Effects on the Enzymatic Activity of OmpLA

(supported by the Austrian Science Fund, Project No. P32514-B20)

expected start of project: October 1, 2019
expected end of project: September 30, 2023

Compartmentalization of biochemical processes is one of the key concepts of life. Nature has realized with the advent of cellular membranes, which are composites of proteins, lipids and carbohydrates. Biological membranes support various physiological processes, including active transport, catalysis and cellular communication. In order to fulfill these manifold functions membranes display distinctly organized and chemically well-controlled structures. On the molecular level these include the formation of distinct lipid/protein domains and an asymmetric transmembrane distribution lipid species. In fact cells spend significant amount of energy to actively maintain this imbalance of lipids. Lipid asymmetry implies several questions of physiological relevance. Here we are interested in the coupling to protein function such as the outer membrane phospholipase A (OmpLA). OmpLA is an integral membrane enzyme located in the outer membrane of Gram-negative bacteria, such as Escherichia coli. Activation leads to a hydrolysis of phospholipids and is coupled to membrane perturbation that cause the formation of OmpLA dimers.

Previous biophysical studies demonstrated that OmpLA readily forms dimers, when reconstituted into artificial bilayers with symmetric lipid composition. The enzyme’s natural environment, however, comprises of an asymmetric bilayer of lipopolysaccharide in the outer leaflet and phosphatidylethanolamine/phosphatidylglycerol in the inner leaflet. We therefore hypothesize that the asymmetric lipid environment serves to maintain the protein’s monomeric dormant state. Membrane perturbation leads to a loss of lipid asymmetric which triggers the enzyme to clear the membrane from lipids flipped to the outer leaflet.

We will reconstitute OmpLA in different symmetric and asymmetric artificial membranes and study its aggregation state and activity as a function of membrane structure. This will be achieved by a coupling of X-ray and neutron scattering techniques with fluorescent energy transfer measurements of labelled proteins. Scattering techniques will allow us to interrogate effects of membrane structure on protein dimerization. Fluorescence assays in turn will give us insight into aggregate form of OmpLA in the diverse lipid membranes.

Research will be performed on chemically well-defined systems, i.e. unlike natural membranes, the lipid and protein composition will be well-known. This entails stringent experimental control and ensures full tractability of methods. Supported by an international network of researches with highly complementary expertise we will derive the significance of lipid asymmetry in OmpLA activation as a role model for the coupling of specific membrane composition to protein function. We expect that the project will form the basis for several follow-up research efforts in the field. Moreover, the designed artificial membranes might serve as prototypical platform for screening of membrane-active antimicrobial drugs.