Site-specific labeling of proteins with NMR-active unnatural amino acids
A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.
KeywordsSite-specific labeling Unnatural amino acids Spin label Metal chelator In-cell NMR
We thank Huiyong Hu for the synthesis of 15N-labeled o-NBTyr, and Hyun Soo Lee for samples of HQ-Ala.
- Fesik SW, Zuiderweg ERP (1988) Heteronuclear three-dimensional NMR spectroscopy. A strategy for the simplification of homonuclear two-dimensional NMR spectra. J Magn Reson 78:588–593Google Scholar
- Gaponenko V, Howarth JW, Columbus L, Gasmi-Seabrook G, Yuan J et al (2000) Protein global fold determination using site-directed spin and isotope labeling. Protein Sci 9:302–309Google Scholar
- Liu CC, Cellitti S, Geierstanger BH, Schultz PG (2009) Efficient expression of tyrosine-sulfated proteins in E. coli using an expanded code. (submitted)Google Scholar
- Neumann H, Peak-Chew SY, Chin JW (2008) Genetically encoding Ne-acetyllysine in recombinant proteins. Nat Chem Biol epublished.Google Scholar
- Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94(23):12366–12371CrossRefADSGoogle Scholar
- Sambrook J, Russell DW (2001) Molecular cloning, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USAGoogle Scholar