The kinetics of chain exchange in two-chain coiled coils: αα- and ββ-tropomyosin

Abstract

Measurements are presented on the time course of chain exchange among two-chain α-helical coiled coils of rabbit tropomyosin. All experiments are in a regime (temperature, protein concentration) in which coiled-coil dimers are the predominant species. Self-exchange in αα-tropomyosin was investigated by mixing αα species with α*α*, the asterisk designating an α-chain whose lone sulfhydryl (C190) has been blocked by carboxyamidomethylation. The overall process αα + α*α* ⇄ 2αα* is followed by measurement of the fraction (h ) of αα* species as a function of time. Similarly, self-exchange in ββ-tropomyosin is examined by measurements of the overall process: ββ + β*β* ⇄ 2ββ*, in which β* signifies a β-chain blocked at both sulfhydryls (C36 and C190). The observed time course for both chains is well fit by the first-order equation: h (t ) = h (∞)(1 − e), with h (∞) ≃ 0.5. This long-time limit is as expected for self-exchange, and agrees with experiments that attain equilibrium after slow cooling of thermally dissociated and unfolded chains. The simplest consonant mechanism is chain exchange by rate-limiting dissociation of dimers followed by random reassociation. Kinetic analysis shows k₁ to be the rate constant for the chain dissociation step, a quantity not previously measured for any coiled coil. This rate constant for ββ species is about an order of magnitude greater than for αα. In both, the activation enthalpy and entropy are very large, suggesting that activation to an extensively (>50%) unfolded species necessarily precedes dissociation. Experiments are also reported for overall processes: αα + β*β* ⇄ 2αβ* and α*α* + ββ ⇄ 2α*β. Results are independent of which chain is blocked. Again h (∞) ≃ 0.5, in agreement with equilibrium experiments, and the time course is first order. The rate constants and activation parameters are intermediate between those for self-exchange.