The goal of the proposed research is to gain an understanding of the molecular basis of visual excitation in vertebrate retinal rod cells. Phototransduction is thought to be mediated by transient decrease of cGMP level on rod outer segments (ROS). Central to this process is a light-activated enzyme cascade which involves the rhodopsin molecule, a regulatory GTP binding protein called transducin and a cGMP phosphodiesterase (PDE). Subunits of transducin and PDE have been purified and functionally reconstituted. Thus provides us an opportunity to study the activation mechanism on molecular level. The structure and function of these subunits, their interaction and regulation in the phototransducin process will be investigated by biochemical and spectroscopic techniques. I propose to carry out the following studies: (1) The role of sulfhydryl groups in the activation of transducin by photolyzed rhodopsin will be studied by chemical modification. Possible involvement of glutathione in the regulation of visual excitation will be examined. (2) Chemical modification on specific sites of rhodopsin, transducin and PDE will be carried out to probe the location and amino acid residues involved in various functional sites. (3) Interaction and relative proximity of components of the cGMP cascade in ROS will be investigated by chemical cross-linking studies using bifunctional reagents and by photoaffinity cross-linking using azido-derivatized transducin and PDE subunits. (4) Fluorescence energy transfer will be used to map the architecture of the T Alpha subunit of transducin. Distances between GTP binding site, the specific sulfhydryl group, the cholera and pertussis toxins ADP-ribosylation sites will be measured. Conformational changes of the T Alpha subunit associated with the binding of GTP, ADP-ribosylation, and chemical modification will be investigated by circular dichorism (CD). It is anticipated that this project will advance our knowledge in understanding the molecular mechanism of visual excitation.
