Department of Anesthesiology

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Real-time Visualization of Phospholipid Degradation by Outer Membrane Phospholipase A using High-Speed Atomic Force Microscopy.

TitleReal-time Visualization of Phospholipid Degradation by Outer Membrane Phospholipase A using High-Speed Atomic Force Microscopy.
Publication TypeJournal Article
Year of Publication2017
AuthorsRangl M, Rima L, Klement J, Miyagi A, Keller S, Scheuring S
JournalJ Mol Biol
Volume429
Issue7
Pagination977-986
Date Published2017 Apr 07
ISSN1089-8638
KeywordsBacterial Outer Membrane Proteins, Calcium, Kinetics, Microscopy, Atomic Force, Models, Biological, Phospholipases A1, Phospholipids
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.

DOI10.1016/j.jmb.2017.03.004
Alternate JournalJ. Mol. Biol.
PubMed ID28283404