Caught in the Act: ATP hydrolysis of an ABC-multidrug transporter followed by 
real-time magic angle spinning NMR

Hellmich, Ute A.; Haase, Winfried; Velamakanni, Saroj; van Veen, Hendrik W.; Glaubitz, Clemens

doi:10.1016/j.febslet.2008.09.033

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Caught in the Act: ATP hydrolysis of an ABC-multidrug transporter followed by real-time magic angle spinning NMR

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Haase, Winfried
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Zitation

Hellmich, U. A., Haase, W., Velamakanni, S., van Veen, H. W., & Glaubitz, C. (2008). Caught in the Act: ATP hydrolysis of an ABC-multidrug transporter followed by real-time magic angle spinning NMR. FEBS Letters, 582(23), 3557-3562. doi:10.1016/j.febslet.2008.09.033.

Zitierlink: https://hdl.handle.net/11858/00-001M-0000-0024-D81D-1

Zusammenfassung

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.