Institut de Biologie StructuraleGrenoble / France

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IrisFP Vis blink

Shedding new light on fluorescent proteins’ dark states

All fluorescent markers used in cell imaging “blink”, switching quickly and stochastically between bright (fluorescent) and dark (non-fluorescent) states. In the case of fluorescent proteins, the molecular and structural origin of blinking remains mysterious. By employing a combination of experimental approaches (crystallography/optical spectroscopy), we demonstrated in 2009 that a transiently dark state of the fluorescent protein IrisFP could be induced by X-rays, characterized by a severe distortion of its chromophore (Adam et al, JACS, 2009, 131, 18063). However, in real imaging conditions, the blinking process results from illumination with visible light, not with X-rays. In the present work, simulations based on a hybrid approach combining quantum mechanics and molecular mechanics (QM/MM) suggest that IrisFP can blink in essentially the same way under illumination with visible light or X-rays. The chromophore distortion at the origin of the fluorescence intermittency can be explained by the reversible transfer of a proton from a nearby arginine residue towards the central part (methylene bridge) of the chromophore in a triplet or a radical state. This distortion of the chromophore disrupts transiently its electronic conjugation and hence stops its fluorescence emission. This work is important for the future development of more photostable fluorescent proteins.

Arijit Roy, Martin J. Field, Virgile Adam and Dominique Bourgeois. The Nature of Transient Dark States in a Photoactivatable Fluorescent Protein
JACS (2011) 133:18586-9

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