Book excerpt: Abstract: Rational design is a powerful tool that can be used to understand the function of a protein, enhance a desired function, or engineer novel functionalities entirely. To this end, we have employed rational design toward the engineering of paraoxonase-1 (PON1). PON1 is able to hydrolyze a broad range of substrates, including toxic organophosphorus (OP) compounds at low levels, which makes it an attractive candidate for rational design. As the means by which PON1 is able to hydrolyze such a broad range of substrates is unknown, we investigated mechanistically-relavant residues along the active site and calcium coordination sphere. While our results mostly support previously proposed mechanistic hypotheses, it appears that hydrolysis of OP substrates may be more complex than originally believed. To provide a foundation for future iterations of rational design, we constructed a comprehensive catalog of mutations along the PON1 active site with the goal of rationalizing substrate specificity. Using the knowledge attained from this study as an input, we constructed rationally-designed directed libraries of PON1. Selected library variants displayed increased activity towards the toxic isomer of GF, further demonstrating the power of rational design. Finally, we report incorporation of unnatural amino acids p-Benzoylphenylalanine, o-nitrobenzyltyrosine, hydroxyquinolinylalanine and bipyridylalanine into proteins in order to rationally incorporate novel functionalities into proteins.