A Purple-blue Chromoprotein from Goniopora tenuidens Belongs to the DsRed Subfamily of GFP-like Proteins

Abstract

A number of recently cloned chromoproteins homologous to the green fluorescent protein show a substantial bathochromic shift in absorption spectra. Compared with red fluorescent protein from Discosoma sp. (DsRed), mutants of these so-called far-red proteins exhibit a clear red shift in emission spectra as well. Here we report that a far-red chromoprotein from Goniopora tenuidens (gtCP) contains a chromophore of the same chemical structure as DsRed. Denaturation kinetics of both DsRed and gtCP under acidic conditions indicates that the red form of the chromophore (absorption maximum at 436 nm) converts to the GFP-like form (384 nm) by a one-stage reaction. Upon neutralization, the 436-nm form of gtCP, but not the 384-nm form, renaturates instantly, implying that the former includes a chromophore in its intact state. gtCP represents a singlechain protein and, upon harsh denaturing conditions, shows three major bands in SDS/PAGE, two of which apparently result from hydrolysis of an acylimine C‫؍‬N bond. Instead of having absorption maxima at 384 nm and 450 nm, which are characteristic for a GFP-like chromophore, fragmented gtCP shows a different spectrum, which presumably corresponds to a 2-keto derivative of imidazolidinone. Mass spectra of the chromophore-containing peptide from gtCP reveal an additional loss of 2 Da relative to the GFP-like chromophore. Tandem mass spectrometry of the chromopeptide shows that an additional bond is dehydrogenated in gtCP at the same position as in DsRed. Altogether, these data suggest that gtCP belongs to the same subfamily as DsRed (in the classification of GFP-like proteins based on the chromophore structure type).A variety of fluorescent proteins from Anthozoa species has been described recently (1-6). These proteins represent the GFP-like family according to their homology to the green fluorescent protein from Aequorea victoria jellyfish. A growing interest in GFP-like proteins was stimulated largely by their potential utilization as biomarkers in living cells (7). Besides their applications to cellular biology, GFP-like proteins are of significant interest as the subjects of basic protein chemistry and enzymology. The main feature distinguishing these proteins from other known chromoproteins is the chromophore moiety composed of modified amino acid residues. Accordingly, the color of GFP-like protein is encoded genetically, and the chromophore center is assembled post-translationally by autocatalytic reactions inside the protein molecule. Thus, GFP-like proteins break the earlier adopted concept that chromoproteins are formed by combining an apoprotein with a prosthetic group.Currently, about 30 members of this family have been described (4); they are conventionally divided into two groups, proteins with inherent fluorescence (FP) 1 and naturally nonfluorescent proteins (CP). Remarkably, all known CPs can be converted to fluorescent analogues by means of mutagenesis (3) or by irradiation with light of fixed wavelength (8, 9). Despite great diversity in the colors of coral pr...

Structural Characterization of Acylimine-Containing Blue and Red Chromophores in mTagBFP and TagRFP Fluorescent Proteins

“…Possibly, a hydration of the N-acylimine C=N bond in the TagRFP chromophore has occurred. Similar hydration of the acylimine bond has been observed for the chromophores in DsRed itself (Gross et al, 2000) and in gtCP (Martynov et al, 2003). In contrast, mTagBFP exhibited a single absorbance maximum at 335 nm in acid, and single maximum at 423 nm in alkali (Figure 3B).…”

“…The same fragment was observed upon MS/MS fragmentation of the species ATSFLYGSKTF. Fragments with the same cleavage site near the C=N bond were observed in the case of the N-acylimine-containing chromopeptides derived from proteolysis of DsRed (Gross et al, 2000) and chromoprotein gtCP (Martynov et al, 2003). Therefore, mass-spectral data are consistent with the presence of the acylimine C=N bond and with the absence of the double bond between hydroxyphenyl ring of Tyr64 and imidazole-5-ol ring formed by the Leu63-Tyr64-Gly65 tripeptide.…”

Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein

“…Positions of molecular mass markers are shown on the Right (sizes in kDa). Upon heating of samples, red fluorescent proteins that carry DsRed-like chromophore often demonstrate partial fragmentation with a break point just before the chromophore (22,23). This well-known effect is also observed for 4D5scFv-KillerRed and results in additional bands corresponding to 31-kDa (4D5scFv with KillerRed C-terminal part) and 24-kDa (KillerRed N-terminal part) fragments.…”

Crystallographic Evidence for Water-assisted Photo-induced Peptide Cleavage in the Stony Coral Fluorescent Protein Kaede

“…18 Peptide cleavage has been reported in several fluorescent proteins such as DsRed, gtCP, zFP538, KFP and EosFP. 17,[19][20][21][22][23] The induction of the break is different among the proteins: DsRed and gtCP upon boiling, zFP538 and KFP by autocatalytic process, Kaede and EosFP by UV irradiation. Nienhaus et al 24 recently reported the crystal structure of EosFP in both the green and red forms.…”