Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, Madrid 28040, Spain.
J Am Chem Soc. 2010 Aug 18;132(32):11183-90. doi: 10.1021/ja103612q.
Proteins that fold over free-energy barriers <or= 3RT are classified as downhill folders. This regime is characterized by equilibrium unfolding that is proportionally broader and more complex the lower the folding barrier. Downhill proteins are also expected to fold up in a few microseconds. However, the relationship between rate and equilibrium signatures is affected by other factors such as protein size and folding topology. Here we perform a direct comparison of the kinetics and equilibrium unfolding of two structural homologues: BBL and PDD. BBL folds-unfolds in just approximately 1 micros at 335 K and displays the equilibrium signatures expected for a protein at the bottom of the downhill folding regime. PDD, which has the same 3D structure and size, folds-unfolds approximately 8 times more slowly and, concomitantly, exhibits all the downhill equilibrium signatures to a lesser degree. Our results demonstrate that the equilibrium signatures of downhill folding are proportional to the changes in folding rate once structural and size-scaling effects are factored out. This conclusion has two important implications: (1) it confirms that the quantitative analysis of equilibrium experiments in ultrafast folding proteins does provide direct information about free-energy barriers, a result that is incompatible with the conventional view of protein folding as a highly activated process, and (2) it advocates for equilibrium-kinetic studies of homologous proteins as a powerful tool to navigate the downhill folding regime via comparative analysis. The latter should prove extremely useful for the investigation of sequence, functional, and evolutionary determinants of protein folding barriers.
折叠自由能势垒<=3RT 的蛋白质被归类为 downhill 折叠者。该机制的特点是平衡展开度随折叠势垒的降低而呈比例增加，变得更加复杂。downhill 蛋白质也有望在几微秒内折叠。然而，速率与平衡特征之间的关系受到其他因素的影响，例如蛋白质大小和折叠拓扑结构。在这里，我们对两个结构同源物：BBL 和 PDD 的动力学和平衡展开进行了直接比较。在 335 K 下，BBL 只需大约 1 微秒即可折叠-展开，并显示出处于 downhill 折叠机制底部的蛋白质的平衡特征。具有相同 3D 结构和大小的 PDD 折叠-展开的速度大约慢 8 倍，并且相应地，以较小的程度表现出所有 downhill 平衡特征。我们的结果表明，一旦消除结构和大小缩放效应，downhill 折叠的平衡特征与折叠速率的变化成正比。这一结论有两个重要的含义：（1）它证实了在超快折叠蛋白质中对平衡实验的定量分析确实提供了有关自由能势垒的直接信息，这一结果与蛋白质折叠作为一个高度激活过程的传统观点不一致，（2）它提倡对同源蛋白质进行平衡-动力学研究，作为通过比较分析探索 downhill 折叠机制的有力工具。后者对于研究蛋白质折叠势垒的序列、功能和进化决定因素应该非常有用。
