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Principal Investigators

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P07  Thomas Prisner
Professor

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Institute of Physical and
Theoretical Chemistry /
Center of Biomolecular Magnetic Resonance
Goethe University Frankfurt a.M.
Max-von-Laue-Str. 7
60438 Frankfurt am Main, Germany

Phone +49 (0)69 798-29406
Fax +49 (0)69 798-29404

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P07  Benesh Joseph
Group Leader

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Institute of Physical and
Theoretical Chemistry
Goethe University Frankfurt a.M.
Max-von-Laue-Str. 7
60438 Frankfurt am Main, Germany

Phone +49 (0)69 798-29423
Fax +49 (0)69 798-29404

> send email
> homepage

> publications

P07 Dipolar EPR spectroscopy on membrane embedded transport complexes


In this project, we will use pulsed EPR spectroscopy to investigate the structure, dynamics and con-formational changes of membrane transport complexes in a detergent, lipid or cellular environment. For this purpose, spin-labels with different spectral and biochemical properties will be attached to membrane protein complexes. The well-established MTSL nitroxide spin-label commonly used for pulsed EPR experiments is covalently bound to cysteine residues and its mobility at room temperature can be used to probe structural and dynamical changes. More recently, gadolinium and trityl spin- labels have been used, due to their stability against reduction under in-cell conditions. The long relaxation times of the trityl spin-label makes it possible to extend the pulsed experiments to room temperature. Additionally its narrow spectral line makes this label ideal for the development of improved dipolar methods. Manganese can replace magnesium and therefore it is a sensitive probe for structural characterizations of nucleotide binding domains of ATP Binding Cassette (ABC) transporters. The dipolar interaction between two paramagnetic centers can be determined up to 8 nm, allowing to investigate long range conformational changes of the transporter proteins in different functional states.

In collaboration with several groups of the CRC we will investigate the TOC-core complex, the fumarate transporter SLC26Dg and the uracil transporter UraA to determine structural changes associated with the transport process, nucleotide loading state, ionic strength and lipid environment. The obtained information will be compared with simulations or modelling studies and experimental data from biochemical, spectroscopic or structural methods. Manganese ions in the nucleotide binding domains will be used to investigate asymmetric nucleotide loading in heterodimeric systems like TmrAB and the TOC-core complex. A major goal in our project is to extend the applicability of the EPR method to more natural conditions. New preparative protocols, pulse sequences and spin-labels will be systematically explored, enabling investigations in whole cells and native membranes as well as extending the experiments to room temperature. We aim to investigate the influence of the membrane surrounding, the interaction with other proteins and freezing effects on the structure and dynamics of the transporter complexes in their different functional states. In-cell EPR investigations will be performed on outer membrane β-barrel proteins BtuB, FecA, FhuA and TolC as well as on the α-helical inner membrane ABC transporters LmrA and BtuCD. 

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Publications

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Joseph B, Sikora A, Bordignon E, Jeschke G, Cafiso DS*, Prisner TF* (2015) Distance measurement on an endogenous membrane transporter in E. coli cells and native membranes using EPR spectroscopy. Angew Chem Int Ed 54, 6196-6199.

Akhmetzyanov D, Plackmeyer J, Endeward B, Denysenkov VP, Prisner TF* (2015) Pulsed electron-electron double resonance spectroscopy between a high-spin Mn2+ ion and a nitroxide spin label. Phys Chem Chem Phys 17, 6760-6. 

Schöps P, Spindler PE, Marko A, Prisner TF* (2015) Broadband spin echoes and broadband SIFTER in EPR. J Magn Reson 250, 55-62. 

Akhmetzyanov D, Schoeps P, Marko A, Kunjir N, Sigurdsson S, Prisner TF (2015) Pulsed EPR dipolar spectroscopy at Q- and G-band on a trityl biradical. Phys Chem Chem Phys 17, 24446-51. 

Spindler PE, Waclawska I, Endeward B, Plackmeyer J, Ziegler C, Prisner TF (2015) Carr-purcell PELDOR with shaped inversion pulses. J Phys Chem Lett 6, 4331-5. 

Lumme C, Altan-Martin H, Dastvan R, Sommer MS, Oreb M, Schuetz D, Hellenkamp B, Mirus O, Kretschmer J, Lyubenova S, Kügel W, Medelnik JP, Dehmer M, Michaelis J, Prisner TF, Hugel T, Schleiff E (2014) Nucleotides and substrates trigger the dynamics of the Toc34 GTPase homodimer involved in chloroplast preprotein translocation. Structure 22, 1-13. 

Joseph B, Korkhov VM, Yulikov M, Jeschke G, Bordignon E (2014) Conformational cycle of the vitamin B12 ABC importer in liposomes detected by double electron-electron resonance (DEER). J Biol Chem 289, 3176-85. 

Marko A, Denysenkov V, Prisner TF (2013) Out-of-Phase PELDOR. Mol Phys 111, 2834-44. 

Spindler PE, Glaser SJ, Skinner TE, Prisner TF (2013) Broadband inversion PELDOR spectroscopy with partially adiabatic shaped pulses. Angew Chem Int Ed 52, 3425-29. 

Hellmich UA, Lyubenova S, Kaltenborn E, Doshi R, Van Veen H, Prisner TF., Glaubitz C (2012) Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR spectroscopy. J Am Chem Soc
134, 5857-5862.


Spindler PE, Zhang Y, Endeward B, Gershernzon N, Skinner TE, Glaser SJ, Prisner TF (2012) Shaped optimal control pulses for increased excitation bandwidth in EPR. J Magn Reson
218, 49-58.

* corresponding authors

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Collaborations

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Glaubitz (P06), Wachtveitl/Bamann (P12), Tampé (P16), Schleiff (P17), Pos (P18),
Hummer (P25), Geertsma (P23)