Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Division of Theoretical Chemistry, Department of Chemistry, Lund University, SE 221 00 Lund, Sweden.
J Am Chem Soc. 2021 Feb 17;143(6):2500-2508. doi: 10.1021/jacs.0c10789. Epub 2021 Feb 2.
Electrostatic forces are important for protein folding and are favored targets of protein engineering. However, interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in p value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions.
静电相互作用对于蛋白质折叠很重要，也是蛋白质工程的重要目标。然而，由于大多数蛋白质中存在复杂的相互作用网络，带电残基之间的相互作用很难研究。我们设计了一个有目的的简单系统来研究这个问题，通过系统地在一个原本不含这些残基的蛋白质中引入单个和成对的带电和可滴定残基。我们使用恒 pH 分子动力学模拟、NMR 光谱和热力学双突变体循环来探测带电残基之间相互作用的结构和热力学。我们发现，表面电荷的部分埋藏有助于 p 值的偏移，导致天冬氨酸在中性 pH 范围内滴定。此外，还发现残基对之间的相互作用高度依赖于上下文，有些对之间没有明显的优先相互作用，而其他对之间则会发生偶联滴定，形成高度稳定的盐桥。我们发现实验和模拟之间有很好的一致性，并使用模拟来合理化我们的观察结果，并提供静电相互作用的详细机制理解。
