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.