Our objective is to understand the visual transduction process in terms of molecular structure of the proteins involved, specifically rhodopsin in this proposal.Understanding of this system is of major significance to the understanding of vision and visual diseases and the design of new therapies. The system is also of fundamental biological significance as the prototype of a family of G-protein coupled receptor systems. We propose to study the structure of rhodopsin and its conformational changes during phototransduction by use of photoaffinity and other chemical labeling approaches in conjunction with tandem mass spectrometry. Photoactivatable retinal analogs will be incorporated into bovine opsin. The reconstituted pigments will be characterized spectrophotometrically and functionally prior to photochemical labeling in the dark adapted, meta I, and meta II states using both continuous irradiation and flash photo-lysis. The protein will then be cleaved and the resulting labeled peptides structurally characterized by tandem mass spectrometry. To cleave the hydrophobic protein without destroying labile photochemically introduced covalent bonds, we will develop new methods for proteolytic cleavage in nonaqueous solvents. Identification of the labeled sites in the protein will show how retinal moves relative to the protein in the photoisomerization process. The conformational changes which enable G-protein binding will be explored by probing the structure of the cytosolic domain of rhodopsin using water soluble photochemical labels and heterobifunctional crosslinking reagents. Salt bridges will be examined using cyanogen induced covalent bonding. We will attempt to observe intermediate conformational states using time resolved photochemical labeling. Flash photo-isomerization at long wavelength will be followed by precisely timed flash photolysis at shorter wavelength in efforts to trap intermediate conformers. These studies will yield distance constraints on the protein structure which will be used in conjunction with distance constraints on the protein structure which will be used in conjunction with computer molecular graphics to develop models or rhodopsin in the dark adapted, meta I, and meta II states. The models will reveal conformational changes which occur in rhodopsin upon light reception to enable G protein binding and the initiation of the photoreception cascade. These studies will increase our understanding of the visual transduction process and the general process of transduction by G-protein coupled receptors.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY008239-02
Application #
3265462
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1989-08-01
Project End
1994-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
2
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Medical University of South Carolina
Department
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
Ablonczy, Zsolt; Kono, Masahiro; Knapp, Daniel R et al. (2006) Palmitylation of cone opsins. Vision Res 46:4493-501
Ablonczy, Zsolt; Crouch, Rosalie K; Knapp, Daniel R (2005) Mass spectrometric analysis of integral membrane proteins at the subpicomolar level: application to rhodopsin. J Chromatogr B Analyt Technol Biomed Life Sci 825:169-75
Ablonczy, Z; Darrow, R M; Knapp, D R et al. (2005) Rhodopsin phosphorylation in rats exposed to intense light. Photochem Photobiol 81:541-7
Wang, Xin; Kim, Sung-Ho; Ablonczy, Zsolt et al. (2004) Probing rhodopsin-transducin interactions by surface modification and mass spectrometry. Biochemistry 43:11153-62
Rohrer, Baerbel; Ablonczy, Zsolt; Znoiko, Sergei et al. (2003) Does constitutive phosphorylation protect against photoreceptor degeneration in Rpe65-/- mice? Adv Exp Med Biol 533:221-7
Knapp, Daniel R; Crouch, Rosalie K; Ball, Lauren E et al. (2002) Mass spectrometric analysis of G protein-coupled receptors. Methods Enzymol 343:157-61
Ablonczy, Zsolt; Crouch, Rosalie K; Goletz, Patrice W et al. (2002) 11-cis-retinal reduces constitutive opsin phosphorylation and improves quantum catch in retinoid-deficient mouse rod photoreceptors. J Biol Chem 277:40491-8
Ablonczy, Zsolt; Goletz, Patrice; Knapp, Daniel R et al. (2002) Mass spectrometric analysis of porcine rhodopsin. Photochem Photobiol 75:316-21
Ablonczy, Z; Kono, M; Crouch, R K et al. (2001) Mass spectrometric analysis of integral membrane proteins at the subnanomolar level: application to recombinant photopigments. Anal Chem 73:4774-9
Gelasco, A; Crouch, R K; Knapp, D R (2000) Intrahelical arrangement in the integral membrane protein rhodopsin investigated by site-specific chemical cleavage and mass spectrometry. Biochemistry 39:4907-14

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