Serial femtosecond crystallography of the photoswitchable fluorescent protein IrisFP
Serial femtosecond crystallography (SFX) at an X-ray free electron laser (XFEL) exploits intense X-ray pulses to provide a diffraction pattern before radiation damage destroys the protein crystal. The sample is replenished millions of times and diffraction data collected in a serial way. SFX permits tiny microcrystals to be studied and enables time-resolved studies of proteins in action down to the femtosecond time scale.
As a proof-of-principle en route to time-resolved crystallographic experiments on fluorescent proteins, the static three-dimensional structure of the photoswitchable fluorescent protein IrisFP in its on state was solved by SFX. The high-quality structure shows no signs of X-ray radiation damage and was determined from a very small amount of crystalline sample. For this project, the IBS DYNAMOP group used the XFEL at SACLA in Japan and teamed up with scientists from SACLA, the Max-Planck Institute in Heidelberg, the Universities of Lille and Rennes and the ESRF in Grenoble.
As a complement to SFX, time-resolved absorption spectroscopy was used to identify picosecond to millisecond intermediate-states of IrisFP during photoswitching, which are assigned to a sequential process of isomerisation and proton transfer. Together, our data lay a solid ground for ultra-fast time-resolved SFX at XFELs of photoswitchable fluorescent proteins that, beyond their fascinating photochemistry, are of major importance for advanced nanoscopy, such as super-resolution microscopy.
Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP. Colletier JP, Sliwa M, Gallat FX, Sugahara M, Guillon V, Schiro G, Coquelle N, Woodhouse J, Roux L, Gotthard G, Royant A, Uriarte LM, Ruckebusch C, Joti Y, Byrdin M, Mizohata E, Nango E, Tanaka T, Tono K, Yabashi M, Adam V, Cammarata M, Schlichting I, Bourgeois D, Weik M (2016) The Journal of Physical Chemistry Letters : 882-887 DOI : 10.1021/acs.jpclett.5b02789