Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding 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. We have developed and applied AFM/Magnetic Tweezers force manipulation combined single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins.1-3 Enzymatic reaction turnovers and the associated structure changes of individual protein molecules under pico-Newton force manipulations were observed simultaneously in real-time by single-molecule FRET detections. We have also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. we have experimentally interrogated direct influence of a mechanical picoNewton (pN) oscillating force, weaker than thermal fluctuation force, on enzymatic activity and the conformational dynamics of the single enzyme under different oscillation frequencies and under enzymatic condition.1-3 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. We have also observed a novel property of protein structure spontaneous ruptures under pN compressive force manipulations. Our new multimodal chemical imaging and manipulation macroscopy have a high and broad impact on applications in studying protein structure-dynamics-function relationship.

Probing and manipulating enzyme activity and conformational dynamics by single-molecule AFM-FRET and magnetic tweezers-FRET ultramicroscopy

2019 Spring National Meeting

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