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Engineering of weak helper interactions for high-efficiency FRET probes

Abstract

Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.

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Figure 1: Design of FRET helper interactions.
Figure 2: Enhanced FRET.
Figure 3: Application of conventional and hiFRET probes to the study of H-Ras–Raf1 signaling.
Figure 4: Detection of a transient Raf1–B-Raf interaction induced by a B-Raf kinase inhibitor.

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References

  1. Miyawaki, A. Development of probes for cellular functions using fluorescent proteins and fluorescence resonance energy transfer. Annu. Rev. Biochem. 80, 357–373 (2011).

    Article  CAS  Google Scholar 

  2. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    Article  CAS  Google Scholar 

  3. Piston, D.W. & Kremers, G.-J. Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem. Sci. 32, 407–414 (2007).

    Article  CAS  Google Scholar 

  4. Lam, A.J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012).

    Article  CAS  Google Scholar 

  5. Zacharias, D.A. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

    Article  CAS  Google Scholar 

  6. Landgraf, D., Okumus, B., Chien, P., Baker, T.A. & Paulsson, J. Segregation of molecules at cell division reveals native protein localization. Nat. Methods 9, 480–482 (2012).

    Article  CAS  Google Scholar 

  7. Nguyen, A.W. & Daugherty, P.S. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Biotechnol. 23, 355–360 (2005).

    Article  CAS  Google Scholar 

  8. Vinkenborg, J.L., Evers, T.H., Reulen, S.W.A., Meijer, E.W. & Merkx, M. Enhanced sensitivity of FRET-based protease sensors by redesign of the GFP dimerization interface. ChemBioChem 8, 1119–1121 (2007).

    Article  CAS  Google Scholar 

  9. Ohashi, T., Galiacy, S.D., Briscoe, G. & Erickson, H.P. An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins. Protein Sci. 16, 1429–1438 (2007).

    Article  CAS  Google Scholar 

  10. Vinkenborg, J.L. et al. Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis. Nat. Methods 6, 737–740 (2009).

    Article  CAS  Google Scholar 

  11. Kotera, I., Iwasaki, T., Imamura, H., Noji, H. & Nagai, T. Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators. ACS Chem. Biol. 5, 215–222 (2010).

    Article  CAS  Google Scholar 

  12. Golynskiy, M.V., Koay, M.S., Vinkenborg, J.L. & Merkx, M. Engineering protein switches: sensors, regulators, and spare parts for biology and biotechnology. ChemBioChem 12, 353–361 (2011).

    Article  CAS  Google Scholar 

  13. Evers, T.H., Appelhof, M.A.M., de Graaf-Heuvelmans, P.T.H.M., Meijer, E.W. & Merkx, M. Ratiometric detection of Zn(II) using chelating fluorescent protein chimeras. J. Mol. Biol. 374, 411–425 (2007).

    Article  CAS  Google Scholar 

  14. Walther, K.A., Papke, B., Sinn, M., Michel, K. & Kinkhabwala, A. Precise measurement of protein interacting fractions with fluorescence lifetime imaging microscopy. Mol. Biosyst. 7, 322–336 (2011).

    Article  CAS  Google Scholar 

  15. Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. Reducing the environmental sensitivity of yellow fluorescent protein. mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001).

    CAS  Google Scholar 

  16. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

  17. Bayle, J.H. et al. Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem. Biol. 13, 99–107 (2006).

    Article  CAS  Google Scholar 

  18. Grünberg, R., Ferrar, T.S., van der Sloot, A.M., Constante, M. & Serrano, L. Building blocks for protein interaction devices. Nucleic Acids Res. 38, 2645–2662 (2010).

    Article  Google Scholar 

  19. Pires, J.R. et al. Solution structures of the YAP65 WW domain and the variant L30 K in complex with the peptides GTPPPPYTVG, N-(n-octyl)-GPPPY and PLPPY and the application of peptide libraries reveal a minimal binding epitope. J. Mol. Biol. 314, 1147–1156 (2001).

    Article  CAS  Google Scholar 

  20. Zarrinpar, A., Park, S.-H. & Lim, W.A. Optimization of specificity in a cellular protein interaction network by negative selection. Nature 426, 676–680 (2003).

    Article  CAS  Google Scholar 

  21. Tonikian, R. et al. Bayesian modeling of the yeast SH3 domain interactome predicts spatiotemporal dynamics of endocytosis proteins. PLoS Biol. 7, e1000218 (2009).

    Article  Google Scholar 

  22. van der Meer, B.W. Kappa-squared: from nuisance to new sense. J. Biotechnol. 82, 181–196 (2002).

    CAS  PubMed  Google Scholar 

  23. Lee, K.C. et al. Application of the stretched exponential function to fluorescence lifetime imaging. Biophys. J. 81, 1265–1274 (2001).

    Article  CAS  Google Scholar 

  24. Ai, H.-w., Henderson, J.N., Remington, S.J. & Campbell, R.E. Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem. J. 400, 531–540 (2006).

    Article  CAS  Google Scholar 

  25. Luo, K.Q., Yu, V.C., Pu, Y. & Chang, D.C. Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells. Biochem. Biophys. Res. Commun. 283, 1054–1060 (2001).

    Article  CAS  Google Scholar 

  26. Goedhart, J. et al. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat. Commun. 3, 751 (2012).

    Article  Google Scholar 

  27. Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. USA 96, 11241–11246 (1999).

    Article  CAS  Google Scholar 

  28. Kato, Y., Ito, M., Kawai, K., Nagata, K. & Tanokura, M. Determinants of ligand specificity in groups I and IV WW domains as studied by surface plasmon resonance and model building. J. Biol. Chem. 277, 10173–10177 (2002).

    Article  CAS  Google Scholar 

  29. Peyker, A., Rocks, O. & Bastiaens, P.I.H. Imaging activation of two Ras isoforms simultaneously in a single cell. ChemBioChem 6, 78–85 (2005).

    Article  CAS  Google Scholar 

  30. Hibino, K. et al. Single- and multiple-molecule dynamics of the signaling from H-Ras to cRaf-1 visualized on the plasma membrane of living cells. ChemPhysChem 4, 748–753 (2003).

    Article  CAS  Google Scholar 

  31. Heidorn, S.J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140, 209–221 (2010).

    Article  CAS  Google Scholar 

  32. Poulikakos, P.I., Zhang, C., Bollag, G., Shokat, K.M. & Rosen, N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464, 427–430 (2010).

    Article  CAS  Google Scholar 

  33. Hatzivassiliou, G. et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464, 431–435 (2010).

    Article  CAS  Google Scholar 

  34. Lavoie, H. et al. Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization. Nat. Chem. Biol. 9, 428–436 (2013).

    Article  CAS  Google Scholar 

  35. Grant, D.M. et al. Multiplexed FRET to image multiple signaling events in live cells. Biophys. J. 95, L69–L71 (2008).

    Article  CAS  Google Scholar 

  36. Gibson, T.J. Cell regulation: determined to signal discrete cooperation. Trends Biochem. Sci. 34, 471–482 (2009).

    Article  CAS  Google Scholar 

  37. Dueber, J.E., Mirsky, E.A. & Lim, W.A. Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat. Biotechnol. 25, 660–662 (2007).

    Article  CAS  Google Scholar 

  38. Lorentzen, A., Kinkhabwala, A., Rocks, O., Vartak, N. & Bastiaens, P.I.H. Regulation of Ras localization by acylation enables a mode of intracellular signal propagation. Sci. Signal. 3, ra68 (2010).

    Article  Google Scholar 

  39. Zhang, Y. & Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res. 33, 2302–2309 (2005).

    Article  CAS  Google Scholar 

  40. Grünberg, R., Nilges, M. & Leckner, J. Biskit—a software platform for structural bioinformatics. Bioinformatics 23, 769–770 (2007).

    Article  Google Scholar 

  41. Schymkowitz, J. et al. The FoldX web server: an online force field. Nucleic Acids Res. 33, W382–W388 (2005).

    Article  CAS  Google Scholar 

  42. Rocchia, W., Alexov, E. & Honig, B. Extending the applicability of the nonlinear Poisson-Boltzmann equation: multiple dielectric constants and multivalent ions. J. Phys. Chem. B 105, 6507–6514 (2001).

    Article  CAS  Google Scholar 

  43. Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  44. Weiner, S.J. et al. A new force field for molecular mechanical simulation of nucleic acids and proteins. J. Am. Chem. Soc. 106, 765–784 (1984).

    Article  CAS  Google Scholar 

  45. Word, J.M., Lovell, S.C., Richardson, J.S. & Richardson, D.C. Asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation. J. Mol. Biol. 285, 1735–1747 (1999).

    Article  CAS  Google Scholar 

  46. Duan, Y. et al. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J. Comput. Chem. 24, 1999–2012 (2003).

    Article  CAS  Google Scholar 

  47. Li, M.Z. & Elledge, S.J. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat. Methods 4, 251–256 (2007).

    Article  CAS  Google Scholar 

  48. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  49. Pace, C.N., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423 (1995).

    Article  CAS  Google Scholar 

  50. Gasteiger, E. et al. in The Proteomics Protocols Handbook Vol. 112 (ed. Walker, J.M.) Ch. 52, 571–607 (Humana Press, 2005).

  51. Abràmoff, M.D., Magalhães, P.J. & Ram, S.J. Image processing with ImageJ. Biophotonics Int. 11, 36–42 (2004).

    Google Scholar 

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Acknowledgements

This work was supported by the Human Frontiers Science Program (LT-fellowship to R.G.) and the European Union (projects EMERGENCE and PROSPECTS, grant agreement number HEALTH-F4-2008-201648, to L.S.) as well as the Spanish Ministry of Education and Science (Juan de la Cierva fellowship to A.M.v.d.S.). We thank M. Therrien for suggesting the B-Raf–Raf1 detection as well as M. Tyers for supporting the project.

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Contributions

R.G. and L.S. conceived the study. R.G. designed experiments and synthetic genes. F.S., R.G., A.M.v.d.S. and L.S. performed modeling and FoldX calculations. R.G. and T.F. performed in vitro experiments. J.V.B. and V.B.-S. performed cell culture experiments. R.G.-O., A.M., X.S., J.V.B. and V.B.-S. measured and analyzed FLIM. T.Z. and R.G. further analyzed FLIM data. X.S. and V.B.-S. performed caspase experiments. R.G., L.S., J.V.B., and T.Z. wrote the paper.

Corresponding authors

Correspondence to Raik Grünberg or Luis Serrano.

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Competing interests

Methods described in this study are subject to two patent applications (PCT/EP2012/075737 and PCT/EP2012/075743) filed by some of the authors (R.G., L.S. and F.S).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18, Supplementary Tables 1–6 and Supplementary Notes 1–4 (PDF 3706 kb)

Supplementary Data 1

Annotated sequences of synthetic proteins and sensors constructs (GenBank format) (ZIP 75 kb)

Supplementary Data 2

In vitro FRET efficiencies (complete data set of FRET measurements on purified proteins) (XLS 45 kb)

Supplementary Software

Python scripts for selection of mutations and cell trace plotting (ZIP 5 kb)

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Grünberg, R., Burnier, J., Ferrar, T. et al. Engineering of weak helper interactions for high-efficiency FRET probes. Nat Methods 10, 1021–1027 (2013). https://doi.org/10.1038/nmeth.2625

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