Membrane Potentials Trigger Molecular-Scale Rearrangements in the Outer Membrane of Gram-Negative Bacteria

The structural complexity of the cell envelope of Gram-negative bacteria limits the fabrication of realistic models of bacterial cell membranes. A vertical Langmuir-Blodgett withdrawing was used to deposit a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) monolayer on the Au(111) surface. The second leaflet composed of di[3-deoxy-D-manno-octulosonyl]-lipid A (KLA) was deposited using Langmuir-Schaefer transfer. The use of an electrode material as a support for the POPE-KLA bilayer allowed electrochemical control of the membrane's stability, compactness, and structure. Capacitance-potential curves showed a typical pattern for the supported lipid bilayers electrochemical characteristic. The minimum membrane capacitance was ∼4 μF cm^-2 and did not change in the following desorption-adsorption cycles, indicating the presence of a stable bilayer structure with an asymmetric composition of both leaflets. However, at a molecular scale, as elucidated in spectroelectrochemical experiments, large differences in the response of both leaflets to electric potentials were observed. The acyl chains in POPE and KLA existed in a liquid state. The quantitative analysis of the CH stretching modes indicated potential-driven reorientations in the hydrophobic fragment of the bilayer, already in the adsorbed state. To assign observed rearrangements to POPE and KLA lipids in both leaflets, per-deuterated d₃₁-POPE was transferred into the inner leaflet. Since no potential-dependent changes of the CD₂ stretching modes in the d₃₁-POPE-KLA bilayer were observed, reorientations in the acyl chain region were assigned to the KLA molecules. Mg²⁺ ions were bound to the polar head groups of KLA. The strength of electrostatic interactions in the polar head group region of KLA was dependent on the direction of the electric field. At negative electric potentials, the binding of divalent cations weakened, which gave the KLA molecules increased orientational flexibility. This behavior in electric fields is peculiar for the outer membrane and indicates that the microbial cell membranes have different electrochemical properties than phospholipid bilayers.