Alpha-helix to random-coil transition of two-chain, coiled coils: experiments on the thermal denaturation of doubly cross-linked beta beta tropomyosin.
Equilibrium thermal denaturation curves (by circular dichroism) are reported for doubly cross-linked beta beta tropomyosin two-chain coiled coils. Cross-linking was performed by reaction of sulfhydryls with either ferricyanide or 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 approximately 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 approximately 40 degrees C. Above approximately 40 degrees 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 the non-cross-linked case. The sag has been ascribed to a "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.(ABSTRACT TRUNCATED AT 250 WORDS)
Holtzer
M E
ME
Askins
K
K
Holtzer
A
A
D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005292:Ferricyanides / Q000494:pharmacology; D006358:Hot Temperature; D046911:Macromolecular Substances; D008433:Mathematics; D009132:Muscles / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013816:Thermodynamics; D014335:Tropomyosin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D005292:Ferricyanides; D046911:Macromolecular Substances; D014335:Tropomyosin; C007931:hexacyanoferrate III; D004228:Dithionitrobenzoic Acid