Synthesis and biophysical analysis of transmembrane domains of a Saccharomyces cerevisiae G protein-coupled receptor

The Ste2p receptor for alpha -factor, a tridecapeptide mating pheromone of the yeast Saccharomyces cerevisiae, belongs to the G protein-coupled family of receptors. In this paper we report on the synthesis of peptides corresp onding to five of the seven transmembrane domains (M1-M5) and two homologue s of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor. The secon dary structures of all new transmembrane peptides and previously synthesize d peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium do decyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers. Tryp tophan fluorescence quenching experiments were used to assess the penetrati on of the membrane peptides into lipid bilayers. All peptides were predomin antly (40-80%) helical in trifluoroethanol and most trifluoroethanol-water mixtures. In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVI FTGDNFKKKG) and M6-31 (KQFDSFHILLIN-leSAQSLLVPSIIFILAYSLK) formed stable be ta -sheet structures in both sodium dodecyl sulfate micelles and DMPC bilay ers. Polyacrylamide gel electrophoresis showed that these two peptides form ed high molecular aggregates in the presence of SDS whereas all other pepti des moved as monomeric species. The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYS LKKKS) corresponding to the sequence in the constitutive mutant was predomi nantly helical under a variety of conditions, whereas the homologous wild-t ype sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to f orm beta -structures. These results demonstrate a connection between a conf ormational shift in secondary structure, as detected by biophysical techniq ues, and receptor function. The aggregation of particular transmembrane dom ains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or m ultimers.