Manual transfer of experimentally-verified manual GO annotation data to orthologs by curator judgment of sequence similarity
Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt
The small protein CP12: a protein linker for supramolecular complex assembly.
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Oxidized CP12 acts as a redox-sensitive linker for assembly of the PRK/GAPDH/CP12 supramolecular complex; reduced CP12 cannot reconstitute the complex.
"oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex"
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The complex has a stoichiometry of 2 PRK dimers, 2 GAPDH tetramers, and CP12. CP12 first binds GAPDH, then PRK is recruited.
"It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12"
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Oxidized CP12 is mainly alpha-helical and flexible; reduced CP12 is mainly unstructured, consistent with intrinsically disordered protein properties.
"Oxidized CP12 is mainly composed of alpha helix and coil segments, and is extremely flexible, while reduced CP12 is mainly unstructured"
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CP12 is a nuclear-encoded chloroplast protein with a transit peptide confirmed by N-terminal sequencing of the mature protein (residues 28-32).
"CP12 is an 8.5-kDa nuclear-encoded chloroplast protein"
Mass spectrometric analysis of the interactions between CP12, a chloroplast protein, and metal ions: a possible regulatory role within a PRK/GAPDH/CP12 complex.
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CP12 specifically binds Cu2+ (Kd = 26 uM) and Ni2+ (Kd = 11 uM) but not Fe2+ or Zn2+, as shown by ESI-MS.
"The oxidized protein bound specifically Cu2+ and Ni2+ (Kd of 26+/-1 microM and 11+/-1 microM, respectively); other cations such as Fe2+ and Zn2+ did not bind"
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Cu2+ catalyzes re-oxidation of reduced CP12, promoting formation of the disulfide bonds required for linker activity.
"Cu2+ catalyzes the re-formation of the disulfide bonds of the reduced CP12, leading to recovery of the fully oxidized CP12"
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CP12 shows sequence similarity to copper chaperones from Arabidopsis thaliana by hydrophobic cluster analysis, suggesting in vivo relevance of metal binding.
"the high similarity between CP12 and copper chaperones from Arabidopsis thaliana, as judged by hydrophobic cluster analysis, provides additional evidence for the relevance of metal binding for the in vivo situation"
Conformational modulation and hydrodynamic radii of CP12 protein and its complexes probed by fluorescence correlation spectroscopy.
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FCS confirms CP12 binding to GAPDH and PRK, with hydrodynamic radius increasing from 3.4 nm (free CP12) to 5.2 nm upon complex formation.
"We quantify a hydrodynamic radius of 3.4 ± 0.2 nm for the CP12 protein with an increase up to 5.2 ± 0.3 nm upon complex formation with GAPDH and PRK"
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N-terminal and C-terminal cysteine mutants show different structural behavior during unfolding, suggesting distinct roles for the two disulfide bonds in mediating GAPDH vs PRK binding.
"The different behavior of the CP12 mutant proteins during hydrophobic collapse transition is a direct clue to different structural orientations of the CP12 mutant proteins"
Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants.
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CRISPR-Cas9 knockout of CP12 in C. reinhardtii results in reduced PRK abundance and activity but normal GAPDH levels and near-normal growth.
"the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics"
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CP12 protects PRK from irreversible inactivation in a redox-independent manner, suggesting functions beyond Calvin cycle regulation.
"in the presence of CP12, the addition of DTT restored the initial PRK activity, indicating that CP12 prevented this irreversible inactivation"
The CP12 protein family: a thioredoxin-mediated metabolic switch?
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Review establishing CP12 as a conserved redox-sensitive protein found across all oxygenic phototrophs whose only clearly defined function is thioredoxin-mediated regulation of the Calvin cycle via GAPDH/PRK/CP12 complex assembly/disassembly.
"The only clearly defined function for CP12 in any organism is in the thioredoxin-mediated regulation of the Calvin-Benson cycle"
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CP12 may have broader regulatory roles beyond Calvin cycle regulation, with evidence suggesting involvement in stress responses and additional metabolic functions.
"CP12 may have a broader role in the regulation of metabolism, over and above the well established role of CP12 in the regulation of the Calvin-Benson cycle"
Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin-Benson cycle.
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Crystal structures of PRK from both Arabidopsis and Chlamydomonas reveal that the regulatory cysteines are connected by a flexible clamp loop unique to eukaryotic PRKs, completing the structural understanding of all redox-regulated Calvin cycle enzymes.
"the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes-the clamp loop-which is believed to be essential for oxidation-induced structural rearrangements"
The primary carbon metabolism in cyanobacteria and its regulation.
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Review positioning CP12 as a principal redox regulator of the Calvin cycle especially in cyanobacteria, noting new metabolomics evidence that CP12 variants affect acclimation to external glucose under diurnal conditions and CO2 fluctuations.
"New results of metabolomic and redox level analyses on strains with Cp12 variants extend the known role of Cp12 regulation towards the acclimation to external glucose supply under diurnal conditions as well as to fluctuations in CO2 levels in the light"
Conformational Disorder Analysis of the Conditionally Disordered Protein CP12 from Arabidopsis thaliana in Its Different Redox States.
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SAXS analysis of Arabidopsis CP12 confirms it is a conditionally disordered protein; reduced CP12 is highly disordered while oxidized CP12 gains partial structural order through disulfide bond formation but remains flexible.
"a small angle X-ray scattering (SAXS) analysis of recombinant Arabidopsis CP12 (AtCP12) in a reduced and oxidized form confirmed the highly disordered nature of this regulatory protein"
Phosphoribulokinase abundance is not limiting the Calvin-Benson-Bassham cycle in Chlamydomonas reinhardtii.
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PRK is present in moderate excess in C. reinhardtii; approximately 86% of wild-type PRK content is sufficient for full photoautotrophic growth, and overexpression does not increase growth.
"Immunoblot and growth assays revealed that a PRK content of ≈86% is sufficient to fully restore photoautotrophic growth"
Structural basis of light-induced redox regulation in the Calvin-Benson cycle in cyanobacteria.
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Cryo-EM structure of the cyanobacterial GAPDH-CP12-PRK ternary complex reveals that CP12's N-terminal disulfide-stabilized helical hairpin plugs the PRK active-site cleft, while the C-terminal region binds GAPDH and influences substrate accessibility of all GAPDH active sites.
"CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes"
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CP12 integrates responses from both the redox state (via disulfide bonds) and metabolite availability (NAD(H)/NADP(H) cofactor preference) to coordinate inhibition of GAPDH and PRK in the dark.
"Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation"