Groningen Biomolecular Sciences & Biotechnology (GBB) Institute, University of Groningen, Groningen, The Netherlands.
Department of Chemistry, KU Leuven, Leuven, Belgium.
Nat Chem. 2020 May;12(5):481-488. doi: 10.1038/s41557-020-0437-0. Epub 2020 Apr 6.
Conformational heterogeneity is emerging as a defining characteristic of enzyme function. However, understanding the role of protein conformations requires their thermodynamic and kinetic characterization at the single-molecule level, which remains extremely challenging. Here we report the ligand-induced conformational changes of dihydrofolate reductase (DHFR) by measuring the modulation of the nanopore currents. The long observation time of the electrical recordings enabled the detection of rare conformational transitions hidden in ensemble measurements. We show that DHFR exists in at least four ground-state configurations or conformers with different affinities for its ligands. Unliganded DHFR adopted low-affinity conformers, whereas the binding of substrates promoted the switch to the high-affinity conformer. Conversion between the conformers was accelerated by molecules that stabilized the transition state of DHFR, which suggests that the reaction lowers the energy barrier for conformer exchange and thus facilitates product release. This mechanism might be a general feature in enzymatic reactions affected by product inhibition or when the release of products is the rate-limiting step.
构象异质性正在成为酶功能的一个决定性特征。然而，要理解蛋白质构象的作用，就需要在单分子水平上对其热力学和动力学特性进行表征，而这仍然极具挑战性。在这里，我们通过测量纳米孔电流的调制来报告二氢叶酸还原酶（DHFR）的配体诱导构象变化。电记录的长时间观测使我们能够检测到在整体测量中隐藏的罕见构象转变。我们表明，DHFR 至少存在四种具有不同配体亲和力的基态构象或构象体。未配位的 DHFR 采用低亲和力构象体，而底物的结合促进了向高亲和力构象体的转变。构象体之间的转换通过稳定 DHFR 过渡态的分子而加速，这表明反应降低了构象体交换的能量障碍，从而促进了产物释放。这种机制可能是受产物抑制或产物释放是限速步骤影响的酶反应的一般特征。
