Simple and efficient system for photoconverting light-sensitive proteins in serial crystallography experiments

• Verfasst von: Schirò, Giorgio [VerfasserIn]
• Verfasst von: Schlichting, Ilme [VerfasserIn]
• Titel: Simple and efficient system for photoconverting light-sensitive proteins in serial crystallography experiments
• Verf.angabe: Giorgio Schirò, Joyce Woodhouse, Martin Weik, Ilme Schlichting and Robert L. Shoeman
• Umfang: 8 S.
• Fussnoten: Gesehen am 12.09.2018
• Titel Quelle: Enthalten in: Journal of applied crystallography
• Jahr Quelle: 2017
• Band/Heft Quelle: 50(2017), 3, S. 932-939
• ISSN Quelle: 1600-5767
• DOI: doi:10.1107/S1600576717006264
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1580893716

Abstract

Proteins that change their structure in response to light absorption regulate many functional processes in living cells. Moreover, biotechnological approaches like optogenetics and super-resolution fluorescence microscopy recently triggered the generation of new genetically modified photosensitive proteins. Light-induced structural changes in photosensitive proteins can be studied by time-resolved serial femtosecond crystallography (SFX), an X-ray diffraction technique that allows the determination of macromolecular structures at X-ray free-electron lasers from a large number of nano- to micro-sized crystals. This article describes a simple and efficient system for converting photosensitive proteins into light-induced semi-stationary states by inline laser illumination prior to sample injection with a gas-focused liquid jet and subsequent optical pump-X-ray probe exposure. The simple setup of this device makes it suitable for integration into other liquid injectors (like electro-spinning and electro-kinetic injectors) and potentially also in high-viscosity extruders, provided that embedding microcrystals in viscous media does not alter protein photophysical properties. The functioning of the device is demonstrated with an example of a photoswitchable fluorescent protein pre-illuminated (photoactivated) for time-resolved SFX experiments. The device can be easily adapted for the conversion in time-resolved SFX experiments of other microcrystalline proteins, such as photosystems, phytochromes and rhodopsins.