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.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Neuhold, Lisa
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Brandeis University
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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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