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