Long Duration, Single-Molecule Monitoring of Lysozyme Kinetics

Abstract

Tethering a single molecule of T4 lysoyzme to a carbon nanotube field effect transistor resulted in a novel technique for monitoring single-molecule enzymatic activity. Continuous electronic monitoring readily resolved lysozyme as a processive enzyme, since substrate release was clearly distinguished from serial hydrolysis of glycosidic bonds. On average, we observed hydrolysis of 100 consecutive bonds at 15 Hz rates when lysozyme processed natural peptidoglycan. Furthermore, long duration measurements allowed statistically-meaningful analysis of thousands of chemical events by the same molecule, uncovering seven independent timescales governing lysozyme's activity, including minute-by-minute dynamic disorder. Stability of the lysozyme-transistor method allowed these timescales to be studied as a function of environmental conditions such as substrate, pH, or temperature, in order to produce a detailed map of factors that affect single molecule processivity. For example, variations in pH do not change any of the rate constants of lysozyme's motions, but rather decrease enzyme activity by increasing the proportion of time stuck in an inactive, closed conformation. As another example, we found that peptidoglycan cross-links are directly responsible for a 50% drop in effective activity. Using a synthetic, linear substrate without cross-links, we observed long, uninterrupted processing runs to the end of individual substrate molecules. By comparison, lysozyme spends approximately half its time traversing the cross-links present in wild-type substrate. Rather than releasing substrate at a cross-link, however, lysozyme instead changes to an alternate, non-processive motion with which it can sidestep the cross-link. The combination of processive and non-processive motions allow lysozyme to remain effective and zigzag through wild-type substrate.