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

Haibo Xie,^‡ Fa-Xiang Ding,^‡ David Schreiber,^‡ Gary Eng,^‡ Shi-feng Liu,^‡^§ Boris Arshava,^‡ Enrique Arevalo, Jeffrey M. Becker, and Fred Naider*^‡

Department of Chemistry and Biochemistry, The College of Staten Island and The Graduate School of The City University of New York, Staten Island, New York 10314, and Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996

Biochemistry, 2000, 39 (50), pp 15462–15474

DOI: 10.1021/bi001432p

Publication Date (Web): November 16, 2000

†

This work was supported by research grants GM22086 and GM22087 from the National Institute of Health and by a grant from the PSC−CUNY Research Award Program. F.N. is currently Varon Visiting Professor at the Weizmann Institute of Science Rehovot.

‡

Department of Chemistry, The College of Staten Island and the Graduate School of The City University of New York.

Current address: Department of Chemistry, University of Waterloo, Ketchener, Ontario N2M 5G4, Canada.

Department of Biochemistry, The Graduate School of The City University of New York.

University of Tennessee.

Correspondence should be addressed to this author. Telephone: (718) 982-3896. Fax: (718) 982-3910. E-mail: naider@postbox.csi.cuny.edu.

Abstract

The Ste2p receptor for α-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 β-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 β-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.

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