Membrane protein selectively oriented on solid support and reconstituted into a lipid membrane.
Mimetic functional membranes on solid support are now emerging for the development of membrane biosensor or for the study of membrane-mediated processes and should have an important impact on biodiagnostics. We established a method to reconstitute a membrane protein into a lipid membrane in a selective orientation on a solid support. Membrane protein OprM, a component of OprM-MexA-MexB multidrug efflux pump, solubilized in detergent was immobilized via its extracellular domain on aminosilane-modified silica surface. The oriented protein was reconstituted into a lipid membrane by detergent removal. The membrane protein reconstitution process carried out on silica nanoparticles and on planar silica surfaces was followed by cryo-electron microscopy (cryo-EM) and quartz crystal microbalance with dissipation monitoring (QCM-D) respectively. The selective protein orientation on aminosilane-modified silica surface was assessed by cryo-EM and was compared to the nonspecific protein deposition on silica surface. Finally, the binding of MexA, a periplasmic component of the tripartite efflux complex, was monitored with QCM-D on the oriented OprM protein monolayer. The large adsorbed mass gave a direct evidence of the high affinity of MexA with the periplasmic helical part of OprM.
Trépout
Sylvain
S
Laboratoire d'Imagerie Moléculaire et Nano-Bio-Technologie, IECB, UMR-CNRS 5471, Université de Bordeaux 1, 2 rue Robert Escarpit, F-33607 Pessac, France.
Mornet
Stéphane
S
Benabdelhak
Houssain
H
Ducruix
Arnaud
A
Brisson
Alain R
AR
Lambert
Olivier
O
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D001703:Biophysics / Q000379:methods; D002627:Chemistry, Physical / Q000379:methods; D020285:Cryoelectron Microscopy; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry; D008566:Membranes / Q000737:chemistry; D008823:Micelles; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D008055:Lipids; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; D008823:Micelles; C402263:OprM protein, Pseudomonas aeruginosa; D012822:Silicon Dioxide