Kindling fluorescent proteins for precise in vivo photolabeling

Chudakov, Dmitriy M.; Belousov, Vsevolod V.; Zaraisky, Andrey G.; Novoselov, Vladimir V.; Staroverov, Dmitry B; Zorov, Dmitry B.; Lukyanov, Sergey; Lukyanov, Konstantin A.

doi:10.1038/nbt778

Cited by 306 publications

(231 citation statements)

References 24 publications

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“…One phenomenon, termed kindling, has been demonstrated for the non-fluorescent protein asFP595 isolated from A. sulcata. This protein becomes fluorescent (kindles) when irradiated with intense green light and has been developed for precise photolabeling in vivo (26). A model of the kindling mechanism has been proposed in which the key event in the transition from the non-fluorescent to fluorescent form of the protein was a trans to cis isomerization of the chromophore (7).…”

Section: Discussionmentioning

confidence: 99%

The 2.0-Å Crystal Structure of eqFP611, a Far Red Fluorescent Protein from the Sea Anemone Entacmaea quadricolor

Petersen

Wilmann

Beddoe

et al. 2003

Journal of Biological Chemistry

169

188

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We have crystallized and subsequently determined to 2.0-Å resolution the crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. The structure of the protomer, which adopts a ␤-can topology, is similar to that of the related monomeric green fluorescent protein (GFP). The quaternary structure of eqFP611, a tetramer exhibiting 222 symmetry, is similar to that observed for the more closely related red fluorescent protein DsRed and the chromoprotein Rtms5. The unique chromophore sequence (Met 63 -Tyr 64 -Gly 65 ) of eqFP611, adopts a coplanar and trans conformation within the interior of the ␤-can fold. Accordingly, the eqFP611 chromophore adopts a significantly different conformation in comparison to the chromophore conformation observed in GFP, DsRed, and Rtms5. The coplanar chromophore conformation and its immediate environment provide a structural basis for the far red, highly fluorescent nature of eqFP611. The eqFP611 structure extends our knowledge on the range of conformations a chromophore can adopt within closely related members of the green fluorescent protein family.

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Section: Discussionmentioning

confidence: 99%

The 2.0-Å Crystal Structure of eqFP611, a Far Red Fluorescent Protein from the Sea Anemone Entacmaea quadricolor

Petersen

Wilmann

Beddoe

et al. 2003

Journal of Biological Chemistry

169

188

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“…Irradiation of the GFP T203H mutant (PA-GFP) with intense blue light (413 nm) induces a 100-fold increase of the green emission upon excitation at 488 nm [19]. Bright green light was shown to increase the red fluorescence of the kindling fluorescent protein mutant asCP A148G 30-fold [20]. Another protein that appears very useful for localized optical marking is the FP "Kaede" isolated from Trachyphyllia geoffroyii [21].…”

Section: Introductionmentioning

confidence: 99%

Targeted Green-Red Photoconversion of EosFP, a Fluorescent Marker Protein

et al. 2005

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Abstract. EosFP is a novel fluorescent protein from the stony coral Lobophyllia hemprichii. Its gene was cloned in Escherichia coli to express the tetrameric wild-type protein. The protein emits strong green fluorescence (516 nm) that shifts toward red (581 nm) upon near-ultraviolet irradiation at ∼390 nm due to a photo-induced modification that involves a break in the peptide backbone next to the chromophore. Using site-directed mutagenesis, dimeric (d1EosFP, d2EosFP) and monomeric (mEosFP) variants were produced with essentially unaltered spectroscopic properties. Here we present a spectroscopic characterization of EosFP and its variants, including room-and low-temperature spectra, fluorescence lifetime determinations, two-photon excitation and two-photon photoconversion. Furthermore, by transfection of a human cancer (HeLa) cell with a fusion construct of a mitochondrial targeting sequence and d2EosFP, we demonstrate how localized photoconversion of EosFP can be employed for resolving intracellular processes.

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“…Remarkably, all known CPs can be converted to fluorescent analogues by means of mutagenesis (3) or by irradiation with light of fixed wavelength (8,9). Despite great diversity in the colors of coral proteins, this variability seems to be achieved in a fairly conservative way.…”

mentioning

confidence: 99%

A Purple-blue Chromoprotein from Goniopora tenuidens Belongs to the DsRed Subfamily of GFP-like Proteins

Martynov

Maksimov

Martynova

et al. 2003

Journal of Biological Chemistry

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A number of recently cloned chromoproteins homologous to the green fluorescent protein show a substantial bathochromic shift in absorption spectra. Compared with red fluorescent protein from Discosoma sp. (DsRed), mutants of these so-called far-red proteins exhibit a clear red shift in emission spectra as well. Here we report that a far-red chromoprotein from Goniopora tenuidens (gtCP) contains a chromophore of the same chemical structure as DsRed. Denaturation kinetics of both DsRed and gtCP under acidic conditions indicates that the red form of the chromophore (absorption maximum at 436 nm) converts to the GFP-like form (384 nm) by a one-stage reaction. Upon neutralization, the 436-nm form of gtCP, but not the 384-nm form, renaturates instantly, implying that the former includes a chromophore in its intact state. gtCP represents a singlechain protein and, upon harsh denaturing conditions, shows three major bands in SDS/PAGE, two of which apparently result from hydrolysis of an acylimine C‫؍‬N bond. Instead of having absorption maxima at 384 nm and 450 nm, which are characteristic for a GFP-like chromophore, fragmented gtCP shows a different spectrum, which presumably corresponds to a 2-keto derivative of imidazolidinone. Mass spectra of the chromophore-containing peptide from gtCP reveal an additional loss of 2 Da relative to the GFP-like chromophore. Tandem mass spectrometry of the chromopeptide shows that an additional bond is dehydrogenated in gtCP at the same position as in DsRed. Altogether, these data suggest that gtCP belongs to the same subfamily as DsRed (in the classification of GFP-like proteins based on the chromophore structure type).A variety of fluorescent proteins from Anthozoa species has been described recently (1-6). These proteins represent the GFP-like family according to their homology to the green fluorescent protein from Aequorea victoria jellyfish. A growing interest in GFP-like proteins was stimulated largely by their potential utilization as biomarkers in living cells (7). Besides their applications to cellular biology, GFP-like proteins are of significant interest as the subjects of basic protein chemistry and enzymology. The main feature distinguishing these proteins from other known chromoproteins is the chromophore moiety composed of modified amino acid residues. Accordingly, the color of GFP-like protein is encoded genetically, and the chromophore center is assembled post-translationally by autocatalytic reactions inside the protein molecule. Thus, GFP-like proteins break the earlier adopted concept that chromoproteins are formed by combining an apoprotein with a prosthetic group.Currently, about 30 members of this family have been described (4); they are conventionally divided into two groups, proteins with inherent fluorescence (FP) 1 and naturally nonfluorescent proteins (CP). Remarkably, all known CPs can be converted to fluorescent analogues by means of mutagenesis (3) or by irradiation with light of fixed wavelength (8, 9). Despite great diversity in the colors of coral pr...

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Kindling fluorescent proteins for precise in vivo photolabeling

Cited by 306 publications

References 24 publications

The 2.0-Å Crystal Structure of eqFP611, a Far Red Fluorescent Protein from the Sea Anemone Entacmaea quadricolor

The 2.0-Å Crystal Structure of eqFP611, a Far Red Fluorescent Protein from the Sea Anemone Entacmaea quadricolor

Targeted Green-Red Photoconversion of EosFP, a Fluorescent Marker Protein

A Purple-blue Chromoprotein from Goniopora tenuidens Belongs to the DsRed Subfamily of GFP-like Proteins

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