Computational and combinatorial design of an anthrax antitoxin

Computer programs have been used to emulate protein-protein interactions and to design novel inhibitors (designer proteins) of the anthrax toxin protein protective antigen (PA). The goal of this project was to design proteins which would bind to protective antigen and inhibit PA oligomerization. In the first part of the two part calculations, a Geometric Recognition Algorithm was used to find orientations of high surface complementarity of the two proteins. In the second part, protein design algorithms were used to computationally mutate the designer protein. Designer proteins bearing the mutations chosen by the protein design algorithms were expressed and purified and tested for binding to protective antigen. Phage display methods were also used to attempt to select improved designer proteins with higher binding affinity. A pull down assay was developed to test the binding of the designer proteins to the protective antigen. Two of the four proteins designed in this study were found to bind with greater affinity than negative controls, to anthrax protective antigen. They did not, however, inhibit the oligomerization of PA as they were designed to do. Instead, the designer proteins enhanced oligomerization of PA in a gel-based in vitro assay. This enhancement of oligomerization may have been due to non-specific binding of the designer proteins to PA. This project was a test of our computer-assisted protein design methods and will help us to further improve these methods in an iterative way.