Rhodopsin, in the dim light vertebrate photoreceptor, is the prototypic and best-studied member of the largest known family of cell surface receptors, the G-protein coupled receptors (GPCRs). Like rhodopsin, these receptors all contain a seven helical transmembrane (TM) domain, a cytoplasmic domain, where all protein-protein interactions occur in signal transduction and an extracellular (intradiscal in rhodopsin) domain. Recently, the dark state structure of rhodopsin has been solved at atomic resolution. This sharpens focus on understanding the precise structural changes that occur in rhodopsin from dark to its activated state. This is important for molecular understanding of visual transduction by rhodopsin and for understanding the corresponding mechanisms in activation of GPCR.
The aim of all the work herein proposed is to understand the conformational changes in the TM and the cytoplasmic domains that occur from the dark to the activated state of rhodopsin. Three experimental approaches are proposed. In the first, single cysteine substitution mutants of every one of the amino acids in the cytoplasmic and TM domains will be prepared and their reactivities to sulfhydryl reagents will be compared in the dark and after illumination. This comprehensive mapping of the changes in accessibilities of the cysteines will define the conformational changes between the dark and the activated states. In the second approach, constitutively active mutants of rhodopsin that can bind both 11-cis- and all-trans-retinal and are active in the dark will be studied for the changes in their conformations from WT rhodopsin. These studies will involve both cysteine scanning and disulfide crosslinking to deduce proximities between different amino acids in their tertiary structures. The third approach will involve crystallization of a triple mutant of rhodopsin that combines three constitutive mutations, is stably bound to all-trans-retinal and displays transducin activation at least at the same level as WT rhodopsin.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY011716-05
Application #
6693753
Study Section
Special Emphasis Panel (ZRG1-VISC (01))
Program Officer
Dudley, Peter A
Project Start
1998-01-01
Project End
2007-11-30
Budget Start
2003-12-01
Budget End
2004-11-30
Support Year
5
Fiscal Year
2004
Total Cost
$289,625
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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