Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1 (Canada).
State Key Laboratory of Precision Measurements Technology and Instruments, School of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072 (China).
Angew Chem Int Ed Engl. 2015 Aug 17;54(34):9921-5. doi: 10.1002/anie.201502938. Epub 2015 Jul 1.
Directly observing protein folding in real time using atomic force microscopy (AFM) is challenging. Here the use of AFM to directly monitor the folding of an α/β protein, NuG2, by using low-drift AFM cantilevers is demonstrated. At slow pulling speeds (<50 nm s(-1)), the refolding of NuG2 can be clearly observed. Lowering the pulling speed reduces the difference between the unfolding and refolding forces, bringing the non-equilibrium unfolding-refolding reactions towards equilibrium. At very low pulling speeds (ca. 2 nm s(-1)), unfolding and refolding were observed to occur in near equilibrium. Based on the Crooks fluctuation theorem, we then measured the equilibrium free energy change between folded and unfolded states of NuG2. The improved long-term stability of AFM achieved using gold-free cantilevers allows folding-unfolding reactions of α/β proteins to be directly monitored near equilibrium, opening the avenue towards probing the folding reactions of other mechanically important α/β and all-β elastomeric proteins.
直接使用原子力显微镜（AFM）实时观察蛋白质折叠具有挑战性。在这里，通过使用低漂移 AFM 悬臂来直接监测α/β 蛋白 NuG2 的折叠。在缓慢的拉伸速度（<50nm/s）下，可以清楚地观察到 NuG2 的重折叠。降低拉伸速度会降低展开和重折叠力之间的差异，使非平衡展开-重折叠反应向平衡状态移动。在非常低的拉伸速度（约 2nm/s）下，观察到展开和重折叠在近平衡状态下发生。基于克劳修斯涨落定理，我们然后测量了 NuG2 的折叠态和展开态之间的平衡自由能变化。使用无金悬臂实现的 AFM 的改进的长期稳定性允许在近平衡状态下直接监测α/β 蛋白的折叠-展开反应，为探测其他机械上重要的α/β 和全-β弹性蛋白的折叠反应开辟了道路。
