A Green Fluorescent Protein Containing a QFG Tri-Peptide Chromophore: Optical Properties and X-Ray Crystal Structure

<div><p>Rtms5 is an deep blue weakly fluorescent GFP-like protein (, 592 nm; , 630nm; Φ_F, 0.004) that contains a ⁶⁶Gln-Tyr-Gly chromophore tripeptide sequence. We investigated the optical properties and structure of two variants, Rtms5^Y67F and Rtms5^Y67F/H146S in which the tyrosine at position 67 was substituted by a phenylalanine. Compared to the parent proteins the optical spectra for these new variants were significantly blue-shifted. Rtms5^Y67F spectra were characterised by two absorbing species (, 440 nm and 513 nm) and green fluorescence emission (, 440 nm; , 508 nm; Φ_F, 0.11), whilst Rtms5^Y67F/H146S spectra were characterised by a single absorbing species (, 440 nm) and a relatively high fluorescence quantum yield (Φ_F, 0.75; , 440 nm; , 508 nm). The fluorescence emissions of each variant were remarkably stable over a wide range of pH (3–11). These are the first GFP-like proteins with green emissions (500–520 nm) that do not have a tyrosine at position 67. The X-ray crystal structure of each protein was determined to 2.2 Å resolution and showed that the benzylidine ring of the chromophore, similar to the 4-hydroxybenzylidine ring of the Rtms5 parent, is non-coplanar and in the <em>trans</em> conformation. The results of chemical quantum calculations together with the structural data suggested that the 513 nm absorbing species in Rtms5^Y67F results from an unusual form of the chromophore protonated at the acylimine oxygen. These are the first X-ray crystal structures for fluorescent proteins with a functional chromophore containing a phenylalanine at position 67.</p>