2 research outputs found
Structural Evidence for a Two-Regime Photobleaching Mechanism in a Reversibly Switchable Fluorescent Protein
Photobleaching,
the irreversible photodestruction of a chromophore,
severely limits the use of fluorescent proteins (FPs) in optical microscopy.
Yet, the mechanisms that govern photobleaching remain poorly understood.
In Reversibly Switchable Fluorescent Proteins (RSFPs), a class of
FPs that can be repeatedly photoswitched between nonfluorescent and
fluorescent states, photobleaching limits the achievable number of
switching cycles, a process known as photofatigue. We investigated
the photofatigue mechanisms in the protein IrisFP using combined X-ray
crystallography, optical <i>in crystallo</i> spectroscopy,
mass spectrometry and modeling approaches. At laser-light intensities
typical of conventional wide-field fluorescence microscopy, an oxygen-dependent
photobleaching pathway was evidenced. Structural modifications induced
by singlet-oxygen production within the chromophore pocket revealed
the oxidation of two sulfur-containing residues, Met159 and Cys171,
locking the chromophore in a nonfluorescent protonated state. At laser-light
intensities typical of localization-based nanoscopy (>0.1 kW/cm<sup>2</sup>), a completely different, oxygen-independent photobleaching
pathway was found to take place. The conserved Glu212 underwent decarboxylation
concomitantly with an extensive rearrangement of the H-bond network
around the chromophore, and an sp<sup>2</sup>-to-sp<sup>3</sup> hybridization
change of the carbon atom bridging the chromophore cyclic moieties
was observed. This two-regime photobleaching mechanism is likely to
be a common feature in RSFPs from Anthozoan species, which typically
share high structural and sequence identity with IrisFP. In addition,
our results suggest that, when such FPs are used, the illumination
conditions employed in localization-based super-resolution microscopy
might generate less cytotoxicity than those of standard wide-field
microscopy at constant absorbed light-dose. Finally, our data will
facilitate the rational design of FPs displaying enhanced photoresistance
Structural Evidence for a Two-Regime Photobleaching Mechanism in a Reversibly Switchable Fluorescent Protein
Photobleaching,
the irreversible photodestruction of a chromophore,
severely limits the use of fluorescent proteins (FPs) in optical microscopy.
Yet, the mechanisms that govern photobleaching remain poorly understood.
In Reversibly Switchable Fluorescent Proteins (RSFPs), a class of
FPs that can be repeatedly photoswitched between nonfluorescent and
fluorescent states, photobleaching limits the achievable number of
switching cycles, a process known as photofatigue. We investigated
the photofatigue mechanisms in the protein IrisFP using combined X-ray
crystallography, optical <i>in crystallo</i> spectroscopy,
mass spectrometry and modeling approaches. At laser-light intensities
typical of conventional wide-field fluorescence microscopy, an oxygen-dependent
photobleaching pathway was evidenced. Structural modifications induced
by singlet-oxygen production within the chromophore pocket revealed
the oxidation of two sulfur-containing residues, Met159 and Cys171,
locking the chromophore in a nonfluorescent protonated state. At laser-light
intensities typical of localization-based nanoscopy (>0.1 kW/cm<sup>2</sup>), a completely different, oxygen-independent photobleaching
pathway was found to take place. The conserved Glu212 underwent decarboxylation
concomitantly with an extensive rearrangement of the H-bond network
around the chromophore, and an sp<sup>2</sup>-to-sp<sup>3</sup> hybridization
change of the carbon atom bridging the chromophore cyclic moieties
was observed. This two-regime photobleaching mechanism is likely to
be a common feature in RSFPs from Anthozoan species, which typically
share high structural and sequence identity with IrisFP. In addition,
our results suggest that, when such FPs are used, the illumination
conditions employed in localization-based super-resolution microscopy
might generate less cytotoxicity than those of standard wide-field
microscopy at constant absorbed light-dose. Finally, our data will
facilitate the rational design of FPs displaying enhanced photoresistance