Structure,biological activity and membrane partitioning of analogs of the isoprenylated a‐factor mating peptide of Saccharomyces cerevisiae

Abstract: Previous biochemical investigations on the Saccharomyces cerevisiae a ‐factor indicated that this lipopeptide pheromone YIIKGVFWDPAC(farnesyl)OMe] might adopt a type II β‐turn at positions 4 and 5 of the peptide sequence. To test this hypothesis, we synthesized five analogs of a ‐factor, in which residues at positions 4 and 5 were replaced with: l ‐Pro⁴(I); d ‐Pro⁴(II); l ‐Pro⁴‐d ‐Ala⁵(III); d ‐Pro⁴‐l ‐Ala⁵(IV); or Nle⁴(V). Analogs were purified to > 99% homogeneity as evidenced by HPLC and TLC and were characterized by mass spectrometry and amino acid analysis. Using a growth arrest assay the conformationally restricted a ‐factor analogs I and III were found to be almost 50‐fold more active than the diastereometric homologs II and IV and were equally active to wild‐type a ‐factor. Replacement of Lys⁴ with the isosteric Nle⁴ almost abolished the activity of the pheromone. Thus, the incorporation of residues that promote a type II β‐turn compensated for the loss of the favorable contribution of the Lys⁴ side chain to pheromone activity. CD spectra on these peptides suggested that they were essentially disordered in both TFE/H₂O and in the presence of DMPC vesicles. There was no correlation between CD peak shape and biological activity. Using fluorescence spectroscopy we measured the interaction of lipid vesicles with these position 4 and 5 analogs as well as with three a ‐factor analogs with a modified farnesyl group. The results indicated that modifications of both the peptide sequence and the lipid moiety affect partitioning into lipid, and that no correlation existed between the propensity of a pheromone to partition into the lipid and its biological activity.