Simple and efficient system for photoconverting light-sensitive proteins in serial crystallography experiments
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 p...
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| Hauptverfasser: | , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
01 June 2017
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| In: |
Journal of applied crystallography
Year: 2017, Jahrgang: 50, Heft: 3, Pages: 932-939 |
| ISSN: | 1600-5767 |
| DOI: | 10.1107/S1600576717006264 |
| Online-Zugang: | Verlag, Volltext: http://dx.doi.org/10.1107/S1600576717006264 Verlag, Volltext: https://onlinelibrary.wiley.com/doi/abs/10.1107/S1600576717006264 
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| Verfasserangaben: | Giorgio Schirò, Joyce Woodhouse, Martin Weik, Ilme Schlichting and Robert L. Shoeman |
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| 520 | |a 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. | ||
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