Organization of reconstituted lipoprotein MexA onto supported lipid membrane
MexA, a periplasmic component of OprM-MexA-MexB tripartite multidrug efflux pump from Pseudomonas aeruginosa, is natively anchored via its fatty acid in the bacteria inner membrane protruding into the periplasm. We used supported lipid bilayer (SLB) to attach the protein to a single leaflet mimicking its perisplamic orientation. For that purpose, we studied the solubilization of DOPC lipid bilayer supported on silica surface with β-octyl glucoside (βOG). First we showed that SLBs resist to βOG concentrations that usually solubilize liposomes. Native form of MexA was directly inserted in the outer leaflet at (βOG concentrations in a range of 20–25 mM). Second, observations by cryo-electron microscopy (cryoEM) revealed a dense protein layer attached to the surface corresponding to a 13-nm layer of MexA proteins. Analysis of protein densities allows proposing a schematic organization of native MexA inserted in lipid membrane. This structural organization provides further insights with respect to the partially solved structure of the soluble form.
KeywordsMembrane protein Multidrug resistance Cryo-electron microscopy Membrane protein on solid support SLB QCM-D
This work has been supported in part by EC grants “NMP4-CT2003-505868- Nanocues”, EC grants “QLR-2000-01339” and program ACI “Dynamique et réactivité des assemblages biologiques” DRAB04/136. The authors wish to thank Xavier Moreel, Dimitri Lerouge and Joséphine Lai Kee Him for technical assistance with MexA and QCM-D. Sylvain Trépout is a recipient of a PhD fellowship of French Ministry of Education and Research and Technology (MENRT).
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