Investigation of the molecular interactions between an anti-peptide antibody and its ligand

Brown, Jennifer Louise (1994) Investigation of the molecular interactions between an anti-peptide antibody and its ligand. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

A detailed understanding of the molecular basis of receptor/ligand interactions is a key component in the rational discovery of novel therapeutic agents. In efforts to develop a model for studying the recognition of flexible ligands by protein receptors, a monoclonal antibody (DB19/1) has been raised which recognizes the highly immunogenic nonapeptide Ac-YPYDVPDYA. Comparison of the primary sequence of DB19/1 with that of other murine antibodies has allowed the identification of the Complementary Determining Regions (CDRs) responsible for binding the peptide antigen. This information, in combination with homology modelling methods, has been used to construct a molecular model of the peptide-binding site in DB19/1. Details of the computational approach are presented, and the structural features of the antibody which may mediate peptide recognition are identified. The backbone conformations of the CDRs in the model structure are compared with those reported in other studies, and the similarities and differences are discussed.

A protocol for the isolation of DB19/1 from ascites fluid has been developed, and the preparation and purification of the Fab fragment using enzymatic cleavage of the intact antibody has been achieved. We sought to probe the bound conformation of the peptide by studying the interaction of the DB19/1 Fab fragment with cyclic peptides in which the antigenic tetrapeptide sequence Tyr-Pro-Tyr-Asp was conformationally restricted. Our successful strategy for the construction of such compounds is illustrated by the synthesis of the cyclic peptide in which a β-alanine unit connects the termini of the antigenic tetrapeptide. (DX 192, 446).

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