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. Only a little more than 1% of all structures currently deposited in the Protein Data Bank are those of membrane proteins, i.e. the structural biology of membrane proteins still lags far behind that of soluble proteins. To fully understand their function, not ony the structure, but also the dynamics of membrane proteins should be known on multiple time-scales. Therefore, this project develops and uses tools of solution NMR spectroscopy for structure determination and dynamical characterization of membrane proteins in multiple lipid environments. Communication to the outside world occurs in Gram-negative bacteria through two membranes where the outer membrane forms the first barrier that nutrients and antibiotics have to cross to gain access to the cell. Pseudomonas aeruginosa is a serious Gram-negative pathogen with a particularly tough outer membrane that is difficult to penetrate by nutrients and antibiotics. Compared to E. coli, Pseudomonas harbors no general and only a relatively small number of specific porins in its outer membrane. In this project, we will determine the dynamic structure, substrate specificity, and the mechanism of transport of the P. aeruginosa OprG porin, which has been hypothesized to be responsible for the uptake of hydrophobic compounds and antibiotics. We will also determine the molecular interactions between OprH and lipopolysaccarides in the outer membrane that contribute to the mechanical strength, high antibiotic resistance, and strong tendency of P. aeruginosa to form biofilms in the lungs of pneumonia patients. Finally, using a combination of NMR and electrophysiology-based approaches, we will engineer the monomeric porin OmpG of E. coli into a practically useful and, compared to previous designs, superior nanopore platform for single molecule biosensing that may be used in the future to detect neurotransmitters, nucleotides, rare metals, or second messengers.

Public Health Relevance

Infections by Gram-negative bacteria cause many difficult to treat diseases. For example, Pseudomonas aeruginosa is a clinically serious pathogen in hospital-acquired infections and the leading cause of death in patients with cystic fibrosis. Antibiotic resistance of P. aeruginosa is partially due to the extreme difficulty for drugs to penetrate its outer membrane. In this research, we will characterize the structures, functions, and drug interactions of three outer membrane proteins from P. aeruginosa and E. coli. The E. coli protein will also be developed into a novel nanopore platform for single molecule biosensing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM051329-17A1
Application #
8879484
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
1997-03-01
Project End
2019-04-30
Budget Start
2015-05-01
Budget End
2016-04-30
Support Year
17
Fiscal Year
2015
Total Cost
$439,910
Indirect Cost
$159,910
Name
University of Virginia
Department
Physiology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Sanganna Gari, Raghavendar Reddy; Seelheim, Patrick; Marsh, Brendan et al. (2018) Quaternary structure of the small amino acid transporter OprG from Pseudomonas aeruginosa. J Biol Chem 293:17267-17277
Liang, Binyong; Tamm, Lukas K (2018) Solution NMR of SNAREs, complexin and ?-synuclein in association with membrane-mimetics. Prog Nucl Magn Reson Spectrosc 105:41-53
Blackburn, Matthew R; Hubbard, Caitlin; Kiessling, Volker et al. (2018) Distinct reaction mechanisms for hyaluronan biosynthesis in different kingdoms of life. Glycobiology 28:108-121
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
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; 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
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 23:2234-2245
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

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