Astbury Centre for Structural Molecular Biology and Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT 06030, USA.
Prog Nucl Magn Reson Spectrosc. 2017 May;100:52-77. doi: 10.1016/j.pnmrs.2016.10.002. Epub 2016 Nov 9.
Protein folding is a highly complex process proceeding through a number of disordered and partially folded nonnative states with various degrees of structural organization. These transiently and sparsely populated species on the protein folding energy landscape play crucial roles in driving folding toward the native conformation, yet some of these nonnative states may also serve as precursors for protein misfolding and aggregation associated with a range of devastating diseases, including neuro-degeneration, diabetes and cancer. Therefore, in vivo protein folding is often reshaped co- and post-translationally through interactions with the ribosome, molecular chaperones and/or other cellular components. Owing to developments in instrumentation and methodology, solution NMR spectroscopy has emerged as the central experimental approach for the detailed characterization of the complex protein folding processes in vitro and in vivo. NMR relaxation dispersion and saturation transfer methods provide the means for a detailed characterization of protein folding kinetics and thermodynamics under native-like conditions, as well as modeling high-resolution structures of weakly populated short-lived conformational states on the protein folding energy landscape. Continuing development of isotope labeling strategies and NMR methods to probe high molecular weight protein assemblies, along with advances of in-cell NMR, have recently allowed protein folding to be studied in the context of ribosome-nascent chain complexes and molecular chaperones, and even inside living cells. Here we review solution NMR approaches to investigate the protein folding energy landscape, and discuss selected applications of NMR methodology to studying protein folding in vitro and in vivo. Together, these examples highlight a vast potential of solution NMR in providing atomistic insights into molecular mechanisms of protein folding and homeostasis in health and disease.
蛋白质折叠是一个高度复杂的过程，经过一系列无序和部分折叠的非天然状态，具有不同程度的结构组织。这些在蛋白质折叠能量景观上短暂且稀疏存在的物种在驱动折叠到天然构象方面起着至关重要的作用，但其中一些非天然状态也可能作为与一系列破坏性疾病（包括神经退行性疾病、糖尿病和癌症）相关的蛋白质错误折叠和聚集的前体。因此，体内蛋白质折叠通常通过与核糖体、分子伴侣和/或其他细胞成分的相互作用在共翻译和翻译后进行重塑。由于仪器和方法的发展，溶液 NMR 光谱学已成为体外和体内复杂蛋白质折叠过程详细特征描述的核心实验方法。NMR 弛豫分散和饱和转移方法为在类似天然的条件下对蛋白质折叠动力学和热力学进行详细特征描述提供了手段，以及对蛋白质折叠能量景观上弱 populate 短寿命构象状态的高分辨率结构进行建模。同位素标记策略和 NMR 方法的不断发展以探测高分子量蛋白质组装，以及细胞内 NMR 的进展，最近使得蛋白质折叠能够在核糖体新生链复合物和分子伴侣的背景下，甚至在活细胞内进行研究。在这里，我们综述了溶液 NMR 方法来研究蛋白质折叠能量景观，并讨论了 NMR 方法在体外和体内研究蛋白质折叠的一些应用。这些例子共同强调了溶液 NMR 在提供对蛋白质折叠和健康与疾病中分子机制的原子水平见解方面具有巨大的潜力。
