van't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam, The Netherlands.
J Phys Chem B. 2009 Dec 17;113(50):16184-96. doi: 10.1021/jp904468q.
The folding mechanism of a protein is determined by its primary sequence. Yet, how the mechanism is changed by a mutation is still poorly understood, even for basic secondary structures such as beta-hairpins. We perform an extensive simulation study of the effects of mutating the GB1 beta-hairpin into Trpzip4 (Y5W, F12W, V14W) on the folding mechanism. While Trpzip4 has a much more stable native state due to very strong hydrophobic interactions of the side chains, its folding rate does not differ significantly from the wild type beta-hairpin. We sample the free-energy landscapes of both hairpins with Replica Exchange Molecular Dynamics (REMD) and identify the four (meta)stable states (U, H, F, and N). Using Transition Path Sampling (TPS), we then harvest ensembles of unbiased pathways between the H and F states and between the F and N states to investigate the unbiased folding mechanisms. In both hairpins, the hydrophobic collapse (U-H) is followed by the middle hydrogen bond formation (H-F), and finally a closing of the strands in a zipper-like fashion (F-N). For the Trpzip4, the path ensembles indicate that the final F-N step is much more difficult than for GB1 and involves partial unfolding, rezipping of hydrogen bonds, and rearrangement of the Trp-14 side chain. For the rate-limiting (H-F) step, the path ensembles show that in GB1 desolvation and strand closure go hand in hand, while in Trpzip4 desolvation is decoupled from strand closure. Nevertheless, likelihood maximization shows that the reaction coordinate for both hairpins remains the interstrand distance. We conclude that the folding mechanism of both hairpins is a combination of hydrophobic collapse and zipping of hydrogen bonds but that the zipper mechanism is more visible in Trpzip4. A major difference between the two hairpins is that in the transition state of the rate-limiting step for Trpzip4 one tryptophan is exposed to the solvent due to steric hindrance, making the folding mechanism more complex and leading to an increased F-N barrier. Thus, our results show in atomistic detail how a mutation leads to a different folding mechanism and results in a more frustrated folding free-energy landscape.
蛋白质的折叠机制由其一级序列决定。然而，即使对于基本的二级结构（如β发夹），突变如何改变机制仍知之甚少。我们对将 GB1β发夹突变为 Trpzip4（Y5W、F12W、V14W）对折叠机制的影响进行了广泛的模拟研究。虽然 Trpzip4 由于侧链的强疏水性相互作用而具有更稳定的天然状态，但它的折叠速率与野生型β发夹没有显著差异。我们使用 Replica Exchange Molecular Dynamics（REMD）对两种发夹的自由能景观进行采样，并确定了四个（亚）稳定状态（U、H、F 和 N）。然后，我们使用过渡路径采样（TPS）从 H 和 F 状态之间以及 F 和 N 状态之间收集无偏途径的集合，以研究无偏折叠机制。在两种发夹中，疏水性塌陷（U-H）之后是中间氢键的形成（H-F），最后以拉链式方式关闭链（F-N）。对于 Trpzip4，路径集合表明最后一步 F-N 比 GB1 更困难，涉及部分展开、氢键重新成键和 Trp-14 侧链的重排。对于限速（H-F）步骤，路径集合表明在 GB1 中去溶剂化和链闭合是齐头并进的，而在 Trpzip4 中去溶剂化与链闭合是解耦的。然而，似然最大化表明，两种发夹的反应坐标仍然是链间距离。我们得出的结论是，两种发夹的折叠机制是疏水性塌陷和氢键成键的组合，但 Trpzip4 中的拉链机制更为明显。两种发夹之间的一个主要区别是，在 Trpzip4 的限速步骤的过渡态中，由于空间位阻，一个色氨酸暴露于溶剂中，使折叠机制更加复杂，并导致 F-N 势垒增加。因此，我们的结果详细显示了突变如何导致不同的折叠机制，并导致更受挫的折叠自由能景观。
