A thiol labelling competition experiment as a probe for sidechain packing in the kinetic folding intermediate of N-PGK
A thiol labelling competition experiment as a probe for sidechain packing in the kinetic folding intermediate of N-PGK
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DOI：
10.1016/j.jmb.2006.09.014
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发表时间：
2006-12-08
影响因子：
5.6
通讯作者：
Waltho, Jonathan P.
中科院分区：
文献类型：
--
作者：
Cliff, Matthew J.;Alizadeh, Tooba;Waltho, Jonathan P.
蛋白质折叠由沿着多肽骨架的侧链序列指导，但尽管如此，折叠过程中侧链相互作用的发展还没有得到很好的理解。沿着多肽骨架的侧链序列指导蛋白质折叠。在这里，硫醇活性试剂，二硫代硝基苯甲酸（DTNB），用于探测暴露的半胱氨酸侧链硫醇的N-末端结构域的磷酸甘油酸激酶（N-PGK）的动力学折叠中间体和一些保守的（I-，L-，或V-到-C）单半胱氨酸变体。在DTNB存在下，将化学变性蛋白质快速稀释到折叠条件下，可以通过分析标记动力学来确定任何快速形成的中间体中的侧链保护程度。中间体的保护因子通常较小（< 25），表明侧链仅部分掩埋。保护的分布与先前报道的NPGK折叠中间体的骨架酰胺保护平行。这些观察结果与这样的中间体类似于熔融球状态的假设一致;即具有天然样骨架氢键和整体三级结构，但具有构成疏水蛋白质核心的侧链动态和间歇性溶剂暴露。成功的竞争技术在表征这种动力学中间邀请应用到其他模型系统。(c)2006爱思唯尔有限公司保留所有权利。
Protein folding is directed by the sequence of sidechains along the polypeptide backbone, but despite this the developement of sidechain interactions during folding is not well understood. Here, the thiol-active reagent, dithio-nitrobenzoic acid (DTNB), is used to probe the exposure of the cysteine sidechain thiols in the kinetic folding intermediates of the N-terminal domain of phosphoglycerate kinase (N-PGK) and a number of conservative (I-, L-, or V-to-C) single cysteine variants. Rapid dilution of chemically denatured protein into folding conditions in the presence of DTNB allowed the degree of sidechain protection in any rapidly formed intermediate to be determined through the analysis of the kinetics of labelling. The protection factors derived for the intermediate (s) were generally small (< 25), indicating only partial burial of the sidechains. The distribution of protection parallels the previously reported backbone amide protection for the folding intermediate of NPGK. These observations are consistent with the hypothesis that such intermediates resemble molten globule states; i.e. with native-like backbone hydrogen bonding and overall tertiary structure, but with the sidechains that make up the hydrophobic protein core dynamic and intermittently solvent exposed. The success of the competition technique in characterizing this kinetic intermediate invites application to other model systems. (c) 2006 Elsevier Ltd. All rights reserved.