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Watching conformational- and photodynamics of single fluorescent proteins in solution

Nature Chemistry volume 2, pages 179–186 (2010)Cite this article

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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.

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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.

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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.

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  1. Department of Chemistry, Stanford University, Stanford, 94305, California

    Randall H. Goldsmith & W. E. Moerner

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  1. Randall H. Goldsmith
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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.

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Correspondence to W. E. Moerner.

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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

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