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Three photoconvertible forms of green fluorescent protein identified by spectral hole-burning

Nature Structural Biology volume 6, pages 557–560 (1999)Cite this article

An Erratum to this article was published on 01 July 1999

Abstract

Several studies have led to the conclusion that, in the green fluorescent protein (GFP) of the jellyfish Aequorea victoria, a photoconversion involving excited-state proton transfer occurs from an A- to a B-form, while an intermediate I-form was held responsible for the green fluorescence. Here we have identified the I-form of wild-type GFP in absorption, located the 0-0 transitions of all three forms A, B and I, and determined vibrational frequencies of the ground and excited states. The intrinsically narrow 0-0 transitions are revealed by the wavelengths at which holes can be burnt. The pathways of photointerconversion are unraveled by excitation, emission and hole-burning spectroscopy. We present an energy-level scheme that has significant implications for GFP-mutants, which likewise can occur in the three photointerconvertible forms.

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Figure 1: Absorption spectra of wild-type green fluorescent protein (GFP) at room temperature (T = 295 K) and low temperature (T = 1.6 K).
Figure 2: Excitation and fluorescence spectra of GFP at a, 295 K and b, 1.6 K.
Figure 3: a, Emission and b, excitation spectra of GFP at 1.6 K recorded at various excitation and emission wavelengths, respectively.
Figure 4: Excitation spectra before (—) and after (– – –) burning (a, c, λdet = 510 nm), and corresponding emission spectra (b,d, λexc = 493 nm) of GFP at 1.6 K.
Figure 5: Proposed energy-level scheme of the three photoconvertible forms A, I and B of wt-GFP.

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Acknowledgements

We thank J.H. van der Waals for critical remarks and helpful suggestions. The high-resolution spectroscopy experiments, carried out in Leiden, were financially supported by the Netherlands Foundation for Physical Research (FOM) and the Council for Chemical Research of the Netherlands Organization for Scientific Research (CW-NWO). V.S is the recipient of a Human Frontiers Science Program long-term fellowship.

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Authors and Affiliations

  1. Center for the Study of the Excited States of Molecules, Huygens and Gorlaeus Laboratories, University of Leiden, 2300 RA Leiden, P.O. Box 9504, The Netherlands

    T.M.H. Creemers, A.J. Lock & S. Völker

  2. Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany

    V. Subramaniam & T.M. Jovin

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  1. T.M.H. Creemers
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  2. A.J. Lock
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  5. S. Völker
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Correspondence to S. Völker.

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Creemers, T., Lock, A., Subramaniam, V. et al. Three photoconvertible forms of green fluorescent protein identified by spectral hole-burning. Nat Struct Mol Biol 6, 557–560 (1999). https://doi.org/10.1038/9335

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