title: Traditional GFP-type cyclization and unexpected fragmentation site in a purple chromoprotein from Anemonia sulcata, asFP595.
abstract: 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 C(alpha) and is a hydrolysis product of another one, with the imino group at Met-65 C(alpha). 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 N(alpha). Our data strongly suggest that both zFP538 and asFP595 could be attributed to the DsRed subfamily of GFP-like proteins.
authors.affiliation: Branch of S&O Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia. vasily@fibkh.serpukhov.su
authors.forename: Vasily E
authors.initials: VE
authors.lastname: Zagranichny
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Rudenko
authors.forename: Andrey Yu
authors.initials: AY
authors.lastname: Gorokhovatsky
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Zakharov
authors.forename: Tamara A
authors.initials: TA
authors.lastname: Balashova
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Arseniev
mesh_terms: D000818:Animals; D037421:Anthozoa; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010447:Peptide Hydrolases / Q000378:metabolism; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D012615:Sea Anemones; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
chemical_list: C430876:FP595 protein, Anemonia sulcata; D008164:Luminescent Proteins; D010456:Peptides, Cyclic; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases