Caught in the act: ATP hydrolysis of an ABC-multidrug transporter followed by real-time magic angle spinning NMR |
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Authors: | Hellmich Ute A Haase Winfried Velamakanni Saroj van Veen Hendrik W Glaubitz Clemens |
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Institution: | Institute for Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, Cluster of Excellence Macromolecular Complexes, J.W. Goethe University, Frankfurt am Main, Germany. |
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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. |
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