期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：52
页码：15006-15011
DOI：10.1073/pnas.1613404114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe exploration of the internal conformational fluctuation dynamics of an enzyme during a catalytic event can have significant implications in the field of enzymology. How an enzyme modulates its conformations under mechanical perturbation while still retaining its activity to a significant extent can be of particular interest in the area of protein engineering. Using single-molecule total internal reflection fluorescence microscopy-guided confocal spectroscopy combined with magnetic tweezers we have interrogated the conformational dynamics of horseradish peroxidase enzyme. We have identified the presence of complex conformations during the product releasing step. However, the complexity is narrowed or even eliminated when the enzyme is deformed or unfolded under magnetic pulling force. Unraveling the conformational details of an enzyme during the essential steps of a catalytic reaction (i.e., enzyme-substrate interaction, enzyme-substrate active complex formation, nascent product formation, and product release) is challenging due to the transient nature of intermediate conformational states, conformational fluctuations, and the associated complex dynamics. Here we report our study on the conformational dynamics of horseradish peroxidase using single-molecule multiparameter photon time-stamping spectroscopy with mechanical force manipulation, a newly developed single-molecule fluorescence imaging magnetic tweezers nanoscopic approach. A nascent-formed fluorogenic product molecule serves as a probe, perfectly fitting in the enzymatic reaction active site for probing the enzymatic conformational dynamics. Interestingly, the product releasing dynamics shows the complex conformational behavior with multiple product releasing pathways. However, under magnetic force manipulation, the complex nature of the multiple product releasing pathways disappears and more simplistic conformations of the active site are populated.
关键词：enzymatic conformational dynamics ; magnetic tweezers ; mechanical force manipulation ; enzymatic product releasing ; fluorogenic substrate
