Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A2 Action at the Single Molecule Level

TL;DR

This study visualized and analyzed the behavior of single phospholipase A2 enzymes on lipid monolayers, revealing how lipid phase heterogeneity influences enzyme activity, diffusion, and hydrolysis at the molecular level.

Contribution

It provides the first direct single-molecule visualization of PLA2 activity on heterogeneous lipid monolayers, linking lipid phase behavior to enzyme dynamics and hydrolysis patterns.

Findings

Enzymes preferentially interact with fluid lipid regions.

Hydrolysis products reduce enzyme mobility significantly.

Enzyme residence times vary between 30 and 220 ms.

Abstract

We monitored the action of phospholipase A2 (PLA2) on L- and D-dipalmitoylphosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA2 molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the build-up of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA2. The mobility of individual enzymes on the monolayers was characterized by single particle tracking (SPT). Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3 mu m^2/s on the L-DPPC and 4.6 mu m^/s on the D-DPPC monolayers. In regions enriched with hydrolysis products the diffusion dropped to approx. 0.2 mu m^2/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between approx. 30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e. enzymes were simply reflected from the gel domains back into solution.