Alpha-helix to random coil transitions of two-chain coiled coils: experiments on the thermal denaturation of beta beta tropomyosin cross-linked selectively at C36.
Electrophoresis, Polyacrylamide Gel
Current ideas on unfolding equilibria in two-chain, coiled-coil proteins are examined by studies of a species of beta beta tropomyosin that is sulfhydryl blocked at C190 and disulfide cross-linked at C36 (.beta-beta.). The desired species is produced by a seven-step process: (1) Rabbit skeletal muscle, comprising predominantly alpha alpha and alpha beta species, is oxidized with ferricyanide, cross-linking both species at C190. (2) The product is carbamylated at C36 of beta chains, using cyanate in denaturing medium at pH 6. (3) All C190 cross-links are reduced with dithiothreitol (DTT). (4) All C190 sulfhydryls are permanently blocked by carboxyamidomethylation. (5) Chromatography on carboxymethylcellulose in denaturing medium is used to separate C190-blocked alpha chains from C190-blocked, C36-carbamylated beta chains. (6) The latter are decarbamylated in denaturing medium by raising the pH to 8.0. (7) The C190-blocked beta chains are renatured and cross-linked at C36 by ferricyanide. The procedure and the quality of the final product are judged by NaDodSO4/polyacrylamide gel electrophoresis, titration of free sulfhydryls, and electrophoretic analysis of trypsin digestion products. Thermal unfolding curves are reported for the resulting pure .beta-beta. species and for its DTT-reduction product. The latter (.beta beta.) show equilibrium thermal unfolding curves that are very similar to those of the parent beta beta noncross-linked species. The .beta-beta. cross-linked species unfolds in a single-phase, cooperative transition with a melting temperature intermediate between the pretransition and posttransition shown by its cross-linked counterpart, the C190 cross-linked, C36-blocked species (.beta-beta.), which was studied earlier. These transitions are compared with one another and with that of the doubly cross-linked species, beta-(-)beta, in the light of two extant physical models for such transitions. The all-or-none segments model successfully rationalizes the data qualitatively for the .beta-beta. and .beta-beta. species if the usual postulates of greater inherent stability of the amino vs the carboxyl end of the molecule and of strain at each cross-link are accepted. However, the same model then requires that the beta-(-)beta species be the least stable of the three, whereas experiment shows the opposite, thus falsifying the all-or-none segments model. The continuum-of-states model is also qualitatively in accord with data on the .beta-beta. and .beta-beta. species.(ABSTRACT TRUNCATED AT 400 WORDS)