Tic62: a protein family from metabolism to protein translocation

BACKGROUND: The function and structure of protein translocons at the outer and inner envelope membrane of chloroplasts (Toc and Tic complexes, respectively) are a subject of intensive research. One of the proteins that have been ascribed to the Tic complex is Tic62. This protein was proposed as a redox sensor protein and may possibly act as a regulator during the translocation process. Tic62 is a bimodular protein that comprises an N-terminal module, responsible for binding to pyridine nucleotides, and a C-terminal module which serves as a docking site for ferredoxin-NAD(P)-oxido-reductase (FNR). This work focuses on evolutionary analysis of the Tic62-NAD(P)-related protein family, derived from the comparison of all available sequences, and discusses the structure of Tic62. RESULTS: Whereas the N-terminal module of Tic62 is highly conserved among all oxyphototrophs, the C-terminal region (FNR-binding module) is only found in vascular plants. Phylogenetic analyses classify four Tic62-NAD(P)-related protein subfamilies in land plants, closely related to members from cyanobacteria and green sulphur bacteria. Although most of the Tic62-NAD(P)-related eukaryotic proteins are localized in the chloroplast, one subgroup consists of proteins without a predicted transit peptide. The N-terminal module of Tic62 contains the structurally conserved Rossman fold and probably belongs to the extended family of short-chain dehydrogenases-reductases. Key residues involved in NADP-binding and residues that may attach the protein to the inner envelope membrane of chloroplasts or to the Tic complex are proposed. CONCLUSION: The Tic62-NAD(P)-related proteins are of ancient origin since they are not only found in cyanobacteria but also in green sulphur bacteria. The FNR-binding module at the C-terminal region of the Tic62 proteins is probably a recent acquisition in vascular plants, with no sequence similarity to any other known motifs. The presence of the FNR-binding domain in vascular plants might be essential for the function of the protein as a Tic component and/or for its regulation

Análisis del proteoma del estroma del cloroplasto del Arabidopsis Thaliana en condiciones de estrés fotooxidativo

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Structural Stability of the PsbQ Protein of Higher Plant Photosystem II

We have characterized the stability and folding behavior of the isolated extrinsic PsbQ protein of photosystem II (PSII) from a higher plant, Spinacia oleracea, using intrinsic protein fluorescence emission and near- and far-UV circular dichroism (CD) spectroscopy in combination with differential scanning calorimetry (DSC). Experimental results reveal that both chemical denaturation using guanidine hydrochloride (GdnHCl) and thermal unfolding of PsbQ proceed as a two-state reversible process. The denaturation free-energy changes (GD) at 20 C extrapolated from GdnHCl (4.0 ± 0.6 kcal mol-1) or thermal unfolding (4.4 ± 0.8 kcal mol-1) are very close. Moreover, the far-UV CD spectra of the denatured PsbQ registered at 90 C in the absence and presence of 6.0 M GdnHCl superimpose, leading us to conclude that both denatured states of PsbQ are structurally and energetically similar. The thermal unfolding of PsbQ has been also characterized by CD and DSC over a wide pH range. The stability of PsbQ is at its maximum at pH comprised between 5 and 8, being wider than the optimal pH for oxygen evolution in the lumen of thylakoid membranes. In addition, no significant structural changes were detected in PsbQ between 50 and 55 C in the pH range of 3-8, suggesting that PsbQ behaves as a soluble and stable particle in the lumen when it detaches from PSII under physiological stress conditions such as high temperature (45-50 C) or low pH (<5.0). Sedimentation experiments showed that, in solution at 20 C, the PsbQ protein is a monomer with an elongated shape.Spanish Minitry of Science and Technology (PB1998-0480 and AGL2004-00045)This work was funded by the Spanish Ministry of Science and Technology (project references PB1998-0480 and AGL2003-0045). M.B. holds a fellowship from the Spanish Ministry of Science and Technology.Peer reviewe

Thioredoxin dependent changes in the redox states of FurA from Anabaena sp. PCC7120

FurA is a multifunctional regulator in cyanobacteria that contains five cysteines, four of them arranged into two CXXC motifs. Lack of a structural zinc ion enables FurA to develop disulfide reductase activity. In vivo, FurA displays several redox isoforms, and the oxidation state of its cysteines determines its activity as regulator and its ability to bind different metabolites. Because of the relationship between FurA and the control of genes involved in oxidative stress defense and photosynthetic metabolism, we sought to investigate the role of type m thioredoxin TrxA as a potential redox partner mediating dithiol-disulfide exchange reactions necessary to facilitate the interaction of FurA with its different ligands. Both in vitro cross-linking assays and in vivo two-hybrid studies confirmed the interaction between FurA and TrxA. Light to dark transitions resulted in reversible oxidation of a fraction of the regulator present in Anabaena sp. PCC7120. Reconstitution of an electron transport chain using E. coli NADPH-thioredoxin-reductase followed by alkylation of FurA reduced cysteines evidenced the ability of TrxA to reduce FurA. Furthermore, the use of site-directed mutants allowed us to propose a plausible mechanism for FurA reduction. These results point to TrxA as one of the redox partners that modulates FurA performance

High-resolution X-ray crystallographic structures expand the repertoire of flavoenzymes in bacteria.

Comunicación oral presentada en el XXX Simposio del Grupo Especializado de Cristalografía y Crecimiento Cristalino (GE3C), celebrado en Benidorm, del 17 al 19 de enero de 2023Peer reviewe

Evolution of the thioredoxin system as a step enabling adaptation to oxidative stres

20 páginas. -- The final version is available at http://www.elsevier.comThioredoxins (Trxs) are low-molecular-weight proteins that participate in the reduction of target enzymes. Trxs contain a redox-active disulfide bond, in the form of a WCGPC amino acid sequence motif, that enables them to perform dithiol-disulfide exchange reactions with oxidized protein substrates. Widely distributed across the three domains of life, Trxs form an evolutionarily conserved family of ancient origin. Thioredoxin reductases (TRs) are enzymes that reduce Trxs. According to their evolutionary history, TRs have diverged, thereby leading to the emergence of variants of the enzyme that in combination with different types of Trxs meet the needs of the cell. In addition to participating in the regulation of metabolism and defense against oxidative stress, Trxs respond to environmental signals—an ability that developed early in evolution. Redox regulation of proteins targeted by Trx is accomplished with a pair of redox-active cysteines located in strategic positions on the polypeptide chain to enable reversible oxidative changes that result in structural and functional modifications target proteins. In this review, we present a general overview of the thioredoxin system and describe recent structural studies on the diversity of its components.MB acknowledges financial support from the Spanish Ministerio de Ciencia, Innovación y Universidades [Project Reference BFU2016_80343-P]Peer reviewe

Redox extends its regulatory reach to chloroplast protein import

7 p.The import of chloroplast proteins synthesized in the cytosol of a plant cell is mediated by two multiprotein complexes or translocons located at the outer and inner membranes of the chloroplast envelope, respectively, TOC and TIC. These complexes integrate different signals to assure the timely transport of proteins into the chloroplast in accordance with the metabolic and developmental needs of the cell. The past few years have witnessed the emergence of redox as a regulator of the protein transport process. Here, we discuss evidence that the metabolic redox state of the chloroplast regulates the import of preproteins by altering either the activity or composition of participating transport components. It appears that, through these redox changes, chloroplasts communicate with other compartments of the plant cell.M.B. acknowledges support from the Spanish National Research Council (200940I204). Support from the Deutsche Forschungsgemeinschaft (SFB 594 and CIPSM) to J.S. is gratefully acknowledged. B.B.B. gratefully acknowledges receipt of a Research Award from the Alexander von Humboldt Foundation and ongoing support from the California Agricultural Experiment Station.Peer reviewe

Protein Import in Chloroplasts: An Emerging Regulatory Role for Redox.

55 p.Chloroplasts as well as mitochondria are surrounded by two envelope membranes that contain protein machineries to ensure well-organized communication and substrate distribution between the organelle and the rest of the cell. Protein import into organelles must be tightly coordinated with the internal protein synthesis machinery to ensure assembly of functional complexes of dual genetic origin. Import must also be coupled to the cellular metabolic state to guarantee requirements of the organelle in response to developmental changes and environmental conditions. Several recently published findings point to a role for redox in regulating chloroplast protein import: light and cysteine-modifying reagents affect import, certain subunits of the protein import machinery contain redox-sensing components, and others are potential thioredoxin targets. Herein we review the recent structural, computational, genetic, and biochemical studies that have begun to identify key characteristics and properties underlying protein import in chloroplasts. The predicted topology of some components is discussed, pointing out conserved cysteines in the protein families that may play a role in linking redox to oxidative stress and changes in the metabolic state of chloroplasts.B. B. B. gratefully acknowledges receipt of a Research Award from the Alexander von Humboldt Foundation. Work in the laboratory of J. S. was supported by Deutsche Forschungsgemeinschaft Grant SFB594.Peer reviewe

Structure and dynamics of the N-terminal loop of PsbQ from photosystem II of Spinacia oleracea

Infrared and Raman spectroscopy were applied to identify restraints for the structure determination of the 20 amino acid loop between two P-sheets of the N-terminal region of the PsbQ protein of the oxygen evolving complex of photosystem 11 from Spinacia oleracea by restraint-based homology modeling. One of the initial models has shown a stable fold of the loop in a 20 ns molecular dynamics simulation that is in accordance with spectroscopic data. Cleavage of the first 12 amino acids leads to a permanent drift in the root means square deviation of the protein backbone and induces major structural changes. (c) 2006 Elsevier Inc. All rights reserved.Supports from the Institutional Research Concept of the Academy of Science of the Czech Republic (No. AVOZ60870520) and from the Ministry of Education of the Czech Republic (No. LC 06010, No. MSM0021620835, and No. MSM6007665808) and the Grant Agency of the Czech Republic (Grant No. 206/03/D082) are gratefully acknowledged. This work was also funded by the Spanish Ministry of Education and Science (Project Ref.: BFU2004-04914-C02-02/BMC).Peer reviewe