Caught in the act: ATP hydrolysis of an ABC-multidrug transporter followed by real-time magic angle spinning NMR.
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.
Hellmich
Ute A
UA
Institute for Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, Cluster of Excellence Macromolecular Complexes, J.W. Goethe University, Frankfurt am Main, Germany.
Haase
Winfried
W
Velamakanni
Saroj
S
van Veen
Hendrik W
HW
Glaubitz
Clemens
C
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
D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010759:Phosphorus Isotopes; C101824:LmrA protein, Lactococcus lactis