Structure Determination of Membrane Proteins in Phospholipid Bilyaers

Summary

Principal Investigator: Stanley J Opella
Abstract: DESCRIPTION (provided by applicant): There are three principal goals. The first is to continue the development of a general method for determining the structures of membrane proteins in phospholipid bilayers under physiological conditions. This goal is important because membrane proteins are high priority targets for structure determination, and existing methods have substantial limitations for this class of proteins. The second goal is to apply the method for structure determination to the mercury transport membrane proteins of the bacterial mercury detoxification system. The structures of MerF and MerE, each of which have two trans membrane (TM) helices, will be determined first, and then the research will proceed to other members of this family with three (MerT) and four (MerC) TM helices. Studying this family of proteins serves a second role in the research by providing protein targets of increasing size and complexity as challenges for the development of the instrumentation and experimental methods of solid-state NMR spectroscopy. Comparisons among these proteins may provide insights into why independent isolates of bacteria capable of detoxifying Hg(II) have varying numbers of transport proteins, the proteins have different numbers of TM helices, and the proteins have different numbers of pairs of cysteine residues that bind mercury. The third goal follows from the development of the technology and the structural findings on members of this family of proteins, which sets the stage for the assembly and structural studies of binary and ternary complexes of examples of the mercury transport membrane proteins with the periplasmic protein, MerP, whose structure we determined previously, and the N-terminal "MerP-like" domain of mercuric reductase, MerA, whose structure has been determined by others. Mercuric reductase (MerA) reduces the highly toxic Hg(II) to the less toxic and volatile Hg(0) that passively diffuses out of the cells. Transporting the Hg(II) from the periplasm to the cytoplasm is a key step and it must be tightly controlled so that the highly reactive Hg(II) is never free in solution and available for reaction with the cysteine residues on essential cellular proteins, which is the source of its toxicity in cells without the mer operon. Our research approach is interdisciplinary and comprehensive, encompassing molecular biology, biochemistry, sample preparation, construction and modification of NMR instrumentation, the development and execution of NMR experiments, and structure calculations. The structures of the mercury transport membrane proteins alone and in their functional complexes set the stage for functional studies of the mechanism of transporting Hg(II) across the bilayer membrane. The results of these studies have the potential to impact the treatment of acute mercury toxicity in humans, and this is one of the first examples of applying the methods of structural biology to environmental research because of the widespread distribution of organ mercurial compounds in the food supply (especially in large fish) and the environment.
Funding Period: 2012-04-01 - 2016-03-31
more information: NIH RePORT

Top Publications

  1. pmc NMR studies of membrane proteins
    Gabriel A Cook
    Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA
    Methods Mol Biol 637:263-75. 2010
  2. pmc Experiments optimized for magic angle spinning and oriented sample solid-state NMR of proteins
    Bibhuti B Das
    University of California, San Diego, 9500 Gilman Drive, 0307 La Jolla, California 92093 0307, United States
    J Phys Chem B 117:12422-31. 2013
  3. pmc Impact of histidine residues on the transmembrane helices of viroporins
    Yan Wang
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92037 0307, USA
    Mol Membr Biol 30:360-9. 2013
  4. pmc Sampling scheme and compressed sensing applied to solid-state NMR spectroscopy
    Eugene C Lin
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 0307, United States
    J Magn Reson 237:40-8. 2013
  5. pmc Resolution and measurement of heteronuclear dipolar couplings of a noncrystalline protein immobilized in a biological supramolecular assembly by proton-detected MAS solid-state NMR spectroscopy
    Sang Ho Park
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92039 0307, USA
    J Magn Reson 237:164-8. 2013
  6. pmc Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR
    George J Lu
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093 0307, USA
    J Biomol NMR 58:69-81. 2014
  7. pmc Covariance spectroscopy in high-resolution multi-dimensional solid-state NMR
    Eugene C Lin
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 0307, USA
    J Magn Reson 239:57-60. 2014
  8. pmc Mechanism of dilute-spin-exchange in solid-state NMR
    George J Lu
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093 0307, USA
    J Chem Phys 140:124201. 2014
  9. pmc Dipolar Assisted Assignment Protocol (DAAP) for MAS solid-state NMR of rotationally aligned membrane proteins in phospholipid bilayers
    Bibhuti B Das
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 0307, United States
    J Magn Reson 242:224-32. 2014
  10. pmc Motion-adapted pulse sequences for oriented sample (OS) solid-state NMR of biopolymers
    George J Lu
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093 0307, USA
    J Chem Phys 139:084203. 2013

Research Grants

  1. Interaction of inhalational anesthestics with macromolecules
    Roderic G Eckenhoff; Fiscal Year: 2013
  2. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
  3. Southeast Regional Centers of Excellence for Biodefense &Emerging Infectious Di
    Philip Frederick Sparling; Fiscal Year: 2013
  4. NMR STUDIES OF HIV-1 PROTEINS
    MICHAEL FINLEY SUMMERS; Fiscal Year: 2013
  5. Center for Gene Therapy of Cystic Firbosis
    John F Engelhardt; Fiscal Year: 2013

Detail Information

Publications21

  1. pmc NMR studies of membrane proteins
    Gabriel A Cook
    Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA
    Methods Mol Biol 637:263-75. 2010
    ..Applications to many helical membrane proteins are underway and promise to add to our understanding of membrane proteins in health and disease...
  2. pmc Experiments optimized for magic angle spinning and oriented sample solid-state NMR of proteins
    Bibhuti B Das
    University of California, San Diego, 9500 Gilman Drive, 0307 La Jolla, California 92093 0307, United States
    J Phys Chem B 117:12422-31. 2013
    ..The techniques are illustrated with data from single crystals both of peptides and of membrane proteins in phospholipid bilayers. ..
  3. pmc Impact of histidine residues on the transmembrane helices of viroporins
    Yan Wang
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92037 0307, USA
    Mol Membr Biol 30:360-9. 2013
    ....
  4. pmc Sampling scheme and compressed sensing applied to solid-state NMR spectroscopy
    Eugene C Lin
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 0307, United States
    J Magn Reson 237:40-8. 2013
    ..A modest increase in signal-to-noise ratio accompanies the reconstruction. ..
  5. pmc Resolution and measurement of heteronuclear dipolar couplings of a noncrystalline protein immobilized in a biological supramolecular assembly by proton-detected MAS solid-state NMR spectroscopy
    Sang Ho Park
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92039 0307, USA
    J Magn Reson 237:164-8. 2013
    ....
  6. pmc Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR
    George J Lu
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093 0307, USA
    J Biomol NMR 58:69-81. 2014
    ....
  7. pmc Covariance spectroscopy in high-resolution multi-dimensional solid-state NMR
    Eugene C Lin
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 0307, USA
    J Magn Reson 239:57-60. 2014
    ....
  8. pmc Mechanism of dilute-spin-exchange in solid-state NMR
    George J Lu
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093 0307, USA
    J Chem Phys 140:124201. 2014
    ..The mechanism of the proton relay in dilute-spin-exchange is crucial for the design of polarization transfer experiments. ..
  9. pmc Dipolar Assisted Assignment Protocol (DAAP) for MAS solid-state NMR of rotationally aligned membrane proteins in phospholipid bilayers
    Bibhuti B Das
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 0307, United States
    J Magn Reson 242:224-32. 2014
    ..Spectra with both dipolar and chemical shift frequency axes assist with resonance assignments. DAAP can be readily extended to three- and four-dimensional experiments and to include both backbone and side chain sites in proteins. ..
  10. pmc Motion-adapted pulse sequences for oriented sample (OS) solid-state NMR of biopolymers
    George J Lu
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093 0307, USA
    J Chem Phys 139:084203. 2013
    ..This approach is applicable to both stationary and magic angle spinning solid-state NMR experiments...
  11. pmc Membrane protein structure determination: back to the membrane
    Yong Yao
    Sanford Burnham Medical Research Institute, La Jolla, CA, USA
    Methods Mol Biol 1063:145-58. 2013
    ..The methods for protein expression and purification, sample preparation, and NMR experiments are described and illustrated with examples from OmpX and Ail, two bacterial outer membrane proteins that function in bacterial virulence...
  12. pmc Three-dimensional structure and interaction studies of hepatitis C virus p7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine by solution nuclear magnetic resonance
    Gabriel A Cook
    Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093, United States
    Biochemistry 52:5295-303. 2013
    ..Changes in the chemical shift frequencies of solution NMR spectra identify the residues taking part in these interactions. ..
  13. pmc Mechanically, magnetically, and "rotationally aligned" membrane proteins in phospholipid bilayers give equivalent angular constraints for NMR structure determination
    Sang Ho Park
    Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093 0307, USA
    J Phys Chem B 114:13995-4003. 2010
    ..The measurement of angular constraints relative to a common external axis system defined by the bilayer normal for all sites in the protein is an essential element of oriented sample (OS) solid-state NMR...
  14. pmc AssignFit: a program for simultaneous assignment and structure refinement from solid-state NMR spectra
    Ye Tian
    Sanford Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
    J Magn Reson 214:42-50. 2012
    ..The methods are demonstrated with data from two integral membrane proteins, one α-helical and one β-barrel, embedded in phospholipid bilayer membranes...
  15. pmc Structure determination of a membrane protein in proteoliposomes
    Bibhuti B Das
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093 0307, USA
    J Am Chem Soc 134:2047-56. 2012
    ..The unique feature of this method is the measurement of orientation restraints that enable the protein structure and orientation to be determined in unoriented proteoliposomes...
  16. pmc Improved 1H amide resonance line narrowing in oriented sample solid-state NMR of membrane proteins in phospholipid bilayers
    George J Lu
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093 0307, USA
    J Magn Reson 220:54-61. 2012
    ....
  17. pmc Improved chemical shift prediction by Rosetta conformational sampling
    Ye Tian
    Sanford Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
    J Biomol NMR 54:237-43. 2012
    ..This approach to chemical shift and structure prediction has the potential to be useful for guiding resonance assignments, especially in solid-state NMR structural studies of membrane proteins in proteoliposomes...
  18. pmc Structure determination of membrane proteins by nuclear magnetic resonance spectroscopy
    Stanley J Opella
    Department of Chemistry and Biochemistry, University of California, San Diego 92093, USA
    Annu Rev Anal Chem (Palo Alto Calif) 6:305-28. 2013
    ..At present, the only method that can work with proteins in liquid crystalline phospholipid bilayers is solid-state NMR spectroscopy...
  19. pmc The structure of the mercury transporter MerF in phospholipid bilayers: a large conformational rearrangement results from N-terminal truncation
    George J Lu
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093 0307, United States
    J Am Chem Soc 135:9299-302. 2013
    ..They provide further evidence of the importance of studying unmodified membrane proteins in near-native bilayer environments in order to obtain accurate structures that can be related to their functions. ..
  20. pmc Structure determination of membrane proteins in their native phospholipid bilayer environment by rotationally aligned solid-state NMR spectroscopy
    Stanley J Opella
    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093 0307, United States
    Acc Chem Res 46:2145-53. 2013
    ..The determination of the structures of several membrane proteins, most prominently the chemokine receptor CXCR1, a 350-residue GPCR, has demonstrated this approach. ..
  21. ncbi Membrane protein structure from rotational diffusion
    Bibhuti B Das
    Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093 0307 USA
    Biochim Biophys Acta 1848:229-45. 2015
    ..This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky. ..

Research Grants30

  1. Interaction of inhalational anesthestics with macromolecules
    Roderic G Eckenhoff; Fiscal Year: 2013
    ..abstract_text> ..
  2. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..
  3. Southeast Regional Centers of Excellence for Biodefense &Emerging Infectious Di
    Philip Frederick Sparling; Fiscal Year: 2013
    ..SERCEB brings new investigators to the biodefense effort through a combination of educational programs, support of innovative new projects, and the synergistic interactions among its world-class investigators. ..
  4. NMR STUDIES OF HIV-1 PROTEINS
    MICHAEL FINLEY SUMMERS; Fiscal Year: 2013
    ..abstract_text> ..
  5. Center for Gene Therapy of Cystic Firbosis
    John F Engelhardt; Fiscal Year: 2013
    ..These efforts have led to numerous basic and applied research findings that have enhanced the utility of gene therapies to both study and treat genetic diseases. ..