The long term goal of these studies is to unravel the molecular mechanism of action of the visual pigment rhodopsin. This work will lead to the design of specific, active-site directed, and/or mechanism-based reagents aimed at treatment of diseases such as retinitis pigmentosa and congenital stationary night blindness. There are two specific aims in this proposal: (i) to identify and elucidate the role of essential amino acid residues in rhodopsin, and (ii) to use this information to design active-site directed reagents that target mutant rhodopsins. The experimental approach will be to use site-directed mutagenesis combined with extensive in vitro functional analysis and chemical modification of the altered rhodopsins. Mutagenesis will be used to identify essential amino acids in the activation and inactivation of rhodopsin and opsin. Crosslinking studies will be used to determine how close together are essential residues in the active site. Transducin activity assays will be performed to determine how closely the activated mutants resemble photoactivated rhodopsin. Finally, the increased understanding of mechanism in the wild-type and mutant rhodopsins will be used to design active-site directed reagents based on retinal analogs that specifically target mutant rhodopsins found in patients with retinitis pigmentosa and congenital stationary night blindness.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY007965-10
Application #
2608612
Study Section
Visual Sciences C Study Section (VISC)
Project Start
1988-12-01
Project End
1998-11-30
Budget Start
1997-12-01
Budget End
1998-11-30
Support Year
10
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Brandeis University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
Trieu, Melissa M; Devine, Erin L; Lamarche, Lindsey B et al. (2017) Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus Blastocladiella emersonii. J Biol Chem 292:10379-10389
Kumar, Ramasamy P; Morehouse, Benjamin R; Fofana, Josiane et al. (2017) Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC. J Biol Chem 292:21578-21589
Lamarche, Lindsey B; Kumar, Ramasamy P; Trieu, Melissa M et al. (2017) Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta. Biochemistry 56:5812-5822
Chakrabarti, Kalyan S; Agafonov, Roman V; Pontiggia, Francesco et al. (2016) Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis. Cell Rep 14:32-42
Devine, Erin L; Theobald, Douglas L; Oprian, Daniel D (2016) Relocating the Active-Site Lysine in Rhodopsin: 2. Evolutionary Intermediates. Biochemistry 55:4864-70
Kumar, Ramasamy P; Ranaghan, Matthew J; Ganjei, Allen Y et al. (2015) Crystal Structure of Recoverin with Calcium Ions Bound to Both Functional EF Hands. Biochemistry 54:7222-8
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, Jörg; Edwards, Patricia C; D'Antona, Aaron et al. (2011) The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471:656-60

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