Key Laboratory of Micro-Nano Materials for Energy Storage and Conversion of Henan Province, Institute of Surface Micro and Nano Materials, College of Chemical and Materials Engineering, Xuchang University, Henan, 461000, PR China.
Key Laboratory of Micro-Nano Materials for Energy Storage and Conversion of Henan Province, Institute of Surface Micro and Nano Materials, College of Chemical and Materials Engineering, Xuchang University, Henan, 461000, PR China.
Anal Chim Acta. 2023 Feb 22;1243:340830. doi: 10.1016/j.aca.2023.340830. Epub 2023 Jan 12.
Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic monitoring approach based on nanopore single-molecule biosensor, which can detect the protein's folding and unfolding transitions by recording a nanopore ionic current. When gradually increasing the concentration of denaturant guanidine hydrochloride (GdmCl), sensitive responses were observed with lysozyme unfolding. The emergence of the featured biphasic-pulse demonstrated the existence of a stable intermediate. It was the first time to experimentally confirm the dynamic equilibrium between the intermediate and the native states at single molecule level, therefore consolidating the standpoint of lysozyme denaturation process following the three-state model. Additionally, we got more insights into the conformation about the intermediate as globular-like structure, larger gyration radius, and enhanced positive charge density. We considered that the manner of denaturant toward lysozyme adopts the "direct" model based on stronger electrostatic and van der Waals forces. Nanopore biosensor exhibited excellent sensitivity with a low detection concentration of 280 pM and reproducibility in analysing the folding intermediate of lysozyme.
蛋白质研究中的不明确问题包括但不限于变性剂存在下的稳定性和变性机制。在此，我们报告了一种基于纳米孔单分子生物传感器的动态监测方法，该方法可以通过记录纳米孔离子电流来检测蛋白质的折叠和展开转变。当逐渐增加变性剂盐酸胍 (GdmCl) 的浓度时，溶菌酶展开时会观察到敏感反应。特征性的双相脉冲的出现表明存在稳定的中间态。这是首次在单分子水平上实验证实中间态与天然态之间的动态平衡，从而巩固了溶菌酶变性过程遵循三态模型的观点。此外，我们对中间态的构象有了更深入的了解，其结构为球状，回转半径更大，正电荷密度增强。我们认为变性剂对溶菌酶的作用方式基于更强的静电和范德华力，采用“直接”模式。纳米孔生物传感器在分析溶菌酶折叠中间态时表现出优异的灵敏度，检测浓度低至 280 pM，重现性好。
