A comparative analysis of the folding and misfolding pathways of the third PDZ domain of PSD95 investigated under different pH conditions

Equilibrium unfolding at neutral pH of the third PDZ domain of PSD95 is well described by the presence of a partly unfolded intermediate that presents association phenomena. After some days' incubation annular and fibrillar structures form from the oligomers. At pH values below 3, however, differential scanning calorimetry shows that PDZ3 seems to unfold under a two-state scheme. Kinetic measurements followed by dynamic light scattering, ThT and ANS fluorescence reveal that the misfolding pathway still exists despite the absence of any populated intermediates and shows an irreversible assembling of the supramacromolecular structures as well as an appreciable lag-phase, contrary to what is found in similar experiments at neutral pH. Moreover, as shown by transmission-electron-microscopy images, the annular structures seen at neutral pH completely disappear from incubated solutions. According to the structural information, this titration behavior appears to be the consequence of a conformational equilibrium that depends on the protonation of some Glu residues located at the C-terminal α3 helix and at the hairpin formed by strands β2 and β3. Our calculations suggest that the enthalpic contribution of these interactions may well be as much as 40 kJ·mol−1. The possible regulatory role of this equilibrium upon PDZ3 functionality and amyloid formation is briefly discussed.

Research highlights► Unfolding at pH > 3.5 of PSD95-PDZ3 reveals the presence of an equilibrium oligomeric intermediate state that self-associates into annular and fibrillar structures reversibly. ► At pH < 3 PDZ3 seems to unfold under a two-state scheme, although the misfolding pathway still exists. ► The supramacromolecular structures organize irreversibly and show a lag-phase at acidic pH. ► This titration behavior is due to a conformational equilibrium that depends on the protonation of some Glu residues. ► The enthalpic contribution of these interactions may well be as much as 40 kJ·mol−1.