Crystallographic Structures of Discosoma Red Fluorescent Protein with Immature and Mature Chromophores:  Linking Peptide Bond Trans−Cis Isomerization and Acylimine Formation in Chromophore Maturation,

Tubbs, J.L.; Tainer, John A.; Getzoff, Elizabeth D.

doi:10.1021/bi0472907

Cited by 79 publications

(90 citation statements)

References 33 publications

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“…The H 2 O 2 release during the formation of the GFP chromophore has been detected experimentally (29). The suggested here mechanism is also supported by the observation that a DsRed/Q66M mutant formed the N-acylimine more efficiently than DsRed (18). That was possible because hydrophobic Met-66 inhibited the formation of hydrogen bond between Glu-215 and Asn-42, resulting in the ability of Glu-215 to protonate the imidazol-5-ol ring (Fig.…”

Section: Discussionmentioning

confidence: 53%

“…The change in the angle after the photoactivation (described later) can possibly slightly increase the chromophore brightness and shift its emission to longer wavelengths because of the improved conjugation of the chromophore with the carbonyl group of N-acylimine. It has been suggested for DsRed that formation of the N-acylimine is coupled to the peptide bond isomerization (18). Thus, electron density of PAmCherry1 in the OFF state is consistent with the post-translational modification of the Met-66-Tyr-67-Gly-68 tripeptide resulting in the formation of the 4-(4-hydroxybenzyl)-imidazol-5-ol and N-acylimine groups.…”

Section: Chromophore and Environment Of Pamcherry1 In The Dark Statementioning

confidence: 54%

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Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states

Subach¹,

Malashkevich²,

Zencheck³

et al. 2009

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

128

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Photoactivatable fluorescent proteins (PAFPs) are required for super-resolution imaging of live cells. Recently, the first red PAFP, PAmCherry1, was reported, which complements the photoactivatable GFP by providing a red super-resolution color. PAmCherry1 is originally ''dark'' but exhibits red fluorescence after UV-violet light irradiation. To define the structural basis of PAmCherry1 photoactivation, we determined its crystal structure in the dark and red fluorescent states at 1.50 Å and 1.65 Å, respectively. The non-coplanar structure of the chromophore in the dark PAmChery1 suggests the presence of an N-acylimine functionality and a single non-oxidized C ␣ -C ␤ bond in the Tyr-67 side chain in the cyclized Met-66-Tyr-67-Gly-68 tripeptide. MS data of the chromophore-bearing peptide indicates the loss of 20 Da upon maturation, whereas tandem MS reveals the C ␣ -N bond in Met-66 is oxidized. These data indicate that PAmCherry1 in the dark state possesses the chromophore N-[(E)-(5-hydroxy-1H-imidazol-2-yl)methylidene]acetamide, which, to our knowledge, has not been previously observed in PAFPs. The photoactivated PAmCherry1 exhibits a non-coplanar anionic DsRed-like chromophore but in the trans configuration. Based on the crystallographic analysis, MS data, and biochemical analysis of the PAmCherry1 mutants, we propose the detailed photoactivation mechanism. In this mechanism, the excited-state PAmCherry1 chromophore acts as the oxidant to release CO2 molecule from Glu-215 via a Koble-like radical reaction. The Glu-215 decarboxylation directs the carbanion formation resulting in the oxidation of the Tyr-67 C ␣ -C ␤ bond. The double bond extends the -conjugation between the phenolic ring of Tyr-67, the imidazolone, and the N-acylimine, resulting in the red fluorescent chromophore.chromophore ͉ localization microscopy ͉ photoconversion S uper-resolution imaging approaches such as photoactivation localization microscopy provide the ability to observe details of cellular and even macromolecular structure that were not previously discernible with less than 40 nm resolution (1). There is significant demand for a broader and more diverse range of photoactivatable fluorescent probes (2), in particular irreversibly photoactivatable fluorescent proteins (PAFPs). To develop PAFPs with new spectral properties, it is essential to gain understanding of the underlying mechanisms of photoactivation.X-ray structures have been reported for a number of irreversible PAFPs, including those that change their fluorescence from green to red upon irradiation with violet light such as EosFP (3), its derivative IrisFP (4), Kaede (5), KikGR (6), and Dendra2 (7). These PAFPs share the same His-Tyr-Gly chromophore-forming tripeptide and the same mechanism of photoactivation. This mechanism is associated with a ␤-elimination reaction, which results in the cleavage of the peptide bond between the amide nitrogen and ␣-carbon of the His residue in the chromophore tripeptide (8).Another group of irreversible PAFPs includes photoactivatable GFP (P...

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Section: Discussionmentioning

confidence: 53%

Section: Chromophore and Environment Of Pamcherry1 In The Dark Statementioning

confidence: 54%

Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states

Subach¹,

Malashkevich²,

Zencheck³

et al. 2009

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

128

View full text Add to dashboard Cite

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“…Only the enhanced thermal motion of the extreme loop segment 184 -190, exposed to bulk solvent and with the atomic B factors 10 -15 Å 2 higher than the mean value, might indirectly indicate a possibility of leakage in this area. It should be noted that another red fluorescent protein, DsRed, despite possessing the same chromophore (27,28), does not show any evidence of phototoxicity (12). An important difference between KillerRed and DsRed (Protein Data Bank code 1GGX) is that, in the latter protein, a water network originating from the end cap does not reach the chromophore.…”

Section: Resultsmentioning

confidence: 99%

Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

Pletnev

Gurskaya

Pletneva

et al. 2009

Journal of Biological Chemistry

127

168

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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 waterfilled channel reaching the chromophore area from the end cap of the ␤-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. The green fluorescent protein (GFP)2 and related proteins have become efficient noninvasive tools in cell biology and biomedicine for visualizing and monitoring the internal processes within cells or whole organisms (1-8). The multicolor labeling technologies, based on fluorescent proteins (FPs), have found important biomedical applications in the studies of various aspects of cancer, including primary tumor growth, tumor cell motility and invasion, metastatic seeding, colonization, and angiogenesis (9 -11).The recent development of the first genetically encoded photosensitizer, KillerRed (SWISS-PROT/TrEMBL data base sequence ID Q2TCH5), a highly phototoxic red fluorescent protein (12, 13), opened a new area of FP application. KillerRed is a red fluorescent protein characterized by excitation and emission maxima at 585 and 610 nm, respectively. This genetic variant was engineered from non-fluorescent and non-phototoxic chromoprotein anm2CP from Hydrozoa jellyfish (sequence ID Q6RYS4). Upon irradiation by green light at the wavelength of 520 -590 nm, KillerRed generates the reactive oxygen species (ROS), accompanied by profound self photobleaching. The ROS-induced phototoxicity of KillerRed is at least 3 orders of magnitude higher than that of other fluorescent proteins exhibiting low background phototoxicity (12). Such a unique property of KillerRed could find use in light-induced inactivation of target proteins and in precise cell killing. Unlike chemical photosensitizers, KillerRed can be directly expressed by a target cell, both individually and in fusion with a target protein. The most exciting future application of KillerRed may be in photodynamic therapy of cancer. This phototoxic agent, precisely delivered to solid tumors by a viral vector, could serve as an intrinsically generated photosensitizer, causing light-induced tumor destruction. Therefore, understanding the relationship between...

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“…Thus, the structure of the DsRed subunit (PDB ID: 1ZGO) was used as the starting structure for MD simulation of mRFP1; the structure file was retrieved from the Protein Data Bank site (http://www.pdb.org) [21,22]. This file contains X ray coordinates of the non hydrogen atoms of the DsRed tetramer [23].…”

Section: Methodsmentioning

confidence: 99%

Mutants of monomeric red fluorescent protein mRFP1 at residue 66: Structure modeling by molecular dynamics and search for correlations with spectral properties

Khrameeva

Друца

Vrzheshch

et al. 2008

Biochemistry Moscow

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Fluorescent proteins are widely used as markers for visualization of processes in intact biological systems. The family of fluorescent proteins includes proteins of various representatives of Coelenterata that are able to absorb light and emit in the visible spectral range. All flu orescent proteins have similar structure represented by 11 strand β barrel with α helix inside, which contains a chromophore in the middle. The chromophore is a het erogroup formed by an autocatalytic posttranslational reaction between three adjacent amino acid residues. The presence of the chromophore imparts color to the protein and induces its possible fluorescence [1].Fluorescence of proteins of this family is influenced by external conditions: pH [2,3], temperature [1], and ionic content of the medium [4,5], but structures of the chromophore and its nearest environment [1,6] are the key factors. The representatives of this family in matura tion rate, stability, spectral properties (absorption and flu orescence spectra, fluorescence quantum yield, photo bleaching, etc.) are described in [1,7]. Theoretical, experimental, and computational studies are carried out to investigate the mechanism of fluorescence of these proteins [8 11].Green fluorescent protein (GFP) was discovered in the early 1960s [12], but its active study began only after cloning of the GFP gene in 1992 [13] and demonstration of its heterologic expression in other organisms. In 1999, another family of colored proteins including the red pro tein DsRed was cloned from corals [14]. However, DsRed is a tetramer, and its use as a fluorescent marker is limit ed because of possible effects of its large molecular mass ISSN 0006 2979, Biochemistry (Moscow), 2008, Vol. 73, No. 10, pp. 1085 1095. © Pleiades Publishing, Ltd., 2008. Original Russian Text © E. E. Khrameeva, V. L. Drutsa, E. P. Vrzheshch, D. V. Dmitrienko, and P. V. Vrzheshch, 2008, published in Biokhimiya, 2008, Vol. 73, No. 10, pp. 1355 1367. Originally published in Biochemistry (Moscow) On Line Papers in Press, as Manuscript BM08 038, September 21, 2008 1085 Abbreviations: DsRed) red fluorescent protein drFP583 from coral Discosoma sp.; GFP) green fluorescent protein from Aequorea victoria; MD) molecular dynamics; mRFP1) monomeric red fluorescent protein, mutant of DsRed; Q66X) mutants of mRFP1 with replacement of residue 66 by residue X. * To whom correspondence should be addressed. Abstract-To study the interrelation between the spectral and structural properties of fluorescent proteins, structures of mutants of monomeric red fluorescent protein mRFP1 with all possible point mutations of Glu66 (except replacement by Pro) were simulated by molecular dynamics. A global search for correlations between geometrical structure parameters and some spectral characteristics (absorption maximum wavelength, integral extinction coefficient at the absorption maximum, excitation maximum wavelength, emission maximum wavelength, and quantum yield) was performed for the chromophore and its 6 A environment in mRFP1, Q66A, Q...

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Crystallographic Structures of Discosoma Red Fluorescent Protein with Immature and Mature Chromophores: Linking Peptide Bond Trans−Cis Isomerization and Acylimine Formation in Chromophore Maturation^,

Cited by 79 publications

References 33 publications

Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states

Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states

Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

Mutants of monomeric red fluorescent protein mRFP1 at residue 66: Structure modeling by molecular dynamics and search for correlations with spectral properties

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