Biosynthesis and biophysical analysis of domains of a yeast G protein-coupled receptor.
The alpha-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 alpha-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.
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins; D019204:GTP-Binding Proteins / Q000737:chemistry*; D005822:Genetic Vectors; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000737:chemistry*; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry; D014157:Transcription Factors
D005656:Fungal Proteins; D010455:Peptides; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D019204:GTP-Binding Proteins; D009584:Nitrogen