The broad, long-term objective of this proposal is to develop techniques for modeling antibody structures that will allow the prediction of mutations affecting the binding and potential catalytic activity of an antibody, and will advance our understanding of the functional and spatial relationships necessary for enzyme catalysis. Techniques developed by this grant will enhance the biomedical uses for antibodies as therapeutic agents and as diagnostic tools, and will help create new uses for antibodies such as highly specific proteases or other catalytic reagents for reactions for which no enzyme is known. Antibody models will be based on analysis and characterization of the database of available crystallographic antibody structures. Enzyme catalytic sites will be examined for shared characteristics to develop motifs that will be used as templates to create or improve catalytic sites in antibodies. Development of these modeling techniques will be driven by the results of mutagenesis experiments performed in the laboratory of Prof. Steve Benkovic on the catalytic antibody NPN43C9.
The Specific aims are 1) to refine the current model of the NPN43C9 antibody by continued analysis of our antibody structural database, analysis of protein structures, and computational techniques; 2) to develop tools for the determination of sequence homology among multiple structures for any set of residues so that structural similarity can be correlated with sequence homology; 3) to develop and test methods for determining the proper geometry for loops in the complementarity determining regions and the proper geometric relationship between variable region heavy and light chains, both factors that determine the size and shape of the antigen binding pocket; 4) to use our antibody structural database to calculate the structural variability of each amino acid residue and to incorporate this information into the model to facilitate model building; 5) to predict the role of specific residues in antigen and substrate binding, catalysis, and antibody structure from the NPN43C9 model and to suggest specific mutations to test these hypotheses; 6) to determine the important geometric and environmental factors that make up catalytic motifs in enzymes, and to use these motifs as templates in the design of antibody catalytic sites. The iterative process of developing hypotheses from the model, testing the hypotheses experimentally by mutagenesis, and applying the experimental results to further development of techniques of antibody structural analysis, building catalytic motifs, and refining the NPN43C9 antibody model will provide a powerful interdisciplinary approach for understanding antibody structure and enzyme catalysis in general and catalytic antibody development in specific.
