Noncanonical Amino Acids in Protein Science and Engineering

Whether guided by computation, intuition, or evolution, recombinant DNA methods have enabled the preparation of an astonishing array of new protein variants of fundamental and practical importance. In recent years, it has become apparent that the power of protein engineering can be extended further through the use of an expanded set of amino acid constituents (Budisa 2006). The use of “noncanonical” amino acids creates new possibilities for protein design, and can be integrated in straightforward fashion into either “rational” or evolutionary design strategies. This chapter describes some of these new possibilities, with emphasis on the use of noncanonical amino acids to interrogate or change the reactivity, stability, or spectral properties of engineered proteins. Examples include the use of “bio-orthogonal” ligation reactions to enable selective dye-labeling and affinity-tagging, the use of aromatic amino acid analogs to alter the emission properties of luminescent proteins, the use of photosensitive amino acids to effect controlled protein cross-linking, and the use of fluorinated amino acids to control protein stability and protein-protein interactions. While many important experiments in this field have utilized in vitro translation, this chapter will focus primarily on cellular synthesis of proteins that contain noncanonical amino acids.