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Significant Conformational Change (significant + conformational_change)
Selected AbstractsIn vitro expansion of DNA triplet repeats with bulge binders and different DNA polymerasesFEBS JOURNAL, Issue 18 2008Di Ouyang The expansion of DNA repeat sequences is associated with many genetic diseases in humans. Simple bulge DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. To probe the possible role of bulged structures in DNA slippage, we designed and synthesized a pair of simple chiral spirocyclic compounds [Xi Z, Ouyang D & Mu HT (2006) Bioorg Med Chem Lett16, 1180,1184], DDI-1A and DDI-1B, which mimic the molecular architecture of the enediyne antitumor antibiotic neocarzinostatin chromophore. Both compounds strongly stimulated slippage in various DNA repeats in vitro. Enhanced slippage synthesis was found to be synchronous for primer and template. CD spectra and UV thermal stability studies supported the idea that DDI-1A and DDI-1B exhibited selective binding to the DNA bulge and induced a significant conformational change in bulge DNA. The proposed mechanism for the observed in vitro expansion of long DNA is discussed. [source] Arginine-induced conformational change in the c -ring/a -subunit interface of ATP synthaseFEBS JOURNAL, Issue 9 2008Thomas Vorburger The rotational mechanism of ATP synthases requires a unique interface between the stator a subunit and the rotating c -ring to accommodate stability and smooth rotation simultaneously. The recently published c -ring crystal structure of the ATP synthase of Ilyobacter tartaricus represents the conformation in the absence of subunit a. However, in order to understand the dynamic structural processes during ion translocation, studies in the presence of subunit a are required. Here, by intersubunit Cys,Cys cross-linking, the relative topography of the interacting helical faces of subunits a and c from the I. tartaricus ATP synthase has been mapped. According to these data, the essential stator arginine (aR226) is located between the c -ring binding pocket and the cytoplasm. Furthermore, the spatially vicinal residues cT67C and cG68C in the isolated c -ring structure yielded largely asymmetric cross-linking products with aN230C of subunit a, suggesting a small, but significant conformational change of binding-site residues upon contact with subunit a. The conformational change was dependent on the positive charge of the stator arginine or the aR226H substitution. Energy-minimization calculations revealed possible modes for the interaction between the stator arginine and the c -ring. These biochemical results and structural restraints support a model in which the stator arginine operates as a pendulum, moving in and out of the binding pocket as the c -ring rotates along the interface with subunit a. This mechanism allows efficient interaction between subunit a and the c- ring and simultaneously allows almost frictionless movement against each other. [source] Site-directed mutagenesis of the active site serine290 in flavanone 3,-hydroxylase from Petunia hybridaFEBS JOURNAL, Issue 3 2000Richard Luka Flavanone 3,-hydroxylase (FHT) catalyzes a pivotal reaction in the formation of flavonoids, catechins, proanthocyanidins and anthocyanidins. In the presence of oxygen and ferrous ions the enzyme couples the oxidative decarboxylation of 2-oxoglutarate, releasing carbon dioxide and succinate, with the oxidation of flavanones to produce dihydroflavonols. The hydroxylase had been cloned from Petunia hybrida and expressed in Escherichia coli, and a rapid isolation method for the highly active, recombinant enzyme had been developed. Sequence alignments of the Petunia hydroxylase with various hydroxylating 2-oxoglutarate-dependent dioxygenases revealed few conserved amino acids, including a strictly conserved serine residue (Ser290). This serine was mutated to threonine, alanine or valine, which represent amino acids found at the corresponding sequence position in other 2-oxoglutarate-dependent enzymes. The mutant enzymes were expressed in E. coli and purified to homogeneity. The catalytic activities of [Thr290]FHT and [Ala290]FHT were still significant, albeit greatly reduced to 20 and 8%, respectively, in comparison to the wild-type enzyme, whereas the activity of [Val290]FHT was negligible (about 1%). Kinetic analyses of purified wild-type and mutant enzymes revealed the functional significance of Ser290 for 2-oxoglutarate-binding. The spatial configurations of the related Fe(II)-dependent isopenicillin N and deacetoxycephalosporin C synthases have been reported recently and provide the lead structures for the conformation of other dioxygenases. Circular dichroism spectroscopy was employed to compare the conformation of pure flavanone 3,-hydroxylase with that of isopenicillin N synthase. A double minimum in the far ultraviolet region at 222 nm and 208,210 nm and a maximum at 191,193 nm which are characteristic for ,-helical regions were observed, and the spectra of the two dioxygenases fully matched revealing their close structural relationship. Furthermore, the spectrum remained unchanged after addition of either ferrous ions, 2-oxoglutarate or both of these cofactors, ruling out a significant conformational change of the enzyme on cofactor-binding. [source] Systematic epitope analysis of the p26 EIAV core proteinJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2007Adriana Soutullo Abstract The major core protein of equine infectious anemia virus (EIAV), p26, is one of the primary immunogenic structural proteins during a persistent infection of horses and is highly conserved among antigenically variants of viral isolates. In order to investigate its immune profile in more detail for a better diagnostic, an epitope mapping was carried out by means of two libraries of overlapping peptide fragments prepared by simultaneous and parallel SPPS on derivatized cellulose membranes (SPOT synthesis). Polyclonal equine sera from infected horses were used for the biological assay. Particularly two promising continuous epitopes (NAMRHL and MYACRD) were localized on the C-terminal extreme of p26, region 194,222. A cyclic synthetic fragment of 29 amino acid residues containing the identified epitopes was designed and studied. A significant conformational change towards a helical structure was observed when the peptide was cyclized by a bridge between Cys198 and Cys218. This observation correlated with an improvement of its ability to be recognized by specific antibodies in an EIA (Enzyme-linked Immunosorbent assay). These results suggest that the conformationally restricted synthetic antigen adequately mimics the native structure of this region of p26 core protein. Copyright © 2007 John Wiley & Sons, Ltd. [source] Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3PROTEIN SCIENCE, Issue 9 2006Mingyue He Abstract The crystal structures of the Fab, fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 104 -fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position. [source] Regulation of ,-Chymotrypsin Catalysis by Ferric Porphyrins and CyclodextrinsCHEMISTRY - AN ASIAN JOURNAL, Issue 4 2008Koji Kano Prof. Abstract Positively charged ,-chymotrypsin (ChT) formed a 1:1 complex with negatively charged 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) (FeTPPS) in phosphate buffer at pH,7.4 through electrostatic interaction. In spite of the large binding constant (K=4.8×105,M,1), FeTPPS could not completely inhibit the catalysis of ChT in the hydrolysis of the model substrate, N -succinyl- L -phenylalanine p -nitroanilide (SPNA). The degree of inhibition (60,%) was saturated at 1.6,equivalents of FeTPPS, which indicates that covering of the active site of ChT by FeTPPS was insufficient. The enzymatic activity lowered by FeTPPS was entirely recovered for the freshly prepared sample when the porphyrin on the protein surface was detached by per- O -methylated ,-cyclodextrin (TMe-,-CD), which formed a stable 1:2 inclusion complex with FeTPPS (K1=1.26×106,M,1, K2=6.3×104,M,1). FeTPPS gradually induced irreversible denaturation of ChT, and the denatured ChT further lost its catalytic ability. No repairing effect of TMe-,-CD was observed with irreversibly denatured ChT. A new reversible inhibitor, 5,10,15,20-tetrakis[4-(3,5-dicarboxyphenylmethoxy)phenyl]porphyrinato iron(III) (FeP8M), was then designed, and its inhibitory behavior was examined. FeP8M formed very stable 1:1 and 1:2 FeP8M/ChT complexes with ChT, the K1 and K2 values being 2.0×108 and 1.0×106,M,1, respectively. FeP8M effectively inhibited the ChT-catalyzed hydrolysis of SPNA (maximum degree of inhibition=85,%), and the activity of ChT was recovered by per- O -methylated ,-cyclodextrin. No irreversible denaturation of ChT occurred upon binding with FeP8M. The kinetic data support the observation that, for nonincubated samples, both inhibitors did not cause significant conformational change in ChT and inhibited the ChT activity by covering the active site of the enzyme. [source] Dye-affinity hollow-fibres and their lysozyme adsorption,desorption characteristicsPOLYMER INTERNATIONAL, Issue 10 2001Serap, enel Abstract Dye-affinity adsorption is increasingly used for protein separation. Hollow-fibres have advantages as adsorbents in comparison to conventional bead supports because they are not compressible and can eliminate internal diffusion limitations. The aim of this study was to explore in detail the performance of polyamide hollow-fibres to which Reactive Green HE-4BD was attached for adsorption of lysozyme. The hollow-fibre was characterized by scanning electron microscopy. These dye-carrying hollow-fibres (26.3,µmol,g,1) were used in the lysozyme adsorption,elution studies. The effect of initial concentration of lysozyme and medium pH on the adsorption efficiency of dye-attached hollow-fibres was studied in a batch system. The non-specific adsorption of lysozyme on the polyamide hollow-fibres was 1.8,mg,g,1. Reactive Green HE-4BD attachment significantly increased the lysozyme adsorption up to 41.1,mg,g,1. Langmuir adsorption model was found to be applicable in interpreting lead adsorption by Reactive Green HE-4BD attached hollow fibres. Significant amount of the adsorbed lysozyme (up to 95%) was eluted in 1,h in the elution medium containing 1.0,M NaSCN at pH 8.0. In order to determine the effects of adsorption conditions on possible conformational changes of lysozyme structure, fluorescence spectrophotometry was employed. We concluded that polyamide dye-affinity hollow-fibres can be applied for lysozyme adsorption without causing any significant conformational changes. Repeated adsorption,elution processes showed that these dye-attached hollow-fibres are suitable for lysozyme adsorption. © 2001 Society of Chemical Industry [source] Structure of Escherichia coli tryptophanaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2006Shao-Yang Ku Pyridoxal 5,-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the ,-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the ,-proton of the substrate for ,-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate,PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal. [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] Nucleosome Immobilization Strategies for Single-Pair FRET Microscopy,CHEMPHYSCHEM, Issue 14 2008Wiepke J. A. Koopmans Abstract All genomic transactions in eukaryotes take place in the context of the nucleosome, the basic unit of chromatin, which is responsible for DNA compaction. Overcoming the steric hindrance that nucleosomes present for DNA-processing enzymes requires significant conformational changes. The dynamics of these have been hard to resolve. Single-pair Fluorescence Resonance Energy Transfer (spFRET) microscopy is a powerful technique for observing conformational dynamics of the nucleosome. Nucleosome immobilization allows the extension of observation times to a limit set only by photobleaching, and thus opens the possibility of studying processes occurring on timescales ranging from milliseconds to minutes. It is crucial however, that immobilization itself does not introduce artifacts in the dynamics. Here we report on various nucleosome immobilization strategies, such as single-point attachment to polyethylene glycol (PEG) or surfaces coated with bovine serum albumin (BSA), and confinement in porous agarose or polyacrylamide gels. We compare the immobilization specificity and structural integrity of immobilized nucleosomes. A crosslinked star polyethylene glycol coating performs best with respect to tethering specificity and nucleosome integrity, and enables us to reproduce for the first time bulk nucleosome unwrapping kinetics in single nucleosomes without immobilization artifacts. [source] | ||||||||||