# ocp  (GE Healthcare)

90

## Structured Review

GE Healthcare ocp

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
https://www.bioz.com/result/ocp/product/GE Healthcare
Average 90 stars, based on 2 article reviews
Price from $9.99 to$1999.99
ocp - by Bioz Stars, 2020-09
90/100 stars

## Images

### 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

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.

### Filtration:

Article Title: Local and global structural drivers for the photoactivation of the orange carotenoid protein
Article Snippet: .. OCP was buffer exchanged by gel filtration before XF-MS and HDX experiments using a Superdex 75 10/300 GL column (GE Healthcare) preequilibrated with 20 mM potassium phosphate, 100 mM NaCl (pH 7.4). .. Analytical SEC was performed on an AKTA Pure M2 chromatography system (GE Healthcare) with triple wavelength absorbance detection.

### 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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## Image Search Results

Journal: Scientific Reports

Article Title: Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein

doi: 10.1038/s41598-020-68463-8

Figure Lengend Snippet: Characteristic time courses of Trp-288 fluorescence intensity in OCP-3FH induced by 100 ms ( A ) and 5 ms ( B ) actinic flashes (blue LED) measured at 20 °C. Trp fluorescence was excited by 262 nm and measured at 340 ± 10 nm in a time-correlated single-photon counting regime. The fluorescence intensity measured before the flash ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${F}_{0}$$\end{document} F 0 ) was subtracted from each point in order to estimate the relative increase of fluorescence intensity as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(F-{F}_{0})/{F}_{0}$$\end{document} ( F - F 0 ) / F 0 . Each time course represents averaging of ~ 2000 photocycles. At least 2 × 10 5 photons were collected for each time step. The signal was recordered with time steps of 25 ms in ( A ) and 1.5 ms in ( B ). Sample concentration was equal to 2 µM. ( C ) A model of the OCP photocycle derived from Trp fluorescence experiments. Numbers indicate kinetic parameters that were used in order to fit experimental data in panels ( A ) and ( B ) (pink lines) by a sequential model. ( D ) Relative concentrations of intermediate states generated after 5 ms flash (experiment in panel B ).

Article Snippet: Analytical size-exclusion spectrochromatography (SESC)The samples containing individual OCP WT or OCP-3FH were loaded on a Superdex 200 Increase 5/150 column (GE Healthcare) equilibrated by a 20 mM Tris–HCl buffer, pH 7.6, containing 150 mM NaCl and operated at a 0.45 ml/min flow rate using a Varian 335/Varian 363 chromatography system (Varian Inc., Melbourne, Australia).

Techniques: Fluorescence, Concentration Assay, Derivative Assay, Generated

Journal: Scientific Reports

Article Title: Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein

doi: 10.1038/s41598-020-68463-8

Figure Lengend Snippet: ( A ) An experimental setup for femtosecond pump-probe fluorescence spectroscopy, see “ Materials and methods ” section for detailed explanation. ( B ) Transient increase of the Trp-288 fluorescence intensity caused by photoexcitation of OCP-3FH by a single 150-fs actinic flash. Each point is an average of three measurements. ( C ) Fluorescence decay of Trp species appearing after excitation of OCP-3FH with the pump pulse averaged for delay times between 100 and 250 ps.

Article Snippet: Analytical size-exclusion spectrochromatography (SESC)The samples containing individual OCP WT or OCP-3FH were loaded on a Superdex 200 Increase 5/150 column (GE Healthcare) equilibrated by a 20 mM Tris–HCl buffer, pH 7.6, containing 150 mM NaCl and operated at a 0.45 ml/min flow rate using a Varian 335/Varian 363 chromatography system (Varian Inc., Melbourne, Australia).

Techniques: Fluorescence, Spectroscopy

Journal: Scientific Reports

Article Title: Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein

doi: 10.1038/s41598-020-68463-8

Figure Lengend Snippet: ( A ) Position of Trp residues within the structure of OCP from Synechocystis sp. PCC 6803. Trp residues are labeled and shown by violet sticks, the carotenoid-coordinating Tyr-201 is shown by blue sticks, the NTE is shown by red color, CTT is shown by green, the NTD and CTD are shown by grey and beige color, respectively, the interdomain linker is shown in pink. ( B ) The UV–Vis absorption spectra of WT OCP (grey) and OCP-3FH (orange) recorded using a diode array detector in the course of the chromatography run and shown normalized at a position corresponding to the peak maximum (retention time 4.5 min). In order to estimate the yield of distinct spectral states (dotted and dashed lines), the absorption spectrum of the photoactivated protein was subtracted from the dark-adapted sample with an appropriate scaling factor, the numbers indicate corresponding yields. This procedure of spectral decomposition shows that ECN absorption in the compact red state and the final photoconverted state are identical. ( C ) Absorption difference spectrum—photoactivated (red) minus dark-adapted sample (orange). See Supplementary Figs. S1 and S2 for information about the temperature dependency of the OCP-3FH photocycle. Analytical SESC profiles of WT OCP ( D ) and OCP-3FH ( E ) variants followed by 280 nm and 460 nm absorbance, with the Mw estimates obtained from column calibration (see “ Materials and methods ” for details). In SESC experiments, protein concentration was about 50 µM.

Article Snippet: Analytical size-exclusion spectrochromatography (SESC)The samples containing individual OCP WT or OCP-3FH were loaded on a Superdex 200 Increase 5/150 column (GE Healthcare) equilibrated by a 20 mM Tris–HCl buffer, pH 7.6, containing 150 mM NaCl and operated at a 0.45 ml/min flow rate using a Varian 335/Varian 363 chromatography system (Varian Inc., Melbourne, Australia).

Techniques: Periodic Counter-current Chromatography, Labeling, Chromatography, Protein Concentration

Journal: Scientific Reports

Article Title: Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein

doi: 10.1038/s41598-020-68463-8

Figure Lengend Snippet: Accumulation of the red state of OCP-3FH as revealed by time-resolved Trp fluorescence spectroscopy. The color-coded image ( A ) represents a sequence of fluorescence decay kinetics of the single Trp in OCP-3FH recorded with an integration time of 5 s. Zero on the horizontal timescale represents the beginning of photoactivation by a 5 mW LED emitting at 445 nm. The vertical orange and red cross-sections indicate intervals (five neighboring decay curves) in which photons were selected to show the characteristic Trp-288 fluorescence decay of OCP-3FH in the dark and light-adapted states. ( B ) Shows these decays on a semilogarithmic scale (logarithm of photon number vs. linear timescale) and corresponds to the color-coded image. The horizontal cross-sections show how the number of photons changes upon photoactivation of the sample at different parts of fluorescence decay and the corresponding time-gated intensity time-courses ( C ): the blue line shows changes at the peak intensity [blue area in panel ( B ), average of 10 time-channels, total width ~ 488 ps] of the decay, while the green line shows changes in time channels which correspond to the slow component of the decay [green area in panel ( B ), average of 100 time-channels, total width ~ 4.8 ns]. Experiments were conducted at 0 °C. Sample concentration was equal to 2 µM. Each point represents an average of four independent experiments. ( D ) Steady-state fluorescence spectra of OCP-3FH in the dark- (orange line) and light-adapted (red line) states. See Supplementary Fig. S4 for additional data analysis and a comparison of Trp-288 fluorescence and ECN absorption changes.

Article Snippet: Analytical size-exclusion spectrochromatography (SESC)The samples containing individual OCP WT or OCP-3FH were loaded on a Superdex 200 Increase 5/150 column (GE Healthcare) equilibrated by a 20 mM Tris–HCl buffer, pH 7.6, containing 150 mM NaCl and operated at a 0.45 ml/min flow rate using a Varian 335/Varian 363 chromatography system (Varian Inc., Melbourne, Australia).

Techniques: Fluorescence, Spectroscopy, Sequencing, Concentration Assay

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

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

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

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

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

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

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

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:

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

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

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

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