Non-Canonical Amino Ac ids as Probes of Protein Synthesis in Complex Biological Systems
This lecture will describe strategies that enable time resolved and state-selective analysis of cellular protein
synthesis. In procedures similar to those used in isotopic labeling, non-canonical amino acids (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 aminoacyl-tRNA synthetases (aaRSs) that
permit incorporation of ncAAs that are not used by the endogenous machinery. Controlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective and state-selectivemetabolic labeling of proteins. Expression of a mutant synthetase in a subset of cells in a complex cellular mixture – 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. Applications in neurobiology, microbiology and infectious disease will be described.