An Oligomeric Equilibrium Intermediate as the Precursory Nucleus of Globular and Fibrillar Supramacromolecular Assemblies in a PDZ Domain

The equilibrium unfolding at neutral pH of the third PDZ domain of PSD95, as followed by DSC, is characterized by the presence of an equilibrium intermediate with clear signs of oligomerization. DLS and SEC measurements indicate that at 60–70°C small oligomers populate, showing a typical β-sheet far-UV CD spectrum. These intermediate species lead to the formation of rodlike particulates of ∼12 nm, which remain in solution after 2 weeks incubation and grow until they adopt annular/spherical shapes of ∼50 nm and protofibrils, which are subsequently fully transformed into fibrils. The fibrils can also disaggregate after the addition of 1:1 buffer dilution followed by cooling to room temperature, thus returning to the initial monomeric state. Growth kinetics, as shown by ThT and ANS fluorescence, show that the organization of the different supramacromolecular structures comes from a common nucleation unit, the small oligomers, which organize themselves before reaching the incubation temperature of 60°C. Our experiments point toward the existence of a well-defined reversible, stepwise, and downhill organization of the processes involved in the association-dissociation of the intermediate. We estimate the enthalpy change accompanying the association-dissociation equilibria to be 130 kJ × mol−1. Furthermore, the coalescence under essentially reversible conditions of different kinds of supramacromolecular assemblies renders this protein system highly interesting for biophysical studies aimed at our further understanding of amyloid pathological conditions.