Direct Single Molecule Imaging of Enhanced Enzyme Diffusion

Recent experimental results have shown that enzymes can diffuse faster when they are in the presence of their reactants (substrate). This faster diffusion has been termed enhanced diffusion. Fluorescence correlation spectroscopy (FCS), which has been employed as the only method to make these measurements, relies on analyzing the fluctuations in fluorescence intensity to measure the diffusion coefficient of particles. Recently, artifacts in FCS measurements due to its sensitivity to environmental conditions have been evaluated, calling prior enhanced diffusion results into question. It behooves us to adopt complementary and direct methods to measure the mobility of enzymes. Herein, we use a technique of direct single molecule imaging to observe the diffusion of individual enzymes in solution. This technique is less sensitive to intensity fluctuations and deduces the diffusion coefficient directly based on the trajectory of the enzyme. Our measurements recapitulate that enzyme diffusion is enhanced in the presence of its substrate and find that the relative increase in diffusion of a single enzyme is even higher than those previously reported using FCS. We also use this complementary method to test if the total enzyme concentration affects the relative increase in diffusion and if the enzyme oligomerization state changes during its catalytic turnover. We find that the diffusion increase is independent of the total concentration of enzymes and the presence of substrate does not change the oligomerization state of enzymes.

Figure 1

(a) Experimental setup for single particle imaging of fluorescent urease (green) using TIRF (blue) in a chamber with Pluronic F127 (black) coating the surface and dilute methylcellulose polymers (orange) to slow down the mobility. Radius of gyration of methylcellulose (dashed red circle, $\sim 30\mathrm{nm}$) was represented [17]. (b) Example time series of single urease diffusing over time (i) without urea and (ii) with 1 mM urea. Scale bar $5\mu \mathrm{m}$. (c) 2D trajectories displayed as time collapsed images with rainbow scale representing diffusion time over 111 frames with (i) 0.13 s frame interval for urease without urea and (ii) 0.08 s frame interval for urease with 1 mM urea. Scale bar $5\mu \mathrm{m}$. (d) Time-averaged MSD plot of each trajectory before, fitting with a linear equation to determine the diffusion coefficient $D$. Inset: Same MSD data plotted on log-log scale. Black lines represent the range of $\alpha$ exponent values: ${\alpha }_{\max }=1.2$, ${\alpha }_{\min }=0.9$. Red squares, urease without urea; blue squares, urease with 1 mM urea; error bars represent the standard error.

Figure 2

(a) Representative probability distribution histograms of log-transformed diffusion data at different urea concentrations: 0 (red region, $N=141$), $10\mu \mathrm{M}$ (green region, $N=97$), 1 mM (blue region, $N=178$), 100 mM (purple region, $N=203$), and corresponding Gaussian fit lines 0 (red line), $10\mu \mathrm{M}$ (green line), 1 mM (blue line), 100 mM (purple line). (b) The normalized relative increase in the diffusion coefficient $(D-D_0)/D_0$, plotted as a function of the urea concentration. Inset: Same data plotted on a logarithmic scale. Solid line shows the hyperbolic fit with a characteristic concentration $K$. (c) (i) Illustration of 40 nM urease with average spacing between molecules of 400 nm. (ii) Illustration of 90 pM urease with average spacing between molecules of $3\mu \mathrm{m}$. (iii) Diffusion coefficients of urease at 40 nM urease concentration (dark gray bars) without urea ($N=31$) and with 1 mM urea ($N=35$), or urease at 90 pM (light gray bars) without urea ($N=30$) and with 1 mM urea ($N=36$). Error bars are determined from the standard errors of the mean of the Gaussian fits. All fit parameters are given in Supplemental Material [18]. PDF: probability distribution function, N.S.: not significant.

Figure 3

(a) Two example intensity traces of fluorescent urease complexes photobleaching over time, showing a one-step bleach (top) and a three-step bleach (bottom). (b) The distributions of photobleaching steps directly report the number of fluorescent urease monomers in each complex in the presence of 0 urea (dark gray bars, $N=100$) and 1 mM urea (light gray bars, $N=100$).