Vallurupalli Pramodh, Chakrabarti Nilmadhab, Pomès Régis, Kay Lewis E
TIFR Centre for Interdisciplinary Sciences , 21 Brundavan Colony, Narsingi , Hyderabad 500075 , India . Email:
Molecular Structure and Function , Hospital for Sick Children , Toronto , ON , Canada M5G 1X8.
Chem Sci. 2016 Jun 1;7(6):3602-3613. doi: 10.1039/c5sc03886c. Epub 2016 Jan 7.
Despite the importance of dynamics to protein function there is little information about the states that are formed as the protein explores its conformational landscape or about the mechanism by which transitions between the different states occur. Here we used a combined NMR spin relaxation and unbiased molecular dynamics (MD) approach to investigate the exchange process by which a cavity in an L99A mutant of T4 lysozyme (T4L 99A) interconverts between an empty and occupied form that involves repositioning of an aromatic residue, Phe114. Although structures of the end-states of the exchange process are available, insight into the mechanism by which the transition takes place cannot be obtained from experiment and the timescales involved are too slow to address using brute force MD. Using spin relaxation NMR methods, we have identified a triple-mutant of T4L that undergoes the same exchange process as T4L L99A but where the minor state lifetime has decreased significantly so that the spontaneous conformational transition to the major state can be studied using all-atom MD simulations. The simulation trajectories were analyzed using Markov state models and the energy landscape so obtained is in good agreement with expectations based on NMR studies. Notably there is no large-scale perturbation of the structure during the transition, multiple intermediates are formed between the two similar exchanging conformations and the free energy barrier between these two well-folded, compact forms is small (6), only slightly larger than for processes considered to be barrierless.
尽管动力学对蛋白质功能很重要，但关于蛋白质在探索其构象景观时形成的状态，或不同状态之间转变发生的机制，几乎没有相关信息。在这里，我们使用了核磁共振自旋弛豫和无偏分子动力学（MD）相结合的方法，来研究T4溶菌酶（T4L 99A）的L99A突变体中一个空腔在空态和占据态之间相互转换的交换过程，该过程涉及一个芳香族残基Phe114的重新定位。虽然交换过程的终态结构是已知的，但无法从实验中获得有关转变发生机制的见解，而且所涉及的时间尺度太慢，无法用蛮力分子动力学来解决。使用自旋弛豫核磁共振方法，我们鉴定出了T4L的一个三重突变体，它经历与T4L L99A相同的交换过程，但其中次要状态的寿命显著缩短，因此可以使用全原子分子动力学模拟来研究向主要状态的自发构象转变。使用马尔可夫状态模型对模拟轨迹进行了分析，由此获得的能量景观与基于核磁共振研究的预期结果高度吻合。值得注意的是，在转变过程中结构没有发生大规模扰动，在两个相似的交换构象之间形成了多个中间体，并且这两种折叠良好、紧密的形式之间的自由能垒很小（6），仅略大于被认为无势垒的过程。
