Direct Single Molecule Imaging of Enhanced Enzyme Diffusion

Abstract

Recent experimental results have shown that active enzymes can diffuse faster when they are in the presence of their substrates. Fluorescence correlation spectroscopy (FCS), which relies on analyzing the fluctuations in fluorescence intensity signal to measure the diffusion coefficient of particles, has typically been employed in most of the prior studies. However, flaws in the FCS method, due to its high sensitivity to the environment, have recently been evaluated, calling the prior diffusion results into question. It behooves us to adopt complementary and direct methods to measure the mobility of enzymes in solution. Herein, we use a novel technique of direct single-molecule imaging to observe the diffusion of single enzymes. This technique is less sensitive to intensity fluctuations and gives the diffusion coefficient directly based on the trajectory of the enzymes. 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 complimentary method to test if the total enzyme concentration affects the relative increase in diffusion and if enzyme oligomerization state changes during catalytic turnover. We find that the diffusion increase is independent of the total background concentration of enzyme and the catalysis of substrate does not change the oligomerization state of enzymes.

Enhanced Diffusion and Oligomeric Enzyme Dissociation

“…This is broadly consistent with the qualitative conclusion from much previous work and helps to clarify the regime of their potential validity. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] In particular, while enhanced diffusion concept needs qualification may not apply in the substrate concentration regime where enzymes dissociate into subunits, they may apply at lesser concentrations, and this is interesting because the regime of lesser concentration is more relevant biologically.…”

Cross-diffusion induced patterns for a single-step enzymatic reaction

“…They differ only by a prefactor (D c − D f )/D f in the second term inside the square root of Eq. (11).…”

“…Most of these experiments relied on fluorescence correlation spectroscopy (FCS) measurements. Although artifacts introduced by this technique have been pointed out 8,9 , recent findings using other techniques 10,11 validated the phenomenon, which is often referred to as "enhanced diffusion". The underlying mechanism is still under debate 12 .…”

“…Some experiments suggest that catalysis plays a key role 3,5,13,14 , while others indicate that enhanced diffusion persists when the substrate is replaced by an inhibitor 1,4,15 . According to the latest experiments performed with the enzyme urease 11,13 , it appears that both the binding and the catalysis step of the reaction scheme contribute to enhanced diffusion.…”

Self-Propulsion Strategies for Artificial Cell-Like Compartments

“…NTA may also be used to track fluorescent particles by detecting the fluorescence signal rather than scattered light by modifying the optical setup. A similar direct single-molecule imaging approach has been used to observe the diffusion of individual enzymes in solution [63]. NTA has been used to study the propulsion of Pt loaded stomatocytes [9].…”