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Resource for NMR Molecular Imaging of Proteins
Contents
Contact Information
University of California, San Diego
Department of Chemistry and Biochemistry
9500 Gilman Drive, 0307
La Jolla, CA 92093-0307
Principal Investigator/Contact
Grant No. EB002031
Research Emphasis
The Resource is focused on the development of NMR spectroscopy for structure determination of proteins in biological supramolecular assemblies. The principal applications are to membrane-associated proteins; however, the approach is generally applicable to polypeptides that cannot be prepared in forms suitable for X-ray crystallography or multidimensional solution NMR spectroscopy. As a result, there are also applications to viruses and other biological systems.
The principal instrumentation consists of high-field NMR spectrometers dedicated to high-resolution solid-state NMR spectroscopy. The spectrometers are capable of the full-range of multiple-resonance experiments on stationary and spinning samples; however, the major emphasis is on methods that utilize mechanically or magnetically oriented samples. Development encompasses preparation of samples, including:
- Expression and purification of membrane proteins
- Design and construction of instrumentation, especially probes
- Implementation of new pulse sequences and other experimental protocols for solid-state NMR spectroscopy
- Calculations for the processing of experimental data and protein structure determination from the orientational constraints derived from these data
Current Research
Most of the current research projects, including those of the core, collaborative, and service research activities, involve determination of the three-dimensional structures of membrane proteins. Methods and instrumentation are being developed so that it is possible to routinely resolve and assign all backbone and side chain resonances in multidimensional solid-state NMR spectra of uniformly isotopically labeled membrane proteins. These spectra lend themselves to measurements of the spectral parameters associated with the chemical shift and heteronuclear dipolar interactions at the 1H, 13C, and 15N sites of the proteins.
Systems currently under investigation include several different proteins that function as ion channels, including the accessory protein Vpu from HIV-1. Other proteins include the mercury transport proteins of the bacterial mercury detoxification system, human copper transporter I, and several G-protein coupled receptors (GPCRs). New systems are constantly being explored as investigators provide an increasing variety of membrane proteins for study.
Resource Capabilities
Instruments
- 900 MHz spectrometer with a Magnex 900/52 standard bore magnet, Bruker Avance console with three high power channels, and double-resonance 1H/15N probes with solenoid and flat coil configurations.
- 750 MHz spectrometer with a Magnex 750/54 standard bore magnet, Bruker Avance console with three high power channels, double-resonance 1H/15N probes with solenoid and flat coil configurations, and a Bruker triple-resonance 1H/13C/15N MAS probe.
- 700 MHz spectrometer with a Magnex 700/62 mid-bore magnet, Tecmag Apollo control unit and high power amplifiers on all three channels, double-resonance 1H/15N probes in both solenoid and flat coil configurations, a 1H/31P double-resonance flat coil probe, and 2H flat-coil probe.
- 550 MHz spectrometer with a Magnex 550/89 wide-bore magnet, Tecmag Apollo control unit and high power amplifiers on all tree channels, double-resonance 1H/15N probes in both solenoid and flat coil configurations and a triple-resonance 1H/13C/15N probe configured with a solenoid coil.
- 500 MHz spectrometer with a Magnex 500/89 wide-bore magnet, Varian Inova console with three high power channels, double-resonance 1H/15N probes in both solenoid and flat coil configurations and a Varian/Chemagnetics triple-resonance 1H/13C /15N probe configured with a solenoid coil.
- A fully equipped electronics shop for the construction, modification, and repair of all spectrometer components, especially probes.
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References
- DeAngelis AA, Nevzorov AA, Park SH, Howell SC, Mrse AA, Opella SJ. High-resolution NMR spectroscopy of membrane proteins in aligned bicelles. Journal of the American Chemical Society 2004;126:15340-15341.
- Opella SJ, Marassi FM. Structure determination of membrane proteins by NMR spectroscopy. Chemical Reviews 2004;104:3587-3606.
- Thiriot DS, Nevzorov AA, Zagyanskiy L, Wu CH, Opella SJ. Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy. Journal of Molecular Biology 2004;341:869-879.
- Mesleh MH, Lee S, Veglia G, Thiriot DS, Marassi FM, Opella SJ. Dipolar waves map the structure and topology of helices in membrane proteins. Journal of the American Chemical Society 2003;125:8928-8935.
- Nevzorov AA, Opella SJ. A "magic sandwich" pulse sequence with reduced offset dependence for high-resolution separated local field spectroscopy. Journal of Magnetic Resonance 2003;164:182-186.
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