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

Abstract

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 degrees C small oligomers populate, showing a typical beta-sheet far-UV CD spectrum. These intermediate species lead to the formation of rodlike particulates of approximately 12 nm, which remain in solution after 2 weeks incubation and grow until they adopt annular/spherical shapes of approximately 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 degrees 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 x 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.

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

“…As we showed in a previous work [4], the thermal unfolding of PDZ3 at neutral pH reveals the presence of an oligomeric equilibrium intermediate that populates maximally at around 60-70 ºC. The DSC traces comprise two well separated unfolding transitions, which can be fully described by a three-state association-dissociation equilibrium model (nN ⇄ I n ⇄ nU).…”

“…PDZ3 experimental conditions were 50 mM buffer (either phosphate at pH 7.5, acetate at pH 4.0 or glycine/HCl at pH 2.0-3.5). DLS, CD and ThT and ANS fluorescence experimental details can be obtained from previous references [4,11]. TEM images of amyloid fibrils were made at the CIC services of the University of Granada.…”

“…The established three-state model including an oligomeric intermediate used to fit DSC traces above pH 3 (nN ⇄ I n ⇄ nU) has also been described previously by us [4]. Linear functions for the heat-capacity functions C pN (T), C pI (T) and C pU (T) were always used.…”

“…In a previous work [4] we explored the misfolding pathway of the third PDZ domain of the PSD95 neuronal protein (PDZ3), comprising residues 302 to 402 of PSD95. At neutral pH at 60-70 ºC in plain water-buffered solutions the PDZ3 domain populates a trimeric β-sheet-rich intermediate state that leads to a stepwise and reversible formation of annular and protofibrillar structures, which remain in solution for various days and finally give rise to fibrils.…”

Application and use of differential scanning calorimetry in studies of thermal fluctuation associated with amyloid fibril formation

“…The thermodynamic data on the stability of amyloid fibrils obtained by DSC (Morel et al 2010) and ITC (Kardos et al 2004) indicate the contribution of entropy increase attained with their assembly, which points toward hydration effects playing a major role in this process. Previous DSC studies have also shown that thermalinduced aggregation of several proteins is accompanied by the exothermic effect of heat (Dzwolak et al 2003;Stirpe et al 2008;Attanasio et al 2009;Murciano-Calles et al 2010). Such an effect may be explained by a nucleation model, i.e., the formation of a stable nucleus that incorporates additional monomeric proteins into a growing aggregate (Dzwolak et al 2003), but the exact molecular basis remains elusive.…”