Structure of segments of a G protein-coupled receptor: CD and NMR analysis of the Saccharomyces cerevisiae tridecapeptide pheromone receptor.
Peptides representing both loop and the sixth transmembrane regions of the alpha-factor receptor of Saccharomyces cerevisiae were synthesized by solid-phase procedures and purified to near homogeneity. CD, nmr, and modeling analysis indicated that in aqueous media the first extracellular loop peptide E1(107-125), the third intracellular loop peptide I3(231-243), and the carboxyl terminus peptide I4(350-372) were mostly disordered. In contrast, the second extracellular loop peptide E2(191-206) assumed a well-defined structure in aqueous medium and the sixth transmembrane domain peptide receptor M6(252-269, C252A) was highly helical in trifluoroethanol/water (4:1), exhibiting a kink at Pro258. A synthetic peptide containing a sequence similar to that of the sixth transmembrane domain of a constitutively active alpha-factor receptor M6(252-269, C252A, P258L) in which Leu replaces Pro258 exhibited significantly different biophysical properties than the wild-type sequence. In particular, this peptide had very low solubility and gave CD resembling that of a beta-sheet structure in hexafluoroacetone/water (1:1) whereas the wild-type peptide was partially helical under identical conditions. These results would be consistent with the hypothesis that the constitutive activity of the mutant receptor is linked to a conformational change in the sixth transmembrane domain. The study of the receptor segments also indicate that peptides corresponding to loops of the alpha-factor receptor do not appear to assume turn structures.
Arshava
B
B
College of Staten Island of the City University of New York 10314, USA.
Liu
S F
SF
Jiang
H
H
Breslav
M
M
Becker
J M
JM
Naider
F
F
D000595:Amino Acid Sequence; D002628:Chemoreceptor Cells / Q000737:chemistry*; D002942:Circular Dichroism; D019204:GTP-Binding Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis*; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014157:Transcription Factors
D010455:Peptides; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D019204:GTP-Binding Proteins