Department of Medical Biochemistry and Microbiology, BMC, Uppsala University, Uppsala, Sweden.
Methods Mol Biol. 2021;2263:105-133. doi: 10.1007/978-1-0716-1197-5_4.
There are multiple examples of protein-protein interactions involving one intrinsically disordered protein region binding to an ordered protein domain in a coupled binding and folding reaction. Similarly to protein folding studies, much effort has been devoted to understanding the mechanisms of such coupled binding and folding reactions. In this chapter, we describe how kinetics can be used to assess binding mechanisms with focus on fluorescence-monitored stopped-flow experiments. The approach can be applied more generally to any protein interaction with or without a coupled conformational change and to other kinetic techniques. Determining binding mechanisms is a great challenge and while "proving" a mechanism may be futile, it is possible to deduce the simplest scenarios, which are consistent with experimental data.
有许多涉及一个固有无序蛋白质区域与一个有序蛋白质结构域在偶联结合和折叠反应中结合的蛋白质-蛋白质相互作用的例子。与蛋白质折叠研究类似，人们付出了大量努力来理解这种偶联结合和折叠反应的机制。在本章中，我们描述了如何使用动力学来评估结合机制，重点是荧光监测的停流实验。该方法可以更一般地应用于任何具有或不具有偶联构象变化的蛋白质相互作用，以及其他动力学技术。确定结合机制是一个巨大的挑战，虽然“证明”一种机制可能是徒劳的，但推断出与实验数据一致的最简单情况是可能的。
