One of the key properties of proteins is that they exhibit remarkable affinities and specificities for small-molecule and peptide binding partners. To improve the success rate of rational, computational protein design and widen the scope of potential applications, it is useful to define generalized strategies and automated methodology to improve and/or alter the affinity and specificity of interactions. I have implemented several strategies for engineering protein-small molecule interactions including: improvement of substrate accessibility, stabilization of the bound state, truncation and surface engineering, and transplantation of residue level, native (or native-like) interactions. Each strategy was applied to one or more model protein, and the resulting changes in affinity, specificity, and activity were characterized experimentally. Finally, we designed a biomolecular tool-kit, consisting of 17 engineered proteins for amino acid side-chain recognition and a single enzyme to catalyze the Edman degradation. We profiled the affinity and specificity of each protein, and implemented a computational framework that demonstrates its utility for amino acid calling in a single molecule protein sequencing assay
