Chromophore Environment Provides Clue to “Kindling Fluorescent Protein” Riddle

Chudakov, Dmitriy M.; Feofanov, Alexei V.; Mudrik, N. N.; Lukyanov, Sergey; Lukyanov, Konstantin A.

doi:10.1074/jbc.m211988200

Cited by 145 publications

(206 citation statements)

References 14 publications

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“…Given that Rtms5 is weakly fluorescent, whereas DsRed and eqFP611 are highly fluorescent, the conformations of the respective chromophores in these structures suggest that coplanarity of the chromophore is required for a high fluorescence quantum yield. The fluorescence quantum yields of DsRed, eqFP611 and Rtms5 are 0.70, 0.45, and Ͻ0.001, respectively (7,8,12).…”

Section: Discussionmentioning

confidence: 99%

“…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). The data presented here together with further mutagenesis and structural analysis of related proteins such as Rtms5 and eqFP611 may help develop a detailed understanding of this phenomenon.…”

Section: Discussionmentioning

confidence: 99%

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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%

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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“…In the cis conformation, the chromophoric p-hydroxyphenyl group is likely to be in equilibrium between a (nonfluorescent) protonated and a (fluorescent) nonprotonated form (11).…”

Section: Discussionmentioning

confidence: 99%

Structure and mechanism of the reversible photoswitch of a fluorescent protein

Andresen

Wahl

Stiel

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

256

341

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Proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state bear enormous potential in diverse fields, such as data storage, in vivo protein tracking, and subdiffraction resolution light microscopy. However, these proteins could hitherto not live up to their full potential because the molecular switching mechanism is not resolved. Here, we clarify the molecular photoswitching mechanism of asFP595, a green fluorescent protein (GFP)-like protein that can be transferred from a nonfluorescent ''off'' to a fluorescent ''on'' state and back again, by green and blue light, respectively. To this end, we establish reversible photoswitching of fluorescence in whole protein crystals and show that the switching kinetics in the crystal is identical with that in solution. Subsequent x-ray analysis demonstrated that upon the absorption of a green photon, the chromophore isomerizes from a trans (off) to a cis (on) state. Molecular dynamics calculations suggest that isomerization occurs through a bottom hula twist mechanism with concomitant rotation of both bonds of the chromophoric methine ring bridge. This insight into the switching mechanism should facilitate the targeted design of photoswitchable proteins. Reversible photoswitching of the protein chromophore system within intact crystals also constitutes a step toward the use of fluorescent proteins in three-dimensional data recording.photoisomerization ͉ asCP ͉ photochromism ͉ optical bistability ͉ asulCP F luorescent proteins have been widely used as genetically encodable tags to monitor protein localizations and dynamics in live cells (1-3). Recently, novel GFP-like fluorescent proteins have been discovered (4-6) that can be reversibly photoswitched between a fluorescent (on) and nonfluorescent (off) state, that is, they are optically bistable and fluorescent. This feature is remarkable, because the reversible photoswitching occurring in photochromic organic compounds, such as in fulgides and diarylethenes, is usually not accompanied by fluorescence (7). Therefore, not surprisingly, these proteins hold great promise in many areas of science reaching out far beyond their prominent use as triggerable protein markers in live cells. For example, the reversible photoswitching of fluorescent markers should provide nanoscale resolution in fluorescence microscopy by using lenses and regular illumination, which was hardly conceivable only a few years ago (8-10). As fluorescence can be sensitively read out from a bulky crystal, the prospect of erasable three-dimensional data storage is equally intriguing.The GFP-like protein asFP595 (asCP or asulCP) from the sea anemone Anemonia sulcata is such a protein: It can be transferred by green light from a nonfluorescent off into a fluorescent on state from which it reverts back eventually, but this transition can also be promptly stimulated by gentle irradiation with blue light (6). The ''on-off'' cycle can be repeated many times. However, with its low quantum yield (Ͻ0.001, ref. 6) and comparatively slo...

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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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Chromophore Environment Provides Clue to “Kindling Fluorescent Protein” Riddle

Cited by 145 publications

References 14 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

Structure and mechanism of the reversible photoswitch of a fluorescent protein

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

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