Probing single molecule protein conformational
dynamics
Peter Lu, Bowling Green State University
Enzymatic reactions are traditionally studied at the ensemble level. Despite significant static and dynamic inhomogeneities, the non-synchronized nature of the enzymatic reactions makes them extremely difficult to obtain stepwise mechanistic and dynamic information. We have applied single-molecule spectroscopy under physiological conditions to study the mechanisms and dynamics of T4 lysozyme enzymatic reactions. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time. We obtained the rates for each step of this complex and non-synchronizable polysaccharide-hydrolysis reaction. The overall reaction rates were found to vary widely from molecule to molecule, and the initial non-specific binding of the enzyme to the substrate was seen to dominate this inhomogeneity. Molecular dynamics simulation has been applied to elucidate the mechanism and intermediate states of the single-molecule enzymatic reaction. Dynamic protein-protein and protein-DNA interactions involve significant conformational motions that initiate chain reactions leading to specific cellular responses. We have observed highly dynamic protein interactions in a number of protein recognition systems, including DNA-protein interactions in DNA damage recognition, GTPase protein interaction signaling, and calmodulin mediated cell signaling. In our study of a GTPase intracellular signaling protein Cdc42 in complex with a downstream effector protein, single molecule fluorescence intensity and polarization measurements have revealed the dynamic and inhomogeneous nature of protein-protein interactions within the Cdc42/WASP complex that is characterized by structured distributions of conformational fluctuation rates. It is the dynamic interactions that ensure the energy-effective and selectivity in cell signaling cascade processes. Recent MD simulations have further provided detailed information about the double-well nature of the protein-protein cooperative binding-folding interaction structure and free energy potential surfaces.