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α‐Helix‐to‐random‐coil transitions of two‐chain coiled coils: Experiments on the thermal denaturation of ββ tropomyosin cross‐linked selectively at C‐190
Author(s) -
Bracken William Clay,
Carey John,
Holtzer Marilyn Emerson,
Holtzer Alfred
Publication year - 1988
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.360270804
Subject(s) - chemistry , tropomyosin , dithiothreitol , iodoacetamide , guanidinium chloride , ferricyanide , denaturation (fissile materials) , guanidine , random coil , size exclusion chromatography , polyacrylamide gel electrophoresis , gel electrophoresis , potassium ferricyanide , chromatography , crystallography , actin , cysteine , organic chemistry , biochemistry , circular dichroism , nuclear chemistry , enzyme
A method is described for preparation of a species of β tropomyosin that is sulfhydryl‐blocked at C36 and disulfide‐cross‐linked at C190. Five steps are involved: (1) Rabbit skeletal muscle tropomyosin, comprising αα and αβ species, is oxidized with ferricyanide, disulfide‐cross‐linking both species at C190. (2) The product is treated with iodoacetamide, blocking the only remaining free sulfhydryl, i.e., C36 of the β‐chains. (3) The C36‐blocked, C190‐cross‐linked product is reduced with dithiothreitol (DTT), unfolded in urea, and α and β chains separated by ion‐exchange chromatography. (4) The C36‐blocked β chains are refolded by dialysis. (5) The refolded, C36‐blocked ββ species are cross‐linked at C190 by ferricyanide oxidation. The resulting C36‐blocked, C190‐cross‐linked ββ product is separated from contaminating species—mostly completely blocked β‐chains and multichain cross‐linked molecules—by size‐exclusion chromatography in denaturing (guanidinium chloride) solvent. The five‐step process and the final product were monitored by titration of free sulfhydryls and by NaDodSO 4 /polyacrylamide gel electrophoresis (PAGE). Thermal unfolding curves from CD are reported for the resulting pure, C36‐blocked, C190‐cross‐linked ββ species and for its DTT‐reduction product, the noncross‐linked C36‐blocked species. The latter shows almost the same thermal unfolding transition as intact, noncross‐linked ββ species. The former shows a pretransition similar to, but larger in extent than, the one well known to occur in the analogous case of C190‐cross‐linked αα tropomyosin. These unfolding transitions are compared with one another and with that previously reported for doubly cross‐linked (at C36 and C190) ββ species. These comparisons are made in the light of current physical models for coiled‐coil unfolding equilibria. It is concluded that although no extent model is demonstrably satisfactory, any successful model must include strain at the cross‐link, loop entropy, and regional nonuniformities as essential parts of the physics.