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Noncovalent Complexes (noncovalent + complex)
Selected AbstractsBJ46a, a snake venom metalloproteinase inhibitorFEBS JOURNAL, Issue 10 2001Isolation, characterization, cloning, insights into its mechanism of action Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4 -reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition. [source] X-ray investigation of gene-engineered human insulin crystallized from a solution containing polysialic acidACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010V. I. Timofeev Attempts to crystallize the noncovalent complex of recombinant human insulin with polysialic acid were carried out under normal and microgravity conditions. Both crystal types belonged to the same space group, I213, with unit-cell parameters a = b = c = 77.365,Å, , = , = , = 90.00°. The reported space group and unit-cell parameters are almost identical to those of cubic insulin reported in the PDB. The results of X-ray studies confirmed that the crystals obtained were cubic insulin crystals and that they contained no polysialic acid or its fragments. Electron-density maps were calculated using X-ray diffraction sets from earth-grown and microgravity-grown crystals and the three-dimensional structure of the insulin molecule was determined and refined. The conformation and secondary-structural elements of the insulin molecule in different crystal forms were compared. [source] A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Mats Ökvist Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10,kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52,kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM,MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM,MtDS complex diffracted to 1.6 and 2.1,Å resolution, respectively. [source] Influence of the Charge State on the Structures and Interactions of Vancomycin Antibiotics with Cell-Wall Analogue Peptides: Experimental and Theoretical StudiesCHEMISTRY - A EUROPEAN JOURNAL, Issue 9 2009Zhibo Yang Dr. Abstract Charge matters! The charge state significantly influences the conformation and the binding energy between vancomycin antibiotic and bacterial cell-wall analogue peptides (see figure). Surface-induced dissociation (SID) studies provide a quantitative comparison between the stabilities of different charge states of the complex. In this study we examined the effect of the charge state on the energetics and dynamics of dissociation of the noncovalent complex between the vancomycin and the cell-wall peptide analogue N,,N, -diacetyl- L -Lys- D -Ala- D -Ala (V,Ac2LKdAdA). The binding energies between the vancomycin and the peptide were obtained from the RRKM (Rice, Ramsperger, Kassel, Marcus) modeling of the time- and energy-resolved surface-induced dissociation (SID) experiments. Our results demonstrate that the stability of the complex towards fragmentation increases in the order: doubly protonated Disruption of structural and functional integrity of ,2 -macroglobulin by cathepsin EFEBS JOURNAL, Issue 6 2003Mitsue Shibata ,2 -Macroglobulin (,2M) is an abundant glycoprotein with the intrinsic capacity for capturing diverse proteins for rapid delivery into cells. After internalization by the receptor- mediated endocytosis, ,2M-protein complexes were rapidly degraded in the endolysosome system. Although this is an important pathway for clearance of both ,2M and biological targets, little is known about the nature of ,2M degradation in the endolysosome system. To investigate the possible involvement of intracellular aspartic proteinases in the disruption of structural and functional integrity of ,2M in the endolysosome system, we examined the capacity of ,2M for interacting with cathepsin E and cathepsin D under acidic conditions and the nature of its degradation. ,2M was efficiently associated with cathepsin E under acidic conditions to form noncovalent complexes and rapidly degraded through the generation of three major proteins with apparent molecular masses of 90, 85 and 30 kDa. Parallel with this reaction, ,2M resulted in the rapid loss of its antiproteolytic activity. Analysis of the N-terminal amino-acid sequences of these proteins revealed that ,2M was selectively cleaved at the Phe811-Leu812 bond in about 100mer downstream of the bait region. In contrast, little change was observed for ,2M treated by cathepsin D under the same conditions. Together, the synthetic SPAFLA peptide corresponding to the Ser808,Ala813 sequence of human ,2M, which contains the cathepsin E-cleavage site, was selectively cleaved by cathepsin E, but not cathepsin D. These results suggest the possible involvement of cathepsin E in disruption of the structural and functional integrity of ,2M in the endolysosome system. [source] Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serumFEBS JOURNAL, Issue 24 2002Surza L. G. Rocha Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human ,1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood. [source] Organization of nucleobase-functionalized ,-peptides investigated by soft electrospray ionization mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2009Nicola Diezemann Abstract The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI-MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) as well as a quadrupole/time-of-flight tandem mass spectrometer (QqToF-MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical ,-peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of ,-peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR-MS and QqToF-MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)-MS/MS and infrared multiphoton dissociation (IRMPD)-MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd. [source] DNA minor groove binders as potential antitumor and antimicrobial agentsMEDICINAL RESEARCH REVIEWS, Issue 4 2004Pier Giovanni Baraldi Abstract DNA minor groove binders constitute an important class of derivatives in anticancer therapy. Some of these compounds form noncovalent complexes with DNA (e.g., distamycin A, Hoechst 33258, and pentamidine) while others DNA-binding compounds (such as CC-1065) cause cleavages in the DNA backbone. In this article, we have reviewed the minor groove binders currently in preclinical evaluation in the last years. Diarylamidines such as DAPI, berenil, and pentamidine; bis-benzimidazoles such as Hoechst 33258; ecteinascidins, pyrrololo [2,1- c]-[1,4]-benzodiazepines (PBDs), CC-1065, and distamycins are the classes discussed in this review article. A special section has been dedicated to hybrid molecules resulted by the combination of two minor groove binders, especially for derivatives of naturally occurring antitumor agents, such as anthramycin or the alkylating unit of the antibiotic CC-1065, and distamycin frames. © 2004 Wiley Periodicals, Inc. Med Res Rev, 24, No. 4, 475,528, 2004 [source] Energy-resolved in-source collisionally induced dissociation for the evaluation of the relative stability of noncovalent complexes in the gas phaseRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 23 2005Nataliya Lyapchenko Energy-resolved in-source collisionally induced dissociation (CID) studies on the complexation of alkali metal cations by some crown ethers, nucleic acid bases, and amino acids have been performed. It has been shown that the cone voltage corresponding to the maximum ion abundance (Vc,Imax) of the breakdown curve is characteristic of a given ion and not influenced by the cone desolvation process or the composition of the solution. Very good agreement of the Vc,Imax value with the bond strength of the ion has been observed. Determination of the Vc,Imax values for different ionic species is a useful, simple, and inexpensive way to obtain their relative stabilities in in-source CID conditions. Copyright © 2005 John Wiley & Sons, Ltd. [source] Homogeneous esterification by lipase from Burkholderia cepacia in the fluorinated solventBIOTECHNOLOGY PROGRESS, Issue 6 2008S. Shipovskov Abstract The formation of noncovalent complexes between lipase from Burkholderia cepacia and the fluorinated ionic surfactant Kryto Development Product 4606 KDP was shown to promote the solubilization of the enzyme in the fluorinated solvent perfluoro(methylcyclohexane) (PFMC) and its operation as a catalyst in the fluorous PFMC/hexane biphasic system (FBS). In the reaction of esterification of 1-phenylethanol and vinyl acetate, the solubilized lipase showed high stereospecificity (ca. 99%) with a catalytic efficiency up to 56 nmol/(U h) with a high operational and storage stability. Temperature modulation of the FBS miscibility allowed the separation and recovery of the solubilized lipase for at least three times. The results are of practical importance for the development of recoverable biocatalysts soluble and active in the FBS. [source]
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