Institut für Angewandte Physik, Universität Tübingen; Institut Max von Laue - Paul Langevin (ILL);
Institut Max von Laue - Paul Langevin (ILL).
J Vis Exp. 2022 Apr 28(182). doi: 10.3791/63664.
Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state. Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer. The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into µm sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.
中子散射提供了一种非破坏性的方法，可以在很宽的能量范围内探测样品中的动力学，而无需除氘以外的其他标记。特别是，中子背散射光谱学同时记录多个散射角的散射信号，非常适合研究 ps-ns 时间尺度上的生物系统动力学。通过使用 D2O 和可能的氘化缓冲成分，该方法可以监测蛋白质在液态中的质心扩散以及骨架和侧链运动（内部动力学）。此外，通过使用 H2O 水合的全氘化蛋白质粉末，可以研究水合动力学。本文介绍了在法国格勒诺布尔的伊莱·朗之万研究所（ILL）的 IN16B 仪器上用于研究蛋白质和水合动力学的工作流程。解释了使用气相交换制备溶液样品和水合蛋白质粉末样品的过程。描述了针对不同类型数据集（准弹性光谱或固定窗口扫描）的蛋白质和水合动力学数据分析程序，这些数据集可以在中子背散射光谱仪上获得。该方法通过两个涉及淀粉样蛋白的研究进行了说明。溶菌酶聚集成 µm 大小的球形聚集体-称为颗粒-被证明在 IN16B 探测的空间和时间范围内以一步过程发生，而内部动力学保持不变。此外，还研究了水合全氘化蛋白质粉末中 tau 的水合动力学。结果表明，在形成淀粉样纤维时，水的平移运动被激活。最后，讨论了协议中的关键步骤，即相对于其他实验生物物理方法，中子散射在动力学研究中的定位。
