Rtms5 is an deep blue weakly fluorescent GFP-like protein (, 592 nm; , 630nm; ΦF, 0.004) that contains a 66Gln-Tyr-Gly chromophore tripeptide sequence. We investigated the optical properties and structure of two variants, Rtms5Y67F and Rtms5Y67F/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. Rtms5Y67F spectra were characterised by two absorbing species (, 440 nm and 513 nm) and green fluorescence emission (, 440 nm; , 508 nm; ΦF, 0.11), whilst Rtms5Y67F/H146S spectra were characterised by a single absorbing species (, 440 nm) and a relatively high fluorescence quantum yield (ΦF, 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 ？ 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 trans conformation. The results of chemical quantum calculations together with the structural data suggested that the 513 nm absorbing species in Rtms5Y67F 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.
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