Computer-aided protein modelling: Applications to antibody and enzyme engineering: Dissertation

Knowledge-based modelling and molecular dynamics were used in designing antibodies and enzymes for which the sequences and the known closely or distantly related crystal structures were available. The hapten binding to structural models of anti-2-phenyloxazolone antibodies in a solvent environment was studied by molecular dynamics simulations. The comparison of modelled antibody-hapten complexes of the idiotype and the high-affinity variant suggested plausible explanations for the experimentally observed increase in affinity of the variant. A structural model of the lignin peroxidase LIII from Phlebia radiata based on a distantly related crystal structure of cytochrome c peroxidase was constructed by using newly developed knowledge-based methods. Comparison of the structural model with a closely related crystal structure of the lignin peroxidase from Phanerochaete chrysosporium showed that the core of the model was predicted rather accurately. However, the last 30 amino acids at the C-terminus were modelled with less certainty owing to a lack of biochemical information about disulfide bridge formation. Cellulose-binding domains of Trichoderma reesei cellulases were homology modelled in order to predict structural correlations that would effect their binding to crystalline cellulose. Comparison of their sequences with sequences of other fungal cellulases predicted an additional disulfide bridge in several cellulose-binding domains. The three-dimensional structures of other fungal cellulose-binding domains can be expected to be similar to those of Trichoderma reesei. Mutations that improved labelling of the anti-human alpha-fetoprotein antibody were predicted on the basis of a structural model of the Fab-fragment of the alpha-fetoprotein antibody. The improved labelling efficiency of the lysine enriched anti-human alpha-fetoprotein antibody Fab-fragment was found to be promising for the development of a more sensitive immunoassays.