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Protein recognition by a pattern-generating fluorescent molecular probe

Nature Nanotechnology volume 12, pages 1161–1168 (2017)Cite this article

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Abstract

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical ‘noses/tongues’). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

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Figure 1: Distinct approaches for recognizing proteins with fluorescent molecular probes.
Figure 2: Chemical structure of the ID-probe.
Figure 3: Identifying MMPs, GSTs and PDGF in mixtures.
Figure 4: Recycling the ID-probe.
Figure 5: Tracking binding interactions and screening for new inhibitors.
Figure 6: Characterizing intracellular states of individual living cells.

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References

  1. Kubota, R. & Hamachi, I. Protein recognition using synthetic small-molecular binders toward optical protein sensing in vitro and in live cells. Chem. Soc. Rev. 44, 4454–4471 (2015).

    Article  CAS  Google Scholar 

  2. Ojida, A. et al. Oligo-Asp tag/Zn(II) complex probe as a new pair for labeling and fluorescence imaging of proteins. J. Am. Chem. Soc. 128, 10452–10459 (2006).

    Article  CAS  Google Scholar 

  3. Mizusawa, K., Takaoka, Y. & Hamachi, I. Specific cell surface protein imaging by extended self-assembling fluorescent turn-on nanoprobes. J. Am. Chem. Soc. 134, 13386–13395 (2012).

    Article  CAS  Google Scholar 

  4. Tsukiji, S., Miyagawa, M., Takaoka, Y., Tamura, T. & Hamachi, I. Ligand-directed tosyl chemistry for protein labeling in vivo. Nat. Chem. Biol. 5, 341–343 (2009).

    Article  CAS  Google Scholar 

  5. Martin, B. R., Giepmans, B. N.G., Adams, S. R. & Tsien, R. Y. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 23, 1308–1314 (2005).

    Article  CAS  Google Scholar 

  6. Hauser, C. T. & Tsien, R. Y. A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure. Proc. Natl Acad. Sci. USA 104, 3693–3697 (2007).

    Article  CAS  Google Scholar 

  7. Halo, T. L., Appelbaum, J., Hobert, E. M., Balkin, D. M. & Schepartz, A. Selective recognition of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid. J. Am. Chem. Soc. 131, 438–439 (2009).

    Article  CAS  Google Scholar 

  8. Sainlos, M., Iskenderian, W. S. & Imperiali, B. A general screening strategy for peptide-based fluorogenic ligands: probes for dynamic studies of PDZ domain-mediated interactions. J. Am. Chem. Soc. 131, 6680–6682 (2009).

    Article  CAS  Google Scholar 

  9. Lata, S., Gavutis, M., Tampé, R. & Piehler, J. Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation. J. Am. Chem. Soc. 128, 2365–2372 (2006).

    Article  CAS  Google Scholar 

  10. Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).

    Article  Google Scholar 

  11. Reinhardt, U. et al. Peptide-templated acyl transfer: a chemical method for the labeling of membrane proteins on live cells. Angew. Chem. Int. Ed. 53, 10237–10241 (2014).

    Article  CAS  Google Scholar 

  12. Vinkenborg, J. L., Mayer, G. & Famulok, M. Aptamer-based affinity labeling of proteins. Angew. Chem. Int. Ed. 51, 9176–9180 (2012).

    Article  CAS  Google Scholar 

  13. Yang, C. J., Jockusch, S., Vicens, M., Turro, N. J. & Tan, W. Light-switching excimer probes for rapid protein monitoring in complex biological fluids. Proc. Natl Acad. Sci. USA 102, 17278–17283 (2005).

    Article  CAS  Google Scholar 

  14. Guignet, E. G., Hovius, R. & Vogel, H. Reversible site-selective labeling of membrane proteins in live cells. Nat. Biotechnol. 22, 440–444 (2004).

    Article  CAS  Google Scholar 

  15. Karpenko, I. A. et al. Red fluorescent turn-on ligands for imaging and quantifying G protein-coupled receptors in living cells. ChemBioChem 15, 359–363 (2014).

    Article  CAS  Google Scholar 

  16. Karpenko, I. A. et al. Fluorogenic squiring dimers with polarity-sensitive folding as bright far-red probes for background-free bioimaging. J. Am. Chem. Soc. 137, 405–412 (2015).

    Article  CAS  Google Scholar 

  17. Unger-Angel, L. et al. Protein recognition by bivalent, ‘turn-on’ fluorescent molecular probes. Chem. Sci. 6, 5419–5425 (2015).

    Article  CAS  Google Scholar 

  18. Baldini, L., Wilson, A. J., Hong, J. & Hamilton, A. D. Pattern-based detection of different proteins using an array of fluorescent protein surface receptors. J. Am. Chem. Soc. 126, 5656–5657 (2004).

    Article  CAS  Google Scholar 

  19. Zhou, H., Baldini, L., Hong, J., Wilson, A. J. & Hamilton, A. D. Pattern recognition of proteins based on an array of functionalized porphyrins. J. Am. Chem. Soc. 128, 2421–2425 (2006).

    Article  CAS  Google Scholar 

  20. Margulies, D. & Hamilton, A. D. Protein recognition by an ensemble of fluorescent DNA G-quadruplexes. Angew. Chem. Int. Ed. 48, 1771–1774 (2009).

    Article  CAS  Google Scholar 

  21. Wright, A. T. et al. Differential receptors create patterns that distinguish various proteins. Angew. Chem. Int. Ed. 44, 6375–6378 (2005).

    Article  CAS  Google Scholar 

  22. Miranda, O. R. et al. Array-based sensing of proteins using conjugated polymers. J. Am. Chem. Soc. 129, 9856–9857 (2007).

    Article  CAS  Google Scholar 

  23. De, M. et al. Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein. Nat. Chem. 1, 461–465 (2009).

    Article  CAS  Google Scholar 

  24. Rana, S. et al. A multichannel nanosensor for instantaneous readout of cancer drug mechanisms. Nat. Nanotech. 10, 65–69 (2014).

    Article  Google Scholar 

  25. Zamora-Olivares, D., Kaoud, T. S., Dalby, K. N. & Anslyn, E. V. In-situ generation of differential sensors that fingerprint kinases and the cellular response to their expression. J. Am. Chem. Soc. 135, 14814–14820 (2013).

    Article  CAS  Google Scholar 

  26. Zamora-Olivares, D. et al. Differential sensing of MAP kinases using SOX-peptides. Angew. Chem. Int. Ed. 53, 14064–14068 (2014).

    Article  CAS  Google Scholar 

  27. Motiei, L., Pode, Z., Koganitsky, A. & Margulies, D. Targeted protein surface sensors as a tool for analyzing small populations of proteins in biological mixtures. Angew. Chem. Int. Ed. 53, 9289–9293 (2014).

    Article  CAS  Google Scholar 

  28. Selvakumar, K., Motiei, L. & Margulies, D. Enzyme−artificial enzyme interactions as a means for discriminating among structurally similar isozymes. J. Am. Chem. Soc. 137, 4892–4895 (2015).

    Article  CAS  Google Scholar 

  29. Stastna, M. & Van Eyk, J. E. Analysis of protein isoforms: can we do it better? Proteomics 12, 2937–2948 (2012).

    Article  CAS  Google Scholar 

  30. Rout, B., Unger, L., Armony, G., Iron, M. A. & Margulies, D. Medication detection by a combinatorial fluorescent molecular sensor. Angew. Chem. Int. Ed. 51, 12477–12481 (2012).

    Article  CAS  Google Scholar 

  31. Rout, B., Milko, P., Iron, M. A., Motiei, L. & Margulies, D. Authorizing multiple chemical passwords by a combinatorial molecular keypad lock. J. Am. Chem. Soc. 135, 15330–15333 (2013).

    Article  CAS  Google Scholar 

  32. Sarker, T., Selvakumar, K., Motiei, L. & Margulies, D. Message in a molecule. Nat. Commun. 7, 11374 (2016).

    Article  Google Scholar 

  33. Tsukiji, S. & Hamachi, I. Ligand-directed tosyl chemistry for in situ native protein labeling and engineering in living systems: from basic properties to applications. Curr. Opin. Chem. Biol. 21, 136–143 (2014).

    Article  CAS  Google Scholar 

  34. Moses, M. A. et al. Increased incidence of matrix metalloproteinases in urine of cancer patients. Cancer Res. 58, 1395–1399 (1998).

    CAS  Google Scholar 

  35. Gersuk, G., Carmel, R. & Pattengale, P. Platelet-derived growth factor concentrations in platelet-poor plasma and urine from patients with myeloproliferative disorders. Blood 74, 2330–2334 (1989).

    CAS  Google Scholar 

  36. Sundberg, A., Appelkvist, E. L., Dallner, G. & Nilsson, R. Glutathione transferases in the urine: sensitive methods for detection of kidney damage induced by nephrotoxic agents in humans. Environ. Health Perspect. 102, 293–296 (1994).

    CAS  Google Scholar 

  37. McIlwain, C. C., Townsend, D. M. & Tew, K. D. Glutathione S-transferase polymorphisms: cancer incidence and therapy. Oncogene 25, 1639–1648 (2006).

    Article  CAS  Google Scholar 

  38. Herszenyi, L. et al. Alterations of glutathione S-transferase and matrix metalloproteinase-9 expressions are early events in esophageal carcinogenesis. World J. Gastroenterol. 13, 676–682 (2007).

    Article  CAS  Google Scholar 

  39. Zhang, B.-B. et al. Diagnostic value of platelet derived growth factor-BB, transforming growth factor-β(1), matrix metalloproteinase-1, and tissue inhibitor of matrix metalloproteinase-1 in serum and peripheral blood mononuclear cells for hepatic fibrosis. World J. Gastroenterol. 9, 2490–2496 (2003).

    Article  CAS  Google Scholar 

  40. Rasmussen, H. S. & McCann, P. P. Matrix metalloproteinase inhibition as a novel anticancer strategy: a review with special focus on batimastat and marimastat. Pharmacol. Ther. 75, 69–75 (1997).

    Article  CAS  Google Scholar 

  41. Klymchenko, A. S. & Mely, Y. in Progress in Molecular Biology and Translational Science Vol. 113 (ed. May, C.M.) 35–58 (Academic, 2013).

    Google Scholar 

  42. Röck, F., Barsan, N. & Weimar, U. Electronic nose: current status and future trends. Chem. Rev. 108, 705–725 (2008).

    Article  Google Scholar 

  43. Lea, W. A. & Simeonov, A. Fluorescence polarization assays in small molecule screening. Expert Opin. Drug Discov. 6, 17–32 (2011).

    Article  CAS  Google Scholar 

  44. Hafner, M. et al. Displacement of protein-bound aptamers with small molecules screened by fluorescence polarization. Nat. Protocols 3, 579–587 (2008).

    Article  CAS  Google Scholar 

  45. Zimmermann, G. R., Lehár, J. & Keith, C. T. Multi-target therapeutics: when the whole is greater than the sum of the parts. Drug Discov. Today 12, 34–42 (2007).

    Article  CAS  Google Scholar 

  46. Gibbons, N. B., Watson, R. W. G., Coffey, R. N. T., Brady, H. P. & Fitzpatrick, J. M. Heat-shock proteins inhibit induction of prostate cancer cell apoptosis. Prostate 45, 58–65 (2000).

    Article  CAS  Google Scholar 

  47. Rao, A. V.S. K. & Shaha, C. Role of glutathione S-transferases in oxidative stress–induced male germ cell apoptosis. Free Radic. Biol. Med. 29, 1015–1027 (2000).

    Article  CAS  Google Scholar 

  48. Mermis, J. et al. Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling. Respir. Res. 12, 113 (2011).

    Article  Google Scholar 

  49. Yang, H.-L. et al. Toona sinensis inhibits LPS-induced inflammation and migration in vascular smooth muscle cells via suppression of reactive oxygen species and NF-B signaling pathway. Oxid. Med. Cell. Longev. 2014, 16 (2014).

    Google Scholar 

  50. Dierickx, P. J. The influence of picolines on glutathione transferase activity and subunit composition in human liver derived Hep G2 cells. Biochem. Pharmacol. 48, 1976–1978 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank I. Sagi for kindly donating MMP-12 and MMP-14. This research was supported by the European Research Council Starting Grant 338265. We thank G. Cohen (Grand Israel National Center for Personalized Medicine) for her help in performing the HTS experiments and O. Matalon (Department Structural Biology) for assisting in cell imaging.

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Authors and Affiliations

  1. Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, 7610001, Israel

    Zohar Pode, Ronny Peri-Naor, Leila Motiei & David Margulies

  2. Department of Structural Biology, Weizmann Institute of Science, Rehovot, 7610001, Israel

    Joseph M. Georgeson & Tal Ilani

  3. Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel

    Vladimir Kiss

  4. Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot, 7610001, Israel

    Tamar Unger

  5. The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot, 761001, Israel

    Barak Markus & Haim M. Barr

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Contributions

Z.P., L.M. and D.M. conceived the research; Z.P. synthesized the probe, performed the experiments and analysed the data. Z.P., R.P.-N. and T.I. carried out the cell-culture work and IF assays. Z.P., J.M.G. and V.K. performed the confocal fluorescence microscopy experiments. T.U. cloned and expressed the GSTs. Z.P. and H.M.B. performed the HTS assays. Z.P., H.M.B. and B.M. analysed the HTS data. The manuscript was written by Z.P., L.M. and D.M.

Corresponding author

Correspondence to David Margulies.

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Pode, Z., Peri-Naor, R., Georgeson, J. et al. Protein recognition by a pattern-generating fluorescent molecular probe. Nature Nanotech 12, 1161–1168 (2017). https://doi.org/10.1038/nnano.2017.175

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