A New Strategy for the Site-Specific Modification of Proteins in Vivo

Abstract

We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-l-phenylalanine into proteins in E. coli. We demonstrate that proteins containing m-acetyl-l-phenylalanine or p-acetyl-l-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells. The labeling reactions are selective and in general proceed with yields of >75%. In specific examples, m-acetyl-l-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes. The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo.

Selective chemical protein modification

“…This residue, once installed, again reacted selectively with a range of fluorescent hydrazines. The ketone amino acids p-acetylphenylalanine 142 and m-acetylphenylalanine 150 were subsequently incorporated into proteins, without the need for chemical acylation, in both E. coli 142,150 and eukaryotic cells 151 .…”

Diels–Alder Ligation of Peptides and Proteins

“…Using this approach, Schultz and co-workers have developed a method for the labeling of proteins in cells via hydrazone ligation using a ketone-modified protein. [58] The same group has reported recently a genetically-encoded incorporation of azide and acetylene tyrosine analogs into proteins that could be modified with dyes by copper(I)-catalysed click chemistry. [59] Furthermore, Bertozzi and co-workers demonstrated the modification of azidohomoalanine-labeled protein through Staudinger ligation with a phosphine reagent bearing an antigenic FLAG peptide ( Figure 10).…”

Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells

“…In this approach, an orthogonal transfer RNA (tRNA)-aminoacyl tRNA synthetase pair is evolved that uniquely recognizes the amino acid of interest and selectively incorporates it into proteins in response to the amber nonsense codon, TAG. This methodology has been used to site-specifically incorporate a variety of unnatural amino acids into proteins with high fidelity and good efficiency, including amino acids with novel functional groups (3)(4)(5)(6), photocrosslinkers (7,8), heavy atoms, sugars (9), and redox active moieties. In addition, new orthogonal tRNA-synthetase pairs have been evolved from leucyl (10), lysyl, glutaminyl (11), aspartyl (12), and tyrosyl (13) tRNA-synthetase pairs to expand the number and structural diversity of amino acids that can be genetically encoded in bacteria and yeast.…”