Monitoring of α-helical secondary structures in peptides by reversed-phase HPLC of replacement sets

Abstract

HPLC studies of systematic double d-amino acid, methionine and methionine sulfoxide replacement sets were applied towards the structural characterization of α-helical peptides. Double d-amino acid replacements disturb the local structure of the helical binding domain, leading to significant reversed-phase HPLC retention time decreases. On the other hand, methionine and methionine sulfoxide replacements locally alter the hydrophobicity along helical peptides, and cause position-dependent retention time differences between methionine and methionine sulfoxide analogs with a 3–4 repeat pattern in amphipathic α-helices. The two approaches yield complementary information on α-helical secondary structure and the hydrophobic interaction domain of peptides with the stationary phase. These HPLC-based methods were successfully applied to determine the potential of α-helix formation in neuropeptide Y and corticotropin releasing hormone. The results clearly demonstrate that hydrophobically induced α-helical structures can be accurately characterized in biologically active peptides by this approach.