Temperature jump kinetic study of the stability of apo-calmodulin

Temperature-jump relaxation spectrometry has been used to study the unfolding properties of Ca(2+)-free Drosophila calmodulin from 278 to 336 K, monitored by absorption of Tyr-138. The T-jump amplitude data are well fitted throughout with a melting temperature T(m) = 315.7 K, deltaH(o)(m) = 140.5 kJ mol(-1) and deltaC(p)(o) = 3.28 kJ K(-1) mol(-1), giving deltaG(o)(293) = 7.36 kJ mol(-1) for the C-domain, in good agreement with other data. The relaxation rate observed (time range 1 micros-1 ms) obeys a simple two-state kinetic mechanism throughout. The activation energy for unfolding is nearly temperature-independent, in contrast to that for refolding, and hence the transition state is relatively compact, resembling the folded state, and the relaxation time, tau, shows complex temperature dependence. The domain unfolding is a two-state process occurring with tau of approximately 100 micros at the T(m). At 296 K, when the C-domain is approximately 6% unfolded, k(unfolding) approximately 305 s(-1), k(refolding) approximately 4660 s(-1) and tau approximately 200 micros. This closely resembles the rate and extent of a reported C-domain exchange process, inferred from NMR line-broadening at 296 K. The inherent instability of the apo-C-domain of calmodulin indicates that the unfolded form significantly contributes to the physical properties of apo-calmodulin at normal temperatures, and this instability is enhanced by low ionic strength conditions.