Yatime Laure
Laboratory "Dynamique des Interactions Membranaires Normales et Pathologiques" (DIMNP)-UMR5235, Inserm, CNRS, University of Montpellier, Montpellier Cedex 5, France.
Methods Mol Biol. 2019;1929:417-435. doi: 10.1007/978-1-4939-9030-6_26.
Ions are important regulators for the cellular function of many proteins. This holds particularly true for S100 proteins whose function is not only calcium-dependent but also appears to be modulated by other divalent cations such as zinc, manganese, or copper. One way ions are thought to influence the function of S100 proteins (and any protein in general) is by changing their three-dimensional organization, through modifications in either their monomeric shape, their oligomeric state, or both. X-ray crystallography is a very powerful technique to study the effect of ions on the 3D architecture of macromolecules since it gives a direct visualization of where ions bind and how the protein structure is affected upon ion binding. Taking the example of human S100A8, I describe here the complete procedure to obtain a highly pure and homogenous S100 protein sample, crystallize it in the presence of divalent cations, and derive a 3D structural model from diffraction images. I further detail computational methods used to determine precisely the nature and position of the divalent cations within S100A8 structure. This methodology can easily be applied to any ion-binding protein, provided that the ion anomalous scattering properties allow to identify it unambiguously.
离子是许多蛋白质细胞功能的重要调节因子。对于S100蛋白来说尤其如此，其功能不仅依赖于钙，而且似乎还受其他二价阳离子如锌、锰或铜的调节。人们认为离子影响S100蛋白（以及一般的任何蛋白质）功能的一种方式是通过改变其三维结构，无论是改变其单体形状、寡聚状态或两者皆有。X射线晶体学是研究离子对大分子三维结构影响的一种非常强大的技术，因为它能直接可视化离子结合的位置以及离子结合后蛋白质结构是如何受到影响的。以人类S100A8为例，我在此描述获得高度纯净且均一的S100蛋白样品、在二价阳离子存在的情况下使其结晶以及从衍射图像推导三维结构模型的完整过程。我还进一步详细介绍了用于精确确定S100A8结构中二价阳离子的性质和位置的计算方法。只要离子的反常散射特性能够明确识别它，这种方法就可以很容易地应用于任何离子结合蛋白。
