Linking Phospholipase Mobility to Activity by Single‐Molecule Wide‐Field Microscopy

Abstract

Many of the biological processes taking place in cells are mediated by enzymatic reactions occurring in the cell membrane. Understanding interfacial enzymatic catalysis is therefore crucial to the understanding of cellular function. Unfortunately, a full picture of the overall mechanism of interfacial enzymatic catalysis, and particularly the important diffusion processes therein, remains unresolved. Herein we demonstrate that single-molecule wide-field fluorescence microscopy can yield important new information on these processes. We image phospholipase enzymes acting upon bilayers of their natural phospholipid substrate, tracking the diffusion of thousands of individual enzymes while simultaneously visualising local structural changes to the substrate layer. We study several enzyme types with different affinities and catalytic activities towards the substrate. Analysis of the trajectories of each enzyme type allows us successfully to correlate the mobility of phospholipase with its catalytic activity at the substrate. The methods introduced herein represent a promising new approach to the study of interfacial/heterogeneous catalysis systems.

The Rylene Colorant Family—Tailored Nanoemitters for Photonics Research and Applications

“…[186] The enzymes labeled with this PDI dye allowed the actions of phospholipase on their natural substrates to be characterized, for example, on phospholipid-supported layers. [185] This approach enabled phospholipase mobilities to be correlated with the catalytic activities for the first time. [185] Furthermore, this assay allowed the validation of the influence of the layer composition and fluidity on both the phospholipase mobility and activity (Figure 22).…”

“…[185] This approach enabled phospholipase mobilities to be correlated with the catalytic activities for the first time. [185] Furthermore, this assay allowed the validation of the influence of the layer composition and fluidity on both the phospholipase mobility and activity (Figure 22). [185] We have shown in previous sections that the combination of the hydrophobic rylene scaffold and polar substituents can lead to the formation of highly amphiphilic molecules.…”

“…[185] Furthermore, this assay allowed the validation of the influence of the layer composition and fluidity on both the phospholipase mobility and activity (Figure 22). [185] We have shown in previous sections that the combination of the hydrophobic rylene scaffold and polar substituents can lead to the formation of highly amphiphilic molecules. These can serve as nanoreporters of the polarity of their direct surroundings, thus allowing the visualization of membranes with high contrast.…”

“…[184] By using the asymmetric PDI chromophore 39, single phospholipase enzymes were functionalized and visualized, even on a fluorescently labeled substrate. [180,185] Under these demanding conditions, the PDI 39 revealed survival times 600 times longer than with ATTO 647N, [177] which is often considered an ultrastable fluorescent reporter. [186] The enzymes labeled with this PDI dye allowed the actions of phospholipase on their natural substrates to be characterized, for example, on phospholipid-supported layers.…”

Single Particle Tracking of ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin Type-1 Repeats) Molecules on Endothelial von Willebrand Factor Strings

“…Here, we aimed to gain insight into the ADAMTS13Ј binding mode on these mobile VWF strings by directly visualizing how and where single inactive ADAMTS13 E225Q enzymes bind to these VWF substrates using SMF microscopy (SMFM). SM measurements have been extensively applied to track proteins in membranes of living cells (24,25) or lipid bilayers (26), following motor proteins along the cytoskeleton (27,28) and DNA-binding proteins on DNA (29 -32). It is important to note that, because the A2 domain is only available once VWF strings are stretched, investigation of the interaction between ADAMTS13 and its substrate is done under flow, which greatly increases the complexity of the system.…”