%0 Journal Article %A Zagranichny, Vasily E. %A Rudenko, Natalia V. %A Gorokhovatsky, Andrey Yu. %A Zakharov, Mikhail V. %A Balashova, Tamara A. %A Arseniev, Alexander S. %D 2004 %T Traditional GFP-Type Cyclization and Unexpected Fragmentation Site in a Purple Chromoprotein from Anemonia sulcata, asFP595 %U 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 %R 10.1021/bi0488247.s001 %2 https://ndownloader.figshare.com/files/5158027 %K polypeptide chain cleavage %K asFP 595 chromopeptides %K Anemonia sulcata %K unsubstituted imino moiety %K asFP 595 chromophore %K Met %K proteolysis products %K asFP 595 %K NMR %K asFP 595 chromophore structure %K Unexpected Fragmentation Site %K DsRed subfamily %K protein %K GFP %X The purple chromoprotein (asFP595) from Anemonia sulcata 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) J. Biol. Chem. 276, 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) Biochemistry 43, 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 p-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 Cα and is a hydrolysis product of another one, with the imino group at Met-65 Cα. The N-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 Nα. Our data strongly suggest that both zFP538 and asFP595 could be attributed to the DsRed subfamily of GFP-like proteins. %I ACS Publications