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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学術論文

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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引用

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, S., 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. (2020). Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy. Nature Communications, 11(1):. doi:10.1038/s41467-020-14537-0.

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

要旨

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.