaceuticals, Spring House, PA

Amino acid profile of plasma and serum is a major interest for investigation of human diseases. Mass spectrometry techniques are very sensitive and powerful for quantitation of metabolites within a large dynamic range; however, accuracy and precision of quantitation depend on careful calibration of the instrument with multiple runs by using a variety of authentic standards. NMR spectroscopy is not as sensitive as mass spectrometry, yet amino acid concentrations in human plasma and serum are sufficiently high to detect by NMR. NMR provides high accuracy and precision without the need for calibration runs with multiple authentic standards. In this study, we established and validated human plasma and serum amino acid assays using NMR and compared them to similar assays.

Session: SOLUTION, poster number: 101

An NMR Method to Estimate 13C Enrichment of Lipid Metabolites in Stable Isotope Tracer Experiments

Penghui Lin1; Li Dai2; Daniel Crooks2; Leonard Neckers2; Teresa Fan1; Andrew Lane1
1University of Kentucky, Lexington, KY; 2Center for Cancer Research, NCI, NIH, Bethesda, MD

Here we report a quick and easy way to estimate the incorporation of 13C into different subunits of complex lipids by NMR using cellular phosphatidylcholine lipids (PCs) as internal standard in stable isotope tracer experiments. The ratios of peak intensities of other species to that of PC methyl groups in both the proton and the HSQC spectrum could be used for enrichment calculation. This method provides a simple tool for generating an overview of 13C incorporation into lipid molecules, which can be utilized as a standalone approach or to compliment targeted mass spectrometry-based lipidomics workflows. Further, with the detection limit as low as 2%, it provides very valuable information other techniques cannot generate.

Session: SOLUTION, poster number: 102

Differentiation of metabolic changes induced by single base mutation in KRAS gene segment from single cell to cell cluster

Yang Chen1; Gaigai Cao1; Yunchao Xu2; Xiaoqiong Tang2; Sanxi Wang3; Jianghua Feng3; Shangyuan Feng2
1Fujian Medical University, Fuzhou, China; 2Fujian Normal University, Fuzhou, China; 3Xiamen University, Xiamen, China

Activation or aberrant expression of oncogene and/or tumor suppressor gene is widely regarded as one of the main causes of tumor. The KRAS is believed to be a major driver during development of kinds of tumor. Based on the concept of object and data consistency, the fusion of metabolomics and Raman spectral data is proposed in this work, the fusion modeling of the valuable parts of the two experimental data can enlarge the detection information from multicellular to single cell and then from dead cell to living cell at the same time. Wild type and mutant type of isogenic colon cancer cell lines were cultured to perform proton nuclear magnetic resonance (NMR) and Raman detection.

Session: SOLUTION, poster number: 103

NMR chemometrics for chemosensory differentiation of sweet and pungent phytochemicals

Ankita Singh; Aruna Singh; Dushyant Kumar; Rama Jayasundar
Department of NMR, AIIMS, New Delhi, India

Recent studies have shown that taste plays a crucial role in nutrition and pharmacology. Pungent compounds have shown anti-obese activity while sweet compounds analgesic effects. In this study, ten phytochemicals and ten medicinal plants containing these phytochemicals from two chemosensory groups (sweet, pungent) were studied using NMR spectroscopy. Chemosensory based discrimination of both taste groups was observed in Partial Least Square Discriminant Analysis for phytochemicals as well as medicinal plants. The discrimination can be attributed to signatory marker resonances like hydroxyl groups (sweet) and phenylamide groups (pungent). 


Session: SOLUTION, poster number: 104

Tracking the Metabolic Fate of Glutamine in MCF-7 Breast Cancer Cells via 13C and 15N NMR Spectroscopy

Asiye Asaadzade
University of Texas at Dallas, Richardson, TX
Glutamine is the most abundant amino acid in the body and is a major source of carbon and nitrogen in cellular metabolism.In this study,we have investigated the metabolic fate of glutamine in MCF7 breast cancer cells using C13 and N15 nuclear magnetic resonance spectroscopy.Due to overexpression of specific glutamine metabolic enzymes,there is an overproduction of glutamine metabolites such as glutamate and ammonia which can be detected by 13C and 15N NMR spectroscopy.For this project,we have utilized[U-13C5,15N2]Lglutamine to be able to see the metabolic fate of glutamine,including its metabolic waste product ammonia 15NH4+.This main goal of this project was to see the fate of glutamine metabolism as its nitrogen arms are stripped off during a series of biochemical reactions in cancer.

Session: SOLUTION, poster number: 105

Intracellular and Extracellular Metabolism of High-Fructose Corn Syrup in Cancer Monitored by 13C NMR Spectroscopy

Fatemeh Khashami
The University of Texas at Dallas, Richardson, TX

Glioblastoma Multiforme is an aggressive type of the Central Nervous System tumor that grows within brain tissue.This type of tumor is fast-growing, characterizing it as one of the most malignant of all primary brain tumor. According to the Surveillance, Epidemiology,and End Results,about 23,820 will be diagnosed with primary brain and other CNS tumors and about 17,760 people will be dead of this disease in the only United States.People with a cancerous brain have the 5-year survival rate of approximately 32.9% from SEER 18 2009-2015.In this study,we have used 13C NMR spectroscopy to probe metabolic effects of high-fructose corn syrup ,which is prevalent in Western diet,on the progression of GBM and another type of cancer (liver cancer) for comparison.

Session: SOLUTION, poster number: 106

Detection of Metabolites of Human Breast Carcinoma Cells Cultured ex-vivo in 2D and 3D Tissue Matrix Scaffolds by HRMAS-NMR

William Hiscox
Washington State University, Pullman, WA

High resolution magic angle spinning (HRMAS) NMR was used for the quantitative evaluation of metabolites released by human breast epithelial cells during ex-vivo culture in 2D and 3D tissue matrix scaffolds. Cells were cultured using growth medium containing biologically relevant concentrations of U-13C(6)-glucose, and NMR data were collected at several time points. Two sets of NMR data were collected for each sample at each time point, one with inverse-gated 13C-decoupling, and the opposing dataset without 13C-decoupling, revealing the incorporation of 13C from glucose, and formation of 13C-labeled metabolites. This approach was used to compare metabolite profiles between MCF10A and MDA-MB-231 breast epithelial cell lines, cell growth between 2D and 3D culture platforms, and diffusion of 13C-labeled metabolites throughout the scaffolds.

Session: SOLUTION, poster number: 107

Strategic approach for NMR spectroscopic identification of metabolites from complex mixtures

Johanna Haerdter
University of Hamburg, Department Chemistry, Hamburg, Germany

A growing field is the analysis of metabolites, which can be used to identify signals of chemical markers. A large number of metabolites, in particular secondary ones, have not yet been recorded.

The challenge in the analysis of metabolites is the identification from extracts. Extracts yield mixture spectra, which is making it difficult to elucidate the structure.

For identification, the polar extract is time fractionated by liquid chromatography reducing the number of metabolites of individual fractions. Each fraction is then measured using mass spectrometry and NMR spectroscopy. MS and NMR signals are correlated by means of their time course. Selective NMR methods are used to identify spin systems and spatial correlations even allowing the structure elucidation of unknown metabolites.

Session: SOLUTION, poster number: 108

Quantitative Analysis of Unstable Metabolites in Biological Samples

G.A. Nagana Gowda; Daniel Raftery
University of Washington, Seattle, WA

Coenzymes including coenzyme A (CoA), acetyl coenzyme A (acetyl-CoA), coenzymes of redox reactions (NAD+, NADH, NADP+, NADPH) and energy (ATP, ADP, AMP), and antioxidants (GSH, GSSG) mediate biochemical reactions fundamental to the functioning of all living cells. The knowledge of cellular levels of these metabolites is critical for assessment of human health and diseases. However, owing to their notoriously unstable nature and the challenges with the conventional analysis methods, to date, no simple method exists to measure them in one step. In this study, we have evaluated sample harvesting, extraction and analysis protocols for tissue and blood specimens and developed optimized analysis methods. The new methods enable reliable analysis of the unstable metabolites in biological samples in a single step.

Session: SOLUTION, poster number: 109

Cocaine induced changes in the metabolic flux of the rat brain

Chongyang Huang; James Collins; Marjory Pompilus; Marcelo Febo; Joanna R. Long
university of florida, Gainesville, FL

NMR based metabolomics profiling technologies were used to study cocaine induced changes in the metabolic flux of the rat brain. Two groups of Long Evans rats were respectively given a single dose of cocaine at concentration of 10 mg/kg/ml (acute cocaine group) or sterile saline solution (control group) via i.p. injection. Data analysis results suggest that cocaine exposure causes disturbance of energy, choline, nucleic acid and neurotransmitter metabolism in the brain to induce the disturbance of brain nervous system activities. Our work provides a comprehensive view of the metabolism disturbance effects induced by cocaine, which is important in cocaine addictive brain study.

Session: SOLUTION, poster number: 110

The Assessment of Mixtures by Spectral Superposition. An Approach for the Field of Metabolomics

René Bachmann; Navid Shakiba; Markus Fischer; Thomas Hackl
University of Hamburg, Hamburg, Germany

We present a model in which mixture spectra are calculated based on the spectral superposition of corresponding pure samples. For the calculation the reduced data (buckets) were used to exclude possible errors due to matrix effects. Considering different mixture ratios exhibited more than 160,000 spectra that were the basis for calculation of assessment curves. Calculated data were compared with real mixtures to validate and assess the suitability of this method. With a suitable sample basis, the limits of the model can be described exactly for valid predictions. In this study we present an approach that saves a tremendous amount of time and resources for all metabolic classification disciplines

Session: SOLUTION, poster number: 111

Analysis of peanut adulteration in food samples by NMR spectroscopy

Caroline Schmitt; Tim Bastek; Alina Stelzer; Tobias Schneider; Markus Fischer; Thomas Hackl
University of Hamburg, Hamburg, Germany

The adulteration of low-cost food products, such as powdered peanuts, to similar matrices is a common problem in the food industry. When peanuts are used as adulterant, there is an additional risk of potential health hazard to consumers due to allergy-induced anaphylaxis. Different series of peanut mixtures with hazelnuts, almonds and walnuts, were produced and analyzed by 1H NMR spectroscopy. For identification an isolated signal at 3.05 ppm in the 1H NMR spectrum of peanut was used as an indicator for adulteration. The marker was identified as (2S,4R)-N-methyl-4-hydroxy-l-proline by synthesis of the compound. The S/N and the integral of the marker can both be used to detect peanut impurities to an approximate limit of detection of 2%.

Session: SOLUTION, poster number: 112

Quantifying binding-induced conformational changes of proteins using hydrodynamic protein size measurements

Aishwarya Mahadevan1; Joanna Deek2; Thomas Weber2; Willie Jeffers1; Ulrich Rant2
1Dynamic Biosensors Inc., San Diego, CA; 2Dynamic Biosensors GmbH, Munich, Germany

We present a novel method for the measurement of the Stokes radii of proteins, and demonstrate how this approach can be used to detect analyte binding-induced conformational changes in proteins. The proteins of interest are immobilized onto DNA-functionalized gold microelectrodes on the biosensor surface. By applying alternating electric fields to the microelectrodes, the fluorescently labeled and electro-switchable DNA nanolevers are actuated through the solution. The speed of this oscillatory movement is observed in real-time by fluorescence energy transfer and correlates to the target protein’s Stokes diameter. The method is particularly sensitive for sizes in the range of 1 – 20 nm. Small molecule-induced conformational changes in the Stokes radius can be detected with an accuracy of 0.1 nm.

Session: SOLUTION, poster number: 113

High-throughput Lipid Testing by Nuclear Magnetic Resonance (NMR) Spectroscopy

Erwin Garcia1; Carisa Crumpler2; Franklin C. Warf1; Margery A. Connelly1; Elias Jeyarajah1; Dennis W. Bennett1; Steven P. Matyus1; David N. Oskardmay1; James D. Otvos1; Randolph M. Young2
1Laboratory Corporation of America® Holdings, Morrisville, NC; 2Laboratory Corporation of America® Holdings, Burlington, NC

The standard lipid panel is essential for risk assessment and management of cardiovascular disease, and is routinely performed by enzymatic/colorimetric methods on chemistry analyzers. A novel NMR-based method was developed to measure total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C) and apolipoprotein B (ApoB) from a simple 1D 1H NMR spectrum (total acquisition time per spectrum = 45 s). The analytical performance of the assay was evaluated using CLSI guidelines and demonstrated excellent characteristics suitable for use in the clinical laboratory.

Session: SOLUTION, poster number: 114

Innovative Capabilities at the EMSL User Facility to Support the Study of Materials and Biosystems with Advanced NMR Spectroscopy

Hardeep Mehta
Pacific Northwest National Laboratory, Richland, WA

The Environmental Molecular Sciences Laboratory (EMSL) is a Department of Energy national scientific user facility, located at Pacific Northwest National Laboratory in Richland, Washington, which houses an array of scientific equipment for research critical to national science needs. The state-of-the-art resources offered by EMSL to researchers worldwide include computation, deposition/microfabrication, mass spectrometry, microscopy, NMR/EPR, spectroscopy/diffraction, and subsurface flow and transport capabilities.  Capabilities and expertise that contribute to a problem-solving research environment in areas including materials development, catalysis, geosciences, structural biology and metabolomics will be discussed during this presentation. 

EMSL instrumentation is available to scientific users via a user proposal system, with no charge for instrument time for non-proprietary research. For more information, visit


Session: SOLUTION, poster number: 115

Improving Field Estimation in High Field Powered Magnets

Waroch Tangbampensountorn1; Xinxing Meng1; Jeffrey L. Schiano1; Ilya Litvak2; Wenping Mao2; William W. Brey2
1Pennsylvania State University, University Park, PA; 2National High Magnetic Field Lab / FSU, Tallahassee, FL

Powered magnets can generate magnetic fields much higher than superconducting persistent magnets and offer unique opportunities for studying NMR phenomena. Nevertheless, the temporal stability and spatial homogeneity are insufficient for high spectral resolution NMR and MRI. We previously demonstrated a field regulation system consisting of an inner loop that uses inductive measurements to attenuate high-frequency field fluctuations arising from the power supply ripple in cascade with an outer loop that uses steady-state free-precision NMR measurements to attenuate low-frequency fluctuations arising from the cooling system. This research aims to increase the bandwidth and SNR of the NMR field estimate by decreasing the time between RF pulses, thereby improving the performance of the 36 T series-connected hybrid for NMR and MRI experiments.

Session: SOLUTION, poster number: 116

Rapid NMR-based Method for Quantifying Abnormal Lipoproteins

Erwin Garcia1; Z. Gordon Jiang2; Irina Shalaurova1; Joe Kirk3; Alan T. Remaley4; James D. Otvos1; Margery A. Connelly1
1Laboratory Corporation of America® Holdings, Morrisville, NC; 2Beth Israel Deaconess Medical Center, Boston, MA; 3Laboratory Corporation of America® Holdings, Burlington, NC; 4National Institutes of Health, Bethesda, MD

Abnormal lipoproteins such as LP-X and LP-Z have been observed in patients with liver disease. There is currently no efficient method to detect and quantify these abnormal lipoproteins. A rapid NMR-based method to detect and quantify LP-X and LP-Z was developed. The assay has analytical characteristics consistent with use in a clinical laboratory. The assay can measure LP-X and LP-Z in a high throughput fashion (~1 minute per sample) potentially as a means of diagnosis and/or prognosis in subjects with liver disease.

Session: SOLUTION, poster number: 117

New NMR-electrochemical cell for in situ measurements using graphite-epoxide electrodes

Pollyana Ferreira Da Silva Vianna1; Bruna Ferreira Gomes2; Luiz Henrique Keng Queiroz Junior3; Luiz Alberto Colnago4
1Universidade de São Paulo, São Carlos, SP; 2Universität Bayreuth, Bayreuth, Germany; 3Universidade Federal de Goiás, Goiânia, Brazil; 4Embrapa Instrumentação, São Carlos, Brazil

Coupling NMR and Electrochemistry (NMR-EC) in situ is a good way to elucidate reaction mechanisms and to obtain information about the concentration of reactants and products in real-time. However, this kind of coupling presents some challenges since NMR interfere in EC measurements and vice versa. This way, the objective of this work was to create a new miniaturized electrochemical system to couple with NMR and minimize these interferences and follow and electrochemical reaction in real-time. It was possible to apply the new NMR-EC cell to follow benzoquinone electro-reduction and the results show that the new system has minimal interference in the NMR spectra since their quality was not significantly compromised and almost 15% of total benzoquinone was converted to hydroquinone.

Session: SOLUTION, poster number: 118

Improving Low Frequency Disturbance Rejection in Powered Magnets

Xinxing Meng1; Jeffrey L. Schiano1; Ilya Litvak2; William Brey2
1The Pennsylvania State University, University Park, PA; 2Florida State University/NHMFL, Tallahassee, FL

The 36-tesla Series-Connected Hybrid (SCH) magnet provides unique opportunities for studying NMR phenomena as it provides much higher magnetic fields than persistent superconducting magnets. A critical component for NMR experiments on the SCH magnet is a cascade control system that reduces temporal field fluctuations. The field regulation system consists of an inner loop that uses inductive measurements to attenuate high-frequency field fluctuations arising from the power supply ripple in cascade with an outer loop that uses steady-state free precession NMR measurements to attenuate low-frequency fluctuations arising from the cooling system. This research aims to improve the rejection of low-frequency disturbances through improving the outer-loop control design by using a dynamic model of the inner loop acquired from experimental measurements.

Session: SOLUTION, poster number: 119

IBM Instruments: why it was formed and why it finally failed

James Cooper1; Robert Johnson2; Willy Shih3
1Food Science Institute, Wilton, CT; 2Golf Performance Center, Ridgefield, CT; 3Harvard Business School, Cambridge, MA

In 1978, three IBM executives got approval to form IBM Instruments as an independent business unit. We have interviewed over 35 of the 150 original employees to study its successes and failures and will report on them. The new company was formed to sell scientific instruments and to improve IBM’s computer position in the laboratory. While nearly all the company’s products were manufactured by Bruker, the most successful ones were designed by IBM, who touted service and support as advantages. While selling OEM machines reduced profit margins, the magnetics group was in the end profitable.  The reasons for the company’s closure are an interesting business study.

Session: SOLUTION, poster number: 120

Development of a HTS-based High-Sensitivity 13C NMR Probe

Omid Sanati1; Ghoncheh Amouzandeh2; Jeremy N. Thomas2; Taylor L. Johnston2; Lawarence A. Hornak1; Jason Kitchen2; Ilya Litvak2; Vijaykumar Ramaswamy3; Nicolas Freytag3; William W. Brey2; Arthur Edison1
1University of Georgia, Athens, GA; 2Florida State University/NHMFL, Tallahassee, FL; 3Bruker Switzerland AG, Fällanden, Switzerland

Low NMR sensitivity is a perpetual problem. Improving NMR probes with high-temperature superconducting coils can help. Our goal was to design and build a probe optimized for 13C detection with ~60% of the active volume of conventional 5 mm probes. Such a probe would be useful for metabolomics or natural product applications using natural abundance 13C as well as macromolecular studies of, for example, disordered or sparsely 13C labeled proteins. The probe will operate at 21.1 T using an HTS 13C coil set and a normal metal coil for 1H and 2H for optimal 13C performance. The coils will be bench-tested by the end of 2019 and built into a probe in early 2020. Progress to date will be reported.

Session: SOLUTION, poster number: 121

HOmonuclear Transfer by HEteronucleAr Decoupling (HOT-HEAD): Novel scheme for establishing correlations in liquid-state NMR by reinstating non-secular J-terms

Mihajlo Novakovic1; Anton Hanopolskyi1; Sungsool Wi2; Lucio Frydman1, 2
1Weizmann Institute of Science, Rehovot, Israel; 2National High Magnetic Field Laboratory, Tallahassee, FL

TOCSY is a well-established solution-state NMR experiment often complicated by high-power demands. This study explores an approach termed HOmonuclear Transfers by HEteronucleAr Decoupling (HOT-HEAD) that can achieve homonuclear correlations based on their J-couplings without pulsing on the nuclei to be correlated. The idea follows from considering H-C1-C2 three-spin system with J-couplings only active among directly-bonded spins. Nutation field w1H applied on C1-bound proton will introduce a modulation in the JCH coupling, that partially reinstates non-secular C1–C2 JCC flip-flop terms. Namely, 1H irradiation creates C1 decoupling sidebands and when these overlap the C2 spectral line efficient C1->C2 transfer can occur. These principles are demonstrated experimentally with different samples. Additional theoretical considerations and possible applications of this method will be presented.

Session: SOLUTION, poster number: 122

Progress of the Cosmic Axion Spin Precession Experiment (CASPEr)

John Blanchard
Helmholtz-Institut Mainz, Mainz, Germany

The nature of dark matter, the invisible substance that makes up over 80% of the matter in the universe, is one of the most intriguing mysteries of modern physics. The Cosmic Axion Spin Precession Experiment (CASPEr) is a multi-faceted research program using NMR techniques to search for dark-matter-driven spin-precession.

I will discuss our recent results using zero- to ultralow-field (ZULF) NMR and efforts to use parahydrogen-enhanced spin polarization to enhance CASPEr-ZULF sensitivity. I will also report on commissioning of and preliminary results from the low-field (10-4 – 10-1 Tesla) experiments. In this field range, we are using superconducting quantum interference devices (SQUIDs) to measure a liquid 129Xe target polarized via spin-exchange optical pumping (SEOP).