.alpha.-Helix to random-coil transition of two-chain, coiled coils: experiments on the thermal denaturation of doubly crosslinked dimeric .beta.-tropomyosin

Abstract

Equilibrium thermal denaturation curves (by circular dichroism) are reported for doubly cross-linked .beta..beta. tropomyosin two-chain coiled coils. Cross-linked was performed by reaction of sulfhydryls with either ferricyanide of 5,5''-dithiobis(2-nitrobenzoate) (NbS2). The extent of reaction was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and either by titration of residual sulfhydryls with NbS2 (ferricyanide cross-linking) or by determination of mixed disulfide (protein-S-SbN) through reaction with dithiothreitol (NbS2 cross-linking). The results indicate .apprx. 90% conversion to molecules with interchain cross-links at both C-36 and C-190. Thermal unfolding curves are compared with those obtained previously for non-cross-linked species. The curves are indistinguishable up to .apprx. 40.degree. C. Above .apprx. 40.degree. C, the doubly cross-linked species is more stable, but the transition is less steep. This relationship is also compared with that found between .alpha..alpha. tropomyosin (a similar coiled coil made of a genetic variant chain having a sulfhydryl only at C-190) and its singly cross-linked derivative. Thermal curves for .alpha..alpha. and .beta..beta. non-cross-linked species are very similar, .alpha..alpha. being somewhat more stable. For cross-linked .alpha..alpha., however, the curve sags at temperatures somewhat below the region of principal cooperative loss of helix, the latter occurring at higher temperature but with the same steepness as in non-cross-linked case. The sag has been ascribed to "pretransition" in the region of C-190. Thus, doubly and singly cross-linked species differ in that the former show no pretransition and decreased steepness in the principal transition. The significance of these findings is discussed in terms of the knonw physical destablization of randomly coiled loops relative to their linear counterparts and the enormous (308-residue) loop that would exist in the completely unfolded doubly cross-linked molecule.