Yu Hao, Siewny Matthew G W, Edwards Devin T, Sanders Aric W, Perkins Thomas T
JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO 80309, USA.
Department of Physics, University of Colorado, Boulder, CO 80309, USA.
Science. 2017 Mar 3;355(6328):945-950. doi: 10.1126/science.aah7124.
Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Using force spectroscopy optimized for 1-microsecond resolution, we reexamined the unfolding of individual bacteriorhodopsin molecules in native lipid bilayers. The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates-many separated by as few as two or three amino acids-exhibited complex dynamics, including frequent refolding and state occupancies of <10 μs. Equilibrium measurements between such states enabled the folding free-energy landscape to be deduced. These results sharpen the picture of the mechanical unfolding of membrane proteins and, more broadly, enable experimental access to previously obscured protein dynamics.
蛋白质折叠是在能量景观中结构状态之间的一系列转变过程。由于仪器分辨率有限，未检测到瞬时存在的状态时，就会出现对折叠过程过于简化的观点。我们使用针对1微秒分辨率优化的力谱技术，重新研究了天然脂质双层中单个细菌视紫红质分子的解折叠过程。实验数据以前所未有的细节揭示了解折叠途径。众多新检测到的中间体——许多仅相隔两三个氨基酸——表现出复杂的动力学，包括频繁的重新折叠和小于10微秒的状态占有率。这些状态之间的平衡测量使得能够推导折叠自由能景观。这些结果使膜蛋白机械解折叠的情况更加清晰，更广泛地说，能够通过实验获取以前难以捉摸的蛋白质动力学信息。
