title: Cooperative effects of mutations in a recombinant Fab on the kinetics of antigen binding.
abstract: Recombinant Fabs, 57P and 174P, recognizing peptide 134-151 of the coat protein of tobacco mosaic virus, differ by 15 amino acid changes in the sequence of their variable region. Kinetic analysis using BIAcore showed that they recognized five peptide variants in the same ranking order, but that Fab 174P consistently dissociated faster from the peptides compared to Fab 57P. In order to identify amino acid substitutions that are responsible for differences in dissociation rates of the two Fabs, six hybrid Fabs have been constructed by exchanging three DNA segments. Four single and five multiple mutants were obtained by site-directed mutagenesis. All Fabs recognized variant peptides in a similar ranking order. The high precision of biosensor measurements made it possible to detect small contributions to dissociation kinetics of at least five substitutions, as well as the presence of small-magnitude non-additive effects of multiple substitutions. Our results demonstrate the cooperative influence on dissociation kinetics of amino acid residues located away from each other and away from the Fab combining site.
authors.affiliation: Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France.
authors.forename: N
authors.initials: N
authors.lastname: Rauffer-Bruyre
authors.forename: J
authors.initials: J
authors.lastname: Chatellier
authors.forename: E
authors.initials: E
authors.lastname: Weiss
authors.forename: M H
authors.initials: MH
authors.lastname: Van Regenmortel
authors.forename: D
authors.initials: D
authors.lastname: Altschuh
mesh_terms: D000941:Antigens / Q000378:metabolism*; D015374:Biosensing Techniques; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D013329:Structure-Activity Relationship
chemical_list: D000941:Antigens; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins