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.

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.

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).

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.

Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in 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 gene to prevent its expression in (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.

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

Carbonic anhydrases (CAs) are a family of ubiquitous enzymes that catalyze the interconversion of CO and HCO. The "iota" class (ι-CA) was first found in the marine diatom (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).

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.

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.

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.

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.

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.

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).

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.

Unlabelled: 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.

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.

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.

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.

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.

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.

A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding.

Although detailed pictures of ribosome structures are emerging, little is known about the structural and cotranslational folding properties of nascent polypeptide chains at the atomic level. Here we used solution-state NMR spectroscopy to define a structural ensemble of a ribosome-nascent chain complex (RNC) formed during protein biosynthesis in Escherichia coli, in which a pair of immunoglobulin-like domains adopts a folded N-terminal domain (FLN5) and a disordered but compact C-terminal domain (FLN6). To study how FLN5 acquires its native structure cotranslationally, we progressively shortened the RNC constructs.

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways.

Archaeal MBF1 binds to 30S and 70S ribosomes via its helix-turn-helix domain.

MBF1 (multi-protein bridging factor 1) is a protein containing a conserved HTH (helix-turn-helix) domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initiation machinery of RNA polymerase II. In addition, MBF1 has been found to be associated with polyadenylated mRNA in yeast as well as in mammalian cells.

Protein folding on the ribosome studied using NMR spectroscopy.

NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.