Comparison of different approaches to single-molecule imaging of enhanced enzyme diffusion*

Enzymes have been shown to diffuse faster in the presence of their substrates. Recently, we revealed new insights into this process of enhanced diffusion using single-particle tracking (SPT). In our prior work, we used a polymer-brush coated surface and a large viscous polymer, methylcellulose, to slow diffusion and facilitate tracking. Here, we simplify our system by using supported lipid bilayer (SLB) coated surface, and a small viscous molecule, glycerol. Using this newly-designed lipid-coated/glycerol chamber, we compare two different analysis approaches for SPT: the mean-squared displacement (MSD) analysis and the jump-length analysis. We find that the MSD analysis requires high viscosity and large particles to accurately report the diffusion coefficient, while jump-length analysis depends less on the viscosity or size limitation. Furthermore, lipid-coated/glycerol chambers fail to reproduce enhanced diffusion due to the presence of glycerol inhibiting enzyme activity. To avoid adding glycerol and to constrain the diffusion to two dimensions, we tether enzymes directly to the lipid bilayer. This approach recovers the 3-fold enhancement in diffusion of enzymes with the presence of their substrate as we observed before.