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The ABC exporter MsbA probed by solid state NMR – challenges and opportunities

  • Hundeep Kaur , Andrea Lakatos , Roberta Spadaccini , Ramona Vogel , Christian Hoffmann , Johanna Becker-Baldus , Olivier Ouari , Paul Tordo , Hassane Mchaourab und Clemens Glaubitz EMAIL logo
Veröffentlicht/Copyright: 3. April 2015

Abstract

ATP binding cassette (ABC) transporters form a superfamily of integral membrane proteins involved in translocation of substrates across the membrane driven by ATP hydrolysis. Despite available crystal structures and extensive biochemical data, many open questions regarding their transport mechanisms remain. Therefore, there is a need to explore spectroscopic techniques such as solid state NMR in order to bridge the gap between structural and mechanistic data. In this study, we investigate the feasibility of using Escherichia coli MsbA as a model ABC transporter for solid state NMR studies. We show that optimised solubilisation and reconstitution procedures enable preparing stable and homogenous protein samples. Depending on the duration of solubilisation, MsbA can be obtained in either an apo- or in a native lipid A bound form. Building onto these optimisations, the first promising MAS-NMR spectra with narrow lines have been recorded. However, further sensitivity improvements are required so that complex NMR experiments can be recorded within a reasonable amount of time. We therefore demonstrate the usability of paramagnetic doping for rapid data acquisition and explore dynamic nuclear polarisation as a method for general signal enhancement. Our results demonstrate that solid state NMR provides an opportunity to address important biological questions related to complex mechanisms of ABC transporters.


Corresponding author: Clemens Glaubitz, Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt/Main, Germany, e-mail:

Acknowledgments

The work was supported by a DFG research grant through SFB 807 ‘Transport and communication across membranes’ and by a DFG equipment grant (GL 307/4–1) to C.G. H.M. acknowledges support by NIH grant U54-GM087519. MALDI-MS data were kindly provided by Dr. Ute Bahr (Institute for Pharmaceutical Chemistry, Goethe University Frankfurt).

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Received: 2015-2-4
Accepted: 2015-3-26
Published Online: 2015-4-3
Published in Print: 2015-9-1

©2015 by De Gruyter

Artikel in diesem Heft

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  2. Meeting Report
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