Structural rearrangements and the unfolding mechanism of a Trigger Factor mutant studied by multiple structural probes.
Structural rearrangements and the unfolding mechanism of a Trigger Factor mutant studied by multiple structural probes.
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DOI：
10.1016/j.bbapap.2009.03.008
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发表时间：
2009-06
期刊：
影响因子：
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通讯作者：
Dong-Jie Fan;Yan-Wei Ding;Jun‐mei Zhou
中科院分区：
文献类型：
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作者：
Dong-Jie Fan;Yan-Wei Ding;Jun‐mei Zhou
触发因子(Trigger Factor，TF)是一种具有三个结构域的伴侣蛋白，催化新生多肽折叠，具有多肽-脯氨酰顺式-反式异构酶活性。Tf的多域结构使其成为研究单个结构域或组合域结构的结构性质和功能行为的有趣和具有挑战性的候选分子。在这里，我们构建了一个Tf突变体NC，将负责Tf伴侣功能的N-结构域和C-结构域结合在一起，通过监测荧光光谱、远紫外CD、化学交联、DSC以及与疏水探针(ANS或BIS-ANS)的结合，比较了NC和野生型Tf的结构变化和去折叠特性。结果表明，与完整的转铁蛋白一样，NC结构可以与疏水探针结合，在溶液中形成二聚体，并且呈现出类似的三态胍诱导的去折叠轮廓。然而，NC片段对胍的展开和热变性的稳定性都降低了，这表明Tf的M结构域的存在有助于完整Tf结构的稳定性。
Trigger Factor (TF) is a three-domain chaperone which catalyzes nascent peptide folding and harbors peptidyl–prolyl cis–trans isomerase activity. The multi-domain structure of TF makes it an interesting and challenging candidate for studies of the structural properties and functional behavior of individual domains or combined domain constructs. Here we constructed a TF mutant, NC, combining the N- and C-domains that are responsible for TF's chaperone function, and compared structural changes and unfolding characteristics of NC with wild-type TF by monitoring fluorescence spectra, far-UV CD, chemical crosslinking, DSC and binding with hydrophobic probes (ANS or bis-ANS). The results showed that the NC construct, like intact TF, could bind to hydrophobic probes, form dimers in solution, and showed a similar 3-state guanidine-induced unfolding profile. However, the NC fragment showed reduced stability towards both guanidine unfolding and thermal denaturation, suggesting that the presence of the M-domain of TF contributes to the stability of the intact TF structure.