Zagranichny, Vasily E. Rudenko, Natalia V. Gorokhovatsky, Andrey Yu. Zakharov, Mikhail V. Balashova, Tamara A. Arseniev, Alexander S. Traditional GFP-Type Cyclization and Unexpected Fragmentation Site in a Purple Chromoprotein from <i>Anemonia sulcata</i>, asFP595<sup>†</sup> The purple chromoprotein (asFP595) from <i>Anemonia sulcata</i> belongs to the family of green fluorescent protein (GFP). Absorption and emission spectra of asFP595 are similar to those of a number of recently cloned GFP-like red proteins of the DsRed subfamily. The earlier proposed asFP595 chromophore structure [Martynov, V. I.; et al. (2001) <i>J. Biol. Chem.</i> <i>276</i>, 21012−21016] was postulated to result from an “alternative cyclization” giving rise to a pyrazine-type six-membered heterocycle. Here we report that the asFP595 chromophore is actually very close in chemical structure to that of zFP538, a yellow fluorescent protein [Zagranichny, V. E.; et al. (2004) <i>Biochemistry</i> <i>43</i>, 4764−4772]. NMR spectroscopic studies of four chromophore-containing peptides (chromopeptides) isolated under mild conditions from enzymatic digests of asFP595 and one chromopeptide obtained from DsRed revealed that all of them contain a <i>p</i>-hydroxybenzylideneimidazolinone moiety formed by Met-65/Gln-66, Tyr-66/67, and Gly-67/68 of asFP595/DsRed, respectively. Two asFP595 chromopeptides are proteolysis products of an isolated full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other asFP595 chromopeptides were isolated as proteolysis products of the purified chromophore-containing C-terminal fragment. One of these has an oxo group at Met-65 C<sup>α</sup> and is a hydrolysis product of another one, with the imino group at Met-65 C<sup>α</sup>. The <i>N</i>-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain cleavage at a very unexpected site, the former peptide bond between Cys-64 C‘ and Met-65 N<sup>α</sup>. Our data strongly suggest that both zFP538 and asFP595 could be attributed to the DsRed subfamily of GFP-like proteins. polypeptide chain cleavage;asFP 595 chromopeptides;Anemonia sulcata;unsubstituted imino moiety;asFP 595 chromophore;Met;proteolysis products;asFP 595;NMR;asFP 595 chromophore structure;Unexpected Fragmentation Site;DsRed subfamily;protein;GFP 2004-10-26
    https://acs.figshare.com/articles/journal_contribution/Traditional_GFP_Type_Cyclization_and_Unexpected_Fragmentation_Site_in_a_Purple_Chromoprotein_from_i_Anemonia_sulcata_i_asFP595_sup_sup_/3319261
10.1021/bi0488247.s001