Probing The Functional Conformation Of The Tridecapeptide Mating Pheromone Of Saccharomyces Cerevisiae Through Study Of Disulfide-Constrained Analogs

Analogs of the Saccharomyces cerevisiae alpha-mating factor, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, where Lys(7) and Gln(10) were replaced with Cys, Cys(CH3), or Ser, were synthesized using solid-phase procedures on a phenylacetamidomethyl resin. CyClo(7,10)[Cys(7),X(9),Cys(10), Nle(12)]alpha-factor, where X=D-Val, D-Ala, L-Ala and Gly, were prepared by on-resin cyclization using thallic trifluoroacetate in yields of 20-30%. Linear sulfhydryl-containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys(7) and Gln(10) with a cysteine residue resulted in an over 100-fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5-10-fold more active than their sulfhydryl-containing homologs, and CyClo(7,10)[Cys(7),L-Ala(9),Cys(10),Nle(12)] alpha-factor was 50-fold more potent than linear analogs containing Ser or Cys(CH3) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the alpha-factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5-aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form beta-turns spanning residues 7-10 than their less active linear counterparts. The results provide strong evidence that this beta-turn is important for optimal signal transduction by alpha-factor.