α-Helix-to-random-coil transition of two-chain, coiled coils. Theory and experiments for thermal denaturation of α-tropomyosin at acidic pH

Abstract

New data are presented for the a-helix content of non-cross-linked and cross-linked a-tropomyosin at acidic pH as a function of temperature (20-80 OC) over a 1000-fold range of protein concentration. Experimental thermal denaturation curves at acidic pH are independent of concentration for both non- cross-linked and cross-linked samples, unlike the case of neutral pH, where it was previously shown that the non-cross-linked protein data are concentration dependent. The a helix in the non-cross-linked case is more stable than in the cross-linked case at low pH and both are considerably more stable than at near-neutral pH. The realized theory developed earlier is applied to these new data. The theory shows that even noninteracting helices are considerably more stable at acidic pH because of substantial increases in the helix propagation parameter s(T) for aspartic and glutamic acids over those for aspartate and glutamate, respectively. The theory for interacting helices fits the data well with an interaction parameter w(T) that is only slightly larger than that required to fit the data at neutral pH. Thus, most of the enhanced tropomyosin helix stability in acid has its origin in enhanced short-range interactions (s(T)) for its acidic residues. The insensitivity of w(T) to pH implies that the salt linkages, which cannot be present in acid, do not contribute appreciably to the helix-helix interaction even when present at neutral pH. The theory also reveals that dissociation to monomers is entirely negligible at low pH over the full range of conditions employed, explaining the lack of dependence of the observed helix content on protein concentration. The reduced helix content of the actual cross-linked dimers, as compared with both theory and experiment for non-cross-linked dimers, confirms an earlier conclusion (drawn less directly from the data on the partially dissociated system at neutral pH) that the actual disulfide cross-link has a locally adverse effect on the helix-stabilizing interactions.