Q-Band FT-EPR Spektrometer
Final Report Abstract
On 23 March 2015 a high-power AWG Q-band EPR spectrometer was installed in my lab at FU Berlin, and since 1 April 2016 it has been relocated to my new lab at Ruhr University Bochum, Germany. This spectrometer has been instrumental for my research activities due to its state-of-the-art performance. ABC transporters: These membrane proteins are fascinating nanomachines which transport in and out of the cell many substrates such as nutrients, toxins and drugs. They are important biomedical targets because some are shown to be the source of hereditary diseases in mammals, for example cystic fibrosis is related to mutations in the Cystic fibrosis transmembrane conductance regulator (CFTR), which is an ABC-transporter class ion channel. They are also known to be the origin of multiple-drug resistance (MDR) in tumor cells and antibiotic resistance in bacteria. Using EPR, we discovered a conformational equilibrium existing between an inward- and an outward- facing conformation of heterodimeric exporters, related to MDR. We also observed how point mutations can affect both equilibrium and ATPase activity, giving new insights in the mechanism of dysregulation of transporters in diseases. Apoptosis: the mitochondrial pathway of cell death is regulated by several proteins belonging to the Bcl-2 class. Using EPR we could unveil the topology of the killer protein Bax at the membrane and its transformation from a soluble into the active dimeric state. Distance measurements allowed to propose the most plausible model of the active dimers. Ton complex: In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. We structurally characterize the Ton complex from Escherichia coli using EPR together with X-ray crystallography, electron microscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Method development: The arbitrary waveform generator (AWG) allowed the optimization of Gaussian pulse sequence which additionally improved fidelity in DEER for interspin distance determination in biomolecules.
Publications
- (2016) Mode of Interaction of the Signal-Transducing Protein EIIAGlc with the Maltose ABC Transporter in the Process of Inducer Exclusion. Biochemistry, 55(38), 5442
Steven Wuttge, Anke Licht, M Hadi Timachi, Enrica Bordignon, Erwin Schneider
(See online at https://doi.org/10.1021/acs.biochem.6b00721) - (2016) Structural insight into the role of the Ton complex in energy transduction. Nature, 538, 7623, 60
Herve Celia, Nicholas Noinaj, Stanislov D Zakarov, Enrica Bordignon, Istvan Botos, Monica Santamaria, Travis J Barnard, William A Cramer, Roland Lloubes, Susan K Buchanan
(See online at https://doi.org/10.1038/nature19757) - (2017) Exploring conformational equilibria of a heterodimeric ABC transporter. eLife, 6, e20236
M Hadi Timachi, Cedric AJ Hutter, Michael Hohl, Tufa Assafa, Simon Böhm, Anshumali Mittal, Markus A Seeger, Enrica Bordignon
(See online at https://doi.org/10.7554/eLife.20236.001) - (2018) Atomistic mechanism of large-scale conformational transition in a heterodimeric ABC exporter. J. Am. Chem. Soc., 140(13), 4543
Hendrik Göddeke, M Hadi Timachi, Cedric AJ Hutter, Laura Galazzo, Markus A Seeger, Mikko Karttunen, Enrica Bordignon, Lars V Schäfer
(See online at https://doi.org/10.1021/jacs.7b12944) - (2018) Improved signal fidelity in 4-pulse DEER with Gaussian pulses, J. Magn. Reson. 296 (103)11
Markus Teucher, Enrica Bordignon
(See online at https://doi.org/10.1016/j.jmr.2018.09.003) - (2018) Light-Driven Domain Mechanics of a Minimal Phytochrome Photosensory Module Studied by EPR. Structure. 26, 1–12
Tufa E. Assafa, Katrin Anders, Uwe Linne, Lars-Oliver Essen, Enrica Bordignon
(See online at https://doi.org/10.1016/j.str.2018.08.003) - (2018) Topology of active, membrane-embedded Bax in the context of a toroidal pore. Cell Death and Diff.
Stephanie Bleicken, Tufa E. Assafa, Carolin Stegmueller, Alice Wittig, Ana J. Garcia-Saez, Enrica Bordignon
(See online at https://doi.org/10.1038/s41418-018-0184-6)
