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.

Public Health Relevance

By developing a general method for determining the structures of membrane proteins in their native phospholipid bilayer environment we will be able to tackle important problems in biomedical research. The development of this technology will have a broad impact since the majority of therapeutic drugs are targeted to protein receptors that reside in cell membranes. The studies described in this proposal are focused on the mercury transport membrane proteins of the bacterial mercury detoxification system. Their structures may assist in the discovery of antidotes to mercury poisoning in humans, and from a broader perspective they provide an opportunity to apply structural biology to an environmental problem.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM099986-02
Application #
8450700
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Wehrle, Janna P
Project Start
2012-04-01
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
2
Fiscal Year
2013
Total Cost
$277,331
Indirect Cost
$93,981
Name
University of California San Diego
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Das, Bibhuti B; Park, Sang Ho; Opella, Stanley J (2015) Membrane protein structure from rotational diffusion. Biochim Biophys Acta 1848:229-45
Park, Sang Ho; Wang, Vivian S; Radoicic, Jasmina et al. (2015) Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA). J Biomol NMR 61:185-96
Lin, Eugene C; Opella, Stanley J (2014) Covariance spectroscopy in high-resolution multi-dimensional solid-state NMR. J Magn Reson 239:57-60
Tian, Ye; Lu, George J; Marassi, Francesca M et al. (2014) Structure of the membrane protein MerF, a bacterial mercury transporter, improved by the inclusion of chemical shift anisotropy constraints. J Biomol NMR 60:67-71
Lu, George J; Opella, Stanley J (2014) Mechanism of dilute-spin-exchange in solid-state NMR. J Chem Phys 140:124201
Wu, Chin H; De Angelis, Anna A; Opella, Stanley J (2014) Magic angle Lee-Goldburg frequency offset irradiation improves the efficiency and selectivity of SPECIFIC-CP in triple-resonance MAS solid-state NMR. J Magn Reson 246:1-3
Radoicic, Jasmina; Lu, George J; Opella, Stanley J (2014) NMR structures of membrane proteins in phospholipid bilayers. Q Rev Biophys 47:249-83
Das, Bibhuti B; Zhang, Hua; Opella, Stanley J (2014) Dipolar Assisted Assignment Protocol (DAAP) for MAS solid-state NMR of rotationally aligned membrane proteins in phospholipid bilayers. J Magn Reson 242:224-32
Lu, George J; Opella, Stanley J (2014) Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR. J Biomol NMR 58:69-81
Das, Bibhuti B; Opella, Stanley J (2014) Multiple acquisition/multiple observation separated local field/chemical shift correlation solid-state magic angle spinning NMR spectroscopy. J Magn Reson 245:98-104

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