Photoswitching mechanism of a fluorescent protein revealed by time-resolved 
crystallography and transient absorption spectroscopy.

Woodhouse, J.; Nass Kovacs, G.; Coquelle, N.; Uriarte, L. M.; Adam, V.; Barends, T. R. M.; Byrdin, M.; de la Mora, E.; Bruce Doak, R.; Feliks, M.; Field, M.; Fieschi, F.; Guillon, V.; Jakobs, S.; Joti, Y.; Macheboeuf, P.; Motomura, K.; Nass, K.; Owada, Sh.; Roome, C. M.; Ruckebusch, C.; Schirò, G.; Shoeman, R. L.; Thepaut, M.; Togashi, T.; Tono, K.; Yabashi, M.; Cammarata, M.; Foucar, L.; Bourgeois, D.; Sliwa, M.; Colletier, J. P.; Schlichting, I.; Weik, M.

doi:10.1038/s41467-020-14537-0

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Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

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Jakobs, S.
Research Group of Mitochondrial Structure and Dynamics, MPI for biophysical chemistry, Max Planck Society;

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Woodhouse, J., Nass Kovacs, G., Coquelle, N., Uriarte, L. M., Adam, V., Barends, T. R. M., et al. (2020). Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy. Nature Communications, 11(1): 741. doi:10.1038/s41467-020-14537-0.

Zitierlink: https://hdl.handle.net/21.11116/0000-0005-A285-D

Zusammenfassung

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.