Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis

Abstract

CONSPECTUS Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed, non-canonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein’s gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell. Because this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a “pulse” in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Non-canonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl transfer RNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other useful ncAAs, including analogs that contain ketones or aryl halides. High-throughput screening strategies can identify aaRS variants that activate a wide range of ncAAs. Controlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective metabolic labeling of proteins. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. Proteins synthesized in cells not expressing the synthetase are neither labeled nor detected. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. In this Account, we summarize the tools and strategies that have been developed for interrogating cellular protein synthesis through residue-specific tagging with ncAAs. We describe the chemical and genetic components of ncAA-tagging strategies and discuss how these methods are being used in chemical biology.

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

“…1A). Although a wide range of synthetic amino acids exists, only a small number are able to exploit the natural translational machinery without the need for genetic modification of the cell (18). To date, the L-methionine (Met) surrogates L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG) (17) have found the widest application (e.g., refs.…”

Cell-specific proteomic analysis in Caenorhabditis elegans

“…Enriching for specific cells is challenging, and researchers cannot systematically identify low-abundance proteins expressed in specific cells from wholeorganism lysates. Cell-selective bioorthogonal noncanonical amino acid tagging (cell-selective BONCAT) offers a way to overcome these limitations (1,2). We have previously engineered a family of mutant Escherichia coli methionyl-tRNA synthetases (MetRSs) capable of appending the azide-bearing L-methionine (Met) analog L-azidonorleucine (Anl) to its cognate tRNA in competition with Met (3,4).…”

Omics in Aid of Host-pathogen Interaction Studies: A Bacterial Perspective

“…Given that selective labeling of bacterial proteins occurs before lysis of infected cells, such approaches do not require a priori physical separation of bacteria from host cells in contrast to the strategies described above [70,71]. For a comprehensive review of labeling approaches, we refer to Tirrell et al [72].…”

“…Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2017 doi:10.20944/preprints201708.0093.v1 11 of 20 Typically, functional groups of non-natural amino acids incorporated into bacterial proteins by the translation machinery enable specific derivatization of labeled proteins and their subsequent isolation [72]. Some of the non-natural amino acids used such as the azide-bearing methionine surrogate azido homoalanine (AHA), can be loaded to methionyl-tRNA following wild-type aminoacyl-tRNA synthetase (aaRS) activation of AHA.…”