307 research outputs found
A thermostable enzyme as an experimental platform to study properties of less stable homologues.
The structural and functional characterization of proteins is frequently hampered by lack of stability or by insufficient assembly of oligomeric proteins in over-expression systems. Using
Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes
Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo. © 2012 The Author
Membrane proteins and proteomics: Love is possible, but so difficult
Despite decades of extensive research, the large-scale analysis of membrane
proteins remains a difficult task. This is due to the fact that membrane
proteins require a carefully balanced hydrophilic and lipophilic environment,
which optimum varies with different proteins, while most protein chemistry
methods work mainly, if not only, in water-based media. Taking this review
[Santoni, Molloy and Rabilloud, Membrane proteins and proteomics: un amour
impossible? Electrophoresis 2000, 21, 1054-1070] as a pivotal paper, the
current paper analyzes how the field of membrane proteomics exacerbated the
trend in proteomics, i.e. developing alternate methods to the historical
two-dimensional electrophoresis, and thus putting more and more pressure on the
mass spectrometry side. However, in the case of membrane proteins, the
incentive in doing so is due to the poor solubility of membrane proteins. This
review also shows that in some situations, where this solubility problem is
less acute, two-dimensional electrophoresis remains a method of choice. Last
but not least, this review also critically examines the alternate approaches
that have been used for the proteomic analysis of membrane proteins
Power and limitations of electrophoretic separations in proteomics strategies
Proteomics can be defined as the large-scale analysis of proteins. Due to the
complexity of biological systems, it is required to concatenate various
separation techniques prior to mass spectrometry. These techniques, dealing
with proteins or peptides, can rely on chromatography or electrophoresis. In
this review, the electrophoretic techniques are under scrutiny. Their
principles are recalled, and their applications for peptide and protein
separations are presented and critically discussed. In addition, the features
that are specific to gel electrophoresis and that interplay with mass
spectrometry (i.e., protein detection after electrophoresis, and the process
leading from a gel piece to a solution of peptides) are also discussed
The CCAAT-binding complex coordinates the oxidative stress response in eukaryotes
The heterotrimeric CCAAT-binding complex is evolutionary conserved in eukaryotic organisms. The corresponding Aspergillus nidulans CCAAT- binding factor (AnCF) consists of the subunits HapB, HapC and HapE. All of the three subunits are necessary for DNA binding. Here, we demonstrate that AnCF senses the redox status of the cell via oxidative modification of thiol groups within the histone fold motif of HapC. Mutational and in vitro interaction analyses revealed that two of these cysteine residues are indispensable for stable HapC/HapE subcomplex formation and high-affinity DNA binding of AnCF. Oxidized HapC is unable to participate in AnCF assembly and localizes in the cytoplasm, but can be recycled by the thioredoxin system in vitro and in vivo. Furthermore, deletion of the hapC gene led to an impaired oxidative stress response. Therefore, the central transcription factor AnCF is regulated at the post-transcriptional level by the redox status of the cell serving for a coordinated activation and deactivation of antioxidative defense mechanisms including the specific transcriptional activator NapA, production of enzymes such as catalase, thioredoxin or peroxiredoxin, and maintenance of a distinct glutathione homeostasis. The underlying fine-tuned mechanism very likely represents a general feature of the CCAAT-binding complexes in eukaryotes
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