Bunching Effect in Single-Molecule T4 Lysozyme Nonequilibrium Conformational Dynamics under Enzymatic Reactions

The bunching effect, implying that conformational motion times tend to bunch in a finite and narrow time window, is observed and identified to be associated with substrate-enzyme complex formation in T4 lysozyme conformational dynamics under enzymatic reactions. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. On the basis of the single-molecule spectroscopic results, molecular dynamics simulation, and a random walk model analysis, multiple intermediate states have been estimated in the evolution of T4 lysozyme enzymatic reaction activ