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Principal Investigator

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P06  Clemens Glaubitz
Professor

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Institute of Biophysical Chemistry,
Center for Biomolecular
Magnetic Resonance
Goethe University Frankfurt a.M.
Max-von-Laue-Str. 9
60438 Frankfurt am Main, Germany

Phone +49 (0)69 798-29927
Fax +49 (0)69 798-29929

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P06 The mechanism of primary-active transporters, light-driven channels and pumps studied by solid-state NMR

 

For translocating substrates across membranes, transport proteins undergo conformational changes at different times and length scales and often require long-range communication between distant domains. Their function is closely coupled to the membrane environment and linked to a complex energetic framework. We will address the interplay between structure, dynamics, transport and catalytic/energetic cycle within the lipid bilayer by applying solid-state NMR methods and biochemical approaches to proteins from the ABC transporter and retinal binding protein families.

The first part of this project will focus on bacterial retinal proteins. In the last funding period, our research efforts were dedicated to proteorhodopsin. Using solid-state NMR and dynamic nuclear polarization, we were able to identify functionally important cross-protomer contacts, reveal essential details of color tuning and developed an approach to fuse this protein from separately expressed fragments using split-inteins. We will now extend these studies towards KR2, which is a pentameric retinal protein complex from the marine flavobacterium Krokinobacter eikastus. It functions as light-driven proton pump but also provides Na+ transport. A single mutation turns it into a K+ pump, which makes it also an attractive target for optogenetic applications. Our aim will be to resolve the structural details of ion transfer and its link to the photocycle, ion selectivity, the role of the oligomeric complex and generally functional relevant structure and dynamic features of this protein.

In a joint effort within the CRC, we will also expand our previous research on channelrhodpsin-2. So far, we were able to trap intermediate states for solid-state NMR analysis providing a first glimpse at the retinal structure. Using more advanced labeling schemes, the retinal structure during the photocycle and its contacts with adjacent residues will be resolved, which will provide an insight how the photocycle is linked with the channel gating mechanism.

The second part of the project is dedicated to the lipid-A flippase MsbA from E. coli, a homo-dimeric ABC exporter. We were able to establish conditions under which well-resolved solid-state NMR spectra of the apo- and of different catalytically trapped states can be acquired. Furthermore, iso-tope labeled lipid-A as substrate could be detected in complex with MsbA. This will now enable us to observe fine structural changes during the catalytic cycle in order to develop a better understanding of the cross-talk between nucleotide-binding and transmembrane domains. This work will be carried out in the context of our recent findings that MsbA follows a much more complex nucleotide-based catalytic mechanism based on a coupled ATPase/kinase activity. This observation adds a new perspective to the energetics and regulation of ABC transporters. 

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Publications

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Mehler M, Eckert CE, Busche A, Kulhei J, Michaelis J, Becker-Baldus J, Wachtveitl J, Dötsch V, Glaubitz C (2015) Assembling a correctly folded and functional heptahelical membrane protein by protein trans-splicing. J Biol Chem 290, 27712-22. 

Becker-Baldus J, Bamann C, Saxena K, Brown LJ, Brown RCD, Reiter C, Bamberg E, Wachtveitl J, Schwalbe H, Glaubitz C (2015) Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR. Proc Natl Acad Sci USA 112, 9896-901.

Maciejko J, Mehler M, Kaur J, Lieblein T, Morgner N, Ouari O, Tordo P, Becker-Baldus J, Glaubitz C (2015) Visualizing specific cross-protomer interactions in the homo-oligomeric membrane protein proteorhodopsin by DNP-enhanced solid-state NMR. J Am Chem Soc 137, 9032-43. 

Kaur H, Lakatos A, Spadaccini R, Vogel R, Hoffmann C, Becker-Baldus J, Ouari O, Tordo P, McHaourab H, Glaubitz C (2015) The ABC exporter MsbA probed by solid state NMR - challenges and opportunities. Biol Chem 396, 1135-49. 

Mao J, Do NN, Scholz F, Reggie L, Mehler M, Lakatos A, Ong YS, Ullrich SJ, Brown LJ, Brown RC, Becker-Baldus J, Wachtveitl J, Glaubitz C (2014) Structural basis of the green-blue color switching in proteorhodopsin as determined by NMR spectroscopy. J Am Chem Soc 136, 17578-17590.

Jakdetchai O, Denysenkov V, Becker-Baldus J, Dutagaci B Prisner TF, Glaubitz C (2014) Dynamic nuclear polarization-enhanced NMR on aligned lipid bilayers at ambient temperature. J Am Chem Soc 136, 15533-15536.

Mao J, Akhmetzyanov D, Ouari O, Denysenkov V, Corzilius B, Plackmeyer J, Tordo P, Prisner TF, Glaubitz C (2014) Host-guest complexes as water-soluble high-performance DNP polarizing agents. J Am Chem Soc 135, 19275-19281.

Ullrich SJ, Hölper S, Glaubitz C (2014) Paramagnetic doping of a 7TM membrane protein in lipid bilayers by Gd³⁺-complexes for solid-state NMR spectroscopy. J Biomol NMR 58, 27-35.

Bamann C, Bamberg E, Wachtveitl J, Glaubitz C (2014) Proteorhodopsin. Biochim Biophys Acta 1837, 614-625.

Ong YS, Lakatos A, Becker-Baldus J, Pos KM, Glaubitz C (2013) Detecting substrates bound to the secondary multidrug efflux pump EmrE by DNP-enhanced solid-state NMR. J Am Chem Soc 135, 15754-15762.

Mehler M, Scholz V, Ullrich SJ, Mao J, Braun M, Brown LJ, Brown RC, Fiedler SA, Becker-Baldus J, Wachtveitl J, Glaubitz C (2013) The EF loop in green proteorhodopsin affects conformation and photocycle dynamics. Biophys J 105, 385-397.

Ullrich SJ, Glaubitz C (2013) Perspectives in enzymology of membrane proteins by solid-state NMR. Acc Chem Res 46, 2164-2171.

Mörs K, Roos C, Scholz F, Wachtveitl J, Dötsch V, Bernhard F, Glaubitz C (2013) Modified lipid and protein dynamics in nanodiscs. Biochim Biophys Acta 1828, 1222-1229.

Mörs K, Hellmich UA, Basting D, Marchand P, Wurm JP, Haase W, Glaubitz C (2012) A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein TBsmr. Biochim Biophys Acta 1828, 561-567.

Lakatos A, Mörs K, Glaubitz C (2012) How to investigate interactions between membrane proteins and ligands by solid-state NMR. Methods Mol Biol 914, 65-86.

Hellmich UA, Lyubenova S, Kaltenborn E, Doshi R, van Veen H, Prisner T, Glaubitz C (2012) Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR spectroscopy. J Am Chem Soc 134, 5827-5862.

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Collaborations

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Tampé (P16), Heilemann (P20), Morgner (P24), Hummer (P25), Wachtveitl/Bamann (P12), 
Prisner/Joseph (P07), Schwalbe (P13), Dötsch/Bernhard (P02), Ernst (P21)