The process of visual excitation requires the specific interaction of several different proteins species. The goal of the proposed research is to determine the 3D (atomic) structural basis of the interactions between the proteins active in visual excitation in order to understand the underlying molecular mechanisms. Synthetic peptide (that are segments of the interacting proteins) are selected for their ability to mimic or block their parent proteins in the protein-protein interactions of interest. The 3D atomic structures of the active peptides, bound to their receptor sites, are determined using high field 2D and 3D liquids nuclear magnetic resonance (NMR) and rotational resonance solid state NMR. The interfacial bound peptide structures are developed using simulated annealing and grid searcher and structures are refined using making amino acid substitution in the synthetic peptides that stabilize or destabilize structural features. Peptide mimetics, with conformations locked into their active shapes, are designed and synthesized in collaboration and tested for activity. The principal providing ground for this approach is the interface between the G protein transduction and rhodopsin. Three transducin alpha subunit peptides (8-23, 311-329,340-350) make up the principal contract interface with light-activated metarhodopsin II (MII) and act to block the MII-G protein complex or the peptides themselves form a complex that stabilizes MII. Three rhodopsin peptides (142-153-231-252,310-321)on the cytoplasmic surface make up the principal contact interfaces with transduction. Initial studies have been carried out in our laboratories and the work proposed promises to reveal the 3D atomic structure of the active MII-transducing interface in the native system and in site- specific mutants that are constitutively active or that bind but do not activate. PDE-transducing and other important protein-protein interfaces will also be studied. The proposed research has broad health implication. The approach appears to be quite general and structural mimetic of active interfacial peptides will specifically modulates steps in the processes of interest. The conformationally locked structure mimetic promise to serve as leads for the discovery of specific new drugs to selectively intervene in biological excitation, regulation, and adhesion processes.
