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Expanding the genetic code in Xenopus laevis oocytes.

TitleExpanding the genetic code in Xenopus laevis oocytes.
Publication TypeJournal Article
Year of Publication2013
AuthorsYe, S, Riou, M, Carvalho, S, Paoletti, P
JournalChembiochem
Volume14
Issue2
Pagination230-5
Date Published2013 Jan 21
ISSN1439-7633
KeywordsAmino Acids, Amino Acyl-tRNA Synthetases, Animals, Genetic Code, Models, Molecular, Oocytes, Protein Conformation, Protein Engineering, Protein Structure, Tertiary, Receptors, N-Methyl-D-Aspartate, RNA, Transfer, Ultraviolet Rays, Xenopus laevis
Abstract

Heterologous expression of ligand-gated ion channels (LGICs) in Xenopus laevis oocytes combined with site-directed mutagenesis has been demonstrated to be a powerful approach to study structure-function relationships. In particular, introducing unnatural amino acids (UAAs) has enabled modifications that are not found in natural proteins. However, the current strategy relies on the technically demanding in vitro synthesis of aminoacylated suppressor tRNA. We report here a general method that circumvents this limitation by utilizing orthogonal aminoacyl-tRNA synthetase (aaRS)/suppressor tRNA(CUA) pairs to genetically encode UAAs in Xenopus oocytes. We show that UAAs inserted in the N-terminal domain of N-methyl-D-aspartate receptors (NMDARs) serve as photo-crosslinkers that lock the receptor in a discrete conformational state in response to UV photo treatment. Our method should be generally applicable to studies of other LGICs in Xenopus oocytes.

DOI10.1002/cbic.201200515
Alternate JournalChembiochem
PubMed ID23292655