Hetero-alpha-helical, two-chain, coiled coils: alpha beta hybrid tropomyosin

The nature of the interhelix interaction in two-chain, α-helical, coiled coils is studied by experiments on the formation of hydrid molecules in which one helical chain is an α-tropomyosin and the other a β-tropomoysin. By means of a recently developed assay, the population of heterohelical (i.e., αβ hybrid) molecules relative to their homohelical (αα and ββ) parent species is determined under a variety of conditions, both equilibrium and nonequilibrium. It is found that mixed intact αα and ββ molecules do not form hybrid species in detectible amounts even after incubation at room temperature (or below) for periods of over one week. That the lack of αβ species in this “native-exchange” system is a result of a kinetic barrier is evident from experiments involving a thermal denaturation–renaturation cycle in which the largely dissociated, unfolded chains at higher temperature are annealed to benign temperatures over a period of 6 h, thus assuring an equilibrium population of two-chain species. In the resulting equilibrium state, the αβ population is one-half the total, indicating that recombination is random. Furthermore, this same (equilibrium) state is reached if the separated, mostly unfolded chains are renatured by a rapid (～ 40 s) quench to benign temperatures. Some implications of these results for the thermodynamics of interhelix interation, for kinetics of chain dissociation and recombination, and for in vivo genesis of two-chain coiled coils are discussed.