Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon.
Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon; Institute for Molecular Biology, University of Oregon, Eugene, Oregon; Oregon Center for Optical, Molecular, & Quantum Science, University of Oregon, Eugene, Oregon.
Biophys J. 2023 May 2;122(9):1600-1612. doi: 10.1016/j.bpj.2023.01.033. Epub 2023 Jan 28.
Mutations introduced into macromolecules often exhibit epistasis, where the effect of one mutation alters the effect of another. Knowing the mechanisms that lead to epistasis is important for understanding how macromolecules work and evolve, as well as for effective macromolecular engineering. Here, we investigate the interplay between "contact epistasis" (epistasis arising from physical interactions between mutated residues) and "ensemble epistasis" (epistasis that occurs when a mutation redistributes the conformational ensemble of a macromolecule, thus changing the effect of the second mutation). We argue that the two mechanisms can be distinguished in allosteric macromolecules by measuring epistasis at differing allosteric effector concentrations. Contact epistasis manifests as nonadditivity in the microscopic equilibrium constants describing the conformational ensemble. This epistatic effect is independent of allosteric effector concentration. Ensemble epistasis manifests as nonadditivity in thermodynamic observables-such as ligand binding-that are determined by the distribution of ensemble conformations. This epistatic effect strongly depends on allosteric effector concentration. Using this framework, we experimentally investigated the origins of epistasis in three pairwise mutant cycles introduced into the adenine riboswitch aptamer domain by measuring ligand binding as a function of allosteric effector concentration. We found evidence for both contact and ensemble epistasis in all cycles. Furthermore, we found that the two mechanisms of epistasis could interact with each other. For example, in one mutant cycle we observed 6 kcal/mol of contact epistasis in a microscopic equilibrium constant. In that same cycle, the maximum epistasis in ligand binding was only 1.5 kcal/mol: shifts in the ensemble masked the contribution of contact epistasis. Finally, our work yields simple heuristics for identifying contact and ensemble epistasis based on measurements of a biochemical observable as a function of allosteric effector concentration.
突变引入大分子后常表现出上位性，即一个突变的效应会改变另一个突变的效应。了解导致上位性的机制对于理解大分子的工作原理和进化以及有效的大分子工程都很重要。在这里，我们研究了“接触上位性”（由突变残基之间的物理相互作用引起的上位性）和“整体上位性”（当一个突变重新分配大分子的构象整体时发生的上位性，从而改变第二个突变的效应）之间的相互作用。我们认为，通过在不同变构效应物浓度下测量上位性，可以将这两种机制区分在变构大分子中。接触上位性表现为描述构象整体的微观平衡常数的非加和性。这种上位性效应与变构效应物浓度无关。整体上位性表现为热力学观测值（如配体结合）的非加和性，这些观测值由整体构象的分布决定。这种上位性效应强烈依赖于变构效应物浓度。使用这个框架，我们通过测量配体结合作为变构效应物浓度的函数，实验研究了三个引入腺嘌呤核糖开关适体结构域的成对突变循环中的上位性起源。我们在所有循环中都发现了接触和整体上位性的证据。此外，我们发现两种上位性机制可以相互作用。例如，在一个突变循环中，我们在微观平衡常数中观察到 6 千卡/摩尔的接触上位性。在同一循环中，配体结合的最大上位性仅为 1.5 千卡/摩尔：整体的变化掩盖了接触上位性的贡献。最后，我们的工作基于对变构效应物浓度下生化观测值的测量，为识别接触和整体上位性提供了简单的启发式方法。
