Cysteinyl-DOPA Crosslinking and the Road to an Expanded Metabolism

Across all domains of life, the proteome is confined to 20 standard amino acids, with just two rare exceptions; selenocysteine and pyrrolysine. Expansion of the genetic code with a growing number of non-canonical amino acids (ncAAs) bearing new amino acid chemistries, has enabled the synthesis of proteins with diverse new functionality. 3,4-dihydroxy-L-phenylalanine (L-DOPA), is a naturally occurring amino acid containing a catechol moiety, a new redox chemistry not occurring in the 20 standard amino acids. Although found widely in nature, 3,4-dihydroxy-L-phenylalanine only occurs in proteins as a post-translational modification of tyrosine (or as a damaging oxidation product). The presence of 3,4-dihydroxy-L-phenylalanine in proteins confers unique properties such as redox dependent crosslinking with nearby nucleophiles or other 3,4-dihydroxy-L-phenylalanine residues, as well as coordination of metal ions. In addition to its use in nature, catechol containing polymers are under investigation as self healing, programmable biomaterials. The ability to harness these properties for protein engineering is of great interest, as proteins are a scalable and programmable scaffold. Although an orthogonal aminoacyl-tRNA synthetase (aaRS):tRNA pair for the incorporation of L-DOPA has previously been reported, it, like many other early aaRS:tRNA pairs, suffers from poor activity and a lack of discrimination between its new substrate and tyrosine. Here we describe the evolution of an aaRS for highly efficient site-specific incorporation of 3,4-dihydroxy-L-phenylalanine, the development of cysteinyl-DOPA crosslinking motifs and our progress towards an expanded metabolism in E. coli. This new aaRS enabled the production of 3,4-dihydroxy-L-phenylalanine containing proteins with yields exceeding 20 mg.L^-1 at over 90% incorporation efficiency. Using a combination of rational design, and refinement by computational modelling, several engineered pairs of 3,4-dihydroxy-L-phenylalanine and cysteine residues were shown to form covalent cysteinyl-DOPA crosslinks in different proteins. This new bioorthogonal crosslinking chemistry and the catechol moiety in general are valuable and highly enabling additions to the protein engineering toolkit.