We wish to understand the molecular basis of visual transduction. In particular, we intend to study (1) the key step in light to chemical energy conversion, the rhodopsin to bathorhodopsin transition, in two separate projects, (2) how the allosteric ligands, GDP and GTP, interact with transducin to better understand the chemical cascade reaction leading to cell hyperpolarization; and (3) the binding properties of retinoids to a class of retinoid binding proteins, some of which are involved in the visual cycle. In order to accomplish this goal, we propose several experiments employing state-of-the-art spectroscopic techniques which yield very detailed molecular information. Those used here are resonance Raman spectroscopy and quite novel non-resonance Raman ultra-sensitive difference spectroscopy as well as ultra-fast visible absorption and infrared kinetic spectroscopies. Four projects are proposed. We shall obtain the resonance Raman spectra of octopus rhodopsin and bathorhodopsin and isotopically labelled derivatives. This will be obtained in order to see if the molecular concepts developed for the well studied bovine system can be generalized to different species. Second, the kinetic steps involved in the rhodopsin to bathorhodopsin transition and aspects of the analogous primary process in bacteriorhodopsin, the bR568 to K photoreaction, will be studied by transient absorption spectroscopy (having a resolution of 0.1 ps) and transient infrared absorption spectroscopy (resolution of 0.5 to 1.0 ps). Thus, we shall be able to examine the kinetic pathway associated with the primary event of the chromophore's electronic changes as well as the vibrational steps of both the chromophore and the apoprotein . Thirdly, we shall obtain the Raman spectrum of GDP and GTP when bound to transducin, the G-protein of vision. The exchange of GDP for GTP at transducin's nucleotide binding pocket, which is catalyzed by excited rhodopsin, transforms transducin into an enzymatically active protein and is the first step in amplification of the light absorption event. In the fourth project, we shall employ pre-resonance Raman spectroscopy to study the binding of retinoids to three proteins (serum retinol binding protein, cytosolic retinol binding protein, and cellular retinaldehyde binding protein) which are specifically designed to bind these insoluble ligands. It is often suggested that the various retinoids are shuttled back and forth between the pigment epithelium and the photoreceptor cells by these type of soluble proteins but little is known how they bind the ligands.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY003142-15
Application #
3257404
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1979-07-01
Project End
1996-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
15
Fiscal Year
1993
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
Huang, L; Deng, H; Koutalos, Y et al. (1997) A resonance Raman study of the C=C stretch modes in bovine and octopus visual pigments with isotopically labeled retinal chromophores. Photochem Photobiol 66:747-54
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
Callender, R; Deng, H (1994) Nonresonance Raman difference spectroscopy: a general probe of protein structure, ligand binding, enzymatic catalysis, and the structures of other biomacromolecules. Annu Rev Biophys Biomol Struct 23:215-45
Weng, G; Chen, C X; Balogh-Nair, V et al. (1994) Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides. Protein Sci 3:22-9
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
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
Yan, M; Manor, D; Weng, G et al. (1991) Ultrafast spectroscopy of the visual pigment rhodopsin. Proc Natl Acad Sci U S A 88:9809-12

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