A fluorescent protein for cryo-nanoscopy
Fluorescence super-resolution microscopy (“nanoscopy”) opens up a considerable research field for integrated structural biology. “PALM” nanoscopy relies on the on and off photoswitching of particular fluorescent proteins called “photoactivatable” fluorescent proteins. The photoswitching mechanisms are linked to significant conformational changes of the chromophore and of the protein matrix, which are typically blocked at low temperature. However, the development of a nanoscopy scheme working at cryogenic temperature could open the door to insightful correlative studies with cryo-electronic microscopy. Thus, it is important to develop markers that can photoswitch at low temperature. In this work, we have studied the photoswitching mechanism of the fluorescent protein Padron, by combining X-ray crystallography, spectroscopy, and molecular dynamics simulations. We discovered that Padron is capable of photoswitching at 100 Kelvin via trans-cis isomerization of its chromophore in an essentially rigid protein matrix. Such a large conformational change had never been observed at such a low temperature in a protein.
Regis-Faro, Aline ; Carpentier, Philippe ; Jonasson, Gabriella ; Pompidor, Guillaume ; Arcizet, Delphine ; Demachy, Isabelle ; Bourgeois, Dominique. "Low-temperature chromophore isomerization reveals the photoswitching mechanism of the fluorescent protein Padron." Journal of the American Chemical Society (2011) 133:16362-5. DOI : 10.1021/ja207001y