The dark side of photoconvertible fluorescent proteins
Photoconvertible fluorescent proteins are markers of choice for PhotoActivated Localization Microscopy (PALM). Notably, these markers allow counting target proteins one by one directly inside cells. Unfortunately, the accuracy of counting is limited by “blinking”, that is, the discontinuous character of light emission by a single fluorescent molecule along time. Indeed, a single molecule that blinks can easily be confounded with an ensemble of distinct molecules that appear successively at the same location. Blinking results from stochastic and reversible transitions between fluorescent and dark states, but the involved mechanisms remain poorly understood. Improving the quantitative analysis of PALM data thus relies on the design of low-blinking variants. By combining X-ray crystallography, optical spectroscopy and PALM microscopy, we discovered that the orientation of a unique, fully conserved, aminoacid located next to the chromophore entirely controls the blinking of photoconvertible fluorescent proteins. The knowledge of the orientation of this aminoacid (arginine 66) is then sufficient to accurately predict blinking properties. This research (that ANR refuses to finance …) brings new knowledge in fundamental photophysics and opens the door to the rational engineering of variants optimized for quantitative PALM.
Romain Berardozzi, Virgile Adam, Alexandre Martins & Dominique Bourgeois Arginine 66 Controls Dark-State Formation in Green-to-Red Photoconvertible Fluorescent Proteins Journal of the American Chemical Society 138, 558-565 (2016). DOI: 10.1021/jacs.5b09923