Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications

Abstract

Residue-specific incorporation of non-canonical amino acids into proteins allows facile alteration and enhancement of protein properties. In this review, we describe recent technical developments and applications of residue-specific incorporation to problems ranging from elucidation of biochemical mechanisms to engineering of protein-based biomaterials. We hope to inform the reader of the ease and broad utility of residue-specific non-canonical amino acid incorporation with the goal of inspiring investigators outside the field to consider applying this tool to their own research.

Figure 1

1A: Schematic for residue-specific incorporation of non-canonical amino acids (ncAAs) into proteins. A natural mRNA contains codons for the 20 canonical amino acids (cAAs). A cAA (purple sphere) assigned to one of those codons (yellow) is replaced with an ncAA (orange star). A medium shift is performed to remove the cAA to be replaced (purple sphere) and to introduce the ncAA (orange star) along with the remaining 19 cAAs (green spheres). The ncAA is charged to the appropriate tRNA (red) by either the wild-type or a mutant aminoacyl-tRNA synthetase (aaRS). The correctly aminoacylated tRNA^cAA (blue with green sphere) and the misacylated tRNA (red with orange star) are processed by the ribosome to give a globally modified protein. The left path depicts normal protein synthesis with 20 cAAs for comparison. 1B: Chemical structures for the ncAAs discussed in the text.

Figure 2

Schematic depictions of BONCAT and FUNCAT technologies. Both begin with metabolic labeling of newly synthesized proteins using an ncAA bearing a reactive handle (blue sphere). Then, a chemoselective reaction is performed (e.g. click chemistry) to append either an enrichment tag (BONCAT) or a fluorophore for visualization (FUNCAT). Taken together, these technologies allow simultaneous imaging and identification of new proteins in cells; proteomic responses to stimuli (changing conditions, infection, cell-cell interaction, etc.) can be monitored.