Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9
Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9
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DOI：
10.1006/jmbi.2001.5001
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发表时间：
2001-09-28
影响因子：
5.6
通讯作者：
Radford, SE
中科院分区：
文献类型：
--
作者：
Gorski, SA;Capaldi, AP;Radford, SE
尽管有60%的序列同源性，但螺旋细菌免疫蛋白Im7和Im9已被证明通过不同复杂的动力学机制进行折叠。在pH为7.0、温度为10℃时，Im7以三态机制折叠，而Im9以明显的两态转变折叠。为了更详细地研究这些蛋白质的折叠机制，研究了Im7和Im9(在0.4M硫酸钠中10℃时)的折叠动力学随pH的变化。对Im7在pH 5.0和8.0之间的折叠和展开数据进行的动力学模拟表明，路径上的中间体在更酸性的条件下是稳定的，而天然状态是不稳定的。PH对这些状态的稳定性的相反影响导致在pH 6.0及以下的平衡时有大量的中间体。在pH为7.0时，Im9的折叠和去折叠动力学可用二态模型进行较好的拟合，与前人的结果一致。但在酸性条件下，折叠速率常数的对数与变性剂浓度的对数曲线有明显的斜率变化，这与折叠早期的一个或多个中间体(S)的数量一致。在这些pH值下，Im9的动力学数据可以符合三态模型，其中随着pH的降低，中间系综稳定，而本征状态不稳定，这合理地解释了先前的结果，即在pH 7.0时实验上没有观察到中间体。这些数据表明，中间产物的形成是免疫蛋白折叠的一般步骤，并证明有必要探索广泛的复性条件，以表明中间产物不会在其他小的单结构域蛋白质的折叠中形成。(C)2001年学术出版社。
The helical bacterial immumity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60% sequence identity. At pH 7.0 and 10 degreesC, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degreesC in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins. (C) 2001 Academic Press.