Department of Physics, ‡Department of Mathematical Sciences, §Department of Computer Science, and ∥Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
J Phys Chem B. 2017 Mar 16;121(10):2220-2229. doi: 10.1021/acs.jpcb.6b10362. Epub 2017 Mar 1.
By applying a controlled mechanical load using optical tweezers, we measured the diffusive barrier crossing in a 49 nt long P5ab RNA hairpin. We find that in the free-energy landscape the barrier height (G) and transition distance (x) are dependent on the loading rate (r) along the pulling direction, x, as predicted by Bell. The barrier shifted toward the initial state, whereas ΔG reduced significantly from 50 to 5 kT, as r increased from 0 to 32 pN/s. However, the equilibrium work (ΔG) during strand separation, as estimated by Crook's fluctuation theorem, remained unchanged at different rates. Previously, helix formation and denaturation have been described as two-state (F ↔ U) transitions for P5ab. Herein, we report three intermediate states I, I, and I located at 4, 11, and 16 nm respectively, from the folded conformation. The intermediates were observed only when the hairpin was subjected to an optimal r, 7.6 pN/s. The results indicate that the complementary strands in P5ab can zip and unzip through complex routes, whereby mismatches act as checkpoints and often impose barriers. The study highlights the significance of loading rates in force-spectroscopy experiments that are increasingly being used to measure the folding properties of biomolecules.
我们使用光学镊子施加受控的机械负载，测量了一个 49 个核苷酸长的 P5ab RNA 发夹的扩散势垒穿越。我们发现，在自由能景观中，势垒高度（G）和跃迁距离（x）与沿着拉伸方向的加载速率（r）有关，这与贝尔的预测一致。势垒向初始状态移动，而ΔG 从 50 到 5 kT 显著降低，r 从 0 增加到 32 pN/s。然而，根据 Crook 的涨落定理，在不同的速率下，链分离过程中的平衡功（ΔG）保持不变。先前，P5ab 的螺旋形成和变性已被描述为两态（F ↔ U）转变。在此，我们报告了三个中间状态 I、I 和 I，分别位于折叠构象的 4、11 和 16nm 处。只有当发夹受到最佳 r（7.6 pN/s）时，才会观察到这些中间体。结果表明，P5ab 中的互补链可以通过复杂的途径进行拉链和解拉链，其中错配充当检查点并经常施加势垒。该研究强调了加载速率在力谱实验中的重要性，力谱实验越来越多地用于测量生物分子的折叠特性。
