The relationship of rhodopsin structure to its functions is to be studied in several ways. The primary sequence of all of rhodopsin's cyanogen bromide peptides is to be completed. Complementary methionine-containing peptides are to be produced and sequenced so that the cyanogen bromide peptides may be placed in a unique order yielding the complete primary sequence of the protein. Membrane-buried helices in the sequence will be predicted using theoretical calculations and experimental data. Tentative tertiary structures will be proposed and tested by model building and further experimentation. Proposed buried carboxyl groups in rhodopsin's retinal-binding pocket will be sought using a hydrophobic carbodiimide. The conformation of rhodopsin's retinyl-binding region will be explored by making spectral measurements on synthetic peptides in vesicles and in hydrocarbon solution. Which of rhodopsin's cysteines are free and which are in disulfide linkage will be determined. Regions of rhodopsin which undergo light-dependent conformational change will be identified by assignment of cysteines which become reactive following bleaching. Posttranslational modifications (acylation, phosphorylation) will be studied. Does rhodopsin, like many other membrane proteins, become acylated with palmitic acid? Recently discovered additional sites of rhodopsin phosphorylation will be identified in the protein sequence. In order to explore structural requirements for phosphorylation, peptides containing rhodopsin's sites of phosphorylation (and their analogs) will be synthesized and tested. Tolerated structural variations in rhodopsin will be examined by phylogenetic studies with site-specific monoclonal antibodies. Such antibodies will be raised against rhodopsin and synthetic peptides from its sequence and characterized for structural specificity.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY006226-02
Application #
3262291
Study Section
(SSS)
Project Start
1985-06-16
Project End
1988-01-31
Budget Start
1986-02-01
Budget End
1987-01-31
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Florida
Department
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611