Cysteine Sulfoxidation Increases the Photostability of Red Fluorescent Proteins.
Photobleaching 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.
Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco , San Francisco, California 94158, United States.
Haiyan
H
Ren
Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco , San Francisco, California 94158, United States.
Bing
B
Yang
Center for Diagnostics & Therapeutics and Department of Chemistry, Georgia State University , Atlanta, Georgia 30303, United States.
Cheng
C
Ma
Nomis Center for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies , La Jolla, California 92037, United States.
Ying S
YS
Hu
Center for Diagnostics & Therapeutics and Department of Chemistry, Georgia State University , Atlanta, Georgia 30303, United States.
Peng George
PG
Wang
Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco , San Francisco, California 94158, United States.
Lei
L
Wang
D003545:Cysteine / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D038761:Photobleaching; D055668:Photochemical Processes; D055550:Protein Stability; D000097573:Red Fluorescent Protein
D003545:Cysteine; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein