Structural basis of rhodopsin/G protein coupling: Biochemical activity of peptide complexes, photo cross-linking, and mass spectrometric analysis

Physiological and sensory mechanisms are primarily controlled by membrane impermeant signals-which are usually detected by membrane-spanning receptor proteins that ultimately trigger intracellular responses. Similarly, development and repair in multi-cellular organisms are controlled by specific adhesion mechanisms that recognize macromolecular binding sites outside cells and control cytoplasmic responses. Understanding of signaling and adhesion mechanisms could be greatly advanced if the structures of the membrane-spanning receptor proteins and their non-covalent interaction partners could be determined at atomic resolution—but these structures can not typically be deduced by x-ray crystallography or NMR.; Mass spectrometry is able to mass analyze high molecular weight species, however, non-covalent protein-ligand or protein-protein interactions are disrupted by the non-aqueous conditions used in conventional mass spectrometry. This thesis describes new mass spectometric methodologies to analyze proteins directly from aqueous solutions, which is an important first step in mass spectrometry of non-covalent protein-ligand complexes. Insight is gained into fundamental mechanisms of laser desorption ionization mass spectrometry from aqueous solutions.; This thesis also describes biochemical and mass spectrometric methods to identify non-covalent interactions between the rhodopsin visual receptor and the G protein, transducin. The contacts between rhodopsin and transducin were studied using synthetic peptides derived from transducin that were characterized by their ability to inhibit rhodopsin catalysis of nucleotide binding by transducin. The peptides were also characterized for their ability to inhibit the homologous formyl peptide receptor's catalysis of nucleotide binding by the Gi protein.; Fluorescent photo-activatable analogs of the most potent peptides were synthesized. Non-covalent rhodopsin-peptide interactions were tested for specificity and covalently stabilized by photo-chemical cross-linking. Rhodopsin-peptide complexes (which are about 42,000 Da) must be cleaved into smaller peptides for mass spectrometric analysis of the amino acid sites cross-linked. Cyanogen bromide cleavage of rhodopsin reduced the size of the hydrophobic fragments to facilitate analysis. Methods are described for mass spectral analysis of all of the cyanogens bromide fragments of rhodopsin at the picomole level, which is an essential first step in identifying the peptide cross-linking sites in rhodopsin. These results represent significant progress in efforts to define the molecular contacts between rhodopsin and transducin and homologous proteins.