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Kinetics of folding and unfolding of ββ‐tropomyosin
Author(s) -
Mo Jianming,
Holtzer Marilyn Emerson,
Holtzer Alfred
Publication year - 1992
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.360321115
Subject(s) - chemistry , tropomyosin , kinetics , folding (dsp implementation) , crystallography , random coil , phase (matter) , protein folding , dilution , coiled coil , biophysics , actin , circular dichroism , thermodynamics , biochemistry , organic chemistry , physics , quantum mechanics , biology , electrical engineering , engineering
The kinetics of folding from random coils to two‐chain coiled coils of ββ‐tropomyosin was studied by stopped‐flow CD (SFCD) in the backbone region (222 nm). Two species were studied: the reduced form and the doubly disulfide cross‐linked form. The proteins were totally unfolded in 6 M urea‐saline buffer, then refolded by tenfold dilution into benign k buffer. In the refolding medium, they spontaneously recover the two‐chain coiled‐coil structure. Reduced ββ refolds in at least two stages: one or more fast phases (<0.04 s), in which an intermediate with 71% of the equilibrium ellipticity forms, followed by a slower time‐resolvable phase that completes the folding. The slow phase is first order, signifying that dimerization occurs in the fast phase. The time constant of the slow phase is 2 s at 20°C and requires activation parameters of Δ S ≠ = –7 ± 0.3 cal/mol · K, Δ H ≠ = 15 ± 1 kcal/ mol. These results are very similar to those previously found for the reduced genetic variant αα‐tropomyosin. In contrast, refolding of doubly disulfide cross‐linked ββ is complete within the dead time (< 0.04 s), whereas the singly cross‐linked αα species also displays a slow phase. The opposite process, unfolding reduced ββ from the coiled‐coil state, is complete within the dead time, as in the αα variant.