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《Process Biochemistry》2010,45(2):292-296
Engineering of DNA-binding domains of regulatory proteins aimed to control gene expression requires a deep knowledge of protein–DNA interactions acquired from structural data on purified species. Most DNA-binding proteins work as dimers establishing multiple protein–protein contacts mainly driven by hydrophobic interactions, being its cleansing a difficult task because of solubility problems. One-step purification of soluble, functional recombinant FurA from the cyanobacterium Anabaena sp. PCC 7120 has been achieved using mild chaotropic conditions. FurA was isolated using a Zn-iminodiacetate chromatography of the crude extract obtained after sonication of Escherichia coli in the presence of 2 M guanidium chloride. CD and 1D NMR spectroscopies demonstrate that FurA conserves the native tertiary structure. Functional analysis reveals FurA ability to recognise and bind target DNAs. We propose that the use of chaotropic agents under mild denaturating conditions might have general application in the purification of DNA-binding proteins and other proteins prone to aggregation. 相似文献
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María Carmen Pallarés Carlos Marcuello Laura Botello-Morte Andrés González María Francisca Fillat Anabel Lostao 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(3):623-631
Fur (ferric uptake regulator) proteins are involved in the control of a variety of processes in most prokaryotes. Although it is assumed that this regulator binds its DNA targets as a dimer, the way in which this interaction occurs remains unknown. We have focused on FurA from the cyanobacterium Anabaena sp. PCC 7120. To assess the molecular mechanism by which FurA specifically binds to “iron boxes” in PfurA, we examined the topology arrangement of FurA–DNA complexes by atomic force microscopy. Interestingly, FurA–PfurA complexes exhibit several populations, in which one is the predominant and depends clearly on the regulator/promoter ratio on the environment. Those results together with EMSA and other techniques suggest that FurA binds PfurA using a sequential mechanism: (i) a monomer specifically binds to an “iron box” and bends PfurA; (ii) two situations may occur, that a second FurA monomer covers the free “iron box" or that joins to the previously used forming a dimer which would maintain the DNA kinked; (iii) trimerization in which the DNA is unbent; and (iv) finally undergoes a tetramerization; the next coming molecules cover the DNA strands unspecifically. In summary, the bending appears when an “iron box” is bound to one or two molecules and decreases when both “iron boxes” are covered. These results suggest that DNA bending contributes at the first steps of FurA repression promoting the recruitment of new molecules resulting in a fine regulation in the Fur-dependent cluster associated genes. 相似文献
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Differential regulation of ssb genes in the nitrogen‐fixing cyanobacterium,Anabaena sp. strain PCC71201
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Anabaena sp. PCC7120 possesses three genes coding for single‐stranded DNA‐binding (SSB) protein, of which ssb1 was a single gene, and ssb2 and ssb3 are the first genes of their corresponding operons. Regulation of the truncated ssb genes, ssb1 (alr0088) and ssb2 (alr7559), was unaffected by N‐status of growth. They were negatively regulated by the SOS‐response regulatory protein LexA, as indicated by the (i) binding of Anabaena LexA to the LexA box of regulatory regions of ssb1 and ssb2, and (ii) decreased expression of the downstream gfp reporter gene in Escherichia coli upon co‐expression of LexA. However, the full‐length ssb gene, ssb3 (all4779), was regulated by the availability of Fe2+ and combined nitrogen, as indicated by (i) increase in the levels of SSB3 protein on Fe2+‐depletion and decrease under Fe2+‐excess conditions, and (ii) 1.5‐ to 1.6‐fold decrease in activity under nitrogen‐fixing conditions compared to nitrogen‐supplemented conditions. The requirement of Fe2+ as a co‐factor for repression by FurA and the increase in levels of FurA under nitrogen‐deficient conditions in Anabaena (Lopez‐Gomollon et al. 2007) indicated a possible regulation of ssb3 by FurA. This was substantiated by (i) the binding of FurA to the regulatory region of ssb3, (ii) repression of the expression of the downstream gfp reporter gene in E. coli upon co‐expression of FurA, and (iii) negative regulation of ssb3 promoter activity by the upstream AT‐rich region in Anabaena. This is the first report on possible role of FurA, an important protein for iron homeostasis, in DNA repair of cyanobacteria. 相似文献
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Jeong-a Kim Martha Nu?ez David?B. Briggs Bethany?L. Laskowski Jimmy?J. Chhun Joseph?K. Eleid Michael?J. Quon Tsu-Shuen Tsao 《Bioscience reports》2012,32(6):641-652
Adiponectin is an adipocyte-secreted hormone that exists as trimers, hexamers and larger species collectively referred to as HMW (high-molecular-weight) adiponectin. Whether hexamers or HMW adiponectin serve as precursors for trimers outside the circulation is currently unknown. Here, we demonstrate that adiponectin trimers can be generated from larger oligomers secreted from primary rat adipose cells or differentiated 3T3-L1 adipocytes. Purified hexameric, but not HMW, adiponectin converted into trimers in conditioned media separated from 3T3-L1 adipocytes or, more efficiently, when enclosed in the dialysis membrane in the presence of adipocytes. Several lines of evidence indicate that the conversion is mediated by an extracellular redox system. First, N-terminal epitope-tagged hexamers converted into trimers without proteolytic removal of the tag. Secondly, appearance of trimers was associated with conversion of disulfide-bonded dimers into monomers. Thirdly, thiol-reactive agents inhibited conversion into trimers. Consistent with a redox-based mechanism, purified hexamers reductively converted into trimers in defined glutathione redox buffer with reduction potential typically found in the extracellular environment while the HMW adiponectin remained stable. In addition, conversion of hexamers into trimers was enhanced by NADPH, but not by NADP+. Collectively, these data strongly suggest the presence of an extracellular redox system capable of converting adiponectin oligomers. 相似文献
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Yohta Kuwahara Satoru Unzai Yoko Hiroaki Hideshi Yokoyama Takahisa Ikegami Hidekazu Hiroaki 《Biophysical journal》2009,97(7):2034-2043
Stomatin, prohibitin, flotillin, and HflK/C (SPFH) domain proteins are membrane proteins that are widely conserved from bacteria to mammals. The molecular functions of these proteins have not been established. In mammals, the domain is often found in raft-associated proteins such as flotillin and podocin. We determined the structure of the SPFH domain of PH0470 derived from Pyrococcus horikoshii using NMR. The structure closely resembles that of the SPFH domain of the paralog PH1511, except for two C-terminal helices. The results show that the SPFH domain forms stable dimers, trimers, tetramers, and multimers, although it lacks the coiled-coil region for oligomerization, which is a highly conserved region in this protein family. The oligomers exhibited unusual thermodynamic behavior, as determined by circular dichroism, NMR, gel filtration, chemical cross-linking, and analytical ultracentrifugation. The oligomers were converted into monomers when they were heated once and then cooled. This transition was one-way and irreversible. We propose a mechanism of domain swapping for forming dimers as well as successive oligomers. The results of this study provide what to our knowledge are new insights into the common molecular function of the SPFH domain, which may act as a membrane skeleton through oligomerization by domain swapping. 相似文献
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We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni2+-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b6f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI. 相似文献
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Márcio H.L Arndt Marcelo M Santoro 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1998,119(4):667-675
The hemoglobin of Biomphalaria glabrata was purified to homogeneity by gel filtration column followed by anion exchange chromatography. The dissociation products were analyzed by a 5–15% gradient polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE) giving a band of 270 kDa and a band of 180 kDa after reduction with β-mercaptoethanol. The same profile was obtained in a 3.5% agarose gel electrophoresis containing SDS (SDS-AGE) but showed additional bands of higher molecular weight. These bands were proposed to be monomers, dimers and trimers, since they showed a good correlation in a plot of Rf versus log Mr. After partial reduction in a two-dimensional SDS-AGE, the proposed monomers and dimers produced two and four bands, respectively, likely indicating one to four chains crosslinked by disulfide bridges. Digestion with four different proteases yielded several equivalent fragments with molecular weights multiples of its minimum molecular weight (17.7 kDa). The circular dichroism spectrum of the protein showed a characteristic high α-helix content (70%). It was proposed that this hemoglobin is a pentamer with a molecular weight of aproximately 1.8×103 kDa, assembled by five 360-kDa subunits, each formed by two 180-kDa chains linked in pairs by disulfide bridges and each of these chains, in turn, comprised by ten heme binding domains linked in tandem. These data are compared to the published information for other planorbid extracellular hemoglobins. 相似文献
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The disulfide bonding pattern in ficolin multimers 总被引:3,自引:0,他引:3
Ficolin is a plasma lectin, consisting of a short N-terminal multimerization domain, a middle collagen domain, and a C-terminal fibrinogen-like domain. The collagen domains assemble the subunits into trimers, and the N-terminal domain assembles four trimers into 12-mers. Two cysteine residues in the N-terminal domain are thought to mediate multimerization by disulfide bonding. We have generated three mutants of ficolin alpha in which the N-terminal cysteines were substituted by serines (Cys4, Cys24, and Cys4/Cys24). The N-terminal cysteine mutants were produced in a mammalian cell expression system, purified by affinity chromatography, and analyzed under nondenaturing conditions to resolve the multimer structure of the native protein and under denaturing conditions to resolve the disulfide-linked structure. Glycerol gradient sedimentation and electron microscopy in nondenaturing conditions showed that plasma and recombinant wild-type protein formed 12-mers. The Cys4 mutant also formed 12-mers, but Cys24 and Cys4/Cys24 mutants formed only trimers. This means that protein interfaces containing Cys4 are stable as noncovalent protein-protein interactions and do not require disulfides, whereas those containing Cys24-Cys24 require the disulfides for stability. Proteins were also analyzed by nonreducing SDS-PAGE to show the covalent structure under denaturing conditions. Wild-type ficolin was covalently linked into 12-mers, whereas elimination of either Cys4 or Cys24 gave dimers and monomers. We present a model in which symmetric Cys24-Cys24 disulfide bonds between trimers are the basis for multimerization. The model may also be relevant to collectin multimers. 相似文献
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Cyanobacteria, contrary to higher plants, have a small psbA gene family encoding the reaction centre D1 protein subunit of photosystem II, the first macromolecular pigment-protein complex of the photosynthetic electron transport chain. Modulation of expression of multiple psbA genes in the family allows cyanobacteria to adapt to changing environmental conditions. To date, two different strategies for regulation of the psbA genes have emerged. One, characterized in Synechocystis PCC6803 and Gloeobacter violaceus PCC7421 involves the increased expression of one type of D1 protein to cope with the increased rate of damage. The other strategy, in Synechococcus PCC7942 and Anabaena PCC7120, is to replace the existing D1 with a new D1 form for the duration of the stress. However, most of the psbA gene families characterized to date contain also a divergent, apparently silent psbA gene of unknown function. This gene, present in Synechocystis, Anabaena and Thermosynechococcus elongatus BP-1 was not induced by any stress condition applied so far. Our data shows a reversible induction of the divergent psbA gene during the onset of argon-induced microaerobic conditions in Synechocystis, Anabaena and Thermosynechococcus elongatus. The unitary functional response of three unrelated cyanobacterial species, namely the induction of the expression of the divergent psbA gene as a reaction to the same environmental cue, indicates that these genes and the protein they encode are part of a specific cellular response to microaerobic conditions. There are no specific primary structure similarities between the different microaerobic inducible D1 forms, designated as D1′. Only three amino acid residues are consistently conserved in D1′. These modifications are: G80 to A, F158 to L and T286 to L. In silico mutation of the published D1 structure from Thermosynechococcus did not reveal major modifications. The point by point effects of the mutations on the local environment of the PSII structure are also discussed. 相似文献
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The DNA-binding protein from stationary phase (Dps) protein family plays an important role in protecting microorganisms from oxidative and nutritional stresses. In silico analysis of the promoter region of alr3808, a dpsA homologue from the cyanobacterium Nostoc sp. PCC7120 shows putative iron-boxes with high homology with those recognized by FurA (ferric uptake regulator). Evidence for the modulation of dpsA by FurA was obtained using in vitro and in vivo approaches. SELEX linked to PCR was used to identify PdpsA as a FurA target. Concurrently, EMSA assays showed high affinity of FurA for the dpsA promoter region. DpsA expression analysis in an insertional mutant of the alr1690-αfurA message (that exhibited an increased expression of FurA) showed a reduced synthesis of DpsA. These studies suggest that FurA plays a significant role in the regulation of the DpsA. 相似文献
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Hongyun Wang Yamin Ma Ying-Hsin Hsieh Hsiuchin Yang Minyong Li Binghe Wang Phang C. Tai 《Biochemical and biophysical research communications》2014
SecA is an essential ATPase in bacterial Sec-dependent protein translocation pathway, and equilibrates between monomers and dimers in solution. The question of whether SecA functions as monomers or dimers in membranes during the protein translocation is controversial. We previously constructed a tail-to-head SecAA tandem dimer, and showed it is fully functional by complementation in vivo and protein translocation in vitro, indicating that SecA can function at least as a dimer in the membrane without dissociating into monomers. In this study, we further constructed genetically a tail-to-head SecAAA trimer, which is functional in complementing a temperature-sensitive secA mutant. The purified SecAAA trimer per protomer is fully active as SecAA tandem dimers in ATPase activity, in protein translocation in vitro and in ion channel activities in the oocytes. With these functional tail-to-head trimer SecAAA and tandem SecAA, we examined their surface topology in the presence of liposomes using AFM. As expected, the soluble SecAAA without lipids are larger than SecAA. However, the ring/pore structures of SecAAA trimers were, surprisingly, almost identical to the SecA 2-monomers and SecAA dimers, raising the intriguing possibility that the SecA may exist and function as hexamer ring-structures in membranes. Cross-linking with formaldehyde showed that SecA, SecAA and SecAAA could form larger oligomers, including the hexamers. The molecular modeling simulation shows that both tail-to-head and tail-to-tail hexamers in the membranes are possible. 相似文献
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Ozbek S Pertz O Schwager M Lustig A Holstein T Engel J 《The Journal of biological chemistry》2002,277(51):49200-49204
The minicollagens found in the inner layer of the Hydra nematocyst walls are the smallest collagens known with 12-16 Gly-X-Y repeats. Minicollagen-1, the best characterized member of this protein family so far, consists of a central collagen triple helix of 12 nm in length flanked at both ends by a polyproline stretch and a conserved cysteine-rich domain. The cysteine-rich tails are proposed to function in the assembly of soluble minicollagen trimers to high molecular structures by a switch of the disulfide linkage from intramolecular to intermolecular bonds. In this study, we investigate the trimeric nature of minicollagen-1 and its capacity to form disulfide-linked polymers in vitro. A fusion protein of minicollagen-1 with maltose-binding protein is secreted as a soluble trimer with only intrachain and no interchain disulfide bridges as confirmed by melting the collagen triple helix under reducing and non-reducing conditions. The conversion of minicollagen-1 trimers to monomers takes place between 40 and 55 degrees C with the melting point being approximately 45 degrees C. Oxidative reshuffling of the minicollagen-1 trimers leads to the formation of high molecular aggregates, which upon reduction show distinct polytrimeric states. Minicollagen trimers in isolated nematocyst capsules proved to be sensitive to SDS and were engaged in polymeric structures with additional cross-links that were resistant to reducing agent. 相似文献