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Biosynthesis and biophysical analysis of domains of a yeast G protein‐coupled receptor
Author(s) -
Arevalo Enrique,
Estephan Racha,
Madeo Jenifer,
Arshava Boris,
Dumont Mark,
Becker Jeffrey M.,
Naider Fred
Publication year - 2003
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.10491
Subject(s) - chemistry , transmembrane domain , transmembrane protein , g protein coupled receptor , saccharomyces cerevisiae , biochemistry , membrane protein , yeast , peptide , receptor , chromatography , biophysics , membrane , biology
The α‐factor receptor(Ste2p) is required for the sexual conjugation of the yeast Saccharomyces cerevisiae. Ste2p belongs to the G protein‐coupled receptor (GPCR) family sharing a common heptahelical transmembrane structure. Biological synthesis of regions of Ste2p fused to a leader protein in Escherichia coli yielded milligram quantities of polypeptides that corresponded to one or two transmembrane domains. Fusion proteins were characterized by polyacrylamide gel electrophoresis, high performance liquid chromatography, and mass spectrometry. CD studies on the fusion proteins in trifluoroethanol:water mixtures indicated the existence of α‐helical structures in the single‐ and the double‐transmembrane domains. NMR experiments were performed in CDCl 3 :CD 3 OH:H 2 O (4:4:1) on the 15 N‐labeled single‐transmembrane peptide showing a clear dispersion of the nitrogen–amide proton correlation cross peaks indicative of a high‐purity, uniformly labeled molecule. The results indicate that single‐ and double‐transmembrane domains of a GPCR can be produced by biosynthetic methods in quantities and purity sufficient for biophysical studies. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 516–531, 2003