Information transmission in biological systems is ofter triggered by interactions between peptides and cells. We propose to investigate the molecular details of this peptide-cell interaction by studying the mechanism of action of the peptide sex pheromone (Alpha-factor) of Saccharomyces cerevisiae. Alpha-Factor labelled to high specific radioactivity will be synthesized and purified using high perrformance liquid chromatography. The radioactive Alpha-factor will be used to develop a binding assay for the interaction of this peptide with the Alpha receptor. Concurrently, fluorescently-labelled and biologically active Alpha-factor will be synthesized and used to develop a binding assay based on polarization of fluorescence. Binding competition studies will be carried out to determine the contribution of various residues of Alpha-factor to the free energy of binding and to relate binding of the pheromone to the physiological responses elicited in yeast cells. A detailed analysis of the conformation of Alpha-factor will be undertaken using circular dichroism and nuclear magnetic resonance spectroscopy. Studies will be conducted in aqueous buffers and in the presence of membrane lipids. In particular, two dimensional NMR techniques will be applied to the total assignment of the proton NMR spectrum and to the determination of internuclear distances. The conformational analyses will be aided by specifically Alpha-deuterated peptides and peptides containing fluorescent chromophores. Analyses will also be conducted on synthetic peptides which are rigidified to decrease their conformational flexibility. Alpha-Factor containing photoaffinity groups will be used to insert radioactive or fluorescent markers into the Alpha-factor receptor. The tagged receptor will be isolated and used to generate antibodies that can be used to follow the purification of the intact receptor. Receptor purification will utilize both classical and affinity chromatography. The latter experiments will employ Alpha-factor attached to soluble and insoluble macromolecules. These studies should increase our understanding of membrane receptors, in general, and may provide insights into the detailed manner by which peptide hormones are recognized by mammalian cells.
Showing the most recent 10 out of 85 publications
