Laboratory of X-ray study

Department of Peptide and Protein Technologies

Head: Vladimir Pletnev, D.Sc
pletnev@ibch.ru+7(495)330-75-10

X-ray study, protein crystallography, three dimensional structure, structure-fuctional relations

The Laboratory is involved in study of three dimensional (3D) structure of the protein-peptide nature compounds by high resolution X-ray methods supported by molecular mechanics/dynamics/graphics and bioinformatics methods. The Laboratory studies proteins of different functional nature with emphasis on the structure-functional aspects of specificity of the ligand/substrate recognition and binding.

The Laboratory collaborates with the Macromolecular Crystallography Laboratory of the National Cancer Institute of USA (Argonne, IL, USA). The laboratory of X-ray study was organized in 1990 from the X-ray group functioning since 1972.

13501646_511786039012539_545356579995775

Laboratory employees.

In the present time, much attention in our lab is paid to structural study of the fluorescent proteins (FP) used as molecular bionanomarkers in cell biology, biotechnology and biomedicine for visualization and monitoring the internal processes within cells or whole organisms. A big series of FPs, emitting in green, yellow, red and far red spectral regions, has been studied in our Lab by high resolution X-ray methods. The following analysis of the structure-functional relation allowed to explain many experimentally observed properties and to design new mutant fluorescent variants with improved photophysical characteristics. The obtained results of the Laboratory are expanding considerably the structural base for rational design of the advanced fluorescent biomarkers for practical application.


The 3D structures of the cyclic depsipeptide ionophores differing by cycle size, nature and configuration of residues helped to understand the important details of the metal ion binding specificity and mechanism of the ion transport through biological membranes.

In collaboration with Hauptman-Woodward Medical Research Institute (Buffalo, USA) a number of research projects has been performed under the title “Rational proteomics of short chain dehydrogenases”. A general approach for identification of the 3D pattern of residues (fingerprints) responsible for the protein fold, cofactor and substrate binding was developed for this family.

A big series of FPs, emitting in green, yellow, red and far red spectral regions, has been studied in our lab by high resolution X-ray methods. The following analysis of the structure-functional relation allowed to explain many experimentally observed properties and to design new mutant fluorescent variants with improved photophysical characteristics.

Selected species of the marine button polyps — the source of fluorescent proteins (FP) (a); The principal structural fold of FPs is an 11-stranded β-barrel and a chromophore (matured from the three residue sequence) embedded in the middle of an internal α-helix going along the β-barrel axis (b).

Crystals of the fluorescent proteins for X-ray study

Unic examples of fluorescent proteins obtained in the laboratory

I. Study of the far red monomeric FP mKate has showed that the observed pH dependence of fluorescence is a consequence of cis-trans isomerization of the internal chromophore. Based on its 3D structure the new gene engineered variant mKate_S158A with almost 2 fold brighter fluorescence has been rationally designed. Currently, the photophysical characteristics of this variant succeed significantly over those of other known monomeric fluorescent biomarkers.

Structural study of the highly toxic red and orange FPs, KillerRed and KillerOrange, allowed to determine the chromophore adjacent key amino-acid residues participating in generation of the active oxygen forms, responsible for the phototoxic effect.

Amino acid environment of the internal chromophore in highly toxic red fluorescent protein KillerRed. 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”.

II. The intermediate form of chromophore biosynthesis has been observed in crystal structure of the colorless nonfluorescent FP, aceGFP-G222E. This new experimentally observed structure of the immature chromophore, characterized by the non-coplanar arrangement of the imidazolinone and phenolic rings, where cyclization of the protein backbone has occurred, but Tyr66 chromophore still stays in the native, non-oxidized form, with Cα and Cβ atoms in sp3 hybridization.

The trapped intermediate state (form III) for GFP chromophore biosynthesis found in the crystal structure of aceGFP-G222E

 III.

Red Fluorescent Protein laRFP from a lancelet. Chromophore Gly58-Tyr59-Gly60 (GYG; shown in yellow) in 2Fo-Fc electron density (density cutoff ρ= 2.0σ). laRFP showing the presence of the covalent bond between chromophore (Tyr59)Cβ and proximal (Tyr62)O. Potential proton acceptor Asp142 forms H-bond (shown in dashed red line), mediated by water molecule (W), with the Tyr62 hydroxyl. Arg89 forms H-bond with the Tyr62 hydroxyl that assumingly may facilitate proton transfer to Asp142 prior to reaction.

IV.

Канал простирающейся вдоль β-бочонка  заполнен цепочкой из связанных водородными связями молекул воды, выполняющей роль транспортной системы для фотогенерируемых токсичных супероксид анионов.

V. For the first time, the three-dimensional structure of the native green and UV irradiated photoconverted red forms of DendFP (Dendronephthya sp.) has been determined,  the latter showing cleavage of the main chain before chromophore

Chromophore structure (His62-Trp63-Gly64) in the green form of DendGFP (a) and the red form of DendRFP (b).

VI. In international collaboration three new bright far-red and near infrared genetically engineered biomarkers (from plant photoreceptors - phytochromes), providing high permeability of emission through biological tissues - miRFP670 (em ~ 670nm ), miRFP703 (703 nm) and miRFP709 (709 nm), have been studied by X-ray method with resolution 1.33, 1.35 and 1.34Å, respectively. Three-dimensional structure and structure-functional relations of these biomarkers have been established

Superimposed structures of miRFP703 (in green) and miRFP709 (in red) showing chromophores in the binding pocket

NamePositionContacts
Vladimir Pletnev, D.Scdepart. dir.pletnev@ibch.ru+7(495)330-75-10
Ekaterina Goryacheva, Ph.D.r. f.goryacheva@ibch.ru+7(499)793-51-22
Igor' Artem'evr. f.artem1@ibch.ru+7(495)335-16-77
Svetlana Arhipovar. f.arhipova@ibch.ru+7(495)335-75-10

Former members:

Igor' Tsygannik, Ph.D.s. r. f.

Selected publications

  1. Baloban M., Shcherbakova D.M., Pletnev S.V., Pletnev V.Z., Lagarias J.C., Verkhusha V.V. (2017). Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies. Chem. Sci. 8, 4546–4557 [+]
    ID:1833
  2. Vajravijayan S., Pletnev S., Pletnev V.Z., Nandhagopal N., Gunasekaran K. (2016). Structural analysis of β-prism lectin from Colocasia esculenta (L.) S chott. Int. J. Biol. Macromol. 91, 518–23 [+]
    ID:1829
  3. Pletneva N.V., Pletnev S., Pakhomov A.A., Chertkova R.V., Martynov V.I., Muslinkina L., Dauter Z., Pletnev V.Z. (2016). Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms. Acta Crystallogr D Struct Biol 72 (Pt 8), 922–32 [+]

    The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C(α)-N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C(α)=C(β) bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

    ID:1587
  4. Плетнёв В.З., Плетнева Н.В., Ефремов Р.Г., Горячева Е.А., Артемьев И.В., Архипова С.Ф., Саркисян К.С., Мишин А.С., Лукьянов К.А., Плетнев С.В. (2016). Пространственная структура рН-зависимого зеленого флуоресцентного белка WASCFP с депротонированным хромофором на основе триптофана. Биоорг. хим. 42 (6), 675–682 ID:1600
  5. Doerr N., Wang Y., Kipp K.R., Liu G., Benza J.J., Pletnev V., Pavlov T.S., Staruschenko A., Mohieldin A.M., Takahashi M., Nauli S.M., Weimbs T. (2016). Regulation of Polycystin-1 Function by Calmodulin Binding. PLoS ONE 11 (8), e0161525 [+]
    ID:1830
  6. Pletnev V.Z., Pletneva N.V., Efremov R.G., Goryacheva E.A., Artemyev I.V., Arkhipova S.F., Sarkisyan K.S., Mishin A.S., Lukyanov K.A., Pletnev S.V. (2016). Three-dimensional structure of pH-dependent fluorescent protein WasCFP with the tryptophan based deprotonated chromophore. Rus. J. Bioorg. Chem. 42 (6), 612–618 [+]
    ID:1832
  7. Pletnev V.Z., Pletneva N.V., Sarkisyan K.S., Mishin A.S., Lukyanov K.A., Goryacheva E.A., Ziganshin R.H., Dauter Z., Pletnev S. (2015). Structure of the green fluorescent protein NowGFP with an anionic tryptophan-based chromophore. Acta Crystallogr. D Biol. Crystallogr. 71 (Pt 8), 1699–707 [+]
    ID:1323
  8. Luker K.E., Pata P., Shemiakina I.I., Pereverzeva A., Stacer A.C., Shcherbo D.S., Pletnev V.Z., Skolnaja M., Lukyanov K.A., Luker G.D., Pata I., Chudakov D.M. (2015). Comparative study reveals better far-red fluorescent protein for whole body imaging. Sci Rep 5, 10332 [+]
    ID:1324
  9. Pletneva N.V., Pletnev V.Z., Sarkisyan K.S., Gorbachev D.A., Egorov E.S., Mishin A.S., Lukyanov K.A., Dauter Z., Pletnev S. (2015). Crystal Structure of Phototoxic Orange Fluorescent Proteins with a Tryptophan-Based Chromophore. PLoS ONE 10 (12), e0145740 [+]

    Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.81 and 1.57 Å resolution, respectively. They are the first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). Same as their red progenitors, both orange photosensitizers have a water-filled channel connecting the chromophore to the β-barrel exterior and enabling transport of ROS. In both proteins, Trp66 of the chromophore adopts an unusual trans-cis conformation stabilized by H-bond with the nearby Gln159. This trans-cis conformation along with the water channel was shown to be a key structural feature providing bright orange emission and phototoxicity of both examined orange photosensitizers.

    ID:1391
  10. Pletneva N.V., Pletnev S.V., Bogdanov A.M., Goriacheva E.A., Artemev I.V., Suslova E.A., Arkhipova S.F., Pletnev V.Z. (2014). Three dimensional structure of the dimeric gene-engineered variant of green fluorescent protein EGFP-K162Q in P6(1) crystal space group. Bioorg. Khim. 40 (4), 414–20 [+]
    ID:1326
  11. Pletnev V.Z., Pletneva N.V., Lukyanov K.A., Souslova E.A., Fradkov A.F., Chudakov D.M., Chepurnykh T., Yampolsky I.V., Wlodawer A., Dauter Z., Pletnev S. (2013). Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine. Acta Crystallogr. D Biol. Crystallogr. 69 (Pt 9), 1850–60 [+]
    ID:1017
  12. Pletneva N.V., Pletnev V.Z., Souslova E., Chudakov D.M., Lukyanov S., Martynov V.I., Arhipova S., Artemyev I., Wlodawer A., Dauter Z., Pletnev S. (2013). Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis. Acta Crystallogr. D Biol. Crystallogr. 69 (Pt 6), 1005–12 [+]

    The yellow fluorescent protein phiYFPv (λem(max) ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow-orange range (535-555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The `yellow' chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.

    ID:850
  13. Pletnev S., Pletneva N.V., Souslova E.A., Chudakov D.M., Lukyanov S., Wlodawer A., Dauter Z., Pletnev V. (2012). Structural basis for bathochromic shift of fluorescence in far-red fluorescent proteins eqFP650 and eqFP670. Acta Crystallogr. D Biol. Crystallogr. 68 (Pt 9), 1088–97 [+]

    The crystal structures of the far-red fluorescent proteins (FPs) eqFP650 (λ(ex)(max)/λ(em)(max) 592/650 nm) and eqFP670 (λ(ex)(max)/λ(em)(max) 605/670 nm), the successors of the far-red FP Katushka (λ(ex)(max)/λ(em)(max) 588/635 nm), have been determined at 1.8 and 1.6 Å resolution, respectively. An examination of the structures demonstrated that there are two groups of changes responsible for the bathochromic shift of excitation/emission bands of these proteins relative to their predecessor. The first group of changes resulted in an increase of hydrophilicity at the acylimine site of the chromophore due to the presence of one and three water molecules in eqFP650 and eqFP670, respectively. These water molecules provide connection of the chromophore with the protein scaffold via hydrogen bonds causing an ∼15 nm bathochromic shift of the eqFP650 and eqFP670 emission bands. The second group of changes observed in eqFP670 arises from substitution of both Ser143 and Ser158 by asparagines. Asn143 and Asn158 of eqFP670 are hydrogen bonded with each other, as well as with the protein scaffold and with the p-hydroxyphenyl group of the chromophore, resulting in an additional ∼20 nm bathochromic shift of the eqFP670 emission band as compared to eqFP650. The role of the observed structural changes was verified by mutagenesis.

    ID:734
  14. Pletneva N.V., Pletnev V.Z., Shemiakina I.I., Chudakov D.M., Artemyev I., Wlodawer A., Dauter Z., Pletnev S. (2011). Crystallographic study of red fluorescent protein eqFP578 and its far-red variant Katushka reveals opposite pH-induced isomerization of chromophore. Protein Sci. 20 (7), 1265–74 [+]

    The wild type red fluorescent protein eqFP578 (from sea anemone Entacmaea quadricolor, λ(ex) = 552 nm, λ(em) = 578 nm) and its bright far-red fluorescent variant Katushka (λ(ex) = 588 nm, λ(em) = 635 nm) are characterized by the pronounced pH dependence of their fluorescence. The crystal structures of eqFP578f (eqFP578 with two point mutations improving the protein folding) and Katushka have been determined at the resolution ranging from 1.15 to 1.85 Å at two pH values, corresponding to low and high level of fluorescence. The observed extinguishing of fluorescence upon reducing pH in eqFP578f and Katushka has been shown to be accompanied by the opposite trans-cis and cis-trans chromophore isomerization, respectively. Asn143, Ser158, His197 and Ser143, Leu174, and Arg197 have been shown to stabilize the respective trans and cis fluorescent states of the chromophores in eqFP578f and Katushka at higher pH. The cis state has been suggested as being primarily responsible for the observed far-red shift of the emission maximum of Katushka relative to that of eqFP578f.

    ID:528
  15. Pletneva N.V., Pletnev V.Z., Lukyanov K.A., Gurskaya N.G., Goryacheva E.A., Martynov V.I., Wlodawer A., Dauter Z., Pletnev S. (2010). Structural evidence for a dehydrated intermediate in green fluorescent protein chromophore biosynthesis. J. Biol. Chem. 285 (21), 15978–84 [+]

    The acGFPL is the first-identified member of a novel, colorless and non-fluorescent group of green fluorescent protein (GFP)-like proteins. Its mutant aceGFP, with Gly replacing the invariant catalytic Glu-222, demonstrates a relatively fast maturation rate and bright green fluorescence (lambda(ex) = 480 nm, lambda(em) = 505 nm). The reverse G222E single mutation in aceGFP results in the immature, colorless variant aceGFP-G222E, which undergoes irreversible photoconversion to a green fluorescent state under UV light exposure. Here we present a high resolution crystallographic study of aceGFP and aceGFP-G222E in the immature and UV-photoconverted states. A unique and striking feature of the colorless aceGFP-G222E structure is the chromophore in the trapped intermediate state, where cyclization of the protein backbone has occurred, but Tyr-66 still stays in the native, non-oxidized form, with C(alpha) and C(beta) atoms in the sp(3) hybridization. This experimentally observed immature aceGFP-G222E structure, characterized by the non-coplanar arrangement of the imidazolone and phenolic rings, has been attributed to one of the intermediate states in the GFP chromophore biosynthesis. The UV irradiation (lambda = 250-300 nm) of aceGFP-G222E drives the chromophore maturation further to a green fluorescent state, characterized by the conventional coplanar bicyclic structure with the oxidized double Tyr-66 C(alpha)=C(beta) bond and the conjugated system of pi-electrons. Structure-based site-directed mutagenesis has revealed a critical role of the proximal Tyr-220 in the observed effects. In particular, an alternative reaction pathway via Tyr-220 rather than conventional wild type Glu-222 has been proposed for aceGFP maturation.

    ID:404
  16. Pletnev S., Gurskaya N.G., Pletneva N.V., Lukyanov K.A., Chudakov D.M., Martynov V.I., Popov V.O., Kovalchuk M.V., Wlodawer A., Dauter Z., Pletnev V. (2009). Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed. J. Biol. Chem. 284 (46), 32028–39 [+]

    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.

    ID:299
  17. Pletnev S., Shcherbo D., Chudakov D.M., Pletneva N., Merzlyak E.M., Wlodawer A., Dauter Z., Pletnev V. (2008). A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore. J. Biol. Chem. 283 (43), 28980–7 [+]

    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.

    ID:33

Vladimir Pletnev

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 52, office. 155
  • Phone: +7(495)330-75-10
  • E-mail: pletnev@ibch.ru








































 

Three-dimensional structure and structure-functional relations of fluorescent proteins (2017-11-21)

Fig. legend: (A) Superimposed structures of miRFP703 (in green) and miRFP709 (in red) showing chromophores in the binding pocket. (B) Emission spectra of miRFP703 (in green), miRFP709 (in red), and miRFP709/Y210F (in magenta)

Three new bright far-red and near infrared genetically engineered biomarkers (from plant photoreceptors - phytochromes), providing high permeability of emission through biological tissues - miRFP670 (lem ~ 670nm ), miRFP703 (703 nm) and miRFP709 (709 nm), have been studied by X-ray method with resolution 1.33, 1.35 and 1.34Å, respectively. Three-dimensional structure and structure-functional relations of these biomarkers have been established

Publications

  1. Baloban M., Shcherbakova D.M., Pletnev S.V., Pletnev V.Z., Lagarias J.C., Verkhusha V.V. (2017). Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies. Chem. Sci. 8, 4546–4557 [+]
    ID:1833

A new phototoxic fluorescent biomarker with Trp-based chromophore (2016-03-03)

Three dimensional structure of two new phototoxic orange fluorescent proteins, dimeric KillerOrange and monomeric m KillerOrange, have been determined by X-ray method at resolution 1.81Å and 1.57 Å, respectively. They are first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). The β-barrel structure of both orange photosensitizers has an internal channel extending along the β-barrel axis. It is filled with a chain of hydrogen-bonded water molecules providing an outlet for the photo-generated reactive oxygen spices. Trp66 of the chromophore in KillerOrange/mKillerOrange adopts an unusual high energy trans-cis conformation stabilized by H-bond with the nearby Gln159. The observed trans-cis isomer of Trp66 presents first example among those found in known Trp-based chromophores. This conformation was suggested a key structural feature for generation of bright orange emission and phototoxicity. 

Publications

  1. Pletneva N.V., Pletnev V.Z., Sarkisyan K.S., Gorbachev D.A., Egorov E.S., Mishin A.S., Lukyanov K.A., Dauter Z., Pletnev S. (2015). Crystal Structure of Phototoxic Orange Fluorescent Proteins with a Tryptophan-Based Chromophore. PLoS ONE 10 (12), e0145740 [+]

    Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.81 and 1.57 Å resolution, respectively. They are the first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). Same as their red progenitors, both orange photosensitizers have a water-filled channel connecting the chromophore to the β-barrel exterior and enabling transport of ROS. In both proteins, Trp66 of the chromophore adopts an unusual trans-cis conformation stabilized by H-bond with the nearby Gln159. This trans-cis conformation along with the water channel was shown to be a key structural feature providing bright orange emission and phototoxicity of both examined orange photosensitizers.

    ID:1391