α‐Helix to random coil transitions of two‐chain coiled coils: Experiments on the thermal denaturation of ββ tropomyosin cross‐linked selectively at C36
Authors: Holtzer Marilyn Emerson , Bracken William Clay , Holtzer Alfred 
Published: December 2004

AbstractCurrent ideas on unfolding equilibria in two‐chain, coiled‐coil proteins are examined by studies of a species of ββ tropomyosin that is sulfhydryl blocked at C190 and disulfide cross‐linked at C36 (\documentclass{article}\pagestyle{empty}\begin{document}$ ^. \beta \_\beta ^. $\end{document}). The desired species is produced by a seven‐step process: (1) Rabbit skeletal muscle, comprising predominantly αα and ββ species, is oxidized with ferricyanide, cross‐linking both species at C190. (2) The product is carbamylated at C36 of β chains, using cyanate in denaturing medium at pH 6. (3) All C190 cross‐links are reduced with dithiothreitol (DTT). (4) All C190 sulfhydryls are permanently blocked by carboxyamidomethylation. (5) Chromatography on carboxymethylcellulose in denaturing medium is used to separate C190‐blocked α chains from C190‐blocked, C36‐carbamylated β chains. (6) The latter are decarbamylated in denaturing medium by raising the pH to 8.0. (7) The C190‐blocked β chains are renatured and cross‐linked at C36 by ferricyanide. The procedure and the quality of the final product are judged by NaDodSO4/polyacrylamide gel electrophoresis, titration of free sulfhydryls, and electrophoretic analysis of trypsin digestion products. Thermal unfolding curves are reported for the resulting pure \documentclass{article}\pagestyle{empty}\begin{document}$ ^. \beta \_\beta ^. $\end{document} species and for its DTT‐reduction product. The latter (\documentclass{article}\pagestyle{empty}\begin{document}$ ^. \beta \beta ^. $\end{document}) show equilibrium thermal unfolding curves that are very similar to those of the parent ββ noncross‐linked species. The \documentclass{article}\pagestyle{empty}\begin{document}$ ^. \beta \_\beta ^. $\end{document} cross‐linked species unfolds in a single‐phase, cooperative transition with a melting temperature intermediate between the pretransition and posttransition shown by its cross‐linked counterpart, the C190 cross‐linked, C36‐blocked species (\documentclass{article}\pagestyle{empty}\begin{document}$ .\beta^- \beta .$\end{document}), which was studied earlier. These transitions are compared with one another and with that of the doubly cross‐linked species,
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, in the light of two extant physical models for such transitions. The all‐or‐none segments model successfully rationalizes the data qualitatively for the \documentclass{article}\pagestyle{empty}\begin{document}$ .\beta^- \beta .$\end{document} and \documentclass{article}\pagestyle{empty}\begin{document}$ ^. \beta \_\beta ^. $\end{document} species if the usual postulates of greater inherent stability of the amino vs the carboxyl end of the molecule and of strain at each cross‐link are accepted. However, the same model then requires that the
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species be the least stable of the three, whereas experiment shows the opposite, thus falsifying the all‐or‐none segments model. The continuum‐of‐states model is also qualitatively in accord with data on the \documentclass{article}\pagestyle{empty}\begin{document}$ .\beta^- \beta .$\end{document} and \documentclass{article}\pagestyle{empty}\begin{document}$ ^. \beta \_\beta ^. $\end{document} species. In fact, the general features of the transition in C36 cross‐linked vs C190 cross‐linked species were predicted by a statistical mechanical theory embodying the continuum‐of‐states model for singly cross‐linked species. Moreover, since the same theory avers that loop entropy greatly stabilizes the large region between cross‐links in the
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species, perhaps offsetting the effect of strain, qualitative considerations alone are insufficient to falsify this model in the face of the data on doubly cross‐linked species. Thus, one model can be eliminated, but the second cannot. However, until quantitative simulations are done and found to agree with these data, the continuum‐of‐states model must still be considered questionable.