title: In vitro investigation of the MexAB efflux pump from Pseudomonas aeruginosa.
abstract: There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.
authors.affiliation: Laboratoire de Cristallographie & RMN Biologiques, CNRS & Université Paris Descartes.
authors.forename: Alice
authors.initials: A
authors.lastname: Verchère
authors.affiliation: Laboratoire de Cristallographie & RMN Biologiques, CNRS & Université Paris Descartes.
authors.forename: Manuela
authors.initials: M
authors.lastname: Dezi
authors.affiliation: Laboratoire de Cristallographie & RMN Biologiques, CNRS & Université Paris Descartes.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Broutin
authors.affiliation: Laboratoire de Cristallographie & RMN Biologiques, CNRS & Université Paris Descartes; martin.picard@parisdescartes.fr.
authors.forename: Martin
authors.initials: M
authors.lastname: Picard
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008081:Liposomes; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; C118597:MexB protein, Pseudomonas aeruginosa