title: Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy.
abstract: Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure-function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2-amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme-substrate interactions, enzyme-substrate active complex formation, and protein folding-binding interactions.
authors.affiliation: Department of Chemistry, Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403.
authors.forename: Qing
authors.initials: Q
authors.lastname: Guo
authors.affiliation: Department of Chemistry, Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403.
authors.forename: Yufan
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Chemistry, Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403 hplu@bgsu.edu.
authors.forename: H Peter
authors.initials: HP
authors.lastname: Lu
mesh_terms: D006735:Horseradish Peroxidase / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation
chemical_list: D006735:Horseradish Peroxidase
keywords: conformational fluctuation; enzymatic conformational dynamics; enzyme; manipulate the conformation; single molecule TIRF-magnetic tweezers microscopy