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An ALuc-Based Molecular Tension Probe for Sensing Intramolecular Protein–Protein Interactions

  • Sung-Bae KimEmail author
  • Ryo Nishihara
  • Koji Suzuki
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1461)

Abstract

Optical imaging of protein–protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. The present protocol demonstrates an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. A unique design of single-chain probes was fabricated, in which a full-length artificial luciferase (ALuc®) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. A molecular tension probe comprising ALuc23 greatly enhances the bioluminescence in response to varying concentrations of rapamycin, and named “tension probe (TP).” The basic probe design can be further modified towards eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named “combinational probe.” TPs may become an important addition to the tool box of bioassays in the determination of protein dynamics of interest in mammalian cells.

Key words

Bioluminescence Protein–protein interactions Tension probe Bioluminescent imaging Combinational probe 

Notes

Acknowledgements

This work was partly supported by JSPS KAKENHI Grants Number 26288088, 16K14051, and 15KK0029.

References

  1. 1.
    Ozawa T, Yoshimura H, Kim SB (2013) Advances in fluorescence and bioluminescence imaging. Anal Chem 85:590–609CrossRefPubMedGoogle Scholar
  2. 2.
    Vogel SS, Thaler C, Koushik SV (2006) Fanciful FRET. Sci STKE 2006(331):re2PubMedGoogle Scholar
  3. 3.
    Hoshino H, Nakajima Y, Ohmiya Y (2007) Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging. Nat Methods 4:637–639CrossRefPubMedGoogle Scholar
  4. 4.
    Luker KE, Smith MC, Luker GD, Gammon ST, Piwnica-Worms H, Piwnica-Worms D (2004) Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci USA 101:12288–12293Google Scholar
  5. 5.
    Kim SB, Awais M, Sato M, Umezawa Y, Tao H (2007) Integrated molecule-format bioluminescent probe for visualizing androgenicity of ligands based on the intramolecular association of androgen receptor with its recognition peptide. Anal Chem 79:1874–1880CrossRefPubMedGoogle Scholar
  6. 6.
    Tarassov K, Messier V, Landry CR, Radinovic S, Serna Molina MM, Shames I, Malitskaya Y, Vogel J, Bussey H, Michnick SW (2008) An in vivo map of the yeast protein interactome. Science 320:1465–1470CrossRefPubMedGoogle Scholar
  7. 7.
    Kim SB, Umezawa Y, Kanno KA, Tao H (2008) An integrated-molecule-format multicolor probe for monitoring multiple activities of a bioactive small molecule. ACS Chem Biol 3:359–372CrossRefPubMedGoogle Scholar
  8. 8.
    Kaihara A, Umezawa Y (2008) Genetically encoded bioluminescent indicator for ERK2 dimer in living cells. Chem Asian J 3:38–45CrossRefPubMedGoogle Scholar
  9. 9.
    Kim SB, Torimura M, Tao H (2013) Creation of artificial luciferases for bioassays. Bioconjug Chem 24:2067–2075CrossRefPubMedGoogle Scholar
  10. 10.
    Paulmurugan R, Gambhir SS (2005) Firefly luciferase enzyme fragment complementation for imaging in cells and living animals. Anal Chem 77:1295–1302CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kim SB, Otani Y, Umezawa Y, Tao H (2007) Bioluminescent indicator for determining protein–protein interactions using intramolecular complementation of split click beetle luciferase. Anal Chem 79:4820–4826Google Scholar
  12. 12.
    Kim SB, Izumi H (2014) Functional artificial luciferases as an optical readout for bioassays. Biochem Biophys Res Commun 448:418–423CrossRefPubMedGoogle Scholar
  13. 13.
    Takenaka Y, Masuda H, Yamaguchi A, Nishikawa S, Shigeri Y, Yoshida Y, Mizuno H (2008) Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica. Gene 425:28–35CrossRefPubMedGoogle Scholar
  14. 14.
    Takenaka Y, Yamaguchi A, Tsuruoka N, Torimura M, Gojobori T, Shigeri Y (2012) Evolution of bioluminescence in marine planktonic copepods. Mol Biol Evol 29:1669–1681CrossRefPubMedGoogle Scholar
  15. 15.
    Kim SB, Nishihara R, Citterio D, Suzuki K (2016) Genetically encoded molecular tension probe for tracing protein–protein interactions in mammalian cells. Bioconjug Chem 27(2):354–362.CrossRefPubMedGoogle Scholar
  16. 16.
    Banaszynski LA, Liu CW, Wandless TJ (2005) Characterization of the FKBP center dot Rapamycin center dot FRB ternary complex. J Am Chem Soc 127:4715–4721Google Scholar
  17. 17.
    Wear MA, Walkinshaw MD (2007) Determination of the rate constants for the FK506 binding protein/rapamycin interaction using surface plasmon resonance: An alternative sensor surface for Ni -nitrilotriacetic acid immobilization of His-tagged proteins. Anal Biochem 371:250–252Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan
  2. 2.Department of Applied Chemistry, Faculty of Science and TechnologyKeio UniversityYokohamaJapan

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