Non-canonical amino acids in the interrogation of cellular protein synthesis

Abstract

Proteins can be rendered susceptible to bio-orthogonal chemistries through metabolic labeling with appropriately designed, noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces, in whole or in part, one of the natural amino acids specified by the protein gene(s) of interest. This approach allows the ncAA to be introduced at a controlled rate into positions normally occupied by the natural amino acid residue. Because this method 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 those used in 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 prior to the pulse through bio-orthogonal ligation of the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. The selectivity of the method can be enhanced through the use of mutant aminoacyltRNA synthetases (aaRSs) that permit incorporation of ncAAs that are not used by the endogenous translational machinery. Controlled expression of mutant synthetases has been combined with ncAA-tagging to permit cell-selective and state-selective metabolic labeling of proteins. Expression of a mutant synthetase in a subset of cells in a complex cellular mixt. - or in a live animal - restricts labeling to that subset; proteins synthesized in cells that do not express the synthetase are neither labeled nor detected.