Noncanonical amino acids in the interrogation of cellular protein synthesis

Abstract

Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical 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. Noncanonical 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 tRNA 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.

Figure 1

Structures of the amino acids discussed in this Account. ncAAs shown in blue are substrates for the natural translational machinery, the analog shown in green requires over-expression of wild-type MetRS, and those shown in red require expression of mutant aaRSs.

Figure 2

ncAA-tagged proteins can be ligated to affinity probes for enrichment and identification, or to dyes for visualization by in-gel fluorescence scanning or fluorescence microscopy.

Figure 3

Imaging of mammalian cells pulsed with Aha in the absence (top) or presence (bottom) of the protein synthesis inhibitor anisomycin. The left-most panel in each row shows the image formed by dye-labeling of newly synthesized proteins (green). Additional panels show mitochondria (red), nuclei (blue), and panel overlays.

Figure 4

(a.) Library screen for MetRS mutants that allow Anl incorporation into cellular proteins.^, (b.) X-ray crystal structure of a MetRS mutant (L13S/Y260L/H301L) bound to Anl (spheres). (c.) Mutations (spheres) that accommodate Anl (sticks and dots) within the MetRS binding pocket.

Figure 5

Cell-selective labeling in mixtures of bacterial and mammalian cells. Macrophages (red) infected by E. coli cells that express GFP (top) or the NLL-MetRS (bottom) labeled with Anl. Proteins expressed in bacterial cells that express the NLLMetRS are labeled with Anl. The control bacterial strain was bound and internalized by macrophages (as confirmed by detection of GFP) but exhibited no Anl incorporation. In neither case were macrophage proteins labeled.