Thermodynamic analysis of point mutations inhibiting high-temperature reversible oligomer of PDZ3

Abstract

Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation exhibiting two endothermic peaks due to the formation of reversible oligomers at high temperature (N{leftrightarrow}I6{leftrightarrow}D). This contrasts sharply with the standard 2-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, 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 2-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. While we designed the mutations based on our previous study showing that a point mutation Val380 to a nonhydrophobic amino acid inhibited RO in DEN4 ED3, the results are nevertheless surprising since high-temperature RO involves proteins in a denatured state, as assessed by circular dichroism. 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. SignificanceDespite being a small globular protein, which normaly undergo a two-state unfolding, the thermal denaturation of PSD95-PDZ3, monitored by DSC, exhibited two endothermic peaks. The second peak resulted from a reversible oligomerization (RO) at high temperatures, which is, on its own, a rare phenomenon. In this study, we show that the substitution of a single hydrophobic residue to an alanine at the interface of the crystallographic tetramers inhibited high-temperature RO, resulting in a single endothermic peak. Future studies are required to determine why mutations designed using crystal structures determined at ambient temperatures can inhibit high-temperature RO, and whether the ROs are precursor of irreversible aggregation, If so, the present observations will provide an entirely new basis for creating aggregation-resistant proteins.