The long-term objectives of this research are: (1) to develop reliable methods to determine structures of membrane protein surfaces that interact with signal transduction or adhesion partners when crystallography is not feasible, (2) to reveal the detailed molecular mechanisms of these interactions, and (3) to provide structural templates for structure-based design of pharmaceutical agents to block the interactions of interest. The present proposal aims to determine the structures of the aqueous surfaces of two G protein-coupled receptors, rhodopsin and the formyl peptide receptor, and to map the sites of interaction of the receptors with their G protein coupling partners. Relatively few membrane protein structures have been solved, since membrane proteins have been difficult to crystallize for x-ray diffraction and are also very challenging to approach by NMR. We use antibody imprints to ascertain the proximity relations and folding of regions of membrane protein surfaces. The peptide epitopes of antibodies to the surfaces of the proteins of interest are mapped, using random peptide libraries displayed on bacteriophage. Discontinuous epitopes show the proximity on the protein surface of two or more segments of the protein that are distant in the primary sequence. The detailed folding of these discontinuous epitopes is studied when bound to the antibody, using transferred-NOESY NMR--or when bound to the Fab domain, using x-ray diffraction analysis. In a second approach, we use photoaffinity labeling to map interacting regions between GPCR and G proteins. Peptides are selected from random libraries or from the sequences of the interacting proteins that block the protein-protein interactions of interest, and photoactivatable residues are scanned over the sequences. Active peptides are cross-linked to the target protein and the detailed cross-linking sites are determined by specific cleavage and mass spectrometry. We plan to time-resolve the interactions of flash-excited rhodopsin with its G protein partner by delayed flash-stimulated cross-linking. The accuracy of photocross-linking reagents will be evaluated using model peptide-protein complexes with known structures.
