Skip to main content

Research Repository

Advanced Search

X-Ray Crystal Structure and Properties of Phanta, a Weakly Fluorescent Photochromic GFP-Like Protein

X-Ray Crystal Structure and Properties of Phanta, a Weakly Fluorescent Photochromic GFP-Like Protein Thumbnail


Abstract

Phanta is a reversibly photoswitching chromoprotein (FF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (FF, 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 FF, 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.

Acceptance Date Mar 2, 2015
Publication Date Apr 29, 2015
Publicly Available Date Mar 28, 2024
Journal PLoS One
Print ISSN 1932-6203
Publisher Public Library of Science
Pages e0123338 - e0123338
DOI https://doi.org/10.1371/journal.pone.0123338
Keywords Chromophores, Fluorescence, Hydrogen bonding, Green fluorescent protein, Protons, Crystal Structure, Ground State, Amino acid substitution
Publisher URL https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123338

Files




Downloadable Citations