title: Real-time Visualization of Phospholipid Degradation by Outer Membrane Phospholipase A using High-Speed Atomic Force Microscopy.
abstract: Phospholipases are abundant in various types of cells and compartments, where they play key roles in physiological processes as diverse as digestion, cell proliferation, and neural activation. In Gram-negative bacteria, outer membrane phospholipase A (OmpLA) is involved in outer-membrane lipid homeostasis and bacterial virulence. Although the enzymatic activity of OmpLA can be probed with an assay relying on an artificial monoacyl thioester substrate, only little is known about its activity on diacyl phospholipids. Here, we used high-speed atomic force microscopy (HS-AFM) to directly image enzymatic phospholipid degradation by OmpLA in real time. In the absence of Ca2+, reconstituted OmpLA diffused within a phospholipid bilayer without revealing any signs of phospholipase activity. Upon the addition of Ca2+, OmpLA was activated and degraded the membrane with a turnover of ~2 phospholipid molecules per second and per OmpLA dimer until most of the membrane phospholipids were hydrolyzed and the protein became tightly packed.
authors.affiliation: Department of Anesthesiology, Physiology and Biophysics, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA; INSERM U1006, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 163 Avenue de Luminy, 13009 Marseille, France.
authors.forename: Martina
authors.initials: M
authors.lastname: Rangl
authors.affiliation: INSERM U1006, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 163 Avenue de Luminy, 13009 Marseille, France; Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland.
authors.forename: Luca
authors.initials: L
authors.lastname: Rima
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Klement
authors.affiliation: Department of Anesthesiology, Physiology and Biophysics, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA; INSERM U1006, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 163 Avenue de Luminy, 13009 Marseille, France.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyagi
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany. Electronic address: mail@sandrokeller.com.
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
authors.affiliation: Department of Anesthesiology, Physiology and Biophysics, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA; INSERM U1006, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 163 Avenue de Luminy, 13009 Marseille, France. Electronic address: sis2019@med.cornell.edu.
authors.forename: Simon
authors.initials: S
authors.lastname: Scheuring
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods*; D008954:Models, Biological; D054466:Phospholipases A1 / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D010743:Phospholipids; D054466:Phospholipases A1; C412876:outer membrane phospholipase A; D002118:Calcium
keywords: Outer membrane protein; Phospholipase; atomic force microscopy; lipid hydrolysis; membrane degradation; membrane protein