The role of position a in determining the stability and oligomerization state of α‐helical coiled coils: 20 amino acid stability coefficients in the hydrophobic core of proteins

Abstract

We describe here a systematic investigation into the role of position a in the hydrophobic core of a model coiled‐coil protein in determining coiled‐coil stability and oligomerization state. We employed a model coiled coil that allowed the formation of an extended three‐stranded trimeric oligomerization state for some of the analogs; however, due to the presence of a Cys‐Gly‐Gly linker, unfolding occurred from the same two‐stranded monomeric oligomerization state for all of the analogs. Denaturation from a two‐stranded state allowed us to measure the relative contribution of 20 different amino acid side chains to coiled‐coil stability from chemical denaturation profiles. In addition, the relative hydrophobicity of the substituted amino acid side chains was assessed by reversed‐phase high‐performance liquid chromatography and found to correlate very highly ( R = 0.95) with coiled‐coil stability. We also determined the effect of position a in specifying the oligomerization state using ultracentrifugation as well as high‐performance size‐exclusion chromatography. We found that nine of the analogs populated one oligomerization state exclusively at peptide concentrations of 50 μM under benign buffer conditions. The Leu‐, Tyr‐, Gln‐, and His‐substituted analogs were found to be exclusively three‐stranded trimers, while the Asn‐, Lys‐, Orn‐, Arg‐, and Trp‐substituted analogs formed exclusively two‐stranded monomers. Modeling results for the Leu‐substituted analog showed that a three‐stranded oligomerization state is preferred due to increased side‐chain burial, while a two‐stranded oligomerization state was observed for the Trp analog due to unfavorable cavity formation in the three‐stranded state.