Temperature-dependent Hammond behavior in a protein-folding reaction: analysis of transition-state movement and ground-state effects.
Temperature-dependent Hammond behavior in a protein-folding reaction: analysis of transition-state movement and ground-state effects.
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蛋白质折叠反应中温度依赖性哈蒙德行为：过渡态运动和基态效应分析。
DOI：
10.1016/j.jmb.2008.02.024
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发表时间：
2008
影响因子：
5.6
通讯作者：
Raleigh,DanielP
中科院分区：
文献类型：
--
作者：
Taskent,Humeyra;Cho,Jae-Hyun;Raleigh,DanielP
过渡态系综的表征和蛋白质折叠自由能垒的性质是激烈的活动和一些争议的领域。近年来出现的一个关键问题是自由能垒的宽度和过渡态对运动的敏感性。在这里，我们报告变性剂诱导和温度依赖性折叠的小混合α-β蛋白，N-末端结构域的L9（NTL 9）的研究。NTL 9的折叠使用在11 ° C和40 °C之间的七个不同温度下进行的荧光检测的停流荧光测量来确定。观察到的一级速率常数与变性剂浓度的对数图，“人字形图”，显示了双态折叠预期的特征V形。即使在最低变性剂浓度下，也没有线性偏离的迹象。由Tanford β参数βT判断的过渡态的相对位置随着温度的升高而向天然态移动。的动力学和平衡m值的温度依赖性的分析表明，该效果是由于过渡态的显着运动，也包括从温度依赖性的基态效应的贡献。对Leffler图、ΔG ε与ΔG°图及其交叉相互作用参数的分析证实了过渡态运动。由于蛋白质在高温下是不稳定的，因此这种转变代表了温度依赖性的哈蒙德效应。这为最近的理论预测提供了独立的证实。NTL 9的温度-变性剂交叉相互作用参数的大小比其他几种研究情况下报道的要大。温度依赖性研究蛋白质折叠的影响进行了讨论。
Characterization of the transition-state ensemble and the nature of the free-energy barrier for protein folding are areas of intense activity and some controversy. A key issue that has emerged in recent years is the width of the free-energy barrier and the susceptibility of the transition state to movement. Here we report denaturant-induced and temperature-dependent folding studies of a small mixed α–β protein, the N-terminal domain of L9 (NTL9). The folding of NTL9 was determined using fluorescence-detected stopped-flow fluorescence measurements conducted at seven different temperatures between 11 and 40 °C. Plots of the log of the observed first-order rate constant versus denaturant concentration, “chevron plots,” displayed the characteristic V shape expected for two-state folding. There was no hint of deviation from linearity even at the lowest denaturant concentrations. The relative position of the transition state, as judged by the Tanford β parameter, βT, shifts towards the native state as the temperature is increased. Analysis of the temperature dependence of the kinetic and equilibrium m values indicates that the effect is due to significant movement of the transition state and also includes a contribution from temperature-dependent ground-state effects. Analysis of the Leffler plots, plots of ΔG‡versus ΔG°, and their cross-interaction parameters confirms the transition-state movement. Since the protein is destabilized at high temperature, the shift represents a temperature-dependent Hammond effect. This provides independent confirmation of a recent theoretical prediction. The magnitude of the temperature-denaturant cross-interaction parameter is larger for NTL9 than has been reported for the few other cases studied. The implications for temperature-dependent studies of protein folding are discussed.
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影响因子：
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影响因子：
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通讯作者：
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