Alternative cyclization in GFP-like proteins family - The formation and structure of the chromophore of a purple chromoprotein from Anemonia sulcata

Anemonia sulcata purple protein (asFP595) belongs to a family of green fluo rescent protein (GFP)-like proteins from the Anthozoa species. Similar to G FP, asFP595 apparently forms its chromophore by modifying amino acids withi n its polypeptide chain. Until now, the GFP-like proteins from Anthozoa wer e thought to contain chromophores with the same imidazolidinone core as GFP . Mass spectral analysis of a chromophore-containing tryptic pentapeptide f rom asFP595 demonstrates that chromophore formation in asFP595 is stoichiom etrically the same as that in GFP: one H2O and two H+ are released while a Schiff base and dehydrotyrosine are formed. However, structural studies of this asFP595 chromopeptide show that in contrast to GFP, the other peptide bond nitrogen and carbonyl carbon are required for chromophore cyclization, a reaction that yields the six-membered heterocycle 2-(4-hydroxybenzyliden e) -6-hydroxy-2,5-dihydropyrazine. Spectrophotometric titration reveals thr ee pH-dependent forms of the asFP595 chromopeptide: yellow (absorption maxi mum = 430 nm) at pH 3.0; red (absorption maximum = 535 nm) at pH 8.0; and c olorless (absorption maximum = 380 nm) at pH 14.0. The pK(a) values for the se spectral transitions (6.8 and 10.9) are consistent with the ionization o f the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation in the chromophore heterocycle, respectively. The amidinium group in asFP595 accounts for the unique absorption spectrum of the protein, which is substantially red-shifted relative to that of GFP, When the asFP595 chro mophore cyclizes, the Cys-Met bond adjacent to the chromophore hydrolyzes, splitting the chromoprotein into 8- and 20-kDa fragments. High performance liquid chromatography analysis of a tryptic digest of denatured asFP595 sho ws that a pentapeptide with the cleaved Cys-Met bond is the only fragment a ssociated with the red-shifted absorbance. These results imply that fragmen tation of asFP595 is a critical step in protein maturation.