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

Abstract

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.

Novel uses of fluorescent proteins

“…5.4 in Ref. [66]), which nicely fits the TA spectra of various RFPs [68 ]. Based on the results of QM/MM calculations this transient was assigned to the unusual open-shell dianionic chromophore (dianion-radical) formed via photoreduction.…”

Extension of the Effective Fragment Potential Method to Macromolecules

“…In a recent study of KillerRed (which has the same chromophore as mPlum) such dianion-radical states were found to be stable. 77 Figs. 5(d), 6(a), 6(b), 6(c) and 6(d) show spin densities for the electron-attached 22 the scheme with two fragments per amino acid (C α -C and C α -N) shows the best performance for this system: it reproduces the full QM result exactly.…”

Advances in fluorescence labeling strategies for dynamic cellular imaging

“…It is clear that when structural and spectroscopic studies are coupled with protein engineering efforts, it is possible to gain molecular insight into factors that influence essential characteristics such as brightness and photoswitching. Although our understanding of some properties (for example, quantum yield) is increasing and engineering efforts targeting these properties have been successful, our understanding of other properties such as dark-state conversion and photobleaching remains very limited 24 . As the biological community demands increasingly photostable proteins, such as those for single-particle tracking of low-copy proteins, or increasingly sophisticated properties, such as those for modulation of dark-state conversion rates for SOFI, a deeper understanding of the mechanism (or mechanisms) of these processes and how these mechanisms are influenced by the protein structure would substantially aid in engineering efforts.…”

“…Mechanistically, photobleaching in FPs may result in part from the low dielectric environment of the protein interior (~4) and large electric fields (10–100 MV cm −1 ). Strong electric fields could promote photoinduced ionization of the chromophore 24 , and electron transfer is a principal pathway for photobleaching in small-molecule fluorophores 25 . Furthermore, electron transfer has been evoked to explain decarboxylation in PA-GFP and DsRed 26 as well as conjugation disruption in IrisFP following X-ray illumination 27 .…”