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Genetically encoding unnatural amino acids for cellular and neuronal studies

Nature Neuroscience volume 10, pages 1063–1072 (2007)Cite this article

Abstract

Proteins participate in various biological processes and can be harnessed to probe and control biological events selectively and reproducibly, but the genetic code limits the building block to 20 common amino acids for protein manipulation in living cells. The genetic encoding of unnatural amino acids will remove this restriction and enable new chemical and physical properties to be precisely introduced into proteins. Here we present new strategies for generating orthogonal tRNA-synthetase pairs, which made possible the genetic encoding of diverse unnatural amino acids in different mammalian cells and primary neurons. Using this new methodology, we incorporated unnatural amino acids with extended side chains into the K+ channel Kv1.4, and found that the bulkiness of residues in the inactivation peptide is essential for fast channel inactivation, a finding that had not been possible using conventional mutagenesis. This technique will stimulate and facilitate new molecular studies using tailored unnatural amino acids for cell biology and neurobiology.

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Figure 1: Efficient expression of bacterial tRNA in mammalian cells using the H1 promoter.
Figure 2: Unnatural-amino-acid-specific synthetases evolved in yeast are functional in mammalian cells.
Figure 3: Genetically encoding unnatural amino acids in neurons.
Figure 4: A model for the N-type inactivation of Kv channels.
Figure 5: Probing N-type inactivation using unnatural amino acid mutagenesis with Kv1.4 channels expressed in mammalian cells.
Figure 6: Diameter of the inactivation peptide affects channel inactivation owing to the size restriction of the side portal.

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Acknowledgements

We thank B.R. Martin and R.Y. Tsien for plasmid pCLHF, E. Cooper for Kv1.4 cDNA, and D. Summerer and P.G. Schultz for plasmid pLeuRSB8T252A before publication. We thank A.R. Parrish, K. Adams, J. Xu, F.D. Winter, R. Nassirpour, Z. Chen for technical support, and P. Aryal for helpful discussions. P.A. Slesinger acknowledges grant support from US National Institutes of Health. L. Wang acknowledges the support of the Searle Scholar Program and the Beckman Young Investigator Program.

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Authors and Affiliations

  1. Jack H. Skirball Center for Chemical Biology and Proteomics, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.,

    Wenyuan Wang, Jeffrey K Takimoto, Gordon V Louie, Thomas J Baiga, Joseph P Noel & Lei Wang

  2. Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, 92037, California, USA

    Kuo-Fen Lee & Paul A Slesinger

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  1. Wenyuan Wang
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Contributions

W.W. conducted experiments for tRNA expression, encoding unnatural amino acids in neurons and probing channel inactivation, and analyzed the data. J.K.T. conducted experiments for encoding unnatural amino acids in other mammalian cells, made constructs for neuronal work, and analyzed the data. G.V.L. conducted the simulation of the inactivation peptide. T.J.B. synthesized DanAla. J.P.N. provided support for computer simulation and chemical synthesis. K.L. provided support for neuronal work. P.A.S. provided support and guidance for the electrophysiology experiments, analyzed the data, and revised the manuscript. L.W. conceived and designed the experiments for unnatural amino acid incorporation and ion channel inactivation, analyzed the data, wrote the manuscript, and supervised the project.

Corresponding authors

Correspondence to Paul A Slesinger or Lei Wang.

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The authors declare no competing financial interests.

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Wang, W., Takimoto, J., Louie, G. et al. Genetically encoding unnatural amino acids for cellular and neuronal studies. Nat Neurosci 10, 1063–1072 (2007). https://doi.org/10.1038/nn1932

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