COOPERATIVE EFFECTS OF MUTATIONS IN A RECOMBINANT FAB ON THE KINETICSOF ANTIGEN-BINDING

Recombinant Fabs, 57P and 174P, recognizing peptide 134-151 of the coa t protein of tobacco mosaic virus, differ by 15 amino acid changes in the sequence of their variable region. Kinetic analysis using BIAcore( TM) showed that they recognized five peptide variants in the same rank ing order, but that Fab 174P consistently dissociated faster from the peptides compared to Fab 57P. In order to identify amino acid substitu tions that are responsible for differences in dissociation rates of th e two Fabs, six hybrid Fabs have been constructed by exchanging three DNA segments. Four single and five multiple mutants were obtained by s ite-directed mutagenesis. All Fabs recognized variant peptides in a si milar 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-magnitu de non-additive effects of multiple substitutions. Our results demonst rate the cooperative influence on dissociation kinetics of amino acid residues located away from each other and away from the Fab combining site. (C) 1997 Elsevier Science Ltd.