Temperature profiling of polypeptides in reversed-phase liquid chromatography -: I.: Monitoring of dimerization and unfolding of amphipathic α-helical peptides

The present study sets out to extend the utility of reversed-phase liquid chromatography (RP-HPLC) by demonstrating its ability to monitor dimerization and unfolding of de novo designed synthetic amphipathic alpha-helical peptides on stationary phases of varying hydrophobicity. Thus, we have compared the effect of temperature (5-80 degreesC) on the RP-HPLC (C-8 or cyano columns) elution behaviour of mixtures of peptides encompassing amphipathic alpha-helical structure, amphipathic alpha-helical structure with L- or D-substitutions or non-amphipathic alpha-helical structure. By comparing the retention behaviour of the helical peptides to a peptide of negligible secondary structure (a random coil), we rationalize that "temperature profiling" by RP-HPLC can monitor association of peptide molecules, either through oligomerization or aggregation, or monitor unfolding of a-helical peptides with increasing temperature. We believe that the conformation-dependent response of peptides to RP-HIPLC under changing temperature has implications both for general analysis and purification of peptides but also for the de novo design of peptides and proteins. (C) 2003 Elsevier B.V. All rights reserved.