The Beauty of Asymmetric Membranes: Reconstitution of the Outer Membrane of Gram-Negative Bacteria.

Laura Paulowski,Annemarie Donoghue,Sabrina Groth,Thomas Gutsmann,Sven O Hagge,Arne Böhling,Mathias Winterhalter,Max Koistinen,Christian Nehls

doi:10.3389/fcell.2020.00586

Abstract

The architecture of the lipid matrix of the outer membrane of Gram-negative bacteria is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is built up by glycolipids. For most Gram-negative species, these glycolipids are lipopolysaccharides (LPS), for a few species, however, glycosphingolipids. We demonstrate experimental approaches for the reconstitution of these asymmetric membranes as (i) solid supported membranes prepared by the Langmuir-Blodgett technique, (ii) planar lipid bilayers prepared by the Montal-Mueller technique, and (iii) giant unilamellar vesicles (GUVs) prepared by the phase transfer method. The asymmetric GUVs (aGUVs) composed of LPS on one leaflet are shown for the first time. They are characterized with respect to their phase behavior, flip-flop of lipids and their usability to investigate the interaction with membrane active peptides or proteins. For the antimicrobial peptide LL-32 and for the bacterial porin OmpF the specificity of the interaction with asymmetric membranes is shown. The three reconstitution systems are compared with respect to their usability to investigate domain formation and interactions with peptides and proteins.

Highlights

Since the introduction of the fluid mosaic model by SINGER and NICOLSON in 1972, the membrane itself and its unique, complex architecture has remained a central pillar of research today (Singer and Nicolson, 1972)
A number of studies have shown how the lipid composition differs in the two leaflets of various membranes, what influence this has on the function (Gupta et al, 2020), localization and orientation of proteins, how asymmetry is established and maintained by active processes and which consequences changes in asymmetry can have
This size range resembles the size of living bacteria and enables the microscopic analysis of asymmetric membranes without solid support

Summary

Introduction

Since the introduction of the fluid mosaic model by SINGER and NICOLSON in 1972, the membrane itself and its unique, complex architecture has remained a central pillar of research today (Singer and Nicolson, 1972). In the early 1970s, KORNBERG and MCCONNELL were the first to successfully resolve the kinetics of lipid flip-flop between the individual membrane leaflets (Kornberg and McConnell, 1971). These two important hallmarks in research were essential steps in membrane biophysics. Many new insights into lipid membranes and their interaction with peptides and proteins have been gained by reconstituted membranes in biophysical experiments. These systems often accept the limitation that the model membranes usually have a symmetrical lipid architecture. Relatively little is known about the importance of axial heterogeneity or lipid asymmetry in membranes

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