Collaboration & Service Projects

For the National Resource for Advanced NMR Technology BTDD, we have identified 23 Collaboration and Service Projects (CSPs), 15 existing and 8 new, encompassing an impressive portfolio of NIH-supported projects.  PIs have the resources needed at their home institutions to make challenging and biomedically relevant samples and to gather preliminary data to inform the experiments they will undertake in our facility. Equally important, these projects will benefit from unique technologies developed in each of the TDPs, including a clear need for improved sensitivity and resolution beyond what conventional NMR instrumentation can provide. The instruments on which our TDP technologies are developed are already integrated into the user program, and as new capabilities are developed and demonstrated they are made immediately available to the users.

Below we provide brief descriptions for these projects and how they will benefit from the TDPs. Many of the projects will use capabilities developed in two and even three of the TDPs. The projects are also distinguished by a clear need for the technologies we are developing. A common theme throughout the projects is a need for sensitivity and resolution beyond what conventional NMR instru­mentation can provide. All the CSP PIs have a clear vision for what they can accomplish as the HTS cryo­probes (TDP1), DNP probes and sample preparation strategies (TDP2), and SCH system and probes (TDP3) are made available through our user program. We will encourage the PIs for each of these CSP to send students and postdoctoral research associates to take advantage of the training workshops spe­cific to the TDP technologies they will be utilizing.

To inquire about starting a collaboration including establishing a Driving Biomedical Project or Collaboration and Service Project please contact principal investigators for the TDP: https://nmrprobe.org/people/leadership/.

Current C&S Projects

	Relationship of CSPs to TDPs (new projects in italics)	TDP1	TDP2	TDP3
CSP1	Hyperpolarized 13C Metabolic MRI of Hypertrophic Cardiomyopathy	X
CSP2	Metabolic Origins of Nonalcoholic Steatohepatitis (NASH)	X
CSP3	Structure and Dynamics of Self-Assembled biomolecules		X	X
CSP4	Mechanistic Investigations of Host Defense Metallopeptide Interactions with Bacterial Cell Membranes		X	X
CSP5	13C NMR measurements of liver samples for development of unified model of hepatic metabolism	X
CSP6	Functionalized gold nanoparticles		X
CSP7	13C NMR measurements of liver samples for development of unified model of hepatic metabolism	X
CSP8	Identification of interfacial bonding environments in functional nanomaterials and biomaterials using high resolution solid state NMR at (ultra)-high fields		X	X
CSP9	Cytotoxic Transthyretin Oligomers and their Interaction with Membranes			X
CSP10	Novel targeted anticancer agents from marine cyanobacteria	X
CSP11	Membrane protein effectors of pathogen interactions with host			X
CSP12	Investigating hereditary and UV light-related aggregation of eye lens proteins			X
CSP13	Probing Zn chemistry in metalloenzymes			X
CSP14	Protein molecular structure, conformational dynamics, and inter-protein interactions in human health and disease		X	X
CSP15	DNP of Membrane Proteins in Lipid Bilayers by ssNMR		X	X
CSP16	The Regulation of Hepatic Metabolic Zonation by the Diabetes Gene TCF7L2	X
CSP17	ssNMR Structural Analysis of Oligomeric Alzheimer’s Beta-Amyloid Peptide		X
CSP18	Structural Studies of FMN domain interactions with cytochrome-P450		X	X
CSP19	Interaction bones/cartilage with extra-cellular medium		X
CSP20	Metabolic Origins of Nonalcoholic Steatohepatitis (NASH)	X
CSP21	Structure and signaling-related changes at protein interfaces in chemotaxis receptor arrays		X
CSP22	NMR Structural Analysis of Calcium Regulation in Muscle			X
CSP23	Dynamic structures of lipid-membrane protein complexes via ssNMR		X	X

1. Hyperpolarized ¹³C Metabolic MRI of Hypertrophic Cardiomyopathy; Roselle Abraham and Peder Larson, University of California, San Francisco Cardiac hypertrophy after exercise training is an adaptive response which increases athlete endurance and performance. Paradoxically, cardiac hypertrophy is also associated with multiple etiologies associated with heart failure. The MyHC genetic mutation is common in humans, and is associated with sudden death due to heart attack. The Abraham lab uses a rodent model of the MyHC mutation, and is collaborating with the Merritt lab to assess myocardial energetics in the context of hypertrophy and ultimate failure. The new R33 grant award to Dr. Larson is for hyperpolarized ¹³C studies in humans displaying heart failure with similar etiologies. The approach will combine multiple different tracers including [U-¹³C]palmitate, [1,6-¹³C₂]glucose, and [1,3-¹³C₂]beta-hydroxybutyrate to estimate the relative sources of acetyl-CoA in the rodent heart. Hypertrophy is often accompanied by a switch between the relative oxidation of fats, glucose, and ketones. A metabolic switch is considered indicative of changes in energy homeostasis associated with heart failure. The rodent heart is an extremely small piece of tissue, and ¹³C analysis of the labeled glutamate produced from the precursors is beset by SNR considerations. The HTS technology is suited for analyzing samples in this context.
2. Metabolic Origins of Nonalcoholic Steatohepatitis (NASH), Shawn Burgess, University of Texas Southwestern Lipogenesis is essential for normal physiology, and its dysregulation is a notable feature of obesity, diabetes, cardiovascular disease, cancer, neurodegeneration and infection. Classical regulation of de novo lipogenesis involves transcriptional regulation of lipogenic gene expression via hormone mediated SREBP activation, and/or carbohydrate sensing via ChREBP activation. The mitochondria, specifically the TCA cycle, is the putative source of acetyl-CoA used for lipid synthesis but before transport to the cytosol, it is converted to citrate, a step that consumes TCA cycle intermediates. The balance between TCA cycle cataplerosis (loss of TCA cycle intermediates) and anaplerosis (replenishment of TCA cycle intermediates) may help to determine the rate at which citrate can be used for lipid synthesis. These pathways are known to be disrupted in many diseases that also have pathological lipid metabolism. We will examine how anaplerotic and cataplerotic pathways of the TCA cycle help to mediate the appropriate lipogenic response to nutrition, by promoting substrate (e.g. citrate) availability and/or cellular energy status necessary for lipogenic reactions. Availability of the HTS ¹³C optimized probes enables state of the art investigation of metabolic fluxes in the rodent liver.
3. Structure and Dynamics of Self-Assembled biomolecules; Bo Chen, University of Central Florida Solution NMR has been highly successful in characterization of the structure and dynamics of molecules tumbling in solution, such as polymers or proteins. However, there are a large group of biomolecules that aggregate and precipitate out of solution, becoming “invisible” to solution NMR techniques. Many of these entities are associated with pathogens or diseases that impact human society, examples are virus capsid shells, amyloid plaques observed in neurodegenative diseases such Alzheimer disease, or mad-cow disease. It is of great value to resolve the structures and dynamics of such aggregates to decipher the assembly mechanism and provide insight to conquer these terrible diseases. Solid-state NMR (ssNMR) is one of the few techniques applicable to these subjects. We are developing and applying ssNMR methods to study the capsid protein assembly of HIV-1 virus and the prions present in yeast Saccharomyces Cerevisiae. We are also working out innovative models for simulations of self-assembly.
4. Mechanistic Investigations of Host Defense Metallopeptide Interactions with Bacterial Cell Membranes; Myriam Cotten, William & Mary Antibiotic resistance, which is called the “other” pandemic, led to 1.2M deaths in 2019, with possibly 10M by 2050. We investigate structure-function relationships in host defense metallopeptides called piscidins that kill drug resistant bacteria and cancer cells. Using natural host defense peptides (HDP) as prototypes to rationally engineer new anti-infective agents requires understanding the molecular features underlying their high potency, specificity, and mechanisms of action. We employ host defense metallopeptides from the piscidin family to investigate HDP action in bacterial cell membranes. Using comparative studies of wild-type and mutated piscidins in the metallated and unmetallated forms, we will perform experiments to discern the chemical and physical membrane effects that underlie their potency. We will use biophysical studies of mutants to identify functionally-important residues in piscidin, and solid-state NMR experiments to establish on a molecular level how these residues contribute to membrane activity. This contribution is significant since it will explore the new paradigm that Cu²⁺ -bound HDPs target physical and chemical vulnerabilities in the membranes of bacteria that can incorporate lipids from the host or natural environment to control virulence. With solid-state NMR, we can explore more complex lipid mixtures that more closely replicate bacterial membranes. However, to characterize the arrangement of the peptide in the bilayers, distance measurements (REDOR) have to be performed. These are notoriously time demanding. The Long lab at UF has showed that REDOR curves can be collected on membrane-bound peptides much more quickly using DNP and interesting changes in structures can be detected thanks to the enhanced sensitivity. Thus, we would like to use a similar approach to investigate the altered structural arrangement of metallated piscidin peptides in bacterial cell membrane mimics.
5. ¹³C NMR measurements of liver samples for development of unified model of hepatic metabolism; Stanislaw Deja, University of Texas Southwestern Dysfunctional liver metabolism is a common feature of obesity, non-alcoholic fatty liver disease and type 2 diabetes and elucidation of disease mechanism and pharmaceutical treatments requires detailed characterization of cellular metabolism. Metabolic flux analysis (MFA) provides critical insights into the basis of metabolic regulation in healthy and pathological states. Flux through the pathways can be tracked by monitoring conversions of isotopically labeled substrates (e.g., tracers containing ²H, ¹³C, ¹⁵N, etc.) into downstream metabolites. The number and position of the isotopic nuclei (isotopomers) in metabolites encodes information of pathway activity that can be deconvoluted using computational models. The complexity of these models is limited by the ability to detect labeling patterns in cell culture and rodent tissue extracts. MFA is most easily accomplished using NMR, as it quantifies metabolite site specific enrichment, allowing creation of more accurate metabolism models. Mass spectrometry only reports on isotopologues, making modeling more difficult. Development of these models depends upon the collection of high quality ¹³C spectra. The experimental plan uses cell culture to develop the models, where sample sizes are limited. The HTS ¹³C probes provided by this resource significantly improve the data quality, potentiating more accurate model construction.
6. Functionalized gold nanoparticles; Terry Gullion, West Virginia University Gold nanoparticles (AuNPs) can be used as non-toxic carriers and can be functionalized to deliver proteins, peptides, mARNs, and drugs. The nanoparticles can be tuned to deliver these in-cell. Correlating function with structure is key in designing the functionalized AuNPs. Obtaining atomic scale geometry and topology is challenging as often the amount of surface material generated, hence sensitivity is very low, hindering the application of conventional solid-state NMR. The enhanced sensitivity provided by DNP combined with multi-dimensional ssNMR would be key to structural determination of the bound carriers in a very short amount of time on material-limited samples. This project uses both ¹³C and ¹⁵N DNP enhanced ssNMR to assess the relationship between AuNPs and the functionalized surface and probe the arrangement of peptides (or other systems) on the surface.
7. ¹³C NMR measurements of liver samples for development of unified model of hepatic metabolism; John Griffith Jones, University of Coimbra Water enriched with ¹⁸O (H₂¹⁸O) is a potential tracer for evaluating the sources of glycerol and glycogen synthesis since it is incorporated into specific sites of triose-phosphate and glucose-6-phosphate via specific enzyme-mediated exchange/addition mechanisms. Unlike ²H, ¹⁸O does not experience significant isotope effects for any of these processes.  Therefore, H₂¹⁸O should provide more precise estimates of endogenous glycerol and glycogen synthesis from dietary glucose and fructose compared to deuterated water (²H₂O). Depending upon the enzymatic reaction, ¹⁸O is incorporated at various positions in the glucose molecule. Carbon-13 NMR spectra display isotope shifts due to the increased mass associated with ¹⁸O. By inspection of the ¹³C spectrum of glucose after labeling with H₂¹⁸O, fractional incorporation is assessed by comparing the amplitudes of the ¹³C-¹⁸O and the ¹³C-¹⁶O peaks. ¹⁸O is expensive; using less H₂¹⁸O is cheaper, but the intensity of the ¹³C-¹⁸O peak can become passingly small. The HTS ¹³C technology provided by the P41 allows small samples with ¹⁸O enrichments as low as 1% to be effectively measured. Direct ¹³C detection is the best method for assessing enrichment, as it is extremely robust and not dependent on any coherence transfers.
8. Identification of interfacial bonding environments in functional nanomaterials and biomaterials using high resolution solid state NMR at (ultra)-high fields; Christian Bonhomme, University of Sorbonne, France Although they are part of the set of the four most abundant metal ions in the human body (with Na⁺ and K⁺), both ⁴³Ca and ²⁵Mg are unreceptive quadrupolar nuclei (both low-γ), making their NMR analysis very challenging for biomaterials research.We investigate the role of Mg²⁺-incorporation in (hydrated) CaCO₃ biominerals, in relation with the recent isolation of a new hydrate CaCO₃.(H₂O)_0.5, and of the structure and dynamics around Ca²⁺-binding sites in proteins, like those of the S100 family, which have been the object of much research (including COVID-19 pandemic research). For such studies, model compounds will be synthesized and analyzed at 32 T (static) and 35.2T (MAS) (in order to optimize acquisition conditions for sensitivity and resolution), before proceeding to the investigations on the actual biominerals and biomolecules.
9. Cytotoxic Transthyretin Oligomers and their Interaction with Membranes, Kwang Hun Lim, East Carolina University Little is known about molecular basis of the diverse misfolding pathways and structural diversity of amyloid, which is implicated in numerous diseases such as amyloidoses, prion and Alzheimer’s diseases. Structural studies of amyloid are essential to understanding the mechanism of amyloid diversity. Effect of the pathogenic mutations on misfolding pathway should also be examined. This research program is aimed at investigating amyloid formation mechanisms of a natively folded protein, transthyretin (TTR), using solid-state NMR. Pathogenic mutant forms of TTR may have distinct misfolding pathways, adopting diverse amyloid conformations with different toxic activities, which may result in diverse disease phenotypes and tissue-selective depositions. The hypothesis will be tested through structural characterization of amyloid derived from wild-type and various pathogenic mutant forms of TTR. In particular, solid-state NMR with innovative labeling schemes will provide valuable insights into amyloid diversity.
10. Novel targeted anticancer agents from marine cyanobacteria; Hendrik Luesch, University of Florida Cyanobacteria have evolved chemical weapons for defensive purposes, which we are exploiting for anticancer drug discovery. Marine cyanobacteria produce compounds with exceptionally potent activity and/or possess unusual or first-in-class inhibitors with novel mechanisms of action. To streamline exploration of marine cyanobacteria for anticancer drug discovery some major challenges are to (1) identify novel unexplored cyanobacterial biodiversity, which presumably translates into novel chemical structures and (2) rapidly obtain in vivo data in model organisms to assist in making a go- or no-go decision for further resource investment. Structures will be determined using NMR and mass spectrometry. We commonly only isolate sub-milligram quantities of purified natural products from the ocean-derived organisms. Key for the success of our drug discovery efforts is availability of high-field NMR spectrometers coupled with the most sensitive probes. The proposed HTS cryoprobes would have the requisite enhanced sensitivity needed to work with these minute sample amounts and enable us to obtain the complete set of NMR data with (both ¹H and ¹³C detection) required for structure determination. Validation of bioactivity and secondary assays will be performed before prioritizing targets for further investigation. Selected compounds with intriguing structure and promising validated bioactivity will be targeted for total synthesis so that a rigorous biological characterization can be performed to pinpoint the molecular changes induced in the cancer cell and to determine potential direct targets.
11. Membrane protein effectors of pathogen interactions with host; Francesca Marassi, Sanford Burnham Prebys Medical Discovery InstituteOur goal developing a framework for understanding the way human host defenses interact with microbial pathogens and age-related stressors. Many pathogens have evolved strategies to evade innate immunity by recruiting complement regulatory factors onto the microbial cell surface. The ectopic deposits that form in age-related macular degeneration and Alzheimer’s disease are also rich in blood proteins involved in innate immunity. Our project centers on understanding the interactions between bacterial membrane proteins and the host. Initial focus is on outer membrane protein Ail (Adhesion invasion locus) from Yersinia pestis. Y. pestis, the causative agent of plague, poses very high risk to public health as it is highly pathogenic, can be easily disseminated, and causes high mortality. The research strategy is multidisciplinary. It relies significantly on structural biology techniques, particularly solution NMR and solid-state NMR. Ail is one of the most highly expressed proteins on the bacterial cell surface during human infection, is critical for promoting the survival of Y. pestis in serum and is a target for therapy development. Structure determination of Ail in lipid bilayer membranes is critical for understanding its molecular function unencumbered by detergent effects.
12. Investigating hereditary and UV light-related aggregation of eye lens proteins; Rachel Martin, UC Irvine Cataract, a major cause of blindness worldwide, results from precipitation of the eye lens proteins (crystallins). Over time, high-molecular weight crystallin aggregates accumulate in the lens, rendering it opaque. We seek to solve the molecular structures of highly concentrated native state and aggregated cataract states representative of both hereditary and UV light-induced cataract. The G18V variant of human γS-crystallin, (γS-G18V), found in childhood-onset cataract, is less thermally stable than wild-type, more aggregation-prone, has more exposed hydrophobic surface area, and forms strong, specific contacts with the holdase chaperone αB-crystallin. Our investigations have focused on its solution behavior and solid-state structural studies, which will be facilitated by access to the proposed instruments, are currently in progress. Future work will focus on more aggregation-prone variants as well as large complexes. These samples are difficult to prepare in large quantities and are less soluble, requiring access to the NHMFL high-temperature superconducting probe, the fast-MAS DNP probe, and the series connected hybrid (SCH) magnet. NMR methodology, primarily in the solid state, will be developed to investigate the structural factors related to gS-crystallin stability and solubility. Differential isotope labeling of peptide binders and variant crystallins will identify residues involved in altered intermolecular interactions and provide structural information. These instruments will enable us to investigate biologically relevant systems that are not feasible to prepare in the quantities and forms required for traditional solution-state and MAS NMR.
13. Probing Zn chemistry in metalloenzymes, Robert McKenna, University of Florida Carbonic anhydrases (CAs) are a family of red blood cell metalloenzymes responsible for the catalysis of the interconversion of carbonic acid as: HCO₃− + H⁺ ⇌  CO₂ + H₂O. The majority of CAs have an active site feature a Zn²⁺ ion, which is a constituent of a zinc prosthetic group that normally features binding with three histidine side chains and one water molecule.  In this “pocket”, a fourth histidine is positioned near the water, which allows for the formation of a Zn-OH bond that binds with CO2 to form a zinc bicarbonate. Our research group has numerous crystalline samples of CAs suitable for investigation by ⁶⁷Zn ssNMR; however, the Zn only constitutes between 0.31-0.34 of the MW of most CAs make such experiments challenging. We will utilize the 32T-SCM platform and our new static HX broadband low-γ probe (operating at low temperatures) to acquire the first ⁶⁷Zn ssNMR spectra of a series of CAs, in order to examine variations in the local environments of the active sites.
14. Protein molecular structure, conformational dynamics, and inter-protein interactions in human health and disease, Dylan Murray, University of California, Davis The dynamic assembly of biomolecules within a living cell is vital for the spatial and temporal organization of biological function. In forming RNA granule membraneless organelles and intermediate filaments cells leverage the self-assembly properties of protein sequences with reduced amino acid diversity. Thirty percent of proteins coded by the human genome contain this type of domain, highlighting these sequences central importance for life. In humans, pathogenic genetic mutations and altered expression levels, in addition to functional post-translational modifications and protein-protein interactions, modulate these proteins assembly processes. This proposal focuses on a mechanistic understanding of how proteins composing RNA granules and intermediate filament networks assemble to achieve the macroscopic behavior observed in living cells. A multifaceted biophysical approach employing cutting-edge nuclear magnetic resonance and cryo-electron microscopy will allow characterization of the molecular structure and conformational dynamics of these proteins in biologically relevant assemblies. These studies, biochemical assays, and protein engineering will form more comprehensive models of how low complexity domain proteins assemble temporally and spatially providing a mechanistic description of how these assembly processes and associated control mechanisms are modulated by point mutations and altered protein expression levels linked to motor neuron disease, dementia, muscular dystrophy, and cancer. The methodologies employed are expected to have applicability and impact on investigations of the proteins in the human genome that contain a low complexity domain.
15. Dynamic Nuclear Polarization of Membrane Proteins in Lipid Bilayers by Solid-State NMR, Alexander Nevzorov, North Carolina State University Solid-state NMR of macroscopically aligned samples or oriented-sample (OS) NMR, provides native-like conditions for the structure-function studies of membrane proteins at high lipid-to-protein ratios (>100) with adequate hydration at physiological temperature and pH. Direct proton detection is often not feasible due to line broadening caused by the densely coupled proton network. Detection using ¹³C or ¹⁵N brings in the problem of low sensitivity. We propose to utilize the newly built SCH magnet in Tallahassee to apply proton-mediated magnetization transfer between the ¹³C and ¹⁵N spin species. Mismatched Hartmann-Hahn (MMHH) conditions establish cross-correlations between the dilute spins over distances ranging from 3 to 7 Å and is applicable to both hetero- and homo-nuclear spin exchange. The MMHH method is suitable for spectroscopic assignment of oriented membrane proteins by establishing cross-correlations between nearest-neighbor (i,i±1) spin sites that are only 2.9 Å apart. NMR assignments of Pf1 coat protein both as phage and reconstituted in magnetically aligned bicelles have been confirmed spectroscopically. However, cross-peak intensity decays rapidly with increasing distance between the spins, especially at low fields. The hybrid 36 T spectrometer at NHMFL together with their highly-efficient E-free probes will provide several-fold boost in terms of resolution and sensitivity, allowing testing the MMHH technique to more complex, polytopic membrane proteins. Samples will include selectively labeled Pf1 coat protein and human Acetylcholine receptor. It is expected the gain in sensitivity afforded by the spectrometer, long distance (>5 Å) cross peaks will be readily established, which will be of primary importance for the elucidation of tertiary structure and helix-helix packing in membrane proteins by the method of OS NMR.
16. The Regulation of Hepatic Metabolic Zonation by the Diabetes Gene TCF7L2, Luke Norton, University of Texas Health San Antonio Liver metabolic dysfunction leads to disturbances in lipid and glucose metabolism, and directly contributes to the development of type 2 diabetes (T2D and nonalcoholic fatty liver disease (NAFLD). The increased prevalence of these metabolic diseases presents a significant threat to public health, and treatment strategies that address the underlying pathological processes are urgently needed. Our proposed studies will reveal novel mechanisms linking the diabetes candidate gene transcription factor 7-like 2 (TCF7L2) to key metabolic pathways in liver that may have significant implications for the treatment of T2D and NAFLD. Carbon-13 tracer technology is an essential approach for assessing metabolic turnover and energy homeostasis in the liver. Using a ¹³C-propionate tracer as well as deuterium NMR of glucose after labeling with D₂O, hepatic gluconeogenesis, glycogenolysis, and Krebs cycle turnover can be read out using NMR. The HTS technology for ¹³C detection will continue to provide the highest mass sensitivity detection of labeled glucose produced in rodent models of diabetes.
17. ssNMR Structural Analysis of Oligomeric Alzheimer’s Beta-Amyloid Peptide; Anant Paravastu, Georgia Tech Alzheimer’s disease (AD) research has traditionally focused on amyloid fibrils found in plaque deposits in the brain. Recent evidence suggests that oligomers of Alzheimer’s beta-amyloid peptides play a more significant role in initiating AD pathology. Our objective is through characterizing Aβ oligomer structures, to answer fundamental questions of Aβ self-assembly pathways and how these pathways could be controlled as strategies for designing therapeutics. We will determine the structure of a 150 kDa Aβ(1-42) oligomer by integrating solid-state nuclear magnetic resonance spectroscopy with computational modeling. Specifically, we will 1) define secondary structure and sidechain proximity across the amino acid sequence; 2) Determine alignments between neighboring beta -strands and conformations of non- beta -strand regions; and 3) Model the structure of the Aβ(1-42) oligomer based on the ssNMR data. The structural information obtained in this project will help characterize early events in Abeta self-assembly and may lead to diagnostic tools for early detection of Alzheimer’s disease and therapeutic strategies that exploit differences in oligomer and fibril assembly pathways.
18. Structural Studies of FMN domain interactions with cytochrome-P450; Ayyalusamy Ramamoorthy, University of Michigan
    Functional reconstitution of membrane proteins has been a major roadblock for the application of biophysical techniques to investigate their high-resolution dynamic structures in a native membrane environment. Our goal is to understand the effects of cytb5 on the enzymatic function of cytP450. Cytochromes P450 (cytsP450) are central to a large enzymatic complex (the “P450monooxygenase system”) that catalyzes the oxidation of a variety of exogenous and endogenous compounds, including drugs, fatty acids, hormones and carcinogens. We will investigate the structure, dynamics and transmembrane domain orientation of full-length mammalian P450s (2B4, 3A4 and 3A5 isoforms) alone and in complex with its redox partner b5 and CPR, incorporated in nanodiscs, using a combination of high-resolution solution and solid-state NMR techniques. The outcome of the proposed studies on P450-redox complexes will provide structure and dynamics/function principles regulating P450 metabolism of a wide variety of substrates. The results obtained from this study will also be useful to design potent drugs to ultimately treat and prevent diseases including cancer.
19. Interaction bones/cartilage with extra-cellular medium; Neeraj Sinha, University of Miami Health Structural stability of various collagen-containing biomaterials such as bones and cartilage is still a mystery. Despite the spectroscopic development of several decades, the detailed mechanism of collagen interaction with citrate in bones and glycosaminoglycans (GAGs) in the cartilage extracellular matrix (ECM) in its native state is unobservable. Solid state NMR combined with Dynamic Nuclear Polarization is critical to provide the sensitivity required to study this interface at natural isotopic abundance. This project uses both 13C and potentially 15N DNP enhanced ssNMR to assess the relationship between the bones/cartilage and its extracellular environment. The increase of sensitivity offered by DNP is needed to study the samples in their native state.
20. Metabolic origins of nonalcoholic steatohepatitis; Nishanth Sunny, University of Maryland
    Nonalcoholic steatohepatitis (NASH) is prevalent in over 70% of the obese and type II diabetes mellitus (T2DM) patients and is a leading cause of liver transplantation. Hepatic insulin resistance and inflammation mirror alterations in mitochondrial oxidative flux (beta-oxidation, tri-carboxylic acid [TCA] cycle and mitochondrial respiration), in rodent models and humans with simple steatosis. One theory of pathogenesis is excess oxidative flux in the liver leads to excessive reactive oxygen species generation, inflammation, and subsequent cirrhosis. Another factor is the impact of branched chain amino acids (BCAAs) on mitochondrial energy homeostasis. Preliminary results indicate that BCAAs are strongly correlated with inflammation in the condition of a high fat diet. This project uses both ¹³C and ²H NMR to assess mitochondrial energy homeostasis as well as lipogenesis in rodent models of NASH. The increased sensitivity of HTS coils enables small amounts of sample to be analyzed, increasing throughput for the experiments substantially.
21. Structure and signaling-related changes at protein interfaces in chemotaxis receptor arrays; Lynmarie Thompson, University of Massachusetts-Amherst Bacterial chemotaxis receptors are an excellent system for investigating the molecular mechanism of transmembrane signaling. These receptors operate in the cell as extended membrane-bound nanoarrays of receptors with two cytoplasmic protein partners, a kinase CheA and a coupling protein CheW. ssNMR is ideally suited to investigate these nanoarrays to determine what structural changes occur between signaling states to transmit the signal that controls the kinase. The signaling mechanism in this system is widely accepted to begin as a 2 Å ligand-induced piston in the periplasmic and transmembrane domains. Current efforts in the field focus on determination of how the signal is propagated through the cytoplasmic domain to control the ac­tivity of the kinase. We employ multiple methods for assembling a cytoplasmic receptor fragment, CheA, and CheW into nanoarrays that preserve native-like architecture assessed by electron cryotomography and native-like function assessed by activity assays. ssNMR measurements of structural constraints in these functional complexes determine how these change with signaling state. This provides an essential complement to crys­tallographic structure determination on nonfunctional complexes, and is a promising approach for illuminating mechanisms of many proteins that operate in the cell within multiprotein complexes. Our ssNMR measure­ments on these homogeneous preparations (receptor:CheA:CheW = 6:1:2) measure receptor-receptor dis­tances to determine structural changes between signaling states. DNP measurements will enable measure­ments at lower abundance interfaces involving CheA and CheW, which are critical to understanding how the receptor modulates activity of the kinase. A protein interface-filtered approach will simplify the spectra via se­lective detection of nuclei at the interface of mixed labeled complexes, for instance arrays that incorporate ¹³C-CheA and ¹⁵N-receptor. Labeling schemes combining uniform, reverse, and selective labeling will be de­signed based on linewidths observed in DNP spectra and predicted chemical shifts for the known structures of CheW and CheA fragments, to enable resolution and assignment of the interface resonances. Comparison of spectra of the nanoarray in the kinase-on and kinase-off signaling states will reveal structural changes initially via chemical shift changes and ultimately via distance changes. This will complement solution NMR studies that have identified resonances at the receptor/CheA and receptor/CheW interfaces but have not investigated functional complexes to determine changes between signaling states.
22. NMR Structural Analysis of Calcium Regulation in Muscle; Gianluigi Veglia, University of Minnesota Calcium (Ca²⁺) is an essential messenger for muscle contractility, and its homeostatic balance is controlled by proteins embedded or peripheral to the sarcoplasmic reticulum (SR) membrane. In cardiac myocytes, the SR Ca²⁺-ATPase (SERCA) regulates diastole by translocating ~70% of the Ca²⁺ ions. In its unphosphorylated state, phospholamban (PLN) reduces SERCA’s affinity for Ca²⁺, whereas phosphorylation of PLN at Ser16 re-establishes basal Ca²⁺ transport. We will focus on the structural analysis of SERCA regulation by two mutants, PLNP21G and PLNM20GP21G, which we designed and currently testing for gene therapy. We will study how these mutants mimic the phosphorylated state of PLN and tune SERCA activity. Studies will be carried out using a combination of molecular biology, biochemical assays, thermocalorimetry, and spectroscopic methods (solution, solid-state NMR and fluorescence) in synthetic and native membranes. We will study the allosteric interactions between the sarcoplasmic reticulum Ca²⁺-ATPase and three regulatory proteins (phosphol- amban, sarcolipin, and HAX-1). Since dysregulation of SERCA is directly linked to cardiac and skeletal muscle diseases, such as hypertrophic and dilated cardiomyopathies, Brody’s disease, and Duchenne muscular dystrophy, understanding the structural details of these interactions is central to designing innovative therapies to treat these devastating diseases.
23. Dynamic structures of lipid-membrane protein complexes via ssNMR; Benjamin Wylie, Texas Tech University Many lipids and membrane proteins associate to form platforms called lipid rafts, which are phase-separated from the surrounding membrane. The dynamic structure and functional importance of these intermediate-sized (5-200 nm), non-crystalline assemblies are difficult to characterize. Many pathogenic bacteria organize lipid rafts which can increase virulence and antibiotic resistance. In humans, rafts form to facilitate multiple signaling processes. Atomic-resolution dynamic structural details of these assemblies will broaden our understanding of signaling processes and inform disease etiology. We will use solid-state NMR to study large (38-150 kDa) membrane proteins and in proteoliposomes and biological membranes. Higher throughput SSNMR requires higher field and ¹H detection using a combination of fractional ¹H/²H side-chain labelling. The functional interactions between lipids and membrane proteins, and their organization into lipid microdomains, regulate key biological signaling processes and are involved in the pathogenesis of HIV, Alzheimer’s, Parkinson’s, and Heart diseases. Our approach will quantify these interactions on the atomic level to understand how these processes occur and how lipid microdomains assemble within biological membranes. Once we understand how functional lipids regulate proteins and how, in turn, proteins order their lipid environment we can develop novel pharmaceuticals and therapeutics to improve human health.

Previous C&S Projects, 2017 – 2022

Identification of pheromones and hormones from C. elegans and other nematodes; Rebecca Butcher, University of Florida This project centers on investi­gating the chemical structures and biosynthesis of an important class of secondary metabolites that is used by Caenorhabditis elegans, as well as other nematode species, to control their development and physiology.  We have identified a complex natural product from C. elegans (termed ‘nemamide’), which is important for starvation-induced larval arrest.  Homologs of the nemamide biosynthetic genes are present in most nematode species, including parasitic ones.  We will purify the nema­mides from select parasitic nematode species and use NMR spectroscopy to elucidate their chemical structures.  Because nemamide molecules likely play conserved roles across many nematode spe­cies, our work will enable the development of new chemical tools and strategies to interfere with the life cycles of parasitic nematodes.

Metabolomics and Natural Products Applications using HTS NMR probes; Art Edison, University of Georgia The most fundamental challenge in metabolomics is the reliable and reproducible identification and quantification of metabolites, and high sensitivity NMR, especially with ¹³C, and improve all of these problems. In many untargeted studies, the number of unassigned features exceeds the number of assigned metabolites. Even for features assigned to metabolites, the degree of confidence of these assignments is often in question. There are many different techniques and analytical platforms that are used for metabolomics, primar­ily various types of liquid or gas chromatography coupled with mass spectrometry (LC-MS or GC-MS) and NMR. With thousands of possible assignments, significant errors can occur when utilizing analytical measurements such as retention time, m/z values, or even 1D ¹H NMR chemical shifts. NMR generally is recognized as more robust for compound identification than MS, but most pub­lished NMR studies rely on heavily overlapped 1D ¹H spectra for metabolite identification and quanti­fication.

Detection of Hyperphosphorylated tau in living cells with different tauopathies; Kendra Frederick, UT Southwestern An outstanding problem with structural studies of metastable proteins is that the conformations that are amplified in vitro for structural work are not those that are propagated in vivo. This is clearly the case for tau in a mammalian system that demonstrates prion-like transmission of tau tangles developed by my colleague Marc Diamond. Despite being able to faithfully replicate tau aggregates through multiple in vivo systems, cel­lular lysates are unable to template the conformational conversion of purified tau (1). This is true even when in vitro prepared fibers are used to start tau aggregation in cells and is possibly because the infectious confor­mation in spontaneously aggregated tau is a minority species that cells can amplify but is energetically unfa­vorable in vitro or perhaps post-translational modification, such as phosphorylation or the involvement of other cellular components, like small chaperone proteins are required. My group is developing methods for in vivo NMR spectroscopy (2) and is working to determine the structure of tau inside cells using DNP NMR. Because tau is hyperphosphorylated in its aggregated form we wish to collect ³¹P-¹³C double CP spectra to determine the identity and extent of phosphorylated residues in aggregated tau. These experiments set a high bar for sensitivity. The dynamic nuclear polarization TR&D mechanism will enable us to detect ³¹P under DNP condi­tions. DNP NMR will be the only way to get structural information about these amorphous aggregated forms that are responsible for devastating neurodegenerative diseases.

The Role of Visceral Fructose Metabolism in the Development of Non-alcoholic Fatty Liver Disease; John Griffith Jones, University of Coimbra Increased fructose intake is implicated in higher incidence of non-alcoholic fatty liver disease (NAFLD) in Western societies.  Alongside its known effects on hepatic lipid metabolism, we hypothesize that fructose metabolism in the intestine, and within adjacent mesenteric and omental adipose tissues (IAT) results in increased hepatic exposure to lipotoxicity and inflammation. These mechanisms will be evaluated in animal models of NAFLD induced by high fructose feeding using novel stable-isotope tracer technologies for monitoring fructose metabolism by IAT and intestinal microbiota as well as noninvasive permeability probes for assessing intestinal integrity.  Since IAT is more prevalent in males than females, gender effects will be also studied. Thiazolidinedieones (TZD) a new class of antidiabetic agents that also improve NAFLD. We will determine if NAFLD resolution is associated with TZD alterations on IAT metabolism and to what extent these are influenced by gender.    

NMR characterization of ligand binding to PPAR-γ and NURR-1; Doug Kojetin, Scripps Research Institute Our first project is characterizing ligands of the nuclear receptor transcription factor PPARγ , a robust mole­cular target for insulin sensi­tizing drugs that improve blood sugar maintenance in patients with type 2 diabetes mellitus (T2DM). A central tenet driving current PPARγ drug development is that synthetic ligands compete with natu­ral endogenous ligands, including fatty acids and lipids, for binding to a canonical ligand-binding pocket (LBP) in the core of the ligand-binding domain (LBD) to pharmacologically regulate PPARγ activity. However, we re­cently identified a second, solvent-accessible alternate ligand-binding site that many current PPARγ -targeted drugs also bind. Very little is known about this alternate binding site, and the overall goal of this project is to study the structure and function of this alternate binding site. Many ligands that bind to the canonical LBP at high affinity (low nM) also bind to the alternate site with affinities ranging from mid-nM to low µM. Our attempts to crystalize a ligand bound to the alternate-site in PPARγ have not been successful.

Optimizing resolution and sensitivity of ssNMR spectra of membrane proteins; Vladimir Ladizhansky, University of Guelph The Ladizhansky lab is inter­ested in the development of solid-state NMR (SSNMR) methods for the elucidation of membrane protein structure and dynamics in their native environments. We study intrinsic membrane proteins of a common seven-helical (7TM) architecture: eubacterial proton pump proteorhodopsin (PR), and a microbial photosensor Anabaena Sensory Rhodopsin (ASR) which is involved in the regulation of gene expression in cyanobacteria. We have developed sample preparation procedures that yield re­solved NMR spectra and obtained extensive spectroscopic assignments for both proteins, and solved high resolution solid-state NMR structure of the oligomeric assembly formed by ASR. Our cur­rent focus is on understanding interactions influencing membrane protein structure in cell mem­branes.

Identification of bioenergetics pathways essential for T-cell effector function; Clayton Mathews, University of Florida Mitochondrial (mt) dysfunction in the T cell compartment has been associated with autoimmune diseases.  In autoimmune Type 1 Diabetes (T1D), studies using murine models have indicated that T cell mitochondrial dysfunction may participate in the pathogenesis.  We have recently discovered that T cells from patients with T1D as well as those at risk for T1D, exhibit mitochondrial inner membrane hyperpolarization (MHP) when compared to controls. Further, mitochondrial respiration and glycolysis are dysregulated in T cells from T1D patients.  Therefore, we hypothesize that T cells from patients with T1D display aberrant metabolism and bioenergetics resulting in pathogenic functional activity including sustained activation, heightened effector function, and resistance to normal mechanisms of peripheral deletion and immune regulation.  Our study is set to define the energy substrate requirements for expansion and effector function of human T cells with or at-risk for developing T1D.  Our data suggest that lymphocyte mitochondria and metabolic pathways are a promising and novel target for reversing autoimmunity in the prevention and cure of T1D.

Long-range spin-spin correlations in polytopic membrane proteins; Alex Nevzorov, North Carolina State University Establishing through-space spin-spin correlations is paramount for both spectroscopic assignment and structure elucidation in protein NMR. This is especially relevant for solid-state NMR of macroscopically aligned samples or oriented-sample (OS) NMR, which provides native-like conditions for the structure-function studies of membrane proteins at high lipid-to-protein ratios (>100) with adequate hydration at physiological tempera­ture and pH. However, direct proton detection in such samples is often not feasible due to the substantial line broadening caused by the densely coupled proton network. Detection using ¹³C or ¹⁵N brings in the problem of low sensitivity. Here we propose to utilize the newly built SCH magnet in Tallahassee to apply proton-mediated magnetization transfer between the non-invasive ¹³C and ¹⁵N spin species. As was previously shown, mis­matched Hartmann-Hahn (MMHH) conditions establish cross-correlations between the dilute spins over dis­tances ranging from 3 to 7 Å. Importantly, the MMHH technique, does not depend on the direct couplings between the dilute spins, and is generally applicable to both hetero- and homo-nuclear spin exchange. The MMHH method is also suitable for spectroscopic assignment of oriented membrane proteins by establishing cross-correlations between nearest-neighbor (i,i±1) spin sites that are only 2.9 Å apart. NMR assignments of Pf1 coat protein both as phage and reconstituted in magnetically aligned bicelles have been confirmed spec­troscopically without the need of preparing multiple selectively labeled samples. However, the cross-peak intensity decays rapidly with the increasing distance between the spins, especially at low fields. The hybrid 36 T spectrometer at NHMFL together with their highly-efficient E-free probes will provide several-fold boost in terms of resolution and sensitivity, which will be essential for testing the applicability of the MMHH technique to more complex, polytopic membrane proteins. Samples will include selectively labeled Pf1 coat protein at differ­ent locations within the protein as well as human Acetylcholine receptor. It is expected that with the gain in sensitivity afforded by the spectrometer, long distance (>5 Å) cross peaks will be readily established, which will be of primary importance for the elucidation of tertiary structure and helix-helix packing in membrane proteins by the method of OS NMR.

Media-free RDCs and RCSAs from high field molecular orientation of cell-surface proteins; James Prestegard, University of Georgia Residual Dipolar Couplings (RDCs) are extensively used in structure determination of molecules of interest to both chemical and biochemical communities. Traditionally this is done with the aid of an alignment medium (liquid crystal or mechanically distorted gel) that has sufficient interior space for molecules to reorient rapidly but still imparts a degree of order (1 in 10³-10⁴). The resulting small RDCs between bonded pairs of nuclei are easily measured as additions to scalar couplings seen in NMR spectra, and show a structurally useful depend­ence on the angle that a bond makes with axes of an alignment frame. Applications to refine protein structures using coupling of ¹⁵N-¹H amide pairs are of particular note. However, the need for a suitable alignment medi­um is problematic. Many proteins associate too strongly with these media to give quality spectra, and some can even be denatured by these interactions. An alternative is field induced orientation of the molecules them­selves. Nearly all molecules orient to some extent in the presence of a magnetic field due to their aniso­tropic magnetic susceptibilities. Since susceptibility tensors are easily predicted from structure, an ability to determine these tensors provides an additional piece of information that should be of particular value in struc­ture deter­mination of molecular assemblies. Cell-surface signaling molecules offer an ideal test case. These often share a terminal IgV-like domain composed of sandwiched b-sheets. The near parallel orientation of amide carbonyls in the b-strands of these structures leads to relatively large anisotropies of mag­netic suscepti­bility tensors making the measurement of field induced RDCs possible. The relative orientation of these do­mains in dimer structures is biologically important and is often modulated by interaction with oligo­saccharides, which is believed to be a regulatory step in activation of B-cells, migration of malignant cells, and guidance during cell development. Adding ex­perimental RDC measurements to prior structural knowledge on iso­lated domains provides an efficient way to characterize the dimerization process. We have in fact measured RDCs up to 1 HZ in a 21T magnetic field for a domain from Siglec-5, but the size of an RDC for a given aniso­tropic susceptibility tensor increases with field squared, making meas­urement at higher fields even more feasi­ble. Using the 36T SCH, RDCs for the example in Figure 1 would be approximately 3 Hz, and measure­ment could become routine and broadly applicable for other systems. Preliminary data may also motivate produc­tion of a higher resolution 36T magnet for future applications.

Reactivity of Selenoproteins; Sharon Rozovsky, University of Delaware The research of the Rozovsky group focuses on structure, dynamics, and function of selenium-containing proteins. Selenium is co­valently incorporated into these proteins in the form of the rare amino acid selenocysteine (Sec, U). Selenium’s biological functions are mediated by selenoproteins, a specialized group of enzymes af­filiated with the management of reactive oxygen species (ROS) and provides critical health benefits associated with protecting the cell against ROS-induced damage.⁷⁷Se is a spin 1/2 nucleus with a wide chemical shielding range of ~3000 ppm.  Its extreme sensitivity to the local environment makes it an excellent reporter of bonding, geometry and electronic structure. My group employs both solid- and solution-phase ⁷⁷Se NMR to study selenoproteins and to develop data interpretation of ⁷⁷Se NMR through close collaborations with theoreticians.

Structural studies of three Toll like receptor TM domains; Hubert Yin, University of Colorado-Boulder The overall goal of this our research is to characterize the backbone structure of three Toll-like Receptor (TLR) transmembrane (TM) domains in high resolution by ssNMR spectroscopy. TLR1, 2 and 6 are important inner membrane proteins that regulate innate and adaptive immunity in the human body. All three TLRs are more than 80KDa and each is predicted to have a single TM helical domain. They recognize pathogen associated molecular patterns (PAMPs) molecules in the bacterial cell wall and viral nucleic acids and activates immune response via NFkB and Myd88 pathways to produce pro-inflammatory cytokines in the host body. Although extensive structural studies have been performed for TLR extracellular domains which are involved in ligand binding and signal transduction, very little is known about their TM domain structures. To date only the TLR3 TMD structure was solved by solution NMR spectroscopy in non native detergent micelle. Here we propose the application of Oriented State solid state NMR spectroscopy (OSssNMR) with Polarization Inversion Spin Exchange at Magic Angle (PISEMA) and SAMPI4 pulse sequences to determine the high resolution backbone structure of the three TLR TMDs in membrane mimetic environment, ie glass slide supported synthetic lipid bilayers. The SCH system equipped with Low-E static ¹H–X probe at the NHMFL is critical for this research for two reasons. First, it will help us to orient the TMD peptides efficiently in the lipid bilayer irrespective of slight sample inhomogenity. Second, it will improve the spectral line widths (<+/- 3ppm) remarkably which is critical to resolve resonance overlap in the TMD region of the PISEMA and SAMPI4 spectra. Also as an effort to im­prove the OSssNMR sample preparation technology we will use thinner glass slides (going from 50µm thick­ness to 30µm thickness) which will help us to load more number of glass slide layers (45-50 glass slides) in­creasing the total protein amount in individual OS samples. This proposed method will also enhance sample sensitivity which is a common bottleneck for membrane protein structural studies by ssNMR. Once the struc­tural restraints are obtained, modeling and computational efforts will be used to refine them for experimentally determined high resolution molecular structures of TLR TMDs in lipid bilayers.