Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland;
Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO 63130.
Proc Natl Acad Sci U S A. 2020 Jun 16;117(24):13480-13489. doi: 10.1073/pnas.1921617117. Epub 2020 Jun 2.
Intrinsically disordered proteins (IDPs) abound in cellular regulation. Their interactions are often transitory and highly sensitive to salt concentration and posttranslational modifications. However, little is known about the effect of macromolecular crowding on the interactions of IDPs with their cellular targets. Here, we investigate the influence of crowding on the interaction between two IDPs that fold upon binding, with polyethylene glycol as a crowding agent. Single-molecule spectroscopy allows us to quantify the effects of crowding on a comprehensive set of observables simultaneously: the equilibrium stability of the complex, the association and dissociation kinetics, and the microviscosity, which governs translational diffusion. We show that a quantitative and coherent explanation of all observables is possible within the framework of depletion interactions if the polymeric nature of IDPs and crowders is incorporated based on recent theoretical developments. The resulting integrated framework can also rationalize important functional consequences, for example, that the interaction between the two IDPs is less enhanced by crowding than expected for folded proteins of the same size.
无规蛋白（IDPs）在细胞调节中大量存在。它们的相互作用通常是短暂的，并且对盐浓度和翻译后修饰高度敏感。然而，对于拥挤效应对 IDP 与其细胞靶标的相互作用的影响知之甚少。在这里，我们研究了拥挤效应对两种折叠蛋白与聚乙二醇作为拥挤剂结合的相互作用的影响。单分子光谱法使我们能够同时定量拥挤对一整套可观察量的影响：复合物的平衡稳定性、缔合和解离动力学以及控制平移扩散的微粘度。我们表明，如果根据最近的理论发展，将 IDP 和拥挤剂的聚合物性质纳入其中，那么在消耗相互作用的框架内，所有可观察量的定量和连贯解释是可能的。由此产生的综合框架还可以合理化重要的功能后果，例如，与相同大小的折叠蛋白相比，两种 IDP 之间的相互作用受拥挤的增强程度较低。
