Gast Klaus, Fiedler Christian
Institut für Biochemie und Biologie, Physikalische Biochemie, Universität Potsdam, Potsdam, Germany.
Methods Mol Biol. 2012;896:137-61. doi: 10.1007/978-1-4614-3704-8_9.
Molecular parameters such as size, molar mass, and intermolecular interactions, which are important to identify and characterize intrinsically disordered proteins (IDPs), can be obtained from light scattering measurements. In this chapter, we discuss the physical basis of light scattering, experimental techniques, sample treatment, and data evaluation with special emphasis on studies on proteins. Static light scattering (SLS) is capable of measuring molar masses within the range 10(3)-10(8) g/mol and is therefore ideal for determining the state of association of proteins in solution. Since proteins are in general too small to obtain the geometric radius of gyration R (G) from SLS, it is more useful to determine the hydrodynamic Stokes radius, R (S), which can be obtained easily and quickly from dynamic light scattering (DLS) experiments. Accordingly, DLS is an appropriate technique to monitor expansion or compaction of protein molecules. This is especially important for IDPs, which can be recognized and characterized by comparing the measured Stokes radii with those calculated for particular reference states, such as the compactly folded and the fully unfolded states. The combined application of DLS and SLS improves measurements of the molar mass and is essential when changes in the molecular dimensions and molecular association/dissociation take place simultaneously.
分子参数，如大小、摩尔质量和分子间相互作用，对于鉴定和表征内在无序蛋白质（IDP）很重要，这些参数可通过光散射测量获得。在本章中，我们将讨论光散射的物理基础、实验技术、样品处理和数据评估，特别着重于蛋白质研究。静态光散射（SLS）能够测量10³ - 10⁸ g/mol范围内的摩尔质量，因此非常适合确定溶液中蛋白质的缔合状态。由于蛋白质通常太小，无法通过SLS获得几何回转半径R(G)，所以确定流体动力学斯托克斯半径R(S)更有用，它可以通过动态光散射（DLS）实验轻松快速地获得。因此，DLS是监测蛋白质分子膨胀或压缩的合适技术。这对于IDP尤为重要，通过将测量的斯托克斯半径与针对特定参考状态（如紧密折叠状态和完全展开状态）计算的半径进行比较，可以识别和表征IDP。DLS和SLS的联合应用可改善摩尔质量的测量，并且当分子尺寸和分子缔合/解离同时发生变化时至关重要。
