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.

Watching conformational- and photodynamics of single fluorescent proteins in solution

Abstract

Observing the dynamics of single biomolecules over prolonged time periods is difficult to achieve without significantly altering the molecule through immobilization. It can, however, be accomplished using the anti-Brownian electrokinetic trap, which allows extended investigation of solution-phase biomolecules—without immobilization—through real-time electrokinetic feedback. Here we apply the trap to study an important photosynthetic antenna protein, allophycocyanin. The technique allows the observation of single molecules of solution-phase allophycocyanin for more than one second. We observe a complex relationship between fluorescence intensity and lifetime that cannot be explained by simple static kinetic models. Light-induced conformational changes are shown to occur and evidence is obtained for fluctuations in the spontaneous emission lifetime, which is typically assumed to be constant. Our methods provide a new window into the dynamics of fluorescent proteins and the observations are relevant for the interpretation of in vivo single-molecule imaging experiments, bacterial photosynthetic regulation and biomaterials for solar energy harvesting.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Structure of APC.
Figure 2: Intensity and lifetime dynamics of single molecules of APC trapped in the ABEL trap.
Figure 3: Intensity level and lifetime histograms.
Figure 4: Correlations between fluorescence intensity and lifetime suggest conformational dynamics.
Figure 5: Fluorescence lifetime fluctuations at near-constant intensity.
Figure 6: Results from state identification by time-order clustering.

References

  1. Moerner, W. E. & Orrit, M. Illuminating single molecules in condensed matter. Science 283, 1670–1676 (1999).

    CAS  Article  Google Scholar 

  2. Myong, S. et al. Cytosolic viral sensor RIG-I Is a 5'-triphosphate-dependent translocase on double-stranded RNA. Science 323, 1070–1074 (2009).

    CAS  Article  Google Scholar 

  3. Hofmann, C., Aartsma, T. J., Michel, H. & Kohler, J. Direct observation of tiers in the energy landscape of a chromoprotein: A single-molecule study. Proc. Natl Acad. Sci. USA 100, 15534–15538 (2003).

    CAS  Article  Google Scholar 

  4. Bopp, M. A., Jia, Y., Li, L., Cogdell, R. J. & Hochstrasser, R. M. Fluorescence and photobleaching dynamics of single light-harvesting complexes. Proc. Natl Acad. Sci. USA 94, 10630–10635 (1997).

    CAS  Article  Google Scholar 

  5. Hofkens, J. et al. Probing photophysical processes in individual multichromophoric dendrimers by single-molecule spectroscopy. J. Am. Chem. Soc. 122, 9278–9288 (2000).

    CAS  Article  Google Scholar 

  6. Rothwell, P. J. et al. Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes. Proc. Natl Acad. Sci. USA 100, 1655–1660 (2003).

    CAS  Article  Google Scholar 

  7. Nie, S., Chiu, D. T. & Zare, R. N. Probing individual molecules with confocal fluorescence microscopy. Science 266, 1018–1021 (1994).

    CAS  Article  Google Scholar 

  8. Shera, E. B., Seitzinger, N. K., Davis, L. M., Keller, R. A. & Soper, S. A. Detection of single fluorescent molecules. Chem. Phys. Lett. 174, 553–557 (1990).

    CAS  Article  Google Scholar 

  9. Rasnik, I., McKinney, S. A. & Ha, T. Surfaces and orientations: Much to FRET About? Acc. Chem. Res. 38, 542–548 (2005).

    CAS  Article  Google Scholar 

  10. Rasnik, I., Myong, S., Cheng, W., Lohman, T. M. & Ha, T. DNA-binding orientation and domain conformation of the E-coli Rep helicase monomer bound to a partial duplex junction: Single-molecule studies of fluorescently labeled enzymes. J. Mol. Biol. 336, 395–408 (2004).

    CAS  Article  Google Scholar 

  11. Okumus, B., Wilson, T. J., Lilley, D. M. J. & Ha, T. Vesicle encapsulation studies reveal that single molecule ribozyme heterogeneities are intrinsic. Biophys. J. 87, 2798–2806 (2004).

    CAS  Article  Google Scholar 

  12. Friedel, M., Baumketner, A. & Shea, J. E. Effects of surface tethering on protein folding mechanisms. Proc. Natl Acad. Sci. USA 103, 8396–8401 (2006).

    CAS  Article  Google Scholar 

  13. Talaga, D. S. et al. Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy. Proc. Natl Acad. Sci. USA 97, 13021–13026 (2000).

    CAS  Article  Google Scholar 

  14. Cohen, A. E. & Moerner, W. E. Suppressing Brownian motion of individual biomolecules in solution. Proc. Natl Acad. Sci. USA 103, 4362–4365 (2006).

    CAS  Article  Google Scholar 

  15. Cohen, A. E. & Moerner, W. E. Controlling Brownian motion of single protein molecules and single fluorophores in aqueous buffer. Opt. Express 16, 6941–6956 (2008).

    CAS  Article  Google Scholar 

  16. Murakoshi, H. et al. Single-molecule imaging analysis of Ras activation in living cells. Proc. Natl Acad. Sci. USA 101, 7317–7322 (2004).

    CAS  Article  Google Scholar 

  17. Lee, S. J. R., Escobedo-Lozoya, Y., Szatmari, E. M. & Yasuda, R. Activation of CaMKII in single dendritic spines during long-term potentiation. Nature 458, 299–304 (2009).

    CAS  Article  Google Scholar 

  18. Berglund, A. J. & Mabuchi, H. Feedback controller design for tracking a single fluorescent molecule. Appl. Phy. B 78, 653–659 (2004).

    CAS  Article  Google Scholar 

  19. Enderlein, J. Tracking of fluorescent molecules diffusing within membranes. Appl. Phy. B 71, 773–777 (2000).

    CAS  Article  Google Scholar 

  20. Cohen, A. E. & Moerner, W. E. Principal-components analysis of shape fluctuations of single DNA molecules. Proc. Natl Acad. Sci. USA 104, 12622–12627 (2007).

    CAS  Article  Google Scholar 

  21. MacColl, R. Allophycocyanin and energy transfer. Biochim. Biophys. Acta 1657, 73–81 (2004).

    CAS  Article  Google Scholar 

  22. Brejc, K., Ficner, R., Huber, R. & Steinbacher, S. Isolation, crystallization, crystal structure analysis and refinement of allophycocyanin from the cyanobacterium Spirulina platensis at 2.3 A resolution. J. Mol. Biol. 249, 424–440 (1995).

    CAS  Article  Google Scholar 

  23. Edington, M. D., Riter, R. E. & Beck, W. F. Evidence for coherent energy-transfer in allophycocyanin trimers. J. Phys. Chem. 99, 15699–15704 (1995).

    CAS  Article  Google Scholar 

  24. Edington, M. D., Riter, R. E. & Beck, W. F. Interexciton-state relaxation and exciton localization in allophycocyanin trimers. J. Phys. Chem. 100, 14206–14217 (1996).

    CAS  Article  Google Scholar 

  25. Beck, W. F. & Sauer, K. Energy-transfer and exciton-state relaxation processes in allophycocyanin. J. Phys. Chem. 96, 4658–4666 (1992).

    CAS  Article  Google Scholar 

  26. Loos, D., Cotlet, M., De Schryver, F. C., Habuchi, S. & Hofkens, J. Single-molecule spectroscopy selectively probes donor and acceptor chromophores in the phycobiliprotein allophycocyanin. Biophys. J. 87, 2598–2608 (2004).

    CAS  Article  Google Scholar 

  27. Ying, L. & Xie, X. S. Fluorescence spectroscopy, exciton dynamics, and photochemistry of single allophycocyanin trimers. J. Phys. Chem. B 102, 10399–10409 (1998).

    CAS  Article  Google Scholar 

  28. Watkins, L. P. & Yang, H. Detection of intensity change points in time-resolved single-molecule measurements. J. Phys. Chem. B 109, 617–628 (2005).

    CAS  Article  Google Scholar 

  29. Vallee, R. A. L. et al. Fluorescence lifetime fluctuations of single molecules probe the local environment of oligomers around the glass transition temperature. J. Chem. Phys. 126 (2007).

  30. Vallee, R. A. L., Van Der Auweraer, M., De Schryver, F. C., Beljonne, D. & Orrit, M. A microscopic model for the fluctuations of local field and spontaneous emission of single molecules in disordered media. Chem. Phys. Chem. 6, 81–91 (2005).

    CAS  Article  Google Scholar 

  31. Lakowicz, J. R. in Principles of Fluorescence Spectroscopy 954 (Springer Science, 2006).

    Book  Google Scholar 

  32. McKinney, S. A., Joo, C. & Ha, T. Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys. J. 91, 1941–1951 (2006).

    CAS  Article  Google Scholar 

  33. Scheer, H. Biliproteins. Angew. Chem. Int. Ed. 20, 241–261 (1981).

    Article  Google Scholar 

  34. Langer, E., Lehner, H., Rudiger, W. & Zickendrahtwendelstadt, B. Circular-dichroism of C-phycoerythrin—a conformational analysis. Z. Naturforsch. C 35, 367–375 (1980).

    Article  Google Scholar 

  35. Bischoff, M. et al. Excited-state processes in phycocyanobilin studied by femtosecond spectroscopy. J. Phys. Chem. B 104, 1810–1816 (2000).

    CAS  Article  Google Scholar 

  36. Braslavsky, S. E., Holzwarth, A. R. & Schaffner, K. Solution conformations, photophysics, and photochemistry of bile-pigments—bilirubin and biliverdin dimethyl esters and related linear tetrapyrroles. Angew. Chem. Int. Ed. 22, 656–674 (1983).

    Article  Google Scholar 

  37. Su-Ping, Z. et al. Generation and identification of the transient intermediates of allophycocyanin by laser photolytic and pulse radiolytic techniques. Int. J. Radiat. Biol. 77, 637–642 (2001).

    CAS  Article  Google Scholar 

  38. Beladakere, N. N. et al. Photovoltaic effects and charge transport studies in phycobiliproteins. Mat. Res. Soc. Symp. Proc. 292, 193–198 (1993).

    CAS  Article  Google Scholar 

  39. Katona, G. et al. Conformational regulation of charge recombination reactions in a photosynthetic bacterial reaction center. Nature Struct. Biol. 12, 630–631 (2005).

    CAS  Article  Google Scholar 

  40. Belcher, J., Sansone, S., Fernandez, N. F., Haskins, W. E. & Brancaleona, L. Photoinduced unfolding of beta-lactoglobulin mediated by a water-soluble porphyrin. J. Phys. Chem. B 113, 6020–6030 (2009).

    CAS  Article  Google Scholar 

  41. Nesvadba, P. & Gossauer, A. Synthesis of Bile-Pigments.14. Synthesis of a bilindionostilbenoparacyclophane as a model for stretched bile pigment chromophores of biliproteins. J. Am. Chem. Soc. 109, 6545–6546 (1987).

    CAS  Article  Google Scholar 

  42. Greene, B. I., Lamola, A. A. & Shank, C. V. Picosecond primary photoprocesses of bilirubin bound to human-serum albumin. Proc. Natl Acad. Sci. USA 78, 2008–2012 (1981).

    CAS  Article  Google Scholar 

  43. Margineanu, A. et al. Visualization of membrane rafts using a perylene monoimide derivative and fluorescence lifetime Imaging. Biophys. J. 93, 2877–2891 (2007).

    CAS  Article  Google Scholar 

  44. Gilson, M. K. & Honig, B. H. The dielectric-constant of a folded protein. Biopolymers 25, 2097–2119 (1986).

    CAS  Article  Google Scholar 

  45. Kartalov, E., Unger, M. & Quake, S. R. Polyelectrolyte surface interface for single-molecule fluorescence studies of DNA polymerase. BioTechniques 34, 505–510 (2003).

    CAS  Article  Google Scholar 

  46. Ong, L. J. & Glazer, A. N. Crosslinking of allophycocyanin. Physiol. Veg. 23, 777–787 (1985).

    CAS  Google Scholar 

  47. Mao, H. B. et al. Effects of glycerol and high temperatures on structure and function of phycobilisomes in Synechocystis sp PCC 6803. FEBS Lett. 553, 68–72 (2003).

    CAS  Article  Google Scholar 

  48. He, J. A., Hu, Y. Z. & Jiang, L. J. Photodynamic action of phycobiliproteins: In situ generation of reactive oxygen species. Biochim. et Biophys. Acta Bioenerg. 1320, 165–174 (1997).

    CAS  Article  Google Scholar 

  49. Zander, C. et al. Detection and characterization of single molecules in aqueous solution. Appl. Phy. B 63, 517–523 (1996).

    CAS  Article  Google Scholar 

  50. Pawitan, Y. in In All Likelihood: Statistical Modeling and Inference Using Likelihood 528 (Clarendon Press, 2001).

    Google Scholar 

Download references

Acknowledgements

We acknowledge support from Y. Jiang, and thank A. Fürstenberg, Q. Wang, S. Bockenhauer, M. Thompson and L. Lau for helpful discussions and A. Cohen for initial trap design and quartz lithography. This work was supported in part by the US Department of Energy Grant No. DE-FG02-07ER15892 and by Grant No. 1R21-RR023149 from the National Center for Research Resources of the National Institutes of Health.

Author information

Authors and Affiliations

Authors

Contributions

R.H.G. and W.E.M. conceived and designed the experiments, R.H.G. performed the experiments, R.H.G. and W.E.M analysed the data and co-wrote the paper.

Corresponding author

Correspondence to W. E. Moerner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 2964 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Goldsmith, R., Moerner, W. Watching conformational- and photodynamics of single fluorescent proteins in solution. Nature Chem 2, 179–186 (2010). https://doi.org/10.1038/nchem.545

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.545

Further reading

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