CeNS Colloquium

Prof. H. Peter Lu, Bowling Green State University

Enzymatic reactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. We applied 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 were observed simultaneously in real-time by single-molecule FRET detections. We obtained the rates for single-molecule conformational active-site open-close fluctuation and correlated enzymatic reactions. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions and protein-protein interactions in cell signaling. Using this approach, we analyzed enzyme-substrate complex formation dynamics to reveal (1) multiple intermediate conformational states, (2) conformational motions involving in active complex formation and product releasing from the enzymatic active site, and (3) conformational memory effects in the chemical reaction process. Furthermore, we have applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions. We obtained the rates for single-molecule conformational active-site open-close fluctuation and correlated enzymatic reactions. We have demonstrated a specific statistical analysis to reveal single-molecule FRET anti-correlated fluctuations from a high background of fluorescence correlated thermal fluctuations. Our new approach is applicable to a wide range of single-molecule AFM-FRET measurements for protein conformational changes under enzymatic reactions, including AFM-FRET control of enzymatic reactivity by mechanical-force manipulating protein conformations. Our recent work on single-molecule imaging studies of ion channel receptor conformational dynamics in living cells will also be discussed.