Laboratory of Chemical Physics, NIDDK , National Institutes of Health , Bethesda , Maryland 20892-0520 , United States.
J Am Chem Soc. 2018 Jul 5;140(26):8096-8099. doi: 10.1021/jacs.8b04833. Epub 2018 Jun 25.
Pressure-jump hardware permits direct observation of protein NMR spectra during a cyclically repeated protein folding process. For a two-state folding protein, the change in resonance frequency will occur nearly instantaneously when the protein clears the transition state barrier, resulting in a monoexponential change of the ensemble-averaged chemical shift. However, protein folding pathways can be more complex and contain metastable intermediates. With a pseudo-3D NMR experiment that utilizes stroboscopic observation, we measure the ensemble-averaged chemical shifts, including those of exchange-broadened intermediates, during the folding process. Such measurements for a pressure-sensitized mutant of ubiquitin show an on-pathway kinetic intermediate whose N chemical shifts differ most from the natively folded protein for strands β5, its preceding turn, and the two strands that pair with β5 in the native structure.
压力跳跃硬件允许在周期性重复的蛋白质折叠过程中直接观察蛋白质 NMR 谱。对于两态折叠蛋白质，当蛋白质清除过渡状态障碍时，共振频率的变化将几乎瞬间发生，导致整体平均化学位移的单指数变化。然而，蛋白质折叠途径可能更复杂，并包含亚稳态中间体。通过利用频闪观察的伪 3D NMR 实验，我们测量折叠过程中的整体平均化学位移，包括交换展宽中间体的化学位移。对于泛素的压力敏感突变体的此类测量显示出一个途径上的动力学中间体，其 N 化学位移与其天然折叠蛋白的差异最大，对于β5 的前一个转角以及与β5 在天然结构中配对的两条链。
