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Project

Crystal structure determination of dynamic Fluorescent proteins

Since the discovery of Green Fluorescent Protein (GFP), a lot of new FPs have been discovered or developed such that today the complete color spectrum is covered. All fluorescent proteins have the same architecture, consisting of an 11-stranded β barrel. An α helix crosses the centre of this barrel and bears the chromophore, which is autocatalitically formed from a tripeptide in aerobic conditions. FPs play a major role in the visualization of cellular events. But for subcellular visualization several super-resolution techniques were developed which all rely on the specific dynamic behavior of fluorescent proteins. This dynamic behavior can be divided into three classes: irreversible photoactivation, reversible photoswitching and irreversible photo conversion. Irreversible photoconvertible and reversible photoswitchable FPs are widely used, and are capable of converting from a green- to a red-emissive state or switching between an on- and off-state, respectively. To date, however, the key factors controlling the photoconversion as well as photoswitching have remained incompletely understood. By crystal structure determination on one hand and a functional characterization on the other hand, a clear structural insight and a thorough understanding of these mechanisms can be provided. Therefore, in collaboration with the group of Peter Dedecker, we are currently mutating a number of fluorescent proteins such as pcDronpa2, ffDronpa, rsEGFP and rsGreen0.7. These mutants will then be crystallized using a crystallization robot. The diffraction data will be collected at a synchrotron source (Soleil, Paris; SLS, Villigen; Elettra, Trieste) and the crystal structures will be fully  characterized. X-ray diffraction, NMR and computational studies have proven that photoswitching is accompanied by a cis-trans isomerization of the chromophore, conformational changes of neighboring amino acids, and differences in β barrel flexibility. The mechanism of this photoswitching will be studied further by simultaneous laser irradiation of the protein crystal during the x-ray data collection. Moreover, both NMR and computational studies have proven the importance of the protonation state of the chromophore and its surrounding amino acids. It is therefore assumed that in its on-state, the chromophore is deprotonated, whereas in the off-state, the chromophore is protonated. However, as it is believed that the hydrogen bonding network around the chromophore plays a leading role in photoswitching, we need to have experimental data showing the hydrogen atoms in a crystal structure. Single crystal neutron diffraction on RSFPs will therefore be tried in order to obtain the very important commentary experimental data about the protonation states and hydrogen networks, needed for a full comprehension about the photoswitching mechanism and its dependence on pH. References Nguyen Bich, N.; Moeyaert, B.; Van Hecke, K.; Dedecker, P.; Mizuno, H.; Hofkens, J.; Van Meervelt, L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2012, 68, 1653-1659. Moeyaert, B.; Nguyen Bich, N.; De Zitter, E.; Rocha, S.; Clays, K.; Mizuno, H.; Van Meervelt, L.; Hofkens, J.; Dedecker, P. ACS Nano 2014, 8, 1664-1673. Duwé, S., De Zitter, E., Gielen, V., Moeyaert, B., Vandenberg, W., Grotjohann; T., Clays, K., Jakobs, J, Van Meervelt, L., Peter Dedecker, P., ACS Nano 2015, 9, 9528-9541. Cloin, B.M.C., De Zitter, E., Salas, D., Gielen, V., Folkers, G.E., Mikhaylova, M., Bergeler, M., Krajnik, B., Harvey, J., Hoogenraad, C.C., Van Meervelt, L., Dedecker, P., Kaptein, L.C., Proc. Nat. Acad. Sci. USA 2017, 114, 7013-7018.

Date:21 Mar 2018 →  21 Mar 2022
Keywords:fluorescent proteins, reversible photoswitching
Disciplines:Biochemistry and metabolism, Systems biology, Medical biochemistry and metabolism
Project type:PhD project