Placing Single-Molecule T4 Lysozyme Enzymes on a Bacterial Cell Surface: Toward Probing Single-Molecule Enzymatic Reaction in Living Cells

Abstract

The T4 lysozyme enzymatic hydrolyzation reaction of bacterial cell walls is an important biological process, and single-molecule enzymatic reaction dynamics have been studied under physiological condition using purified Escherichia coli cell walls as substrates. Here, we report progress toward characterizing the T4 lysozyme enzymatic reaction on a living bacterial cell wall using a combined single-molecule placement and spectroscopy. Placing a dye-labeled single T4 lysozyme molecule on a targeted bacterial cell wall by using a hydrodynamic microinjection approach, we monitored single-molecule rotational motions during binding, attachment to, and dissociation from the cell wall by tracing single-molecule fluorescence intensity time trajectories and polarization. The single-molecule attachment duration of the T4 lysozyme to the cell wall during enzymatic reactions was typically shorter than the photobleaching time under physiological conditions. Applying single-molecule fluorescence polarization measurements to characterize the binding and motions of the T4 lysozyme molecules, we observed that the motions of wild-type and mutant T4 lysozyme proteins are essentially the same whether under an enzymatic reaction or not. The changing of the fluorescence polarization suggests that the motions of the T4 lysozyme are associated with orientational rotations. This observation also suggests that the T4 lysozyme binding-unbinding motions on cell walls involve a complex mechanism beyond a single-step first-order rate process.