Mori Toshifumi, Saito Shinji
Institute for Molecular Science , Myodaiji, Okazaki, Aichi 444-8585, Japan.
School of Physical Sciences, The Graduate University for Advanced Studies , Okazaki, Aichi 444-8585, Japan.
J Phys Chem B. 2016 Nov 17;120(45):11683-11691. doi: 10.1021/acs.jpcb.6b08066. Epub 2016 Nov 3.
Proteins involve motions over a wide range of spatial and temporal scales. While the large conformational changes, such as folding and functioning, are slow and appear to occur in a highly cooperative manner, how the hierarchical dynamics over different time scales play a role during these slow transitions has been of great interest over the decades. Here we study the folding mechanism of the villin headpiece subdomain (HP35) to understand the molecular mechanism behind this prototypical fast-folding protein. The ∼400 μs molecular dynamics (MD) trajectories obtained by Piana et al. [ Piana , S. ; Lindorff-Larsen , K. ; Shaw , D. E. Proc. Natl. Acad. Sci. U.S.A. 2012 , 109 , 17845 ] are analyzed in detail. By extracting the slowest mode from the trajectories, which is responsible for the folding/unfolding transitions, and by analyzing the transition events along this mode, we find that the transitions occur in a heterogeneous manner. Detailed analysis of the individual transition events shows that the folding/unfolding transitions occur via two qualitatively different pathways, i.e., the unfolding triggered from the C-terminal (α helix) and from the N-terminal (α-α loop). Non-native contacts are also found to contribute in slowing down the transitions. The folding of HP35 thus proceeds in a segmental manner rather than cooperatively at the submicrosecond time scale. The Lys→Nle mutation is found to speed up the transitions by rigidifying the α helix, i.e., suppressing one transition pathway. The analysis of the microsecond dynamics in the single-molecule Förster resonance energy transfer efficiency trajectories, which are calculated from the MD data, reveals that the folding/unfolding transitions in the NleNle mutant can be fitted with a two-state model, whereas those in WT appear to be more complex and involves multiple time scales. This is due to the coupling between the folding/unfolding transitions and conformational transitions within the unfolded and intermediate states. The present study demonstrates that a protein as small as HP35 already involves heterogeneous characters during folding/unfolding transitions when the hierarchical dynamics at the molecular level is considered, thus heterogeneity can be a general characteristic in protein folding.
蛋白质涉及广泛的空间和时间尺度上的运动。虽然诸如折叠和功能发挥等大的构象变化较为缓慢，且似乎以高度协同的方式发生，但在过去几十年中，不同时间尺度上的层次动力学在这些缓慢转变过程中如何发挥作用一直备受关注。在此，我们研究了绒毛蛋白头部结构域（HP35）的折叠机制，以了解这种典型的快速折叠蛋白背后的分子机制。我们详细分析了皮阿纳等人[皮阿纳，S.；林多夫 - 拉森，K.；肖，D. E.《美国国家科学院院刊》2012年，109卷，17845页]获得的约400微秒的分子动力学（MD）轨迹。通过从轨迹中提取负责折叠/去折叠转变的最慢模式，并分析沿此模式的转变事件，我们发现转变以异质性方式发生。对各个转变事件的详细分析表明，折叠/去折叠转变通过两种性质不同的途径发生，即从C端（α螺旋）和从N端（α - α环）引发的去折叠。还发现非天然接触有助于减缓转变。因此，HP35的折叠在亚微秒时间尺度上以分段方式进行，而非协同进行。发现赖氨酸→正亮氨酸突变通过使α螺旋刚性化来加速转变，即抑制一种转变途径。对由MD数据计算得到的单分子福斯特共振能量转移效率轨迹中的微秒动力学分析表明，正亮氨酸突变体中的折叠/去折叠转变可以用两态模型拟合，而野生型中的转变似乎更复杂，涉及多个时间尺度。这是由于折叠/去折叠转变与未折叠态和中间态内的构象转变之间的耦合。本研究表明，当考虑分子水平的层次动力学时，像HP35这样小的蛋白质在折叠/去折叠转变过程中已经涉及异质性特征，因此异质性可能是蛋白质折叠的一个普遍特征。
