site directed mutagenesis for improving biophysical properties of vh domains
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
... 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 ... by replacement of this residue with aspartate To the best of our knowledge, this is the first report in which the substrate specificity of arginase was altered by using sitedirected mutagenesis Experimental ... of human arginase II, was kindly supplied by S Cederbaum (University of California, Los Angeles) Synthetic nucleotide primers were obtained from Invitrogen and the QuickChange site- directed mutagenesis...
- 9
- 651
- 0
Báo cáo khoa học: Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55-1 pptx
... group of l-aspartate forms hydrogen-bonding interactions with the side chains of Thr101, Asn142 and His188 [23] In the present study, we performed site- directed mutagenesis experiments on all of ... effect of this mutation is on the value of kcat The mutation of Thr101 to an alanine has a large effect on the catalytic efciency of AspB For the T101A mutant, a plot of various concentrations of ... prole of the T141A mutant of AspB that Thr141 is not the acidic group responsible for the descending limb of the pH-rate prole of wild-type AspB Therefore, we conclude that the protonation state of...
- 14
- 354
- 0
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
... Fig The 3D structures of PAI-1 and uPA The gure shows ribbon diagrams of the structure of the active conformation of PAI-1 [53] (pdb le 1B3K) and the structure of the SPD of uPA [27] (pdb le 1LMW) ... background For analyses of the binding of complexes of uPA with PAI-1 mutants, we rst mutated groups of three residues to Ala If a triple mutation resulted in more than a twofold reduction in binding of ... the use of the biaevaluation 3.0 software (global tting) resulted in a Kd value for binding of the wild-type uPAwild-type PAI-1 complex to either of the three receptors of % nm, while Kd for the...
- 17
- 269
- 0
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
... catalytic residues of IPU Also, Trp425 of Dex49A, which is the corresponding residue of Trp402 of IPU, is reportedly located in the vicinity of the active site and could form a binding site for a glucosyl ... subsite )1 (Fig 4B) Together with the observations from the site- directed mutagenesis, Trp402 of IPU appears to be a residue participating in subsite )1 The residues that form potential subsites ... 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 structures of the model of IPU (Fig 4A) and...
- 8
- 551
- 0
Tài liệu Báo cáo khoa học: Identification of critical active-site residues in angiotensin-converting enzyme-2 (ACE2) by site-directed mutagenesis docx
... provides the first investigation of the importance of key active -site residues of ACE2 through site- directed mutagenesis, with the aim of providing practical evidence for their role in substrate binding ... Fig Schematic view of 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 ... chain of Arg273 is therefore 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...
- 9
- 789
- 2
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
... intact A detailed study of the limited proteolysis of the E1 component of B stearothermophilus PDH complex [20] identified Tyr281 (for chymotrypsin) and Arg282 (for trypsin) of the E1a subunit, two ... Biochem 270) the 2-oxo group of the substrate [for a detailed formulation of the mechanism to date, see [44]) The importance of this region is emphasized by the reports of clinically deleterious ... might not function 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...
- 10
- 459
- 0
Tài liệu Báo cáo Y học: Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling ppt
... appropriate equations Site- directed mutagenesis The D153N mutant form of E coli agmatinase was obtained by a two-step PCR [20], using the plasmid pKA5 containing the speB gene of E coli agmatinase ... superposition matrix calculated for manganese ions, in order to conserve the ligand-manganese distances RESULTS AND DISCUSSION By site- directed mutagenesis, a D153N mutant form of E coli agmatinase was ... supporting the presence of a binuclear metal center in the active site of fully activated agmatinase [9] The Km for agmatine (1.6 ± 0.1 mM) and the Ki for competitive Fig Effect of added Mn2+ and EDTA...
- 5
- 475
- 0
Tài liệu Báo cáo Y học: Study of substrate specificity of human aromatase by site directed mutagenesis pdf
... lipophilicity values [44] and a distance-dependent function [45] Site- directed mutagenesis This step was performed with the QuickChangeTM SiteDirected Mutagenesis method from Stratagene Briefly, this was based ... Binding characteristics of seven inhibitors of human aromatase: a site- directed mutagenesis study Cancer Res 56, 3451–3460 22 Chen, S & Zhou, D (1992) Functional domains of aromatase cytochrome ... aromatase following site- directed mutagenesis Biochim Biophys Acta 1163, 195–200 52 Zhou, D.J., Korzekwa, K.R., Poulos, T & Chen, S.A (1992) A site- directed mutagenesis study of human placental...
- 13
- 506
- 0
Báo cáo khoa học: Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2-producing ligninolytic enzyme pot
... resulting vector (pALAAO) was used for site- directed mutagenesis, and transformation of E nidulans biA1, metG1, argB2 (IJFM A729), as described below [17] Site- directed mutagenesis AAO variants were ... substrate-binding site of AAO were modified by site- directed mutagenesis Tyr78 did not seem to be involved in catalysis, as the kinetic properties of the Y78A variant were not very different from those of wild-type ... 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 of some selected residues...
- 11
- 471
- 0
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
... 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 ... figures Sequence numbers refer to the locations of the transcriptional start site of hbpC (B) Alignments of the sequences of three HbpR-binding sites within the hbp gene region [19] The lane ‘cons-1’ ... of the hbpC promoter in Escherichia coli Results Mutagenesis of the HbpR-binding sites Sequence alignment of the UASs found in the HbpR operators in the hbpC and hbpD promoters defined a set of...
- 11
- 468
- 0
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
... expressed in lmặmg)1ặmin)1 Site- directed mutagenesis, protein expression and purication Presteady-state kinetic parameters for the acetylation of NAT Site- directed mutagenesis of the hamster NAT2 Tyr190 ... 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 k4 for ... 0.6 0.1 for Y190F, bnuc = 0.4 0.1 for Y190I, bnuc = 0.5 0.1 for Y190A) for the Brứnsted plot for pKNH3+ (or pKH3O+) ranging from )1.7 to 5.34 (Fig 4) These results indicate that for the mutants,...
- 14
- 350
- 0
Báo cáo khoa học: Functional significance of five noncanonical Ca2+-binding sites of human transglutaminase 2 characterized by site-directed mutagenesis potx
... Ca2+-binding sites of TG2 ´ly R Kira et al Fig Multiple sequence alignment of Ca2+-binding sites of transglutaminases Sequence alignments of Ca2+-binding sites of TG2 compared with the other members of ... binding site of wild typeTG2 0.0 Weak binding sites of wild type TG2 Weak binding sites of S1 TG2 0.0 0.00 –0.1 –0.3 n = 0.52 ± 0.63 Kd = 0.1 ± 0.03 µM –0.4 kcal·mol–1 of injectant –0.2 kcal·mol–1 of ... serve as Ca2+-binding sites [19] The aim of the present study was to identify the exact Ca2+-binding sites of human TG2, by using sitedirected mutagenesis and targeting sites homologous to FXIIIa...
- 14
- 378
- 0
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
... projection of their side-chains into the location of this site Consequently, ACE2 only binds a chloride ion at one CL1 site [24] Previous mutagenesis studies of sACE have shown that the CL2 site residue ... changed by 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 ... cycle (50 °C for h); one cycle (94 °C for min); 30 cycles (94 °C for 30 s, 65 °C for 30 s, 68 °C for min); one cycle (68 °C for min) Amplicons were sequenced to confirm the integrity of the product,...
- 10
- 362
- 1
Báo cáo khoa học: Investigation of the substrate specificity of a b-glycosidase from Spodoptera frugiperda using site-directed mutagenesis and bioenergetics analysis pdf
... with 6-OH [7] The purpose of the present study was to substitute residues Q39 and E451 of Sfbgly50, through site- directed mutagenesis, for residues presenting hydrogen bond-forming side-chains Steady-state ... from Sigma or Merck Site- directed mutagenesis Site- directed mutagenesis was performed using a plasmid pT7-7 [13] containing an inserted DNA fragment coding for the mature Sfbgly50 (pT7b50) [14] ... are a sum of DGà Each DGà value represents the disruption of a noncovalent interaction owing to the substitution of residues 39 or 451 for an alanine residue or because of the lack of a glycone...
- 9
- 371
- 0
Báo cáo khoa học: Probing the unfolding region of ribonuclease A by site-directed mutagenesis potx
... Madison, WI, USA) for the gift of the plasmid pBXR, to Professor G Vriend (University of Nijmegen, the Netherlands) for molecular modeling, and to Y Markert for providing the plasmids for the variants ... at room temperature for 24 h], it was puried on a Mono S column (50 mM Tris/HCl, pH 7.5, with a linear gradient of 0500 mM NaCl) Table Oligonucleotides for site- directed mutagenesis The replaced ... is the signal of the substrate before the addition of enzyme, Fend is the signal after cleavage of all substrate, and [E] is the concentration of RNase A CD spectroscopy CD spectra of RNase A and...
- 10
- 491
- 0
Báo cáo khoa học: Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site pot
... apparent Kd for [Mg2+] of 2.06 ± 0.38 mM 597 Mutagenesis of the active site of E coli KARI residues in the E coli enzyme reacting within a few seconds, a further three reacting over a period of several ... attempted to understand these roles by mutagenesis of E coli KARI FEBS Journal 272 (2005) 593–602 ª 2005 FEBS Mutagenesis of the active site of E coli KARI Most of the mutations abolish the overall ... HE (1969) Purification and properties of the acetohydroxy acid isomeroreductase of Salmonella typhimurium J Biol Chem 244, 1118–1127 601 Mutagenesis of the active site of E coli KARI Primerano...
- 10
- 339
- 0
Báo cáo khoa học: Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis Tobias Hefner and Joachim Stockigt ¨ ppt
... of the site- directed mutagenesis experiments presented here The question concerning the mechanism of one of the basic reactions in cells, the transfer of a sugar moiety during the formation of ... Probing catalytic domains of NRD glycosyltransferases (Eur J Biochem 270) 537 the nucleophile We were able to prove for the first time the suggested model by site- directed mutagenesis of arbutin synthase ... Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 Bradford, M.M (1976) A rapid and sensitive method for quantitation of microgram quantities of...
- 6
- 282
- 0
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
... role of the Y238 side chain in substrate/product exchange to the active site of RgDAAO A superimposition of the active sites of yeast and mammalian DAAO [24] shows that the side chain of Y223 of ... obtained by subtraction of the absorbance spectrum of the free oxidized form of DAAOs to the spectrum of the same enzyme after addition of a saturating concentration (% 20 mM) of sodium anthranilate ... has been associated with formation of the EFlox D-alanine complex in the reductive half-reaction of any of the Y238 mutants As shown for Y238F in Fig 5A, the formation of the spectral intermediate,...
- 10
- 496
- 0
Báo cáo khoa học: Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis potx
... obtained from Perkin-Elmer (Boston, MA, USA) Site- directed mutagenesis Site- directed mutagenesis was carried out using the QuikChange Site- Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) ... concentrations used for individual mutants were: 12 nm for K699S, 10 nm for N257D, 150 nm for R210A, 38 nm for Y700F, 67 nm for N519D, 20 nm for N519V, nm for R534L, and 32 nm for wt rhGCPII Enzyme ... of all mutant forms of rhGCPII were performed essentially as previously described [19] only the induction conditions were altered (to mm CuSO4) Purification of mutant forms of rhGCPII Mutant forms...
- 11
- 484
- 0
Báo cáo khoa học: Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA ppt
... the interaction of the homeodomain of HAKN1, a sunflower class I KNOX protein [28], with DNA Based on studies of wild-type and mutant forms of the homeodomain, we propose a model for the complex ... critical for binding (Fig 1A, lanes 4, 6A and 5) Regarding position 7, the change of A for T does not seem to affect binding, while the introduction of C partially reduces the amount of complex formed ... part of the binding sites of the barley KNOX protein Hooded [23] and of maize Knotted1 [26] This element is also present in the sequence GTNAC, postulated to be important for the binding of the...
- 13
- 558
- 0
Tìm thêm: hệ việt nam nhật bản và sức hấp dẫn của tiếng nhật tại việt nam xác định các mục tiêu của chương trình xác định các nguyên tắc biên soạn khảo sát chương trình đào tạo gắn với các giáo trình cụ thể tiến hành xây dựng chương trình đào tạo dành cho đối tượng không chuyên ngữ tại việt nam điều tra đối với đối tượng giảng viên và đối tượng quản lí điều tra với đối tượng sinh viên học tiếng nhật không chuyên ngữ1 khảo sát các chương trình đào tạo theo những bộ giáo trình tiêu biểu xác định mức độ đáp ứng về văn hoá và chuyên môn trong ct mở máy động cơ lồng sóc các đặc tính của động cơ điện không đồng bộ hệ số công suất cosp fi p2 đặc tuyến hiệu suất h fi p2 đặc tuyến tốc độ rôto n fi p2 đặc tuyến dòng điện stato i1 fi p2 động cơ điện không đồng bộ một pha sự cần thiết phải đầu tư xây dựng nhà máy từ bảng 3 1 ta thấy ngoài hai thành phần chủ yếu và chiếm tỷ lệ cao nhất là tinh bột và cacbonhydrat trong hạt gạo tẻ còn chứa đường cellulose hemicellulose chỉ tiêu chất lượng theo chất lượng phẩm chất sản phẩm khô từ gạo của bộ y tế năm 2008 chỉ tiêu chất lượng 9 tr 25