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

✍ Scribed by Sumio Ozeki; Tadashi Kato; Marilyn Emerson Holtzer; Alfred Holtzer

• Publisher: Wiley (John Wiley & Sons)
• Year: 1991
• Tongue: English
• Weight: 960 KB
• Volume: 31
• Category: Article
• ISSN: 0006-3525
• DOI: 10.1002/bip.360310805

✦ Synopsis

SYNOPSIS

Measurements are presented on the time course of chain exchange among two-chain ahelical 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 rue-tropomyosin was investigated by mixing aa species with a*a*, the asterisk designating a n a-chain whose lone sulfhydryl (C190) has been blocked by carboxyamidomethylation. T h e overall process aa + a*a* 2 2aa* is followed by measurement of the fraction ( h ) of aa* species as a function of time. Similarly, self-exchange in PP-tropomyosin is examined by measurements of the overall process: /7/3 + p*P* F? 2pp*, in which p* signifies a P-chain blocked a t both sulfhydryls (C36 and C190). T h e observed time course for both chains is well fit by the first-order equation:

with h ( z ) Y 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 k , to be the rate constant for the chain dissociation step, a quantity not previously measured for any coiled coil. This rate constant for p/3 species is about a n order of magnitude greater than for aa. In both, the activation enthalpy and entropy are very large, suggesting that activation to a n extensively ( >50% ) unfolded species necessarily precedes dissociation. Experiments are also reported for overall processes: aa + @*P* * 2aP* and a*a* + F? 2a*o. Results are independent of which chain is blocked. Again h ( a ) Y 0.5, in agreement with equilibrium experiments, and the time course is first order. T h e rate constants and activation parameters are intermediate between those for self-exchange.