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Biomedical Technology Resources Directory

Magnetic Resonance Spectroscopy

 
National Biomedical Center for Advanced ESR Technology (ACERT)
National Magnetic Resonance Facility at Madison
Southwestern Biomedical Magnetic Resonance Facility

 
National Biomedical Center for Advanced ESR Technology (ACERT)
Cornell University
Department of Chemistry and Chemical Biology
Baker Laboratory
Ithaca, NY 14853
www.acert.cornell.edu

Grant No. P41 RR016292
Principal Investigator
Jack H. Freed, Ph.D.
607-255-3647; Fax: 607-255-6969
E-mail: JHF@ccmr.cornell.edu

Research Emphasis

The National Biomedical Center for Advanced ESR Technology (ACERT) is an outgrowth of extensive experience in developing the methods of modern electron spin resonance (ESR), which are now ripe for dissemination in the biomedical community. In addition to making equipment and facilities available to outside scientists, publishing and advertising results, and running workshops on the new methodologies, ACERT also will address the need to bring these new technologies to other laboratories. The center’s collaborators cover a wide range of biomedical research areas, emphasizing the central role that modern ESR methods can play. The principal areas of core research are: Distance measurements in proteins and aggregates using double quantum coherence (DQC) ESR: To advance our knowledge of structure and function of biomolecules using longer distance constraints, provided by site-directed spin labeling and pulsed ESR distance measurements. Pulsed two-dimensional ESR: To further develop 2D-ELDOR in study of the dynamic structure of membranes and of proteins, as well as lipid-protein interactions. Functional dynamics of proteins: To study protein folding and unfolding and transient structural changes by 2D-FT ESR and DQC ESR. Fourier transform ESR imaging: To establish FT ESR imaging for biomedical studies of macroscopic translational diffusion and for 3D spatial imaging. Instrumental developments in high-frequency high-field (HFHF) ESR: To further develop quasi-optical techniques for HFHF-ESR to unravel the complex dynamics in biological systems. High-power pulsed HFHF ESR: To extend high power pulsed ESR to the mm-wave regime, to benefit from the improved orientational resolution and faster “snapshot” feature at higher frequencies. Multifrequency studies of dynamics: To advance knowledge of dynamics in proteins, DNA, and membranes by a mixture of multifrequency and multidimensional ESR. Theoretical and computational methods: To improve the methods for interpreting multifrequency spectra in terms of powerful algorithms based on the stochastic Liouville equation and on molecular dynamics simulations.

Current Research

Distance measurements: Structure of membrane proteins. Pulsed 2D-ESR: Dynamics of proteins and membrane domains. Functional dynamics: Integration of pulsed 2D-ESR with continuous and stopped-flow techniques. FT-ESR imaging: Rapid pulsed field gradient techniques. HFHF ESR: Wide-band, quasi-optical reflection bridge and multifrequency studies of dynamics. Pulsed HFHF ESR: High-power 95 GHz spectrometer.

Resource Capabilities

ACERT occupies about 6,000 square feet of space in Baker Laboratory. It includes facilities for conducting cw multifrequency ESR from 1 to 250 GHz; 2-D and FT ESR and electron-spin-echo spectrometers at 9, 17.3, and 95 GHz; field gradient equipment; workstations for theoretical simulation of ESR spectra; wet chemistry facilities; electronics and machining facilities.

  1. Borbat, P. P., Mchaourab, H. S., and Freed, J. H., Protein structure determination using long-distance constraints from double-quantum coherence ESR: Study of T4-lysozyme. Journal of American Chemical Society 124:5304–5314, 2002.
  2. Lou, Y., Ge, M., and Freed, J. H., A multifrequency ESR study of the complex dynamics of membranes. Journal of Physical Chemistry B 105:11053–11056, 2001.
  3. Borbat, P. P., Costa-Filho, A. J., Earle, K. A., Moscicki, J. K., and Freed, J. H., ESR in studies of membranes and proteins. Science 291:266–269, 2001.
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National Magnetic Resonance Facility at Madison
University of Wisconsin-Madison
433 Babcock Drive
Madison, WI 53706-1544
www.nmrfam.wisc.edu/

Grant No. P41 RR002301
Principal Investigator and Contact
John L. Markley, Ph.D.
608-263-9349; Fax: 608-262-3759
E-mail: markley@nmrfam.wisc.edu

Contacts
William M. Westler, Ph.D.
608-263-9599
E-mail: milo@nmrfam.wisc.edu

Craig S. Newman, Ph.D.
608-262-3173
E-mail: csnewman@nmrfam.wisc.edu

Research Emphasis

The major focus of this resource is on developing multinuclear, multidimensional nuclear magnetic resonance (NMR) approaches to solution-state studies of biological macromolecules, such as proteins, nucleic acids, carbohydrates, and lipids. Related collaborative projects converge on structure-function investigations of proteins and nucleic acids and structural genomics. The resource has considerable experience in labeling proteins with stable isotopes and in studying chemical and dynamic properties of biomolecules, such as binding equilibria, conformational equilibria, internal mobility, and protonation steps. Data collection for oxygen-sensitive samples and proteins containing paramagnetic centers is a specialty. Methods for efficient NMR data collection, processing, structure determination, validation, and data deposition are being developed through a collaboration with the Center for Eukaryotic Structural Genomics. This includes continued development of a versatile web-based project management system (the Sesame software project). Instrumentation developed here permits preparation of NMR samples under pressure and collection of NMR data under variable pressure and temperature. The resource also supports collaborations and user projects on micro-imaging and noninvasive analysis of the biochemistry of cells, tissues, organs, and intact living organisms. Computational resources include not only hardware and software, but also expertise in ab initio calculations of chemical shifts and J-couplings from structural modeling.

Resource Capabilities

Seven NMR spectrometers for data collection at 1H NMR frequencies: 400 MHz, 500 MHz (two units), 600 MHz (two units), 750 MHz, and 800 MHz. Two additional spectrometers (one at 600 MHz and one at 900 MHz) are expected to be operational by the middle of 2003. Triple-resonance probes (5 mm OD sample diameter; 1H, 15N, 13C, with 2H-lock) are available on all spectrometers. Cryogenic triple-resonance NMR probes are on order for seven spectrometers (between 500 and 900 MHz); one such probe (at 500 MHz) is operational, and two more are expected to be installed in 2003. Specialty probes include a quadruple-resonance probe (1H, 13C, 15N, 31P with 2H-lock) at 400 MHz, large (8 mm), and small (2.5 mm) diameter triple-resonance probes with single-axis gradient, several inverse, and direct observe variable frequency probes, and a surface coil probe for in vivo studies. Home-built probes have been developed for perifusion of tissue samples, perfused organs, and high-pressure NMR (up to 4.2 kbar). A server and network of computer workstations are equipped with a wide range of software packages relevant to NMR spectroscopy and structural biology. Complete details on the current status and specifications of equipment, including the availability of high-sensitivity triple-resonance cryogenic probes, are available on the facility’s web site.

  1. Dmitriev, O. Y., Abildgaard, F., Markley, J. L., and Fillingame, R. H., Structure of Ala24/Asp61->Asp24/Asn61 substituted subunit c of Escherichia coli ATP synthase: Implications for the mechanism of proton transport and rotary movement in the Fo complex. Biochemistry 41:5537–5547, 2002.
  2. Wilkens, S. J., Westler, W. M., Weinhold, F., and Markley, J. L., Trans-hydrogen-bond h2JNN and h1JNH couplings in the DNA A–T base pair: Natural bond orbital analysis. Journal of the American Chemical Society 124:1190–1191, 2002.
  3. Machonkin, T. E., Westler, W. M., and Markley, J. L., 13C{13C} 2D NMR: A novel strategy for the study of paramagnetic proteins with slow electronic relaxation rates. Journal of the American Chemical Society 124:3204–3205, 2002.
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Southwestern Biomedical Magnetic Resonance Facility
University of Texas Southwestern Medical Center
Mary Nell and Ralph B. Rogers Magnetic Resonance Center
5801 Forest Park Road
Dallas, TX 75235-9085
www.swmed.edu/home_pages/rogersmr

Grant No. P41 RR002584
Principal Investigator and Contact
Craig R. Malloy, M.D.
214-648-5886; Fax: 214-648-5881
E-mail: cmallo@mednet.swmed.edu

Research Emphasis

The facility’s core research efforts focus on three projects. The first is to develop and refine the use of 13C NMR isotopomer analysis for the investigation of intermediary metabolism in intact, functioning tissues, and to apply these methods to current questions in metabolism. Techniques to assess human hepatic metabolism by 13C NMR of body fluids following ingestion of stable carbon isotopes and metabolite-conjugation agents have been developed.

The second project aims to develop and refine mathematical models to estimate the flux through different metabolic pathways using isotopomer analysis of 13C NMR and/or mass spectroscopy (MS) data. Three programs are under development: tcaSIM, a simulation of the tricarboxylic acid (TCA) cycle that generates 13C isotopomer data for use in the design of experiments; tcaCALC, a model that estimates relative pathway fluxes from NMR spectra or MS data obtained at metabolic and isotopic steady state; and tcaFLUX, a kinetic analysis that allows measurement of absolute flux from systems at metabolic, but not isotopic, steady state. tcaSIM and tcaCALC are available free of charge to researchers.

The third project aims to develop new techniques and agents for monitoring intercellular cations, particularly sodium, magnesium, and calcium, in perfused tissues and in vivo. Synthetic chemistry and characterization of new agents is ongoing at the University of Texas at Dallas. Evaluation and application of the new agents for perfused organ and in vivo studies is carried out at the Rogers Magnetic Resonance Center at the University of Texas Southwestern Medical Center.

Resource Capabilities

A range of NMR instruments (200, 300, 400, 500, and 600 MHz, including vertical- and horizontal-bore systems) is available with capabilities for both gradient-based experiments (imaging, spatially localized spectroscopy, metabolite-specific spectroscopy) and standard spectroscopy (decoupling, magnetization transfer, multiple quantum, 2-D). A bench-top gas chromatograph–mass spectrometer capable of tandem MS complements the NMR data and provides an additional approach to 13C isotopomer analysis. Special expertise is available in isotopomer analysis of 13C NMR spectra, working heart perfusion preparations, synthesis of shift reagents, and in vivo MR studies.

1. Jones, J. G., Sherry, A. D., and Malloy, C. R., Quantitation of gluconeogenesis by 2H nuclear magnetic resonance analysis of plasma glucose following ingestion of 2H2O. Analytical Biochemistry 277:121–126, 2000.
2. Batista, A., Corbett, R., Tidor, S., Castleman, E., Laptook, A., and Sherry, A. D., Comparison of the distribution of magnesium in plasma determined by size exclusion chromatography and 31P NMR spectroscopy. Magnesium Research 13:3–9, 2000.
3. Corbett, R., Batista, A., Laptook, A., and Sherry, A. D., A macrocyclic reporter ligand for Mg2+: Analytical implications for clinical magnesium determinations. Magnesium Research 12:79–88, 1999.
4. Colet, J.-M., Bansal, N., Malloy, C. R., and Sherry, A. D., Multiple quantum filtered 23Na NMR spectroscopy of the isolated, perfused rat liver. Magnetic Resonance in Medicine 41:1127–1135, 1999.
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