1. Efficient and stable reconstitution of the ABC transporter BmrA for solid-state NMR studies
- Author
-
Anja Böckmann, Britta Kunert, Denis Lacabanne, Beat H. Meier, François Penin, Carole Gardiennet, Pierre Falson, Daniel Calles-Garcia, Jean-Michel Jault, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
- Subjects
[SDV]Life Sciences [q-bio] ,ATP-binding cassette transporter ,Bacillus subtilis ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,ABC-transporter ,Sample preparation ,Molecular Biosciences ,Original Research Article ,isotope labeling ,lcsh:QH301-705.5 ,Molecular Biology ,Protein secondary structure ,biology ,Chemistry ,B. subtilis lipids ,Transporter ,biology.organism_classification ,3. Good health ,Membrane ,BmrA ,lcsh:Biology (General) ,Solid-state nuclear magnetic resonance ,Membrane protein ,Solid-state NMR ,Isotope labeling ,Biophysics ,solid-state NMR - 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. ISSN:2296-889X
- Published
- 2014
- Full Text
- View/download PDF