Journal articles on the topic 'Dimers Purification'
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Tellinghuisen, Timothy L., Rushika Perera, and Richard J. Kuhn. "In Vitro Assembly of Sindbis Virus Core-Like Particles from Cross-Linked Dimers of Truncated and Mutant Capsid Proteins." Journal of Virology 75, no. 6 (March 15, 2001): 2810–17. http://dx.doi.org/10.1128/jvi.75.6.2810-2817.2001.
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ABSTRACT A nucleic acid-bound capsid protein dimer was previously identified using a Sindbis virus in vitro nucleocapsid assembly system and cross-linking reagents. Cross-link mapping, in combination with a model of the nucleocapsid core, suggested that this dimer contained one monomer from each of two adjacent capsomeres. This intercapsomere dimer is believed to be the initial intermediate in the nucleocapsid core assembly mechanism. This paper presents the purification of cross-linked dimers of a truncated capsid protein and the partial purification of cross-linked dimers of a full-length assembly-defective mutant. The assembly of core-like particles from these cross-linked capsid protein dimers is demonstrated. Core-like particles generated from cross-linked full-length mutant CP(19-264)L52D were examined by electron microscopy and appeared to have a morphology similar to that of wild-type in vitro-assembled core-like particles, although a slight size difference was often visible. Truncated cross-linked CP(81-264) dimers generated core-like particles as well. These core-like particles could subsequently be disassembled when reversible cross-linking reagents were used to form the dimers. The ability of the covalent intercapsomere cross-link to rescue capsid proteins with assembly defects or truncations in the amino-terminal region of the capsid protein supports the previous model of assembly and suggests a possible role for the amino-terminal region of the protein.
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Croucher, David R., Mary Iconomou, Jordan F. Hastings, Sean P. Kennedy, Jeremy Z. R. Han, Robert F. Shearer, Jessie McKenna, et al. "Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers." Science Signaling 9, no. 436 (July 12, 2016): ra69. http://dx.doi.org/10.1126/scisignal.aaf0793.
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Singh, S. V., T. Leal, G. A. Ansari, and Y. C. Awasthi. "Purification and characterization of glutathione S-transferases of human kidney." Biochemical Journal 246, no. 1 (August 15, 1987): 179–86. http://dx.doi.org/10.1042/bj2460179.
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Several forms of glutathione S-transferase (GST) are present in human kidney, and the overall isoenzyme pattern of kidney differs significantly from those of other human tissues. All the three major classes of GST isoenzymes (α, mu and pi) are present in significant amounts in kidney, indicating that GST1, GST2 and GST3 gene loci are expressed in this tissue. More than one form of GST is present in each of these classes of enzymes, and individual variations are observed for these classes. The structural, immunological and functional properties of GST isoenzymes of three classes differ significantly from each other, whereas the isoenzymes belonging to the same class have similar properties. All the cationic GST isoenzymes of human kidney except for GST 9.1 are heterodimers of 26,500-Mr and 24,500-Mr subunits. GST 9.1 is a dimer of 24,500-Mr subunits. All the cationic isoenzymes of kidney GST cross-react with antibodies raised against a mixture of GST α, beta, gamma, delta and epsilon isoenzymes of liver. GST 6.6 and GST 5.5 of kidney are dimers of 26,500-Mr subunits and are immunologically similar to GST psi of liver. Unlike other human tissues, kidney has at least two isoenzymes (pI 4.7 and 4.9) associated with the GST3 locus. Both these isoenzymes are dimers of 22,500-Mr subunits and are immunologically similar to GST pi of placenta. Some of the isoenzymes of kidney do not correspond to known GST isoenzymes from other human tissues and may be specific to this tissue.
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Cunha Lima, Suzana T., and Edson D. Rodrigues. "The oligomeric state of thyroid receptor regulates hormone binding kinetics." Journal of Endocrinology 210, no. 1 (April 20, 2011): 125–34. http://dx.doi.org/10.1530/joe-11-0019.
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We previously reported that mutations in the thyroid hormone receptor (TR) surface that mediates dimer and heterodimer formation do not alter affinity for cognate hormone (triiodothyronine (T3)) yet dramatically enhance T3 association and dissociation rates. This study aimed to show that TR oligomeric state influences binding and dissociation kinetics. We performed binding assays using marked hormone (125I-T3) and TRs expressed and purified by different methods. We find that T3 associates with TRs with biphasic kinetics in solution; a rapid step (half-life ±0.1 h) followed by a slower second step (half-life ±5 h) and that purification of monomers suggests that biphasic kinetics are due to the presence of monomers and dimers in our preparations. In support of this idea, incubation of TR ligand binding domain monomers with corepressor peptide induces dimer formation and decreases association rates and T3 binds to, and dissociates from, a TRβ mutant that only forms dimers (TRβD355R) with slow single-phase kinetics. In addition, heterodimer formation with retinoid X receptors also influences ligand binding kinetics. Together, these results suggest that the dimer/heterodimer surface is allosterically coupled to the hormone binding pocket and that different interactions at this surface exert different effects on ligand binding that may be relevant for TR actions in the cell.
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Guo, Xiangxue, Robert W. Harrison, and Phang C. Tai. "Nucleotide-Dependent Dimerization of the C-Terminal Domain of the ABC Transporter CvaB in Colicin V Secretion." Journal of Bacteriology 188, no. 7 (April 1, 2006): 2383–91. http://dx.doi.org/10.1128/jb.188.7.2383-2391.2006.
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ABSTRACT The cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC constitute the bacteriocin colicin V secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter, and its C-terminal domain (CTD) contains typical motifs for the nucleotide-binding and Walker A and B sites and the ABC signature motif. To study the role of the CvaB CTD in the secretion of colicin V, a truncated construct of this domain was made and overexpressed. Different forms of the CvaB CTD were found during purification and identified as monomer, dimer, and oligomer forms by gel filtration and protein cross-linking. Nucleotide binding was shown to be critical for CvaB CTD dimerization. Oligomers could be converted to dimers by nucleotide triphosphate-Mg, and nucleotide release from dimers resulted in transient formation of monomers, followed by oligomerization and aggregation. Site-directed mutagenesis showed that the ABC signature motif was involved in the nucleotide-dependent dimerization. The spatial proximity of the Walker A site and the signature motif was shown by disulfide cross-linking a mixture of the A530C and L630C mutant proteins, while the A530C or L630C mutant protein did not dimerize on its own. Taken together, these results indicate that the CvaB CTD formed a nucleotide-dependent head-to-tail dimer.
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Hannon, Clare, Abimael Cruz-Migoni, Olga Platonova, Robin L. Owen, Joanne E. Nettleship, Ami Miller, Stephen B. Carr, Gemma Harris, Terence H. Rabbitts, and Simon E. V. Phillips. "Cloning, purification and structure determination of the HIV integrase-binding domain of lens epithelium-derived growth factor." Acta Crystallographica Section F Structural Biology Communications 74, no. 3 (February 26, 2018): 143–49. http://dx.doi.org/10.1107/s2053230x18001553.
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Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase in human cells. The crystal structure of the HIV integrase-binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed inEscherichia coli, purified and crystallized by sitting-drop vapour diffusion. X-ray diffraction data were collected at Diamond Light Source to a resolution of 2.05 Å. The crystals belonged to space groupP21, with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain-swapped IBD dimers. IBD exists as a mixture of monomers and dimers in concentrated solutions, but the dimers are unlikely to be biologically relevant.
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Spencer, J. B., and P. M. Jordan. "Purification and properties of 6-methylsalicylic acid synthase from Penicillium patulum." Biochemical Journal 288, no. 3 (December 15, 1992): 839–46. http://dx.doi.org/10.1042/bj2880839.
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6-Methylsalicylic acid synthase has been isolated in homogeneous form from Penicillium patulum grown in liquid culture from a spore inoculum. The enzyme is highly susceptible to proteolytic degradation in vivo and in vitro, but may be stabilized during purification by incorporating proteinase inhibitors in the buffers. The enzyme exists as a homotetramer of M(r) 750,000, with a subunit M(r) of 180,000. 6-Methylsalicyclic acid synthase also accepts acetoacetyl-CoA as an alternative starter molecule to acetyl-CoA. The enzyme also catalyses the formation of small amounts of triacetic acid lactone as an oligatory by-product of the reaction. In the absence of NADPH, triacetic acid lactone is the exclusive enzymic product, being formed at 10% of the rate of 6-methylsalicylic acid. The enzyme is inactivated by 1,3-dibromopropan-2-one, leading to the formation of cross-linked dimers similar to that observed with type I fatty acid synthases. Acetyl-CoA protects the enzyme against the inactivation and inhibits dimer formation. An adaptation of the purification method for 6-methylsalicylic acid synthase may be used for the isolation of fatty acid sythase from Penicillium patulum.
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Gómez, J. "Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes." International Journal of Biochemistry & Cell Biology 35, no. 7 (July 2003): 1109–18. http://dx.doi.org/10.1016/s1357-2725(03)00007-4.
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Brennan, Bradley J., Jaro Arero, Paul A. Liddell, Thomas A. Moore, Ana L. Moore, and Devens Gust. "Selective oxidative synthesis of meso-beta fused porphyrin dimers." Journal of Porphyrins and Phthalocyanines 17, no. 04 (April 2013): 247–51. http://dx.doi.org/10.1142/s1088424613500363.
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An efficient route to meso-β doubly connected fused porphyrin dimers was developed. Synthesis of the dimers incorporated two successive C–C bond-forming steps selectively coupling unsubstituted meso- and β-positions. Using Cu(BF4)2 as an oxidant in nitromethane solvent, the radical coupling of Cu(II) -porphyrins occurred in high yield and without side-products, allowing chromatography-free purification. Efficient demetalation of the product yielded free-base derivatives and the possibility to incorporate other metals into the macrocycles. The absorption and electrochemical properties vary with the inserted metal, showing broad UV-visible-NIR absorption and multiple one-electron oxidations/reductions in a relatively narrow electrochemical window.
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FAMULSKI, Konrad S., Michel LIUZZI, Saber BASHIR, Razmik MIRZAYANS, and Malcolm C. PATERSON. "Purification and characterization of a novel human acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase." Biochemical Journal 345, no. 3 (January 25, 2000): 583–93. http://dx.doi.org/10.1042/bj3450583.
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A novel N-glycosylated, mannose-rich protein has been purified approx. 4000-fold from human liver in a seven-step procedure including ion-exchange chromatography and fractionation on concanavalin A-Sepharose, Sephadex G-75 and oligo(dT)-cellulose matrices. The molecular mass of the protein is 46 kDa when measured by gel filtration (i.e. under non-denaturing conditions) and 60 kDa by SDS/PAGE (i.e. under denaturing conditions). The protein possesses two DNA backbone-incising activities, namely, the random introduction of single-strand breaks in native DNA and the rupture of the phosphodiester linkage internal to cyclobutyl pyrimidine dimers, the major class of DNA lesions induced by solar UV rays. Both activities are optimal at pH 5.0 in vitro, although the non-specific nuclease displays appreciable activity at neutral pH, depending on the buffer composition. The protein has been named acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase (AN/IDP). As a nuclease, the protein ‘prefers#x2019; a linear DNA structure over a covalently closed circular molecule and is more proficient at digesting single-stranded than double-stranded DNA. The polynucleotide cleavage products of the nuclease contain 5ʹ-OH and 3ʹ-PO4 termini, which are refractory to direct rejoining by DNA ligases. Depending on the substrate, the nuclease activity exhibits a temperature optimum of 50 °C or greater, and is neither stimulated by Mg2+ or Ca2+ nor inhibited by Zn2+. AN/IDP is present in human liver and in cultured human cells of both fibroblastic and lymphocytic origins. Intracellularly, the protein can be readily detected in both the cytosolic and nuclear fractions, although much more (approx. 3-fold) is found in the latter fraction. We propose that this bifunctional enzyme may be involved in both apoptotic DNA digestion and metabolism of cyclobutyl pyrimidine dimers in UV-irradiated human cells.
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Sugimoto, Shinya, Hiroyuki Yoshida, Yoshimitsu Mizunoe, Keigo Tsuruno, Jiro Nakayama, and Kenji Sonomoto. "Structural and Functional Conversion of Molecular Chaperone ClpB from the Gram-Positive Halophilic Lactic Acid Bacterium Tetragenococcus halophilus Mediated by ATP and Stress." Journal of Bacteriology 188, no. 23 (September 22, 2006): 8070–78. http://dx.doi.org/10.1128/jb.00404-06.
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ABSTRACT In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB Tha ) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB Tha forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45°C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB Tha reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE Tha ) and ATP. Interestingly, the mixture of dimer and monomer ClpB Tha , which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB Tha forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE Tha and ATP under poststress conditions.
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Bidault, Sébastien, and Albert Polman. "Water-Based Assembly and Purification of Plasmon-Coupled Gold Nanoparticle Dimers and Trimers." International Journal of Optics 2012 (2012): 1–5. http://dx.doi.org/10.1155/2012/387274.
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We describe a simple one-pot water-based scheme to produce gold nanoparticle groupings with short interparticle spacings. This approach combines a cross-linking molecule and a hydrophilic passivation layer to control the level of induced aggregation. Suspensions of dimers and trimers are readily obtained using a single electrophoretic purification step. The final interparticle spacings allow efficient coupling of the particle plasmon modes as verified in extinction spectroscopy.
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Cadet, Jean, Lucienne Voituriez, Frank E. Hruska, Lou-Sing Kan, Frank A. A. M. de Leeuw, and Cornelis Altona. "Characterization of thymidine ultraviolet photoproducts. Cyclobutane dimers and 5,6-dihydrothymidines." Canadian Journal of Chemistry 63, no. 11 (November 1, 1985): 2861–68. http://dx.doi.org/10.1139/v85-477.
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We describe the preparation of the six configurationally distinct cyclobutane-type photodimers generated by the acetone-sensitized uv irradiation of thymidine in aqueous solution. Also prepared as minor photoproducts are the 5R and 5S di-astereoisomers of 5,6-dihydrothymidine, and two novel molecules, the 5R and 5S diastereoisomers of 5-acetonyl-5,6-dihydrothymidine. The purification of the ten molecules by chromatographic techniques (hplc, tlc) is described, along with their extensive characterization by uv, ir, cd, FAB-ms and 1H nmr. The proton chemical shifts and coupling constants are discussed in terms of the geometry of the pyrimidine, cyclobutane, and sugar rings.
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KRISHNA RAO, J. V., Junutula R. JAGATH, Balasubramanya SHARMA, N. APPAJI RAO, and H. S. SAVITHRI. "Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase." Biochemical Journal 343, no. 1 (September 24, 1999): 257–63. http://dx.doi.org/10.1042/bj3430257.
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Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5′-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 μM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5% active. The apo-D89N mutant tetramer was a dimer unlike the apo-form of the wild-type enzyme which was present predominantly as a tetramer. Furthermore the apo mutant dimer could not be reconstituted to the holo-form by the addition of excess PLP, suggesting that dimer-dimer interactions are weak in this mutant. The recently published crystal structure of human liver cytosolic recombinant SHMT indicates that this residue (D90 in the human enzyme) is located at the N-terminal end of the fourth helix of one subunit and packs against K39 from the second N-terminal helix of the other symmetry related subunit forming the tight dimer. D89 is at the interface of tight dimers where the PLP 5′-phosphate is also bound. Mutation of D89 could lead to weakened ionic interactions in the tight dimer interface, resulting in decreased affinity of the enzyme for the cofactor.
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Kaeswurm, Julia A. H., Bettina Nestl, Sven M. Richter, Max Emperle, and Maria Buchweitz. "Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1." Methods and Protocols 4, no. 1 (December 27, 2020): 3. http://dx.doi.org/10.3390/mps4010003.
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Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.
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Harhammer, R., B. Nürnberg, K. Spicher, and G. Schultz. "Purification of the G-protein G13 from rat brain membranes." Biochemical Journal 303, no. 1 (October 1, 1994): 135–40. http://dx.doi.org/10.1042/bj3030135.
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Significant amounts of G13, a member of the recently described G12-subfamily of heterotrimeric G-proteins, have been detected in rat brain membranes by specific antisera. The alpha-subunits of G13 (G alpha 13) were purified by using a combination of conventional and subunit-exchange chromatography. Purification was facilitated by the fact that the initial anion-exchange chromatography separated G13 from most of the other G-proteins, including Gq/11. Moreover, G alpha 13-enriched fractions obtained from this chromatographic step were devoid of beta gamma-dimers, despite the absence of G-protein-activating agents. Nevertheless, the purified G alpha 13 retained its ability to interact with beta gamma-dimers under appropriate conditions, i.e. the addition of Lubrol PX instead of cholate as detergent and the omission of ethylene glycol routinely used as a protecting additive. The interaction was demonstrated by (i) the binding of G alpha 13 to immobilized beta gamma-complexes and (ii) the formation of stable heterotrimers during sucrose-density-gradient centrifugation. Furthermore, our studies on G alpha 13 provide evidence for an extremely slow basal GDP/GTP exchange rate. The purified protein showed negligible binding of guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S]). Accordingly, dissociation of G alpha 13 from immobilized beta gamma-complexes was achieved by AlF4-/Mg2+, but not by GTP[S]. These data indicate that G13 exhibits properties highly distinct from those of other G-proteins.
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Mieczkowski, Carl, Soheila Bahmanjah, Yao Yu, Jeanne Baker, Gopalan Raghunathan, Daniela Tomazela, Mark Hsieh, Mark McCoy, Corey Strickland, and Laurence Fayadat-Dilman. "Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies." Antibodies 9, no. 4 (November 22, 2020): 66. http://dx.doi.org/10.3390/antib9040066.
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We report the novel crystal structure and characterization of symmetrical, homodimeric humanized heavy-chain-only antibodies or dimers (HC2s). HC2s were found to be significantly coexpressed and secreted along with mAbs from transient CHO HC/LC cotransfection, resulting in an unacceptable mAb developability attribute. Expression of full-length HC2s in the absence of LC followed by purification resulted in HC2s with high purity and thermal stability similar to conventional mAbs. The VH and CH1 portion of the heavy chain (or Fd) was also efficiently expressed and yielded a stable, covalent, and reducible dimer (Fd2). Mutagenesis of all heavy chain cysteines involved in disulfide bond formation revealed that Fd2 intermolecular disulfide formation was similar to Fabs and elucidated requirements for Fd2 folding and expression. For one HC2, we solved the crystal structure of the Fd2 domain to 2.9 Å, revealing a highly symmetrical homodimer that is structurally similar to Fabs and is mediated by conserved (CH1) and variable (VH) contacts with all CDRs positioned outward for target binding. Interfacial dimer contacts revealed by the crystal structure were mutated for two HC2s and were found to dramatically affect HC2 formation while maintaining mAb bioactivity, offering a potential means to modulate novel HC2 formation through engineering. These findings indicate that human heavy-chain dimers can be secreted efficiently in the absence of light chains, may show good physicochemical properties and stability, are structurally similar to Fabs, offer insights into their mechanism of formation, and may be amenable as a novel therapeutic modality.
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Wientjes, Emilie, and Roberta Croce. "The light-harvesting complexes of higher-plant Photosystem I: Lhca1/4 and Lhca2/3 form two red-emitting heterodimers." Biochemical Journal 433, no. 3 (January 14, 2011): 477–85. http://dx.doi.org/10.1042/bj20101538.
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The outer antenna of higher-plant PSI (Photosystem I) is composed of four complexes [Lhc (light-harvesting complex) a1–Lhca4] belonging to the light-harvesting protein family. Difficulties in their purification have so far prevented the determination of their properties and most of the knowledge about Lhcas has been obtained from the study of the in vitro reconstituted antennas. In the present study we were able to purify the native complexes, showing that Lhca2/3 and Lhca1/4 form two functional heterodimers. Both dimers show red-fluorescence emission with maxima around 730 nm, as in the intact PSI complex. This indicates that the dimers are in their native state and that LHCI-680, which was previously assumed to be part of the PSI antenna, does not represent the native state of the system. The data show that the light-harvesting properties of the two dimers are functionally identical, concerning absorption, long-wavelength emission and fluorescence quantum yield, whereas they differ in their high-light response. Implications of the present study for the understanding of the energy transfer process in PSI are discussed. Finally, the comparison of the properties of the native dimers with those of the reconstituted complexes demonstrates that all of the major properties of the Lhcas are reproduced in the in vitro systems.
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Buchanan,, Malcolm S., Melvyn Gill, and Jin Yu. "Pigments of Fungi. XLV The Cardinalins 8 - 12, Unique Pre-naphthoquinone Dehydro Dimers from the New Zealand Toadstool Dermocybe cardinalis." Australian Journal of Chemistry 50, no. 11 (1997): 1081. http://dx.doi.org/10.1071/c97110.
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Five new pre-naphthoquinone dehydro dimers (3)–(7) belonging to the unique cardinalin class of pyranonaphthoquinonoid natural products have been isolated and characterized from the ethanolic extracts of the fruit bodies of the New Zealand toadstool Dermocybe cardinalis. Four other cardinalin derivatives (8) and (10)–(12) are believed to be artefacts of the purification procedure. The structures and relative stereochemistry of these new cardinalins are deduced from spectroscopic data.
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Borrega, Marc, Klaus Niemelä, and Herbert Sixta. "Effect of hydrothermal treatment intensity on the formation of degradation products from birchwood." Holzforschung 67, no. 8 (December 1, 2013): 871–79. http://dx.doi.org/10.1515/hf-2013-0019.
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Abstract Hydrothermal treatments (HT) of birchwood were conducted at various intensities to extract the hemicelluloses before pulping. The amount of hemicellulose-derived sugars in the hydrolysates, including xylulose, an isomerization product of xylose, reached first a maximum and then decreased with further increasing treatment intensity. The hydrolysates also contained furanic compounds, carboxylic acids, and a large variety of aromatics, the amounts of which were dependent on HT intensity. At high treatment intensities, furfural and acetic acid were the main products quantified. Numerous nonvolatile, low molar mass carboxylic acids were also formed, with 3-deoxypentonic acid being the most abundant. Additionally, almost 40 aromatic monomers and up to 30 dimers were detected. Syringaldehyde was the main monomer and syringaresinol was the main dimer. Some aromatic compounds could not be identified. The complexity of the hydrolysates, particularly after high-intensity HT, requires selective filtration and purification methods before the hydrolysates can be utilized in downstream processes.
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RAMJEESINGH, Mohabir, Francisca UGWU, Canhui LI, Sonja DHANI, Ling Jun HUAN, Yanchun WANG, and Christine E. BEAR. "Stable dimeric assembly of the second membrane-spanning domain of CFTR (cystic fibrosis transmembrane conductance regulator) reconstitutes a chloride-selective pore." Biochemical Journal 375, no. 3 (November 1, 2003): 633–41. http://dx.doi.org/10.1042/bj20030774.
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Structural information is required to define the molecular basis for chloride conduction through CFTR (cystic fibrosis transmembrane conductance regulator). Towards this goal, we expressed MSD2, the second of the two MSDs (membrane-spanning domains) of CFTR, encompassing residues 857–1158 in Sf9 cells using the baculovirus system. In Sf9 plasma membranes, MSD2 migrates as expected for a dimer in non-dissociative PAGE, and confers the appearance of an anion permeation pathway suggesting that dimeric MSD2 mediates anion flux. To assess directly the function and quaternary structure of MSD2, we purified it from Sf9 cells by virtue of its polyhistidine tag and nickel affinity. Reconstitution of MSD2 into liposomes conferred a 4,4′-di-isothiocyanostilbene-2,2′-disulphonate-inhibitable, chloride-selective electrodiffusion pathway. Further, this activity is probably mediated directly by MSD2 as reaction of its single cysteine residue (Cys866) with the thiol modifying reagent, Nα(3-maleimidylpropionyl)biocytin, inhibited chloride flux. Only MSD2 dimers were labelled by Nα(3-maleimidylpropionyl)biocytin, supporting the idea that only dimeric MSD2 can mediate anion flux. As a further test of this hypothesis, we conducted a second purification procedure, wherein purified dimeric and monomeric MSD2 proteins were reconstituted separately. Only proteoliposomes containing stable MSD2 dimers mediated chloride electrodiffusion, providing direct evidence that dimeric MSD2 mediates chloride channel function. In summary, we have shown that the second membrane domain of CFTR can be purified and functionally reconstituted as a chloride channel, providing a tool for probing the structural basis of chloride conduction through CFTR.
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Jorba, Núria, Estela Area, and Juan Ortín. "Oligomerization of the influenza virus polymerase complex in vivo." Journal of General Virology 89, no. 2 (February 1, 2008): 520–24. http://dx.doi.org/10.1099/vir.0.83387-0.
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The influenza virus polymerase is a heterotrimer formed by the PB1, PB2 and PA subunits and is responsible for virus transcription and replication. We have expressed the virus polymerase complex by co-transfection of the subunit cDNAs, one of which was tandem affinity purification (TAP)-tagged, into human cells. The intracellular polymerase complexes were purified by the TAP approach, involving two affinity chromatography steps, IgG–Sepharose and calmodulin–agarose. Gel-filtration analysis indicated that, although most of the purified polymerase behaved as a heterotrimer, a significant proportion of the purified material migrated as polymerase dimers, trimers and higher oligomers. Co-purification of polymerase complexes alternatively tagged in the same subunit confirmed that the polymerase complex might form oligomers intracellularly. The implications of this observation for virus infection are discussed.
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Kern, Petra, Rebecca E. Hussey, Rebecca Spoerl, Ellis L. Reinherz, and Hsiu-Ching Chang. "Expression, Purification, and Functional Analysis of Murine Ectodomain Fragments of CD8αα and CD8αβ Dimers." Journal of Biological Chemistry 274, no. 38 (September 17, 1999): 27237–43. http://dx.doi.org/10.1074/jbc.274.38.27237.
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Heitlinger, E., M. Peter, M. Häner, A. Lustig, U. Aebi, and E. A. Nigg. "Expression of chicken lamin B2 in Escherichia coli: characterization of its structure, assembly, and molecular interactions." Journal of Cell Biology 113, no. 3 (May 1, 1991): 485–95. http://dx.doi.org/10.1083/jcb.113.3.485.
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Chicken lamin B2, a nuclear member of the intermediate-type filament (IF) protein family, was expressed as a full-length protein in Escherichia coli. After purification, its structure and assembly properties were explored by EM, using both glycerol spraying/low-angle rotary metal shadowing and negative staining for preparation, as well as by analytical ultracentrifugation. At its first level of structural organization, lamin B2 formed "myosin-like" 3.1S dimers consisting of a 52-nm-long tail flanked at one end by two globular heads. These myosin-like molecules are interpreted to represent two lamin polypeptides interacting via their 45-kD central rod domains to form a segmented, parallel and unstaggered 52-nm-long two-stranded alpha-helical coiled-coil, and their COOH-terminal end domains folding into globular heads. At the second level of organization, lamin B2 dimers associated longitudinally to form polar head-to-tail polymers. This longitudinal mode of association of laminin dimers is in striking contrast to the lateral mode of association observed previously for cytoplasmic IF dimers. At the third level of organization, these polar head-to-tail polymers further associated laterally, in an approximately half-staggered fashion, to form filamentous and eventually paracrystal-like structures revealing a pronounced 24.5-nm axial repeat. Finally, following up on recent studies implicating the mitotic cdc2 kinase in the control of lamin polymerization (Peter, M., J. Nakagawa, M. Dorée, J. C. Labbé, and E. A. Nigg. 1990. Cell. 61:591-602), we have examined the effect of phosphorylation by purified cdc2 kinase on the assembly properties and molecular interactions of the bacterially expressed lamin B2. Phosphorylation of chicken lamin B2 by cdc2 kinase interferes with the head-to-tail polymerization of the lamin dimers. This finding supports the notion that cdc2 kinase plays a major, direct role in triggering mitotic disassembly of the nuclear lamina.
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Chien, Chia-Hui, Li-Hao Huang, Chi-Yuan Chou, Yuan-Shou Chen, Yu-San Han, Gu-Gang Chang, Po-Huang Liang, and Xin Chen. "One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins." Journal of Biological Chemistry 279, no. 50 (September 24, 2004): 52338–45. http://dx.doi.org/10.1074/jbc.m406185200.
Full textAbstract:
DPP-IV is a prolyl dipeptidase, cleaving the peptide bond after the penultimate proline residue. It is an important drug target for the treatment of type II diabetes. DPP-IV is active as a dimer, and monomeric DPP-IV has been speculated to be inactive. In this study, we have identified the C-terminal loop of DPP-IV, highly conserved among prolyl dipeptidases, as essential for dimer formation and optimal catalysis. The conserved residue His750on the loop contributes significantly for dimer stability. We have determined the quaternary structures of the wild type, H750A, and H750E mutant enzymes by several independent methods including chemical cross-linking, gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Wild-type DPP-IV exists as dimers both in the intact cell andin vitroafter purification from human semen or insect cells. The H750A mutation results in a mixture of DPP-IV dimer and monomer. H750A dimer has the same kinetic constants as those of the wild type, whereas the H750A monomer has a 60-fold decrease inkcat. Replacement of His750with a negatively charged Glu (H750E) results in nearly exclusive monomers with a 300-fold decrease in catalytic activity. Interestingly, there is no dynamic equilibrium between the dimer and the monomer for all forms of DPP-IVs studied here. This is the first study of the function of the C-terminal loop as well as monomeric mutant DPP-IVs with respect to their enzymatic activities. The study has important implications for the discovery of drugs targeted to the dimer interface.
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Goodwin, Sophie R., Amy Stimpson, Richard Moon, Lauren Cowie, Najib Aragrag, Sorin V. Filip, Andrew G. Smith, and Derek J. Irvine. "Facile Synthesis of Functionalised Hyperbranched Polymers for Application as Novel, Low Viscosity Lubricant Formulation Components." Polymers 14, no. 18 (September 14, 2022): 3841. http://dx.doi.org/10.3390/polym14183841.
Full textAbstract:
A novel, previously unreported, method for synthesising hyperbranched (HB) materials is detailed. Their use as additives to produce lubricant formulations that exhibit enhanced levels of wear protection and improved low-temperature oil viscosity and flow is also reported. The lubricant formulations containing HB additives were found to exhibit both significantly lower viscosities and improved in-use film-forming properties than the current industry standard formulations. To achieve this, alkyl methacrylate oligomers (predominantly dimers and trimers) were synthesised using catalytic chain transfer polymerisation. These were then used as functional chain transfer agents (CTA) to control the polymerisation of divinyl benzene (DVB) monomers to generate highly soluble, high polydispersity HB polymers. The level of dimer/trimer purification applied was varied to define its influence on both these HB resultant structures and the resultant HB additives’ performance as a lubricant additive. It was shown that, while the DVB acted as the backbone of the HB, the base oil solubility of the additive was imparted by the presence of the alkyl chains included in the structure via the use of the oligomeric CTAs.
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Mavrides, Charalampos, and Guylaine Nadeau. "Purification and properties of the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase from rat heart." Biochemistry and Cell Biology 65, no. 3 (March 1, 1987): 239–44. http://dx.doi.org/10.1139/o87-031.
Full textAbstract:
The present work describes the purification from rat heart of the mitochondrial and cytosolic forms of the enzymes of the malate–aspartate shuttle, aspartate aminotransferase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37), by a single procedure after the preparation of the original crude extract. In 10 purification steps, the four enzymes were obtained electrophoretically pure in yields ranging from 6 to 54% of their respective isoenzyme levels in the crude extract. Apoenzymes were formed from the aminotransferases by reacting them with cysteine sulfinate and dialyzing. Complete reconstitution was obtained after a brief incubation with pyridoxal phosphate. All four enzymes are dimers. The mitochondrial isoenzymes are of slightly lower molecular weight than their respective cytosolic forms. Michaelis constants and maximal velocities were derived by the use of primary and secondary plots. In general, the properties of the enzymes from rat heart are similar to the properties of the enzymes from other animal sources.
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Morton, H. C., J. D. Atkin, R. J. Owens, and J. M. Woof. "Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells." Journal of Immunology 151, no. 9 (November 1, 1993): 4743–52. http://dx.doi.org/10.4049/jimmunol.151.9.4743.
Full textAbstract:
Abstract Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fc alpha receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.
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Yu, Wen, Jian-Hao Huang, and Chung-Sung Tan. "Purification of Polybutylene Terephthalate by Oligomer Removal Using a Compressed CO2 Antisolvent." Polymers 11, no. 7 (July 23, 2019): 1230. http://dx.doi.org/10.3390/polym11071230.
Full textAbstract:
In this study, the cyclic oligomers in the highly chemically resistant polyester polybutylene terephthalate (PBT) were effectively removed using a compressed CO2 antisolvent technique in which 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was used as the solvent. In addition to the oligomers, tetrahydrofuran was completely removed because of its low molecular weight and liquid state. The effects of the operating variables, including temperature, pressure, and the PBT concentration in HFIP, on the degree of removal of the oligomers were systematically studied using experimental design and the response surface methodology. The most appropriate operating conditions for the purification of PBT were 8.3 MPa and 23.4 °C when using 4.5 wt % PBT in HFIP. Under these conditions, the cyclic trimers and dimers could be removed by up to 81.4% and 95.7%, respectively, in a very short operating time.
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Pangas, SA, and TK Woodruff. "Production and purification of recombinant human inhibin and activin." Journal of Endocrinology 172, no. 1 (January 1, 2002): 199–210. http://dx.doi.org/10.1677/joe.0.1720199.
Full textAbstract:
Inhibin and activin are protein hormones with diverse physiological roles including the regulation of pituitary FSH secretion. Like other members of the transforming growth factor-beta gene family, they undergo processing from larger precursor molecules as well as assembly into functional dimers. Isolation of inhibin and activin from natural sources can only produce limited quantities of bioactive protein. To purify large-scale quantities of recombinant human inhibin and activin, we have utilized stably transfected cell lines in self-contained bioreactors to produce protein. These cells produce approximately 200 microg/ml per day total recombinant human inhibin. Conditioned cell media can be purified through column chromatography resulting in dimeric mature 32-34 kDa inhibin A and 28 kDa activin A. The purified recombinant proteins maintain their biological activity as measured by traditional in vitro assays including the regulation of FSH in rat anterior pituitary cultures and the regulation of promoter activity of the activin-responsive promoter p3TP-luc in tissue culture cells. These proteins will be valuable for future analysis of inhibin and activin function and have been distributed to the US National Hormone and Peptide Program.
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OURY, Tim D., James D. CRAPO, Zuzana VALNICKOVA, and Jan J. ENGHILD. "Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: a simplified, high-yield purification of extracellular superoxide dismutase." Biochemical Journal 317, no. 1 (July 1, 1996): 51–57. http://dx.doi.org/10.1042/bj3170051.
Full textAbstract:
Studies examining the biochemical characteristics and pharmacological properties of extracellular superoxide dismutase (EC SOD) have been severely limited because of difficulties in purifying the enzyme. Recently EC SOD was found to exist in high concentrations in the arteries of most mammals examined and it is the predominant form of SOD activity in many arteries. We now describe a three-step, high-yield protocol for the purification of EC SOD from human aorta. In the first step, the high affinity of EC SOD for heparin is utilized to obtain a fraction in which EC SOD constitutes roughly 13% of the total protein compared with only 0.3% of that of the starting material. In addition, over 80% of the original EC SOD activity present in the aortic homogenate was retained after the first step of purification. EC SOD was further purified using a combination of cation- and anion-exchange chromatography. The overall yield of EC SOD from this purification procedure was 46%, with over 4 mg of EC SOD obtained from 230 g of aorta. Purified EC SOD was found to exist predominantly as a homotetramer composed of two disulphide-linked dimers. However, EC SOD was also found to form larger multimers when analysed by native PAGE. It was shown by urea denaturation that the formation of multimers increased the thermodynamic stability of the protein. Limited proteolysis of EC SOD suggested that there is one interchain disulphide bond covalently linking two subunits. This disulphide bond involves cysteine-219 and appears to link the heparin-binding domains of the two subunits.
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Brophy, P. M., and J. Barrett. "Blocking factors and the isolation of glutathione transferases from Hymenolepis diminuta (Cestoda: Cyclophyllidea)." Parasitology 100, no. 1 (February 1990): 137–41. http://dx.doi.org/10.1017/s0031182000060212.
Full textAbstract:
SummaryFour acidic glutathione (GSH) transferase forms were isolated from the cytosol of the adult cestode Hymenolepis diminuta by hydroxylapatite chromatography, glutathione-affinity chromatography and chromatofocusing, pH 7–5. The enzymes were dimers of subunit size approximately 24 kDa and accounted for at least 3% of the total soluble protein. The major GSH transferase had limited catalytic activity but may interact with a range of ligands and function as a binding/passive detoxification protein. An endogenous factor interfered with the binding of the crude cytosolic GSH transferase activity to glutathione-dependent affinity matrices but, following partial purification, the GSH transferase activity successfully interacted with the glutathione affinity matrix.
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Shraddha, Ravi Shekher, Simran Sehgal, Mohit Kamthania, and Ajay Kumar. "Laccase: Microbial Sources, Production, Purification, and Potential Biotechnological Applications." Enzyme Research 2011 (June 21, 2011): 1–11. http://dx.doi.org/10.4061/2011/217861.
Full textAbstract:
Laccase belongs to the blue multicopper oxidases and participates in cross-linking of monomers, degradation of polymers, and ring cleavage of aromatic compounds. It is widely distributed in higher plants and fungi. It is present in Ascomycetes, Deuteromycetes and Basidiomycetes and abundant in lignin-degrading white-rot fungi. It is also used in the synthesis of organic substance, where typical substrates are amines and phenols, the reaction products are dimers and oligomers derived from the coupling of reactive radical intermediates. In the recent years, these enzymes have gained application in the field of textile, pulp and paper, and food industry. Recently, it is also used in the design of biosensors, biofuel cells, as a medical diagnostics tool and bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases have received attention of researchers in the last few decades due to their ability to oxidize both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants. It has been identified as the principal enzyme associated with cuticular hardening in insects. Two main forms have been found: laccase-1 and laccase-2. This paper reviews the occurrence, mode of action, general properties, production, applications, and immobilization of laccases within different industrial fields.
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Chen, Bing, Yifan Cheng, Lesley Calder, Stephen C. Harrison, Ellis L. Reinherz, John J. Skehel, and Don C. Wiley. "A Chimeric Protein of Simian Immunodeficiency Virus Envelope Glycoprotein gp140 and Escherichia coli Aspartate Transcarbamoylase." Journal of Virology 78, no. 9 (May 1, 2004): 4508–16. http://dx.doi.org/10.1128/jvi.78.9.4508-4516.2004.
Full textAbstract:
ABSTRACT The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R2) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C6R6 ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.
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Scott, C. A., K. C. Garcia, F. R. Carbone, I. A. Wilson, and L. Teyton. "Role of chain pairing for the production of functional soluble IA major histocompatibility complex class II molecules." Journal of Experimental Medicine 183, no. 5 (May 1, 1996): 2087–95. http://dx.doi.org/10.1084/jem.183.5.2087.
Full textAbstract:
Structural studies of cellular receptor molecules involved in immune recognition require the production of large quantities of the extracellular domains of these glycoproteins. The murine major histocompatibility complex (MHC) class II-restricted response has been extensively studied by functional means, but the engineering and purification of the native, empty form of the most-studied murine MHC class II molecule, IA, has been difficult to achieve. IA molecules, which are the murine equivalent of human histocompatibility leukocyte antigen-DQ molecules, have a low efficiency of chain pairing, which results in poor transport to the cell surface and in the appearance of mixed isotype pairs. We have engineered soluble IA molecules whose pairing has been forced by the addition of leucine zipper peptide dimers at their COOH-terminus. The molecules are secreted "empty" into the extracellular medium and can be loaded with single peptide after purification. These IA molecules have been expressed in milligram quantity for crystallization as well as for activation of T cells and measurement of MHC class II-T cell receptor interactions.
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Dikshit, Archana, Manjula Chaddha, R. K. Singh, and Krishna Misra. "Naphthaloyl group: a new selective amino protecting group for deoxynucleosides in oligonucleotide synthesis." Canadian Journal of Chemistry 66, no. 12 (December 1, 1988): 2989–94. http://dx.doi.org/10.1139/v88-464.
Full textAbstract:
The naphthaloyl group has been found to be a selective amino protecting group for deoxycytidine, deoxyadenosine, and deoxyguanosine in oligodeoxyribonucleotide synthesis. All three protected monomers obtained (78–85%), being six-membered cyclic imides, were fairly stable. These protected monomers were used successfully for the preparation of dimers (phosphodiester approach) and tetramers (phosphotriester approach) in solution as well as solid phase, respectively. The group acted as a purification tool due to its high lipophilicity. No adverse effect has been observed either on the glycosidic bond (depurination) or the internucleotidic bond during its removal. The monomeric units were characterized by UV, NMR, and elemental analyses whereas the tetramers were characterized by enzymatic hydrolyses with snake venom phosphodiesterase followed by alkaline phosphatase.
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Ye, Fuzhou, Chao Wang, Qinqin Fu, Lian-hui Zhang, and Yong-gui Gao. "Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, fromAgrobacterium tumefaciens." Acta Crystallographica Section F Structural Biology Communications 71, no. 9 (August 25, 2015): 1139–45. http://dx.doi.org/10.1107/s2053230x15012881.
Full textAbstract:
Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts byAgrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen–host interaction.
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Avraham, Orly, Yael Levi-Kalisman, and Oded Livnah. "Generating a High Valency Biotin Binder by Selecting Uniform Protein Assemblies via Crystallization." Crystals 9, no. 7 (July 11, 2019): 353. http://dx.doi.org/10.3390/cryst9070353.
Full textAbstract:
Crystallization is a common practice in the purification process in small molecule synthesis while selecting the wanted product. For proteins it is rarely applied due to the methodological predicaments in obtaining crystals. Our observation of the stabilized octamers in the crystal structure of hoefavidin, a novel dimeric member of the avidin family, led to the notion of developing a novel biotechnological tool via covalent crosslinking. The avidin–biotin system has been exploited for decades utilizing the ultra-high affinity between avidin and biotin as a basis for numerous applications. Optimizing the system led to the discovery of a novel group of dimeric avidins including hoefavidin. Hoefavidin has a dynamic quaternary structure, where a dimer is the basis for generating the octamer via crystallographic symmetry operation. Upon biotin binding in solution hoefavidin dissociates solely into dimers. In order to stabilize the octamer, we designed the P61C mutant to form a disulfide bridge stabilizing the octamer and preventing dissociation upon biotin binding. The process of selecting P61C hoefavidin uniform octamers includes crystallization followed by dissolving the crystals. The P61C modified hoefavidin octamer can have a substantial added value to the various biotechnological applications and advances of the biotin based high affinity systems.
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Hong, Chenyang, and Kevin Y. Yip. "Flexible k-mers with variable-length indels for identifying binding sequences of protein dimers." Briefings in Bioinformatics 21, no. 5 (November 5, 2019): 1787–97. http://dx.doi.org/10.1093/bib/bbz101.
Full textAbstract:
Abstract Many DNA-binding proteins interact with partner proteins. Recently, based on the high-throughput consecutive affinity-purification systematic evolution of ligands by exponential enrichment (CAP-SELEX) method, many such protein pairs have been found to bind DNA with flexible spacing between their individual binding motifs. Most existing motif representations were not designed to capture such flexibly spaced regions. In order to computationally discover more co-binding events without prior knowledge about the identities of the co-binding proteins, a new representation is needed. We propose a new class of sequence patterns that flexibly model such variable regions and corresponding algorithms that identify co-bound sequences using these patterns. Based on both simulated and CAP-SELEX data, features derived from our sequence patterns lead to better classification performance than patterns that do not explicitly model the variable regions. We also show that even for standard ChIP-seq data, this new class of sequence patterns can help discover co-bound events in a subset of sequences in an unsupervised manner. The open-source software is available at https://github.com/kevingroup/glk-SVM.
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Yu, Nuo, and Niels Galjart. "TAPping into the treasures of tubulin using novel protein production methods." Essays in Biochemistry 62, no. 6 (November 14, 2018): 781–92. http://dx.doi.org/10.1042/ebc20180033.
Full textAbstract:
Microtubules are cytoskeletal elements with important cellular functions, whose dynamic behaviour and properties are in part regulated by microtubule-associated proteins (MAPs). The building block of microtubules is tubulin, a heterodimer of α- and β-tubulin subunits. Longitudinal interactions between tubulin dimers facilitate a head-to-tail arrangement of dimers into protofilaments, while lateral interactions allow the formation of a hollow microtubule tube that mostly contains 13 protofilaments. Highly homologous α- and β-tubulin isotypes exist, which are encoded by multi-gene families. In vitro studies on microtubules and MAPs have largely relied on brain-derived tubulin preparations. However, these consist of an unknown mix of tubulin isotypes with undefined post-translational modifications. This has blocked studies on the functions of tubulin isotypes and the effects of tubulin mutations found in human neurological disorders. Fortunately, various methodologies to produce recombinant mammalian tubulins have become available in the last years, allowing researchers to overcome this barrier. In addition, affinity-based purification of tagged tubulins and identification of tubulin-associated proteins (TAPs) by mass spectrometry has revealed the ‘tubulome’ of mammalian cells. Future experiments with recombinant tubulins should allow a detailed description of how tubulin isotype influences basic microtubule behaviour, and how MAPs and TAPs impinge on tubulin isotypes and microtubule-based processes in different cell types.
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RAMJEESINGH, Mohabir, Jackie F. KIDD, Ling Jun HUAN, Yanchun WANG, and Christine E. BEAR. "Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane." Biochemical Journal 374, no. 3 (September 15, 2003): 793–97. http://dx.doi.org/10.1042/bj20030683.
Full textAbstract:
CFTR (cystic fibrosis transmembrane conductance regulator) mediates chloride conduction across the apical membrane of epithelia, and mutations in CFTR lead to defective epithelial fluid transport. Recently, there has been considerable interest in determining the quaternary structure of CFTR at the cell surface, as such information is a key to understand the molecular basis for pathogenesis in patients harbouring disease-causing mutations. In our previous work [Ramjeesingh, Li, Kogan, Wang, Huan and Bear (2001) Biochemistry 40, 10700–10706], we showed that monomeric CFTR is the minimal functional form of the protein, yet when expressed in Sf 9 cells using the baculovirus system, it also exists as dimers. The purpose of the present study was to determine if dimeric CFTR exists at the surface of mammalian cells, and particularly in epithelial cells. CFTR solubilized from membranes prepared from Chinese-hamster ovary cells stably expressing CFTR and from T84 epithelial cells migrates as predicted for monomeric, dimeric and larger complexes when subjected to sizing by gel filtration and analysis by non-dissociative electrophoresis. Purification of plasma membranes led to the enrichment of CFTR dimers and this structure exists as the complex glycosylated form of the protein, supporting the concept that dimeric CFTR is physiologically relevant. Consistent with its localization in plasma membranes, dimeric CFTR was labelled by surface biotinylation. Furthermore, dimeric CFTR was captured at the apical surface of intact epithelial cells by application of a membrane-impermeable chemical cross-linker. Therefore it follows from the present study that CFTR dimers exist at the surface of epithelial cells. Further studies are necessary to understand the impact of dimerization on the cell biology of wild-type and mutant CFTR proteins.
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SOUSA, Vítor L., Maria Teresa COSTA, Angelina S. PALMA, Francisco ENGUITA, and Júlia COSTA. "Localization, purification and specificity of the full-length membrane-bound form of human recombinant α1,3/4-fucosyltransferase from BHK-21B cells." Biochemical Journal 357, no. 3 (July 25, 2001): 803–10. http://dx.doi.org/10.1042/bj3570803.
Full textAbstract:
Fucosyltransferase III [galactoside 3(4)-l-fucosyltransferase; EC 2.4.1.65] (FT3) is a Golgi type II membrane protein that catalyses the synthesis of fucosylated Lewis motifs that are associated with cell-adhesion events and are differentially expressed during cell differentiation. In the present work, the full-length membrane bound form of FT3 has been expressed in baby hamster kidney cells. The enzyme has been found in the trans-Golgi and trans-Golgi network (TGN) of the transfected cells, where it appeared as monomers and dimers, but not as oligomers with high molecular masses. Therefore oligomerization is not the basis for correct localization of FT3 in the Golgi. The enzyme has been purified, with a final yield of 2% and a total purification of 2900-fold, by DEAE-Sepharose, SP-Sepharose, GDP-Fractogel and Superdex 200 chromatography. The purified enzyme showed a clear preference for the Galβ3GlcNAc motif in oligosaccharides conjugated with the hydrophobic tail (CH2)3-NHCO-(CH2)5-NH-biotin. Substitution of galactose with α2-linked fucose or α2,3-linked N-acetylneuraminic acid yielded a 1.9-fold increase or a 43% decrease in activity respectively. The enzyme showed no activity towards asialofetuin, a glycoprotein containing the Galβ3GlcNAc acceptor motif. Therefore it has been concluded that the membrane-bound form of FT3 is present in the Golgi and the TGN in an equilibrium of monomers ↔ dimers, which might fucosylate glycans from glycolipids, but not from glycoproteins.
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Pieri, Marina, Evgeny Fominskiy, Pasquale Nardelli, Matteo A. Bonizzoni, and Anna M. Scandroglio. "CytoSorb purification in critically ill SARS-CoV-2 patients." International Journal of Artificial Organs 45, no. 2 (October 26, 2021): 216–20. http://dx.doi.org/10.1177/03913988211052572.
Full textAbstract:
Objective: To describe the experience with CytoSorb treatment in patients with refractory acute respiratory distress syndrome (ARDS) following SARS-CoV-2 infection. Methods: Retrospective observational study on 15 patients treated in a University Hospital. Results: All patients were male, with a mean age of 55 ± 14 years; eight patients (53%) were on venovenous extracorporeal membrane oxygenation (VV ECMO) due to refractory ARDS and all (100%) under mechanical ventilation at the time of CytoSorb use. We observed reduction in the level of C reactive protein (−52%, p = 0.002), total bilirubin (−46%, p = 0.03), direct bilirubin (−50%, p = 0.02), and D-dimers (−39%, p = 0.04) during CytoSorb treatment and a trend toward reduction in lactate dehydrogenase (−20%, p = 0.2), creatine phosphokinase (−38%, p = 0.1), and fibrinogen (−15%, p = 0.07). Eight patients died (53%) and seven (47%) were discharged from the ICU, of which five had recovery of the native lung function and two were successfully bridged to lung transplantation on VV ECMO support. No difference between survivors and non-survivors was present at baseline. Patients received three CytoSorb cycles on average: mean duration of CytoSorb cycle was 17 h 21 min, but premature circuit clotting despite appropriate level of systemic anticoagulation was frequently observed. Conclusions: CytoSorb treatment was effective in improving several laboratory parameters and inflammation in our experience and no treatment-related adverse effects were recorded. In the light of the unique pathophysiology of SARS-CoV-2 infection, CytoSorb treatment is extremely promising, since it might both reduce inflammation and activation of coagulation.
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Melasniemi, H. "Purification and some properties of the extracellular α-amylase-pullulanase produced by Clostridium thermohydrosulfuricum." Biochemical Journal 250, no. 3 (March 15, 1988): 813–18. http://dx.doi.org/10.1042/bj2500813.
Full textAbstract:
The novel alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum E 101-69 was purified as two forms (I and II) from culture medium, by using gel filtration in 6 M-guanidine hydrochloride as the final step. Renatured alpha-amylase-pullulanase I and II had apparent Mr values of 370,000 +/- 85,000 and 330,000 +/- 85,000 respectively, as determined by native polyacrylamide-gradient-gel electrophoresis. Both forms appear to be dimers of two similar subunits, with Mr values of 190,000 +/- 30,000 for enzyme I and 180,000 +/- 30,000 for enzyme II according to SDS/polyacrylamide-gradient-gel electrophoresis. The two forms had similar amino acid compositions, the same N-terminal sequence (Glu-Ile-Asp-Thr-Ala-Pro-Ala-Ile) and the same pI of 4.25. Both forms contained sugars having mobilities identical with those of rhamnose, glucose, galactose and mannose. The amount of neutral hexoses relative to protein was 11-12% (w/w) for both forms.
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TAKEUCHI, Ryo, Masahiko OSHIGE, Makiyo UCHIDA, Gen ISHIKAWA, Kei-ichi TAKATA, Kaori SHIMANOUCHI, Yoshihiro KANAI, Tatsushi RUIKE, Hiroshi MORIOKA, and Kengo SAKAGUCHI. "Purification of Drosophila DNA polymerase ζ by REV1 protein-affinity chromatography." Biochemical Journal 382, no. 2 (August 24, 2004): 535–43. http://dx.doi.org/10.1042/bj20031833.
Full textAbstract:
Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol ζ (DNA polymerase ζ) from Drosophila melanogaster (Dmpol ζ). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol ζ was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2′,3′-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)10:1 as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol ζ showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol ζ were not detected. Drosophila REV7 interacted with Dmpol ζ in vitro, but did not influence the DNA synthesis activity of Dmpol ζ. The present study is the first report about characterization of purified pol ζ from multicellular organisms, and the second concerning the characterization of yeast pol ζ.
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Gil, Fernando, Santiago Ramón-Maiques, Alberto Marina, Ignacio Fita, and Vicente Rubio. "N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the recombinant enzyme and preliminary X-ray analysis of the free and ligand-bound forms." Acta Crystallographica Section D Biological Crystallography 55, no. 7 (July 1, 1999): 1350–52. http://dx.doi.org/10.1107/s0907444999005351.
Full textAbstract:
The gene for Escherichia coli N-acetyl-L-glutamate kinase (NAGK) was cloned in a plasmid and expressed in E. coli, allowing enzyme purification in three steps. NAGK exhibits high specific activity (1.1 µmol s−1 mg−1), lacks Met1 and forms dimers (shown by cross-linking). Crystals of unliganded NAGK diffract to 2 Å and belong to space group P6122 or its enantiomorph P6522 (unit-cell parameters a = b = 78.6, c = 278.0 Å) with two monomers in the asymmetric unit. Crystals of NAGK with acetylglutamate and the ATP analogue AMPPNP diffract to 1.8 Å and belong to space group C2221 (unit-cell parameters a = 60.0, b = 71.9, c = 107.4 Å), with one monomer in the asymmetric unit. NAGK crystallization will allow the determination of proposed structural similarities to carbamate kinase.
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Gorbachev, Stanislav A., and yacheslav V. Zuev. "Synthesis and Mesomorphic Properties of Biphenyl Derivatives with Central Unit Based on 1,6-Hexamethylene Diisocyanate Oligomers." Liquid Crystals and their Application 22, no. 4 (December 22, 2022): 27–36. http://dx.doi.org/10.18083/lcappl.2022.4.27.
Full textAbstract:
A number of compounds with biphenyl as a mesogenic fragment and a structure simulating LC dimers, polymers with mesogenic side chain groups and a star-shaped structure have been synthesized. An approach for the synthesis of such compounds under mild conditions and without a complex purification procedure with a quantitative yield, has been developed. The approach is the implementation of “click chemistry” methods for the synthesis of LC compounds. It has been shown that the LC state is observed only if there are three biphenyls in one molecule, i.e., the compounds appear to be similar to polyesters with biphenyl as a mesogenic moiety. The determining factor in the formation of the LC state and particularly smectic mesomorphism of compounds with such a potentially “weak” mesogen as biphenyl is the formation of intermolecular hydrogen bonds.
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Bujacz, Anna, Maria Rutkiewicz-Krotewicz, Karolina Nowakowska-Sapota, and Marianna Turkiewicz. "Crystal structure and enzymatic properties of a broad substrate-specificity psychrophilic aminotransferase from the Antarctic soil bacteriumPsychrobactersp. B6." Acta Crystallographica Section D Biological Crystallography 71, no. 3 (February 26, 2015): 632–45. http://dx.doi.org/10.1107/s1399004714028016.
Full textAbstract:
Aminotransferases (ATs) are enzymes that are commonly used in the chemical and pharmaceutical industries for the synthesis of natural and non-natural amino acids by transamination reactions. Currently, the easily accessible enzymes from mesophilic organisms are most commonly used; however, for economical and ecological reasons the utilization of aminotransferases from psychrophiles would be more advantageous, as their optimum reaction temperature is usually significantly lower than for the mesophilic ATs. Here, gene isolation, protein expression, purification, enzymatic properties and structural studies are reported for the cold-active aromatic amino-acid aminotransferase (PsyArAT) fromPsychrobactersp. B6, a psychrotrophic, Gram-negative strain from Antarctic soil. Preliminary computational analysis indicated dual functionality of the enzyme through the ability to utilize both aromatic amino acids and aspartate as substrates. This postulation was confirmed by enzymatic activity tests, which showed that it belonged to the class EC 2.6.1.57. The first crystal structures of a psychrophilic aromatic amino-acid aminotransferase have been determined at resolutions of 2.19 Å for the native enzyme (PsyArAT) and 2.76 Å for its complex with aspartic acid (PsyArAT/D). Both types of crystals grew in the monoclinic space groupP21under slightly different crystallization conditions. ThePsyArAT crystals contained a dimer (90 kDa) in the asymmetric unit, which corresponds to the active form of this enzyme, whereas the crystals of thePsyArAT/D complex included four dimers showing different stages of the transamination reaction.
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Ithayaraja, M., N. Janardan, Rik K. Wierenga, H. S. Savithri, and M. R. N. Murthy. "Crystal structure of a thiolase fromEscherichia coliat 1.8 Å resolution." Acta Crystallographica Section F Structural Biology Communications 72, no. 7 (June 22, 2016): 534–44. http://dx.doi.org/10.1107/s2053230x16008451.
Full textAbstract:
Thiolases catalyze the Claisen condensation of two acetyl-CoA molecules to give acetoacetyl-CoA, as well as the reverse degradative reaction. Four genes coding for thiolases or thiolase-like proteins are found in theEscherichia coligenome. In this communication, the successful cloning, purification, crystallization and structure determination at 1.8 Å resolution of a homotetramericE. colithiolase are reported. The structure ofE. colithiolase co-crystallized with acetyl-CoA at 1.9 Å resolution is also reported. As observed in other tetrameric thiolases, the presentE. colithiolase is a dimer of two tight dimers and probably functions as a biodegradative enzyme. Comparison of the structure and biochemical properties of theE. colienzyme with those of other well studied thiolases reveals certain novel features of this enzyme, such as the modification of a lysine in the dimeric interface, the possible oxidation of the catalytic Cys88 in the structure of the enzyme obtained in the presence of CoA and active-site hydration. The tetrameric enzyme also displays an interesting departure from exact 222 symmetry, which is probably related to the deformation of the tetramerization domain that stabilizes the oligomeric structure of the protein. The current study allows the identification of substrate-binding amino-acid residues and water networks at the active site and provides the structural framework required for understanding the biochemical properties as well as the physiological function of thisE. colithiolase.
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LANIŠNIK RIŽNER, Tea, Gabriele MOELLER, Hubert H. THOLE, Marija ŽAKELJ-MAVRIČ, and Jerzy ADAMSKI. "A novel 17β-hydroxysteroid dehydrogenase in the fungus Cochliobolus lunatus: new insights into the evolution of steroid-hormone signalling." Biochemical Journal 337, no. 3 (January 25, 1999): 425–31. http://dx.doi.org/10.1042/bj3370425.
Full textAbstract:
17β-Hydroxysteroid dehydrogenase (17β-HSD) from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) catalyses the reduction of steroids and of several o- and p-quinones. After purification of the enzyme, its partial amino acid sequence was determined. A PCR fragment amplified with primers derived from peptide sequences was generated for screening the Coch. lunatus cDNA library. Three independent full-length cDNA clones were isolated and sequenced, revealing an 810-bp open reading frame encoding a 270-amino-acid protein. After expression in Escherichia coli and purification to homogeneity, the enzyme was found to be active towards androstenedione and menadione, and was able to form dimers of Mr 60000. The amino acid sequence of the novel 17β-HSD demonstrated high homology with fungal carbonyl reductases, such as versicolorin reductase from Emericella nidulans (Aspergillus nidulans; VerA) and Asp. parasiticus (Ver1), polyhydroxynaphthalene reductase from Magnaporthe grisea, the product of the Brn1 gene from Coch. heterostrophus and a reductase from Colletotrichum lagenarium, which are all members of the short-chain dehydrogenase/reductase superfamily. 17β-HSDcl is the first discovered fungal 17β-hydroxysteroid dehydrogenase belonging to this family. The primary structure of this enzyme may therefore help to elucidate the evolutionary history of steroid dehydrogenases.