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Designer proteins: applications of genetic code expansion in cell biology

Key Points

  • A large number of unnatural amino acids can now be incorporated into proteins using 'orthogonal' aminoacyl-tRNA synthetase–tRNA pairs. The pyrrolysyl-tRNA synthetase (PylRS)–tRNACUA pair is currently the most versatile, and it has been used in bacteria, yeast, mammalian cells and Caenorhabditis elegans.

  • Photocrosslinking amino acids allows protein interactions to be defined in vitro and in bacteria, yeast and mammalian cells.

  • Post-translational modifications, including lysine acetylation, monomethylation and dimethylation, and ubiquitylation, can be quantitatively directed into proteins by genetic code expansion.

  • Photocaged amino acids allow rapid activation of protein function inside living cells. This approach can be used to dissect signalling pathways.

  • Biophysical probes can be incorporated into proteins. Infrared probes have been used to examine conformational changes in G protein-coupled receptors. Other probes will probably have similar uses.

  • Bio-orthogonal chemistry provides a promising approach for labelling proteins for a range of applications, including imaging, but more rapid methods are needed for cellular imaging.

  • More sophisticated methods for incorporating multiple distinct amino acids, including orthogonal ribosome evolution and the generation of new synthetase–tRNA pairs are likely to expand the range of applications that are possible in the future.

Abstract

Designer amino acids, beyond the canonical 20 that are normally used by cells, can now be site-specifically encoded into proteins in cells and organisms. This is achieved using 'orthogonal' aminoacyl-tRNA synthetase–tRNA pairs that direct amino acid incorporation in response to an amber stop codon (UAG) placed in a gene of interest. Using this approach, it is now possible to study biology in vitro and in vivo with an increased level of molecular precision. This has allowed new biological insights into protein conformational changes, protein interactions, elementary processes in signal transduction and the role of post-translational modifications.

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Figure 1: Expanding the genetic code.
Figure 2: In vivo photocrosslinking of membrane proteins and study of protein interactions.
Figure 3: Applications of genetically encoded post-translational modifications.
Figure 4: Genetically encoded photocaged amino acids allow insights into cell biology in real time.
Figure 5: Use of genetically encoded infrared probes to dissect local GPCR conformational changes during activation.

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Acknowledgements

We are grateful to the UK Medical Research Council for funding(U105181009 and MC_UP_A024_1008) and to K. Lang and S. Hancock for help with the manuscript.

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DATABASES

Protein Databank

1AAR

1KX5

1GZM

1S9J

2CPL

2JF5

2W9N

2XEW

2XK5

3DIN

3DQB

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Glossary

Selective pressure incorporation

Bacteria that are auxotrophic for a natural amino acid are used in conjunction with a related unnatural amino acid, leading to the incorporation of the unnatural amino acid throughout the cell's proteome.

TAP tagging

(Tandem affinity purification tagging). A process in which a protein is carboxy-terminally tagged with a peptide containing a calmodulin-binding peptide, a TEV protease cleavage site and protein A. The protein is first purified using Immunoglobulin G-coated beads that bind protein A. The protein fusion is then cleaved from the gene of interest by the TEV protease and purified.

Chaperones

Proteins that assist other macromolecules in folding and/or unfolding and assembly and/or disassembly.

Elongation factor-Tu

(EF-Tu). A protein that binds to aminoacylated tRNAs and delivers them to the ribosome for protein synthesis.

Nucleosome

A unit of DNA packaging in eukaryotes in which a length of DNA is wrapped around an octamer of histone proteins.

SWI/SNF

(Switch/sucrose nonfermentable). An ATP-dependent multiprotein nucleosome-remodelling complex that is found in yeast.

RSC

(Remodels the structure of chromatin). An ATP-dependent multiprotein nucleosome-remodelling complex.

Prolyl isomerase

An enzyme that is responsible for the cis–trans isomerization of peptide bonds that are on the amino-terminal side of Pro residues.

Enthalpy

A measure (in thermodynamics) of the total internal energy of a system plus its pressure multiplied by its volume.

Entropy

A thermodynamic property that can be used to determine the energy that is not available for work.

E1, E2 and E3

The enzymes by which ubiquitin is added to cellular proteins. The E1 activates ubiquitin by attaching the molecule to its active site Cys residue. The E2 then binds the ubiquitin molecule, also by a Cys residue. The E2 then binds an E3 ligase which binds the target protein and catalyses the transfer of the ubiquitin to a Lys residue of the target protein.

Atypical ubiquitin chains

Ubiquitin chains that are not linked by the common Lys48 or Lys63 linkages but by one of the other Lys residues of ubiquitin.

Sumoylation

The covalent attachment of small ubiquitin-like modifier (SUMO) to a substrate protein.

Neddylation

A process, analogous to ubiquitylation, in which ubiquitin-like protein NEDD8 is conjugated to a protein substrate.

Distributive

A mode of activation in which two or more events occur independently. In the case of ERK2, which is diphosphorylated when activated, the MEK1–ERK2 complex dissociates after each phosphorylation event.

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Davis, L., Chin, J. Designer proteins: applications of genetic code expansion in cell biology. Nat Rev Mol Cell Biol 13, 168–182 (2012). https://doi.org/10.1038/nrm3286

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