Chromophore Aspartate Oxidation−Decarboxylation in the Green-to-Red Conversion of a Fluorescent Protein from Zoanthus sp. 2

Abstract

The red fluorescence of a Discosoma coral protein is the result of an additional autocatalytic oxidation of a green fluorescent protein (GFP)-like chromophore. This reaction creates an extra pi-electron conjugation by forming a C=N-C=O substituent. Here we show that the red fluorescence of a protein from Zoanthus sp. 2 (z2FP574) arises from a coupled oxidation-decarboxylation of Asp-66, the first amino acid of the chromophore-precursory DYG sequence. Comparative mutagenesis of highly homologous green (zFP506) and red (z2FP574) fluorescent proteins from Zoanthus species reveals that an aspartate at position 66 is critical for the development of red fluorescence. The maturation kinetics of wild-type z2FP574 and the zFP506 N66D mutant indicates that the "green" GFP-like form is the actual intermediate in producing the red species. Furthermore, via maturation kinetics analysis of zFP506 N66D, combined with mass spectrometry, we determined that the oxidation-decarboxylation of Asp-66 occurs without detectable intermediate products. According to mass spectral data, the minor "red" chromophore of the z2FP574 D66E mutant appears to be oxidized and completely decarboxylation deficient, indicating that the side chain length of acidic amino acid 66 is critical in controlling efficient oxidation-decarboxylation. Substitutions with aspartate at the equivalent positions of a Condylactis gigantea purple chromoprotein and Dendronephthya sp. green fluorescent protein imply that additional oxidation of a GFP-like structure is a prerequisite for chromophore decarboxylation. In summary, these results lead to a mechanism that is related to the chemistry of beta-keto acid decarboxylation.