ArticleNature communications2020
Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.
Article in Nature communications, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 44 papers.
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Who cites it
44 citing papers in PubMed.
- Recent developments of fluorescent proteins and biosensors driven by cross-disciplinary insights.Nature methods · 2026Review
- Review
- A Bayesian Approach to Interpret Time-Resolved Experiments Using Molecular Simulations.ACS physical chemistry Au · 2026Article
- A Versatile Tool to Predict and Guide RESOLFT Images Based on Photoswitching, Labelling and Optical Properties.Chemphyschem : a European journal of chemical physics and physical chemistry · 2026Article
- Non-traditional fluorescence in quadruple hydrogen bonded supramolecular polymers.Nature communications · 2026Article
- All-optical strategies to minimize photobleaching in reversibly switchable fluorescent proteins.Nature communications · 2025Article
- Sample delivery methods for protein X-ray crystallography with a special focus on sample consumption.Nature communications · 2025Review
- Standard Sample Preparation for Serial Femtosecond Crystallography.Biomolecules · 2025Review
- Predicting Excited-State Absorption Spectra from Non-Aufbau Configurations.Journal of chemical theory and computation · 2025Article
- Ultrafast photophysics of a positive reversibly switchable fluorescent protein.Chemical science · 2025Article
- Article
- Exploring the dynamics of allostery through multi-dimensional crystallography.Biophysical reviews · 2024Review
- XFEL Beamline Optical Instrumentation for Ultrafast Science.The journal of physical chemistry. B · 2024Review
- A snapshot love story: what serial crystallography has done and will do for us.Acta crystallographica. Section D, Structural biology · 2024Review
- Kinetic isotope effect reveals rate-limiting step in green-to-red photoconvertible fluorescent proteins.Protein science : a publication of the Protein Society · 2024Article
- The time revolution in macromolecular crystallography.Structural dynamics (Melville, N.Y.) · 2024Article
- From femtoseconds to minutes: time-resolved macromolecular crystallography at XFELs and synchrotrons.Acta crystallographica. Section D, Structural biology · 2024Article
- Review of serial femtosecond crystallography including the COVID-19 pandemic impact and future outlook.Structure (London, England : 1993) · 2023Review
- Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism.Journal of the American Chemical Society · 2023Article
- Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy.International journal of molecular sciences · 2023Article
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Authors and funding
34 authors.
Funding
No grant is acknowledged in the PubMed record.
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 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.
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