Alpha-helix to random-coil transitions of two-chain coiled coils: the use of physical models in treating thermal denaturation equilibria of isolated subsequences of alpha alpha-tropomyosin

Abstract

Two extant models of thermal folding/unfolding equilibria in two-chain, alpha-helical coiled coils are tested by comparison with experimental results on excised, isolated subsequences of rabbit alpha alpha-tropomyosin (Tm). These substances are designated iTmj where i and j are, respectively, the residue numbers (in the 284-residue parent chain) of the N- and C-terminal residues of the subsequence. One model postulates that a coiled coil consists of segments, each denaturing in an all-or-none manner, like small globular proteins. Thus this model yields a small number of populated molecular species. In an extant calorimetry study of 11Tm127 and of 190Tm284, each required only two all-or-none-segments, and their enthalpies and transition temperatures were assigned. These assignments are shown here to yield the concentration of all molecular species, and therefore the helix content, as a function of temperature. Such calculations for 190Tm284 are in tolerable agreement with CD experiments, but those for 11Tm127 are in gross disagreement. Thus, either the model itself or the calorimetric assignment is faculty. In the second model, all conformational states are counted and weighted, as in the Zimm-Bragg theory for single-chain polypeptides. This theory has been extended (by Skolnick) to two-chain coiled coils and is here used to fit CD data for 11Tm127, 142Tm281, and 190Tm284. The fit is tolerable for 11Tm127, good for 142Tm281, and quantitative for 190Tm284. Thus this comparison does not falsify this second model. The helix-helix interaction free energy, obtainable from the fit, shows nonadditivity when isolated subsequences are compared with the parent. This suggests that removal of a region from a long coiled coil allows energetically substantial adjustments in side-chain packing in the helix-helix interface. Thus, the helix-helix interaction in long coiled coils is characteristic of a global free energy minimum and not just of the regional constellation of side chains.