Functional characterization of an anti-estradiol antibody by site-directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the VLdomain in estradiol recognition

2002 ◽  
Vol 15 (1) ◽  
pp. 6-18 ◽  
Author(s):  
Stéphane Coulon ◽  
Jean-Luc Pellequer ◽  
Thierry Blachère ◽  
Martine Chartier ◽  
Elisabeth Mappus ◽  
...  
Keyword(s):  
Molecular Modelling ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Properties

Functional characterization of two anti-estradiol antibodies as deduced from modelling and site-directed mutagenesis experiments

2001 ◽  
Vol 14 (2) ◽  
pp. 99-109 ◽  
Author(s):  
Florence Bettsworth ◽  
C�line Monnet ◽  
B�n�dicte Watelet ◽  
Nicole Battail-Poirot ◽  
Bernard Gilquin ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals The mechanism of action of dd-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 dd-peptidase

1994 ◽  
Vol 301 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J M Wilkin ◽  
A Dubus ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peptide Substrate ◽  
Binding Properties ◽  
Beta Lactamases ◽  
Penicillin Binding Proteins ◽  
Active Site Cavity

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

scholarly journals Site-directed mutagenesis of the Actinomadura R39 DD-peptidase

1997 ◽  
Vol 327 (2) ◽  
pp. 377-381 ◽  
Author(s):  
Guo-Hua ZHAO ◽  
Colette DUEZ ◽  
Sophie LEPAGE ◽  
Christine FORCEILLE ◽  
Noureddine RHAZI ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Catalytic Properties ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Structural Elements ◽  
Penicillin Binding Protein ◽  
Peptide Substrate ◽  
Binding Properties ◽  
Km Value

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive DD-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the ‘SDN loop’ by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 DD-peptidase and Escherichia coli penicillin-binding protein (PBP) 5.

Characterization of Recombinant von Willebrand Factors Mutated on Cysteine 509 or 695

1996 ◽  
Vol 76 (03) ◽  
pp. 453-459 ◽  
Author(s):  
Virginie Siguret ◽  
Anne-Sophie Ribba ◽  
Olivier Christophe ◽  
Ghislaine Chérel ◽  
Bernadette Obert ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Mutant Proteins ◽  
Von Willebrand ◽  
High Molecular Weight Multimers

SummaryThe interacting domain of vWF with platelet GPIb has been shown to overlap the large A1 loop formed by the intra-chain disulfide bond linking Cys 509 to Cys 695. In order to further investigate the role of the conformation of this region, we have expressed in COS-7 cells three mutated full-length recombinant vWFs (rvWFs) in which the substitutions Cys509Gly, Cys509Arg or Cys695Gly have been introduced by site-directed mutagenesis. SDS-agarose gel electrophoresis demonstrated an impaired multimerization of the mutants with undetectable high molecular weight multimers and a decrease of the relative amounts of the intermediate sized multimers. Binding analysis showed that rvWFC509G and rvWFC509R did not interact with botrocetin but spontaneously interacted with GPIb; the latter binding remained unchanged in the presence of ristocetin. This indicates that the substitution of Cys509 by Gly or Arg creates a conformation of vWF that increases its binding to GPIb. In contrast, rvWFC695G which did not react with botrocetin was also unable to interact with GPIb even in the presence of ristocetin, indicating that sequences interacting with GPIb are masked and/or disrupted. In conclusion, the substitution of each of the Cys509 and 695 results in mutant proteins which may be “locked” into active or inactive conformations in regard to the binding to platelet GPIb receptor.

Sign in / Sign up

Export Citation Format

Share Document