Advertisement

Site-Specific Incorporation of Unnatural Amino Acids as Probes for Protein Conformational Changes

  • Jennifer C. Peeler
  • Ryan A. MehlEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 794)

Abstract

Site-specific in vivo incorporation of unnatural amino acids provides powerful tools for the study of protein interaction and dynamics. Here, we provide a protocol for the incorporation of six such UAA probes into a GFP reporter system, expressed in Escherichia coli from both arabinose and lactose-inducible expression plasmids using an autoinduction media.

Key words

Unnatural amino acid probe Autoinduction GFP reporter Amber codon suppression pDule2 plasmid 

References

  1. 1.
    Liu C C, Schultz P G (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79, 413–444.PubMedCrossRefGoogle Scholar
  2. 2.
    Xie J, Schultz P G (2005) An expanding genetic code. Methods 36, 227–238.PubMedCrossRefGoogle Scholar
  3. 3.
    Miyake-Stoner S J et al. (2010) Generating permissive site-specific unnatural aminoacyl-tRNA synthetases. Biochemistry 49, 1667–1677.PubMedCrossRefGoogle Scholar
  4. 4.
    Li C G et al. (2010) Protein F-19 NMR in Escherichia coli. J Am Chem Soc 132, 321–327.PubMedCrossRefGoogle Scholar
  5. 5.
    Jackson J C, Hammill J T, Mehl R A (2007) Site-specific incorporation of a F-19-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity. J Am Chem Soc129, 1160–1166.PubMedCrossRefGoogle Scholar
  6. 6.
    Hammill J T et al. (2007) Preparation of site-specifically labeled fluorinated proteins for F-19-NMR structural characterization. Nat Protoc 2, 2601–2607.PubMedCrossRefGoogle Scholar
  7. 7.
    Taskent-Sezgin H et al. (2009) Interpretation of p-cyanophenylalanine fluorescence in proteins in terms of solvent exposure and contribution of side-chain quenchers: a combined fluorescence, IR and molecular dynamics study. Biochemistry 48, 9040–9046.PubMedCrossRefGoogle Scholar
  8. 8.
    Miyake-Stoner S J et al. (2009) Probing protein folding using site-specifically encoded unnatural amino acids as FRET Donors with tryptophan. Biochemistry 48, 5953–5962.PubMedCrossRefGoogle Scholar
  9. 9.
    Chin J W et al. (2002) Addition of p-Azido-l-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc 124, 9026–9027.PubMedCrossRefGoogle Scholar
  10. 10.
    Stokes A L et al. (2009) Enhancing the utility of unnatural amino acid synthetases by manipulating broad substrate specificity. Mol Biosyst 5, 1032–1038.PubMedCrossRefGoogle Scholar
  11. 11.
    Chin J W et al. (2002) Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc Natl Acad Sci USA 99, 11020–11024.PubMedCrossRefGoogle Scholar
  12. 12.
    Studier F W (2005) Protein production by auto-induction in high density shaking cultures. Protein Express Purif 41, 207–234.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of ChemistryFranklin & Marshall CollegeLancasterUSA

Personalised recommendations