Department of Physics, Beckman Institute, §Department of Chemistry, and ‡Center for Biophysics and Computational Biology, University of Illinois , Urbana, Illinois 61801, United States.
J Am Chem Soc. 2014 Mar 19;136(11):4265-72. doi: 10.1021/ja412639u. Epub 2014 Feb 3.
Density is an easily adjusted variable in molecular dynamics (MD) simulations. Thus, pressure-jump (P-jump)-induced protein refolding, if it could be made fast enough, would be ideally suited for comparison with MD. Although pressure denaturation perturbs secondary structure less than temperature denaturation, protein refolding after a fast P-jump is not necessarily faster than that after a temperature jump. Recent P-jump refolding experiments on the helix bundle λ-repressor have shown evidence of a <3 μs burst phase, but also of a ~1.5 ms "slow" phase of refolding, attributed to non-native helical structure frustrating microsecond refolding. Here we show that a λ-repressor mutant is nonetheless capable of refolding in a single explicit solvent MD trajectory in about 19 μs, indicating that the burst phase observed in experiments on the same mutant could produce native protein. The simulation reveals that after about 18.5 μs of conformational sampling, the productive structural rearrangement to the native state does not occur in a single swift step but is spread out over a brief series of helix and loop rearrangements that take about 0.9 μs. Our results support the molecular time scale inferred for λ-repressor from near-downhill folding experiments, where transition-state population can be seen experimentally, and also agrees with the transition-state transit time observed in slower folding proteins by single-molecule spectroscopy.
密度是分子动力学（MD）模拟中易于调整的变量。因此，如果压力跃变（P-jump）诱导的蛋白质重折叠能够足够快地发生，它将非常适合与 MD 进行比较。尽管压力变性对二级结构的干扰小于温度变性，但快速 P-jump 后的蛋白质重折叠并不一定比温度跃变后的重折叠更快。最近对螺旋束 λ-阻遏物的 P-jump 重折叠实验表明存在 <3 μs 的爆发相，但也存在 ~1.5 ms 的“慢”重折叠相，归因于非天然螺旋结构阻碍微秒重折叠。在这里，我们表明 λ-阻遏物突变体仍然能够在单个显式溶剂 MD 轨迹中在大约 19 μs 内重折叠，这表明在相同突变体的实验中观察到的爆发相可以产生天然蛋白质。模拟表明，在大约 18.5 μs 的构象采样后，到天然状态的生产性结构重排不会在单个快速步骤中发生，而是分散在一系列短的螺旋和环重排中，这些重排需要大约 0.9 μs。我们的结果支持从近下坡折叠实验推断出的 λ-阻遏物的分子时间尺度，其中可以在实验中观察到过渡态种群，并且与单分子光谱法观察到的较慢折叠蛋白质的过渡态迁移时间一致。
