Structured Review

GE Healthcare ocp
Changes in Absorption Spectra of <t>OCP</t> and <t>RCP</t> Illuminated in the Presence of Methylene Blue. (A) and (B) OCP was illuminated for 20 min with 1000 μmol quanta m −2 s −1 white light (A) or orange-red light (filter cut on, 600 nm) (B) in the presence of 5 μM methylene blue (MB). (C) RCP was illuminated with 1000 μmol quanta m −2 s −1 white light in the presence of 5 μM methylene blue for 20 min.
Ocp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher [C]The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher [C] [W]"

Article Title: The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher [C]The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher [C] [W]

Journal: The Plant Cell

doi: 10.1105/tpc.114.123802

Changes in Absorption Spectra of OCP and RCP Illuminated in the Presence of Methylene Blue. (A) and (B) OCP was illuminated for 20 min with 1000 μmol quanta m −2 s −1 white light (A) or orange-red light (filter cut on, 600 nm) (B) in the presence of 5 μM methylene blue (MB). (C) RCP was illuminated with 1000 μmol quanta m −2 s −1 white light in the presence of 5 μM methylene blue for 20 min.
Figure Legend Snippet: Changes in Absorption Spectra of OCP and RCP Illuminated in the Presence of Methylene Blue. (A) and (B) OCP was illuminated for 20 min with 1000 μmol quanta m −2 s −1 white light (A) or orange-red light (filter cut on, 600 nm) (B) in the presence of 5 μM methylene blue (MB). (C) RCP was illuminated with 1000 μmol quanta m −2 s −1 white light in the presence of 5 μM methylene blue for 20 min.

Techniques Used:

Related Articles

Isolation:

Article Title: The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher [C]The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher [C] [W]
Article Snippet: .. In this study, OCP and RCP proteins were isolated using an Ni-ProBond resin and a Whatman DE-52 cellulose column, as described previously ( ). .. The FRP protein was isolated using the method described previously ( ).

Injection:

Article Title: Local and global structural drivers for the photoactivation of the orange carotenoid protein
Article Snippet: .. One hundred microliters of OCP (1.3 mg/mL) were injected onto a high resolution Sepharose 200 column (GE Healthcare) with a flow rate of 50 μL/min in 20 mM Na3 PO4 (pH 7.4), 150 mM NaCl, 0.02% NaN3 , 1 mM EDTA. .. The flow from the column passed through a UV detector cell, followed by 15 cm of clear tubing leading to the inlet of the quartz capillary cell.

Flow Cytometry:

Article Title: Local and global structural drivers for the photoactivation of the orange carotenoid protein
Article Snippet: .. One hundred microliters of OCP (1.3 mg/mL) were injected onto a high resolution Sepharose 200 column (GE Healthcare) with a flow rate of 50 μL/min in 20 mM Na3 PO4 (pH 7.4), 150 mM NaCl, 0.02% NaN3 , 1 mM EDTA. .. The flow from the column passed through a UV detector cell, followed by 15 cm of clear tubing leading to the inlet of the quartz capillary cell.

Purification:

Article Title:
Article Snippet: .. The OCP was further purified on a Whatman DE-52-cellulose column. .. Cell absorbance was monitored with an UVIKONXL spectrophotometer (SECOMAN, Alès).

Article Title: A photoactive carotenoid protein acting as light intensity sensor
Article Snippet: .. The OCP was further purified on a Whatman DE-52 cellulose column. .. To quantify the OCP present in the different strains, membrane-phycobilisome fractions were isolated ( ) and were analyzed by SDS page on 12%/2M urea in a Tris/Mes system ( ).

Article Title: Influence of zeaxanthin and echinenone binding on the activity of the orange carotenoid protein.
Article Snippet: .. In most cyanobacteria high irradiance induces a photoprotective mechanism that downregulates photosynthesis by increasing thermal dissipation of the energy absorbed by the phycobilisome, the water-soluble antenna. .. In most cyanobacteria high irradiance induces a photoprotective mechanism that downregulates photosynthesis by increasing thermal dissipation of the energy absorbed by the phycobilisome, the water-soluble antenna.

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    GE Healthcare ocp forms
    Analysis of the ∆NTE O interaction with oxFRPcc. a A fixed concentration of ∆NTE O was titrated by increasing amounts of oxFRPcc (indicated in µM per dimer); the samples (100 µl) were analyzed using a Superdex 200 Increase 10/300 column in the absence of reducing agents. Arrows indicate the direction of titration. b The binding curve obtained upon quantification of the amplitude of the ∆NTE O –oxFRPcc peak presented in a , in comparison with the curve for FRPwt (identical conditions). c Pairwise distance distribution functions for ∆NTE O , oxFRPcc dimer, and their complex obtained using GNOM. d One of the possible conformations of the ∆NTE O –oxFRPcc complex (1:2) consistent with the SAXS data and complementary information, shown as the CORAL-derived atomistic model overlaid with the best fitting GASBOR-derived ab initio bead model. Dashed circle in d marks the tentative <t>FRP</t> binding site located on the β-sheet of the <t>OCP-CTD,</t> normally occupied by NTE in OCP O . e The fit of the CORAL model to the SAXS data with the associated residuals (∆/σ). f Hypothetical 2:2 binding on top of the 1:2 complex suggested by crosslinking experiments. Although two tentative OCP-binding sites on the head domains of FRP may coexist, the 2:2 binding leads to a clash between OCP molecules (marked by a red dashed circle). In the dissociable FRPwt, such a binding may provoke FRP monomerization and formation of the 1:1 heterocomplexes to relieve tension caused by the clashing OCP molecules. In oxFRPcc, this is not possible because of the covalent interface stabilization by disulfides
    Ocp Forms, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GE Healthcare recombinant gfp ocp fusion protein
    Carotenoid transfer from COCP to <t>GFP-OCP</t> Apo chimera. ( A ) Given here are absorption spectra of GFP-OCP chimera and related species. Upon addition of 4.4 μ M of COCP (line 1 ) to a 6.3 μ M solution of GFP-OCP Apo ( 2 ), absorption of COCP gradually decreases. After equilibration of COCP-GFP-OCP Apo interactions, the resulting spectrum of the system ( 3 ) represents the sum of GFP, OCP O , and COCP absorption ( dashed line ). Obtained orange fraction is photoactive and, upon illumination of the sample by actinic light (450 nm, 200 MW), reversibly converts to the red state ( 4 ). Difference ( 5 ) between the spectra of the red and orange states is typical for all known OCP species. ( B ) Given here are the GFP fluorescence decay kinetics of GFP-OCP chimera in the absence (GFP-OCP Apo ) and in the presence of canthaxanthin (GFP-OCP CAN ). COCP to GFP-OCP Apo ratio was equal to three. ( Insets ) and schematic representation of GFP-OCP chimera. ( C ) Given here are the kinetics of carotenoid transfer monitored by measurements of O.D. at 550 nm and intensity of GFP fluorescence at 510 nm, simultaneously. Experiment was conducted at 20°C and with constant stirring. To see this figure in color, go online.
    Recombinant Gfp Ocp Fusion Protein, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of the ∆NTE O interaction with oxFRPcc. a A fixed concentration of ∆NTE O was titrated by increasing amounts of oxFRPcc (indicated in µM per dimer); the samples (100 µl) were analyzed using a Superdex 200 Increase 10/300 column in the absence of reducing agents. Arrows indicate the direction of titration. b The binding curve obtained upon quantification of the amplitude of the ∆NTE O –oxFRPcc peak presented in a , in comparison with the curve for FRPwt (identical conditions). c Pairwise distance distribution functions for ∆NTE O , oxFRPcc dimer, and their complex obtained using GNOM. d One of the possible conformations of the ∆NTE O –oxFRPcc complex (1:2) consistent with the SAXS data and complementary information, shown as the CORAL-derived atomistic model overlaid with the best fitting GASBOR-derived ab initio bead model. Dashed circle in d marks the tentative FRP binding site located on the β-sheet of the OCP-CTD, normally occupied by NTE in OCP O . e The fit of the CORAL model to the SAXS data with the associated residuals (∆/σ). f Hypothetical 2:2 binding on top of the 1:2 complex suggested by crosslinking experiments. Although two tentative OCP-binding sites on the head domains of FRP may coexist, the 2:2 binding leads to a clash between OCP molecules (marked by a red dashed circle). In the dissociable FRPwt, such a binding may provoke FRP monomerization and formation of the 1:1 heterocomplexes to relieve tension caused by the clashing OCP molecules. In oxFRPcc, this is not possible because of the covalent interface stabilization by disulfides

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Analysis of the ∆NTE O interaction with oxFRPcc. a A fixed concentration of ∆NTE O was titrated by increasing amounts of oxFRPcc (indicated in µM per dimer); the samples (100 µl) were analyzed using a Superdex 200 Increase 10/300 column in the absence of reducing agents. Arrows indicate the direction of titration. b The binding curve obtained upon quantification of the amplitude of the ∆NTE O –oxFRPcc peak presented in a , in comparison with the curve for FRPwt (identical conditions). c Pairwise distance distribution functions for ∆NTE O , oxFRPcc dimer, and their complex obtained using GNOM. d One of the possible conformations of the ∆NTE O –oxFRPcc complex (1:2) consistent with the SAXS data and complementary information, shown as the CORAL-derived atomistic model overlaid with the best fitting GASBOR-derived ab initio bead model. Dashed circle in d marks the tentative FRP binding site located on the β-sheet of the OCP-CTD, normally occupied by NTE in OCP O . e The fit of the CORAL model to the SAXS data with the associated residuals (∆/σ). f Hypothetical 2:2 binding on top of the 1:2 complex suggested by crosslinking experiments. Although two tentative OCP-binding sites on the head domains of FRP may coexist, the 2:2 binding leads to a clash between OCP molecules (marked by a red dashed circle). In the dissociable FRPwt, such a binding may provoke FRP monomerization and formation of the 1:1 heterocomplexes to relieve tension caused by the clashing OCP molecules. In oxFRPcc, this is not possible because of the covalent interface stabilization by disulfides

    Article Snippet: Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques: Concentration Assay, Titration, Binding Assay, Derivative Assay

    Validation of the proposed topology of the OCP–FRP complexes. a The SAXS-derived structural model of the 1:2 ∆NTE O . OCP is shown in light-violet with the carotenoid in orange. Note high conservation on the concave side of the FRP dimer and that (i) binding of the first head domain of FRP occurs on the OCP–CTD in place of the NTE (shown in yellow), (ii) presumable contact area includes F299 of OCP and K102 and F76 of FRP, whereas (iii) the second head domain of FRP is open for the interaction with another OCP molecule and (iv) the dimer interface of FRP is not directly involved in OCP binding. b Distribution of the regions with positive (+3 k T e −1 ; blue) and negative (−3 k T e −1 ; red) electrostatic potentials on surface of FRP and OCP suggesting extended multisite binding, in agreement with the scaffolding role of FRP. c Functional interaction of Cys mutants of OCP and FRP assessed by the ability of FRP variants to accelerate the R–O conversion of the photoactivated OCP–F299C at 25 °C. Insert shows the color of the OCP–F299C sample in the dark and under actinic light. d Schematic picture of the 1:2 complex with the positions chosen for Cys mutagenesis and disulfide trapping. The dashed circle indicates the tentative OCP–FRP interface. e The ability of Cys mutants to form disulfide crosslinked heterocomplexes upon mild oxidation by GSH/GSSG of the OCP–F299C mixtures with either FRP–K102C or FRP–F76C mutants. M w markers (M) are indicated in kDa. Ox and Red designate the absence or presence of βME in the sample buffer. Arrowhead marks the 46 kDa band corresponding to the OCP–FRP complex fixed by disulfide bond and disappearing upon reduction

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Validation of the proposed topology of the OCP–FRP complexes. a The SAXS-derived structural model of the 1:2 ∆NTE O . OCP is shown in light-violet with the carotenoid in orange. Note high conservation on the concave side of the FRP dimer and that (i) binding of the first head domain of FRP occurs on the OCP–CTD in place of the NTE (shown in yellow), (ii) presumable contact area includes F299 of OCP and K102 and F76 of FRP, whereas (iii) the second head domain of FRP is open for the interaction with another OCP molecule and (iv) the dimer interface of FRP is not directly involved in OCP binding. b Distribution of the regions with positive (+3 k T e −1 ; blue) and negative (−3 k T e −1 ; red) electrostatic potentials on surface of FRP and OCP suggesting extended multisite binding, in agreement with the scaffolding role of FRP. c Functional interaction of Cys mutants of OCP and FRP assessed by the ability of FRP variants to accelerate the R–O conversion of the photoactivated OCP–F299C at 25 °C. Insert shows the color of the OCP–F299C sample in the dark and under actinic light. d Schematic picture of the 1:2 complex with the positions chosen for Cys mutagenesis and disulfide trapping. The dashed circle indicates the tentative OCP–FRP interface. e The ability of Cys mutants to form disulfide crosslinked heterocomplexes upon mild oxidation by GSH/GSSG of the OCP–F299C mixtures with either FRP–K102C or FRP–F76C mutants. M w markers (M) are indicated in kDa. Ox and Red designate the absence or presence of βME in the sample buffer. Arrowhead marks the 46 kDa band corresponding to the OCP–FRP complex fixed by disulfide bond and disappearing upon reduction

    Article Snippet: Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques: Derivative Assay, Binding Assay, Scaffolding, Functional Assay, Mutagenesis

    Functional characterization of FRP variants with predefined oligomeric state. a Characteristic time-courses of OCP R -OCP O relaxation in the absence or presence of FRP species [a fixed ratio of ~1.7 FRP per OCP; monomeric FRP concentration (mFRP) was chosen] followed by changes of optical density (O.D.) at 550 nm after the actinic light is turned off. Maximal O.D. changes at 550 nm which could be obtained in the presence of FRP species under constant illumination by the actinic light ( b ) – normalized to such values in the absence of FRP species, and, thus, representing the maximal concentration of OCP R normalized to values between 0 and 1 for dimeric FRP variants to show at which FRP/OCP ratio half-saturation occurs (insert). c Corresponding R-O conversion rates in the presence of different concentrations of FRP species. All experiments were conducted at 10 °C to reduce the rate of OCP R -OCP O conversion, which is otherwise extremely high in the presence of FRPwt

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Functional characterization of FRP variants with predefined oligomeric state. a Characteristic time-courses of OCP R -OCP O relaxation in the absence or presence of FRP species [a fixed ratio of ~1.7 FRP per OCP; monomeric FRP concentration (mFRP) was chosen] followed by changes of optical density (O.D.) at 550 nm after the actinic light is turned off. Maximal O.D. changes at 550 nm which could be obtained in the presence of FRP species under constant illumination by the actinic light ( b ) – normalized to such values in the absence of FRP species, and, thus, representing the maximal concentration of OCP R normalized to values between 0 and 1 for dimeric FRP variants to show at which FRP/OCP ratio half-saturation occurs (insert). c Corresponding R-O conversion rates in the presence of different concentrations of FRP species. All experiments were conducted at 10 °C to reduce the rate of OCP R -OCP O conversion, which is otherwise extremely high in the presence of FRPwt

    Article Snippet: Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques: Functional Assay, Concentration Assay

    Stoichiometric analysis of the ∆NTE O –FRP interaction by GA crosslinking. a SDS-PAGE analysis of the results of GA crosslinking of ∆NTE O with either FRPwt, or oxFRPcc, or of individual proteins. Protein concentrations, the presence or absence of GA, M w markers and the contents of the crosslinked samples are shown. The OCP:FRP stoichiometries corresponding to the main bands observed on the gel are given on the left, and the corresponding apparent M w are shown on the right. b Schematic depiction of ∆NTE O (beige oval), the FRP dimer (tints of green) stabilized by disulfides (yellow bars), and their complexes crosslinked at different stoichiometries, relevant for c and d . Triangle, open circle, and star additionally mark the heterocomplexes with 1:1, 1:2, and 2:2 stoichiometries, respectively. c Kinetics of the crosslinking of the ∆NTE O mixture with oxFRPcc by 0.3% GA (final concentration) incubated at room temperature and analyzed by SEC on a Superdex 200 Increase 5/150 column upon loading 30 µl aliquots of the reaction mixture after different incubation times. The decrease of the 1:2 complex peak and a concomitant increase of the 2:2 complex peak are marked by arrows, the void volume is indicated ( V o ). d Chromatograms showing positions of the ∆NTE O –FRP complexes with different stoichiometries. SEC was followed by carotenoid-specific absorbance (500 nm). The Arthrospira

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Stoichiometric analysis of the ∆NTE O –FRP interaction by GA crosslinking. a SDS-PAGE analysis of the results of GA crosslinking of ∆NTE O with either FRPwt, or oxFRPcc, or of individual proteins. Protein concentrations, the presence or absence of GA, M w markers and the contents of the crosslinked samples are shown. The OCP:FRP stoichiometries corresponding to the main bands observed on the gel are given on the left, and the corresponding apparent M w are shown on the right. b Schematic depiction of ∆NTE O (beige oval), the FRP dimer (tints of green) stabilized by disulfides (yellow bars), and their complexes crosslinked at different stoichiometries, relevant for c and d . Triangle, open circle, and star additionally mark the heterocomplexes with 1:1, 1:2, and 2:2 stoichiometries, respectively. c Kinetics of the crosslinking of the ∆NTE O mixture with oxFRPcc by 0.3% GA (final concentration) incubated at room temperature and analyzed by SEC on a Superdex 200 Increase 5/150 column upon loading 30 µl aliquots of the reaction mixture after different incubation times. The decrease of the 1:2 complex peak and a concomitant increase of the 2:2 complex peak are marked by arrows, the void volume is indicated ( V o ). d Chromatograms showing positions of the ∆NTE O –FRP complexes with different stoichiometries. SEC was followed by carotenoid-specific absorbance (500 nm). The Arthrospira

    Article Snippet: Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques: SDS Page, Concentration Assay, Incubation, Size-exclusion Chromatography

    Physical interaction of the FRP mutants with various OCP forms studied by analytical SEC. Either redFRPcc ( a , b , c ) or FRP-L49E ( d , e , f ) were pre-incubated alone or in the presence of either OCP AA ( a , d ), ∆NTE O ( b , e ), or COCP ( c , f ) and then analyzed by SEC on a Superdex 200 Increase 10/300 column by following either protein-specific or carotenoid-specific absorbance (wavelengths are indicated). Distinct peaks of the complexes are marked by C. Load concentrations of FRP species, OCP AA , ∆NTE O , and COCP were equal to 50, 37, 6, and 8 µM, respectively

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Physical interaction of the FRP mutants with various OCP forms studied by analytical SEC. Either redFRPcc ( a , b , c ) or FRP-L49E ( d , e , f ) were pre-incubated alone or in the presence of either OCP AA ( a , d ), ∆NTE O ( b , e ), or COCP ( c , f ) and then analyzed by SEC on a Superdex 200 Increase 10/300 column by following either protein-specific or carotenoid-specific absorbance (wavelengths are indicated). Distinct peaks of the complexes are marked by C. Load concentrations of FRP species, OCP AA , ∆NTE O , and COCP were equal to 50, 37, 6, and 8 µM, respectively

    Article Snippet: Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques: Size-exclusion Chromatography, Incubation

    Absorption spectra of OCP ( A ) and OCP-TMR complexes ( B ) in the dark-adapted state (1) and after 2 minutes of actinic illumination (2) by a 150 mW blue LED. The difference (3) between absorption of the samples in photoactivated and dark-adapted states. The black line (4) shows absorption spectra of the TMR-labeled Apo-OCP, the dashed line (5) represents absorption of free TMR in ethanol. Absorption was measured at 2°C in order to reduce the rate of OCP R -OCP O conversion. ( C ) Chemical structure of tetramethylrhodamine-5-maleimide (TMR) with maleimide group, used for its attachment to OCP, looking upwards. ( D ). The protein is shown in cartoon representation, the ECN molecule is shown as blue spheres and Cys residues are shown as green sticks and are labeled corresponding to Synechocystis sp. PCC 6803 amino acid numbering. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

    doi: 10.1016/j.bpj.2016.11.3193

    Figure Lengend Snippet: Absorption spectra of OCP ( A ) and OCP-TMR complexes ( B ) in the dark-adapted state (1) and after 2 minutes of actinic illumination (2) by a 150 mW blue LED. The difference (3) between absorption of the samples in photoactivated and dark-adapted states. The black line (4) shows absorption spectra of the TMR-labeled Apo-OCP, the dashed line (5) represents absorption of free TMR in ethanol. Absorption was measured at 2°C in order to reduce the rate of OCP R -OCP O conversion. ( C ) Chemical structure of tetramethylrhodamine-5-maleimide (TMR) with maleimide group, used for its attachment to OCP, looking upwards. ( D ). The protein is shown in cartoon representation, the ECN molecule is shown as blue spheres and Cys residues are shown as green sticks and are labeled corresponding to Synechocystis sp. PCC 6803 amino acid numbering. To see this figure in color, go online.

    Article Snippet: First, a 5–10 molar excess of fresh dithiothreitol was added for 15 min at room temperature to ensure Cys reduction, and then the OCP preparation was again buffer-exchanged into buffer L using NAP-10 columns (GE Healthcare) to remove the excess of dithiothreitol.

    Techniques: Labeling, Periodic Counter-current Chromatography

    Analysis of the ∆NTE O interaction with oxFRPcc. a A fixed concentration of ∆NTE O was titrated by increasing amounts of oxFRPcc (indicated in µM per dimer); the samples (100 µl) were analyzed using a Superdex 200 Increase 10/300 column in the absence of reducing agents. Arrows indicate the direction of titration. b The binding curve obtained upon quantification of the amplitude of the ∆NTE O –oxFRPcc peak presented in a , in comparison with the curve for FRPwt (identical conditions). c Pairwise distance distribution functions for ∆NTE O , oxFRPcc dimer, and their complex obtained using GNOM. d One of the possible conformations of the ∆NTE O –oxFRPcc complex (1:2) consistent with the SAXS data and complementary information, shown as the CORAL-derived atomistic model overlaid with the best fitting GASBOR-derived ab initio bead model. Dashed circle in d marks the tentative FRP binding site located on the β-sheet of the OCP-CTD, normally occupied by NTE in OCP O . e The fit of the CORAL model to the SAXS data with the associated residuals (∆/σ). f Hypothetical 2:2 binding on top of the 1:2 complex suggested by crosslinking experiments. Although two tentative OCP-binding sites on the head domains of FRP may coexist, the 2:2 binding leads to a clash between OCP molecules (marked by a red dashed circle). In the dissociable FRPwt, such a binding may provoke FRP monomerization and formation of the 1:1 heterocomplexes to relieve tension caused by the clashing OCP molecules. In oxFRPcc, this is not possible because of the covalent interface stabilization by disulfides

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Analysis of the ∆NTE O interaction with oxFRPcc. a A fixed concentration of ∆NTE O was titrated by increasing amounts of oxFRPcc (indicated in µM per dimer); the samples (100 µl) were analyzed using a Superdex 200 Increase 10/300 column in the absence of reducing agents. Arrows indicate the direction of titration. b The binding curve obtained upon quantification of the amplitude of the ∆NTE O –oxFRPcc peak presented in a , in comparison with the curve for FRPwt (identical conditions). c Pairwise distance distribution functions for ∆NTE O , oxFRPcc dimer, and their complex obtained using GNOM. d One of the possible conformations of the ∆NTE O –oxFRPcc complex (1:2) consistent with the SAXS data and complementary information, shown as the CORAL-derived atomistic model overlaid with the best fitting GASBOR-derived ab initio bead model. Dashed circle in d marks the tentative FRP binding site located on the β-sheet of the OCP-CTD, normally occupied by NTE in OCP O . e The fit of the CORAL model to the SAXS data with the associated residuals (∆/σ). f Hypothetical 2:2 binding on top of the 1:2 complex suggested by crosslinking experiments. Although two tentative OCP-binding sites on the head domains of FRP may coexist, the 2:2 binding leads to a clash between OCP molecules (marked by a red dashed circle). In the dissociable FRPwt, such a binding may provoke FRP monomerization and formation of the 1:1 heterocomplexes to relieve tension caused by the clashing OCP molecules. In oxFRPcc, this is not possible because of the covalent interface stabilization by disulfides

    Article Snippet: Analytical SEC Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques: Concentration Assay, Titration, Binding Assay, Derivative Assay

    Proposed mechanism of the FRP scaffold terminating OCP-mediated photoprotection in cyanobacteria. Stages of the process are numbered from 1 to 6 and described in the text. The proteins are color-coded as in Fig. 5d , the carotenoid is shown as an orange dumbbell. OCP photoactivation is depicted by sun symbol. Yellow circle designates NTE. Individual FRP monomers are shown partially unfolded. Stoichiometry of the heterocomplexes formed between OCP and FRP are indicated. The yellow star designates the tentative clash between two bound OCP molecules destabilizing OCP–FRP complexes with 2:2 stoichiometry

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Proposed mechanism of the FRP scaffold terminating OCP-mediated photoprotection in cyanobacteria. Stages of the process are numbered from 1 to 6 and described in the text. The proteins are color-coded as in Fig. 5d , the carotenoid is shown as an orange dumbbell. OCP photoactivation is depicted by sun symbol. Yellow circle designates NTE. Individual FRP monomers are shown partially unfolded. Stoichiometry of the heterocomplexes formed between OCP and FRP are indicated. The yellow star designates the tentative clash between two bound OCP molecules destabilizing OCP–FRP complexes with 2:2 stoichiometry

    Article Snippet: Analytical SEC Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques:

    Validation of the proposed topology of the OCP–FRP complexes. a The SAXS-derived structural model of the 1:2 ∆NTE O –FRP complex with FRP residues colored by a gradient from conserved (purple) to variable (cyan) using Consurf 65 . OCP is shown in light-violet with the carotenoid in orange. Note high conservation on the concave side of the FRP dimer and that (i) binding of the first head domain of FRP occurs on the OCP–CTD in place of the NTE (shown in yellow), (ii) presumable contact area includes F299 of OCP and K102 and F76 of FRP, whereas (iii) the second head domain of FRP is open for the interaction with another OCP molecule and (iv) the dimer interface of FRP is not directly involved in OCP binding. b Distribution of the regions with positive (+3 k T e −1 ; blue) and negative (−3 k T e −1 ; red) electrostatic potentials on surface of FRP and OCP suggesting extended multisite binding, in agreement with the scaffolding role of FRP. c Functional interaction of Cys mutants of OCP and FRP assessed by the ability of FRP variants to accelerate the R–O conversion of the photoactivated OCP–F299C at 25 °C. Insert shows the color of the OCP–F299C sample in the dark and under actinic light. d Schematic picture of the 1:2 complex with the positions chosen for Cys mutagenesis and disulfide trapping. The dashed circle indicates the tentative OCP–FRP interface. e The ability of Cys mutants to form disulfide crosslinked heterocomplexes upon mild oxidation by GSH/GSSG of the OCP–F299C mixtures with either FRP–K102C or FRP–F76C mutants. M w markers (M) are indicated in kDa. Ox and Red designate the absence or presence of βME in the sample buffer. Arrowhead marks the 46 kDa band corresponding to the OCP–FRP complex fixed by disulfide bond and disappearing upon reduction

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Validation of the proposed topology of the OCP–FRP complexes. a The SAXS-derived structural model of the 1:2 ∆NTE O –FRP complex with FRP residues colored by a gradient from conserved (purple) to variable (cyan) using Consurf 65 . OCP is shown in light-violet with the carotenoid in orange. Note high conservation on the concave side of the FRP dimer and that (i) binding of the first head domain of FRP occurs on the OCP–CTD in place of the NTE (shown in yellow), (ii) presumable contact area includes F299 of OCP and K102 and F76 of FRP, whereas (iii) the second head domain of FRP is open for the interaction with another OCP molecule and (iv) the dimer interface of FRP is not directly involved in OCP binding. b Distribution of the regions with positive (+3 k T e −1 ; blue) and negative (−3 k T e −1 ; red) electrostatic potentials on surface of FRP and OCP suggesting extended multisite binding, in agreement with the scaffolding role of FRP. c Functional interaction of Cys mutants of OCP and FRP assessed by the ability of FRP variants to accelerate the R–O conversion of the photoactivated OCP–F299C at 25 °C. Insert shows the color of the OCP–F299C sample in the dark and under actinic light. d Schematic picture of the 1:2 complex with the positions chosen for Cys mutagenesis and disulfide trapping. The dashed circle indicates the tentative OCP–FRP interface. e The ability of Cys mutants to form disulfide crosslinked heterocomplexes upon mild oxidation by GSH/GSSG of the OCP–F299C mixtures with either FRP–K102C or FRP–F76C mutants. M w markers (M) are indicated in kDa. Ox and Red designate the absence or presence of βME in the sample buffer. Arrowhead marks the 46 kDa band corresponding to the OCP–FRP complex fixed by disulfide bond and disappearing upon reduction

    Article Snippet: Analytical SEC Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques: Derivative Assay, Binding Assay, Scaffolding, Functional Assay, Mutagenesis

    Functional characterization of FRP variants with predefined oligomeric state. a Characteristic time-courses of OCP R -OCP O relaxation in the absence or presence of FRP species [a fixed ratio of ~1.7 FRP per OCP; monomeric FRP concentration (mFRP) was chosen] followed by changes of optical density (O.D.) at 550 nm after the actinic light is turned off. Maximal O.D. changes at 550 nm which could be obtained in the presence of FRP species under constant illumination by the actinic light ( b ) – normalized to such values in the absence of FRP species, and, thus, representing the maximal concentration of OCP R normalized to values between 0 and 1 for dimeric FRP variants to show at which FRP/OCP ratio half-saturation occurs (insert). c Corresponding R-O conversion rates in the presence of different concentrations of FRP species. All experiments were conducted at 10 °C to reduce the rate of OCP R -OCP O conversion, which is otherwise extremely high in the presence of FRPwt

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Functional characterization of FRP variants with predefined oligomeric state. a Characteristic time-courses of OCP R -OCP O relaxation in the absence or presence of FRP species [a fixed ratio of ~1.7 FRP per OCP; monomeric FRP concentration (mFRP) was chosen] followed by changes of optical density (O.D.) at 550 nm after the actinic light is turned off. Maximal O.D. changes at 550 nm which could be obtained in the presence of FRP species under constant illumination by the actinic light ( b ) – normalized to such values in the absence of FRP species, and, thus, representing the maximal concentration of OCP R normalized to values between 0 and 1 for dimeric FRP variants to show at which FRP/OCP ratio half-saturation occurs (insert). c Corresponding R-O conversion rates in the presence of different concentrations of FRP species. All experiments were conducted at 10 °C to reduce the rate of OCP R -OCP O conversion, which is otherwise extremely high in the presence of FRPwt

    Article Snippet: Analytical SEC Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques: Functional Assay, Concentration Assay

    Stoichiometric analysis of the ∆NTE O –FRP interaction by GA crosslinking. a SDS-PAGE analysis of the results of GA crosslinking of ∆NTE O with either FRPwt, or oxFRPcc, or of individual proteins. Protein concentrations, the presence or absence of GA, M w markers and the contents of the crosslinked samples are shown. The OCP:FRP stoichiometries corresponding to the main bands observed on the gel are given on the left, and the corresponding apparent M w are shown on the right. b Schematic depiction of ∆NTE O (beige oval), the FRP dimer (tints of green) stabilized by disulfides (yellow bars), and their complexes crosslinked at different stoichiometries, relevant for c and d . Triangle, open circle, and star additionally mark the heterocomplexes with 1:1, 1:2, and 2:2 stoichiometries, respectively. c Kinetics of the crosslinking of the ∆NTE O mixture with oxFRPcc by 0.3% GA (final concentration) incubated at room temperature and analyzed by SEC on a Superdex 200 Increase 5/150 column upon loading 30 µl aliquots of the reaction mixture after different incubation times. The decrease of the 1:2 complex peak and a concomitant increase of the 2:2 complex peak are marked by arrows, the void volume is indicated ( V o ). d Chromatograms showing positions of the ∆NTE O –FRP complexes with different stoichiometries. SEC was followed by carotenoid-specific absorbance (500 nm). The Arthrospira homolog of FRP was taken because of its ability to form almost exclusively 1:1 complexes with OCP forms 25

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Stoichiometric analysis of the ∆NTE O –FRP interaction by GA crosslinking. a SDS-PAGE analysis of the results of GA crosslinking of ∆NTE O with either FRPwt, or oxFRPcc, or of individual proteins. Protein concentrations, the presence or absence of GA, M w markers and the contents of the crosslinked samples are shown. The OCP:FRP stoichiometries corresponding to the main bands observed on the gel are given on the left, and the corresponding apparent M w are shown on the right. b Schematic depiction of ∆NTE O (beige oval), the FRP dimer (tints of green) stabilized by disulfides (yellow bars), and their complexes crosslinked at different stoichiometries, relevant for c and d . Triangle, open circle, and star additionally mark the heterocomplexes with 1:1, 1:2, and 2:2 stoichiometries, respectively. c Kinetics of the crosslinking of the ∆NTE O mixture with oxFRPcc by 0.3% GA (final concentration) incubated at room temperature and analyzed by SEC on a Superdex 200 Increase 5/150 column upon loading 30 µl aliquots of the reaction mixture after different incubation times. The decrease of the 1:2 complex peak and a concomitant increase of the 2:2 complex peak are marked by arrows, the void volume is indicated ( V o ). d Chromatograms showing positions of the ∆NTE O –FRP complexes with different stoichiometries. SEC was followed by carotenoid-specific absorbance (500 nm). The Arthrospira homolog of FRP was taken because of its ability to form almost exclusively 1:1 complexes with OCP forms 25

    Article Snippet: Analytical SEC Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques: SDS Page, Concentration Assay, Incubation, Size-exclusion Chromatography

    Physical interaction of the FRP mutants with various OCP forms studied by analytical SEC. Either redFRPcc ( a , b , c ) or FRP-L49E ( d , e , f ) were pre-incubated alone or in the presence of either OCP AA ( a , d ), ∆NTE O ( b , e ), or COCP ( c , f ) and then analyzed by SEC on a Superdex 200 Increase 10/300 column by following either protein-specific or carotenoid-specific absorbance (wavelengths are indicated). Distinct peaks of the complexes are marked by C. Load concentrations of FRP species, OCP AA , ∆NTE O , and COCP were equal to 50, 37, 6, and 8 µM, respectively

    Journal: Nature Communications

    Article Title: OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

    doi: 10.1038/s41467-018-06195-0

    Figure Lengend Snippet: Physical interaction of the FRP mutants with various OCP forms studied by analytical SEC. Either redFRPcc ( a , b , c ) or FRP-L49E ( d , e , f ) were pre-incubated alone or in the presence of either OCP AA ( a , d ), ∆NTE O ( b , e ), or COCP ( c , f ) and then analyzed by SEC on a Superdex 200 Increase 10/300 column by following either protein-specific or carotenoid-specific absorbance (wavelengths are indicated). Distinct peaks of the complexes are marked by C. Load concentrations of FRP species, OCP AA , ∆NTE O , and COCP were equal to 50, 37, 6, and 8 µM, respectively

    Article Snippet: Analytical SEC Oligomeric state of FRP species and their interaction with various OCP forms were analyzed by SEC on either Superdex 200 Increase 10/300 or Superdex 200 Increase 5/150 columns (both GE Healthcare) operated using a ProStar 325 chromatographic system (Varian) with simultaneous UV/vis detection.

    Techniques: Size-exclusion Chromatography, Incubation

    Carotenoid transfer from COCP to GFP-OCP Apo chimera. ( A ) Given here are absorption spectra of GFP-OCP chimera and related species. Upon addition of 4.4 μ M of COCP (line 1 ) to a 6.3 μ M solution of GFP-OCP Apo ( 2 ), absorption of COCP gradually decreases. After equilibration of COCP-GFP-OCP Apo interactions, the resulting spectrum of the system ( 3 ) represents the sum of GFP, OCP O , and COCP absorption ( dashed line ). Obtained orange fraction is photoactive and, upon illumination of the sample by actinic light (450 nm, 200 MW), reversibly converts to the red state ( 4 ). Difference ( 5 ) between the spectra of the red and orange states is typical for all known OCP species. ( B ) Given here are the GFP fluorescence decay kinetics of GFP-OCP chimera in the absence (GFP-OCP Apo ) and in the presence of canthaxanthin (GFP-OCP CAN ). COCP to GFP-OCP Apo ratio was equal to three. ( Insets ) and schematic representation of GFP-OCP chimera. ( C ) Given here are the kinetics of carotenoid transfer monitored by measurements of O.D. at 550 nm and intensity of GFP fluorescence at 510 nm, simultaneously. Experiment was conducted at 20°C and with constant stirring. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: The Unique Protein-to-Protein Carotenoid Transfer Mechanism

    doi: 10.1016/j.bpj.2017.06.002

    Figure Lengend Snippet: Carotenoid transfer from COCP to GFP-OCP Apo chimera. ( A ) Given here are absorption spectra of GFP-OCP chimera and related species. Upon addition of 4.4 μ M of COCP (line 1 ) to a 6.3 μ M solution of GFP-OCP Apo ( 2 ), absorption of COCP gradually decreases. After equilibration of COCP-GFP-OCP Apo interactions, the resulting spectrum of the system ( 3 ) represents the sum of GFP, OCP O , and COCP absorption ( dashed line ). Obtained orange fraction is photoactive and, upon illumination of the sample by actinic light (450 nm, 200 MW), reversibly converts to the red state ( 4 ). Difference ( 5 ) between the spectra of the red and orange states is typical for all known OCP species. ( B ) Given here are the GFP fluorescence decay kinetics of GFP-OCP chimera in the absence (GFP-OCP Apo ) and in the presence of canthaxanthin (GFP-OCP CAN ). COCP to GFP-OCP Apo ratio was equal to three. ( Insets ) and schematic representation of GFP-OCP chimera. ( C ) Given here are the kinetics of carotenoid transfer monitored by measurements of O.D. at 550 nm and intensity of GFP fluorescence at 510 nm, simultaneously. Experiment was conducted at 20°C and with constant stirring. To see this figure in color, go online.

    Article Snippet: Recombinant GFP-OCP fusion protein was purified on a HisTrap HP column (GE Healthcare Life Sciences, Little Chalfont, UK), dialyzed and stored at +4°C in the presence of sodium azide.

    Techniques: Fluorescence