Our long-term objectives are to understand the molecular mechanisms of visual transduction and adaptation. This proposal is focused on the molecular mechanism of rhodopsin action. After rhodopsin absorbs a photon it proceeds through a series of spectroscopically defined conformation states. One or more of these conformational states (R*) catalyzes the exchange of GTP for GDP in a disk associated G-protein resulting in the activation of the latter. Similar, if not identical , informational transducing steps occur as a consequences of the interaction of many hormones and drugs with their receptors. The proposal is largely concerned with defining the molecular changes in rhodopsin which enable activated rhodopsin R* to be reached. A major interest here is to establish a structural link between R* and the spectroscopically defined intermediates. This work will be of relevance both to our understanding of vision and the nature of drug-receptor interactions. The fact that Schiff base of rhodopsin can be protonated is of immense importance in the functioning of this protein. The protonated Schiff base of the chromophore, interacting with negatively charge amino acids at the active-site, is thought to be of primary importance in wavelength regulation and energization of rhodopsin. The ultimate deprotonation of the Schiff base is assumed also to be important in the formation of metarhodopsin II, the presumed spectroscopic signature of R*. Along these lines, the active-site lysine of opsin must also be deprotonated to form a Schiff base with 11-cis-retinal. The experiments described here are designed to prove the role of charge and charge movement in the functioning of rhodopsin and, to a much lesser extent, bacteriorhodopsin. The approaches used here are bio-organic chemical and biochemical in nature and will involve modification of the protein along with retinal analog studies. By introducing chemical probes, such as a methyl group, at the active-site lysine we will determine if a full positive charge on this lysine is required for photochemical energy storage, wavelength regulation and the formation of R*. Furthermore the pK of the active-site lysine of rhodopsin will be determined. The amino acid counterions which critically interact with the chromophore will be identified by structural studies designed to use the active-site lysine of opsin to direct a pseudo cross-linking reagent to these counterions. Finally, novel retinal analogs which form pigments with opsin will be used to probe the mechanism of energy storage.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY003624-10
Application #
3258024
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1980-12-01
Project End
1991-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
10
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Mondal, M S; Ruiz, A; Hu, J et al. (2001) Two histidine residues are essential for catalysis by lecithin retinol acyl transferase. FEBS Lett 489:14-8
Mondal, M S; Ruiz, A; Bok, D et al. (2000) Lecithin retinol acyltransferase contains cysteine residues essential for catalysis. Biochemistry 39:5215-20
Parish, C A; Rando, R R (2000) Isoprenylation/methylation and transducin function. Methods Enzymol 316:451-64
Tok, J B; Cho, J; Rando, R R (1999) Aminoglycoside antibiotics are able to specifically bind the 5'-untranslated region of thymidylate synthase messenger RNA. Biochemistry 38:199-206
Hamasaki, K; Killian, J; Cho, J et al. (1998) Minimal RNA constructs that specifically bind aminoglycoside antibiotics with high affinities. Biochemistry 37:656-63
Perez-Sala, D; Gilbert, B A; Rando, R R et al. (1998) Analogs of farnesylcysteine induce apoptosis in HL-60 cells. FEBS Lett 426:319-24
Cho, J; Hamasaki, K; Rando, R R (1998) The binding site of a specific aminoglycoside binding RNA molecule. Biochemistry 37:4985-92
Hamasaki, K; Rando, R R (1998) A high-throughput fluorescence screen to monitor the specific binding of antagonists to RNA targets. Anal Biochem 261:183-90
Wang, Y; Hamasaki, K; Rando, R R (1997) Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region. Biochemistry 36:768-79
Hamasaki, K; Rando, R R (1997) Specific binding of aminoglycosides to a human rRNA construct based on a DNA polymorphism which causes aminoglycoside-induced deafness. Biochemistry 36:12323-8

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