Yuji Goto

X-ray solution scattering studies of protein folding

Protein folding is a reaction in which an extended polypeptide chain acquires maximal packing through formation of secondary and tertiary structures. Compactness and shape are, therefore, critical properties characterizing the process of protein folding. Because the stability of the native state is determined by the subtle free energy balance between the native and denatured states, the characterization of the denatured state is also essential to understand the conformational stability of the native state. We show that solution X-ray scattering is the best technique available today to address these problems. Although the structural resolution of the unfolded or compact denatured states elucidated from solution X-ray scattering is low, it provides a variety of information complementary to that obtained by NMR or X-ray crystallography.

Acid-induced folding of heme proteins.

Publisher Summary The acid-induced unfolding and refolding reactions are readily examined by using spectroscopic methods such as circular dichroism (CD) and fluorescence; it is straightforward to explore these intermediate states of proteins. In particular, by applying this method to heme proteins, it is possible to estimate the contribution of the heme group in stabilizing the intermediate states. Acid-induced refolding in combination with acid-induced unfolding is a useful method to examine the behavior of proteins under acidic conditions. In particular with heme proteins, the comparison of apo- and holo-proteins may elucidate the role of the heme group in the stability of intermediate states. Various acids differ in their potential to stabilize the intermediate state. Titration with hydrochloric acid (HCl) is a standard method to follow the maximum acid-induced unfolding and subsequent acid-induced refolding. In a case in which the acid-induced refolding is not observed with HCl, the effects of perchloric acid should be checked, because it is more efficient at inducing refolding, and the far-ultraviolet (UV) absorbance is extremely low.

Salt-induced formation of the molten globule state of apomyoglobin studied by isothermal titration calorimetry

Abstract Whereas apomyoglobin is unfolded at pH 2 by HCl in the absence of salt, the addition of anions either from salts or acids stabilizes the molten globule state, i.e. a compact denatured state with a significant native-like secondary structure but with largely flexible side-chains. To clarify the thermodynamic mechanism responsible for the conformational stability of the molten globule state of apomyoglobin, we studied the salt-induced formation of the molten globule state of horse apomyoglobin at pH 2 by isothermal titration calorimetry (ITC). By titrating the acid-unfolded apomyoglobin with NaClO 4 or Na 2 SO 4 , an exothermic reaction was observed. The titration curve obtained from the heat was cooperative and consistent with the conformational transition curve measured by circular dichroism at 222 nm. This suggested that the salt-induced conformation change can be approximated by a two-state transition between the acid-unfolded and molten globule states. However, the heat for formation of the molten globule state estimated by ITC was slightly larger in magnitude than the enthalpy change for unfolding of the salt-stabilized molten globule state at pH 2, suggesting a relatively small contribution of heat other than the conformational change. These results support a view that the conformational transition of apomyoglobin at pH 2 can be represented, as a first approximation, by a two-state transition between the molten globule state and the fully unfolded state.