Peptide hormones are involved in numerous biological processes including growth, reproduction, basal metabolism, memory, and pain perception. We propose to investigate the interaction of peptides with cells using the Saccharomyces cerevisiae mating factors (alpha-factor and a-factor) as model peptide hormones. The alpha-factor (WHWLQLKPGQPMY) is a simple tridecapeptide whereas the a-factor (YIIKGVFWDPAC[farnesyl]OMe) is post- translationally modified and representative of a newly discovered class of isoprenylated lipopeptides which serve an important role in both signal transduction and growth regulation. The conformation of the mating factors favored for receptor binding will be examined by synthesizing rigidified and constrained analogs and investigating their biological and spectroscopic properties. Spectroscopic analyses will be carried out using NMR and CD on organic, organic/aqueous and lipid-containing solutions and by application of rotational-echo double-resonance (REDOR) spectroscopy on solid or solid-like samples. The REDOR analyses are aimed at developing procedures which will eventually be used to directly study the conformation of receptor-bound pheromones. Photoactivatable alpha- factor analogs will be cross-linked into the alpha-factor receptor to map the specific residues which bind this mating factor. Concurrently, molecular biological approaches will be used to generate mutations in the alpha-factor and the a-factor receptors that cause them to respond to weak agonists and antagonists synthesized in our laboratory. The mutant genes will be sequenced to reveal information concerning regions of these receptor proteins involved in ligand binding and signal transduction. The a-factor will be investigated using biophysical approaches such as microcalorimetry, fluorescence spectroscopy and neutron diffraction to learn about the molecular interactions of isoprenylated peptides with membranes. The role of a-factor degradation in the mating response pathway will be examined by cloning the gene encoding a-factorase, an alpha-cell specific protease. Overall, these studies will provide insights into receptor recognition and membrane biophysics of peptides and lipopeptides involved in signal transduction and cell-cell communication.
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