Thermodynamic Stability of the Molten Globule States of Apomyoglobin

Whereas horse apomyoglobin is fully unfolded at pH 2 in the absence of salt, addition of a salt such as sodium chloride or sodium trichloroacetate stabilizes the molten globule state. Thermal unfolding of the salt-stabilized molten globule states of horse apomyoglobin at pH 2 measured by far-UV circular dichroism occurs not only on heating (i.e. heat-denaturation) but also on cooling (i.e. cold-denaturation). This demonstrates that a hydrophobic interaction contributes to the stability of the molten globule state and suggests that the unfolding transition can be represented by a cooperative two-state mechanism. To clarify the mechanism of conformational transition, we investigated the thermal unfolding of the chloride-stabilized molten globule state by differential scanning calorimetry. We observed a broad but distinct excess heat capacity peak, which is consistent with the unfolding transition measured by circular dichroism. To further characterize the molten globule states, we examined by far-UV circular dichroism the denaturant-induced unfolding transitions of the molten globule states stabilized by sodium chloride or sodium trichloroacetate. The urea-induced unfolding transitions of the molten globule states were explained by the two-state mechanism. The guanidine-hydrochloride-induced unfolding experiments clarified that the trichloroacetate-stabilized molten globule state is distinct from the chloride-stabilized one and that the former involves additional helical segment(s). These results support a view that the thermal unfolding of the molten globule states at pH 2 can be approximated by a two-state transition. However, several results suggested that a combined mechanism incorporating the two-state transition and a gradual structural change would be more general in describing the conformational transition of the molten globule states. f2 f2 Abbreviations used: H/D, hydrogen-deuterium; NaTCA, sodium trichloroacetate; Gdn-HCl, guanidine hydrochloride; CD, circular dichroism; DSC, differential scanning calorimetry; SAXS, small angle X-ray scattering; R gradius of gyration.