The green cupredoxin CopI is a multicopper protein able to oxidize Cu(I).

Anthropogenic activities in agriculture and health use the antimicrobial properties of copper. This has led to copper accumulation in the environment and contributed to the emergence of copper resistant microorganisms. Understanding bacterial copper homeostasis diversity is therefore highly relevant since it could provide valuable targets for novel antimicrobial treatments. The periplasmic CopI protein is a monodomain cupredoxin comprising several copper binding sites and is directly involved in copper resistance in bacteria. However, its structure and mechanism of action are yet to be determined. To study the different binding sites for cupric and cuprous ions and to understand their possible interactions, we have used mutants of the putative copper binding modules of CopI and spectroscopic methods to characterize their properties. We show that CopI is able to bind a cuprous ion in its central histidine/methionine-rich region and oxidize it thanks to its cupredoxin center. The resulting cupric ion can bind to a third site at the N-terminus of the protein. Nuclear magnetic resonance spectroscopy revealed that the central histidine/methionine-rich region exhibits a dynamic behavior and interacts with the cupredoxin binding region. CopI is therefore likely to participate in copper resistance by detoxifying the cuprous ions from the periplasm.

Monitoring galactolipid digestion and simultaneous changes in lipid-bile salt micellar organization by real-time NMR spectroscopy.

The use of Nuclear Magnetic Resonance spectroscopy for studying lipid digestion in vitro most often consists of quantifying lipolysis products after they have been extracted from the reaction medium using organic solvents. However, the current sensitivity level of NMR spectrometers makes possible to avoid the extraction step and continuously quantify the lipids directly in the reaction medium. We used real-time 1H NMR spectroscopy and guinea pig pancreatic lipase-related protein 2 (GPLRP2) as biocatalyst to monitor in situ the lipolysis of monogalactosyl diacylglycerol (MGDG) in the form of mixed micelles with the bile salt sodium taurodeoxycholate (NaTDC). Residual substrate and lipolysis products (monogalactosyl monoacylglycerol (MGMG); monogalactosylglycerol (MGG) and octanoic acid (OA) were simultaneously quantified throughout the reaction thanks to specific proton resonances. Lipolysis was complete with the release of all MGDG fatty acids. These results were confirmed by thin layer chromatography (TLC) and densitometry after lipid extraction at different reaction times. Using diffusion-ordered NMR spectroscopy (DOSY), we could also estimate the diffusion coefficients of all the reaction compounds and deduce the hydrodynamic radius of the lipid aggregates in which they were present. It was shown that MGDG-NaTDC mixed micelles with an initial hydrodynamic radius rH of 7.3 ± 0.5 nm were changed into smaller micelles of NaTDC-MGDG-MGMG of 2.3 ± 0.5 nm in the course of the lipolysis reaction, and finally into NaTDC-OA mixed micelles (rH of 2.9 ± 0.5 nm) and water soluble MGG. These results provide a better understanding of the digestion of galactolipids by PLRP2, a process that leads to the complete micellar solubilisation of their fatty acids and renders their intestinal absorption possible.

Location of the photosynthetic carbon metabolism in microcompartments and separated phases in microalgal cells.

Carbon acquisition, assimilation and storage in eukaryotic microalgae and cyanobacteria occur in multiple compartments that have been characterised by the location of the enzymes involved in these functions. These compartments can be delimited by bilayer membranes, such as the chloroplast, the lumen, the peroxisome, the mitochondria or monolayer membranes, such as lipid droplets or plastoglobules. They can also originate from liquid-liquid phase separation such as the pyrenoid. Multiple exchanges exist between the intracellular microcompartments, and these are reviewed for the CO2 concentration mechanism, the Calvin-Benson-Bassham cycle, the lipid metabolism and the cellular energetic balance. Progress in microscopy and spectroscopic methods opens new perspectives to characterise the molecular consequences of the location of the proteins involved, including intrinsically disordered proteins.

In situ monitoring of galactolipid digestion by infrared spectroscopy in both model micelles and spinach chloroplasts.

Galactolipids are the main lipids from plant photosynthetic membranes and they can be digested by pancreatic lipase related protein 2 (PLRP2), an enzyme found in the pancreatic secretion in many animal species. Here, we used transmission Fourier-transform infrared spectroscopy (FTIR) to monitor continuously the hydrolysis of galactolipids by PLRP2, in situ and in real time. The method was first developed with a model substrate, a synthetic monogalactosyl diacylglycerol with 8-carbon acyl chains (C8-MGDG), in the form of mixed micelles with a bile salt, sodium taurodeoxycholate (NaTDC). The concentrations of the residual substrate and reaction products (monogalactosylmonoglyceride, MGMG; monogalactosylglycerol, MGG; octanoic acid) were estimated from the carbonyl and carboxylate vibration bands after calibration with reference standards. The results were confirmed by thin layer chromatography analysis (TLC) and specific staining of galactosylated compounds with thymol and sulfuric acid. The method was then applied to the lipolysis of more complex substrates, a natural extract of MGDG with long acyl chains, micellized with NaTDC, and intact chloroplasts isolated from spinach leaves. After a calibration performed with α-linolenic acid, the main fatty acid (FA) found in plant galactolipids, FTIR allowed quantitative measurement of chloroplast lipolysis by PLRP2. A full release of FA from membrane galactolipids was observed, that was not dependent on the presence of bile salts. Nevertheless, the evolution of amide vibration band in FTIR spectra suggested the interaction of membrane proteins with NaTDC and lipolysis products.

A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12.

The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO2 assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have.

Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants.

The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin-Benson-Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.

Structural Contour Map of the Iota Carbonic Anhydrase from the Diatom Thalassiosira pseudonana Using a Multiprong Approach.

Carbonic anhydrases (CAs) are a family of ubiquitous enzymes that catalyze the interconversion of CO2 and HCO3-. The "iota" class (ι-CA) was first found in the marine diatom Thalassiosira pseudonana (tpι-CA) and is widespread among photosynthetic microalgae and prokaryotes. The ι-CA has a domain COG4875 (or COG4337) that can be repeated from one to several times and resembles a calcium-calmodulin protein kinase II association domain (CaMKII-AD). The crystal structure of this domain in the ι-CA from a cyanobacterium and a chlorarachniophyte has been recently determined. However, the three-dimensional organization of the four domain-containing tpι-CA is unknown. Using biophysical techniques and 3-D modeling, we show that the homotetrameric tpι-CA in solution has a flat "drone-like" shape with a core formed by the association of the first two domains of each monomer, and four protruding arms formed by domains 3 and 4. We also observe that the short linker between domains 3 and 4 in each monomer confers high flexibility, allowing for different conformations to be adopted. We propose the possible 3-D structure of a truncated tpι-CA containing fewer domain repeats using experimental data and discuss the implications of this atypical shape on the activity and metal coordination of the ι-CA.

Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts.

In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment created in the light by electrons from the photosystems. In the dark, these enzymes are inhibited, mainly caused by oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent, measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges is simultaneous. In oxidized CP12, the C23-C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66-C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment: a cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66-C75 pair than for the C23-C31 pair. The redox mid-potentials for the two cysteine pairs differ and are similar to those found for glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase, consistent with the regulatory role of CP12.

A new type of flexible CP12 protein in the marine diatom Thalassiosira pseudonana.

BACKGROUND: CP12 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. METHODS: A 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. RESULTS: Here, 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. CONCLUSIONS: These 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. Video Abstract.

Regulation of Carbon Metabolism by Environmental Conditions: A Perspective From Diatoms and Other Chromalveolates.

Diatoms belong to a major, diverse and species-rich eukaryotic clade, the Heterokonta, within the polyphyletic chromalveolates. They evolved as a result of secondary endosymbiosis with one or more Plantae ancestors, but their precise evolutionary history is enigmatic. Nevertheless, this has conferred them with unique structural and biochemical properties that have allowed them to flourish in a wide range of different environments and cope with highly variable conditions. We review the effect of pH, light and dark, and CO2 concentration on the regulation of carbon uptake and assimilation. We discuss the regulation of the Calvin-Benson-Bassham cycle, glycolysis, lipid synthesis, and carbohydrate synthesis at the level of gene transcripts (transcriptomics), proteins (proteomics) and enzyme activity. In contrast to Viridiplantae where redox regulation of metabolic enzymes is important, it appears to be less common in diatoms, based on the current evidence, but regulation at the transcriptional level seems to be widespread. The role of post-translational modifications such as phosphorylation, glutathionylation, etc., and of protein-protein interactions, has been overlooked and should be investigated further. Diatoms and other chromalveolates are understudied compared to the Viridiplantae, especially given their ecological importance, but we believe that the ever-growing number of sequenced genomes combined with proteomics, metabolomics, enzyme measurements, and the application of novel techniques will provide a better understanding of how this important group of algae maintain their productivity under changing conditions.

Probing the dynamic stalk region of the ribosome using solution NMR.

We describe an NMR approach based on the measurement of residual dipolar couplings (RDCs) to probe the structural and motional properties of the dynamic regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the mobile C-terminal domain (CTD) of the stalk protein bL12. A structural refinement of this domain using the observed RDCs did not show large changes relative to the isolated protein in the absence of the ribosome, and we also found that alignment of the CTD was almost independent of the presence of the core ribosome particle, indicating that the inter-domain linker has significant flexibility. The nature of this linker was subsequently probed in more detail using a paramagnetic alignment strategy, which revealed partial propagation of alignment between neighbouring domains, providing direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome-nascent chain complexes.

Orchestration of algal metabolism by protein disorder.

Intrinsically disordered proteins (IDPs) are proteins that provide many functional advantages in a large number of metabolic and signalling pathways. Because of their high flexibility that endows them with pressure-, heat- and acid-resistance, IDPs are valuable metabolic regulators that help algae to cope with extreme conditions of pH, temperature, pressure and light. They have, however, been overlooked in these organisms. In this review, we present some well-known algal IDPs, including the conditionally disordered CP12, a protein involved in the regulation of CO2 assimilation, as probably the best known example, whose disorder content is strongly dependent on the redox conditions, and the essential pyrenoid component 1 that serves as a scaffold for ribulose-1, 5-bisphosphate carboxylase/oxygenase. We also describe how some enzymes are regulated by protein regions, called intrinsically disordered regions (IDRs), such as ribulose-1, 5-bisphosphate carboxylase/oxygenase activase, the A2B2 form of glyceraldehyde-3-phosphate dehydrogenase and the adenylate kinase. Several molecular chaperones, which are crucial for cell proteostasis, also display significant disorder propensities such as the algal heat shock proteins HSP33, HSP70 and HSP90. This review confirms the wide distribution of IDPs in algae but highlights that further studies are needed to uncover their full role in orchestrating algal metabolism.

Cyclophilin A allows the allosteric regulation of a structural motif in the disordered domain 2 of NS5A and thereby fine-tunes HCV RNA replication.

Implicated in numerous human diseases, intrinsically disordered proteins (IDPs) are dynamic ensembles of interconverting conformers that often contain many proline residues. Whether and how proline conformation regulates the functional aspects of IDPs remains an open question, however. Here, we studied the disordered domain 2 of nonstructural protein 5A (NS5A-D2) of hepatitis C virus (HCV). NS5A-D2 comprises a short structural motif (PW-turn) embedded in a proline-rich sequence, whose interaction with the human prolyl isomerase cyclophilin A (CypA) is essential for viral RNA replication. Using NMR, we show here that the PW-turn motif exists in a conformational equilibrium between folded and disordered states. We found that the fraction of conformers in the NS5A-D2 ensemble that adopt the structured motif is allosterically modulated both by the cis/trans isomerization of the surrounding prolines that are CypA substrates and by substitutions conferring resistance to cyclophilin inhibitor. Moreover, we noted that this fraction is directly correlated with HCV RNA replication efficiency. We conclude that CypA can fine-tune the dynamic ensemble of the disordered NS5A-D2, thereby regulating viral RNA replication efficiency.

Author Correction: Exploring intrinsically disordered proteins in Chlamydomonas reinhardtii.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Interaction between adenylate kinase 3 and glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii.

The critical and ubiquitous enzyme adenylate kinase (ADK) catalyzes the nucleotide phosphoryl exchange reaction: 2ADP ↔ ATP + AMP. The ADK3 in the chloroplasts of the green alga Chlamydomonas reinhardtii, bears an unusual C-terminal extension that is similar to the C-terminal end of the intrinsically disordered protein CP12. In this study, we report that this enzyme, when oxidized but not when reduced, is able to interact with the chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forming a stable complex as shown by native electrophoresis and mass spectrometry. In this bienzyme complex, the activity of ADK3 is unchanged while the NADPH-dependent activity of GAPDH is significantly inhibited. Moreover ADK3, like CP12, can protect GAPDH against thermal inactivation and aggregation. The ADK3-GAPDH bienzyme complex is unable to recruit phosphoribulokinase (PRK), in contrast with the ternary complex formed between GAPDH-CP12 and PRK. The interaction between ADK3 and GAPDH might be a mechanism to regulate the crucial ATP: NADPH ratio within chloroplasts to optimize the Calvin-Benson cycle during rapid fluctuation in environmental resources. ENZYMES: Adenylate kinase (EC 2.7.4.3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13), phosphoribulokinase (PRK, EC 2.7.1.19).

Exploring intrinsically disordered proteins in Chlamydomonas reinhardtii.

The content of intrinsically disordered protein (IDP) is related to organism complexity, evolution, and regulation. In the Plantae, despite their high complexity, experimental investigation of IDP content is lacking. We identified by mass spectrometry 682 heat-resistant proteins from the green alga, Chlamydomonas reinhardtii. Using a phosphoproteome database, we found that 331 of these proteins are targets of phosphorylation. We analyzed the flexibility propensity of the heat-resistant proteins and their specific features as well as those of predicted IDPs from the same organism. Their mean percentage of disorder was about 20%. Most of the IDPs (~70%) were addressed to other compartments than mitochondrion and chloroplast. Their amino acid composition was biased compared to other classic IDPs. Their molecular functions were diverse; the predominant ones were nucleic acid binding and unfolded protein binding and the less abundant one was catalytic activity. The most represented proteins were ribosomal proteins, proteins associated to flagella, chaperones and histones. We also found CP12, the only experimental IDP from C. reinhardtii that is referenced in disordered protein database. This is the first experimental investigation of IDPs in C. reinhardtii that also combines in silico analysis.

Cryptic Disorder Out of Disorder: Encounter between Conditionally Disordered CP12 and Glyceraldehyde-3-Phosphate Dehydrogenase.

Among intrinsically disordered proteins, conditionally disordered proteins undergo dramatic structural disorder rearrangements upon environmental changes and/or post-translational modifications that directly modulate their function. Quantifying the dynamics of these fluctuating proteins is extremely challenging but paramount to understanding the regulation of their function. The chloroplast protein CP12 is a model of such proteins and acts as a redox switch by formation/disruption of its two disulfide bridges. It regulates the Calvin cycle by forming, in oxidized conditions, a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and then phosphoribulokinase. In this complex, both enzymes are inactive. The highly dynamic nature of CP12 has so far hindered structural characterization explaining its mode of action. Thanks to a synergistic combination of small-angle X-ray scattering, nuclear magnetic resonance and circular dichroism that drove the molecular modeling of structural ensembles, we deciphered the structural behavior of Chlamydomonas reinhardtii oxidized CP12 alone and in the presence of GAPDH. Contrary to sequence-based structural predictions, the N-terminal region is unstable, oscillates at the ms timescale between helical and random conformations, and is connected through a disordered linker to its C-terminus, which forms a stable helical turn. Upon binding to GAPDH, oxidized CP12 undergoes an induced unfolding of its N-terminus. This phenomenon called cryptic disorder contributes to decrease the entropy cost and explains CP12 unusual high affinity for its partners.

NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B.

Non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase that catalyzes replication of the hepatitis C virus (HCV) RNA genome and therefore is central for its life cycle. NS5B interacts with the intrinsically disordered domain 2 of NS5A (NS5A-D2), another essential multifunctional HCV protein that is required for RNA replication. As a result, these two proteins represent important targets for anti-HCV chemotherapies. Despite this importance and the existence of NS5B crystal structures, our understanding of the conformational and dynamic behavior of NS5B in solution and its relationship with NS5A-D2 remains incomplete. To address these points, we report the first detailed NMR spectroscopic study of HCV NS5B lacking its membrane anchor (NS5BΔ21). Analysis of constructs with selective isotope labeling of the δ1 methyl groups of isoleucine side chains demonstrates that, in solution, NS5BΔ21 is highly dynamic but predominantly adopts a closed conformation. The addition of NS5A-D2 leads to spectral changes indicative of binding to both allosteric thumb sites I and II of NS5BΔ21 and induces long-range perturbations that affect the RNA-binding properties of the polymerase. We compared these modifications with the short- and long-range effects triggered in NS5BΔ21 upon binding of filibuvir, an allosteric inhibitor. We demonstrate that filibuvir-bound NS5BΔ21 is strongly impaired in the binding of both NS5A-D2 and RNA. NS5A-D2 induces conformational and functional perturbations in NS5B similar to those triggered by filibuvir. Thus, our work highlights NS5A-D2 as an allosteric regulator of the HCV polymerase and provides new insight into the dynamics of NS5B in solution.

A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy.

During biosynthesis on the ribosome, an elongating nascent polypeptide chain can begin to fold, in a process that is central to all living systems. Detailed structural studies of co-translational protein folding are now beginning to emerge; such studies were previously limited, at least in part, by the inherently dynamic nature of emerging nascent chains, which precluded most structural techniques. NMR spectroscopy is able to provide atomic-resolution information for ribosome-nascent chain complexes (RNCs), but it requires large quantities (≥10 mg) of homogeneous, isotopically labeled RNCs. Further challenges include limited sample working concentration and stability of the RNC sample (which contribute to weak NMR signals) and resonance broadening caused by attachment to the large (2.4-MDa) ribosomal complex. Here, we present a strategy to generate isotopically labeled RNCs in Escherichia coli that are suitable for NMR studies. Uniform translational arrest of the nascent chains is achieved using a stalling motif, and isotopically labeled RNCs are produced at high yield using high-cell-density E. coli growth conditions. Homogeneous RNCs are isolated by combining metal affinity chromatography (to isolate ribosome-bound species) with sucrose density centrifugation (to recover intact 70S monosomes). Sensitivity-optimized NMR spectroscopy is then applied to the RNCs, combined with a suite of parallel NMR and biochemical analyses to cross-validate their integrity, including RNC-optimized NMR diffusion measurements to report on ribosome attachment in situ. Comparative NMR studies of RNCs with the analogous isolated proteins permit a high-resolution description of the structure and dynamics of a nascent chain during its progressive biosynthesis on the ribosome.

Absence of residual structure in the intrinsically disordered regulatory protein CP12 in its reduced state.

The redox switch protein CP12 is a key player of the regulation of the Benson-Calvin cycle. Its oxidation state is controlled by the formation/dissociation of two intramolecular disulphide bridges during the day/night cycle. CP12 was known to be globally intrinsically disordered on a large scale in its reduced state, while being partly ordered in the oxidised state. By combining Nuclear Magnetic Resonance and Small Angle X-ray Scattering experiments, we showed that, contrary to secondary structure or disorder predictions, reduced CP12 is fully disordered, with no transient or local residual structure likely to be precursor of the structures identified in the oxidised active state and/or in the bound state with GAPDH or PRK. These results highlight the diversity of the mechanisms of regulation of conditionally disordered redox switches, and question the stability of oxidised CP12 scaffold.