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Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / BYRDIN Martin
Contact person(s) related to this article / BYRDIN Martin

Eos FP photoconversion

A Complex Mechanism Explains How a Fluorescent Protein Changes Colour



_Fluorescent proteins of the GFP family are the object of intense study due to their inherent bioluminescence, and have proved to be excellent markers for cellular imaging. In the last few years, "photoactivatable" fluorescent proteins have been developed whose fluorescence properties change as a function of their illumination conditions. These novel proteins are crucial to the new "super-resolution" fluorescence imaging methods that permit images of living cells to be obtained at nanometre resolution. One of the most popular photoactivatable proteins in nanoscopy is EosFP. This protein normally fluoresces in green, but when illuminated with violet light its fluorescence changes to red. This "photoconversion" process involves the rupture of the peptide chain next to the chromophore and formation of an enlarged conjugated system, but its mechanism has proved elusive. Starting from X-ray crystallographic structures of the green and red forms of EosFP, we have employed QC/MM simulation methods to investigate possible reaction pathways and have been able to propose a mechanism for the photoconversion. Absorption of a violet photon by the protein promotes it to its first singlet excited state. Approximately once in a thousand times, the singlet undergoes a forbidden transition to a triplet state. Once there, a proton transfer occurs that leads to a cascade of events that results in rupture of the peptide backbone, elongation of the conjugated system and red fluorescence. This work is an advance in our understanding of the function of photoactivatable fluorescent proteins, and could allow the development of variants with improved photophysical properties.

Mickael Lelimousin, Virgile Adam, G. Ulrich Nienhaus, Dominique Bourgeois and Martin J. Field. Photoconversion of the Fluorescent Protein EosFP: A Hybrid Potential Simulation Study Reveals Intersystem Crossings. JACS (2009) 131:16814-23.

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