Under blue light irradiation green FPs can act as light-induced electron donors in photochemical reactions with various electron acceptors. These reactions are accompanied by green-to-red photoconversion (oxidative redding) which is among the processes significantly contributing to the photostability of many GFP family proteins. We suppose that inhibition of redding will have similar consequences in super-resolution microscopy (PALM) as it does in epifluorescent microscopy, namely, enhanced photostability and average fluorophore brightness. Understanding of the primary mechanisms of electron transfer (ET) occurring in excited state allows to control the photostability. In our recent work [1] we consider two mechanistic hypotheses:

1) ET proceeds via direct ET to a surface-docked oxidant (tunnel mechanism)

2) Photoinduced ET proceeds via a hopping mechanism (in which the first step is ET from the chromophore to some intermediate acceptor, and the second step is ET from this acceptor to an outside oxidant molecule)

Hopping mechanism appears to be the most likely process according to the quantum-mechanical calculations. One of the approaches to reduce ET via hopping mechanism is an elimination of the potential internal electron acceptors, located in the side chain of fluorescent protein. We determined such critical residues in the polypeptide chain of PAGFP using quantum-chemical simulations, and changed them to amino acid that is not able to be an efficient electron acceptor, namely, leucine. According to both epifluorescent and TIRF-experiments’ data, this substitution leads to the highly improved photostability in comparison with the original PAGFP. It has also been shown that in TIRF-experiments PAGFP Y145L had significantly lower blinking rate than PAGFP (see Fig.1). This property could be potentially used in SPT (single particle tracking) technique, where transitions to the dark states impose serious constraints and appear to be a drawback.

[1]. Bogdanov et al., JACS, 2016, in press

Figures:

Figure 1. Microscopy of the single molecules of PA-GFP-integrin and PA-GFP-Y145L-integrin. Distribution of localization precision of individual fluorophores (16ms for the frame). As it turned out, the photoactivated forms of mutant PA-GFP-Y145L, unlike PA-GFP, does not have detectable reversible long-living states leading to "blink" of the single molecule fluorescence in the investigated time scale (0.016 – 100 sec).

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EMC Abstracts - https://emc-proceedings.com/abstract/turning-off-photoinduced-electron-transfer-in-green-fluorescent-proteins-for-super-resolution-microscopy/