Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9
Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9
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DOI：
10.1006/jmbi.1998.2548
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发表时间：
1999-03-12
影响因子：
5.6
通讯作者：
Radford, SE
中科院分区：
文献类型：
--
作者：
Ferguson, N;Capaldi, AP;Radford, SE
同源四螺旋蛋白Im7和Im9在PH7.0和10℃下折叠的动力学和热力学特征已被表征。这些蛋白质在序列上60%相同，具有相同的三维结构，但似乎通过不同的动力学机制折叠。Im9的折叠和解折叠速率的对数与尿素浓度呈线性关系，并符合描述两态机制的方程(折叠速率为1500 S(-1)，解折叠速率为0.01 S(-1)，高度致密的过渡态，其埋藏的天然表面积接近95%)。相比之下，有明显的证据表明，在Im7的复性过程中有中间体的存在，这表明折叠速度对尿素的依赖性发生了变化，并且在复性动力学中存在显著的猝发相幅度。在稳定条件下(0.25M Na2SO4，pH 7.0和10℃)，Im9的折叠保持两态，而在类似的条件下(0.4M Na2SO4，pH 7.0和10℃)，Im7重折叠过程中填充的中间体显著稳定(K-UI=125)。平衡变性实验表明，在动力学测定的条件下，Im7的稳定性明显不如Im9(Delta G为9.3kJ/mol)，所测得的Delta G和m值与动力学数据的拟合值相吻合。因此，结果表明，免疫蛋白结构重折叠中中间体的数量是由精确的氨基酸序列决定的，而不是由蛋白质的整体稳定性决定的。我们讨论了由于Im7和Im9中螺旋倾向的差异而填充Im7中间体的可能性，以及这些数据与螺旋蛋白质折叠的一般相关性。(C)1999年学术出版社。
The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and lm9 have been characterised at PH 7.0 and 10 degrees C. These proteins are 60% identical in sequence and have the same three-dimensional structure, yet appear to fold by different kinetic mechanisms. The logarithm of the folding and unfolding rates of Im9 change linearly as a function of urea concentration and fit well to an equation describing a two-state mechanism (with a folding rate of 1500 s(-1) an unfolding rate of 0.01 s(-1), and a highly compact transition state that has similar to 95% of the native surface area buried). By contrast, there is clear evidence for the population of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a significant burst phase amplitude in the refolding kinetics. Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10 degrees C) the folding of Im9 remains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 10 degrees C) the intermediate populated during Im7 refolding is significantly stabilised (K-UI=125). Equilibrium denaturation experiments, under the conditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (Delta Delta G 9.3 kJ/mol) and the Delta G and m values determined accord with those obtained from the fit to the kinetic data. The results show, therefore, that the population of an intermediate in the refolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein. We discuss the possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general. (C) 1999 Academic Press.