Religa T L, Markson J S, Mayor U, Freund S M V, Fersht A R
MRC Centre for Protein Engineering and Cambridge University Chemical Laboratories, MRC Centre, Hills Road, Cambridge CB2 2QH, UK.
Nature. 2005 Oct 13;437(7061):1053-6. doi: 10.1038/nature04054.
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 or progress from populated transition states, 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.
蛋白质折叠中最具争议的领域涉及其最初阶段。诸如是否存在真正的折叠中间体，以及最初阶段的事件仅仅是变性状态的重排还是从大量存在的过渡态进展而来等问题，仍未得到解决。问题在于，在有利于折叠的条件下，缺乏关于早期折叠中间体和变性状态的实验性高分辨率结构信息，因为有活性的状态会自发快速折叠。在此，我们通过核磁共振直接解析了在通常有利于折叠的条件下真实变性状态的溶液结构，并直接研究了其平衡和动力学行为。我们构建了果蝇Engrailed同源结构域的一个突变体L16A（Leucine 16 changed to Alanine），它仅通过改变离子强度就能可逆地折叠和解折叠。在高离子强度下，突变体L16A就像野生型蛋白一样，是一种超快折叠的天然蛋白；然而，在生理离子强度下它是变性的。变性状态是一种有序的折叠中间体，准备通过对接螺旋并打破一些非天然相互作用来进行折叠。随着变性条件的增加，它相对逐步地展开，因此表面上类似于一种性质随条件变化的变性状态。这种定义不明确的展开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关于中间体与紧密变性状态存在如此多的争议。
