Expanding the genetic code to study metalloproteins and protein posttranslational modifications

Genetic code expansion is a useful method to study metalloproteins and protein posttranslational modifications. We constructed several tyrosine and pyrrolysine tRNA synthetase libraries based on tyrosine and pyrrolysine tRNA synthetase/tRNA pairs to screen unnatural amino acids (UAAs) with similar chemical structure to tyrosine or lysine. We had incorporated several unnatural amino acids into proteins, including those with electron and proton transfer mediators, metal-chelating, bio-orthogonal reaction groups, and played a series of bio-orthogonal reactions in vitro and in vivo, such as copper click of azide and alkyne, copper free click of azide and cycloalkyne, photoclick of alkene and tetrazole, which laid the foundation for proteins specifically labeling, protein-protein interaction probing, and biological processes photo-regulation. Besides that, we applied this method to incorporate redox tyrosine analogs to improve the property of fluorescent proteins. We directed N-formyllysine into histones to study the impact of protein formylation naturally generated by oxidative damage. We also explored the activities of proteins and downstream signaling pathways by using sulfur or fluorine mimetics of acetylated lysine as probes of protein posttranslational modifications.