Strukturelle Untersuchungen an Atrazin- und 2,4-Dichlorphenoxyessigsäure-spezifischen Antikörperfragmenten mittels molecular modelling und ortspezifischer Mutagenese [article]

Wien Kusharyoto, Universität Stuttgart, Universität Stuttgart
2001
In this study, the binding properties of an atrazine specific Fab fragment K411B and a 2,4-Dichlorphenoxyacetic acid (2,4-D)-specific Fab fragment Fab24D were investigated by means of molecular modelling and site-directed mutagenesis. Based on the homology to antibodies of known structures and by the identification of canonical structure classes of the complementarity determining regions (CDRs), a structure model for each of the variable fragments of the antibodies was constructed. Molecular
more » ... ucted. Molecular mechanics and dynamics as well as the cross-reactivity patterns of the respective antibodies were used to identify amino acid residues responsible for the binding of the hapten. The reliability of the models was scrutinized by mutations of amino acid residues in the binding site of the respective antibodies. In the case of the Fab-fragment K411B, a five-fold improvement in affinity due to a combination of point mutations GlnL89Glu, ValH37Ile and GluL3Val could be obtained, as indicated by surface plasmone resonance (SPR) measurement using BIAcore. However, the increased affinity did not lead to an improvement in sensitivity of a direct competitive ELISA for the determination of atrazine. An eight-fold improvement in sensitivity of a direct competitive ELISA for atrazine could be achieved by taking advantage of the increased affinity and the low cross-reactivity of the mutant towards the hapten 6-{[4-amino-6-chloro-1,2,3-triazin-2-yl]aminohexanoic acid. Decreasing the spacer recognition due to the mutations PheL32Leu and GlnL89Glu led to a three-fold improvement in sensitivity of a direct competitive atrazine-ELISA. In the case of the Fab-fragment Fab24D, a change in specificity from 2,4-D to 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) could be achieved by either the mutation HisL34Phe or HisL34Tyr. However, the change in specificity led to a decrease in affinity towards 2,4,5-T.
doi:10.18419/opus-696 fatcat:fiqjzyyguvfkhbppueikfil5h4