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首页> 外文期刊>Biochemistry >New approach to the study of transient protein conformations: the formation of a semiburied salt link in the folding pathway of barnase.
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New approach to the study of transient protein conformations: the formation of a semiburied salt link in the folding pathway of barnase.

机译：研究瞬时蛋白质构象的新方法：在Barnase的折叠途径中形成半埋式盐键。

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摘要

We use in this study a novel kinetic approach to determine the H+ titration properties of a semiburied salt link in the transition state for unfolding of barnase. The approach is based on changes in the pH dependence of the kinetics upon mutation of a target residue. This makes it relatively insensitive to the absolute value of the stability and, thereby, to artifacts caused by structural rearrangements around the site of mutation. The semiburied salt bridge studied here is between Asp93 and Arg69. Mutation of either residue significantly destabilized the protein, and the pKa value of Asp93 is severely lowered in the native state to below 1 because of the ionic interaction with Arg69. The Asp93-Arg69 salt link appears to be formed early in the folding process; the pKa value of Asp93 in the transition state (approximately 1) is similar to that in the native state, and deletion of the ionic interaction with Arg69 substantially destabilizes the folding intermediate and changes the kinetic behavior from multistate to two-state or close to two-state, depending on the conditions. The results suggest that the formation of ionic interactions within clusters of hydrophobic residues can be important for early folding events and can control kinetically the folding pathway. This is not because of the inherent stability of the salt link but because the presence of two unpaired charges is very unfavorable. The data reveal also that fractional phi values are consistent with a uniformly expanded transition state or one with closely spaced energy levels and not with parallel folding pathways.
机译：在这项研究中，我们使用了一种新颖的动力学方法来确定半掩埋盐键在过渡状态下的H +滴定特性，以释放barnase。该方法基于目标残基突变后动力学的pH依赖性变化。这使其对稳定性的绝对值相对不敏感，从而对突变位点周围的结构重排所引起的伪像不敏感。此处研究的半埋式盐桥位于Asp93和Arg69之间。任一残基的突变都会使蛋白质不稳定，并且由于与Arg69发生离子相互作用，Asp93的pKa值在天然状态下严重降低至1以下。 Asp93-Arg69盐键似乎在折叠过程的早期形成;过渡态（大约1）的Asp93的pKa值与天然态相似，缺失与Arg69的离子相互作用会大大破坏折叠中间体的稳定性，并将动力学行为从多态变为两态或接近两态状态，具体取决于条件。结果表明，疏水残基簇中离子相互作用的形成对于早期折叠事件可能很重要，并且可以动力学控制折叠路径。这不是因为盐键的固有稳定性，而是因为两个不成对电荷的存在是非常不利的。数据还表明，分数phi值与均匀扩展的过渡状态或具有紧密间隔的能级且不具有平行折叠路径的phi相一致。

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