Chromophore Photoreduction in Red Fluorescent Proteins Is Responsible for Bleaching and Phototoxicity

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• 作者：Russell B. Vegh ; Ksenia B. Bravaya ; Dmitry A. Bloch ; Andreas S. Bommarius ; Laren M. Tolbert ; Michael Verkhovsky ; Anna I. Krylov ; Kyril M. Solntsev
• 刊名：Journal of Physical Chemistry B
• 出版年：2014
• 出版时间：May 1, 2014
• 年：2014
• 卷：118
• 期：17
• 页码：4527-4534
• 全文大小：330K
• 年卷期：v.118,no.17(May 1, 2014)
• ISSN：1520-5207

文摘

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鈥搈illisecond time scale. This intermediate is characterized by red-shifted absorption at 1.68鈥?.72 eV (位b>maxb> = 720鈥?40 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.