Solution structure of a protein denatured state and folding intermediate
Solution structure of a protein denatured state and folding intermediate
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DOI：
10.1038/nature04054
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发表时间：
2005-10-13
期刊：
影响因子：
64.8
通讯作者：
Fersht, AR
中科院分区：
文献类型：
--
作者：
Religa, TL;Markson, JS;Fersht, AR
蛋白质折叠中最有争议的领域涉及其最早阶段。诸如是否存在真正的折叠中间体，以及最早阶段的事件是否只是变性状态的重排（1）或从填充的过渡状态（2）的进展等问题仍然没有解决。问题是，缺乏实验的高分辨率结构信息的早期折叠中间体和变性状态下，有利于折叠，因为主管国家自发折叠迅速。在这里，我们已经解决了直接的解决方案结构的一个真正的变性状态的核磁共振条件下，通常有利于折叠，并直接研究其平衡和动力学行为。我们设计了一个果蝇Engrailed同源结构域的突变体，它可以通过改变离子强度可逆地折叠和展开。在高离子强度下，突变体L16 A是一种超快速折叠的天然蛋白质，就像野生型蛋白质一样;然而，在生理离子强度下，它会变性。变性状态是一种有序的折叠中间体，通过对接螺旋和破坏一些非天然相互作用来折叠。随着变性条件的增加，它相对渐进地展开，因此表面上类似于变性状态，其性质随条件而变化。这种不明确的去折叠是早期折叠中间态的一个共同特征，也解释了为什么在蛋白质折叠中中间态与紧凑变性态之间存在如此多的争议。
The most controversial area in protein folding concerns its earliest stages. Questions such as whether there are genuine folding intermediates, and whether the events at the earliest stages are just rearrangements of the denatured state(1) or progress from populated transition states(2), remain unresolved. The problem is that there is a lack of experimental high-resolution structural information about early folding intermediates and denatured states under conditions that favour folding because competent states spontaneously fold rapidly. Here we have solved directly the solution structure of a true denatured state by nuclear magnetic resonance under conditions that would normally favour folding, and directly studied its equilibrium and kinetic behaviour. We engineered a mutant of Drosophila melanogaster Engrailed homeodomain that folds and unfolds reversibly just by changing ionic strength. At high ionic strength, the mutant L16A is an ultra-fast folding native protein, just like the wild-type protein; however, at physiological ionic strength it is denatured. The denatured state is a well-ordered folding intermediate, poised to fold by docking helices and breaking some non-native interactions. It unfolds relatively progressively with increasingly denaturing conditions, and so superficially resembles a denatured state with properties that vary with conditions. Such ill-defined unfolding is a common feature of early folding intermediate states and accounts for why there are so many controversies about intermediates versus compact denatured states in protein folding.