The overall goal of this project is to elucidate molecular mechanisms of membrane excitation events occurring in visual transduction. The present program explores a new approach to the study of structure and function in membrane proteins. The strategy is to place a cysteine residue at any chosen site in the protein using site-directed mutagenesis. Advantage is taken of the unique chemistry of cysteine to selectively attach a nitroxide spin-label. The recent development of the loop-gap resonator for Electron Spin Resonance makes it possible to record spectra of the spin-labeled mutants with high sensitivity using advanced techniques such as Electron-Electron Double Resonance (ELDOR), Pulse Saturation Recovery and CW Saturation. Data analysis on a sufficiently large set of labeled mutant gives detailed information on protein secondary and tertiary structure as well as dynamics. Applications of this technique will be rhodopsin, bacteriorhodopsin and colicin E1. Rhodopsin and bacteriorhodopsin are helical-bundle proteins, and structural features such as the origin, termination and orientation of the transmembrane helices can now be determined.; Solvation of the helices by lipids and the role of the lipid in determining the stability of the helical bundle will be investigated. In rhodopsin, a goal of particular interest will be to describe the conformational changes which occur as a consequence of photo-excitation. Colicin E1, a voltage-gated ion channel, will be investigated to probe molecular mechanisms of gating.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37EY005216-17
Application #
2888162
Study Section
Special Emphasis Panel (NSS)
Project Start
1983-07-01
Project End
2000-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
17
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Davydov, Dmitri R; Yang, Zhongyu; Davydova, Nadezhda et al. (2016) Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy. Biophys J 110:1485-1498
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