Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Yoshida Konoe-cho, Sakyo-Ku, Kyoto 606-8501, Japan.
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku Katsura, Nishikyo-Ku, Kyoto 615-8510, Japan.
Methods. 2018 Sep 15;148:28-38. doi: 10.1016/j.ymeth.2018.04.026. Epub 2018 Apr 26.
Among the tools of structural biology, NMR spectroscopy is unique in that it not only derives a static three-dimensional structure, but also provides an atomic-level description of the local fluctuations and global dynamics around this static structure. A battery of NMR experiments is now available to probe the motions of proteins and nucleic acids over the whole biologically relevant timescale from picoseconds to hours. Here we focus on one of these methods, relaxation dispersion, which resolves dynamics on the micro- to millisecond timescale. Key biological processes that occur on this timescale include enzymatic catalysis, ligand binding, and local folding. In other words, relaxation-dispersion-resolved dynamics are often closely related to the function of the molecule and therefore highly interesting to the structural biochemist. With an astounding sensitivity of ∼0.5%, the method detects low-population excited states that are invisible to any other biophysical method. The kinetics of the exchange between the ground state and excited states are quantified in the form of the underlying exchange rate, while structural information about the invisible excited state is obtained in the form of its chemical shift. Lastly, the population of the excited state can be derived. This diversity in the information that can be obtained makes relaxation dispersion an excellent method to study the detailed mechanisms of conformational transitions and molecular interactions. Here we describe the two branches of relaxation dispersion, R and R, discussing their applicability, similarities, and differences, as well as recent developments in pulse sequence design and data processing.
在结构生物学的工具中，NMR 光谱学是独特的，因为它不仅可以得出静态的三维结构，还可以提供围绕该静态结构的局部波动和整体动力学的原子水平描述。现在有一系列 NMR 实验可用于探测蛋白质和核酸在从皮秒到小时的整个生物学相关时间尺度上的运动。在这里，我们重点介绍其中一种方法，即弛豫弥散，它可以解析微秒到毫秒时间尺度上的动力学。在此时间尺度上发生的关键生物过程包括酶催化、配体结合和局部折叠。换句话说，弛豫弥散分辨动力学通常与分子的功能密切相关，因此对结构生物化学家非常有兴趣。该方法的灵敏度惊人地达到了 ∼0.5%，可以检测到任何其他生物物理方法都无法检测到的低人口激发态。处于基态和激发态之间交换的动力学以基础交换速率的形式进行量化，而关于不可见激发态的结构信息则以其化学位移的形式获得。最后，可以推导出激发态的种群。这种可以获得的信息多样性使弛豫弥散成为研究构象转变和分子相互作用详细机制的极好方法。在这里，我们描述了弛豫弥散的两个分支，R 和 R，讨论了它们的适用性、相似性和差异，以及脉冲序列设计和数据处理方面的最新进展。
