Ong, Y.S., et al., Detecting substrates bound to the secondary multidrug efflux pump EmrE by DNP-enhanced solid-state NMR. J Am Chem Soc, 2013. 135(42): p. 15754-62.
Escherichia coli EmrE, a homodimeric multidrug antiporter, has been suggested to offer a convenient paradigm for secondary transporters due to its small size. It contains four transmembrane helices and forms a functional dimer. We have probed the specific binding of substrates TPP(+) and MTP(+) to EmrE reconstituted into 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes by (31)P MAS NMR. Our NMR data show that both substrates occupy the same binding pocket but also indicate some degree of heterogeneity of the bound ligand population, reflecting the promiscuous nature of ligand binding by multidrug efflux pumps. Direct interaction between (13)C-labeled TPP(+) and key residues within the EmrE dimer has been probed by through-space (13)C-(13)C correlation spectroscopy. This was made possible by the use of solid-state NMR enhanced by dynamic nuclear polarization (DNP) through which a 19-fold signal enhancement was achieved. Our data provide clear evidence for the long assumed direct interaction between substrates such as TPP(+) and the essential residue E14 in transmembrane helix 1. Our work also demonstrates the power of DNP-enhanced solid-state NMR at low temperatures for the study for secondary transporters, which are highly challenging for conventional NMR detection.
Detecting Substrates Bound to the Secondary Multidrug Efflux Pump EmrE by DNP-Enhanced Solid-State NMR
Detecting Substrates Bound to the Secondary Multidrug Efflux Pump EmrE by DNP-Enhanced Solid-State NMR
Yean Sin Ong, Andrea Lakatos, Johanna Becker-Baldus, Klaas M. Pos and Clemens Glaubitz
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Journal of the American Chemical Society
DOI: 10.1021/ja402605s
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[NMR paper] Ligand-Induced Conformational Changes of the Multidrug Resistance Transporter EmrE Probed by Oriented Solid-State NMR Spectroscopy.
Ligand-Induced Conformational Changes of the Multidrug Resistance Transporter EmrE Probed by Oriented Solid-State NMR Spectroscopy.
Ligand-Induced Conformational Changes of the Multidrug Resistance Transporter EmrE Probed by Oriented Solid-State NMR Spectroscopy.
Angew Chem Int Ed Engl. 2013 Aug 12;
Authors: Gayen A, Banigan JR, Traaseth NJ
Abstract
An EmrE-ging market: Oriented solid-state NMR spectroscopy and biochemical cross-linking experiments were used to show that the ligand-free membrane protein transporter EmrE forms...
Dynamic Nuclear Polarization-Enhanced Solid-State NMR of a 13C-Labeled Signal Peptide Bound to Lipid-Reconstituted Sec Translocon
Dynamic Nuclear Polarization-Enhanced Solid-State NMR of a 13C-Labeled Signal Peptide Bound to Lipid-Reconstituted Sec Translocon
Lenica Reggie, Jakob J. Lopez, Ian Collinson, Clemens Glaubitz and Mark Lorch
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja209378h/aop/images/medium/ja-2011-09378h_0002.gif
Journal of the American Chemical Society
DOI: 10.1021/ja209378h
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NMR and fluorescence spectroscopy approaches to secondary and primary active multidrug efflux pumps.
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Authors: Lorch M, Lehner I, Siarheyeva A, Basting D, Pfleger N, Manolikas T, Glaubitz C
Multidrug efflux pumps are found in all major transporter families. Along with a lack of three-dimensional structure information, the mechanism of drug recognition, energy coupling with drug...
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Control of the pump cycle in bacteriorhodopsin: mechanisms elucidated by solid-state NMR of the D85N mutant.
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By varying the pH, the D85N mutant of bacteriorhodopsin provides models for several photocycle intermediates of the wild-type protein in which D85 is protonated. At pH 10.8, NMR spectra of...
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[NMR paper] NMR investigation of the multidrug transporter EmrE, an integral membrane protein.
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Authors: Schwaiger M, Lebendiker M, Yerushalmi H, Coles M, Gröger A, Schwarz C, Schuldiner S, Kessler H
EmrE is an Escherichia coli multidrug transport protein that confers resistance to a wide range of toxicants by active transport across the bacterial cell membrane. The highly hydrophobic polytopic integral membrane...
Detecting substrates bound to the secondary multidrug efflux pump EmrE by DNP-enhanced solid-state NMR
Linear Mode
