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. 2008 Oct 24;283(43):28980-7.
doi: 10.1074/jbc.M800599200. Epub 2008 Aug 4.

A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore

Sergei Pletnev  1 , Dmitry Shcherbo, Dmitry M Chudakov, Nadezhda Pletneva, Ekaterina M Merzlyak, Alexander Wlodawer, Zbigniew Dauter, Vladimir Pletnev
Affiliations

A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore

Sergei Pletnev et al. J Biol Chem. .

Abstract

The far-red fluorescent protein mKate (lambda(ex), 588 nm; lambda(em), 635 nm; chromophore-forming triad Met(63)-Tyr(64)-Gly(65)), originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75-2.6 A. The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. The weakly fluorescent state of the protein at pH 4.2 is characterized by a mixture of trans and cis isomers. The chromophore in a highly fluorescent state at pH 7.0/9.0 adopts the cis form. Three key residues, Ser(143), Leu(174), and Arg(197) residing in the vicinity of the chromophore, have been identified as being primarily responsible for the far-red shift in the spectra. A group of residues consisting of Val(93), Arg(122), Glu(155), Arg(157), Asp(159), His(169), Ile(171), Asn(173), Val(192), Tyr(194), and Val(216), are most likely responsible for the observed monomeric state of the protein in solution.

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Figures

FIGURE 1.
FIGURE 1.
Sequence alignment of the wild-type eqFP578 and its mutant far-red variants, Katushka and mKate. Residue positions in three-dimensional structure: $, chromophore forming residues. Black and red font (highlighted in yellow), chromophore nearest environment (see Figs. 4 and 5). Red, chromophore environment; apparently responsible for the far-red shift. White font (highlighted in black), β-barrel caps area; presumably silent mutations or covariant with those in red. White font (highlighted in green), crystal intratetramer interfaces; apparently responsible for monomerization in solution. White font (highlighted in blue), presumably silent mutations in random mutagenesis process.
FIGURE 2.
FIGURE 2.
mKate and rationally designed structure-based variant mKate_S158A. a, pH dependence of the fluorescence; b, fluorescence spectra.
FIGURE 3.
FIGURE 3.
A stereoview of the chromophores in a difference Fo-Fc electron density (cutoff ρ = 3. 0σ). This was calculated with the phases omitting the contribution of the chromophore in the mKate crystal structures at pH 2.0 (a), pH 4.2 (subunit A) (b), pH 7.0 (c). This figure was produced with PYMOL (44).
FIGURE 4.
FIGURE 4.
A schematic diagram illustrating the environment of the trans chromophore in the mKate_pH2.0 structure (a) and the cis chromophore in the mKate_pH7.0 structure (b). Hydrogen bonds (≤3.3 Å) are shown as blue dashed lines, waters (W) as red spheres, and van der Waals contacts (≤3.9 Å) as black “eyelashes.” This figure was produced with LIGPLOT/HBPLUS (45, 46).
FIGURE 5.
FIGURE 5.
Stereoview of the H-bond network in the vicinity of the trans chromophore (shown in green) in the mKate_pH2.0 structure (a) and the cis chromophore in the mKate_pH7.0 structure (b). Mediating waters are shown by red spheres. This figure was produced with SETOR (47).
See this image and copyright information in PMC

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