cb6b00579_si_001.pdf (6.01 MB)
Cysteine Sulfoxidation Increases the Photostability of Red Fluorescent Proteins
journal contribution
posted on 2016-09-07, 00:00 authored by Haiyan Ren, Bing Yang, Cheng Ma, Ying S. Hu, Peng George Wang, Lei WangPhotobleaching of fluorescent proteins
(FPs) is a major limitation
to their use in advanced microscopy, and improving photostability
remains highly challenging due to limited understanding of its molecular
mechanism. Here we discovered a new mechanism to increase FP photostability.
Cysteine oxidation, implicated in only photobleaching before, was
found to drastically enhance FP photostability to the contrary. We
generated a far-red FP mStable by introducing a cysteine proximal
to the chromophore. Upon illumination, this cysteine was oxidized
to sulfinic and sulfonic acids, enabling mStable more photostable
than its ancestor mKate2 by 12-fold and surpassing other far-red FPs.
mStable outperformed in laser scanning confocal imaging and super-resolution
structured illumination microscopy. Moreover, photosensitization to
oxidize a cysteine similarly introduced in another FP mPlum also increased
its photostability by 23-fold. This postfolding cysteine sulfoxidation
cannot be simply substituted by the isosteric aspartic acid, representing
a unique mechanism valuable for engineering better photostability
into FPs.
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FP mPlumisosteric aspartic acidancestor mKate 2far-red FP mStablemechanismillumination microscopysulfonic acidsCysteine Sulfoxidation Increasespostfolding cysteine sulfoxidationlaser scanning confocal imagingFP photostabilityincrease FP photostabilityCysteine oxidationProteins Photobleachingfar-red FPs
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