We wish to understand the molecular basis of visual transduction. In particular, we intend to study in depth (1) the key light to chemical energy conversion step, the rhodopsin to bathorhodopsin transition, and (2) the first steps in the transducin chemical cascade reaction leading to rod cell hyperpolarization which is catalyzed by excited rhodopsin. In order to accomplish these goals, we propose here several experiments employing state-of- the-art spectroscopic techniques which yield very detailed molecular information. Resonance Raman and very fast (subpicosecond and picosecond) absorption and fluorescence techniques will be used to study the rhodopsin to bathorhodopsin photoreaction. The resonance Raman spectra of octopus rhodopsin and bathorhodopsin, and isotopically labelled derivatives, and the insect rhodopsin, Ascalaphus macaronius, will be obtained in order to see if the molecular concepts developed for the well studied bovine system can be generalized to different species. The rhodopsin to bathorhodopsin transition has not been kinetically resolved. Knowledge of whether or not proton translocation accompanies the rhodopsin to bathorhodopsin photoreaction and understanding the molecular dynamics of this step can only be obtained by kinetic measurements. We believe our 0.2 psec absorption spectrometer should be able to resolve this key step. The most novel work described in this proposal is the application of very sensitive classical Raman difference techniques, recently developed in our laboratory, to obtain the Raman spectra of GDP and GTP when bound to transducin, T. The exchange reaction T.GDP+GTP yields T.GTP+GDP, catalyzed by excited rhodopsin, is the first step in amplification of the light absorption event. The Raman spectra of bound GDP and GTP should provide a great deal of information on how the reaction takes place.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY003142-13
Application #
3257403
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1979-07-01
Project End
1992-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
13
Fiscal Year
1991
Total Cost
Indirect Cost
Name
City College of New York
Department
Type
Schools of Arts and Sciences
DUNS #
603503991
City
New York
State
NY
Country
United States
Zip Code
10031
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Huang, L; Deng, H; Weng, G et al. (1996) A resonance Raman study of the C=N configurations of octopus rhodopsin, bathorhodopsin, and isorhodopsin. Biochemistry 35:8504-10
Deng, H; Huang, L; Callender, R et al. (1994) Evidence for a bound water molecule next to the retinal Schiff base in bacteriorhodopsin and rhodopsin: a resonance Raman study of the Schiff base hydrogen/deuterium exchange. Biophys J 66:1129-36
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Manor, D; Callender, R; Noy, N (1993) The interactions of retinoids with retinol-binding protein. A resonance Raman spectroscopic study. Eur J Biochem 213:413-8
Manor, D; Weng, G Z; Deng, H et al. (1991) An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21. Biochemistry 30:10914-20
Deng, H; Manor, D; Weng, G et al. (1991) Resonance Raman studies of the HOOP modes in octopus bathorhodopsin with deuterium-labeled retinal chromophores. Biochemistry 30:4495-502
Deng, H; Manor, D; Weng, G et al. (1991) A resonance Raman study of octopus bathorhodopsin with deuterium labeled retinal chromophores. Photochem Photobiol 54:1001-7
Pande, J; McDermott, M J; Callender, R H et al. (1991) The calf gamma crystallins--a Raman spectroscopic study. Exp Eye Res 52:193-7

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