Structure of a red fluorescent protein from Zoanthus, zRFP574, reveals a novel chromophore

The three-dimensional structure of the red fluorescent protein (RFP) zRFP574 from the button polyp Zoanthus sp. (two dimers per asymmetric unit, 231 × 4 amino acids) has been determined at 2.4 Å resolution in space group C222₁. The crystal structure, refined to a crystallographic R factor of 0.203 (R_free = 0.249), adopts the β-barrel fold composed of 11 strands similar to that of the yellow fluorescent protein zYFP538. The zRFP574 chromophore, originating from the protein sequence Asp66-Tyr67-Gly68, has a two-ring structure typical of GFP-like proteins. The bond geometry of residue 66 shows the strong tendency of the corresponding C^α atom to sp² hybridization as a consequence of N-acylimine bond formation. The zRFP574 chromophore contains the 65–66 cis-peptide bond characteristic of red fluorescent proteins. The chromophore phenolic ring adopts a cis conformation coplanar with the imidazolinone ring. The crystallographic study has revealed an unexpected chemical feature of the internal chromophore. A decarboxylated side chain of the chromophore-forming residue Asp66 has been observed in the structure. This additional post-translational modification is likely to play a key role in the bathochromic shift of the zRFP574 spectrum.