Institute of Biological Information Processing (IBI-7), Forschungszentrum Jülich; Institute of Physical Biology, Faculty of Mathematics and Natural Sciences, Heinrich Heine University Düsseldorf.
Institute of Biological Information Processing (IBI-7), Forschungszentrum Jülich; Institute of Physical Biology, Faculty of Mathematics and Natural Sciences, Heinrich Heine University Düsseldorf;
J Vis Exp. 2024 Nov 1(213). doi: 10.3791/67088.
Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard N relaxation measurements overview a protein's internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.
核磁共振（NMR）光谱学允许在溶液中和生理温度下研究蛋白质。通常，蛋白质骨架的酰胺基团或侧链中的甲基基团被用作蛋白质结构动力学的报告者。对 N 标记和完全质子化样品的球状蛋白质骨架的结构动力学研究通常适用于分子量高达 50 kDa 的蛋白质。当与横向弛豫优化光谱学（TROSY）结合使用侧链氘代时，该限制可以扩展到 200 kDa 的球状蛋白质，并且当重点放在侧链上时，可以扩展到 1 MDa。当研究天然无序蛋白质（IDPs）或具有天然无序区域（IDRs）的蛋白质时，这些重量限制不适用，但可以远远超出。原因是 IDPs 或 IDRs 具有高度的内部灵活性，通常是动态解耦的。各种 NMR 方法提供了广泛的时间尺度上的结构蛋白质动力学的原子分辨率见解，从皮秒到小时不等。标准的 N 弛豫测量概述了蛋白质的内部灵活性，并在快速皮秒到纳秒时间尺度上描述了蛋白质骨架动力学。本文提供了一个实用的协议，用于设置和记录 NMR N R1、R2 和异核 Overhauser 效应（hetNOE）实验。我们展示了示例数据，并在进行任何更复杂的分析之前，简单地定性解释如何解释它们。
