site directed mutagenesis in the research of protein kinases the case of protein kinase ck2
Báo cáo khoa học: Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA ppt
... both binding sites in the complexes formed by KNOX and Bell proteins Sequences outside the homeodomain may also in uence the binding properties of the protein Indeed, a stretch of 16 amino acids ... the right of each panel indicate the DNA sequence (5¢ end in the upper part) of the corresponding strand in this region Below the footprints, the sequence of the corresponding binding site is shown ... calculate the position of the footprint Letters to the right of each panel indicate the DNA sequence (5¢ end in the upper part) of the corresponding strand in this region In the lower part, the sequence...
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Tài liệu Báo cáo khoa học: nsights into the reaction mechanism of glycosyl hydrolase family 49 Site-directed mutagenesis and substrate preference of isopullulanase doc
... Trp402 of IPU, is reportedly located in the vicinity of the active site and could form a binding site for a glucosyl unit in subsite )1 (Fig 4B) Together with the observations from the site- directed ... for binding of Glc +2 of the a-(1fi4)-linked glucose units of pullulan (Fig 4A) IPU is an anomer-inverting enzyme Comparison of the active sites of the model of IPU and Dex49A The active site ... (activating the nucleophilic water molecule) in the first step of the reaction A carbonium ion intermediate subsequently forms, and is further attacked by the water molecule The study of site- directed...
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Báo cáo Y học: Role of tyrosine 238 in the active site of Rhodotorula gracilis D-amino acid oxidase A site-directed mutagenesis study docx
... potentials The Y238 RgDAAO mutants were puried as holoenzymes (retaining their FAD prosthetic group) The mutants, in their oxidized state, show the typical spectrum of the FADcontaining avoproteins (line ... following the binding of anthranilate (Fig 3, inset) and the shoulder at % 500 nm following the binding of benzoate (De497nm of 7500ặM)1ặcm)1 vs a gure of 2000 4000ặM)1ặcm)1 observed with the other ... in binding were observed for Y238 mutants with the ligands tested (Table 2) These results indicate that the mode of ligand binding is retained in the two mutants, and that the alteration of the...
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Báo cáo khoa học: Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis potx
... evolution of nucleotide-binding protein Nature 250, 194–199 Wierenga RK, Terpstra P & Hol WG (1986) Prediction of the occurrence of the ADP-binding bab-fold in proteins, using an amino acid sequence ... value in the absence of the other substrate In the presence of oxoglutarate, however, coenzyme binding was tighter and therefore measurable and, in particular, gave measurable Kd values for binding ... However, in this latter case, the interaction of enzyme and coenzyme caused a very small change in fluorescence The measurements with NADH in the absence of oxoglutarate show a weakening of binding...
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Tài liệu Báo cáo khoa học: Identification of critical active-site residues in angiotensin-converting enzyme-2 (ACE2) by site-directed mutagenesis docx
... Subdomain I (N-terminal) contains the zinc-binding site, which faces into the deep cleft formed by the two subdomains connected at the base of the cleft The hinge-bending motion observed upon inhibitor ... critical for binding of the substrate Maintaining the positive charge at this position (R273K) is not sufficient for docking of the peptide into the ACE2 active site In fact, the distance of this positive ... in the side chain at this position while maintaining most of the hydrophobic surface area For comparative purposes, the arginine residue was also replaced with a lysine in order to maintain the...
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Tài liệu Báo cáo khoa học: Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies Alteration of substrate specificity by replacement of Asn149 with Asp docx
... residues known to be involved in binding and hydrolysis of the guanidino group of l-arginine by arginase are strictly conserved in the active site of the agmatinases [19] Moreover, modeling studies have ... agmatine hydrolysis, in studying the inhibitory effect of agmatine on arginine hydrolysis, reactions were followed by measuring the formation of ornithine, determined by the method of Chinard ... Differences in the binding of the a-carboxylate and a-amino groups of BEC were also ascribed to the larger volume of the active site cleft of arginase II, which allows more water-mediated enzyme–inhibitor...
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Tài liệu Báo cáo khoa học: Site-directed mutagenesis of a loop at the active site of E1 (a2b2) of the pyruvate dehydrogenase complex A possible common sequence motif docx
... inactivated Binding of E1 to the peripheral subunit-binding domain of E2 The E1 component of the PDH complex of B stearothermophilus is bound to the E2 core mainly by interaction of its Table Kinetic ... properly as a lid for the active site, thereby granting easier access for the cofactor ThDP to its binding site The lower thermal stability of the mutant E1s and the increased Vmax in the DCPIP assay, ... crystal structures in that the arginine residues corresponding to E1aR282 of the B stearothermophilus E1 are not pointing towards the active site, identified by the C2-carbon of the cofactor ThDP Although...
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Tài liệu Báo cáo Y học: Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling ppt
... deletions in the agmatinase sequence were modelled using the DGLOOP set of options in WHATIF; the whole loops and the two connecting amino acids at the beginning and the end, were mutated to glycines ... liver arginase (yellow) and the modelled structure of E coli agmatinase (blue) Note the shorter extension, in the case of E coli agmatinase, of the loop indicated by the letter a differed in these ... coordination interaction with one of the manganese ions This, together with the fact that the positions of other Fig Scheme of the binuclear manganese cluster and the localization of the side-chains...
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Báo cáo khoa học: Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2-producing ligninolytic enzyme pot
... localize the active site (substrate-binding pocket) of AAO by molecular docking The enzyme consists of two domains, the FAD-binding domain (bottom part) and the substratebinding domain (top part), ... 50% of them clustered together in front of the rectus (re)-face of the isoalloxazine ring of the FAD cofactor This substrate location is shown in Fig 2A, which includes the 10 molecules of veratryl ... clustering together after docking The putative substrate-binding pocket is connected to the protein surface by a main channel providing direct access to the re-side of the isoalloxazine ring, near...
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Báo cáo khoa học: Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain pot
... contacts the DNA The objectives of this work were to identify critical motifs in the binding site for HbpR and to examine the necessity of the sensing A-domain of HbpR for DNA binding The role of individual ... larger than the sequences of the palindromes [19,21], but the exact contribution of nucleotides in the binding site contacted by the proteins is not known The current hypothesis of hexameric ... A-domain of HbpR is important in forming the oligomeric structure and for DNA binding, by cloning an hbpR gene devoid of the A-domain, purifying this protein, and analyzing its DNA-binding characteristics...
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Báo cáo khoa học: Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190 pot
... the imidazole of the catalytic triad His159 in the papain-like Cys proteinase [44] Mutations of the Trp to either Tyr, Phe, Ile or Ala (the strength of the Hisaromatic interaction decreases in ... The reaction rate was determined at 25 C by measuring the linear increase in absorbance at 400 nm during the initial Control experiments were carried out in the absence of NAT2 The slope of the ... On the basis of the Ping Pong mechanism, the acetylation rate (k2) of NAT2 by the acetyl donor is independent of the transacetylation rate (k4) of the arylamine substrate Because the values of...
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Báo cáo khoa học: Residues affecting the chloride regulation and substrate selectivity of the angiotensin-converting enzymes (ACE and ACE2) identified by site-directed mutagenesis pot
... for each of the mutant variants Protein determination Protein concentrations were determined using the bicinchoninic acid assay with bovine serum albumin as standard [32] Site- directed mutagenesis ... stabilizing complex formation for the residues in subdomain II involved directly in substrate binding [20] The CL2 site of ACE has previously been suggested to contain the mechanistically binding ... R514 that has the greatest in uence on the chloride regulation of activity, and that the CL2 site of ACE does indeed contain the mechanistically binding chloride ion The ACE2 CL2 site residue...
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Báo cáo khoa học: Investigation of the substrate specificity of a b-glycosidase from Spodoptera frugiperda using site-directed mutagenesis and bioenergetics analysis pdf
... Bioscience) The nonretained proteins were washed out in the dialysis buffer, while the retained proteins were eluted from the column using an NaCl gradient prepared in the same buffer The presence of the ... calculated, strengthening the importance of E451 in determining Sfbgly50 specificity The large length of /6 interaction is incompatible with a hydrogen bond In addition, the replacement of Q39 with ... fine tuning of the active site structure, in order to increase the catalytic activity of the mutant b-glycosidases However, the identification of these residues is not an easy task owing to the large...
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Báo cáo khoa học: Probing the unfolding region of ribonuclease A by site-directed mutagenesis potx
... to the thermodynamic stability of the entire RNase A molecule The coincidence of the degree of destabilization in these variants points to an effect on the stability of the entire region rather ... to the transition state, underlining the predominant importance of this region for maintaining the natively folded structure of the RNase A molecule Interestingly, the hydrophobic nature of residues ... accessibility of amino acid residues of wild-type RNase A The relative accessibility was calculated using the program WHAT IF [32] and relates the accessibility of the side chain of the residue in the protein...
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Báo cáo khoa học: Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site pot
... when the crystal structure of the spinach enzyme was determined, first in the presence of the inhibitor IpOHA [8] and later with product bound in the active site [9] There are several interesting ... a single active site [1] One of the main purposes of this study was to try to dissect the two reactions by mutagenesis of active site residues, expecting to find mutants of the E coli enzyme in ... followed the published procedure [14] for performing and analyzing NADPH binding experiments In addition, we examined the use of fluorescence resonance energy transfer to monitor NADPH binding In these...
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Báo cáo khoa học: Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis potx
... distortion of the coordination sphere of the active -site zincs Our results indicate that the binding of the glutarate part of the inhibitor in the S1¢ pocket contributes to the inhibition effect of the ... maintaining productive architecture of the S1¢ site of GCPII, including the positioning of the Tyr552 side chain Despite similarities between lysine and arginine residues, the lysine side chain ... acids delineating the substrate binding cavity of GCPII were designed and introduced into the GCPII ectodomain (rhGCPII; amino acids 44–750) using site- directed mutagenesis Individual amino acid...
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Báo cáo khoa học: Defining the QP-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy doc
... cluster of SdhB [1,6] In the case of FrdABCD, the membrane-intrinsic domain does not contain heme, but instead contains two menaquinones at discreet sites in the crystallized form of the enzyme ... Q -site) , is located in the interface region between the FrdCD subunits and the [3Fe-4S] cluster coordinating region of FrdB on the cytoplasmic side of the membrane The other site, the ˚ QD site ... would therefore be of interest to examine the effects of a range of sitedirected mutants on the HOQNO binding properties and enzymology of the enzyme In this paper, we evaluate the effects of mutation...
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Báo cáo Y học: Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis ppt
... of the wild-type, indicating that these cysteines might not have a big in uence on the binding of the substrate In conclusion, we have identified by site- directed mutagenesis the residues of the ... serine protease inhibitors The next clear information obtained was the necessity of histidine groups which proved the active role of at least one histidine in substrate binding The following site- directed ... Nevertheless, the specific activities of each of the muteins decreased With the exchange of Cys132, there was a dramatic decrease (fourfold) in the specific activity, and in the case of Cys170, the...
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Báo cáo khoa học: The phosphate site of trehalose phosphorylase from Schizophyllum communeprobed by site-directed mutagenesis and chemical rescue studies pptx
... experiments, in which a small molecule compensates for the missing side chain of a relevant site- directed mutant, often provide valuable insights into the role of active -site arginine [3236] and lysine ... However, even in the absence of correction for the inuence of molecular volume and hydrophobicity of the rescue reagent, Table shows clearly that the effect of the pKa of the amine and guanidine derivatives ... concentration of G1P released against the incubation time was linear in all cases, indicating that enzyme inactivation did not interfere with determination of the initial rate under the conditions used Enzymatic...
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Báo cáo khoa học: Binding areas of urokinase-type plasminogen activator– plasminogen activator inhibitor-1 complex for endocytosis receptors of the low-density lipoprotein receptor family, determined by site-directed mutagenesis doc
... proteaseserpin complexes and for the rst time implicate the SPD of uPA in the binding The residues that mediate the binding afnity of the A-chain of uPA remain to be established In the long term, ... numbering from the N-terminus of the native protein [27]) The presence of the antibody reduced the binding signicantly (Fig 3) We therefore studied the effect of Ala substitution of clusters of ... domain and the kringle contribute to the binding, but putative endocytosis receptor-binding residues in the kringle remain unknown The involvement of the growth factor domain readily explains the previously...
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