Single-molecule conformational manipulation provides unique methods for studying the relationshipbetween the function and structure of biomolecules, and for exploring novel properties of biomolecules under complex local environments. Enzymatic reactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. We have developed and applied AFM/Magnetic Tweezers force manipulation combined with single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structural changes of individual protein molecules under pico-Newton force manipulations were observed simultaneously in real-time by single-molecule FRET detections. By a repetitive pulling-releasing manipulation of Cy3-Cy5 dye-labeled kinase molecules under the conditions with and without enzymatic substrates, we observed and analyzed the enzymatic conformational dynamics. We demonstrated that the enzyme conformational flexibility can be regulated by enzyme-substrate interaction, experimental evidence of the protein-substrate folding-binding interaction mechanism. Our new approach is applicable to a wide range of single-molecule AFM-FRET measurements for protein conformational changes under enzymatic reactions, including controls of enzymatic reactivity by mechanical-force manipulating protein conformations.

Single-molecule spectroscopy and force manipulation studies of protein dynamics and signaling

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