The native state conformational ensemble of the SH3 domain from alpha-spectrin.
The native state conformational ensemble of the SH3 domain from alpha-spectrin.
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来自 α-血影蛋白的 SH3 结构域的天然状态构象集合。
DOI：
10.1021/bi990413g
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Freire,E
中科院分区：
文献类型：
--
作者：
Sadqi,M;Casares,S;Abril,MA;Lopez-Mayorga,O;Conejero-Lara,F;Freire,E
通过NMR检测的氢/氘交换和差示扫描量热法测量了α-血影蛋白SH 3结构域的折叠/解折叠平衡。在天然条件下，已经获得了超过一半的残基在域中的保护因子对交换。大多数受保护的残基位于β链、短310螺旋和长RT环的一部分，而连接二级结构元件的环没有显示出可测量的保护。每个残基的表观稳定常数和它们相应的吉布斯自由能已从交换实验计算。SH 3结构域的最稳定区域由β链的中心部分限定。另一方面，肽结合区由一个高度稳定的区域（残基53 - 57）和一个高度不稳定的区域组成，即残基34 - 41之间的环（n-Src环）。域中的所有残基具有低于通过差示扫描量热法测量的全局展开吉布斯能的表观吉布斯能，表明在我们的实验条件下，SH 3域中的所有残基的酰胺交换主要通过局部展开反应发生。一个基于结构的热力学分析，使我们能够正确地预测热力学的全球展开的域和定义的构象状态，定量占所观察到的模式的氢交换保护的合奏。这些结果表明，在天然条件下的SH 3域需要被认为是一个整体的构象和在这些条件下获得的氢交换数据不能被解释为一个两态平衡。蛋白质的特定区域能够进行独立的局部折叠/解折叠反应的观察表明，在天然条件下，合作相互作用的规模是区域性的，而不是全球性的。
The folding/unfolding equilibrium of the α-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the β-strands, the short 310helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the β-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53−57) and a highly unstable region, the loop between residues 34−41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential scanning calorimetry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reactions. A structure-based thermodynamic analysis has allowed us to predict correctly the thermodynamics of the global unfolding of the domain and to define the ensemble of conformational states that quantitatively accounts for the observed pattern of hydrogen exchange protection. These results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independent local folding/unfolding reactions indicates that under native conditions the scale of cooperative interactions is regional rather than global.