Photoswitchable fluorescent proteins are used as molecular markers in super-resolution light microscopy that images life biological cells at a resolution of a few tens of nanometers. These proteins can be reversibly toggled between a non-fluorescent (off) state and a fluorescent (on) state by irradiation with light at specific wavelengths. Photoswitching between on and off states involves ultra-fast excited-state processes that have been recently characterized structurally. Conformational changes on the slower time scale, however, have remained elusive, hampering a comprehensive description of the photoswitching mechanism at the molecular level. Using time-resolved serial crystallography at the X-ray free electron laser (XFEL) at SACLA, Japan, in combination with transient absorption spectroscopy, researchers from the IBS, the ILL and the Universities of Lille and Rennes, in collaboration with colleagues from the Max-Planck Institutes in Heidelberg and Göttingen, Germany, have now clarified the photoswitching mechanism of rsEGFP2 by determining the three-dimensional structure of a key photo-intermediate (i.e. the one populated 10 nanoseconds after photoexcitation of the off state, see figure). This study clarifies the order of events during the off-to-on photoswitching and is anticipated to facilitate rational improvement of reversibly photoswitchable fluorescent proteins for applications in super-resolution light microscopy of biological cells.
Photoswitching mechanism of a fluorescent protein revealed by time-resolved serial femtosecond crystallography and transient absorption spectroscopy. Woodhouse J, Nass-Kovacs G, Coquelle N, Uriarte LM, Adam V, Barends TRM, Byrdin M,. de la Mora E, Doak RB, Feliks M, Field M, Fieschi F, Guillon V, Jakobs S, Joti Y, Macheboeuf P, Motomura K, Nass K, Owada S, Roome CM, Ruckebusch C, Schirò G, Shoeman RL, Thepaut M, Togashi T, Tono K, Yabashi M, Cammarata M, Foucar L, Bourgeois D, Sliwa M, Colletier JP, Schlichting I, Weik M. Nature Communications ; 11, 741