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首页> 外文期刊>Journal of Molecular Biology >Stiffness of the distal loop restricts the structural heterogeneity of the transition state ensemble in SH3 domains.
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Stiffness of the distal loop restricts the structural heterogeneity of the transition state ensemble in SH3 domains.

机译：远端环的刚度限制了SH3域中过渡态集合的结构异质性。

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Protein engineering experiments and Phi(F)-value analysis of SH3 domains reveal that their transition state ensemble (TSE) is conformationally restricted, i.e. the fluctuations in the transition state (TS) structures are small. In the TS of src SH3 and alpha-spectrin SH3 the distal loop and the associated hairpin are fully structured, while the rest of the protein is relatively disordered. If native structure predominantly determines the folding mechanism, the findings for SH3 folds raise the question: What are the features of the native topology that determine the nature of the TSE? We propose that the presence of stiff loops in the native state that connect local structural elements (such as the distal hairpin in SH3 domains) conformationally restricts TSE. We validate this hypothesis using the simulations of a "control" system (16 residue beta-hairpin forming C-terminal fragment of the GBl protein) and its variants. In these fragments the role of bending rigidity in determining the nature of the TSE can be directly examined without complications arising from interactions with the rest of the protein. The TSE structures in the beta-hairpins are determined computationally using cluster analysis and limited Phi(F)-value analysis. Both techniques prove that the conformational heterogeneity decreases as the bending rigidity of the loop increases. To extend this finding to SH3 domains a measure of bending rigidity based on loop curvature, which utilizes native structures in the Protein Data Bank (PDB), is introduced. Using this measure we show that, with few exceptions, the ordering of stiffness of the distal, n-src, and RT loops in the 29 PDB structures of SH3 domains is conserved. Combining the simulation results for beta-hairpins and the analysis of PDB structures for SH3 domains, we propose that the stiff distal loop restricts the conformational fluctuations in the TSE. We also predict that constraining the distal loop to be preformed in the denatured ensemble should not alter the nature of TSE. On the other hand, if the amino and carboxy terminals are cross-linked to form a circular polypeptide chain, the pathways and TSs are altered. These contrasting scenarios are illustrated using simulations of cross-linked WT beta-hairpin fragments. Computations of bending rigidities for immunoglobulin-like domain proteins reveal no clear separation in the stiffness of their loops. In the beta-sandwich proteins, which have large fractions of non-local native contacts, the nature of the TSE cannot be apparently determined using purely local structural characteristics. Nevertheless, the measure of loop stiffness still provides qualitative predictions of the ordered regions in the TSE of Ig27 and TenFn3.
机译：蛋白质工程实验和SH3结构域的Phi（F）值分析表明，它们的过渡态整体（TSE）受构象限制，即过渡态（TS）结构的波动很小。在src SH3和α-spectrinSH3的TS中，远端环和相关的发夹结构完整，而其余的蛋白质则相对无序。如果自然结构主要决定了折叠机制，那么SH3折叠的发现就引发了一个问题：自然拓扑的哪些特征决定了TSE的性质？我们提出在原始状态下连接局部结构元件（例如SH3域中的远端发夹）的刚性环的构象限制了TSE。我们使用“对照”系统（16个残基β-发夹形成GB1蛋白的C-末端片段）的模拟来验证该假设。在这些片段中，可以直接检查抗弯刚度在确定TSE性质方面的作用，而不会因与其余蛋白质相互作用而引起并发症。使用-类分析和有限的Phi（F）值分析计算确定β-发夹中的TSE结构。两种技术都证明，构象异质性随环的弯曲刚度的增加而降低。为了将此发现扩展到SH3域，引入了一种基于环曲率的弯曲刚度测量方法，该方法利用了蛋白质数据库（PDB）中的天然结构。使用此度量，我们表明，除少数例外，SH3域的29个PDB结构中的远端，n-src和RT循环的刚度顺序是保守的。结合β-发夹的模拟结果和SH3域的PDB结构的分析，我们建议刚性远端环限制TSE中的构象波动。我们还预测，约束远端环在变性集合中执行不应改变TSE的性质。另一方面，如果氨基和羧基末端被交联形成环状多肽链，则途径和TS被改变。这些相反的场景使用交联的WTβ-发夹片段的模拟进行了说明。免疫球蛋白样结构域蛋白的抗弯刚度计算表明其环的刚度没有明确的分离。在具有大量非局部天然接触的β夹心蛋白中，无法使用纯局部结构特征明显确定TSE的性质。尽管如此，环路刚度的度量仍然提供了Ig27和TenFn3的TSE中有序区域的定性预测。

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