School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, University College Dublin Conway Institute, University College Dublin, Dublin, Ireland.
Biophys J. 2012 Apr 4;102(7):1636-45. doi: 10.1016/j.bpj.2012.02.048. Epub 2012 Apr 3.
The majority of pK(a) values in protein unfolded states are close to the amino acid model pK(a) values, thus reflecting the weak intramolecular interactions present in the unfolded ensemble of most proteins. We have carried out thermal denaturation measurements on the WT and eight mutants of HEWL from pH 1.5 to pH 11.0 to examine the unfolded state pK(a) values and the pH dependence of protein stability for this enzyme. The availability of accurate pK(a) values for the folded state of HEWL and separate measurements of mutant-induced effects on the folded state pK(a) values, allows us to estimate the pK(a) values of seven acidic residues in the unfolded state of HEWL. Asp-48 and Asp-66 display pK(a) values of 2.9 and 3.1 in our analysis, thus representing the most depressed unfolded state pK(a) values observed to date. We observe a strong correlation between the folded state pK(a) values and the unfolded state pK(a) values of HEWL, thus suggesting that the unfolded state of HEWL possesses a large degree of native state characteristics.
在蛋白质去折叠状态下，大多数 pK(a) 值接近氨基酸模型 pK(a) 值，因此反映了大多数蛋白质去折叠聚集体中存在的弱分子内相互作用。我们已经在 pH 1.5 到 pH 11.0 的范围内对 HEWL 的 WT 和 8 个突变体进行了热变性测量，以研究该酶的去折叠状态 pK(a) 值和蛋白质稳定性的 pH 依赖性。对于 HEWL 的折叠状态，我们获得了准确的 pK(a) 值，并且对突变体对折叠状态 pK(a) 值的影响进行了单独测量，这使我们能够估计 HEWL 去折叠状态下 7 个酸性残基的 pK(a) 值。在我们的分析中，Asp-48 和 Asp-66 的 pK(a) 值分别为 2.9 和 3.1，因此代表了迄今为止观察到的最受抑制的去折叠状态 pK(a) 值。我们观察到 HEWL 的折叠状态 pK(a) 值与去折叠状态 pK(a) 值之间存在很强的相关性，因此表明 HEWL 的去折叠状态具有很大程度的天然状态特征。
