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

FEBS Lett. 2008 Oct 15;582(23-24):3557-62. doi: 10.1016/j.febslet.2008.09.033. Epub 2008 Sep 24.

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / chemistry*
  • Bacterial Proteins / chemistry*
  • Hydrolysis
  • Kinetics
  • Microscopy, Electron
  • Multidrug Resistance-Associated Proteins / chemistry*
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Phosphorus Isotopes / chemistry
  • Time Factors

Substances

  • Bacterial Proteins
  • LmrA protein, Lactococcus lactis
  • Multidrug Resistance-Associated Proteins
  • Phosphorus Isotopes
  • Adenosine Triphosphate