The side chain of aspartic acid 69 dictates the folding mechanism of Bacillus subtilis HPr.
The side chain of aspartic acid 69 dictates the folding mechanism of Bacillus subtilis HPr.
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天冬氨酸 69 的侧链决定了枯草芽孢杆菌 HPr 的折叠机制。
DOI：
10.1021/bi0357412
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Scholtz,JMartin
中科院分区：
文献类型：
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作者：
Schmittschmitt,JasonP;Scholtz,JMartin
许多小的单结构域蛋白质表现出平衡和动力学折叠机制，似乎可以充分描述为两种状态。双态模型做出了几个可以通过实验验证的预测。首先，在一组参比条件下测定或外推至一组参比条件的构象稳定性应与测量方法（热或溶剂变性或氢交换）无关。其次，从直接量热法确定的主要热力学参数（Tm，ΔH）的模型独立测量值应与从热去折叠数据的双态分析确定的那些相同。第三，动力学折叠和去折叠速率常数的比值应等于在相同条件下由平衡测量确定的Keq。在这里，我们表明，野生型HPr蛋白从枯草芽孢杆菌不符合所有这些标准，在我们的标准条件下。然而，如果我们替换Asp69的侧链，或者添加中等浓度的盐，我们发现在平衡和动力学折叠中都有极好的双态行为。因此，对于这种蛋白质和可能的其他蛋白质，一级结构或溶液条件的非常细微的变化可以显著改变天然中间体的相对稳定性，并且未折叠的集合体可以引起折叠机制性质的可观察变化。
Many small, single-domain proteins show equilibrium and kinetic folding mechanisms that appear to be adequately described as two state. The two-state model makes several predictions that can be tested experimentally. First, the conformational stability determined at or extrapolated to a set of reference conditions should be independent of the measurement method (thermal or solvent denaturation or hydrogen exchange). Second, model-independent measures of the cardinal thermodynamic parameters (Tm, ΔH) as determined from direct calorimetric means should be identical to those determined from the two-state analysis of thermal unfolding data. Third, the ratio of the kinetic folding and unfolding rate constants should be equal toKeqdetermined from an equilibrium measurement under the same conditions. Here, we show that the wild-type HPr protein fromBacillus subtilisdoes not meet all of these criteria under our standard conditions. However, if we replace the side chain of Asp69, or add moderate concentrations of salt, we find excellent two-state behavior in both equilibrium and kinetic folding. Thus, for this protein and possibly others, very subtle changes in the primary structure or in the solution conditions can dramatically alter the relative stabilities of the native intermediate, and unfolded ensembles can cause an observable change in the nature of the folding mechanism.