Abstract
BackgroundCP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background.MethodsA combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12.ResultsHere, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions.ConclusionsThese findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle.Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta.While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism.5h3nzM1M4bjv4RCQxTYJACVideo
Highlights
Chloroplast protein of 12 kDa (CP12) is a small nuclear encoded protein of about 80 amino acid residues, originally described by Pohlmeyer et al [1] that occurs in many photosynthetic organisms [2, 3] and in Plantae and cyanobacteria is associated to the redox signaling pathway involved in the switch on/off of the light and dark metabolisms [4]
Green and red algae, Shao et al Cell Commun Signal (2021) 19:38 and cyanobacteria, it is associated with two enzymes, phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the Calvin Benson Bassham (CBB) cycle that is responsible for CO2 assimilation [5]
In silico analysis of CP12 sequences The sequence of this CP12 protein was aligned with other representative CP12 sequences from the angiosperms A. thaliana, Pisum sativum and Spinacia oleracea, the green alga C. reinhardtii, two red algae Cyanidioschyzon merolae and Galdieria sulphuraria, and the cyanobacterium S. elongatus (PCC 7942) (Fig. 2)
Summary
Chloroplast protein of 12 kDa (CP12) is a small nuclear encoded protein of about 80 amino acid residues, originally described by Pohlmeyer et al [1] that occurs in many photosynthetic organisms [2, 3] and in Plantae and cyanobacteria is associated to the redox signaling pathway involved in the switch on/off of the light and dark metabolisms [4]. Green and red algae, Shao et al Cell Commun Signal (2021) 19:38 and cyanobacteria, it is associated with two enzymes, phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the Calvin Benson Bassham (CBB) cycle that is responsible for CO2 assimilation [5]. This ternary complex has been well-studied and its structure has been recently solved using cryo-electron microscopy in the cyanobacterium Synechococcus elongatus [6] and using X-ray diffraction in the model higher plant Arabidopsis thaliana [7]. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background
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