STRUCTURE-FUNCTION ANALYSIS OF THE SACCHAROMYCES-CEREVISIAE TRIDECAPEPTIDE PHEROMONE USING ALANINE-SCANNED ANALOGS

Twenty-six peptide analogs of the Saccharomyces cerevisiae alpha-facto r, a tridecapeptide mating pheromone )W(3)L(4)Q(5)L(6)K(7)P(8)G(9)Q(10 )P(11)M(12)Y(13)) with either L- or D-alanine replacement of each amin o acid residue (Ala-scanned) and with the isosteric replacement of met hionine at position 12 by norleucine, were synthesized, purified to ho mogeneity and assayed for biological activity and receptor binding. Tw o new and effective antagonists, [D-Ala(3),Nle(12)]alpha-factor and [D -Ala(4),Nle(12)]alpha-factor, were found among the series, and the [D- Ala(10),Nle(12)]alpha-factor demonstrated a marked ability to increase the biological activity of [Nle(12)]alpha-factor without having any e ffect by itself. One analog, the [L-Ala(1) alpha-factor, showed a 3-fo ld increase in bioactivity over the [Nle(12)]alpha-factor, although it s binding to the alpha-factor receptor was about 70-fold less than [Nl e(12)]alpha-factor. Residues near the carboxyl terminus contributed mo re strongly to receptor binding than other residues, whereas those nea r the amine terminus of the alpha-factor played an important role in s ignal transduction. The effect of insertion of D-Ala residues at posit ions 7, 8, 9 and 10 on bioactivity and receptor binding of the peptide suggested a specific positioning role of the central loop in establis hing optimal contacts between the receptor and the ends of the pheromo ne. We conclude that the alpha-factor may be divided into segments wit h dominant roles in forming the biologically active pheromone conforma tion, in receptor binding and in initiating signal transduction. The d iscovery of such relationships was made possible by the systematic var iation of each residue in the peptide and by the testing of each analo g in highly defined biological and binding assays.