The possibility of photoconvertible fluorescent proteins (PCFPs) to convert from a green- to a red-emitting state is widely used in single-molecule localization microscopy. However, single-molecule imaging is highly disturbed by ‘blinking’ (ie transient loss of fluorescence), which is a consequence of individual PCFPs visiting « dark states ». Blinking has generally been described in the photoconverted red state, but in this paper, we focused on green-state blinking.
In a collaboration between IBS/DYNAMOP, KU Leuven (Belgium) and University Paris-Saclay, we studied dark-state formation in green mEos4b, a popular PCFP, by using UV-VIS and Raman spectroscopy, X-ray crystallography and MD simulations. We discovered that formation of one main dark state originates from cis-trans isomerization of the chromophore, similarly to what happens in so-called reversibly switchable fluorescent proteins (RSFPs). However, we found that mEos4b cannot be completely switched off (ie its « switching contrast » is low), because its chromophore wiggles a lot in the dark state and easily returns to the on fluorescent state. By comparing the number of H-bonding interactions maintained by the chromophore in different fluorescent proteins in their on- and off-states, we could suggest that the switching contrast in RSFPs is tuned by the relative strength by which the chromophore is anchored to the protein matrix in the dark compared to the bright state.
Mechanistic Investigations of Green mEos4b Reveal a Dynamic Long-Lived Dark State. Elke De Zitter, Jacqueline Ridard, Daniel Thedie, Virgile Adam, Bernard Levy, Martin Byrdin, Guillaume Gotthard, Luc Van Meervelt, Peter Dedecker, Isabelle Demachy, Dominique Bourgeois. Journal of the American Chemical Society, 2020, ja-2020-01880m (10.1021/jacs.0c01880)
Contact : Dominique Bourgeois