Project 9 is concerned with the elucidation of the structural and dynamic basis for antibody catalysis. Knowledge of the structure and dynamics of the complementary-determining regions and active sites of catalytic antibodies should allow us to understand the details of their catalytic mechanism and the origins of the differences in substrate specificity between enzymes and antibodies which catalyze similar reactions. Catalytic antibodies provide an excellent system to examine the factors that influence the rates and specificities of enzymatic reactions. In addition,, catalytic antibodies offer unique opportunities as designed biocatalysts because they are capable of accepting a far broader range of substrates than most enzymes. This project will focus on the interplay of structure and dynamics in the mechanism of catalysis by antibody aldolases elicited by reactive immunization. These antibody aldolases have exceptionally broad scope and are able to catalyze efficiently more than 100 different aldol additions or condensations. The first specific aim will be to examine changes in the local structure and dynamics of the Fv fragment of the catalytic antibody 33F12 in complex with substrate analogs and inhibitors. These studies should provide novel insights into the role of dynamics in substrate recognition and are fully complementary to the X-ray structural studies proposed in Project 10. In the second specific aim, the evolution of dynamics during affinity maturation will be examined, to test the hypothesis that both local structure and flexibility of the polypeptide are refined during affinity maturation. Mutant aldolase antibodies will be examined in specific aim 3, in order to distinguish structural and dynamic factors that lead to changes in substrate specificity and efficiency or catalysis by the antibodies. Finally, the Fab fragments of a new set of aldolase catalytic antibodies generated in Project 11 will be used in a rapid screening for differences in substrate and inhibitor affinity, using newly available NMR techniques. The most interesting interactions will be chosen for detailed study by crystallography (Project 10) and NMR (Project 9) and NMR (Project 9), following cloning and expression of the relevant Fv polypeptide chain and side chains in the active site are an important component of the catalytic process. The experiments proposed for Project II are designed to test this hypothesis.
Showing the most recent 10 out of 155 publications
