Abstract

The focus of this proposal is on the structure and function of the visual pigment rhodopsin. There are three Specific Aims: 1. To use disulfide cross-linking of rhodopsin to map tertiary contacts in the protein in both the active and inactive states. These studies will foster a better understanding of the structure of the protein and how the structure changes after exposure to light. As part of Specific Aim I the biosynthesis of split rhodopsin mutants will be explored in transfected COS cells. The formation of rhodopsin dimers and in vitro re-folding of the denatured protein will also be investigated. 2. To use a recently developed assay for transphosphorylation of rhodopsin to determine if phosphorylation of dark rhodopsin by rhodopsin kinase is a viable mechanism for the phenomenon of high-gain phosphorylation. 3. To develop a high-affinity, highly specific inhibitor of constitutively active mutants of rhodopsin found in patients with autosomal dominant retinitis pigmentosa.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY007965-11
Application #
2758434
Study Section
Special Emphasis Panel (ZRG1-VISB (07))
Project Start
1988-12-01
Project End
2003-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
11
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
Brandeis University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454

  Publications

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

Showing the most recent 10 out of 37 publications