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Directed molecular evolution to design advanced red fluorescent proteins

Nature Methods volume 8, pages 1019–1026 (2011)Cite this article

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Abstract

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

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Figure 1: Steps in the directed molecular evolution of fluorescent probes.
Figure 2: Major chemical transformations of the chromophores in red fluorescent proteins.
Figure 3: Methods that could improve molecular evolution of fluorescent proteins.
Figure 4: Possible FACS-based screening approaches for red-shifted fluorescent proteins.

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References

  1. Chudakov, D.M., Matz, M.V., Lukyanov, S. & Lukyanov, K.A. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 90, 1103–1163 (2010).

    Article  CAS  Google Scholar 

  2. Piatkevich, K.D. & Verkhusha, V.V. Advances in engineering of fluorescent proteins and photoactivatable proteins with red emission. Curr. Opin. Chem. Biol. 14, 23–29 (2010).

    Article  CAS  Google Scholar 

  3. Piatkevich, K.D., Malashkevich, V.N., Almo, S.C. & Verkhusha, V.V. Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large Stokes shift. J. Am. Chem. Soc. 132, 10762–10770 (2010).

    Article  CAS  Google Scholar 

  4. Pletnev, S., Subach, F.V., Dauter, Z., Wlodawer, A. & Verkhusha, V.V. Understanding blue-to-red conversion in monomeric fluorescent timers and hydrolytic degradation of their chromophores. J. Am. Chem. Soc. 132, 2243–2253 (2010).

    Article  CAS  Google Scholar 

  5. Subach, O.M. et al. Structural characterization of acylimine-containing blue and red chromophores in mTagBFP and TagRFP fluorescent proteins. Chem. Biol. 17, 333–341 (2010).

    Article  CAS  Google Scholar 

  6. Subach, O.M. et al. Conversion of red fluorescent protein into a bright blue probe. Chem. Biol. 15, 1116–1124 (2008).

    Article  CAS  Google Scholar 

  7. Subach, F.V. et al. Monomeric fluorescent timers that change color from blue to red report on cellular trafficking. Nat. Chem. Biol. 5, 118–126 (2009).

    Article  CAS  Google Scholar 

  8. Subach, F.V. et al. Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states. Proc. Natl. Acad. Sci. USA 106, 21097–21102 (2009).

    Article  CAS  Google Scholar 

  9. Subach, F.V. et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat. Methods 6, 153–159 (2009).

    Article  CAS  Google Scholar 

  10. Subach, F.V., Patterson, G.H., Renz, M., Lippincott-Schwartz, J. & Verkhusha, V.V. Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. J. Am. Chem. Soc. 132, 6481–6491 (2010).

    Article  CAS  Google Scholar 

  11. Gunewardene, M.S. et al. Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells. Biophys. J. 101, 1522–1528 (2011).

    Article  CAS  Google Scholar 

  12. Wang, Q. et al. Molecular mechanism of a green-shifted, pH-dependent red fluorescent protein mKate variant. PLoS ONE 6, e23513 (2011).

    Article  CAS  Google Scholar 

  13. Kremers, G.J., Hazelwood, K.L., Murphy, C.S., Davidson, M.W. & Piston, D.W. Photoconversion in orange and red fluorescent proteins. Nat. Methods 6, 355–358 (2009).

    Article  CAS  Google Scholar 

  14. Mishin, A.S. et al. The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore. Biochemistry 47, 4666–4673 (2008).

    Article  CAS  Google Scholar 

  15. Bogdanov, A.M. et al. Green fluorescent proteins are light-induced electron donors. Nat. Chem. Biol. 5, 459–461 (2009).

    Article  CAS  Google Scholar 

  16. Subach, O.M. et al. A photoswitchable orange-to-far-red fluorescent protein, PSmOrange. Nat. Methods 8, 771–777 (2010).

    Article  Google Scholar 

  17. Chica, R.A., Moore, M.M., Allen, B.D. & Mayo, S.L. Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries. Proc. Natl. Acad. Sci. USA 107, 20257–20262 (2010).

    Article  CAS  Google Scholar 

  18. Strack, R.L. et al. A rapidly maturing far-red derivative of DsRed-Expres2 for whole-cell labeling. Biochemistry 48, 8279–8281 (2009).

    Article  CAS  Google Scholar 

  19. Lin, M.Z. et al. Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem. Biol. 16, 1169–1179 (2009).

    Article  CAS  Google Scholar 

  20. Morozova, K.S. et al. Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy. Biophys. J. 99, L13–L15 (2010).

    Article  CAS  Google Scholar 

  21. Hoi, H. et al. A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization. J. Mol. Biol. 401, 776–791 (2010).

    Article  CAS  Google Scholar 

  22. Subach, F.V. et al. Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET. Chem. Biol. 17, 745–755 (2010).

    Article  CAS  Google Scholar 

  23. Davis, J.N. & van den Pol, A.N. Viral mutagenesis as a means for generating novel proteins. J. Virol. 84, 1625–1630 (2010).

    Article  CAS  Google Scholar 

  24. Arakawa, H. et al. Protein evolution by hypermutation and selection in the B cell line DT40. Nucleic Acids Res. 36, e1 (2008).

    Article  Google Scholar 

  25. Corish, P. & Tyler-Smith, C. Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng. 12, 1035–1040 (1999).

    Article  CAS  Google Scholar 

  26. Cava, F., Hidalgo, A. & Berenguer, J. Thermus thermophilus as biological model. Extremophiles 13, 213–231 (2009).

    Article  CAS  Google Scholar 

  27. Kwon, W.S., Da Silva, N.A. & Kellis, J.T. Jr. Relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of Escherichia coli. Protein Eng. 9, 1197–1202 (1996).

    Article  CAS  Google Scholar 

  28. Martin, A., Schmid, F.X. & Sieber, V. Proside: a phage-based method for selecting thermostable proteins. Methods Mol. Biol. 230, 57–70 (2003).

    CAS  PubMed  Google Scholar 

  29. Pavoor, T.V., Cho, Y.K. & Shusta, E.V. Development of GFP-based biosensors possessing the binding properties of antibodies. Proc. Natl. Acad. Sci. USA 106, 11895–11900 (2009).

    Article  Google Scholar 

  30. Daugherty, P.S. Protein engineering with bacterial display. Curr. Opin. Struct. Biol. 17, 474–480 (2007).

    Article  CAS  Google Scholar 

  31. Bergquist, P.L., Hardiman, E.M., Ferrari, B.C. & Winsley, T. Applications of flow cytometry in environmental microbiology and biotechnology. Extremophiles 13, 389–401 (2009).

    Article  Google Scholar 

  32. Telford, W.G., Subach, F.V. & Verkhusha, V.V. Supercontinuum white light lasers for flow cytometry. Cytometry A 75, 450–459 (2009).

    Article  Google Scholar 

  33. Goddard, G. et al. Single particle high resolution spectral analysis flow cytometry. Cytometry A 69, 842–851 (2006).

    Article  Google Scholar 

  34. Houston, J.P., Naivar, M.A. & Freyer, J.P. Digital analysis and sorting of fluorescence lifetime by flow cytometry. Cytometry A 77, 861–872 (2010).

    Article  Google Scholar 

  35. Kim, J., Kwon, D., Lee, J., Pasquier, H. & Grailhe, R. The use of Cyan Fluorescent Protein variants with a distinctive lifetime signature. Mol. Biosyst. 5, 151–153 (2009).

    Article  CAS  Google Scholar 

  36. Goedhart, J. et al. Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat. Methods 7, 137–139 (2010).

    Article  CAS  Google Scholar 

  37. Buschke, D.G. et al. Multiphoton flow cytometry to assess intrinsic and extrinsic Fluorescence in cellular aggregates: applications to stem cells. Microsc. Microanal. 17, 540–554 (2010).

    Article  Google Scholar 

  38. Drobizhev, M., Makarov, N.S., Tillo, S.E., Hughes, T.E. & Rebane, A. Two-photon absorption properties of fluorescent proteins. Nat. Methods 8, 393–399 (2011).

    Article  CAS  Google Scholar 

  39. Godin, J. et al. Microfluidics and photonics for Bio-System-on-a-Chip: a review of advancements in technology towards a microfluidic flow cytometry chip. J. Biophotonics 1, 355–376 (2008).

    Article  CAS  Google Scholar 

  40. Heng, X., Hsiung, F., Sadri, A. & Patt, P. Serial line scan encoding imaging cytometer for both adherent and suspended cells. Anal. Chem. 83, 1587–1593 (2011).

    Article  CAS  Google Scholar 

  41. Pai, J.H., Xu, W., Sims, C.E. & Allbritton, N.L. Microtable arrays for culture and isolation of cell colonies. Anal. Bioanal. Chem. 398, 2595–2604 (2010).

    Article  CAS  Google Scholar 

  42. Kim, J. et al. Cell lysis on a microfluidic CD (compact disc). Lab Chip 4, 516–522 (2004).

    Article  CAS  Google Scholar 

  43. Zhang, C., Xing, D. & Li, Y. Micropumps, microvalves, and micromixers within PCR microfluidic chips: advances and trends. Biotechnol. Adv. 25, 483–514 (2007).

    Article  CAS  Google Scholar 

  44. Barbulovic-Nad, I., Au, S.H. & Wheeler, A.R. A microfluidic platform for complete mammalian cell culture. Lab Chip 10, 1536–1542 (2010).

    Article  CAS  Google Scholar 

  45. Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S.W. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl. Acad. Sci. USA 102, 17565–17569 (2005).

    Article  CAS  Google Scholar 

  46. Lippincott-Schwartz, J. & Patterson, G.H. Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol. 19, 555–565 (2009).

    Article  CAS  Google Scholar 

  47. Wu, B., Piatkevich, K.D., Lionnet, T., Singer, R.H. & Verkhusha, V.V. Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics. Curr. Opin. Cell Biol. 23, 310–317 (2011).

    Article  CAS  Google Scholar 

  48. Davidson, M.W. & Campbell, R.E. Engineered fluorescent proteins: innovations and applications. Nat. Methods 6, 713–717 (2009).

    Article  CAS  Google Scholar 

  49. Nienhaus, G.U. & Wiedenmann, J. Structure, dynamics and optical properties of fluorescent proteins: perspectives for marker development. ChemPhysChem 10, 1369–1379 (2009).

    Article  CAS  Google Scholar 

  50. Post, J.N., Lidke, K.A., Rieger, B. & Arndt-Jovin, D.J. One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos. FEBS Lett. 579, 325–330 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health grants GM073913 and CA164468 to V.V.V.

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Author notes

  1. Fedor V Subach and Kiryl D Piatkevich: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA

    Fedor V Subach, Kiryl D Piatkevich & Vladislav V Verkhusha

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  1. Fedor V Subach
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Correspondence to Vladislav V Verkhusha.

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Subach, F., Piatkevich, K. & Verkhusha, V. Directed molecular evolution to design advanced red fluorescent proteins. Nat Methods 8, 1019–1026 (2011). https://doi.org/10.1038/nmeth.1776

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