Long-range order in the src SH3 folding transition state
Long-range order in the src SH3 folding transition state
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DOI：
10.1073/pnas.97.13.7084
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发表时间：
2000-06-20
影响因子：
11.1
通讯作者：
Baker, D
中科院分区：
文献类型：
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作者：
Grantcharova, VP;Riddle, DS;Baker, D
蛋白质折叠中的一个突出问题涉及折叠过渡态系综的异质性程度：蛋白质是通过大量不同的“途径”折叠，还是天然结构的元素以明确的顺序组装？在这里，我们建立在以前的点突变研究的src SH 3直接调查的结构元素和骨干构象熵的损失在折叠过程中的协会。双突变分析的极性残基在远端β-发夹和发散的转折表明，这些元素之间的氢键网络主要是在折叠过渡态形成。10-甘氨酸插入的n-src环（其连接的远端发夹和发散的转弯）和二硫键交联的基础上的远端P-发夹排他地影响折叠速率，显示这些结构元素几乎是有序的折叠过渡状态中的天然状态。相比之下，交联的RT环或N和C末端的基础显着减慢展开速率，这表明末端的解离和RT环的开放先于展开中的限速步骤。总而言之，这些结果表明，折叠过渡态系综中的基本上所有构象都形成了中心三链β折叠，这表明对于SRC同源3结构域，折叠期间的结构组装存在离散顺序。
One of the outstanding questions in protein folding concerns the degree of heterogeneity in the folding transition state ensemble: does a protein fold via a large multitude of diverse "pathways," or are the elements of native structure assembled in a well defined order'? Herein, we build on previous point mutagenesis studies of the src SH3 by directly investigating the association of structural elements and the loss of backbone conformational entropy during folding. Double-mutant analysis of polar residues in the distal beta-hairpin and the diverging turn indicates that the hydrogen bond network between these elements is largely formed in the folding transition state. A 10-glycine insertion in the n-src loop (which connects the distal hairpin and the diverging turn) and a disulfide crosslink at the base of the distal P-hairpin exclusively affect the folding rate, showing that these structural elements are nearly as ordered in the folding transition state as in the native state. In contrast, crosslinking the base of the RT loop or the N and C termini dramatically slows down the unfolding rate, suggesting that dissociation of the termini and opening of the RT loop precede the rate-limiting step in unfolding. Taken together, these results suggest that essentially all conformations in the folding transition state ensemble have the central three-stranded beta-sheet formed, indicating that, for the src homology 3 domain, there is a discrete order to structure assembly during folding.