An engineered disulfide cross-link accelerates the refolding rate of calcium-free subtilisin by 850-fold.
An engineered disulfide cross-link accelerates the refolding rate of calcium-free subtilisin by 850-fold.
复制标题
工程化二硫键交联可将无钙枯草杆菌蛋白酶的重折叠速度加快 850 倍。
DOI：
10.1021/bi00090a012
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Bryan,P
中科院分区：
文献类型：
--
作者：
Strausberg,S;Alexander,P;Wang,L;Gallagher,T;Gilliland,G;Bryan,P
摘要：枯草杆菌蛋白酶的成熟形式是单体蛋白质的一个不寻常的例子，它对折叠和展开具有很高的动力学障碍。使用枯草杆菌蛋白酶BPN '的定点突变，我们试图确定动力学屏障的物理和能量性质。高亲和力的钙结合位点A已被证明可以产生一个大的解折叠屏障。通过删除氨基酸75-83从枯草杆菌蛋白酶中去除钙结合位点A大大加速了解折叠和重折叠反应。这里，在75-83枯草杆菌蛋白酶中的残基22和87之间引入二硫键交联。这样做是为了探测折叠过渡态的构象熵。报道了该突变体的1.8-X射线结构和交联对展开和重折叠动力学的影响。与预期的由于交联而导致的未折叠蛋白质熵损失一致，二硫键相对于未交联形式加速折叠。折叠速率的加速幅度（在25 ℃下为700-850倍）表明残基22和87在过渡态中是有序的，使得二硫键不影响其总熵。虽然围绕氨基酸22和87的结构的早期组织大大加速了折叠，但我们不知道在没有交联的情况下，该区域的早期折叠是否是高度聚集的折叠途径。75-83枯草杆菌蛋白酶折叠反应中的缓慢步骤可能是形成能够传播折叠反应的初始结构。因此，任何稳定天然拓扑结构的突变（或离子条件）都可能加速其折叠速率。
Revised Manuscript Received July 19, 1993® abstract: The mature form of subtilisin is an unusual example of a monomeric protein with a high kinetic barrier to folding and unfolding. Using site-directed mutagenesis of subtilisin BPN', we are attempting to determine the physical and energetic nature of the kinetic barrier. The high-affinity calcium-binding site A has been shown to create a large enthalpicbarrier to unfolding. Removing the calcium-binding site A from subtilisin by deleting amino acids 75-83 greatly accelerates both unfolding and refolding reactions. Here a disulfide cross-link is introduced between residues 22 and 87 in 75-83 subtilisin. This was done to probe the conformational entropy of the transition state for folding. The 1.8-Á X-ray structure of this mutant and the effects of the cross-link on the kinetics of unfolding and refolding are reported. Consistent with an expected loss of entropy of the unfolded protein due to the cross-link, the disulfide accelerates folding relative to the uncross-linkedform. The magnitude of the acceleration of folding rate (700-850-fold at 25 C) indicates that residues 22 and 87 are ordered in the transition state such that the disulfide does not affect its total entropy. Although early organization of structure around amino acids 22 and 87 greatly accelerates folding, we do not know whether the earlyfolding of this region is a highly populated folding pathway in the absence of the cross-link. The slow step in the 75-83 subtilisin folding reaction may be forming initial structures capable of propagating the folding reaction. Any mutation (or ionic condition) which stabilizes a native-like topology may therefore accelerate its folding rate.