Effect of mobile phase on the oligomerization state of alpha-helical coiled-coil peptides during high-performance size-exclusion chromatography.

Important structural motifs involving amphipathic helices include two-stranded and multiple-stranded coiled-coils. High-performance size-exclusion chromatography (HPSEC) is a useful tool to examine both the oligomerization state of coiled-coils as well as the stability of such motifs, due to the facile manipulation of the mobile phase and the lack of interaction of the peptide solutes with the stationary phase. In the present study, HPSEC was applied to two series of de novo designed model amphipathic alpha-helical peptides with the sequences (1) Ac-(E-A-L-K-A-E-I)n-E-A-C-K-A-amide, where n = 1 or 3, Ac-E-I-(E-A-L-K-A-E-I)4-E-A-C-K-A-amide and (2) Ac-(K-L-E-A-L-E-A)n-amide, where n = 1, 2 or 4. Observation of the retention behaviour of Series 1 under both denaturing and non-denaturing conditions at pH 7.0 offered insights into the effect of polypeptide chain length and disulphide bridge formation on the stability of alpha-helical coiled-coils. In contrast, the Series 2 peptides showed promise as peptide standards to monitor the effect of environment on the multi-strandedness of coiled-coils, since the 28-residue peptide of this series was eluted as a monomer, dimer or trimer depending on mobile phase conditions.