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Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation, exhibiting two endothermic peaks because of the formation of reversible oligomers at high temperature (N↔I(6)↔D). This contrasts sharply with the standard two-state denaturation model observed for small, globular proteins. We performed an alanine scanning analysis by individually mutating three hydrophobic residues at the crystallographic oligomeric interface (Phe340, Leu342, and Ile389) and one away from the interface (Leu349, as a control). DSC analysis indicated that PDZ3-F340A and PDZ3-L342A exhibited a single endothermic peak. Furthermore, PDZ3-L342A underwent a perfect two-state denaturation, as evidenced by the single endothermic peak and confirmed by detailed DSC analysis, including global fitting of data measured at different protein concentrations. Reversible oligomerization (RO) at high temperatures by small globular proteins is a rare event. Furthermore, our present study showing that a point mutation, L342A, designed based on the crystal structure inhibited RO is surprising because RO occurs at a high-temperature. Future studies will determine how and why mutations designed using crystal structures determined at ambient temperatures influence the formation of RO at high temperatures, and whether high-temperature ROs are related to the propensity of proteins to aggregate or precipitate at lower temperatures, which would provide a novel and unique way of controlling protein solubility and aggregation.

The e subunit of F1-ATPase acts as an endogenous inhibitor of the ATPase activity in F1-ATPase. Biochemical and structural studies have shown that the e subunit from the thermophilic Bacillus strain PS3 (TF1) binds ATP specifically, and the ATP binding induces the conformational change of the C-terminal domain of the e subunit from the extended form to the folded form that allows ATP hydrolysis in F1 motor. The mechanism of how ATP binding induces the conformational change of the e subunit remains unclear, because the atomic detail of the conformational ensemble of the ATP-free form is still not known.In this study, to address the above question, we used molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS) experiments. Analysis of the SAXS data measured at SPring-8 has shown that the overall structural characteristics of the ATP-bound form in solution are consistent with the crystal structure, while the molecular shape determined for the ATP-free form shows a more expanded conformation. We performed MD simulations for both the ATP-bound and ATP-free forms to obtain conformational ensemble of these forms, and the validity of the calculated ensembles was checked by a comparison of simulation-derived SAXS profiles with the experimentally observed profiles.