Kinetic isotope effects reveal the presence of significant secondary structure in the transition state for the folding of the N-terminal domain of L9.
Kinetic isotope effects reveal the presence of significant secondary structure in the transition state for the folding of the N-terminal domain of L9.
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动力学同位素效应揭示了 L9 N 端结构域折叠的过渡态中存在显着的二级结构。
DOI：
10.1016/j.jmb.2007.02.084
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发表时间：
2007
影响因子：
5.6
通讯作者：
Raleigh,DanielP
中科院分区：
文献类型：
--
作者：
Sato,Satoshi;Raleigh,DanielP
我们目前对蛋白质折叠过渡态性质的理解主要取决于通过突变分析(ϕ值分析)绘制出单个侧链贡献的研究。这种方法虽然非常强大，但通常不能提供有关主链氢键形成的直接信息。在这里，我们报告了酰胺H/D同位素效应的研究结果，该研究探索了一个小的α−β蛋白(L9的N-末端结构域)在折叠过程中过渡态的氢键相互作用的发展。在恒定同位素组成的溶剂中用氚取代酰胺质子会破坏结构域的稳定，降低其展开的Tm和ΔG0。折叠率也有所下降。参数NTL9 H/D，即同位素取代对活化自由能的影响与平衡自由能之比，在D2O背景下为0.6，在H2O背景下为0.75，表明Φ在折叠的过渡态中发生了显著的蛋白质内氢键相互作用。这一值与更传统的过渡态位置测量方法非常一致，后者报告的是表面积的相对埋藏。这些结果提供了一幅包含重要二级结构的紧凑折叠过渡态的图片。间接分析认为，大部分动力学同位素效应来自蛋白质的β-折叠富集区，并表明该区域蛋白质内氢键的发展在NTL9的折叠过程中起着关键作用。
Our present understanding of the nature of the transition state for protein folding depends predominantly on studies where individual side-chain contributions are mapped out by mutational analysis (ϕ value analysis). This approach, although extremely powerful, does not in general provide direct information about the formation of backbone hydrogen bonds. Here, we report the results of amide H/D isotope effect studies that probe the development of hydrogen bonded interactions in the transition state for the folding of a small α−β protein, the N-terminal domain of L9. Replacement of amide protons by deuterons in a solvent of constant isotopic composition destabilized the domain, decreasing both its Tmand ΔG0of unfolding. The folding rate also decreased. The parameter ΦH/D, defined as the ratio of the effect of isotopic substitution upon the activation free energy to the equilibrium free energy was determined to be 0.6 in a D2O background and 0.75 in a H2O background, indicating that significant intraprotein hydrogen bond interactions are developed in the transition state for the folding of NTL9. The value is in remarkably good agreement with more traditional measures of the position of the transition state, which report on the relative burial of surface area. The results provide a picture of a compact folding transition state containing significant secondary structure. Indirect analysis argues that the bulk of the kinetic isotope effect arises from the β-sheet-rich region of the protein, and suggests that the development of intraprotein hydrogen bonds in this region plays a critical role in the folding of NTL9.