PDF(Pubmed)
Abstract：
Proteins are constantly undergoing folding and unfolding transitions, with rates that determine their homeostasis in vivo and modulate their biological function. The ability to optimize these rates without affecting overall native stability is hence highly desirable for protein engineering and design. The great challenge is, however, that mutations generally affect folding and unfolding rates with inversely complementary fractions of the net free energy change they inflict on the native state. Here we address this challenge by targeting the folding transition state (FTS) of chymotrypsin inhibitor 2 (CI2), a very slow and stable two-state folding protein with an FTS known to be refractory to change by mutation. We first discovered that the CI2\'s FTS is energetically taxed by the desolvation of several, highly conserved, charges that form a buried salt bridge network in the native structure. Based on these findings, we designed a CI2 variant that bears just four mutations and aims to selectively stabilize the FTS. This variant has >250-fold faster rates in both directions and hence identical native stability, demonstrating the success of our FTS-centric design strategy. With an optimized FTS, CI2 also becomes 250-fold more sensitive to proteolytic degradation by its natural substrate chymotrypsin, and completely loses its activity as inhibitor. These results indicate that CI2 has been selected through evolution to have a very unstable FTS in order to attain the kinetic stability needed to effectively function as protease inhibitor. Moreover, the CI2 case showcases that protein (un)folding rates can critically pivot around a few key residues-interactions, which can strongly modify the general effects of known structural factors such as domain size and fold topology. From a practical standpoint, our results suggest that future efforts should perhaps focus on identifying such critical residues-interactions in proteins as best strategy to significantly improve our ability to predict and engineer protein (un)folding rates.
摘要：
蛋白质不断经历折叠和展开转变，以决定其体内稳态并调节其生物学功能的速率。因此,优化这些速率而不影响整体天然稳定性的能力对于蛋白质工程和设计是高度期望的。最大的挑战是,然而,突变通常会影响折叠和展开速率，而它们对天然状态造成的净自由能变化的反向互补部分。在这里，我们通过靶向胰凝乳蛋白酶抑制剂2（CI2）的折叠过渡态（FTS）来解决这一挑战，一种非常缓慢和稳定的双态折叠蛋白，具有已知难以通过突变改变的FTS。我们首先发现，CI2的FTS被几个人的去溶剂化所消耗，高度保守,在原生结构中形成掩埋盐桥网络的电荷。基于这些发现,我们设计了aCI2变体，该变体仅带有四个突变，旨在选择性稳定FTS。该变体在两个方向上具有>250倍的快速度，因此具有相同的天然稳定性，展示了我们以FTS为中心的设计策略的成功。有了优化的FTS，CI2对其天然底物胰凝乳蛋白酶的蛋白水解降解也变得敏感250倍，并完全失去其作为抑制剂的活性。这些结果表明，CI2已通过进化选择为具有非常不稳定的FTS，以获得有效发挥蛋白酶抑制剂作用所需的动力学稳定性。此外，CI2案例表明，蛋白质(非)折叠速率可以关键地围绕几个关键残基-相互作用，它可以强烈修改已知结构因素的一般影响，如畴大小和折叠拓扑。从实际的角度来看，我们的研究结果表明,未来的工作或许应该集中在确定蛋白质中的关键残基-相互作用,作为显著提高我们预测和改造蛋白质(非)折叠速率的能力的最佳策略.
