Effect of preferred binding domains on peptide retention behavior in reversed-phase chromatography: amphipathic alpha-helices.

A nonpolar environment, such as the hydrophobic stationary phase of a reversed-phase chromatographic packing, may induce helical structures in potentially helical molecules. If a molecule becomes helical on binding and contains a preferred binding domain, as in the case of an amphipathic helix, then some residues may not be contributing to the same extent to the overall hydrophobicity of the peptide. Amphipathic alpha-helical structures may play an important role in protein folding and the interaction of amphipathic alpha-helices with a hydrophobic surface during RPC is likely to be a good mimic of their hydrophobic interactions with other hydrophobic regions in the folded protein. We have designed and synthesized two sets of model peptides of 7, 14, 21, 28 and 35 residues having the same composition but different sequences (Ac-Lys-Cys-Ala-Glu-Gly-Glu-Leu-[Lys-Leu-Glu-Ala-Gly-Glu-Leu]n-amide and Ac-Lys-Cys-Ala-Glu-Leu-Glu-Gly-[Lys-Leu-Glu-Ala-Leu-Glu-Gly]n-amide, where n = 1-4). Circular dichroism studies demonstrated that both sets of peptides had a high potential to form alpha-helical structure in a nonpolar environment, one set representing amphipathic alpha-helical structures and the other set representing non-amphipathic alpha-helical structures. Size-exclusion chromatography confirmed that all of the peptides in both sets were monomeric when bound to a reversed-phase matrix and also under the conditions used for circular dichroism measurements. Peptides with the same amino acid composition and similar secondary structure could be separated by reversed-phase chromatography. The difference in retention time between peptides of the same length increased with the peptide chain length, ranging from a difference of 2.9 min on a C8 column for the two 14-residue peptides up to a maximum difference of 7.3 min for the 35-residue peptides. From the observed and predicted retention times of these two sets of peptides during reversed-phase chromatography, we have demonstrated that it is possible not only to predict the retention behavior of amphipathic alpha-helices during reversed-phase chromatography, but also to deduce the presence of amphipathic alpha-helical structure in peptides based upon their retention data. If from studies such as these we are eventually able to predict, from only amino acid sequence information, the secondary structure of a peptide on binding to a hydrophobic matrix, we may be able to extrapolate this predictive facility to the conformation of the same sequence in larger polypeptides or proteins.