The ΔH associated with the thermal unfolding of G-actin has been determined by differential scanning calorimetry (DSC) to be 142 ± 5 kcal/mol, with the Tm (melting temperature) at 57.2 ± 0.5 °C, at pH 8.0 (heating rate 0.5 K/min). The transition is broad and cannot be treated as a single transition that mimics a two-state process, suggesting the existence of domains. Deconvolution is done to fit it into two quasi-independent two-state transitions. For F-actin, the transition is more cooperative, with a cooperative ratio (the ratio of van’t Hoff enthalpy and calorimetric enthalpy) of 1.4, indicating intermonomer interaction. The ΔH of the thermal unfolding of F-actin is 162 ± 10 kcal/mol with a Tm at 67.0 ± 0.5 °C. A state of G-actin similar to that of the heat-denatured form, designated D-actin, is obtained by removing tightly bound Ca2+ with EGTA. The DSC-detectable cooperative transition is completely lost when the free calcium concentration of the medium is 1 X 10‒11 M or lower, using a Ca2+/EGTA buffer system. However, circular dichroism (CD) shows that the helix content of actin, 32% in the G-form, is only partially reduced to 19% in this apo form. The CD spectrum and the helix content of the calcium-depleted actin are almost identical with those of the heat-denatured D form. This loss of 40% of the native helical content is irreversible in both cases. The remaining 60% of the native helical content cannot be further eliminated by heating to 95 °C. A complete and reversible unfolding of the D-actin can be obtained by 5 M guanidinium chloride or 8 M urea. The heat denaturation as well as chemical denaturant unfolding have also been followed by the intrinsic fluorescence of tryptophans. A red shift of the emission maximum from 325 to 335 nm is observed with heat and EGTA denaturation. Completely unfolded actin has an emission maximum at 345 nm. The accessibility of hydrophobic binding sites upon heat and EGTA denaturation is detected by ANS (anili-nonaphthalenesulfonate) binding; the total number of binding sites increases by about 5-fold upon denaturation. These findings suggest a two-step pathway for the complete unfolding of G-actin, N→D ⇌ U, where N, D, and U denote the native, a denatured but compact, and the completely unfolded states, respectively. The first irreversible step is characterized by a large enthalpic change and involves the dissociation of the high-affinity Ca2+. This transition involves melting of two independent domains. The second reversible step is purely entropie and cannot be induced by heating. The structural characteristics of D-actin are intermediate between N- and U-actin. ΔG, ΔH, and ΔS of each conformational transition are given. © 1990, American Chemical Society. All rights reserved.
