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Disulfide Bridges (disulfide + bridge)
Selected AbstractsLimited tendency of ,-helical residues to form disulfide bridges: a structural explanationJOURNAL OF PEPTIDE SCIENCE, Issue 12 2006Alfonso De Simone Abstract Disulfide bridges have an enormous impact on the structure of a large number of proteins and polypeptides. Understanding the structural basis that regulates their formation may be important for the design of novel peptide-based molecules with a specific fold and stability. Here we report a statistical analysis of the relationships between secondary structure and disulfide bond formation, carried out using a large database of protein structures. Our analyses confirm the observation sporadically reported in previous investigations that cysteine residues located in ,-helices display a limited tendency to form disulfide bridges. The very low occurrence of the disulfide bond in all ,-chains compared to all ,-chains indicates that this property is also evident when proteins with different topologies are investigated. Taking advantage of the large database that endorsed the analysis on relatively rare motifs, we demonstrate that cysteine residues embedded in 310 helices present a good tendency to form disulfide bonds. This result is somewhat surprising since 310 helices are commonly assimilated into ,-helices. A plausible structural explanation for the observed data has been derived combining analyses of disulfide bond sequence separation and of the length of the different secondary structure elements. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [source] Screening for disulfide-rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 17 2001Susanne Neitz Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57,u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116,u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate. Copyright © 2001 John Wiley & Sons, Ltd. [source] Structurally Minimized ,-Conotoxin Analogues as Sodium Channel Blockers: Implications for Designing Conopeptide-Based TherapeuticsCHEMMEDCHEM, Issue 3 2009Tiffany Abstract Transforming the neuroactive toxins of cone snails into small-size compounds poses a challenge due to the presence of multiple disulfide bridges. Herein we describe our successful efforts in minimizing the size of ,-conotoxin while retaining its biological activity. Disulfide bridges that stabilize the native conformation of conotoxins pose a challenge in the synthesis of smaller conotoxin analogues. Herein we describe the synthesis of a minimized analogue of the analgesic ,-conotoxin KIIIA that blocks two sodium channel subtypes, the neuronal NaV1.2 and skeletal muscle NaV1.4. Three disulfide-deficient analogues of KIIIA were initially synthesized in which the native disulfide bridge formed between either C1C9, C2C15, or C4C16 was removed. Deletion of the first bridge only slightly affected the peptide's bioactivity. To further minimize this analogue, the N-terminal residue was removed and two nonessential serine residues were replaced by a single 5-amino-3-oxapentanoic acid residue. The resulting "polytide" analogue retained the ability to block sodium channels and to produce analgesia. Until now, the peptidomimetic approach applied to conotoxins has progressed only modestly at best; thus, the disulfide-deficient analogues containing backbone spacers provide an alternative advance toward the development of conopeptide-based therapeutics. [source] The Janus-faced atracotoxins are specific blockers of invertebrate KCa channelsFEBS JOURNAL, Issue 16 2008Simon J. Gunning The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca2+ -activated K+ (KCa) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked KCa channels in cockroach unpaired dorsal median neurons with an IC50 of 2 nm, but it did not significantly affect a wide range of other voltage-activated K+, Ca2+ or Na+ channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca2+ -activated K+ (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC50 , 10 ,m) and its lack of activity on rat dorsal root ganglion neuron KCa channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate KCa channels and suggests that KCa channels might be potential insecticide targets. [source] Native and subunit molecular mass and quarternary structure of the hemoglobin from the primitive branchiopod crustacean Triops cancriformisFEBS JOURNAL, Issue 17 2006Morgane Rousselot Many branchiopod crustaceans are endowed with extracellular, high-molecular-weight hemoglobins whose exact structural characteristics have remained a matter of conjecture. By using a broad spectrum of techniques, we provide precise and coherent information on the hemoglobin of one of the phylogenetically ,oldest' extant branchiopods, the tadpole shrimp Triops cancriformis. The hemoglobin dissociated under reducing conditions into two subunits, designated TcHbA and TcHbB, with masses of 35 775 ± 4 and 36 055 ± 4 Da, respectively, determined by ESI-MS. Nonreducing conditions showed only two disulfide-bridged dimers, a homodimer of TcHbA, designated D1 (71 548 ± 5 Da), and the heterodimer D2 (71 828 ± 5 Da). Carbamidomethylation of free SH groups revealed the presence of three cysteines per subunit and indicated one intrasubunit and one intersubunit disulfide bridge. Ultracentrifugation and light-scattering experiments under nondenaturating conditions yielded mass estimates that suggested an uneven number of 17 subunits forming the native hemoglobin. This unrealistic number resulted from the presence of two size classes (16-mer and 18-mer), which were recognized by native PAGE and Ferguson plot analysis. ESI-MS revealed three hemoglobin isoforms with masses of 588.1 kDa, 662.0 kDa, and 665.0 kDa. The 16-mer and the smaller 18-mer species are supposed to be composed of TcHbA only, given the dominance of this subunit type in SDS/PAGE. Transmission electron microscopy of negatively stained specimens showed a population of compact molecules with geometrical extensions of 14, 16 and 9 nm. The proposed stoichiometric model of quarternary structure provides the missing link to achieve a mechanistic understanding of the structure,function relationships among the multimeric arthropodan hemoglobins. [source] Analysis of the thyrotropin-releasing hormone-degrading ectoenzyme by site-directed mutagenesis of cysteine residuesFEBS JOURNAL, Issue 9 2000Cys68 is involved in disulfide-linked dimerization Thyrotropin-releasing hormone-degrading ectoenzyme is a member of the M1 family of Zn-dependent aminopeptidases and catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH2). Cloning of the cDNA of this enzyme and biochemical studies revealed that the large extracellular domain of the enzyme with the catalytically active site contains nine cysteine residues that are highly conserved among species. To investigate the functional role of these cysteines in TRH-DE we used a site-directed mutagenesis approach and replaced individually each cysteine by a serine residue. The results revealed that the proteolytically truncated and enzymatically fully active enzyme consists of two identical subunits that are associated noncovalently by protein,protein interactions but not via interchain S-S bridges. The eight cysteines contained within this region are all important for the structure of the individual subunit and the enzymatic activity, which is dramatically reduced in all mutant enzymes. This is even true for the four cysteines that are clustered within the C-terminal domain remote from the Zn-binding consensus sequence HEICH. In contrast, Cys68, which resides within the stalk region seven residues from the end of the hydrophobic membrane-spanning domain, can be replaced by serine without a significant change in the enzymatic activity. Interestingly, this residue is involved in the formation of an interchain disulfide bridge. Covalent dimerization of the subunits, however, does not seem to be essential for efficient biosynthesis, enzymatic activity and trafficking to the cell surface. [source] Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxideFEBS JOURNAL, Issue 5 2000Evidence for a dithiol, disulfide equilibrium, implications for redox regulation Calcineurin (CaN) is a Ca2+ -and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe,Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3,8 µm and the inactivation was reversed by 2,3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+,Zn2+ dinuclear centre. Upon H2O2 -mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN. [source] Assignment of a single disulfide bridge in rat liver methionine adenosyltransferaseFEBS JOURNAL, Issue 1 2000María L. Martínez-Chantar Rat liver methionine adenosyltransferase incorporated 8 mol of N -ethylmaleimide per mol of subunit upon denaturation in the presence of 8 m urea, whereas 10 such groups were labelled when dithiothreitol was also included. This observation prompted a re-examination of the state of the thiol groups, which was carried out using peptide mapping, amino acid analysis and N-terminal sequencing. The results obtained revealed a disulfide bridge between Cys35 and Cys61. This disulfide did not appear to be conserved because cysteines homologous to residue 61 do not exist in methionine adenosyltransferases of other origins, therefore suggesting its importance for the differential aspects of the liver-specific enzyme. [source] Thermomyces lanuginosus: properties of strains and their hemicellulasesFEMS MICROBIOLOGY REVIEWS, Issue 1 2003Suren Singh Abstract The non-cellulolytic Thermomyces lanuginosus is a widespread and frequently isolated thermophilic fungus. Several strains of this fungus have been reported to produce high levels of cellulase-free ,-xylanase both in shake-flask and bioreactor cultivations but intraspecies variability in terms of ,-xylanase production is apparent. Furthermore all strains produce low extracellular levels of other hemicellulases involved in hemicellulose hydrolysis. Crude and purified hemicellulases from this fungus are stable at high temperatures in the range of 50,80°C and over a broad pH range (3,12). Various strains are reported to produce a single xylanase with molecular masses varying between 23 and 29 kDa and pI values between 3.7 and 4.1. The gene encoding the T. lanuginosus xylanase has been cloned and sequenced and is shown to be a member of family 11 glycosyl hydrolases. The crystal structure of the xylanase indicates that the enzyme consists of two ,-sheets and one ,-helix and forms a rigid complex with the three central sugars of xyloheptaose whereas the peripheral sugars might assume different configurations thereby allowing branched xylan chains to be accepted. The presence of an extra disulfide bridge between the ,-strand and the ,-helix, as well as to an increase in the density of charged residues throughout the xylanase might contribute to the thermostability. The ability of T. lanuginosus to produce high levels of cellulase-free thermostable xylanase has made the fungus an attractive source of thermostable xylanase with potential as a bleach-boosting agent in the pulp and paper industry and as an additive in the baking industry. [source] Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variantsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2006A. J. GALE Summary.,Background:,The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664,C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa. Aims:,Here, we characterize the functional parameters of C664,C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662,C1828 FVIII). Methods:,In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex. Results:,Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662,C1828 FVIIIa had normal activity while C664,C1826 FVIIIa had reduced activity. Both C664,C1826 FVIIIa and C662,C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different. Conclusion:,Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding. [source] Active TEM-1 ,-lactamase mutants with random peptides inserted in three contiguous surface loopsPROTEIN SCIENCE, Issue 10 2006Pascale Mathonet Abstract Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 ,-lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two ,-strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well-conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N-terminal helix and the first ,-strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final ,-strand to the C-terminal helix, particularly if the wild-type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium-sized loops. The combination of these activity-selected libraries generated a huge library containing active hybrid enzymes with all three loops modified. [source] Conversion of trypsin to a functional threonine proteasePROTEIN SCIENCE, Issue 6 2006Teaster T. Baird Jr Abstract The hydroxyl group of a serine residue at position 195 acts as a nucleophile in the catalytic mechanism of the serine proteases. However, the chemically similar residue, threonine, is rarely used in similar functional context. Our structural modeling suggests that the Ser 195 , Thr trypsin variant is inactive due to negative steric interaction between the methyl group on the ,-carbon of Thr 195 and the disulfide bridge formed by cysteines 42 and 58. By simultaneously truncating residues 42 and 58 and substituting Ser 195 with threonine, we have successfully converted the classic serine protease trypsin to a functional threonine protease. Substitution of residue 42 with alanine and residue 58 with alanine or valine in the presence of threonine 195 results in trypsin variants that are 102,104 -fold less active than wild type in kcat/KM but >106 -fold more active than the Ser 195 , Thr single variant. The substitutions do not alter the substrate specificity of the enzyme in the P1,, P4, positions. Removal of the disulfide bridge decreases the overall thermostability of the enzyme, but it is partially rescued by the presence of threonine at position 195. [source] The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venomPROTEIN SCIENCE, Issue 7 2000Juan J. Calvete Abstract The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed. [source] Removal of the N-terminal hexapeptide from human ,2-microglobulin facilitates protein aggregation and fibril formationPROTEIN SCIENCE, Issue 5 2000G. Esposito Abstract The solution structure and stability of N-terminally truncated ,2-microglobulin (,N6,2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that ,N6,2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at ,M concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that ,N6,2-m is significantly less protected than its wild-type counterpart. Analysis of ,N6,2-m by NMR shows that this loss of protection occurs in , strands I, III, and part of II. At mM concentration gel filtration analysis shows that ,N6,2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of ,2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that ,N6,2-m could be a key intermediate of a proteolytic pathway of ,2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of ,2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between ,-strands far removed from this constrain is greatly perturbed. [source] Screening for disulfide-rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 17 2001Susanne Neitz Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57,u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116,u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate. Copyright © 2001 John Wiley & Sons, Ltd. [source] A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavityACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2009Daniel A. Breustedt Tear lipocalin (TLC) with the bound artificial ligand 1,4-butanediol has been crystallized in space group P21 with four protein molecules in the asymmetric unit and its X-ray structure has been solved at 2.6,Å resolution. TLC is a member of the lipocalin family that binds ligands with diverse chemical structures, such as fatty acids, phospholipids and cholesterol as well as microbial siderophores and the antibiotic rifampin. Previous X-ray structural analysis of apo TLC crystallized in space group C2 revealed a rather large bifurcated ligand pocket and a partially disordered loop region at the entrace to the cavity. Analysis of the P21 crystal form uncovered major conformational changes (i) in ,-strands B, C and D, (ii) in loops 1, 2 and 4 at the open end of the ,-barrel and (iii) in the extended C-terminal segment, which is attached to the ,-barrel via a disulfide bridge. The structural comparison indicates high conformational plasticity of the loop region as well as of deeper parts of the ligand pocket, thus allowing adaptation to ligands that differ vastly in size and shape. This illustrates a mechanism for promiscuity in ligand recognition which may also be relevant for some other physiologically important members of the lipocalin protein family. [source] Structure of recombinant human cyclophilin J, a novel member of the cyclophilin familyACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2005Li-Li Huang Cyclophilins (CyPs) are a large class of highly conserved ubiquitous peptidyl-prolyl cis,trans isomerases. CyPs have also been identified as being a specific receptor for the immunosuppressive drug cyclosporin A and are involved in a variety of biological functions. CyPJ is a novel member of the CyP family and human CyPJ (hCyPJ) is the protein encoded by a cyclophilin-like gene from human foetal brain, which shows 50% sequence identity to human cyclophilin A (hCyPA). Recombinant hCyPJ was expressed in Escherichia coli and purified. The three-dimensional structure of hCyPJ has been determined by molecular replacement using the hCyPA structure as the search model and has been refined at 2.6,Å resolution. The hCyPJ molecule contains four helices and one ,-barrel composed of eight antiparallel ,-strands. The overall secondary and tertiary structures of hCyPJ are similar to those of hCyPA, but hCyPJ contains an additional disulfide bridge and four segments with conformations that are strikingly different from those of hCyPA. His43 and Gln52 of hCyPJ are expected to be the active sites based on sequence alignment with hCyPA. The hCyPJ structure shows a conserved water molecule close to His43 and Gln52 which appears to support the solvent-assisted mechanism. [source] The structure of a triple mutant of pI258 arsenate reductase from Staphylococcus aureus and its 5-thio-2-nitrobenzoic acid adductACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2004Joris Messens Structural insights into formation of the complex between the ubiquitous thiol,disulfide oxidoreductase thioredoxin and its oxidized substrate are under-documented owing to its entropical instability. In vitro, it is possible via a reaction with 5,5,-dithiobis-(2-nitrobenzoic acid) to make a stable mixed-disulfide complex between thioredoxin from Staphylococcus aureus and one of its substrates, oxidized pI258 arsenate reductase (ArsC) from S. aureus. In the absence of the crystal structure of an ArsC,thioredoxin complex, the structures of two precursors of the complex, the ArsC triple mutant ArsC C10SC15AC82S and its 5-thio-2-nitrobenzoic acid (TNB) adduct, were determined. The ArsC triple mutant has a structure very similar to that of the reduced form of wild-type ArsC, with a folded redox helix and a buried catalytic Cys89. In the adduct form, the TNB molecule is buried in a hydrophobic pocket and the disulfide bridge between TNB and Cys89 is sterically inaccessible to thioredoxin. In order to form a mixed disulfide between ArsC and thioredoxin, a change in the orientation of the TNB,Cys89 disulfide in the structure is necessary. [source] Crystallization and preliminary X-ray analysis of candoxin, a novel reversible neurotoxin from the Malayan krait Bungarus candidusACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2003Palasingam Paaventhan Candoxin, a novel three-finger toxin from Bungarus candidus, is a reversible antagonist of muscle (,,,,) but a poorly reversible antagonist of neuronal ,7 nicotinic acetylcholine receptors. It has a molecular weight of 7344,Da, with 66 amino-acid residues including ten half-cystines. The fifth disulfide bridge is located at the tip of loop I (Cys6,Cys11) instead of in loop II as found in other ,-neurotoxins. Interestingly, candoxin lacks the segment cyclized by the fifth disulfide bridge at the tip of the middle loop of long-chain neurotoxins, which was reported to be critical for binding to ,7 receptors. As a first step to determining its three-dimensional structure, candoxin was crystallized by the hanging-drop vapour-diffusion technique in conditions around 1.5,M sodium chloride, 10%(v/v) ethanol. The crystals formed belonged to the hexagonal system, space group P6222, with unit-cell parameters a = 54.88, b = 54.88, c = 75.54,Å, , = , = 90, , = 120°, and diffract to a resolution of 1.80,Å. The crystallographic asymmetric unit contains one molecule of candoxin, with an estimated solvent content of 44.6%. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated. [source] Crystallization and preliminary X-ray studies of a galactose-specific lectin from the seeds of bitter gourd (Momordica charantia)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Thyageshwar Chandran A galactose-specific lectin from the seeds of bitter gourd (Momordica charantia) is a four-chain type II ribosome-inactivating protein (RIP) resulting from covalent association through a disulfide bridge between two identical copies of a two-chain unit. The available structural information on such four-chain RIPs is meagre. The bitter gourd lectin was therefore crystallized for structural investigation and the crystals have been characterized. It is anticipated that the structure of the orthorhombic crystals will be analysed using molecular replacement by taking advantage of its sequence, and presumably structural, homology to normal two-chain type II RIPs. [source] A C-terminal disulfide bond in the copper-containing amine oxidase from pea seedlings violates the twofold symmetry of the molecular dimerACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2006Anthony P. Duff The structure of a newly crystallized form of the copper-dependent amine oxidase from pea seedlings has been refined at a resolution of 2.2,Å to a final R factor of 0.181. The structure (form II) was originally discovered during a study of xenon binding to copper-dependent amine oxidases as a probe for dioxygen-binding sites [Duff et al. (2004), J. Mol. Biol.344, 599,607]. The form II crystals belong to space group P21, with two dimers in the asymmetric unit. The overall structure is very similar to the crystals of form I in space group P212121 with a dimer in the asymmetric unit [Kumar et al. (1996), Structure, 4, 943,955]. In form I the last three residues (644,647) observable in the two subunits were apparently splayed apart. It was noted that the absence of a disulfide bond between the Cys647 residues of the two subunits was inconsistent with chemical evidence for the absence of free sulfhydryl groups. In both of the crystallographically independent dimers of form II the two subunits are clearly joined by a disulfide bridge between the C-terminal cysteine residues. This is only possible if the two polypeptide chains in the dimer adopt different conformations near the C-terminus so that the twofold symmetry is lost. A proline residue (645) two residues before the cysteine has a cis conformation in one chain and a trans conformation in the other. As a result, the disulfide bond lies more than 5,Å from the twofold axis. The loss of local twofold symmetry in form II can be explained by intermolecular contacts, which provide an asymmetric environment. [source] Structurally Minimized ,-Conotoxin Analogues as Sodium Channel Blockers: Implications for Designing Conopeptide-Based TherapeuticsCHEMMEDCHEM, Issue 3 2009Tiffany Abstract Transforming the neuroactive toxins of cone snails into small-size compounds poses a challenge due to the presence of multiple disulfide bridges. Herein we describe our successful efforts in minimizing the size of ,-conotoxin while retaining its biological activity. Disulfide bridges that stabilize the native conformation of conotoxins pose a challenge in the synthesis of smaller conotoxin analogues. Herein we describe the synthesis of a minimized analogue of the analgesic ,-conotoxin KIIIA that blocks two sodium channel subtypes, the neuronal NaV1.2 and skeletal muscle NaV1.4. Three disulfide-deficient analogues of KIIIA were initially synthesized in which the native disulfide bridge formed between either C1C9, C2C15, or C4C16 was removed. Deletion of the first bridge only slightly affected the peptide's bioactivity. To further minimize this analogue, the N-terminal residue was removed and two nonessential serine residues were replaced by a single 5-amino-3-oxapentanoic acid residue. The resulting "polytide" analogue retained the ability to block sodium channels and to produce analgesia. Until now, the peptidomimetic approach applied to conotoxins has progressed only modestly at best; thus, the disulfide-deficient analogues containing backbone spacers provide an alternative advance toward the development of conopeptide-based therapeutics. [source] Anomalous electrophoretic behavior of a very acidic protein: Ribonuclease U2ELECTROPHORESIS, Issue 18 2005Lucía García-Ortega Abstract Ribonuclease U2 is a low-molecular-weight acidic protein with three disulfide bridges. This protein displays an anomalous electrophoretic behavior on standard SDS-PAGE. The electrophoretic mobility of the nonreduced protein roughly corresponds to its molecular mass while the migration of the reduced protein would be in accordance with the expected molecular mass of the protein dimer. This study reveals that the protein does not bind SDS under the SDS-PAGE conditions, its electrophoretic mobility being only determined by its electrostatic charge and hydrodynamic properties. In addition, the nonreduced protein cannot be blotted to a membrane. Unfolding of the protein upon reduction of its disulfide bridges enables electrotransference to membranes due to a restricted diffusion along the electrophoresis gel. [source] Cloning and Characterization of the cDNA Encoding the Masquerade-like Serine Proteinase Homologue Gene of the Silkworm, Bombyx moriENTOMOLOGICAL RESEARCH, Issue 3 2002Doo-Sang PARK ABSTRACT From Bombyx mori larvae, RT-PCR and cDNA library screening isolated masquerade-like serine proteinase homologue cDNA gene, proposed to be related to insect immunity and its characteristics were examined. The isolated gene is composed of 1.3 kb of nucleotide and 420 amino acid residues were encoded. According to the results of database search, the isolated gene showed high sequence homology with Holotrichia and Tenebrio's 45 kDa protein, Drosophila CG5390 gene. Moreover, it is composed of regulatory domain and catalytic domain, which is characteristic of serine proteinase that can be found in the insect immune reaction and embryonic development processes. Enzyme activation site by proteolytic cleavage and the sequence of three amino acids participate in the catalytic triad of enzyme and 14 cystein residues used in disulfide bridges are well conserved with the compared genes. The mRNA expression was increased following E. coli injection and constitutive expression was also observed before injection by Northern blot analysis. [source] Molecular cloning and functional expression of a gene encoding an antiarrhythmia peptide derived from the scorpion toxinFEBS JOURNAL, Issue 18 2002Fang Peng From a cDNA library of Chinese scorpion Buthus martensii Karsch, full-length cDNAs of 351 nucleotides encoding precursors (named BmKIM) that contain signal peptides of 21 amino acid residues, a mature toxin of 61 residues with four disulfide bridges, and an extra Gly-Lys-Lys tail, were isolated. The genomic sequence of BmKIM was cloned and sequenced; it consisted of two exons disrupted by an intron of 1622 bp, the largest known in scorpion toxin genomes, inserted in the region encoding the signal peptide. The cDNA was expressed in Escherichia coli. The recombinant BmKIM was toxic to both mammal and insects. This is the first report that a toxin with such high sequence homology with an insect-specific depressant toxin group exhibits toxicity to mammals. Using whole cell patch-clamp recording, it was discovered that the recombinant BmKIM inhibited thesodium current in rat dorsal root ganglion neurons andventricular myocytes and protected against aconitine- induced cardiac arrhythmia. [source] The solution structure of gomesin, an antimicrobial cysteine-rich peptide from the spiderFEBS JOURNAL, Issue 4 2002Nicolas Mandard Gomesin is the first peptide isolated from spider exhibiting antimicrobial activities. This highly cationic peptide is composed of 18 amino-acid residues including four cysteines forming two disulfide linkages. The solution structure of gomesin has been determined using proton two-dimensional NMR (2D-NMR) and restrained molecular dynamics calculations. The global fold of gomesin consists in a well-resolved two-stranded antiparallel ,,sheet connected by a noncanonical ,,turn. A comparison between the structures of gomesin and protegrin-1 from porcine and androctonin from scorpion outlines several common features in the distribution of hydrophobic and hydrophilic residues. The N- and C-termini, the ,,turn and one face of the ,,sheet are hydrophilic, but the hydrophobicity of the other face depends on the peptide. The similarities suggest that the molecules interact with membranes in an analogous manner. The importance of the intramolecular disulfide bridges in the biological activity of gomesin is being investigated. [source] Stepwise proteolytic removal of the , subdomain in ,-lactalbuminFEBS JOURNAL, Issue 15 2001The protein remains folded, can form the molten globule in acid solution Bovine ,-lactalbumin (,-LA) is an ,/, protein which adopts partly folded states when dissolved at low pH (A-state), by removal of the protein-bound calcium at neutral pH and low salt concentration (apo-state), as well as in aqueous trifluoroethanol. Previous spectroscopic studies have indicated that the A-state of ,-LA at pH 2.0, considered a prototype molten globule, has a native-like fold in which the helical core is mostly retained, while the , subdomain is less structured. Here, we investigate the conformational features of three derivatives of ,-LA characterized by a single peptide bond fission or a deletion of 12 or 19/22 amino-acid residues of the , subdomain of the native protein (approximately from residue 34 to 57). These ,-LA derivatives were obtained by limited proteolysis of the protein in its partly folded state(s). A nicked ,-LA species consisting of fragments 1-,3,40 and 41,123 (nicked-LA) was prepared by thermolytic digestion of the 123-residue chain of ,-LA in 50% (v/v) aqueous trifluoroethanol. Two truncated or gapped protein species given by fragments 1,40 and 53,123 (des,1-LA) or fragments 1,34 and 54-,57,123 (des,2-LA) were obtained by digestion of ,-LA with pepsin in acid or with proteinase K at neutral pH in its apo-state, respectively. The two protein fragments of nicked or gapped ,-LA are covalently linked by the four disulfide bridges of the native protein. CD measurements revealed that, in aqueous solution at neutral pH and in the presence of calcium, the three protein species maintain the helical secondary structure of intact ,-LA, while the tertiary structure is strongly affected by the proteolytic cleavages of the chain. Temperature effects of CD signals in the far- and near-UV region reveal a much more labile tertiary structure in the ,-LA derivatives, while the secondary structure is mostly retained even upon heating. In acid solution at pH 2.0, the three ,-LA variants adopt a conformational state essentially identical to the molten globule displayed by intact ,-LA, as demonstrated by CD measurements. Moreover, they bind strongly the fluorescent dye 8-anilinonaphthalene-1-sulfonate, which is considered a diagnostic feature of the molten globule of proteins. Therefore, the , subdomain can be removed from the ,-LA molecule without impairing the capability of the rest of the chain to adopt a molten globule state. The results of this protein dissection study provide direct experimental evidence that in the ,-LA molten globule only the , domain is structured. [source] Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-,-1,4-glucanase from blue mussel, Mytilus edulisFEBS JOURNAL, Issue 16 2000Bingze Xu A cellulase (endo-,-1,4- d -glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 °C. Another unusual feature is that the enzyme retains 55,60% of its maximum activity at 0 °C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 °C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication). [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] Probing ligand-induced conformational changes of human CD38FEBS JOURNAL, Issue 10 2000Valérie Berthelier The lymphoid surface antigen CD38 is basically a NAD+glycohydrolase, which is also involved in the metabolism of cyclic ADP-ribose. Besides, this ecto-enzyme has potential signalling roles in T- and B-cells. Such multiple functions prompted us to study the molecular dynamics of the CD38 protein and especially the relationship between its ecto-enzymatic active site and its epitope, i.e. the binding site of most known anti-CD38 monoclonal antibodies. Both epitopic and enzymatic sites were shown to be degraded by proteases, such as trypsin or chymotrypsin. This sensitivity was almost entirely suppressed in the presence of substrates or inhibitors. Both sites were also degraded in the presence of reducing agents, as dithiothreitol. Inhibitory ligands induced the same resistance of both sites against reducing attack. The binding of CD38 ligands to the active site triggers therefore conformational changes that shield some backbone bonds and disulfide bridges against, respectively, proteolytic cleavage or reduction. This transconformation was found moreover to irreversibly take place after incubation with substrates such as NAD+ in the presence of dithiothreitol. The epitope remained preserved, while the enzymatic activity was lost. This inactivation probably resulted from the covalent trapping of the catalytically reactive intermediate in the active site (i.e. paracatalytic inactivation). These data have major implications in the knowledge of the CD38 structure, especially with regard to the location of disulfide bridges and their accessibility. Potential consequences of the conformational plasticity of CD38 should also be considered in its physiological functions such as signalling. [source] | |||||||||||||||||||||||||||||||