Abstract
Stopped flow CD (SFCD) kinetic studies of self–assembly of coiled coils of rabbit αα–tropomyosin and of nonpolymerizable αα–tropomyosin (NPTm) are reported. The protein was denatured in 6__M__ urea buffer, then renatured by 10‐fold dilution into benign saline buffer. Folding was monitored by SFCD in the backbone region (222 nm). Protein chains are shown to be totally unfolded (and separated in the reduced species) in the initial denaturing medium and fully folded as two‐chain coiled coils in the final benign medium. In all cases of folding in benign buffer of totally unfolded chains, two phases were found in the folding process: a fast phase (<0.04 s, the SFCD dead time), in which an intermediate state with about 70% of the equilibrium ellipticity forms; followed by a slower, observable phase that completes the folding. The slow phase is first order (k ^−1^ = 1.6 s at 20°C), signifying that chain association for reduced samples occurs in the fast phase. In contrast, folding in benign buffer from an initial state with 70% of the equilibrium ellipticity is all fast, suggesting that the folding intermediate is not an equilibrium species. Cross‐linking at Cys‐190 increases the helix content of the fast‐formed intermediate state to about 85% of the equilibrium value, but leaves the rate constant of the slow phase unchanged. In NPTm, which does not form high aggregates at low ionic strength, the rate of the observable phase is almost independent of ionic strength in the range of ∼ 0.15‐0.6__M__, but is reduced one to two orders of magnitude by further reduction to 0.026__M__. In folding from totally unfolded chains, the rate is reduced less than one order of magnitude by changing the final state to about 50% folded. In contrast to folding, unfolding of αα‐tropomyosin from the native state is all fast.