Kinetics of folding and unfolding of ββ‐tropomyosin

AbstractThe 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 6M 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 ΔS≠ = –7 ± 0.3 cal/mol · K, ΔH≠ = 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.