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. 2009 Nov 13;284(46):32028-39.
doi: 10.1074/jbc.M109.054973. Epub 2009 Sep 8.

Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

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Free PMC article

Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

Sergei Pletnev et al. J Biol Chem. .
Free PMC article

Abstract

KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the beta-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu(68) and Ser(119), located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.

Figures

FIGURE 1.
FIGURE 1.
The environment of the chromophore in KillerRed. A, stereoview of a chain of hydrogen-bonded waters (red spheres in the upper part of the drawing) flowing from the outside to the phenolic ring hydroxyl of the QYG chromophore (shown in orange) through a pore located on the cylindrical β-barrel surface composed of the backbone of residues 142–144 and 199–201. A fraction of the other water chain (lower part of the drawing; see the legend for B) and the key residues Asn145 and Thr201 and the catalytic Arg94 and Glu218, interacting with chromophore, are also shown. B, stereoview of the water-filled channel with a chain of seven consecutive hydrogen-bonded water molecules (red spheres) traversing along the β-barrel from Pro192 at the cap loop to the catalytic Glu218 in the chromophore area. Two other water molecules are shown at the assumed entry point outside of the β-barrel (figure created with SETOR (55)).
FIGURE 2.
FIGURE 2.
Oligomerization of KillerRed. A, stereoview of the interface between the A and B subunits in the KillerRed dimer that are related by a 2-fold non-crystallographic symmetry axis. B, KillerRed oligomeric assembly in the crystal lattice showing three consecutive dimers composed from the β-barrel monomeric units (figure created with SETOR (55)).
FIGURE 3.
FIGURE 3.
Stereoviews of the QYG chromophore with a preceding Ile residue in the weighted omit mFoDFc electron density map (18) (cut-off ρ = 3.0σ). A, active fluorescent state; B, photobleached non-fluorescent state after 5 h of green laser light irradiation at λ = 532 nm. The latter density indicates considerable damage to the Gln65 and Tyr66 side chains of the chromophore (figure created with PyMOL (56)).
FIGURE 4.
FIGURE 4.
A schematic diagram illustrating the environment of the chromophore in the KillerRed structure. Hydrogen bonds (≤3.3 Å) are shown as blue dashed lines, water molecules (W) are shown as red spheres, and van der Waals contacts (≤3.9 Å) are shown as blackeyelashes” (figure prepared with LIGPLOT/HBPLUS (57)).
FIGURE 5.
FIGURE 5.
Spectral transitions of KillerRed in solution. A, reduction of red fluorescence intensity of KillerRed immobilized on beads upon the addition of 100 mm βME and its recovery after βME has been washed out. B, normalized photobleaching curves for KillerRed immobilized on beads in the absence and presence of βME upon excitation by mercury arc lamp (540–580 nm, 0.2 watt/cm2). C, absorbance spectrum changes in the presence of βME at different concentrations (shown in mm above each curve). An inset shows the dependence of absorbance at 585 nm on βME concentration. D, absorbance spectrum changes during photobleaching with a green laser at 532 nm.
FIGURE 6.
FIGURE 6.
The key residues adjacent to the chromophore. Glu68 is hydrogen-bonded via two waters to Ser119, in a mostly hydrophobic pocket. The side chain of Ser119 shows two alternative orientations (created with SETOR (55)).
FIGURE 7.
FIGURE 7.
Absorption, excitation, and emission spectra of the mutants of KillerRed. A, KillerRed-E68Q; B, KillerRed-S119A; C, KillerRed-A220T. D, decrease of viable bacterial counts (in colony-forming units (CFU)) after white light illumination of E. coli expressing the designated fluorescent proteins.
FIGURE 8.
FIGURE 8.
Structure-based sequence alignment of KillerRed, its wild-type progenitor anm2CP, DsRed, and avGFP. The numbering of residues in KillerRed and avGFP is given above and below the corresponding sequences. Buried residues are highlighted in gray. The chromophore-forming sequence is underlined. In red are shown residues residing in the nearest environment of the chromophore (≤3.9 Å); among them are the catalytic residues, Arg94 and Glu218 (highlighted in yellow), whereas the KillerRed/anm2CP differences are highlighted in blue.
FIGURE 9.
FIGURE 9.
A comparison of KillerRed with other FPs. A, stereoview showing the distribution of the amino acid differences (see Fig. 8) between the phototoxic, fluorescent KillerRed (element C in green) and the non-toxic, non-fluorescent anm2CP (element C in orange). B, the neighborhood of the chromophore in the superimposed three-dimensional structures of KillerRed (element C in green) and DsRed (element C in orange) (created with SETOR (55)).

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