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Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222

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Abstract

Wild type green fluorescent protein (GFP) from Aequorea victoria absorbs predominantly at 398 nm. Illumination with UV (254 nm) or visible (390 nm) light transforms this state (GFP398) into one absorbing at 483 nm (GFP483). Here we show that this photoconversion of GFP is a one-photon process that is paralleled by decarboxylation of Glu 222. We propose a mechanism in which decarboxylation is due to electron transfer between the γ-carboxylate of Glu 222 and the p-hydroxybenzylidene-imidazolidinone chromophore of GFP, followed by reverse transfer of an electron and a proton to the remaining carbon side chain atom of Glu 222. Oxidative decarboxylation of a γ-carboxylate represents a new type of posttranslational modification that may also occur in enzymes with high-potential reaction intermediates.

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Figure 1: Spectroscopic properties of GFP phototransformation.
Figure 2: Phototransformation results in a mass reduction of 44 Da.
Figure 3: Photo-induced conformational changes in the chromophore vicinity.
Figure 4: Proposed mechanism for light-induced decarboxylation of Glu 222.

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Acknowledgements

We acknowledge E. Garman for helping establish the extent of X-ray induced structural damage and T. Davies for help with crystallographic computing. We thank A. Watts for help with ESR measurements, and C. Valance, R. Wilmouth and R. Aplin for access to their facilities. J.J.v.T. acknowledges financial support from the European Molecular Biology Organization and the Human Frontier Science Program Organization.

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Authors and Affiliations

  1. Laboratory of Molecular Biophysics, University of Oxford, The Rex Richards Building, South Parks Road, Oxford, OX1 3QU, UK

    Jasper J. van Thor & Louise N. Johnson

  2. Institute for Biological Information Processing IBI-1, Research Centre Jülich, Jülich, D-52425, Germany

    Thomas Gensch

  3. Laboratory for Microbiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, WV, 1018, Amsterdam, The Netherlands

    Klaas J. Hellingwerf

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  1. Jasper J. van Thor
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  2. Thomas Gensch
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  4. Louise N. Johnson
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Correspondence to Jasper J. van Thor.

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van Thor, J., Gensch, T., Hellingwerf, K. et al. Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222. Nat Struct Mol Biol 9, 37–41 (2002). https://doi.org/10.1038/nsb739

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