Guo, Qing; He, Yufan; Lu, Peter
Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 112:13904-13909, NOV 10 2015

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.

DOI:10.1073/pnas.1506405112

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