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Intramolecular Disulfide Bonds (intramolecular + disulfide_bond)
Selected AbstractsDetection and Function of the Intramolecular Disulfide Bond in Arginine Racemase: An Enzyme with Broad Substrate SpecificityCHEMISTRY & BIODIVERSITY, Issue 6 2010Daisuke Matsui Abstract We found that a single intramolecular disulfide bond between the cysteines C47 and C73 exists in the primary structure of arginine racemase (ArgR) from Pseudomonas taetrolens NBRC 3460, and this is the first example of a pyridoxal phosphate (PLP)-dependent amino acid racemase that contains a disulfide bond. The amino acid racemase activity was still detected, when the disulfide bond of ArgR was disrupted by site-directed mutagenesis or reduced with dithiothreitol (DTT). The thermal and pH profiles and the quaternary structure of ArgR did not change when the disulfide bond of ArgR was disrupted by site-directed mutagenesis. The substrate specificity and the overall structure did not change when the disulfide bond of ArgR was reduced with DTT after the protein was matured. However, these properties changed when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before protein maturation. The total activity of ArgR decreased when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before the protein was matured or when ArgR was expressed in the cytoplasm. Based on these results, we can conclude that the disulfide bond of ArgR is essential for ArgR to fold and mature as an amino acid racemase with broad substrate specificity. [source] Studies on structural and functional divergence among seven WhiB proteins of Mycobacterium tuberculosis H37RvFEBS JOURNAL, Issue 1 2009Md. Suhail Alam The whiB -like genes (1-7) of Mycobacterium tuberculosis are involved in cell division, nutrient starvation, pathogenesis, antibiotic resistance and stress sensing. Although the biochemical properties of WhiB1, WhiB3 and WhiB4 are known, there is no information about the other proteins. Here, we elucidate in detail the biochemical and biophysical properties of WhiB2, WhiB5, WhiB6 and WhiB7 of M. tuberculosis and present a comprehensive comparative study on the molecular properties of all WhiB proteins. UV,Vis spectroscopy has suggested the presence of a redox-sensitive [2Fe,2S] cluster in each of the WhiB proteins, which remains stably bound to the proteins in the presence of 8 m urea. The [2Fe,2S] cluster of each protein was oxidation labile but the rate of cluster loss decreased under reducing environments. The [2Fe,2S] cluster of each WhiB protein responded differently to the oxidative effect of air and oxidized glutathione. In all cases, disassembly of the [2Fe,2S] cluster was coupled with the oxidation of cysteine-thiols and the formation of two intramolecular disulfide bonds. Both CD and fluorescence spectroscopy revealed that WhiB proteins are structurally divergent members of the same family. Similar to WhiB1, WhiB3 and WhiB4, apo WhiB5, WhiB6 and WhiB7 also reduced the disulfide of insulin, a model substrate. However, the reduction efficiency varied significantly. Surprisingly, WhiB2 did not reduce the insulin disulfide, even though its basic properties were similar to those of others. The structural and functional divergence among WhiB proteins indicated that each WhiB protein is a distinguished member of the same family and together they may represent a novel redox system for M. tuberculosis. [source] Effects of proline mutations in the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activityFEBS JOURNAL, Issue 22 2000Toshiro Takai Der f 2 is the major group 2 allergen from house dust mite Dermatophagoides farinae and is composed of 129 amino-acid residues. Wild-type and six proline mutants of Der f 2 (P26A, P34A, P66A, P79A, P95A, and P99A) expressed in Escherichia coli were refolded and purified. Formations of intramolecular disulfide bonds in the purified proteins were confirmed correct. The apparent molecular masses analyzed by gel-filtration were 14,15 kDa. The IgE-binding capacity in the sera of seven mite-allergic patients, inhibitory activity for IgE-binding to immobilized wild-type Der f 2, and activity to stimulate peripheral blood basophils to release histamine in two volunteers were analyzed. P95A and P99A, which slightly differed from the wild-type Der f 2 in their CD spectrum, showed reduced IgE-binding, reduced inhibitory activity, and less histamine-releasing activity than the wild-type. P34A also showed reduced allergenicity. Considering that Pro95, Pro99 and Pro34 are closely located in loops at one end of the tertiary structure of Der f 2, we concluded that these loop regions included an IgE-binding site common to all tested patients. P66A showed reduced IgE-binding in two sera out of seven. P26A and P79A showed no reduced allergenicity. However, in immunoblot analysis after SDS/PAGE under reduced conditions, P79A showed no or markedly reduced IgE-binding while the other mutants showed IgE-binding corresponding to that in the assay using correctly refolded proteins. This suggests that Pro79 is involved in refolding of Der f 2. The findings in this study are important for the understanding of the antigenic structure of mite group 2 allergens and for manipulation of the allergens for specific immunotherapy. [source] Mass spectrometry study of ecto-5,-nucleotidase from bull seminal plasmaFEBS JOURNAL, Issue 16 2000Carlo Fini The structure of ecto-5,-nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry. MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for ,,6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N -glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high-mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl-phosphatidylinositol core contained EtN(P)Man3GlcNH2 -myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S,S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51,Cys57, Cys353,Cys358, Cys365,Cys387 and Cys476,Cys479. This work resolves details of the structure of ecto-5,-nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinositol anchor. [source] A description of the structural determination procedures of a gap junction channel at 3.5,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Michihiro Suga Intercellular signalling is an essential characteristic of multicellular organisms. Gap junctions, which consist of arrays of intercellular channels, permit the exchange of ions and small molecules between adjacent cells. Here, the structural determination of a gap junction channel composed of connexin 26 (Cx26) at 3.5,Å resolution is described. During each step of the purification process, the protein was examined using electron microscopy and/or dynamic light scattering. Dehydration of the crystals improved the resolution limits. Phase refinement using multi-crystal averaging in conjunction with noncrystallographic symmetry averaging based on strictly determined noncrystallographic symmetry operators resulted in an electron-density map for model building. The amino-acid sequence of a protomer structure consisting of the amino-terminal helix, four transmembrane helices and two extracellular loops was assigned to the electron-density map. The amino-acid assignment was confirmed using six selenomethionine (SeMet) sites in the difference Fourier map of the SeMet derivative and three intramolecular disulfide bonds in the anomalous difference Fourier map of the native crystal. [source] Structure of acostatin, a dimeric disintegrin from Southern copperhead (Agkistrodon contortrix contortrix), at 1.7,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2008Natalia Moiseeva Disintegrins are a family of small (4,14,kDa) proteins that bind to another class of proteins, integrins. Therefore, as integrin inhibitors, they can be exploited as anticancer and antiplatelet agents. Acostatin, an ,, heterodimeric disintegrin, has been isolated from the venom of Southern copperhead (Agkistrodon contortrix contortrix). The three-dimensional structure of acostatin has been determined by macromolecular crystallography using the molecular-replacement method. The asymmetric unit of the acostatin crystals consists of two heterodimers. The structure has been refined to an Rwork and Rfree of 18.6% and 21.5%, respectively, using all data in the 20,1.7,Å resolution range. The structure of all subunits is similar and is well ordered into N-terminal and C-terminal clusters with four intramolecular disulfide bonds. The overall fold consists of short ,-sheets, each of which is formed by a pair of antiparallel ,-strands connected by ,-turns and flexible loops of different lengths. Conformational flexibility is found in the RGD loops and in the C-terminal segment. The interaction of two N-terminal clusters via two intermolecular disulfide bridges anchors the ,, chains of the acostatin dimers. The C-terminal clusters of the heterodimer project in opposite directions and form a larger angle between them in comparison with other dimeric disintegrins. Extensive interactions are observed between two heterodimers, revealing an ,,,, acostatin tetramer. Further experiments are required to identify whether the ,,,, acostatin complex plays a functional role in vivo. [source] Changes in protein conformation and dynamics upon complex formation of brain-derived neurotrophic factor and its receptor: Investigation by isotope-edited Fourier transform IR spectroscopyBIOPOLYMERS, Issue 1 2002Tiansheng Li Abstract The interactions of brain-derived neurotrophic factor (BDNF) with the extracellular domain of its receptor (trkB) are investigated by employing isotope-edited Fourier transform IR (FTIR) spectroscopy. The protein secondary structures of individual BDNF and trkB in solutions are compared with those in their complex. The temperature dependence of the secondary structures of BDNF, trkB, and their complex is also investigated. Consistent with the crystal structure, we observe by FTIR spectroscopy that BDNF in solution contains predominantly , strands (,53%) and relatively low contents of other secondary structures including , turns (,16%), disordered structures (,12%), and loops (,18%) and is deficient in , helix. We also observe that trkB in solution contains mostly , strands (52%) and little , helix. Conformational changes in both BDNF and trkB are observed upon complex formation. Specifically, upon binding of BDNF, the conformational changes in trkB appear to involve mostly , turns and disordered structures while the majority of the ,-strand conformation remains unchanged. The IR data indicate that some of the disordered structures in the loop regions are likely converted to , strands upon complex formation. The FTIR spectral data of BDNF, trkB, and their complex indicate that more amide NH groups of trkB undergo H,D exchange within the complex than those of the ligand-free receptor and that the thermal stability of trkB is decreased slightly upon binding of BDNF. The FT-Raman spectra of BDNF, trkB, and their complex show that the six intramolecular disulfide bonds of trkB undergo significant conformational changes upon binding of BDNF as a result of changes in the tertiary structure of trkB. Taken together, the FTIR and Raman data are consistent with the loosening of the tertiary structure of trkB upon binding of BDNF, which leads to more solvent exposure of the amide NH group and decreased thermal stability of trkB. This finding reveals an intriguing structural property of the neurotrophin ligand,receptor complex that is in contrast to other ligand,receptor complexes such as a cytokine,receptor complex that usually shows protection of the amide NH group and increased thermal stability upon complex formation. © 2002 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 67: 10,19, 2002; DOI 10.1002/bip.10038 [source] Vascular endothelial growth factor receptor-2: Its unique signaling and specific ligand, VEGF-ECANCER SCIENCE, Issue 9 2003Masabumi Shibuya Vascular endothelial growth factor receptor-2 (VEGFR-2/KDR/Flk-1) is a high-affinity receptor for vascular endothelial growth factor-A (VEGF-A), and mediates most of the endothelial growth and survival signals from VEGF-A. VEGFR-2 has a typical tyrosine kinase receptor structure with seven immunoglobulin (Ig)-like domains in the extracellular region, as well as a long kinase insert in the tyrosine kinase domain. It utilizes a unique signaling system for DNA synthesis in vascular endothelial cells, i.e. a phospholipase C,-protein kinaseC-Raf-MAP kinase pathway. Although VEGF-A binds two receptors, VEGFR-1 and -2, a newly isolated ligand VEGF-E (Orf-virus-derived VEGF) binds and activates only VEGFR-2. Transgenic mice expressing VEGF-ENZ-7 showed a dramatic increase in angiogenesis with very few side effects (such as edema and hemorrhagic spots), suggesting strong angiogenic signaling and a potential clinical utility of VEGF-E. VEGF family members bear three loops produced via three intramolecular disulfide bonds, and cooperation between loop-1 and loop-3 is necessary for the specific binding and activation of VEGFR-2 for angiogenesis. As it directly upregulates tumor angiogenesis, VEGFR-2 is an appropriate target for suppression of solid tumor growth using exogenous antibodies, small inhibitory molecules and in vivo stimulation of the immune system. [source] | ||||||||||