Single-molecule fluorescence spectroscopy to study structure and conformational dynamics of model proteins and the transporter BetP from Corynebacterium glutamicum
Final Report Abstract
Overall, we achieved very good results for the task 1 (establishment of a fluorescence spectroscopic toolkit) and for task 2 (Monitoring conformational exchange in selected model proteins by fluorescence). The fluorescence methods could be published in very good journals and are well cited. We successfully applied them to nine proteins to study their conformational dynamics in the time range between 80 ns and sub-milliseconds. The findings for T4L and the Klenow fragment were surprising. For both proteins we found a more complex conformational dynamics and identified new previously hidden states. So far it was general belief that both systems were well understood (e.g. > 400 crystal structures for T4L), so that we had to perform a lot of control experiments to convince ourselves and the colleagues that the found novel states are no methodological artefacts but functionally relevant enzyme states. The achieved progress was a crucial technological boost for the Seidel group, which put me in the position to submit the project proposal "hybridFRET" for an ERC Advanced Grant 2014 which will be actually funded.
Publications
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A toolkit and benchmark study for FRET-restrained highprecision structural modeling. Nat. Methods 9, 1218-1225 (2012)
Kalinin, S., Peulen, Th.-O., Sindbert, S., Rothwell, P. J., Berger, S., Restle, T., Goody, R. S., Gohlke, H., Seidel, C. A. M.
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Combining MFD and PIE for accurate single-pair Förster Resonance Energy Transfer measurements. ChemPhysChem 13, 1060-1078 (2012)
Kudryavtsev, V., Sikor, M., Kalinin, S., Mokranjac, D., Seidel, C. A. M., Lamb, D. C.
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Filtered FCS: Species auto and cross correlation functions highlight binding and dynamics in biomolecules. ChemPhysChem 13, 1036-1053 (2012)
Felekyan, S., Kalinin, S., Sanabria, H., Valeri, A., Seidel, C. A. M.
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The supertertiary structure of the synaptic MAGuK scaffold proteins is conserved. Proc. Natl. Acad. Sci. USA. 109, 15775-15780 (2012)
McCann, J. J., Zheng, L. Rohrbeck, D., Felekyan, S., Kühnemuth, R., Sutton, R. B., Seidel, C. A. M., Bowen, M. E.
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Analyzing Förster resonance energy transfer with fluctuation algorithms. Methods in Enzymology 519 (Fluorescence Fluctuation Spectroscopy (FFS) Part B, Ed. Sergey Y. Tetin; UK, Academic Press, ISBN: 978-0-12-405539-1) Chapter 2, 39-86 (2013)
Felekyan, S., Sanabria, H., Kalinin, S., Kühnemuth, R., Seidel, C. A. M.
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Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy. Nat. Commun. 5, Article Number: 5206 (2014)
Olofsson, L., Felekyan S., Doumazane, E., Scholler, P., Fabre, L., Zwier, J. M., Philippe Rondard, P., Seidel, C. A. M., Pin, J.-P., Margeat, E.
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Untersuchung von funktionellen Dynamiken in DNA-Strukturen und DNA-Polymerasen mittels Einzelmolekül-Fluoreszenzspektroskopie, Dissertation Heinrich-Heine- Universität 10.8.2014
Richert, Markus
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Outcome of the First wwPDB Hybrid/Integrative Methods Task Force Workshop. Structure. 23
Sali, A., Helen M. Berman, T. Schwede, J. Trewhella, G. Kleywegt, Stephen K. Burley, J. Markley, H. Nakamura, P. Adams, Alexandre M. J. J. Bonvin, W. Chiu, Matteo D. Peraro, F. Di Maio, Thomas E. Ferrin, K. Grünewald, A. Gutmanas, R. Henderson, G. Hummer, K. Iwasaki, G. Johnson, Catherine L. Lawson, J. Meiler, Marc A. Marti-Renom, Gaetano T. Montelione, M. Nilges, R. Nussinov, A. Patwardhan, J. Rappsilber, Randy J. Read, H. Saibil, Gunnar F. Schröder, Charles D. Schwieters, Claus A. M. Seidel, D. Svergun, M. Topf, Eldon L. Ulrich, S. Velankar, and John D. Westbrook
