Kiss Bence, Ecsédi Péter, Simon Márton, Nyitray László
Department of Biochemistry, ELTE Eötvös Loránd University, Budapest, Hungary.
Methods Mol Biol. 2019;1929:325-338. doi: 10.1007/978-1-4939-9030-6_21.
S100 proteins are small, mostly dimeric, EF-hand Ca-binding proteins. Upon Ca binding, a conformational change occurs resulting in the exposure of a shallow hydrophobic binding groove in each subunit. Interestingly, S100 proteins can interact with their partners in two ways: symmetrically, when the two partners identically bind into each groove, or asymmetrically, when only one partner binds to the S100 dimer occupying both binding pockets. Here we present a heterologous expression and purification protocol for all known human S100 proteins as well as for their partner peptides. Moreover, we provide a detailed description of three in vitro methods to determine the affinity, stoichiometry, and kinetics of S100 protein-protein interactions.
S100蛋白是一类小分子蛋白，大多为二聚体，属于EF手型钙结合蛋白。结合钙离子后，会发生构象变化，导致每个亚基中一个浅的疏水结合凹槽暴露出来。有趣的是，S100蛋白可以通过两种方式与它们的伙伴相互作用：对称方式，即两个伙伴以相同方式结合到每个凹槽中；非对称方式，即只有一个伙伴结合到占据两个结合口袋的S100二聚体上。在此，我们介绍了一种针对所有已知人类S100蛋白及其伙伴肽的异源表达和纯化方案。此外，我们还详细描述了三种体外方法，用于确定S100蛋白-蛋白相互作用的亲和力、化学计量比和动力学。
