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Caught in the Act: ATP hydrolysis of an ABC‐multidrug transporter followed by real‐time magic angle spinning NMR
Author(s) -
Hellmich Ute A.,
Haase Winfried,
Velamakanni Saroj,
van Veen Hendrik W.,
Glaubitz Clemens
Publication year - 2008
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2008.09.033
Subject(s) - magic angle spinning , atp binding cassette transporter , atp hydrolysis , solid state nuclear magnetic resonance , nuclear magnetic resonance spectroscopy , lactococcus lactis , chemistry , crystallography , nuclear magnetic resonance , stereochemistry , biochemistry , transporter , biology , enzyme , atpase , bacteria , physics , gene , lactic acid , genetics
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 31 P‐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 31 P 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.