Chromophore photoreduction in red fluorescent proteins is responsible for bleaching and phototoxicity.
Red fluorescent proteins (RFPs) are indispensable tools for deep-tissue imaging, fluorescence resonance energy transfer applications, and super-resolution microscopy. Using time-resolved optical spectroscopy this study investigated photoinduced dynamics of three RFPs, KillerRed, mRFP, and DsRed. In all three RFPs, a new transient absorption intermediate was observed, which decays on a microsecond-millisecond time scale. This intermediate is characterized by red-shifted absorption at 1.68-1.72 eV (λmax = 720-740 nm). On the basis of electronic structure calculations, experimental evidence, and published literature, the chemical nature of the intermediate is assigned to an unusual open-shell dianionic chromophore (dianion-radical) formed via photoreduction. A doubly charged state that is not stable in the isolated (gas phase) chromophore is stabilized by the electrostatic field of the protein. Mechanistic implications for photobleaching, blinking, and phototoxicity are discussed.
School of Chemistry and Biochemistry, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
Russell B
RB
Vegh
Ksenia B
KB
Bravaya
Dmitry A
DA
Bloch
Andreas S
AS
Bommarius
Laren M
LM
Tolbert
Michael
M
Verkhovsky
Anna I
AI
Krylov
Kyril M
KM
Solntsev
D007700:Kinetics; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry* / Q000633:toxicity*; D008958:Models, Molecular; D038761:Photobleaching; D011487:Protein Conformation; D013816:Thermodynamics; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein