Signal transduction in vision involves a series of proteins that are sequentially activated via protein-protein interactions, thus passing the signal from the receptor to a final target. The long-term objectives of this project are to elucidate the molecular mechanisms of activation and signal propagation in three proteins of the relay: rhodopsin, transducin and arrestin. Of particular interest is the role of dynamics in function, i.e. the roles of backbone flexibility in protein-protein interactions and of conformational flexibility in activation. To obtain information on structure and dynamics in the complex membrane environment of signal transduction, the technique of site-directed spin labeling (SDSL) will be further developed. In this approach, nitroxide side chains are introduced at specific sites in a protein and analyzed by electron paramagnetic resonance (EPR) spectroscopy.
Specific aims i nclude: (1) analysis of nitroxide side chain interactions and dynamics via X-ray crystallography and spectral simulations, (2) evaluation of multifrequency EPR methods and new nitroxide side chains for analysis of backbone flexibility, and (3) evaluation of SDSL and high pressure EPR for detection of conformational flexibility in proteins. Modern pulse-EPR methods including saturation recovery, p-ELDOR, 2D-ELDOR and DEER will play important roles. These and existing SDSL tools will be applied to elucidate structure and dynamics in: (1) the conformational substates of activated rhodopsin (R*), (2) transducin in the R*-transducin complex and the mechanism of its activation, and (3) arrestin in solution and in the R*-arrestin complex, and the transformations leading to the high-affinity binding form. A growing number of pathological conditions have been traced to genetic defects in the proteins of the visual and other signaling systems. In order to design pharmacological approaches for altering such pathological states, it is imperative to understand molecular mechanism involved in the signal relay. Success in this project is anticipated to contribute to this understanding.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY005216-24
Application #
7038991
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Mariani, Andrew P
Project Start
1983-07-01
Project End
2010-04-30
Budget Start
2006-05-01
Budget End
2007-04-30
Support Year
24
Fiscal Year
2006
Total Cost
$519,227
Indirect Cost
Name
University of California Los Angeles
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Kintzer, Alexander F; Green, Evan M; Dominik, Pawel K et al. (2018) Structural basis for activation of voltage sensor domains in an ion channel TPC1. Proc Natl Acad Sci U S A 115:E9095-E9104
Bergdoll, Lucie A; Lerch, Michael T; Patrick, John W et al. (2018) Protonation state of glutamate 73 regulates the formation of a specific dimeric association of mVDAC1. Proc Natl Acad Sci U S A 115:E172-E179
Stadtmueller, Beth M; Bridges, Michael D; Dam, Kim-Marie et al. (2018) DEER Spectroscopy Measurements Reveal Multiple Conformations of HIV-1 SOSIP Envelopes that Show Similarities with Envelopes on Native Virions. Immunity 49:235-246.e4
Gu, Xin; Bridges, Michael D; Yan, Yan et al. (2018) Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK). J Biol Chem 293:16994-17007
Van Eps, Ned; Altenbach, Christian; Caro, Lydia N et al. (2018) Gi- and Gs-coupled GPCRs show different modes of G-protein binding. Proc Natl Acad Sci U S A 115:2383-2388
Van Eps, Ned; Caro, Lydia N; Morizumi, Takefumi et al. (2017) Conformational equilibria of light-activated rhodopsin in nanodiscs. Proc Natl Acad Sci U S A 114:E3268-E3275
Zhou, X Edward; He, Yuanzheng; de Waal, Parker W et al. (2017) Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors. Cell 170:457-469.e13
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
Stadtmueller, Beth M; Yang, Zhongyu; Huey-Tubman, Kathryn E et al. (2016) Biophysical and Biochemical Characterization of Avian Secretory Component Provides Structural Insights into the Evolution of the Polymeric Ig Receptor. J Immunol 197:1408-14
Yang, Zhongyu; Bridges, Michael D; López, Carlos J et al. (2016) A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement. J Magn Reson 269:50-54

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