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.

Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy

Abstract

Tracing the transient atomic motions that lie at the heart of chemical reactions requires high-resolution multidimensional structural information on the timescale of molecular vibrations, which commonly range from 10 fs to 1 ps. For simple chemical systems, it has been possible to map out in considerable detail the reactive potential-energy surfaces describing atomic motions and resultant reaction dynamics1, but such studies remain challenging for complex chemical and biological transformations2. A case in point is the green fluorescent protein (GFP)3,4,5 from the jellyfish Aequorea victoria, which is a widely used gene expression marker owing to its efficient bioluminescence. This feature is known to arise from excited-state proton transfer (ESPT)6,7,8, yet the atomistic details of the process are still not fully understood. Here we show that femtosecond stimulated Raman spectroscopy9,10 provides sufficiently detailed and time-resolved vibrational spectra of the electronically excited chromophore of GFP to reveal skeletal motions involved in the proton transfer that produces the fluorescent form of the protein. In particular, we observe that the frequencies and intensities of two marker bands, the C–O and C = N stretching modes at opposite ends of the conjugated chromophore, oscillate out of phase with a period of 280 fs; we attribute these oscillations to impulsively excited low-frequency phenoxyl-ring motions, which optimize the geometry of the chromophore for ESPT. Our findings illustrate that femtosecond simulated Raman spectroscopy is a powerful approach to revealing the real-time nuclear dynamics that make up a multidimensional polyatomic reaction coordinate.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Ultrafast structural response of the photoexcited wild-type GFP chromophore.
Figure 2: FSRS spectra of wild-type GFP in D 2 O from -100 fs to 1 ps following 396-nm excitation.
Figure 3: Time-dependent vibrational structural features of the excited wild-type GFP chromophore in D 2 O.
Figure 4: Deconvolved marker-band frequency oscillations reveal multidimensionality of the reaction coordinate of wild-type GFP.

References

  1. Zewail, A. H. Femtochemistry: Ultrafast Dynamics of the Chemical Bond 217–326 (World Scientific, 1994)

    Google Scholar 

  2. Levine, B. G. & Martínez, T. J. Isomerization through conical intersections. Annu. Rev. Phys. Chem. 58, 613–634 (2007)

    Article  ADS  CAS  Google Scholar 

  3. Shimomura, O., Johnson, F. H. & Saiga, Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59, 223–239 (1962)

    Article  CAS  Google Scholar 

  4. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994)

    Article  ADS  CAS  Google Scholar 

  5. Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998)

    Article  CAS  Google Scholar 

  6. Brejc, K. et al. Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc. Natl Acad. Sci. USA 94, 2306–2311 (1997)

    Article  ADS  CAS  Google Scholar 

  7. Chattoraj, M., King, B. A., Bublitz, G. U. & Boxer, S. G. Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc. Natl Acad. Sci. USA 93, 8362–8367 (1996)

    Article  ADS  CAS  Google Scholar 

  8. 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  Google Scholar 

  9. Kukura, P., McCamant, D. W. & Mathies, R. A. Femtosecond stimulated Raman spectroscopy. Annu. Rev. Phys. Chem. 58, 461–488 (2007)

    Article  ADS  CAS  Google Scholar 

  10. McCamant, D. W., Kukura, P., Yoon, S. & Mathies, R. A. Femtosecond broadband stimulated Raman spectroscopy: apparatus and methods. Rev. Sci. Instrum. 75, 4971–4980 (2004)

    Article  ADS  CAS  Google Scholar 

  11. Ormo, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996)

    Article  ADS  CAS  Google Scholar 

  12. Yang, F., Moss, L. G. & Phillips, G. N. The molecular structure of green fluorescent protein. Nature Biotechnol. 14, 1246–1251 (1996)

    Article  CAS  Google Scholar 

  13. Kasha, M. Proton-transfer spectroscopy. Perturbation of the tautomerization potential. J. Chem. Soc. Faraday Trans. II 82, 2379–2392 (1986)

    Article  CAS  Google Scholar 

  14. Mandal, D., Tahara, T. & Meech, S. R. Excited-state dynamics in the green fluorescent protein chromophore. J. Phys. Chem. B 108, 1102–1108 (2004)

    Article  CAS  Google Scholar 

  15. 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  ADS  CAS  Google Scholar 

  16. Kukura, P., McCamant, D. W., Yoon, S., Wandschneider, D. B. & Mathies, R. A. Structural observation of the primary isomerization in vision with femtosecond-stimulated Raman. Science 310, 1006–1009 (2005)

    Article  ADS  CAS  Google Scholar 

  17. McCamant, D. W., Kukura, P. & Mathies, R. A. Femtosecond stimulated Raman study of excited-state evolution in bacteriorhodopsin. J. Phys. Chem. B 109, 10449–10457 (2005)

    Article  CAS  Google Scholar 

  18. Dasgupta, J., Frontiera, R. R., Taylor, K. C., Lagarias, J. C. & Mathies, R. A. Ultrafast excited-state isomerization in phytochrome revealed by femtosecond stimulated Raman spectroscopy. Proc. Natl Acad. Sci. USA 106, 1784–1789 (2009)

    Article  ADS  CAS  Google Scholar 

  19. Kennis, J. T. M. et al. Uncovering the hidden ground state of green fluorescent protein. Proc. Natl Acad. Sci. USA 101, 17988–17993 (2004)

    Article  ADS  CAS  Google Scholar 

  20. Gaussian. 03 (Gaussian Inc., Wallingford, Connecticut, 2004)

  21. Humphrey, W., Dalke, A. & Schulten, K. VMD – visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996)

    Article  CAS  Google Scholar 

  22. Zhu, L., Sage, J. T. & Champion, P. M. Observation of coherent reaction dynamics in heme proteins. Science 266, 629–632 (1994)

    Article  ADS  CAS  Google Scholar 

  23. Wang, Q., Schoenlein, R. W., Peteanu, L. A., Mathies, R. A. & Shank, C. V. Vibrationally coherent photochemistry in the femtosecond primary event of vision. Science 266, 422–424 (1994)

    Article  ADS  CAS  Google Scholar 

  24. Kukura, P., Frontiera, R. & Mathies, R. A. Direct observation of anharmonic coupling in the time domain with femtosecond stimulated Raman scattering. Phys. Rev. Lett. 96, 238303 (2006)

    Article  ADS  Google Scholar 

  25. Coe, J. D., Levine, B. G. & Martínez, T. J. Ab initio molecular dynamics of excited-state intramolecular proton transfer using multireference perturbation theory. J. Phys. Chem. A 111, 11302–11310 (2007)

    Article  CAS  Google Scholar 

  26. Agmon, N. The Grotthuss mechanism. Chem. Phys. Lett. 244, 456–462 (1995)

    Article  ADS  CAS  Google Scholar 

  27. Maddalo, S. L. & Zimmer, M. The role of the protein matrix in green fluorescent protein fluorescence. Photochem. Photobiol. 82, 367–372 (2006)

    Article  CAS  Google Scholar 

  28. Cerullo, G. et al. Photosynthetic light harvesting by carotenoids: detection of an intermediate excited state. Science 298, 2395–2398 (2002)

    Article  ADS  CAS  Google Scholar 

  29. Shim, S. & Mathies, R. A. Generation of narrow-bandwidth picosecond visible pulses from broadband femtosecond pulses for femtosecond stimulated Raman. Appl. Phys. Lett. 89, 121124 (2006)

    Article  ADS  Google Scholar 

Download references

Acknowledgements

Plasmids were provided by R. Wachter. We thank M. Marletta for the use of his laboratory facilities to express and purify GFP samples. We thank J. Dasgupta for discussions. This work was supported by the Mathies Royalty Fund.

Author Contributions C.F., R.R.F. and R.A.M. designed the research. C.F. and R.R.F. performed the spectroscopic measurements and DFT calculations. C.F. analysed the data and simulated the spectra. R.T. expressed and purified the protein samples. C.F. and R.A.M. wrote the paper. All authors discussed and edited the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Richard A. Mathies.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary References and Supplementary Figures S1-S3 with Legends. (PDF 551 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Fang, C., Frontiera, R., Tran, R. et al. Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy. Nature 462, 200–204 (2009). https://doi.org/10.1038/nature08527

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08527

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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