Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
J Chem Phys. 2013 Sep 28;139(12):121930. doi: 10.1063/1.4820490.
The dimensions of intrinsically disordered and unfolded proteins critically depend on the solution conditions, such as temperature, pH, ionic strength, and osmolyte or denarurant concentration. However, a quantitative understanding of how the complex combination of chain-chain and chain-solvent interactions is affected by the solvent is still missing. Here, we take a step towards this goal by investigating the combined effect of pH and denaturants on the dimensions of an unfolded protein. We use single-molecule fluorescence spectroscopy to extract the dimensions of unfolded cold shock protein (CspTm) in mixtures of the denaturants urea and guanidinium chloride (GdmCl) at neutral and acidic pH. Surprisingly, even though a change in pH from 7 to 2.9 increases the net charge of CspTm from -3.8 to +10.2, the radius of gyration of the chain is very similar under both conditions, indicating that protonation of acidic side chains at low pH results in additional hydrophobic interactions. We use a simple shared binding site model that describes the joint effect of urea and GdmCl, together with polyampholyte theory and an ion cloud model that includes the chemical free energy of counterion interactions and side chain protonation, to quantify this effect.
无规和去折叠蛋白质的尺寸严重依赖于溶液条件，如温度、pH 值、离子强度、渗透压或变性剂浓度。然而，对于溶剂如何影响链-链和链-溶剂相互作用的复杂组合，我们仍然缺乏定量的理解。在这里，我们通过研究 pH 值和变性剂对去折叠冷休克蛋白（CspTm）尺寸的综合影响，朝着这个目标迈出了一步。我们使用单分子荧光光谱法在中性和酸性 pH 值下，在脲和盐酸胍（GdmCl）变性剂的混合物中提取去折叠冷休克蛋白（CspTm）的尺寸。令人惊讶的是，尽管 pH 值从 7 增加到 2.9 会使 CspTm 的净电荷从-3.8 增加到+10.2，但在这两种条件下，链的回转半径非常相似，这表明低 pH 值下酸性侧链的质子化会导致额外的疏水相互作用。我们使用一个简单的共享结合位点模型来描述脲和 GdmCl 的联合效应，同时使用聚两性电解质理论和一个包含抗衡离子相互作用和侧链质子化的化学自由能的离子云模型来量化这种效应。
