Probing and Manipulating Enzyme Activity and Conformational Dynamics by Single-Molecule Afm-FRET and Magnetic Tweezers-FRET Ultramicroscopy

Abstract

Single-molecule conformational manipulation provides unique methods for studying the relationship between 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 single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. 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. By a repetitive pulling-releasing manipulation of a 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, an experimental evidence of 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.Reference:1. Qing Guo, Yufan He, H. Peter Lu, “Repetitive Force Pulling-Releasing Manipulation of Enzymatic Conformational Fluctuations and Enzyme-Substrate Interactions by Single-Molecule FRET-Magnetic Tweezers Microscopy,” Phys. Chem. Chem. Phys., 16, 13052 −13058 (2014).2. H. Peter Lu, “Sizing up single-molecule enzymatic conformational dynamics,” Chem. Soc. Rev., Invited Review Article, 43, 1118-1143 (2014).3. Yufan He, Maolin Lu, H. Peter Lu, “Single-Molecule Photon Stamping FRET Spectroscopy Study of Enzymatic Conformational Dynamics,” Phys. Chem. Chem. Phys., 15, 770-775 (2013) (Cover page).4. Yufan He, Maolin Lu, Jin Cao, H. Peter Lu, “Manipulating Protein Conformations by Single-Molecule AFM-FRET Nanoscopy,” ACS Nano, 6, 1221-1229 (2012).