Traditional GFP-type cyclization and unexpected fragmentation site in a purple chromoprotein from Anemonia sulcata, asFP595.
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.
Zagranichny
Vasily E
VE
Branch of S&O Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia. vasily@fibkh.serpukhov.su
Rudenko
Natalia V
NV
Gorokhovatsky
Andrey Yu
AY
Zakharov
Mikhail V
MV
Balashova
Tamara A
TA
Arseniev
Alexander S
AS
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
C430876:FP595 protein, Anemonia sulcata; D008164:Luminescent Proteins; D010456:Peptides, Cyclic; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases