Membrane proteins constitute about 30 % of all prokaryotic and eukaryotic proteomes. They are responsible for ion conduction, chemical transport, energy conversion, signal transduction, hormone- and photo-reception, cell adhesion, and many other functions. The long-held tenet of structural biology is that to fully understand the function of biomolecules their structure must be known. Membrane proteins are no exception in this regard. However, the science aimed at elucidating their structures has lagged far behind similar science on soluble proteins, mostly for technical reasons. The goal of the research proposed in this application is two-fold: (1) to advance solution NMR spectroscopy as a method for structure determination of membrane proteins and (2) to further the understanding of the functions of four prokaryotic outer membrane proteins by studying their structures by solution NMR. Specifically: OmpA from E. coli primarily will be used as a model membrane protein for NMR methods development, for studying lipid-protein interactions, and for examining the stability of membrane proteins in lipid bilayers;OmpG from E. coli is becoming the preferred biological nanopore for biosensor development and NMR will be used to understand the mechanism, by which it gates from the open to the closed state;and OprG and OprH are two outer membrane proteins from Pseudomonas aeruginosa, whose contributions to the severe antibiotic resistance of this microorganism will be investigated by determining their structures and studying their functions.

Public Health Relevance

This project will push the frontier of determining the structures and understanding the functions of membrane-bound proteins by nuclear magnetic resonance spectroscopy. The specific membrane proteins that will be examined have biotechnological applications in biosensor development or are components of the cell envelope of Pseudomonas aeruginosa, i.e. a clinically important pathogen in hospital-induced infections and complications of cystic fibrosis. The research may ultimately contribute to better treatments of these infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051329-14
Application #
7821440
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
1997-03-01
Project End
2013-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
14
Fiscal Year
2010
Total Cost
$395,136
Indirect Cost
Name
University of Virginia
Department
Physiology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Kucharska, Iga; Tamm, Lukas K (2017) Solution NMR Provides New Insight into Lipid-Protein Interaction. Biochemistry 56:4291-4292
Lee, Joonseong; Patel, Dhilon S; Kucharska, Iga et al. (2017) Refinement of OprH-LPS Interactions by Molecular Simulations. Biophys J 112:346-355
Kucharska, Iga; Liang, Binyong; Ursini, Nicholas et al. (2016) Molecular Interactions of Lipopolysaccharide with an Outer Membrane Protein from Pseudomonas aeruginosa Probed by Solution NMR. Biochemistry 55:5061-72
Liang, Binyong; Tamm, Lukas K (2016) NMR as a tool to investigate the structure, dynamics and function of membrane proteins. Nat Struct Mol Biol 23:468-74
Kucharska, Iga; Seelheim, Patrick; Edrington, Thomas et al. (2015) OprG Harnesses the Dynamics of its Extracellular Loops to Transport Small Amino Acids across the Outer Membrane of Pseudomonas aeruginosa. Structure 23:2234-45
Kucharska, Iga; Edrington, Thomas C; Liang, Binyong et al. (2015) Optimizing nanodiscs and bicelles for solution NMR studies of two ?-barrel membrane proteins. J Biomol NMR 61:261-74
Marcoux, Julien; Politis, Argyris; Rinehart, Dennis et al. (2014) Mass spectrometry defines the C-terminal dimerization domain and enables modeling of the structure of full-length OmpA. Structure 22:781-90
Zhuang, Tiandi; Tamm, Lukas K (2014) Control of the conductance of engineered protein nanopores through concerted loop motions. Angew Chem Int Ed Engl 53:5897-902
Gregory, Sonia M; Larsson, Per; Nelson, Elizabeth A et al. (2014) Ebolavirus entry requires a compact hydrophobic fist at the tip of the fusion loop. J Virol 88:6636-49
Moissoglu, Konstadinos; Kiessling, Volker; Wan, Chen et al. (2014) Regulation of Rac1 translocation and activation by membrane domains and their boundaries. J Cell Sci 127:2565-76

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