title: Caught in the act: ATP hydrolysis of an ABC-multidrug transporter followed by real-time magic angle spinning NMR.
abstract: The ATP binding cassette (ABC) transporter LmrA from Lactococcus lactis transports cytotoxic molecules at the expense of ATP. Molecular and kinetic details of LmrA can be assessed by solid-state nuclear magnetic resonance (ssNMR), if functional reconstitution at a high protein-lipid ratio can be achieved and the kinetic rate constants are small enough. In order to follow ATP hydrolysis directly by 31P-magic angle spinning (MAS) nuclear magnetic resonance (NMR), we generated such conditions by reconstituting LmrA-dK388, a mutant with slower ATP turnover rate, at a protein-lipid ration of 1:150. By analysing time-resolved 31P spectra, protein activity has been directly assessed. These data demonstrate the general possibility to perform ssNMR studies on a fully active full length ABC transporter and also form the foundation for further kinetic studies on LmrA by NMR.
authors.affiliation: Institute for Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, Cluster of Excellence Macromolecular Complexes, J.W. Goethe University, Frankfurt am Main, Germany.
authors.forename: Ute A
authors.initials: UA
authors.lastname: Hellmich
authors.forename: Winfried
authors.initials: W
authors.lastname: Haase
authors.forename: Saroj
authors.initials: S
authors.lastname: Velamakanni
authors.forename: Hendrik W
authors.initials: HW
authors.lastname: van Veen
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008854:Microscopy, Electron; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010759:Phosphorus Isotopes / Q000737:chemistry; D013997:Time Factors
chemical_list: D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010759:Phosphorus Isotopes; C101824:LmrA protein, Lactococcus lactis