title: Efficient and stable reconstitution of the ABC transporter BmrA for solid-state NMR studies.
abstract: We present solid-state NMR sample preparation and first 2D spectra of the Bacillus subtilis ATP-binding cassette (ABC) transporter BmrA, a membrane protein involved in multidrug resistance. The homodimeric 130-kDa protein is a challenge for structural characterization due to its membrane-bound nature, size, inherent flexibility and insolubility. We show that reconstitution of this protein in lipids from Bacillus subtilis at a lipid-protein ratio of 0.5 w/w allows for optimal protein insertion in lipid membranes with respect to two central NMR requirements, high signal-to-noise in the spectra and sample stability over a time period of months. The obtained spectra point to a well-folded protein and a highly homogenous preparation, as witnessed by the narrow resonance lines and the signal dispersion typical for the expected secondary structure distribution of BmrA. This opens the way for studies of the different conformational states of the transporter in the export cycle, as well as on interactions with substrates, via chemical-shift fingerprints and sequential resonance assignments.
authors.affiliation: Labex Ecofect, Bases Moleculaires et Structurales des Systemes Infectieux, UMR 5086 CNRS, IBCP, Université de Lyon 1 Lyon, France.
authors.forename: Britta
authors.initials: B
authors.lastname: Kunert
authors.affiliation: Labex Ecofect, Bases Moleculaires et Structurales des Systemes Infectieux, UMR 5086 CNRS, IBCP, Université de Lyon 1 Lyon, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Gardiennet
authors.affiliation: Labex Ecofect, Bases Moleculaires et Structurales des Systemes Infectieux, UMR 5086 CNRS, IBCP, Université de Lyon 1 Lyon, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Lacabanne
authors.affiliation: Labex Ecofect, Bases Moleculaires et Structurales des Systemes Infectieux, UMR 5086 CNRS, IBCP, Université de Lyon 1 Lyon, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Calles-Garcia
authors.affiliation: Labex Ecofect, Bases Moleculaires et Structurales des Systemes Infectieux, UMR 5086 CNRS, IBCP, Université de Lyon 1 Lyon, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Falson
authors.affiliation: Labex Ecofect, Bases Moleculaires et Structurales des Systemes Infectieux, UMR 5086 CNRS, IBCP, Université de Lyon 1 Lyon, France.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Jault
authors.affiliation: Physical Chemistry, ETH Zürich Zurich, Switzerland.
authors.forename: Beat H
authors.initials: BH
authors.lastname: Meier
authors.affiliation: Labex Ecofect, Bases Moleculaires et Structurales des Systemes Infectieux, UMR 5086 CNRS, IBCP, Université de Lyon 1 Lyon, France.
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.affiliation: Labex Ecofect, Bases Moleculaires et Structurales des Systemes Infectieux, UMR 5086 CNRS, IBCP, Université de Lyon 1 Lyon, France.
authors.forename: Anja
authors.initials: A
authors.lastname: Böckmann
keywords: ABC-transporter; B. subtilis lipids; BmrA; isotope labeling; solid-state NMR