Unknown

Dataset Information

0

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


ABSTRACT: 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 ?s 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.

SUBMITTER: Woodhouse J 

PROVIDER: S-EPMC7005145 | BioStudies | 2020-01-01

REPOSITORIES: biostudies

Similar Datasets

2007-01-01 | S-EPMC1783997 | BioStudies
2008-01-01 | S-EPMC2274881 | BioStudies
2014-01-01 | S-EPMC4104898 | BioStudies
2008-01-01 | S-EPMC2587625 | BioStudies
2019-01-01 | S-EPMC7036281 | BioStudies
2005-01-01 | S-EPMC1201575 | BioStudies
2015-01-01 | S-EPMC4574475 | BioStudies
2010-01-01 | S-EPMC2863246 | BioStudies
2017-01-01 | S-EPMC5677571 | BioStudies
2007-01-01 | S-EPMC1941826 | BioStudies