Don Paul, C, Traore, DAK ORCID: https://orcid.org/0000-0003-1001-4716, Olsen, S, Devenish, RJ, Close, DW, Bell, TDM, Bradbury, A, Wilce, MCJ and Prescott, M (2015) X-Ray Crystal Structure and Properties of Phanta, a Weakly Fluorescent Photochromic GFP-Like Protein. PLoS One, 10 (4). e0123338 - e0123338.

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Abstract

Phanta is a reversibly photoswitching chromoprotein (ΦF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (ΦF, 0.8). We have investigated the contribution of substitutions at positions His193, Thr69 and Gln62, individually and in combination, to the optical properties of Phanta. Single amino acid substitutions at position 193 resulted in proteins with very low ΦF, indicating the importance of this position in controlling the fluorescence efficiency of the variant proteins. The substitution Thr69Val in Phanta was important for supressing the formation of a protonated chromophore species observed in some His193 substituted variants, whereas the substitution Gln62Met did not significantly contribute to the useful optical properties of Phanta. X-ray crystal structures for Phanta (2.3 Å), eCGP123T69V (2.0 Å) and eCGP123H193Q (2.2 Å) in their non-photoswitched state were determined, revealing the presence of a cis-coplanar chromophore. We conclude that changes in the hydrogen-bonding network supporting the cis-chromophore, and its contacts with the surrounding protein matrix, are responsible for the low fluorescence emission of eCGP123 variants containing a His193 substitution.

Item Type: Article
Additional Information: © 2015 Don Paul et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Uncontrolled Keywords: Chromophores, Fluorescence, Hydrogen bonding, Green fluorescent protein, Protons, Crystal Structure, Ground State, Amino acid substitution
Subjects: Q Science > Q Science (General)
Q Science > QH Natural history
Divisions: Faculty of Natural Sciences > School of Life Sciences
Depositing User: Symplectic
Date Deposited: 14 Aug 2019 10:30
Last Modified: 16 Aug 2019 14:00
URI: http://eprints.keele.ac.uk/id/eprint/6700

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