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

Enzymes have been shown to diffuse faster in the presence of their reactants. Recently, we revealed new insights into this process of enhanced diffusion using single-particle tracking (SPT) with total internal reflection fluorescence (TIRF) microscopy. We found that the mobility of individual enzymes was enhanced three fold in the presence of the substrate, and the motion remained Brownian. In this work, we compare different experimental designs, as well as different data analysis approaches, for studying single enzyme diffusion. We first tether enzymes directly on supported lipid bilayers (SLBs) to constrain the diffusion of enzymes to two dimensions. This experimental design recovers the 3-fold enhancement in enzyme diffusion in the presence of the substrate, as we observed before. We also simplify our system by replacing the bulky polymers used in the prior chamber design with a SLB-coated surface and glycerol. Using this newly-designed SLB/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. Furthermore, the SLB-glycerol chamber fails to reproduce the enhanced diffusion of enzymes because glycerol inhibits enzyme activity.