Synthesis and Biophysical Analysis of Transmembrane Domains of a Saccharomyces cerevisiae G Protein-Coupled Receptor

Abstract

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 corresponding to five of the seven transmembrane domains (M1-M5) and two homologues of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor. The secondary structures of all new transmembrane peptides and previously synthesized peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers. All peptides were predominantly (40-80%) helical in trifluoroethanol and most trifluoroethanol-water mixtures. In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31 (KQFDSFHILLINleSAQSLLVPSIIFILAYSLK) formed stable beta-sheet structures in both sodium dodecyl sulfate micelles and DMPC bilayers. Polyacrylamide gel electrophoresis showed that these two peptides formed high molecular aggregates in the presence of SDS whereas all other peptides moved as monomeric species. The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence in the constitutive mutant was predominantly helical under a variety of conditions, whereas the homologous wild-type sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form beta-structures. These results demonstrate a connection between a conformational shift in secondary structure, as detected by biophysical techniques, and receptor function. The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers.

The Chain Length Dependence of Helix Formation of the Second Transmembrane Domain of a G Protein-coupled Receptor ofSaccharomyces cerevisiae

“…The sequence of the 8 amino acids of the N terminus was confirmed by 1 H NMR spectroscopy. The longest peptide with 35 residues had previously been confirmed by amino acid analysis (19). The peptide synthesized in this study was identical with the authentic sample as judged by coinjection on analytical HPLC.…”

“…This peptide includes the entire transmembrane hydrophobic region plus 6 extracellular and 5 cytoplasmic residues (19). The complete peptide with the wild-type sequence was synthesized previously and found to form a highly ␣-helical secondary structure in aqueous TFE, SDS micelles, DMPC vesicles, and multilayers (19,20).…”

“…We synthesized seven peptide fragments with 10, 14, 18, 22, 26, 30, and 35 residues, respectively, starting from the carboxyl-terminal serine (Ser 108 ). These peptide fragments were named M2-10, M2-14, M2-18, M2-22, M2-26, M2-30, and M2-35, respectively, in which M2 represents the second transmembrane domain and the number following M2-represents the chain length of these peptide fragments (19). The peptides were prepared using solid-phase peptide chemistry.…”

“…This G protein-coupled receptor recognizes the tridecapeptide phero-mone (WHWLQLKPGQPMY-␣-factor) and triggers a cascade of intracellular events ultimately resulting in sexual conjugation and diploid formation (16,17). Studies on fragments of the receptor using CD (18,19), IR (20), and NMR spectroscopies (21,22) have revealed the conformational tendencies of the seven transmembrane domains and, in the case of IR analysis, gave information on the orientation of the peptides in lipid bilayers.…”

Oligomerization, Biogenesis, and Signaling Is Promoted by a Glycophorin A-like Dimerization Motif in Transmembrane Domain 1 of a Yeast G Protein-coupled Receptor

“…By modeling TM1 as an ␣-helix (37), consistent with structural analyses of TM1 peptides (38,39), we compared the potential structure of this motif with that of the glycophorin A transmembrane domain (Fig. 1A) determined by NMR (40,41).…”