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 synthesize d by solid-phase procedures and purified to near homogeneity. CD, nmr, and modeling analysis indicated that in aqueous media the first extra celluar loop peptide E1(107-125) the third intracellular loop peptide 13(231-243) and the carboxyl terminus peptide I4(350-372) were mostly disordered. In contrast the second extracellular loop peptide E2(191-2 06) assumed a well-defined structure in aqueous medium and the sixth t ransmembrane domain peptide receptor M6(252-269, C252A) was highly hel ical in trifluoroethanol/water (4:1), exhibiting a kink at Pro258. A s ynthetic peptide containing a sequence similar to that of the sixth tr ansmembrane domain of a constitutively active alpha-factor receptor M6 (252-269, C252A, P258L) in which Leu replaces Pro258 exhibited signifi cantly different biophysical properties than the wild-type sequence. I n particular; this peptide had very low solubility and gave CD resembl ing that of a beta-sheet structure in hexafluoroacetone water (1:1) wh ereas the wild-type peptide was partially helical under identical cond itions. These results would be consistent with the hypothesis that the constitutive activity of the mutant receptor is linked to a conformat ional change in the sixth transmembrane domain. The study of the recep tor segments also indicate that peptides corresponding to loops of the alpha-factor receptor do not appear to assume turn structures. (C) 19 98 John Wiley & Sons, Inc.