The long-term goal of this work is to elucidate the molecular mechanism of phototransduction in the vertebrate visual pigment rhodopsin. The studies are geared toward an understanding of the protein in terms of its mechanism of activation, its interaction with downstream proteins of the phototransduction cascade, and its function and dysfunction in health and disease. The proposal is based upon strong progress in the previous grant period where an x-ray crystal structure was determined for a rhodopsin mutant isolated from transfected monkey kidney cells in culture. That achievement is significant because it was the first instance in which an x-ray crystal structure was determined for a recombinant G protein-coupled receptor, and as such provides a proof-of-principle study for crystallographic studies of other, more interesting, rhodopsin mutants. There are three Specific Aims: 1. To determine the 3-dimensional structure of the constitutively active rhodopsin mutant E113Q in both the inactive (11-cis-retinal) and active (all-trans-retinal) states using x-ray crystallography. We currently have crystals of active-state E113Q which diffract to 10? resolution; 2. To prepare, biochemically characterize, and investigate the molecular mechanism of formation and decay of an activated complex of rhodopsin and its downstream G protein transducin.
This Aim i s based upon preliminary results in which we have prepared and purified large quantities of the activated complex using recombinant rhodopsin. The studies will exploit the large number of functional mutants that exist for rhodopsin. 3. To prepare, biochemically characterize, and investigate the molecular mechanism of formation and decay of an activated complex of rhodopsin and rhodopsin kinase.
This Aim i s based upon preliminary results in which we have prepared and purified an activated complex of rhodopsin and rhodopsin kinase, as well as a complex of rhodopsin and the RGS domain of the kinase. As with Aim 2, Aim 3 exploits the use of rhodopsin mutants in preparation of the complex and investigation of its formation and decay.

Public Health Relevance

This study will have an impact on our fundamental understanding of the molecular mechanism of vertebrate phototransduction. Specifically, the studies are geared toward elucidation of the atomic changes undergone by the visual pigment rhodopsin upon activation by light, and upon binding of the activated rhodopsin to downstream signal transduction partners in the visual response.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY007965-24
Application #
8389554
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Neuhold, Lisa
Project Start
1988-12-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2014-11-30
Support Year
24
Fiscal Year
2013
Total Cost
$360,240
Indirect Cost
$132,240
Name
Brandeis University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
D'Antona, Aaron M; Xie, Guifu; Sligar, Stephen G et al. (2014) Assembly of an activated rhodopsin-transducin complex in nanoscale lipid bilayers. Biochemistry 53:127-34
Devine, Erin L; Oprian, Daniel D; Theobald, Douglas L (2013) Relocating the active-site lysine in rhodopsin and implications for evolution of retinylidene proteins. Proc Natl Acad Sci U S A 110:13351-5
Deupi, Xavier; Edwards, Patricia; Singhal, Ankita et al. (2012) Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II. Proc Natl Acad Sci U S A 109:119-24
Standfuss, Jorg; Edwards, Patricia C; D'Antona, Aaron et al. (2011) The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471:656-60
Xie, Guifu; D'Antona, Aaron M; Edwards, Patricia C et al. (2011) Preparation of an activated rhodopsin/transducin complex using a constitutively active mutant of rhodopsin. Biochemistry 50:10399-407
Standfuss, Jorg; Xie, Guifu; Edwards, Patricia C et al. (2007) Crystal structure of a thermally stable rhodopsin mutant. J Mol Biol 372:1179-88
Bayburt, Timothy H; Leitz, Andrew J; Xie, Guifu et al. (2007) Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins. J Biol Chem 282:14875-81
Tam, Beatrice M; Xie, Guifu; Oprian, Daniel D et al. (2006) Mislocalized rhodopsin does not require activation to cause retinal degeneration and neurite outgrowth in Xenopus laevis. J Neurosci 26:203-9
Xie, Guifu; Gross, Alecia K; Oprian, Daniel D (2003) An opsin mutant with increased thermal stability. Biochemistry 42:1995-2001
Jin, Shengnan; McKee, Timothy D; Oprian, Daniel D (2003) An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis. FEBS Lett 542:142-6

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