NMR and EPR Related User Projects

• COMBINED EPR, NMR, MAGNETIC SUSCEPTIBILITY AND COMPUTATIONAL DETERMINATION OF RARE EARTH HYPERFINE EFFECTS IN CERAMICS.
  Project Lead: Ian Farnan
  Project Lead Institution: University of Cambridge
  Abstract
  The first successful high-resolution solid-state NMR experiments on plutonium containing ceramics were carried out at EMSL. As these breakthrough experiments are exploited in the radiological NMR facility the transferred hyperfine field effects from 5f electrons must be understood in the spectra of actinide containing natural (contaminated) materials and actinide immobilization media (ceramics). These hyperfine effects can be used in principle to discriminate molecular sites close to actinides through nuclear spin relaxation effects and shifts induced by Fermi contact shifts and the electron-nucleus dipolar coupling. The latter may be used to determine distance information including lattice relaxation effects around dilute actinide nuclei in many materials. Similar multiple unpaired f electrons are present as unpaired 4f electrons in lanthanide rare earth elements. The proof of principle experiments on 4f systems proposed here should provide the background to develop a powerful spectroscopic tool to examine actinide environments in molecular and extended solids with applications in the Geo-BioGeochemistry and Sub-Surface Science theme.
• The Role of Interfacial Processes on Mineral Transformations in Wet Supercritical CO2
  Project Lead: Andrew Felmy
  Project Lead Institution: Environmental Molecular Sciences Laboratory
  Abstract
  We propose a comprehensive experimental and theoretical investigation of the role of interfacial water and CO2 on the energies, mechanisms, and rates of reactivity of a series of orthosilicate and phyllosilicate minerals in contact with supercritical-CO2 (scCO2) containing variable H2O. The molecular mechanisms for carbonation reactions, as well as the distribution of H2O between scCO2 and mineral surfaces, will be followed using molecular simulations and surface spectroscopy as a function of T, P, mineral composition and structure, and aH2O. The dynamics of water reactivity will be simulated using newly developed molecular dynamics models and reaction barriers will be calculated using density functional and molecular orbital theory electronic structure methods. The theoretical studies will be coupled with high-resolution spectroscopic measurements (e.g., IR/FTIR, NMR) to investigate the formation and structure of water layers at mineral surfaces and in the interlayer region of phyllosilicates, and identify potential interfacial carbonate and silicate species. The macroscopic reactivity of the orthosilicates and the changes in interlayer water structure and reactivity of the phyllosilicates will be determined experimentally using high pressure cells. The proposed research will provide new insights into mineral transformations under extreme conditions and help establish a basis for assessing the effectiveness of CO2 sequestration in geologic disposal sites.
• Solid State NMR Characterization of Hydrogenase
  Project Lead: Wendy Shaw
  Project Lead Institution: Pacific Northwest National Laboratory
  Abstract
  Hydrogenase is a naturally occurring enzyme that efficiently converts H2->2H+ + 2e- and is reversible. Producing synthetic catalysts with these properties is highly desirable, but current analogs cannot match the low overpotentials and the fastest rates of the enzyme. Understanding how the enzyme works is an essential first step in the intelligent development of synthetic catalysts. A thorough understanding of the structure will likely reveal key details in functional properties of the enzyme. This work proposes to investigate the full structure of hydrogenase using solid state NMR, including the unique 900 MHz capability housed in the EMSL facility.
• A Systems Biology Approach to Infectious Diseases Research
  Project Lead: Joshua Adkins
  Project Lead Institution: Pacific Northwest National Laboratory
  Abstract
  We describe an integrated program for coupling advanced capabilities in high-throughput transcriptomics, proteomics, and metabolomics with a comprehensive informatics infrastructure and a sophisticated approach to computational reconstruction and modeling of metabolic and gene regulatory networks. These powerful tools will be directed at delineating the mechanisms by which two related pathogens, Salmonella and Yersinia, adapt to the intracellular environment upon infecting macrophages and then manipulate that environment to facilitate their own survival and replication. The insights derived from modeling these behaviors may lead to the identification of novel therapeutic targets. The overall objectives are to: 1) Develop genome-scale metabolic and regulatory network reconstructions for Salmonella and Yersinia to provide the computational foundation for our systems biology approach; 2) Develop and disseminate 'sample matched' global datasets for Salmonella and Yersinia using transcriptomics, proteomics, and metabolomics technologies to provide the experimental foundation for our systems biology approach; 3) Produce and characterize knock-out mutations in regulatory genes predicted to be essential for systemic infection in mouse models for Salmonella enterica and Yersinia pestis; and 4) Use the network reconstructions, omics results, and phenotype changes in knock-out mutants to refine computational models of pathogenesis for Salmonella and Yersinia. EMSL world class capabilities in proteomics, NMR for structure and metabolomics, and advanced computation are well recognized. While all of these areas are included in the proposal the real benefit of using EMSL's capabilities is the team oriented nature of the operations; the project described above requires multiple-disciplines and multiple-strategies. These capabilities with the high quality team are why we request to perform this research in collaboration with EMSL.
• Metal Ion Binding and Substrate Specificity in Hydroquinone Dioxygenases
  Project Lead: Timothy Machonkin
  Project Lead Institution: Whitman College
  Abstract
  Bacterial pathways for the catabolism of aromatic hydrocarbons are of interest for use in bioremediation of pollutants such as chlorinated arenes. The oxidative ring cleavage step is often the major bottleneck. One such class of oxidative ring-cleaving enzymes are the hydroquinones dioxygenases (HQDOs). These enzymes have been little studied, and the factors that determine their unique substrate specificity remain unknown. The HQDOs are of particular interest for three reasons. (1) The nature of the substrate precludes bidentate coordination, indicating that there must be significant differences in substrate binding and cleavage compared to the well-studied catechol extradiol dioxygenases (EDOs). (2) While in the EDOs, chlorinated catechols usually lead to mechanism-based inactivation and thus are not substrates, in the HQDOs, chlorinated rings are the preferred (and in some cases, the only) substrates. (3) Since these enzymes are involved in the catabolism of xenobiotic compounds such as pentachlorophenol, this enzymatic function presumably evolved recently from some other enzyme class. We propose to use paramagnetic NMR as a direct spectroscopic probe for measuring Fe(II) and substrate/inhibitor binding to the active site of two hydroquione dioxygenases, PcpA and LinE, as well as a structurally characterized homolog that lacks ring-cleaving ability. We will measure Fe(II)-binding by monitoring the appearance of hyperfine-shifted peaks in a 1D 1H-NMR experiment. Similar paramagnetic 1D 1H-NMR will be used to measure the binding affinity of substrates and inhibitors to the Fe(II) center in these enzymes. Additionally, we will label the substrate with 2H and acquire 2H-NMR spectra. One issue of great interest is if monosubstituted hydroquinones bind in two different orientations, since this may partly explain the difference in substrate specificity between PcpA and LinE. Detailed analyses of the hyperfine shifts may allow assignment of the peaks to specific ring orientations. Understanding of how PcpA and LinE are able to specifically recognize and oxidatively cleave chlorinated aromatic rings could lead to important insights that would enable the redesign of other ring-cleaving dioxygenases with improved activity on chlorinated rings, which in turn could lead to improved bacterial bioremediation pathways for chlorinated organic pollutants. Access to the NMR spectrometers at EMSL (mostly the 600 MHz spectrometer, but possibly the 750 or 800 MHz instrument as well) is necessary for this work, since an NMR spectrometer is not currently available at Whitman College.
• MICROSCALE METABOLIC, REDOX AND ABIOTIC REACTIONS IN HANFORD 300 AREA SUBSURFACE SEDIMENTS
  Project Lead: Haluk Beyenal
  Project Lead Institution: Washington State University
  Abstract
  This proposal is the NMR component of a funded DOE/ERSP program project in which we proposed to use the Hanford 300 Area as a site for identifying microscale metabolic, abiotic and redox reactions in the subsurface and in the microbial communities. We plan to measure metabolites, redox chemicals and uranyl concentrations at the microscale using nuclear magnetic resonance (NMR) spectroscopy and various microelectrodes (UO22+, H2S, O2, SO4-, NO3-, Eh and pH). In practice, uranium transformation from the liquid phase to the solid phase occurs in established communities of cells growing on mineral surfaces. Development of bacterial communities on surfaces results in dense, highly metabolically active cells along with extracellular polymeric substances (EPS). A recent discovery showed that precipitated uranium nanoparticles can associate with the extracellular matrix material, which supports the importance of these community processes and the potential increased reactive potential of EPS. Hence, microbial processes and redox and abiotic reactions which operate at the microscale are critical to understanding factors controlling the macroscopic fate and transport of contaminants in the subsurface. This proposal addresses the development and implementation of biofilm based methods to understand uranium mobilitiy in subsurface biofilms. A NMR compatible biofilm reactor will be used to characterize metabolite concentrations (lactate, acetate and pyruvate), alternative electron acceptor concentration (fumarate) with/without uranium. NMR data will be combined the microsensors data produced at Washington State University to determine microscale factors affecting uranium mobility and the data will be used further biofilm modeling.
• Seattle Structural Genomics Center for Infectious Diseases - April 1, 2009 - Oct.1 2009.
  Project Lead: Garry Buchko
  Project Lead Institution: Pacific Northwest National Laboratory
  Abstract
  The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium of four institutions (Seattle Biomedical Research Institue (SBRI), deCODE biostructures, University of Washington (UW), and Battelle Memorial Institute) funded by NIAID in response to RFP-NIH-NIAID-DMID-07-19. This center is lead by Dr. Peter Myler at SBRI, a world leader on the genomics of three related parasites that cause leishmaniasis, Chagas disease, and African sleeping sickness. SSGCIDâ??s primary mission is to determine the structure of ~400 protein targets over a period of five years, from NIAID Category A-C organisms as well as emerging and re-emerging infectious disease organisms. Pro-active engagement of the infectious disease research and drug therapy communities in the target selection process will help ensure that the resulting protein structures provide a blueprint for structure-based drug design of new therapeutics to combat infectious diseases. This goal will be facilitated by the annual selection of a small number of high-impact targets for a fragment-based drug lead discovery campaign within SSGCID. The consortium is also committed to providing structural genomics service to the research community and publicly disseminating all structure information and material resources generated as part of the NIAID contract. While the primary method of structure solution will be XRD-based, targets that fail to crystallize will be directed to the NMR group in the consortium, composed of Drs. G.W. Buchko (PNNL) and G. Varani (University of Washington). Together, the NMR group is mandated to determine 30-40 structures over the five-year period
• Probing the Dynamics of a Protein Hydrophobic Core at Low Temperatures
  Project Lead: Liliya Vugmeyster
  Project Lead Institution: University of Alaska at Anchorage
  Abstract
  Hydrophobic cores are found in the interior of globular proteins and are essential for the formation of the folded state. The core represents a complex dynamical medium reflecting the existence of a conformational ensemble. Our goal is to investigate dynamical features typical in hydrophobic cores by looking at model globular proteins. Our major experimental tool will be deuteron NMR spectroscopy, which is ideally suited for investigation of dynamics over a wide range of time scales. We choose chicken villin subdomain (HP36), which is one of the smallest examples of a cooperatively folding protein. Its small size makes it possible to use solid-state peptide synthesis for sample preparation. We can thus easily obtain several samples with mutations and isotopic labels of our choice. Preliminary data on the wild-type HP36 sample with the deuteron label on methyl of Leuicine-69, located in the key position of the hydrophobic core, allowed us to generate a precise motional model that governs the behavior of this methyl group. The data were obtained in collaboration with PNNL staff members and Prof. R.L. Vold laboratory at the College of William and Mary. The main temperature range covered was between 110K-298K. At temperatures below 100K the methyl group undergoes a switch to a new motional regime and eventually reaches the limit when deuteron tunneling becomes important. Thus, the low temperature regime opens a new realm for dynamical studies. Yet, low temperatures proved to be difficult due to low signal to noise. To alleviate the signal-to-noise constraints we propose to implement Quadrupolar Echo CPMG detection scheme for T1 relaxation times measurements. We will then use this technique to obtained T1 measurements for the wild-type HP36 sample, F58L mutant, and FMOC-leucine amino acid between 5-100K. The resulting relaxation data will be used to extract activation energies, tunneling temperatures, and heat capacities. The comparison among the three samples will allow for the elucidation of the dynamical features specific to the hydrophobic core of HP36. To our knowledge this will be the first detailed study probing the dynamics of the core at this temperature regime by deuteron relaxation. The cryogenic probes developed at PNNL make a unique opportunity for this study. The results will advance our understanding of complex dynamical behavior of globular proteins. Proposal Type: Open call, standard access, non-proprietary, general
• Mechanistic understanding of zeolite deactivation pathways
  Project Lead: Ronald Silver
  Project Lead Institution: Caterpillar, Inc.
  Abstract
  Three zeolite powders (Fe-MFI, Cu-MFI, and Fe-BEA) commonly used in SCR NOx reduction will be hydrothermally aged in air using a variety of temperatures, aging times and water concentrations in a Caterpillar Inc. bench oven. It is proposed that the aged powders will be characterized at PNNL-EMSL using High Resolution Transmission Electron Microscopy (HR-TEM) and solid state Nuclear Magnetic Resonance (NMR). HR-TEM will examine the physical migration of alumina and metals to aggregate species on the exterior of the zeolites. Solid-state NMR, specifically 27Al NMR, will be used to examine changes in the Al co-ordination in order to determine the relative amount of dealumination compared to a fresh analog and subsequently the degree to which the incorporated Cu or Fe is affected by the aging. Critical to the implementation of urea-SCR catalysts is a detailed understanding of the deactivation processes of these catalysts in order to develop robust and market acceptable catalytic systems. Urea-SCR catalysts are the optimal technology advocated for the control of NOx emission from heavy duty diesel engines and for Tier 2 Bin 5 emission requirements for light duty diesel engines. This fundamental research will enable NOx emissions reduction in vehicles for the future and is a topic mentioned in the current developing economic stimulus package and a means to create future green jobs. EMSL is uniquely positioned at the cutting edge in the characterization of materials/catalysts with all the necessary components: the scientific experts, the latest characterization tools and support from scientists in the Institute of Interfacial Catalysis. It was in a paper in Catalysis Today that Ja Hun Kwak and Chuck Peden (at EMSL) demonstrated for the first time an elligant means to utilize NMR to provide semi-quantitative numbers on de-alumination of Cu-zeolites. Fe zeolites have the additional challenge of the possibility to form paramagnetic species that would accelerate the relaxation of the NMR spins so much as to render NMR spectra problematic.
• Radical Mechanisms for Antibiotic and Herbicide Biosynthesis
  Project Lead: Susan Wang
  Project Lead Institution: Washington State University
  Abstract
  Antibiotic resistance is a major public health issue; hospital-acquired infections are now the fourth-leading cause of death in the United States. To address this problem, new antibiotics for clinical use are needed, and industrial processes for designing and synthesizing these drugs must be performed more efficiently at lower cost. The goal of our research is to elucidate enzyme mechanisms in antibiotic biosynthesis so that they can be engineered to generate novel compounds. We are particularly interested in methyltransferases from the radical SAM (S-adenosyl-L-methionine) protein superfamily. To date, none of the methyltransferases in this smaller group of radical SAM proteins has been characterized. In general, radical SAM enzymes catalyze difficult chemical reactions through the use of a 5-deoxyadenosyl radical. The deoxyadenosyl radical is generated upon homolytic cleavage of SAM, an equilibrium process that occurs when a reduced iron-sulfur cluster ([4Fe-4S]+1) in the protein provides SAM with an electron. We are investigating four related radical SAM methyltransferases. We hypothesize that all four proteins are radical SAM enzymes that also require a cobalamin (vitamin B12) for their methylation activity. We are interested in characterizing the iron-sulfur clusters of these proteins and identifying cobalamin intermediates/products or radical intermediates in order to establish the mechanism of these enzymes. These experiments are ideally performed using electron paramagnetic resonance (EPR) spectroscopy, so we are hopeful that we can collaborate with the NMR and EPR facility at EMSL on this research.