proteoglycan site mapping and site directed mutagenesis

... 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 mutagenesis, Trp402 of IPU appears ... participating in subsite )1 The residues that form potential subsites )1 and )2 are reportedly more conserved in GH family 49 than the residues in subsites +1 and +2 [17] Comparison of the active sites of ... K.H., Armand, ˚ S., Visser, J & Dijkstra, B.W (1999) 1.68-A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site- directed mutagenesis...

Ngày tải lên: 19/02/2014, 16:20

... HCl pH 7.5 Site- directed mutagenesis The His120Asn, His145Asn and Asn149Asp mutant forms of human arginase II were obtained by a two-step PCR [40], using the QuickChange site- directed mutagenesis ... of human arginase II with substrate and metal ions was examined by sitedirected mutagenesis and kinetic studies Selection of target residues (Asp149, His120 and His145) was based on the roles ... S-(2-boronoethyl)-l-cysteine (BEC) His120 and His145, and the corresponding His101 and His126 in arginase I, were described among the ligands for coordination of Mn2+A and Mn2+B, respectively [9,24] However,...

Ngày tải lên: 20/02/2014, 02:21

... ligand-manganese distances RESULTS AND DISCUSSION By site- directed mutagenesis, a D153N mutant form of E coli agmatinase was obtained The mutant enzyme retained about 5% of wild-type activity and ... (AUH) and rat liver arginase (1rla) H, S, T and stands for a-helix, strand, turn and 310-helix Conserved residues are marked by an asterisk Identical behaviour was previously described, and confirmed ... ACCTATGCG-3¢) and the 3¢ complementary primer 5¢-AT TAATGGCATGCTTTACCCGT-3¢ Using the PCR products of agmatinase with the GfiA and CfiT substitutions in the coding and noncoding strands, respectively, and...

Ngày tải lên: 21/02/2014, 01:21

... binding site for HbpR and to examine the necessity of the sensing A-domain of HbpR for DNA binding The role of individual nucleotides and nucleotide motifs was characterized by site- directed mutagenesis ... slightly between the proximal and the distal sites Some were located 1757 HbpR-binding site D Tropel and J R van der Meer Fig DNA sequence of the different mutants and the HbpR concentration required ... mutants and (each with 3· GC and five mutations) Both mutants and were bound equally well by HbpR under the conditions of EMSA Changing, more or less randomly, other sets of bp (mutants 10, 11 and...

Ngày tải lên: 07/03/2014, 17:20

... site- directed mutagenesis, and the effects on chloride sensitivity, substrate selectivity and inhibitor potency were observed Results PCR mutagenesis of CL1 and CL2 site residues of tACE and ACE2 ... of Glu398 and Ser511 projecting into this region Table tACE and ACE2 residues subjected to site- directed mutagenesis For all residues subjected to PCR mutagenesis, the chloride-binding site at ... CL1 and CL2 sites of tACE and ACE2 are shown in Fig In tACE, the residues that coordinate the chloride ion at the CL1 site are Arg186, Trp485 and Arg489, which are conserved as Arg169, Trp477 and...

Ngày tải lên: 16/03/2014, 04:20

... All reagents, unless otherwise specified, were purchased from Sigma or Merck Site- directed mutagenesis Site- directed mutagenesis was performed using a plasmid pT7-7 [13] containing an inserted ... interactions (4 and 6) and different active -site residues at positions 39 and 451 (/4e, /4a, /6, g4e, g4a, g6; Tables and 6) were calculated by isolating each DGà, as described in the Materials and methods ... (Sfbgly50) active site The interactions are identified as g6, g4e, /4e, /6 and /3 Residue 451 is represented by g, and / represents residue 39, e denotes an equatorial hydroxyl, and 6, and indicate...

Ngày tải lên: 16/03/2014, 18:20

... concentration and activity Protein concentrations were measured using the method of Bradford [5] and a standard curve derived from bovine serum albumin For testing the activity and determining ... amino acid sequence showing the NRD1bS (boxed) and NRD1bL (dot boxed) domains, putative DxD motifs (written white on black), and the H–E site in position and position (marked with arrows) of the appropriate ... of earlier suggestions concerning catalytic amino acids and proposed reaction mechanisms of glycosyltransferases [8] Because site- directed mutagenesis studies have not so far been performed for...

Ngày tải lên: 17/03/2014, 09:20

... Sweeden) and 3H-NAAG substrate was obtained from Perkin-Elmer (Boston, MA, USA) Site- directed mutagenesis Site- directed mutagenesis was carried out using the QuikChange Site- Directed Mutagenesis ... Arg534, and Arg536 (Fig 2B) To verify this model experimentally, the N519D, N519V, R534L, and R536L mutants were constructed and kinetically characterized and the results are summarized in Table and ... cells and large scale expression of mutant forms of rhGCPII Conclusions In conclusion, we report a detailed analysis of the active site of glutamate carboxypeptidase II using sitedirected mutagenesis...

Ngày tải lên: 23/03/2014, 07:20

... (A) and I50K–HAKN1 (B) bound to their preferred binding sites After binding and hydroxyl radical attack, free (F) and bound (B) DNA were separated and analysed The left and right panels in (A) and ... expression and purification of recombinant proteins HAKN1 homeodomain and C-terminal sequences were amplified and cloned in-frame into the EcoRI and PstI sites of the expression vector pMAL-c2 (New England ... of Ile50 and Arg55 must have been particularly important Other homeodomains that contain Ile50 and Arg55 are those of the TGIF, Meis and Bell families [22] and yeast MATa1 [36] TGIF and Meis...

Ngày tải lên: 23/03/2014, 13:20

... spectroscopy and X-ray crystallography Initial mutagenesis studies suggested that 314 R A Rothery et al there may be two Q-sites present – a polar QB site (equivalent to the QP site) , and an apolar QA site ... observed at the QP -site in the structure of FrdABCD: T205, F206, Q225 and K228 from FrdB; R28, E29, W86, L89 and A93 from FrdC; and W14, F17, G18, H80, R81 and H84 from FrdD [3,4,10] Site- directed mutants ... electron density has been identified recently between the QP and QD sites (the ‘M’ site) , ˚ and is centered approximately 13 A from each Q -site [4] If this electron density corresponds to an additional...

Ngày tải lên: 23/03/2014, 13:20

... from AppliChem (Darmstadt, Germany) Site- directed mutagenesis Site- directed mutagenesis of (His)6PNAE was achieved using the QuikChangeTM in vitro Site- Directed Mutagenesis Kit, according to the ... (His)6PNAE Site- directed mutagenesis In this study, we have produced wild-type (His)6PNAE and 10 muteins of it Four of six cysteine residues in positions 20, 132, 170 and 257 were changed to alanine and ... of PNAE In order to gain evidence for these suggestions, inhibitor studies, site- directed mutagenesis experiments and modelling of PNAE were performed Inhibitor studies Several known inhibitors...

Ngày tải lên: 24/03/2014, 04:21

... (1989) Site- directed mutagenesis by overlap extension using the polymerase chain reaction Gene 77, 5159 37 Braman J, Papworth C & Greener A (1996) Site- directed mutagenesis using double-stranded ... crystal structure of AspA and site- directed mutagenesis studies, two positively charged residues (Arg29 and Lys327) were proposed to bind the two carboxylate groups of (C4 and C1, respectively) [12,20,21] ... active site of AspB [23] The roles of the key active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) and their interactions are discussed in the text Sufxes A, B and...

Ngày tải lên: 29/03/2014, 23:20

... phosphorylase scanning mutagenesis of the active site of trehalose phosphorylase [16] and report on results of steadystate kinetic analysis and chemical rescue studies for site- directed ScTPase mutants ... used sitedirected replacement by Ala and detailed kinetic comparison of wild-type and mutant enzymes to portray the tasks fullled by Arg507 and Lys512 in the interaction network of active site ... stabilization in a pre-organized polar active site: the catalytic role of Lys-80 in Candida tenuis xylose reductase (AKR2B5) probed by site- directed mutagenesis and functional complementation studies...

Ngày tải lên: 30/03/2014, 04:20

... respective ÔsitesÕ in StP As in other GPs, the catalytic site and the PLP binding site of StP are virtually identical to the corresponding sites in rmGP and EcMalP By contrast, the regulatory sites ... and frequently show interaction with oxyanion ligands such as phosphate or sulfate, the ÔTOWER argininesÕ of StP were selected as candidates to be replaced by alanine-scanning site- directed mutagenesis ... for site- directed mutagenesis, and their PCRbased replacement by alanine was chosen to eliminate all electrostatic interactions at the respective position It is worth noting that Arg234 and Arg242...

Ngày tải lên: 31/03/2014, 01:20

... forward strand and R- designates it is on the reverse strand) DNA sequence was obtained from both forward and reverse strands around the site- directed mutations and for the entire strand for random ... iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif We have conducted site- directed and random mutagenesis in the S cerevisiae wild-type strain SCY876 Site- Directed mutagenesis ... Auxotrophic Strains in Candida glabrata & Random and SiteDirected Mutagenesis of ERG25 in Saccharomyces cerevisiae Major Professor: Dr Martin Bard Candida albicans and Candida glabrata are opportunistic...

Ngày tải lên: 24/08/2014, 12:11

... the active site of ACE2 and tACE Binding interactions of the inhibitor (A) MLN-4760 at the active site of ACE2 and (B) lisinopril at the active site of tACE Hydrogen bonds to the ligand are shown ... to ACE2 (yellow) and tACE (white) (A) Binding site of CL1 in ACE2 and tACE; (B) binding site of CL2 in ACE2 and tACE Residue numbering for ACE2 is first The chloride ion is green and the zinc ion ... relative to Arg522 in ACE and is somewhat closer to the zinc-binding site The CL2 binding site is in close proximity to the catalytic site ˚ (10.4 A away from the zinc) and is located at the interface...

Ngày tải lên: 20/02/2014, 01:20

... sequence motif at the active site is underlined P1 and P2 mark phosphorylation sites and (serine residues) in the E1a chains of eukaryotic PDH and BCDH complexes ODPA and ODPT, E1a chain of PDH ... that the two active sites of pigeon breast muscle E1p work in an Ôalternating site mechanismÕ and that intersubunit interactions may play an essential part in the catalysis by, and regulation of, ... was from New England Nuclear (Boston, MA, USA) Centrifugal filter devices were purchased from Millipore Bacterial strains and plasmids E coli strain TG1recO and plasmids pKBstE1a and pKBstE1b, expressing...

Ngày tải lên: 20/02/2014, 23:20

... partial lipophilicity values [44] and a distance-dependent function [45] Site- directed mutagenesis This step was performed with the QuickChangeTM SiteDirected Mutagenesis method from Stratagene ... interaction between F134 and the Fig Relative position of residues toward androstenedione in the active site D476 and D309 may stabilize androstenedione by interacting with the C(16) and C(2) hydrogens, ... residues K130 and F134 appear to form a hydrophobic surface close to the heme and control the position of the substrate inside the active site (differences for norandrogens and androgens) These...

Ngày tải lên: 21/02/2014, 15:20

... docking on the above A Site- directed mutagenesis of aryl-alcohol oxidase model, site- directed mutagenesis and kinetic studies were used to identify the enzyme active site and evaluate the role ... FAD and conserved AAO His502 and His546, and glucose oxidase His516 and His559 (re-side of the FAD ring), are also shown (glucose oxidase residues in italics) From AAO and glucose oxidase 1GAL and ... strains and plasmids cDNA encoding P eryngii AAO with its own signal peptide was cloned into plasmid palcA, and the resulting vector (pALAAO) was used for site- directed mutagenesis, and transformation...

Ngày tải lên: 07/03/2014, 11:20

... calculated and expressed in lmặmg)1ặmin)1 Site- directed mutagenesis, protein expression and purication Presteady-state kinetic parameters for the acetylation of NAT Site- directed mutagenesis ... acetyl-coenzyme A (AcCoA) in the absence of amine [21] and the active site Cys68 or Cys69 has been further identied through thiol-specic modication and site- directed mutagenesis [2224] The rst crystal structure ... Ile (Y190I) and to Ala (Y190A), was carried out using the pPH70D vector and QuickChange site- directed mutagenesis kit (Stratagene)[52] The oligonucleotide primers used for Y190F, Y190I and Y190A...

Ngày tải lên: 16/03/2014, 00:20