Wu Shaowen, Zhang Wenyang, Li Wenyan, Huang Wenjie, Kong Qian, Chen Zhongjian, Wei Wenkang, Yan Shijuan
Guangdong Key Laboratory for Crop Germplasm Resources Preservation and Utilization, Agro-biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, Guangdong, China.
Biochemistry. 2022 Mar 15;61(6):433-445. doi: 10.1021/acs.biochem.1c00771. Epub 2022 Feb 28.
Protein-ligand interactions are crucial to many biological processes. Ligand binding and dissociation are the basic steps that allow proteins to function. Protein conformational dynamics have been shown to play important roles in ligand binding and dissociation. However, it is challenging to determine the ligand binding kinetics of dynamic proteins. Here, we undertook comprehensive single-molecule FRET (smFRET) measurements and kinetic model analysis to characterize the conformational dynamics coupled ligand binding of glutamine-binding protein (GlnBP). We showed that hinge and T118A mutations of GlnBP affect its conformational dynamics as well as the ligand binding affinity. Based on smFRET measurements, the kinetic model of ligand-GlnBP interactions was constructed. Using experimentally measured parameters, we solved the rate equations of the model and obtained the undetectable parameters of the model which allowed us to understand the ligand binding kinetics fully. Our results demonstrate that modulation of the conformational dynamics of GlnBP affects the ligand binding and dissociation rates. This study provides insights into the binding kinetics of ligands, which are related to the protein function itself.
蛋白质-配体相互作用对许多生物过程至关重要。配体结合和解离是蛋白质发挥功能的基本步骤。蛋白质构象动力学已被证明在配体结合和解离中起重要作用。然而，确定动态蛋白质的配体结合动力学具有挑战性。在此，我们进行了全面的单分子荧光共振能量转移（smFRET）测量和动力学模型分析，以表征谷氨酰胺结合蛋白（GlnBP）的构象动力学耦合配体结合。我们表明，GlnBP的铰链和T118A突变影响其构象动力学以及配体结合亲和力。基于smFRET测量，构建了配体-GlnBP相互作用的动力学模型。使用实验测量的参数，我们求解了模型的速率方程，并获得了模型中不可检测的参数，这使我们能够充分理解配体结合动力学。我们的结果表明，GlnBP构象动力学的调节影响配体结合和解离速率。这项研究为与蛋白质功能本身相关的配体结合动力学提供了见解。
