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

Abstract

comparative analysis of the folding and misfolding pathways of the third PDZ domain of PSD95 investigated under different pH conditions. Biophysical Chemistry, Elsevier, 2011, 158 (2-3) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT AbstractEquilibrium 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-electronmicroscopy 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. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT IntroductionProtein aggregation is considered today to be a generic property of polypeptides and is thus a considerable problem for living organisms, which must develop response strategies to avoid its harmful effects. These effects span from arthritis to serious neurodegenerative diseases. At the molecular and energetic level, the self-organization mechanism of proteins relies on many properties related to both the protein system (hydrophobicity, β-sheet propensities etc.) and the solvent conditions (pH, ionic strength, organic reagents etc.). These properties can modulate the misfolding pathway in ways ranging from downhill to nucleationcooperative aggregation mechanisms.In spite of extensive reports in the literature, our knowledge of the basic processes by which polypeptide chains can give rise to well ordered supramacromolecular structures is still poorly understood [1]. It has been sug...

Post-Translational Modifications Modulate Ligand Recognition by the Third PDZ Domain of the MAGUK Protein PSD-95

“…As discussed above, Glu334 appears to play an important role in KKETAV binding via interactions with Lys-5 in the ligand. In a previous report, we proposed that these two salt-bridges are responsible for the pH dependency of the unfolding of the PDZ3 domain, which changes dramatically below pH 3.5 due to salt-bridge relaxation associated with the titration of solvent-exposed Glu residues [32]. The results presented here support the concept that the Glu401-Lys355 and Arg399-Glu334 salt-bridges play a critical role in binding linked to the α3 helix region.…”

“…This interaction also occurs during binding of the CRIPT peptide to PSD-95-PDZ3 [30] and is considered a key interaction in class I PDZ domains [31]; however, to our knowledge, the energetic consequences of pH changes on this interaction have not yet been evaluated. Considering the lack of other titratable residues within this pH range in the vicinity of the PSD-95-PDZ3 binding site, and the fact that no substantial differences in stability and folding behaviour have been observed for the domain between pH 4.0 and pH 7.5 [32], it is reasonable to argue that entropic differences might arise from changes in the conformational entropy of ligand side chains interacting with His372 and not from significant conformational changes in the PDZ3 domain.…”

The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

“…We carried out experiments at 1.3 mg·mL −1 protein concentration under different pH conditions; within the range 2.5–3.5 we used Glycine/HCl and at pH 4.0 acetic/acetate buffer. The behaviour is qualitatively similar to PDZ3, since at pH values 4.0–7.5 traces are biphasic (Figure 2) and at more acidic conditions both transitions approach to a single transition but, differently, we could not properly fit any of them to the two-state model as done in PDZ3 [23]. In fact, fittings in Figure 2 were performed using whether the four-state model described above for pH 4.0, or the three-state model previously described for PDZ3 for traces at lower pH values.…”

“…Since a change in the conformational equilibrium of PDZ3 below pH 3 exists [23], we have studied here the pH behaviour of the Δ10ct-PDZ3 domain by DSC. We carried out experiments at 1.3 mg·mL −1 protein concentration under different pH conditions; within the range 2.5–3.5 we used Glycine/HCl and at pH 4.0 acetic/acetate buffer.…”

“…This titration behaviour might be attributed to the protonation equilibriums of some Glu and/or Asp residues, the only ones whose pK a values are within the range of pH 3–4 [23]. This evidence strongly pointed to some of the above mentioned Glu residues, concretely to Glu334 and Glu401, since these are the main responsible for the packing of the extra α3-helix to the domain through salt-bridges with residues Lys355 and Arg399, respectively [18].…”