Traditional GFP-Type Cyclization and Unexpected Fragmentation Site in a Purple Chromoprotein from <i>Anemonia sulcata</i>, asFP595<sup>†</sup> Vasily E. Zagranichny Natalia V. Rudenko Andrey Yu. Gorokhovatsky Mikhail V. Zakharov Tamara A. Balashova Alexander S. Arseniev 10.1021/bi0488247.s001 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 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. 2004-10-26 00:00:00 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