Tracking single lipase molecules on a trimyristin substrate surface using quantum dots

The mobility of single lipase molecules has been analyzed using single molecule tracking on a trimyristin substrate surface. This was achieved by conjugating lipases to quantum dots and imaging on spin-coated trimyristin surfaces by means of confocal laser scanning microscopy. Image series of single lipase molecules were collected, and the diffusion coefficient was quantified by analyzing the mean square displacement of the calculated trajectories. During no-flow conditions, the lipase diffusion coefficient was (8.0+/-5.0)x10(-10) cm2/s. The trajectories had a "bead on a string" appearance, with the lipase molecule restricted in certain regions of the surface and then migrating to another region where the restricted diffusion continued. This gave rise to clusters in the trajectories. When a flow was applied to the system, the total distance and average step length between the clusters increased, but the restricted diffusion in the cluster regions was unaffected. This can be explained by the lipase operating in two different modes on the surface. In the cluster regions, the lipase is likely oriented with the active site toward the surface and hydrolyzes the substrate. Between these regions, a diffusion process is proposed where the lipase is in contact with the surface but affected by the external flow.