- Authors: Woodhouse, J., Nass Kovacs, G., Coquelle, N., Uriarte, L.M. Adam, V., Barends, T.R.M., Byrdin, M., de la Mora, E., Doak, R.B., Feliks, M., Field, M., Fieschi, F., Guillon, V., Jakobs, S., [...], Weik, M.
- Title: Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy
- Journal: Nature Communications
- Volume: 11
- Issue: 741
- DOI: 10.1038/s41467-020-14537-0
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.