Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Infrared spectroscopy reveals multi-step multi-timescale photoactivation in the photoconvertible protein archetype dronpa

Nature Chemistry volume 10, pages 845–852 (2018)Cite this article

Subjects

Abstract

Photochromic fluorescent proteins play key roles in super-resolution microscopy and optogenetics. The light-driven structural changes that modulate the fluorescence involve both trans-to-cis isomerization and proton transfer. The mechanism, timescale and relative contribution of chromophore and protein dynamics are currently not well understood. Here, the mechanism of off-to-on-state switching in dronpa is studied using femtosecond-to-millisecond time-resolved infrared spectroscopy and isotope labelling. Chromophore and protein dynamics are shown to occur on multiple timescales, from picoseconds to hundreds of microseconds. Following excitation of the trans chromophore, a ground-state primary product is formed within picoseconds. Surprisingly, the characteristic vibrational spectrum of the neutral cis isomer appears only after several tens of nanoseconds. Further fluctuations in protein structure around the neutral cis chromophore are required to form a new intermediate, which promotes the final proton-transfer reaction. These data illustrate the interplay between chromophore dynamics and the protein environment underlying fluorescent protein photochromism.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Structure and interactions for the dronpa chromophore in off and on states.
Fig. 2: TRIR data and analysis for dronpa2 over the picosecond to 100 ms timescale.
Fig. 3: DFT-calculated infrared transition wavenumbers and intensities for the three most prominent bands of the cis and trans forms of the neutral chromophore in its natural, 1-13C- and 3-13C-labelled forms.
Fig. 4: Steady-state infrared spectra for the dronpa chromophore in pure cis and cis + trans photostationary states, providing an experimental check of the calculated spectral shifts in hydrogen-bonding and non-hydrogen-bonding solvents.
Fig. 5: Isotope effect on dronpa2 EADS recovered from a global analysis with a common set of rate constants, showing that the 1,702 cm−1 transient is associated with the chromophore C=O stretch.
Fig. 6: Proposed mechanism for off to on switching in dronpa2.

References

  1. Ando, R., Mizuno, H. & Miyawaki, A. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306, 1370–1373 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Shcherbakova, D. M., Sengupta, P., Lippincott-Schwartz, J. & Verkhusha, V. V. Photocontrollable fluorescent proteins for superresolution imaging. Ann. Rev. Biophys. 43, 303–329 (2014).

    Article  CAS  Google Scholar 

  3. Zhou, X. X. & Lin, M. Z. Photoswitchable fluorescent proteins: ten years of colorful chemistry and exciting applications. Curr. Opin. Chem. Biol. 17, 682–690 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Rodriguez, E. A. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci. 42, 111–129 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Zhou, X. X., Chung, H. K., Lam, A. J. & Lin, M. Z. Optical control of protein activity by fluorescent protein domains. Science 338, 810–814 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhou, X. X., Fan, L. Z., Li, P., Shen, K. & Lin, M. Z. Optical control of cell signaling by single-chain photoswitchable kinases. Science 355, 836 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Korpany, K. V. et al. Conductance switching in the photoswitchable protein dronpa. J. Am. Chem. Soc. 134, 16119–16122 (2012).

    Article  CAS  PubMed  Google Scholar 

  8. Andresen, M. et al. Structural basis for reversible photoswitching in dronpa. Proc. Natl Acad. Sci. USA 104, 13005–13009 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Andresen, M. et al. Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proc. Natl Acad. Sci. USA 102, 13070–13074 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Mizuno, H. et al. Light-dependent regulation of structural flexibility in a photochromic fluorescent protein. Proc. Natl Acad. Sci. USA 105, 9227–9232 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Fron, E. et al. Ultrafast excited-state dynamics of the photoswitchable protein dronpa. J. Am. Chem. Soc. 129, 4870 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Warren, M. M. et al. Ground-state proton transfer in the photoswitching reactions of the fluorescent protein dronpa. Nat. Commun. 4, 1461 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Lukacs, A. et al. Protein photochromism observed by ultrafast vibrational spectroscopy. J. Phys. Chem. B 117, 11954–11959 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Yadav, D. et al. Real-time monitoring of chromophore isomerization and deprotonation during the photoactivation of the fluorescent protein dronpa. J. Phys. Chem. B 119, 2404–2414 (2015).

    Article  CAS  PubMed  Google Scholar 

  15. Colletier, J.-P. et al. Serial femtosecond crystallography and ultrafast absorption spectroscopy of the photoswitchable fluorescent protein IrisFP. J. Phys. Chem. Lett. 7, 882–887 (2016).

    Article  CAS  PubMed  Google Scholar 

  16. Coquelle, N. et al. Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography. Nat. Chem. 10, 31–37 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. Greetham, G. M. Time-resolved multiple probe spectroscopy. Rev. Sci. Instrum. 83, 103107 (2012).

    Article  CAS  PubMed  Google Scholar 

  18. Ando, R., Flors, C., Mizuno, H., Hofkens, J. & Miyawaki, A. Highlighted generation of fluorescence signals using simultaneous two-color irradiation on dronpa mutants. Biophys. J. 92, L97–L99 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Stoner-Ma, D. et al. Proton relay reaction in green fluorescent protein (GFP): polarization-resolved ultrafast vibrational spectroscopy of isotopically edited GFP. J. Phys. Chem. B 110, 22009–22018 (2006).

    Article  CAS  PubMed  Google Scholar 

  20. van Thor, J. J., Ronayne, K. L., Towrie, M. & Sage, J. T. Balance between ultrafast parallel reactions in the green fluorescent protein has a structural origin. Biophys. J. 95, 1902–1912 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. He, X., Bell, A. F. & Tonge, P. J. Isotopic labeling and normal-mode analysis of a model green fluorescent protein chromophore. J. Phys. Chem. B 106, 6056–6066 (2002).

    Article  CAS  Google Scholar 

  22. Snellenburg, J. J., Laptenok, S. P., Seger, R., Mullen, K. M. & van Stokkum, I. H. M. Glotaran: a Java-based graphical user interface for the R package TIMP. J. Stat. Softw. 49, 1–22 (2012).

    Article  Google Scholar 

  23. Barth, A. Infrared spectroscopy of proteins. Biochim. Biophys. Acta Bioenergetics 1767, 1073–1101 (2007).

    Article  CAS  Google Scholar 

  24. Kaucikas, M., Tros, M. & van Thor, J. J. Photoisomerization and proton transfer in the forward and reverse photoswitching of the fast-switching M159T mutant of the dronpa fluorescent protein. J. Phys. Chem. B 119, 2350–2362(2015).

    Article  CAS  PubMed  Google Scholar 

  25. Wachter, R. M. Chromogenic cross-link formation in green fluorescent protein. Acc. Chem. Res. 40, 120–127 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Weber, W., Helms, V., McCammon, J. A. & Langhoff, P. W. Shedding light on the dark and weakly fluorescent states of green fluorescent proteins. Proc. Natl Acad. Sci. USA 96, 6177–6182 (1999).

    Article  CAS  PubMed  Google Scholar 

  27. Conyard, J. et al. A new twist in the photophysics of the GFP chromophore: a volume-conserving molecular torsion couple. Chem. Sci. 9, 1803–1812 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Voliani, V. et al. Cis–trans photoisomerization of fluorescent-protein chromophores. J. Phys. Chem. B 112, 10714–10722 (2008).

    Article  CAS  PubMed  Google Scholar 

  29. Snyder, J. W., Fales, B. S., Hohenstein, E. G., Levine, B. G. & Martínez, T. J. A direct-compatible formulation of the coupled perturbed complete active space self-consistent field equations on graphical processing units. J. Chem. Phys. 146, 174113 (2017).

    Article  CAS  PubMed  Google Scholar 

  30. Lukacs, A. et al. Photoexcitation of the blue light using FAD photoreceptor AppA results in ultrafast changes to the protein matrix. J. Am. Chem. Soc. 133, 16893–16900 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. 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 

  32. Wallace, A. C., Laskowski, R. A. & Thornton, J. M. LIGPLOT—a program to generate schematic diagrams of protein ligand interactions. Protein Eng. 8, 127–134(1995).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

S.R.M. acknowledges EPSRC for financial support (EP/N033647/1 and EP/M001997/1). P.J.T. acknowledges NSF for financial support (CHE-1223819). A.M. acknowledges the Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-aid for Scientific research on Innovative Areas: Resonance Bio. The authors acknowledge STFC for access to the Central Laser Facility. Calculations were performed on the High Performance Computing Cluster at the University of East Anglia.

Author information

Author notes

  1. Sergey P. Laptenok

    Present address: Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia

  2. Agnieszka A. Gil

    Present address: Department of Molecular Biology, Princeton University, Princeton, NJ, USA

  3. Christopher R. Hall

    Present address: ARC Centre of Excellence in Exciton Science, School of Chemistry, The University of Melbourne, Parkville, Victoria, Australia

Authors and Affiliations

  1. School of Chemistry, University of East Anglia, Norwich, UK

    Sergey P. Laptenok, Christopher R. Hall, Garth A. Jones & Stephen R. Meech

  2. Department of Chemistry, Stony Brook University, Stony Brook, NY, USA

    Agnieszka A. Gil, James N. Iuliano & Peter J. Tonge

  3. Department of Biophysics, Medical School, University of Pecs, Pecs, Hungary

    Andras Lukacs

  4. Central Laser Facility, Harwell Science and Innovation Campus, Didcot, Oxfordshire, UK

    Gregory M. Greetham & Paul Donaldson

  5. Laboratory for Cell Function Dynamics, RIKEN Center for Brain Science, Wako, Saitama, Japan

    Atsushi Miyawaki

Authors
  1. Sergey P. Laptenok
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Agnieszka A. Gil
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christopher R. Hall
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andras Lukacs
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. James N. Iuliano
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Garth A. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gregory M. Greetham
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Paul Donaldson
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Atsushi Miyawaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Peter J. Tonge
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Stephen R. Meech
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.P.L., A.A.G., C.R.H., A.L. and J.N.I. measured and collected the data. S.P.L. and C.R.H. analysed the data. A.A.G. and J.N.I. grew and purified the samples. G.A.J. performed the DFT calculations. G.M.G. and P.D. built, developed and managed the ULTRA and LifeTime apparatus used in the measurements. A.M. designed the dronpa2 protein. P.J.T. and S.R.M. designed the experiment and wrote the paper, with discussion and editorial input from all authors

Corresponding authors

Correspondence to Peter J. Tonge or Stephen R. Meech.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary information

Supplementary Figures 1–9, Supplementary Tables 1–5, Supplementary Methods, Supplementary Data, Supplementary Analysis

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Laptenok, S.P., Gil, A.A., Hall, C.R. et al. Infrared spectroscopy reveals multi-step multi-timescale photoactivation in the photoconvertible protein archetype dronpa. Nature Chem 10, 845–852 (2018). https://doi.org/10.1038/s41557-018-0073-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-018-0073-0

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing