Wednesday 12.8. in YAA305 at 1 o'clock with coffee, talk starts 13:15
University of Zürich
Kinetic and dynamic responses of proteins utilizing time-resolved IR spectroscopy and single-molecule fluorescence spectroscopy
By covalently linking an azobenzene photoswitch across the binding groove of an allosteric protein domain, a conformational transition can be initiated by a laser pulse . This transition mimics the conformational change of the unmodified domain upon ligand binding. We have studied this light induced transition by time-resolved IR spectroscopy. So far, we have probed two IR absorption bands: First, the amide I band which arises from the carbonyl stretch vibration of all amide groups in the protein and is sensitive to overall structural changes, and second, a vibration localized on the photoswitch, which is sensitive to the local environment of the photoswitch, namely the opening of the binding groove. We have found that the binding groove opens on a timescale of 100 ns in a non-exponential manner, once the binding groove has equilibrated, the protein conformation still continues to change elsewhere. Currently, we are incorporating site-specific IR labels, to learn more about the response of the protein to the perturbation of the binding groove.
In a second project we have studied the dynamics in an unfolded state of a protein. We have labeled the protein with two fluorescent dyes at different positions in the protein and used single-molecule Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to reveal the dynamics in the unfolded state . This allowed us to quantify the extent of internal friction in unfolded protein chains.
1. Buchli B., Waldauer S. A., Walser R., Donten M., Pfister R., Blöchliger N., Steiner S., Caflisch A., Zerbe O., Hamm P. (2013) Kinetic response of a photoperturbed allosteric protein. Proc. Natl. Acad. Sci. U.S.A. 110, 11725-11730.
2. Soranno A., Buchli B., Nettels D., Cheng R. R., Müller-Späth S., Pfeil S. H., Hoffmann A., Lipman E. A., Makarov D. E., Schuler B. (2012) Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 109, 17800-17806.
NANOSEMINARS AUTUMN 2015
|12.8||Kinetic and dynamic responses of proteins utilizing time-resolved IR spectroscopy and single-molecule fluorescence spectroscopy||Brigitte Buchli
University of Zürich
|11.9||Nanomaster seminars||Johannes Lehmuskoski
|18.9||Nanomaster seminars||Niko-Ville Hakkola
Department of Physics