Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520
Proc Natl Acad Sci U S A. 2018 May 1;115(18):E4169-E4178. doi: 10.1073/pnas.1803642115. Epub 2018 Apr 16.
In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. N and C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state.
一般来说，小蛋白质可以在毫秒或更短的时间尺度上快速折叠。对于折叠态和未折叠态之间体积差异较大的蛋白质，通过改变静水压力可以改变其热力学平衡。我们使用泛素的压力敏感突变体证明，在 NMR 样品室内快速切换压力，可以在天然条件下研究未折叠的蛋白质，反之亦然，在变性条件下研究天然蛋白质。这种方法使得在大气压力下记录未折叠蛋白质的 2D 和 3D NMR 谱成为可能，为折叠过程提供残基特异性信息。在将压力从 2.5kbar（有利于展开）降低到 1bar（天然）后立即测量的 N 和 C 化学位移接近该蛋白质的大的、无规卷曲肽片段的无规卷曲化学位移。然而，N 弛豫数据显示在大约 100μs 的时间尺度上，未折叠状态与不稳定的、结构化的状态之间存在快速交换的证据，这些状态可以被认为是折叠失败的事件。NMR 数据还提供了平行折叠途径的直接证据，大约一半的蛋白质分子通过途径上的动力学中间体有效地折叠，而另一半则以单步折叠。在蛋白质浓度高于约 300μM 时，与天然状态折叠竞争的是无途径的寡聚中间体。
