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Photoswitching of E222Q GFP mutants: “concerted” mechanism of chromophore isomerization and protonation

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Abstract

Photochromic (i.e. reversibly photoswitchable) fluorescent proteins increasingly find applications as biomarkers for advanced bioimaging applications. From a mechanistic point of view, photochromicity usually stems from the reversible cis-trans photoisomerization of the chromophore. We demonstrated experimentally that cis-trans photoisomerization constitutes a very efficient deactivation pathway of isolated chromophores upon visible light excitation. Nonetheless, this intrinsic property is seldom displayed by chromophores in the folded protein structure. We found that the E222Q amino acid replacement restores efficient photochromicity in otherwise poorly switchable green fluorescent protein variants of different optical properties. Glutamic acid 222 is known to play a pivotal role in the inner proton wires that involve the GFP chromophore and the surrounding residues. Hence its substitution with an isosteric but non-ionizable residue presumably leads to a extensive rewiring of proton pathways around the chromophore, which has a deep effect also on the photochromic properties. In this work, we review and discuss the main photophysical properties of photochromic E222Q GFP mutants. Additionally we show, by means of flash-photolysis experiments, that chromophore cis to trans photoswitching involves a molecular mechanism where stereochemical isomerization and chromophore protonation occur in a coordinated way. Such a “concerted” mechanism is, in our opinion, at the basis of efficient photochromic behavior and might be activated by the E222Q mutation.

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Authors and Affiliations

  1. Dipartimento di Fisica, Università di Parma, viale G. P. Usberti 7A, 43100, Parma, Italy

    Stefania Abbruzzetti

  2. Dipartimento di Biotecnologie, Università di Verona, cà Vignal 1, Strada Le Grazie 15, 37134, Verona, Italy

    Stefania Abbruzzetti

  3. NEST, Istituto Nanoscienze-CNR, piazzale S. Silvestro 12, 56127, Pisa, Italy

    Stefania Abbruzzetti, Ranieri Bizzarri, Riccardo Nifosì & Cristiano Viappiania

  4. NEST, Scuola Normale Superiore, piazzale S. Silvestro 12, 56127, Pisa, Italy

    Ranieri Bizzarri, Stefano Luin, Barbara Storti & Fabio Beltram

  5. IIT@NEST, Center for Nanotechnology Innovation, Piazza S. Silvestro 12, I-56127, Pisa, Italy

    Ranieri Bizzarri & Fabio Beltram

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  1. Stefania Abbruzzetti
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Correspondence to Ranieri Bizzarri.

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This article is published as part of a themed issue on photofunctional proteins: from understanding to engineering.

‡ Stefania Abbruzzetti and Ranieri Bizzarri contributed equally to this work.

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Abbruzzetti, S., Bizzarri, R., Luin, S. et al. Photoswitching of E222Q GFP mutants: “concerted” mechanism of chromophore isomerization and protonation. Photochem Photobiol Sci 9, 1307–1319 (2010). https://doi.org/10.1039/c0pp00189a

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