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Individual Subunits (individual + subunit)
Selected AbstractsStructure of a Nudix protein from Pyrobaculum aerophilum reveals a dimer with two intersubunit ,-sheetsACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2002Shuishu Wang Nudix proteins, formerly called MutT homolog proteins, are a large family of proteins that play an important role in reducing the accumulation of potentially toxic compounds inside the cell. They hydrolyze a wide variety of substrates that are mainly composed of a nucleoside diphosphate linked to some other moiety X and thus are called Nudix hydrolases. Here, the crystal structure of a Nudix hydrolase from the hyperthermophilic archaeon Pyrobaculum aerophilum is reported. The structure was determined by the single-wavelength anomalous scattering method with data collected at the peak anomalous wavelength of an iridium-derivatized crystal. It reveals an extensive dimer interface, with each subunit contributing two strands to the ,-sheet of the other subunit. Individual subunits consist of a mixed highly twisted and curved ,-sheet of 11 ,-strands and two ,-helices, forming an ,,,,, sandwich. The conserved Nudix box signature motif, which contains the essential catalytic residues, is located at the first ,-helix and the ,-strand and loop preceding it. The unusually short connections between secondary-structural elements, together with the dimer form of the structure, are likely to contribute to the thermostability of the P. aerophilum Nudix protein. [source] Analysis of the thyrotropin-releasing hormone-degrading ectoenzyme by site-directed mutagenesis of cysteine residuesFEBS JOURNAL, Issue 9 2000Cys68 is involved in disulfide-linked dimerization Thyrotropin-releasing hormone-degrading ectoenzyme is a member of the M1 family of Zn-dependent aminopeptidases and catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH2). Cloning of the cDNA of this enzyme and biochemical studies revealed that the large extracellular domain of the enzyme with the catalytically active site contains nine cysteine residues that are highly conserved among species. To investigate the functional role of these cysteines in TRH-DE we used a site-directed mutagenesis approach and replaced individually each cysteine by a serine residue. The results revealed that the proteolytically truncated and enzymatically fully active enzyme consists of two identical subunits that are associated noncovalently by protein,protein interactions but not via interchain S-S bridges. The eight cysteines contained within this region are all important for the structure of the individual subunit and the enzymatic activity, which is dramatically reduced in all mutant enzymes. This is even true for the four cysteines that are clustered within the C-terminal domain remote from the Zn-binding consensus sequence HEICH. In contrast, Cys68, which resides within the stalk region seven residues from the end of the hydrophobic membrane-spanning domain, can be replaced by serine without a significant change in the enzymatic activity. Interestingly, this residue is involved in the formation of an interchain disulfide bridge. Covalent dimerization of the subunits, however, does not seem to be essential for efficient biosynthesis, enzymatic activity and trafficking to the cell surface. [source] Molecular dissection of photosystem I in higher plants: topology, structure and functionPHYSIOLOGIA PLANTARUM, Issue 3 2003Poul Erik Jensen Photosystem I catalyses the light driven electron transfer from plastocyanin/cytochrome c6 on the lumenal side of the thylakoid membrane to ferredoxin/flavodoxin at the stromal side. Photosystem I of higher plants consists of 18 different protein subunits. Fourteen of these make up the chlorophyll a -containing core, which also contains the cofactors involved in the electron transfer reactions, and four make up the peripheral chlorophyll a/b -containing antenna. Arabidopsis plants devoid of the nuclear-encoded photosystem I subunits have been obtained either by different suppression techniques or by insertional knock-out of the genes. This has allowed a detailed analysis of the role and function of the individual subunits. This review is focused on recent developments in the role of the individual subunit in the structure and function of photosystem I of higher plants. [source] Focused proteomics: Monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stainELECTROPHORESIS, Issue 15 2004James Murray Abstract We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue. [source] RelB/p50 regulates CCL19 production, but fails to promote human DC maturationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2009Chiara Gasparini Abstract DC, when fully matured are the APC best able to activate naïve T cells. Recently, we demonstrated using adenoviruses overexpressing I,B, and proteosome inhibitors that NF-,B is involved in DC activation, but the role of the individual subunits is still not clear. We investigated the role of the NF-,B subunits RelB and p50 in human DC activation using adenoviral vectors expressing RelB or p50. Nuclear RelB, in the form of RelB/p50, was active only in DC infected with both viruses, this induced the production of the soluble homeostatic chemokine CCL19, but not other homeostatic chemokines, particularly in LPS-matured DC. However, RelB/p50 did not affect the expression of costimulatory and antigen-presenting molecules, and increased the allogeneic mixed lymphocyte reaction only in LPS-matured DC. This enhanced mixed lymphocyte reaction is most likely due to enhanced CCL19 production, which sustains the interaction between mature DC and naïve T cells. In conclusion, we demonstrated that RelB/p50 was active only in DC expressing both RelB and p50, and induced CCL19 production, but not DC maturation. [source] The crystal structure of a plant 2C -methyl- D -erythritol 4-phosphate cytidylyltransferase exhibits a distinct quaternary structure compared to bacterial homologues and a possible role in feedback regulation for cytidine monophosphateFEBS JOURNAL, Issue 5 2006Mads Gabrielsen The homodimeric 2C -methyl- d -erythritol 4-phosphate cytidylyltransferase contributes to the nonmevalonate pathway of isoprenoid biosynthesis. The crystal structure of the catalytic domain of the recombinant enzyme derived from the plant Arabidopsis thaliana has been solved by molecular replacement and refined to 2.0 Å resolution. The structure contains cytidine monophosphate bound in the active site, a ligand that has been acquired from the bacterial expression system, and this observation suggests a mechanism for feedback regulation of enzyme activity. Comparisons with bacterial enzyme structures, in particular the enzyme from Escherichia coli, indicate that whilst individual subunits overlay well, the arrangement of subunits in each functional dimer is different. That distinct quaternary structures are available, in conjunction with the observation that the protein structure contains localized areas of disorder, suggests that conformational flexibility may contribute to the function of this enzyme. [source] Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutantsFEBS JOURNAL, Issue 18 2005Lucia Forzi Ech hydrogenase from Methanosarcina barkeri is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). The sequence of the enzyme predicts the binding of three [4Fe-4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 105 Pa of H2 at pH 7 giving rise to two distinct S½ electron paramagnetic resonance (EPR) signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH-dependent midpoint potential indicating a function in ion pumping. To assign these signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g = 1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH-dependence of these two [4Fe-4S] clusters suggests that they simultaneously mediate electron and proton transfer and thus could be an essential part of the proton-translocating machinery. [source] Molecular dissection of photosystem I in higher plants: topology, structure and functionPHYSIOLOGIA PLANTARUM, Issue 3 2003Poul Erik Jensen Photosystem I catalyses the light driven electron transfer from plastocyanin/cytochrome c6 on the lumenal side of the thylakoid membrane to ferredoxin/flavodoxin at the stromal side. Photosystem I of higher plants consists of 18 different protein subunits. Fourteen of these make up the chlorophyll a -containing core, which also contains the cofactors involved in the electron transfer reactions, and four make up the peripheral chlorophyll a/b -containing antenna. Arabidopsis plants devoid of the nuclear-encoded photosystem I subunits have been obtained either by different suppression techniques or by insertional knock-out of the genes. This has allowed a detailed analysis of the role and function of the individual subunits. This review is focused on recent developments in the role of the individual subunit in the structure and function of photosystem I of higher plants. [source] A recombinant multimeric immunoglobulin expressed in rice shows assembly-dependent subcellular localization in endosperm cellsPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2005Liz Nicholson Summary To investigate the role of subunit assembly in the intracellular deposition of multimeric recombinant proteins, we expressed a partially humanized secretory immunoglobulin in rice endosperm cells and determined the subcellular locations of the assembled protein and its individual components. Transgenic rice plants expressing either individual subunits or all the subunits of the antibody were generated by particle bombardment, and protein localization was determined by immunoelectron microscopy. Assembly of the antibody was confirmed by immunoassay and coimmunoprecipitation. Immunolocalization experiments showed no evidence for secretion of the antibody or any of its components to the apoplast. Rather, the nonassembled light chain, heavy chain and secretory component accumulated predominantly within endoplasmic reticulum-derived protein bodies, while the assembled antibody, with antigen-binding function, accumulated specifically in protein storage vacuoles. These results show that the destination of a complex recombinant protein within the plant cell is influenced by its state of assembly. [source] Modeling of possible subunit arrangements in the eukaryotic chaperonin TRiCPROTEIN SCIENCE, Issue 6 2006Erik J. Miller Abstract The eukaryotic cytosolic chaperonin TRiC (TCP-1 Ring Complex), also known as CCT (Cytosolic Chaperonin containing TCP-1), is a hetero-oligomeric complex consisting of two back-to-back rings of eight different subunits each. The general architecture of the complex has been determined, but the arrangement of the subunits within the complex remains an open question. By assuming that the subunits have a defined arrangement within each ring, we constructed a simple model of TRiC that analyzes the possible arrangements of individual subunits in the complex. By applying the model to existing data, we find that there are only four subunit arrangements consistent with previous observations. Our analysis provides a framework for the interpretation and design of experiments to elucidate the quaternary structure of TRiC/CCT. This in turn will aid in the understanding of substrate binding and allosteric properties of this chaperonin. [source] Structure of the heterotrimeric PCNA from Sulfolobus solfataricusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2006Gareth J. Williams PCNA is a ring-shaped protein that encircles DNA, providing a platform for the association of a wide variety of DNA-processing enzymes that utilize the PCNA sliding clamp to maintain proximity to their DNA substrates. PCNA is a homotrimer in eukaryotes, but a heterotrimer in crenarchaea such as Sulfolobus solfataricus. The three proteins are SsoPCNA1 (249 residues), SsoPCNA2 (245 residues) and SsoPCNA3 (259 residues). The heterotrimeric protein crystallizes in space group P21, with unit-cell parameters a = 44.8, b = 78.8, c = 125.6,Å, , = 100.5°. The crystal structure of this heterotrimeric PCNA molecule has been solved using molecular replacement. The resulting structure to 2.3,Å sheds light on the differential stabilities of the interactions observed between the three subunits and the specificity of individual subunits for partner proteins. [source] | |||||||||||||