02266nas a2200217   4500008004100000022001400041245010100055210006900156260000800225300001100233490000800244520150100252653003101753653003001784653002801814653002201842100001901864700001701883700002401900856012401924       2021                        eng d  a0301-462200apH-dependent protein stability switch coupled to the perturbed pKa of a single ionizable residue0 apHdependent protein stability switch coupled to the perturbed pK  cJUL  a1065910 v2743 a<p>The contribution of electrostatic interactions in protein stability has not been fully understood. Burial of an ionizable amino acid inside the hydrophobic protein core can affect its ionization equilibrium and shift its pKa differentially in the native (N) and unfolded (U) states of a protein and this coupling between the folding/ unfolding cycle and the ionization equilibria of the ionizable residue can substantially influence the protein stability. Here, we studied the coupling of the folding/unfolding cycle with the ionization of a buried ionizable residue in a multi-domain protein, Human Serum Albumin (HSA) using fluorescence spectroscopy. A pHdependent change in the stability of HSA was observed in the near native pH range (pH 6.0-9.0). The protonation-deprotonation equilibrium of a single thiol residue that is buried in the protein structure was identified to give rise to the pH-dependent protein stability. We quantified the pKa of the thiol residue in the N and the U states. The mean pKa of the thiol in the N state was upshifted by 0.5 units to 8.7 due to the burial of the thiol in the protein structure. Surprisingly, the mean pKa of the thiol in the U state was observed to be downshifted by 1.3 units to 6.9. These results indicate that some charged residues are spatially proximal to the thiol group in the U state. Our results suggest that, in addition to the N state, electrostatic interactions in the U state are important determinants of protein stability.</p>10aelectrostatic interactions10aFluorescence spectroscopy10aThermodynamic stability10aUnfolded proteins1 aMishra, Prajna1 aPatni, Divya1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/ph-dependent-protein-stability-switch-coupled-perturbed-pka-single-ionizable-residue02040nas a2200157   4500008004100000022001400041245011600055210006900171260000900240300001400249490000800263520144100271100001701712700002401729856012901753       2021                        eng d  a1520-610600aProtonation-deprotonation switch controls the amyloid-like misfolding of nucleic-acid-binding domains of TDP-430 aProtonationdeprotonation switch controls the amyloidlike misfold  cAUG   a8383-83940 v1253 aNutrient starvation stress acidifies the cytosol and leads to the formation of large protein assemblies and misfolded aggregates. However, how starvation stress is sensed at the molecular level and leads to protein misfolding is poorly understood. TDP-43 is a vital protein, which, under stress-like conditions, associates with stress granule proteins via its functional nucleic-acid-binding domains (TDP-43(t)(RRM)) and misfolds to form aberrant aggregates. Here, we show that the monomeric N form of TDP-43(t)(RRM) forms a misfolded amyloid-like protein assembly, beta form, in a pH-dependent manner and identified the critical protein side-chain residue whose protonation triggers its misfolding. We systematically mutated the three buried ionizable residues, D105, H166, and H256, to neutral amino acids to block the pH-dependent protonation-deprotonation titration of their side chain and studied their effect on the N-to-beta transition. We observed that D105A and H2S6Q resembled TDP-43(t)(RRM) in their pH-dependent misfolding behavior. However, H166Q retains the N-like secondary structure under low-pH conditions and does not show pH-dependent misfolding to the beta form. These results indicate that H166 is the critical side-chain residue whose protonation triggers the misfolding of TDP-43(t)(RRM) and shed light on how stress-induced misfolding of proteins during neurodegeneration could begin from site-specific triggers.1 aPatni, Divya1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/protonation-deprotonation-switch-controls-amyloid-misfolding-nucleic-acid-binding-domains