pH dependent thermodynamic and amide exchange studies of the C-terminal domain of the ribosomal protein L9: implications for unfolded state structure.
pH dependent thermodynamic and amide exchange studies of the C-terminal domain of the ribosomal protein L9: implications for unfolded state structure.
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核糖体蛋白 L9 C 端结构域的 pH 依赖性热力学和酰胺交换研究：对未折叠状态结构的影响。
DOI：
10.1021/bi052534o
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Raleigh,DanielP
中科院分区：
文献类型：
--
作者：
Li,Ying;Horng,Jia-Cherng;Raleigh,DanielP
现在人们认识到球状蛋白的未折叠状态不是随机卷曲，而是可以包含大量的残余结构。在这里，我们结合酰胺 H/D 交换研究和热力学测量来探测小型混合 α-β 蛋白 CTL9 未折叠状态下的 pH 依赖性结构。通过尿素变性测量的值强烈依赖于 pD，从 pD 7.5 增加到 4.85，增加了 40%。同样，展开时热容的变化 ΔCp° 从 pD 7.5 显着增加到 5.5。这些研究认为，展开状态包含相互作用，可能本质上是疏水性的，这会导致在高 pH 值下形成更紧凑的状态。较低 pH 下的扩增与 CTL9 中三个组氨酸的估计未折叠状态 pKa 值相关，以及较低 pH 下酸性侧链的额外贡献。在 pD 5.0、6.0 和 7.0 下进行酰胺 H/D 交换研究。在 pD 5.0 时，可以测量 44 个残基的交换率，其中 29 个残基通过全局展开进行交换。没有发现任何超级受保护站点的证据，即交换速率低于全球交换预期速率的站点。实验的估计精度将检测范围限制为比内在汇率高 2.3 倍的受保护残留物。在 pD 6 处可追踪 37 个残基，在 pD 7 处可追踪 27 个残基。同样没有观察到显着的超级保护结构的证据。 CTL911 的特性与其他结构变性状态进行了比较。
It is now recognized that unfolded states of globular proteins are not random coils but instead can contain significant amounts of residual structure. Here, we combine amide H/D exchange studies and thermodynamic measurements to probe pH dependent structure in the unfolded state of the small, mixed α-β protein CTL9. Themvalue measured by urea denaturation is strongly dependent upon pD, increasing by 40% from pD 7.5 to 4.85. Likewise, the change in heat capacity upon unfolding, ΔCp°, increases significantly from pD 7.5 to 5.5. These studies argue that the unfolded state contains interactions, presumably hydrophobic in nature, that lead to a more compact state at high pH. The expansion at lower pH correlates with the estimated unfolded state pKavalues of the three histidines in CTL9 with additional contributions from acid side chains at the lower pH. Amide H/D exchange studies were conducted at pD 5.0, 6.0, and 7.0. At pD 5.0, the exchange rates could be measured for 44 residues, 29 of which exchanged by global unfolding. No evidence was found for any super protected sites, that is, sites that exchange at rates slower than those expected for global exchange. The estimated precision for the experiments limits detection to residues that are protected 2.3-fold above the intrinsic exchange rate. Thirty-seven residues could be followed at pD 6 and 27 residues at pD 7. Again no evidence for a significant super protected structure was observed. The properties of CTL911are compared to other structured denatured states.