Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation

Abstract

We report evolved orthogonal pyrrolysyl-tRNA synthetase/tRNA_CUA pairs that direct the efficient, site-specific incorporation of N^ε-l-thiaprolyl-l-lysine, N^ε-d-cysteinyl-l-lysine, and N^ε-l-cysteinyl-l-lysine into recombinant proteins in Escherichia coli. We demonstrate that the unique 1,2-aminothiol introduced by our approach can be efficiently, rapidly, and specifically labeled via a cyanobenzothiazole condensation to quantitatively introduce biophysical probes into proteins. Moreover, we show that, in combination with cysteine labeling, this approach allows the dual labeling of proteins with distinct probes at two distinct, genetically defined sites.