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首页> 外文期刊>Biochemistry >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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pH dependent thermodynamic and amide exchange studies of the C-terminal domain of the ribosomal protein L9: Implications for unfolded state structure

机译：pH依赖的核糖体蛋白L9 C末端结构域的热力学和酰胺交换研究：对未折叠状态结构的影响

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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 alpha-beta protein CTL9. The m value 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, Delta Cp degrees, 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 pK(a) values 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 CTL9(11) are compared to other structured denatured states.
机译：现在已经认识到，球形蛋白质的未折叠状态不是随机的卷曲，而是可以包含大量的残留结构。在这里，我们结合酰胺H / D交换研究和热力学测量，以探测小的，混合的α-β蛋白CTL9处于未折叠状态的pH依赖性结构。通过尿素变性测量的m值在很大程度上取决于pD，从pD 7.5到4.85增加40％。同样，展开时的热容变化Delta Cp度从pD 7.5显着增加到5.5。这些研究认为，未折叠的状态包含相互作用，可能是疏水性的，在高pH值下会导致更紧密的状态。在较低pH下的扩增与CTL9中三个组氨酸的估计的未折叠状态pK（a）值相关，而在较低pH下酸侧链的贡献更大。酰胺H / D交换研究分别在pD 5.0、6.0和7.0进行。在pD 5.0时，可以测量44个残基的汇率，其中29个残基通过全局展开进行交换。没有发现任何超级保护站点的证据，即那些站点的交换速率低于全球交换所预期的速率。实验估计的精确度将检测范围限制为被保护为高于固有交换速率2.3倍的残基。在pD 6处可追踪37个残基，在pD 7处追踪27个残基。再次未观察到显着的超级保护结构的证据。将CTL9（11）的属性与其他结构化变性状态进行比较。

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