研究目的
To 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.
研究成果
The study establishes that off-to-on photoswitching in rsEGFP2 involves excited-state isomerization on the ps timescale followed by μs conformational changes in the ground state that trigger proton transfer on the ms timescale. This provides a detailed mechanism of photoswitching in rsEGFP2.
研究不足
The study's limitations include the technical challenges associated with time-resolved serial crystallography and transient absorption spectroscopy, such as the need for precise synchronization between the pump and probe beams and the difficulty in capturing transient intermediate states.
1：Experimental Design and Method Selection:
The study combined time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump–probe UV-visible spectroscopy to investigate the photoswitching mechanism in rsEGFP
2：Sample Selection and Data Sources:
rsEGFP2 microcrystals were used, and data were collected at the Spring-8 Angstrom Compact Laser (SACLA) XFEL.
3：List of Experimental Equipment and Materials:
The study utilized a Ti:sapphire laser for photoexcitation, an octal-MPCCD detector for diffraction data, and a GDVN injector for sample delivery.
4：Experimental Procedures and Operational Workflow:
Microcrystals were photoswitched from their on-state to their off-state before data collection. TR-SFX data were collected using an optical pump–X-ray probe scheme with a pump–probe delay of 10 ns.
5：Data Analysis Methods:
Data processing was performed using NanoPeakCell for hit-finding and CrystFEL for indexing and integration. Structure solution and refinement were carried out using Phaser, Phenix, and Coot.
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