Abstract
Nonnatural amino acid mutagenesis using expanded genetic codes allows us to introduce artificial functional groups such as fluorophores at specific sites of proteins. By applying this technique, site-specific fluorescence labeling has been achieved. This method enables to generate fluorescent protein probes that show ligand-dependent fluorescence change based on fluorescence quenching of the incorporated fluorophores by endogenous Trp residues. This strategy has been proved to be effective using ligand-binding proteins and antibodies. Moreover, site-specific double-labeling of ligand-binding proteins with two fluorophores using two expanded genetic codons has achieved ratiometric detection of the ligand binding based on FRET and fluorescence quenching. These achievements demonstrate the usefulness of the site-specific incorporation of fluorescent nonnatural amino acids and their potential utility as research and diagnostic tools.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Wang L, Schultz PG (2005) Expanding the genetic code. Angew Chem Int Ed 44:34
Budisa N (2004) Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. Angew Chem Int Ed 43:6426
Link J, Mock ML, Tirrell DA (2003) Non-canonical amino acids in protein engineering. Curr Opin Biotechnol 14:603
Hendrickson TL, de Crécy-Lagard V, Schimmel P (2004) Incorporation of nonnatural amino acids into proteins. Annu Rev Biochem 73:147
Zhang WH, Otting G, Jackson CJ (2013) Protein engineering with unnatural amino acids. Curr Opin Struct Biol 23:581
Hohsaka T, Sisido M (2002) Incorporation of non-natural amino acids into proteins. Curr Opin Chem Biol 6:809
Hohsaka T, Ashizuka Y, Murakami H, Sisido M (1996) Incorporation of nonnatural amino acids into streptavidin through in vitro frame-shift suppression. J Am Chem Soc 118:9778
Hohsaka T, Kajihara D, Ashizuka Y, Murakami H, Sisido M (1999) Efficient incorporation of nonnatural amino acids with large aromatic groups into streptavidin in in vitro protein synthesizing systems. J Am Chem Soc 121:34
Hohsaka T, Ashizuka Y, Taira H, Murakami H, Sisido M (2001) Incorporation of nonnatural amino acids into proteins by using various four-base codons in an E. coli in vitro translation system. Biochemistry 40:11060
Robertson SA, Ellman JA, Schultz PG (1991) A general and efficient route for chemical aminoacylation of transfer RNAs. J Am Chem Soc 113:2722
Hecht SM, Alford BL, Kuroda Y, Kitano S (1978) “Chemical aminoacylation” of tRNA’s. J Biol Chem 253:4517
Kajihara D, Abe R, Iijima I, Komiyama C, Sisido M, Hohsaka T (2006) FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids. Nat Methods 3:923
Abe R, Shiraga K, Ebisu S, Takagi H, Hohsaka T (2010) Incorporation of fluorescent non-natural amino acids into N-terminal tag of proteins in cell-free translation and its dependence on position and neighboring codons. J Biosci Bioeng 110:32
Miyawaki A et al (1997) Fluorescent indicators for Ca based on green fluorescent proteins and calmodulin. Nature 388:882
Miyawaki A (2003) Visualization of the spatial and temporal dynamics of intracellular signaling. Dev Cell 4:295
Hohsaka T, Ashizuka Y, Sasaki H, Murakami H, Sisido M (1999) Incorporation of two different nonnatural amino acids independently into a single protein through extension of the genetic code. J Am Chem Soc 121:12194
Taki M, Hohsaka T, Murakami H, Taira K, Sisido M (2002) Position-specific incorporation of a fluorophore-quencher pair into a single streptavidin through orthogonal four-base codon/anticodon pairs. J Am Chem Soc 124:14586
Marme N, Knemeyer J-P, Sauer M, Wolfrum J (2003) Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan. Bioconjug Chem 14:1133
Iijima I, Hohsaka T (2009) Position-specific incorporation of fluorescent non-natural amino acids into maltosebinding protein for detection of ligand binding by FRET and fluorescence quenching. ChemBioChem 17:999
Quiocho FA, Spurlino JC, Rodseth LE (1997) Extensive features of tight oligosaccharide binding revealed in high-resolution structures of the maltodextrin transport/chemosensory receptor. Structure 5:997
Li IT, Pham E, Truong K (2006) Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics. Biotechnol Lett 28:1971
Otsuji T, Okuda-Ashitaka E, Kojima S, Akiyama H, Ito S, Ohmiya Y (2004) Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase. Anal Biochem 329:230
Gammon ST, Villalobos VM, Roshal M, Samrakandi M, Piwnica-Worms D (2009) Rational design of novel redshifted BRET pairs: platforms for real-time single-chain protease biosensors. Biotechnol Prog 25:559
Charest PG, Terrillon S, Bouvier M (2005) Monitoring agonist-promoted conformational changes of beta-arrestin in living cells by intramolecular BRET. EMBO Rep 6:334
Yamaguchi A, Hohsaka T (2012) Synthesis of novel BRET/FRET protein probes containing light-emitting proteins and fluorescent nonnatural amino acids. Bull Chem Soc Jpn 85:576
Verhaegent M, Christopoulos TK (2002) Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal Chem 74(4378)
Gabius H-J, André S, Jimenez-Barbero J, Romeo A, Solís D (2011) From lectin structure to functional glycomics: principles of the sugar code. Trends Biochem Sci 36:298
Krishnamoorthy L, Mahal LK (2009) Glycomic analysis: an array of technologies. ACS Chem Biol 4:715
Yabe R, Suzuki R, Kuno A, Fujimoto Z, Jigami Y, Hirabayashi J (2007) Tailoring a novel sialic acid-binding lectin from a ricin-B chain-like galactose-binding protein by natural evolution-mimicry. J Biochem 141:389
Taira H, Matsushita Y, Kojima K, Shiraga K, Hohsaka T (2008) Comprehensive screening of amber suppressor tRNAs suitable for incorporation of non-natural amino acids in a cell-free translation system. Biochem Biophys Res Commun 374:304
Ito Y, Hohsaka T (2013) Incorporation of fluorescent nonnatural amino acid into sialic acid-binding lectin for fluorescence detection of ligand-binding. Bull Chem Soc Jpn 86:729
Kuno A, Uchiyama N, Koseki-Kuno S, Ebe Y, Takashima S, Yamada M, Hirabayashi J (2005) Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling. Nat Methods 2:851
Ueda H et al (1996) Open sandwich ELISA: a novel immunoassay based on the interchain interaction of antibody variable region. Nat Biotechnol 14:1714
Abe R, Ohashi H, Iijima I, Ihara M, Takagi H, Hohsaka T, Ueda H (2011) “Quenchbodies”: quench-based antibody probes that show antigen-dependent fluorescence. J Am Chem Soc 133:17386
Wang J, Xie J, Schultz PG (2006) A genetically encoded fluorescent amino acid. J Am Chem Soc 128:8738
Summerer D, Chen S, Wu N, Deiters A, Chin JW, Schultz PG (2006) A genetically encoded fluorescent amino acid. Proc Natl Acad Sci U S A 103:9785
Speight LC, Muthusamy AK, Goldberg JM, Warner JB, Wissner RF, Willi TS, Woodman BF, Mehl RA, Petersson EJ (2013) Efficient synthesis and in vivo incorporation of acridonylalanine, a fluorescent amino acid for lifetime and Förster resonance energy transfer/luminescence resonance energy transfer studies. J Am Chem Soc 135:18806
Acknowledgments
The author deeply appreciates the contributions of all the coworkers in this research project. This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas (20107005) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Japan
About this chapter
Cite this chapter
Hohsaka, T. (2016). Site-Specific Incorporation of Fluorescent Nonnatural Amino Acids into Proteins and Its Application to Fluorescence Analysis of Proteins. In: Terazima, M., Kataoka, M., Ueoka, R., Okamoto, Y. (eds) Molecular Science of Fluctuations Toward Biological Functions . Springer, Tokyo. https://doi.org/10.1007/978-4-431-55840-8_5
Download citation
DOI: https://doi.org/10.1007/978-4-431-55840-8_5
Published:
Publisher Name: Springer, Tokyo
Print ISBN: 978-4-431-55838-5
Online ISBN: 978-4-431-55840-8
eBook Packages: Chemistry and Materials ScienceChemistry and Material Science (R0)