Structural modeling of SoxF protein from Chlorobium tepidum: an approach to understand the molecular basis of thiosulfate oxidation

Microbial redox reactions of inorganic sulfur compounds play a vital role in balancing the turnover of this element in the environment. These vital reactions are carried out by the enzyme system encoded by the sox operon. The central player of the sulfur oxidation biochemistry is the SoxY–Z protein complex. Another protein called SoxF having sulfide dehydrogenase activity has the ability to reactivate the inactivated SoxY–Z protein complex. This SoxF protein is obtained from the sox operon of Chlorobium tepidium. In the present work an attempt has been made to understand the structural details of the activity of SoxF protein. A plausible biochemical mechanism has been predicted regarding the involvement of the SoxF protein in biological sulfur anion oxidation process. Since this is the first report regarding the structural biology of SoxF protein this study may shed light in the hitherto unknown molecular biochemistry of sulfur anion oxidation by sox operon.     

Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana

N-acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi enzyme involved in the biosynthesis of glycoprotein-bound N-linked oligosaccharides, catalysing an essential step in the conversion of oligomannose-type to complex N-glycans. GnTII activity has been detected in both animals and plants. However, while cDNAs encoding the enzyme have already been cloned from several mammalian sources no GnTII homologue has been cloned from plants so far. Here we report the molecular cloning of an Arabidopsis thalianaGnTII cDNA with striking homology to its animal counterparts. The predicted domain structure of A. thalianaGnTII indicates a type II transmembrane protein topology as it has been established for the mammalian variants of the enzyme. Upon expression of A. thalianaGnTII cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited GnTII activity.     

Changes in protein kinase and protein phosphatase properties during the cycle of asparaginase activity in Leptosphaeria michotti

Regulation of the cyclic activity of asparaginase (obtained as a purified protein complex) by a reversible auto-phosphorylation process has been previously reported in the fungus Leptosphaeria michotii (West) Sacc. In the present study, the protein complex was purified in the presence of either a mixture of 3 protein phosphatase inhibitors (fluoride, vanadate and molybdate) or EGTA, during the cycle of asparaginase activity, and the protein kinase and protein phosphatase activities characterized. (I) At the phase of increasing asparaginase activity, a Ca²⁺/calmodulin-dependent kinase activity was identified by (a) its inhibition by calmidazolium, reversed by calmodulin, and its inhibition by EGTA, but not by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole or polylysine (b) an increasing level of calmodulin bound to the complex, as estimated by enzyme-linked immunosorbent assay (ELISA). (2) At the phase of decreasing asparaginase activity, the Ca²⁺-calmodulin-dependent kinase activity disappeared and a little calmodulin remained associated with the complex: phosphorylation of the complex was increased several-fold by 1 nM okadaic acid and 25 nM inhibitor-2, and was not affected by EGTA, indicating a protein phosphatase-2A-like activity. (3) When asparaginase activity was low, a little calmodulin was bound to the complex. The kinase could phosphorylate casein and phosvitin. was inhibited by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole and heparin, stimulated by polylysine and not affected by calmidazolium or EGTA, just as a casein kinase 2. A Ca²⁺-dependent but calmodulin-independent protein phosphatase activity, not affected by okadaic acid and inhibitor-2. was then identified. We postulate the presence in the complex, of (a) only one protein kinase and one protein phosphatase, whose properties could change during the cycle of asparaginase activity: (b) two Ca²⁺/-binding proteins: first calmodulin, which could bind to Ca²⁺ and the casein kinase-2 form to give a Ca²⁺/calmodulin-dependent kinase, which could become Ca²⁺/calmodulin-independent following an auto-phosphorylation process: second a protein homologous to calmodulin, able to bind to the protein phosphatase-2A catalytic subunit to give a protein phosphatase-2B catalytic subunit.     

Problems in Obtaining Diffraction-quality Crystals of Hetero-oligomeric Integral Membrane Proteins

The major barrier responsible for the slow pace of structure determination of integral membrane proteins is the difficulty of crystallizing detergent-solubilized hydrophobic proteins, particularly hetero-oligomeric integral membrane proteins. For the latter class of multi-subunit proteins, we have encountered the following problems in addition to the ubiquitous problem of detergent compatibility: (i) instability caused by over-purification that results in delipidation; (ii) protease activity degrading exposed loops and termini of subunits of the complex that could not be inhibited; (iii) poor protein–protein contacts presumably arising from masking by the detergent micelle. Problem (i) could be ameliorated in crystallization of the cytochrome b₆f complex by augmenting the delipidated complex with synthetic lipid. Problem (ii) has not been solved. Problem (iii) has been solved in other systems by the use of monoclonal antibodies (or other protein ligands) to increase the probability of protein–protein contacts. In the case of the complex formed by the cobalamin and colicin receptor, BtuB, and the receptor binding domain of colicin E3, the latter served as a ligand for protein–protein contacts that facilitated crystallization.     

Minireview: The NADH: Ubiquinone Oxidoreductase (Complex I) of the Mammalian Respiratory Chain and the cAMP Cascade

Recent work has revealed cAMP-dependent phosphorylation of the 18-kDa IP subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene (chromosome 5). Phosphorylation of this protein has been shown to take place in fibroblast cultures in vivo, as well as in isolated mitochondria, which in addition to the cytosol also contain, in the inner-membrane matrix fraction, a cAMP-dependent protein kinase. Mitochondria appear to have a Ca²⁺-inhibited phosphatase, which dephosphorylates the 18-kDa phosphoprotein. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18-kDa protein is associated with potent stimulation of complex I and overall respiratory activity with NAD-linked substrates. Mutations in the human NDUFS4 gene have been found, which in the homozygous state are associated with deficiency of complex I and fatal neurological syndrome. In one case consisting of a 5 bp duplication, which destroyed the phosphorylation site, cAMP-dependent activation of complex I was abolished in the patient's fibroblast cultures. In another case consisting of a nonsense mutation, leading to termination of the protein after only 14 residues of the putative mitochondria targeting peptide, a defect in the assembly of complex I was found in fibroblast cultures.     

A Psb27 homologue in Arabidopsis thaliana is required for efficient repair of photodamaged photosystem II

Psb27 has been identified as a lumenal protein associated with photosystem II (PSII). To gain insight into the function of Psb27, we isolated a mutant Arabidopsis plant with a loss of psb27 function. The quantity of PSII complexes and electron transfer within PSII remained largely unaffected in the psb27 mutant. Our results also showed that under high-light-illumination, PSII activity and the content of the PSII reaction center protein D1 decreased more significantly in the psb27 mutant than in wild-type (WT) plant. Treatment of leaves with a chloroplast protein synthesis inhibitor resulted in similar light-induced PSII inactivation levels and D1 protein degradation rates in the WT and psb27 mutant plants. Recovery of PSII activity after photoinhibition was delayed in the psb27 mutant, suggesting that Psb27 is required for efficient recovery of the photodamaged PSII complex. Overall, these results demonstrated that Psb27 in Arabidopsis is not essential for oxygenic photosynthesis and PSII formation. Instead, our results provide evidence for the involvement of this lumenal protein in the recovery process of PSII. Hua Chen and Dongyuan Zhang contribute equally to this work.     

Cell-free synthesis of cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase,a multicomponent membrane protein

Cell-free protein synthesis is a useful technique that can site-specifically incorporate isotope-labeled amino acids into proteins. This incorporation is essential for infrared analyses of the electronic state of a specific amino acid residue used to elucidate protein function. Although 17 membrane proteins have been synthesized in their active state by cell-free systems, to date no hetero-subunit protein has been synthesized with this technique, suggesting that there are serious technical limitations. Here we report the cell-free synthesis of Paracoccus denitrificans cytochrome c oxidase, a membrane protein complex composed of three distinct subunits that contain two heme A molecules and two redox-active copper centers. The synthesized protein exhibited normal Soret/vis absorption spectra and ferrocytochrome c oxidation activity.     

Cofractionation of hela cell replication proteins with Ors-binding activity

Ors (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV-1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE-Sephadex and then on a Affi-Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band-shift elution experiments of the specific protein-DNA complex detect by silver-staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases α and δ, topoisomerase II, and replication protein A, (RP-A).     

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: Purification and characterization of the inhibitory complex

Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% β-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K_A) between yeast AZ and yeast ODC is 6 × 10⁷ (M⁻¹). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.     

GC (Guanine‐Cytosine)‐Selective DNA‐Binding and Antitumor Activity of a Quercetin?Manganese(II) Complex

The DNA binding and cleavage properties of quercetin? manganese(II) complexes have been studied, but little attention has been devoted to the relationship between the antitumor activity of these complexes and the DNA‐binding properties. Here, the DNA binding properties of the quercetin? manganese(II) complex [Mn(Que)₂(H₂O)₂] were studied using UV/VIS and fluorescence spectroscopy and viscosity measurements. The results indicate that the complex was preferentially bound to DNA in the GC (guanine? cytosine)‐rich regions via an intercalative mode. Furthermore, the cytotoxicity experiments confirmed its apoptosis‐inducing activity. We also demonstrated that the levels of survivin protein expression in HepG2 cells decreased and that the relative activity of caspase‐3 significantly increased after treatment with the complex. Hence, our results suggest that the antitumor activity of the [Mn(Que)₂(H₂O)₂] complex might be related to its intercalation into DNA and its DNA‐binding selectivity.     

GreenCut protein CPLD49 of Chlamydomonas reinhardtii associates with thylakoid membranes and is required for cytochrome b6f complex accumulation

The GreenCut encompasses a suite of nucleus‐encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non‐photosynthetic/heterotrophic organisms. In Chlamydomonas reinhardtii, CPLD49 (C onserved in P lant L ineage and D iatoms49 ) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that a cpld49 mutant has impaired photoautotrophic growth under high‐light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochrome b₆f complex (Cytb₆f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore, CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein, CPLD38; a mutant null for CPLD38 also impacts Cytb₆f complex accumulation. We investigated several potential functions of CPLD49, with some suggested by protein homology. Our findings are congruent with the hypothesis that CPLD38 and CPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of the Cytb₆f complex. Based on motifs of CPLD49 and the activities of other CPLD49‐like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule or cofactor that influences the assembly and activity of Cytb₆f.     

Crystal Structures of S120G Mutant and Wild Type of Human Nucleoside Diphosphate Kinase A in Complex with ADP

Nm23 was the first metastasis suppressor gene identified. This gene encodes a NDP kinase that also exhibits other properties like histidine protein kinase and interactions with proteins and DNA. The S120G mutant of NDPK-A has been identified in aggressive neuroblastomas and has been found to reduce the metastasis suppressor effect of Nm23. In order to understand the differences between the wild type and the S120G mutant, we have determined the structure of both mutant and wild type NDPK-A in complex with ADP. Our results reveal that there are no significant changes between the two enzyme versions even in the surroundings of the catalytic histidine that is required for NDP kinase activity. This suggests that the S120G mutation may affect an other protein property than NDP kinase activity.     

紫外交联合并串联亲和纯化高效鉴定蛋白复合物组分的RNA结合活性

RNA结合蛋白在RNA的生成与代谢中发挥着重要作用.我们在近年报道的PAR-CLIP(photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation)技术的基础上建立了一套快速、有效鉴定RNA结合蛋白的实验方法:以串联亲和纯化替代一步免疫沉淀获得高纯度蛋白-RNA复合物;将Sypro Ruby蛋白染色与RNA放射自显影相结合判断复合物中哪种或哪些组分为RNA结合蛋白,该方法命名为紫外交联合并的串联亲和纯化(cross-linkingand tandem affinity purification,CLiTAP).运用该方法对布氏锥虫的三种锌指蛋白ZC3H7、ZC3H34和ZC3H5进行分析,发现ZC3H7作为帽结合蛋白复合物的核心组分具有很强的RNA结合能力;ZC3H34结合RNA能力较弱,但其互作蛋白具有强的RNA结合活性;相比之下,ZC3H5及其复合物组分皆无RNA结合活性.这些结果表明,CLiTAP与蛋白质鉴定方法相结合,能够有效鉴定靶蛋白复合物中的RNA结合蛋白种类,也为进一步定位RNA结合位点、研究RNA结合蛋白的结构及作用机制奠定了基础.     

Assembly of Deletion Mutants of the Rieske Iron–Sulfur Protein into the Cytochrome bc 1 Complex of Yeast Mitochondria

The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc ₁ complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc ₁ complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212–16218]. The deletion mutants lacking amino acid residues 55–66 or residues 161–180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161–180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc ₁ complex; however, the protein lacking residues 55–66 was assembled in vitro into the bc ₁ complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55–66. This deletion mutant protein was also assembled into the bc ₁ complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55–66, is not required for assembly of the iron-sulfur protein into the bc ₁ complex; however, this association did not lead to enzymatic activity of the bc ₁ complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.     

Azocasein assay for alkaline protease in complex fermentation broth

Summary An azocasein assay has been developed for determination of alkaline protease in fermentation broth from a complex substrate containing ca. 50 g/l protein which to a high degree interferes with azocasein. Methods described in the literature have been found inaccurate as results deviate ca. 50% from true activity, so one of the methods is modified in order to eliminate interference. Proportionality between the relative azocasein concentration and the deviation from true activity is found. An azocasein concentration of 350 mg azocasein per ml sample gives satisfactory results with an accuracy of ±5%. Application of a standard addition method also improves the accuracy, but is laborious and less precise.Abbreviations a true enzyme activity - a _s activity determined from standard curve - AU Anson unit - c _A/T relative azocasein concentration (mg azocasein per mg total protein) - r ratio between measured and true enzyme activity - RD relative deviation from true value (%) - RSD relative standard deviation (%) - TCA trichloroacetic acid     

Cellulase production with Penicillium iriense (n. sp.)

Summary A new cellulase producing species of penicillium, named Penicillium iriense, has been isolated. Cultures of this fungus in liquid media containing cellulose as carbon source. excrete into the medium an enzyme complex able to degrade both soluble and insoluble forms of cellulose. This complex has been separated into five protein fractions. Three of them are endowed with CM-cellulase activity, one contains a cellobiase and one contains a C₁-like factor. These fractions show a moderate synergism in the attack of cotton fibres.     

Directed inactivation of the psbl gene does not affect Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

PsbI is a small, integral membrane protein component of photosystem II (PSII), a pigment-protein complex in cyanobacteria, algae and higher plants. To understand the function of this protein, we have isolated the psbI gene from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and determined its nucleotide sequence. Using an antibiotic-resistance cartridge to disrupt and replace the psbI gene, we have created mutants of Synechocystis 6803 that lack the PsbI protein. Analysis of these mutants revealed that absence of the PsbI protein results in a 25–30% loss of PSII activity. However, other PSII polypeptides are present in near wild-type amounts, indicating that no significant destabilization of the PSII complex has occurred. These results contrast with recently reported data indicating that PsbI-deficient mutants of the eukaryotic alga Chlamydomonas reinhardtii are highly light-sensitive and have a significantly lower (80–90%) titer of the PSII complex. In Synechocystis 6803, PsbI-deficient cells appear to be slightly more photosensitive than wild-type cells, suggesting that this protein, while not essential for PSII biogenesis or function, plays a role in the optimization of PSII activity.     

Identification of GroEL as a constituent of an mRNA-protection complex in Escherichia coli

An RNA-binding activity has been identified in Escherichia coli that provides physical protection of RNA against ribonucleases in an ATP- and Mg²⁺-dependent manner. This binding activity is stimulated under growth conditions known to cause a decrease in the rate of mRNA decay. RNA protection is mediated by a protein complex that contains a modified form of the chaperonin GroEL as an indispensable constituent. These results suggest a new role for GroEL as an RNA chaperone.