Control of Outer Membrane Phospholipase a (OmpLA) Functions on an Artificial Lipid Bilayer Membrane

Abstract

Giant lipid vesicles consisted of a lipid bilayer membrane are used to investigate the function of several membrane proteins in a living cell. The membrane protein-reconstituted giant lipid vesicles, which emulated cellular functions, was created. However, to occur the complex functions into the giant lipid vesicles, the generation of giant lipid vesicles containing a diverse set of membrane proteins at sufficient concentrations is difficult.In this study, we observe dual functions (small molecule transportation and budding of small vesicle) on an artificial lipid bilayer using Outer membrane phospholipase A (OmpLA) expressed in E.Coli outer membranes. OmpLA has not only enzyme activity but also the function of molecular transportation. In the presence of calcium ions, OmpLA catalyzes phospholipids to lysolipid and fatty acid. By measuring ion currents of OmpLA nanopores using a patch-clamp method of the artificial lipid bilayer, the reconstitution of OmpLA into artificial lipid bilayers was promoted by adding anionic and cationic lipids. To the best of our knowledge, we have revealed for the first time that the lipid composition controlled OmpLA orientation into the lipid bilayer because hydrophilic region of OmpLA has asymmetric distribution of charge. In addition, we observed the transportation of small molecules through OmpLA pore on giant lipid vesicles. Finally, by control of OmpLA orientation, we showed a budding of small vesicles from giant lipid vesicles by changing a curvature of the lipid membranes due to OmpLA enzyme activity. The giant lipid vesicles containing OmpLA will be contributed to regulate cell functions and recognize the living cells by changing the external environment of the artificial cell model.