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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Liu C C, Schultz P G (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79, 413–444.
Xie J, Schultz P G (2005) An expanding genetic code. Methods 36, 227–238.
Miyake-Stoner S J et al. (2010) Generating permissive site-specific unnatural aminoacyl-tRNA synthetases. Biochemistry 49, 1667–1677.
Li C G et al. (2010) Protein F-19 NMR in Escherichia coli. J Am Chem Soc 132, 321–327.
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.
Hammill J T et al. (2007) Preparation of site-specifically labeled fluorinated proteins for F-19-NMR structural characterization. Nat Protoc 2, 2601–2607.
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.
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.
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.
Stokes A L et al. (2009) Enhancing the utility of unnatural amino acid synthetases by manipulating broad substrate specificity. Mol Biosyst 5, 1032–1038.
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.
Studier F W (2005) Protein production by auto-induction in high density shaking cultures. Protein Express Purif 41, 207–234.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Peeler, J.C., Mehl, R.A. (2012). Site-Specific Incorporation of Unnatural Amino Acids as Probes for Protein Conformational Changes. In: Pollegioni, L., Servi, S. (eds) Unnatural Amino Acids. Methods in Molecular Biology, vol 794. Humana Press. https://doi.org/10.1007/978-1-61779-331-8_8
Download citation
DOI: https://doi.org/10.1007/978-1-61779-331-8_8
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-330-1
Online ISBN: 978-1-61779-331-8
eBook Packages: Springer Protocols