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Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis

Ngo, John T. and Tirrell, David A. (2011) Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis. Accounts of Chemical Research, 44 (9). pp. 677-685. ISSN 0001-4842. PMCID PMC3178009. https://resolver.caltech.edu/CaltechAUTHORS:20111115-121540025

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/ar200144yDOIArticle
http://pubs.acs.org/doi/abs/10.1021/ar200144yPublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178009PubMed CentralArticle
ORCID:
AuthorORCID
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2011 American Chemical Society. Published In Issue September 20, 2011; Article ASAP August 04, 2011; Received: May 24, 2011. Work on noncanonical amino acids at Caltech is supported by the National Institutes of Health (Grant GM 62523) and by the Army Research Office through the Institute for Collaborative Biotechnologies.
Funders:
Funding AgencyGrant Number
NIHGM62523
Army Research Office (ARO)UNSPECIFIED
Issue or Number:9
PubMed Central ID:PMC3178009
Record Number:CaltechAUTHORS:20111115-121540025
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20111115-121540025
Official Citation:Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis John T. Ngo and David A. Tirrell and Accounts of Chemical Research 2011 44 (9), 677-685
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:27786
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:15 Nov 2011 21:24
Last Modified:03 Oct 2019 03:26

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