Temperature jump kinetic study of the stability of apo-calmodulin Original Research Article

Temperature-jump relaxation spectrometry has been used to study the unfolding properties of Ca2+-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 Tm=315.7 K, ΔHom=140.5 kJ mol−1 and ΔCpo=3.28 kJ K−1 mol−1, giving ΔGo293=7.36 kJ mol−1 for the C-domain, in good agreement with other data. The relaxation rate observed (time range 1 μs–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, τ, shows complex temperature dependence. The domain unfolding is a two-state process occurring with τ of ∼100 μs at the Tm. At 296 K, when the C-domain is ∼6% unfolded, kunfolding≈305 s−1, krefolding≈4660 s−1 and τ≈200 μs. 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.