Alternative Cyclization in GFP-like Proteins Family

THE FORMATION AND STRUCTURE OF THE CHROMOPHORE OF A PURPLE CHROMOPROTEIN FROM ANEMONIA SULCATA*

Vladimir I. MartynovDagger , Alexander P. Savitsky§, Natalya Y. MartynovaDagger , Pavel A. Savitsky§, Konstantin A. LukyanovDagger , and Sergey A. LukyanovDagger

From the Dagger  Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia, and § Institute of Biochemistry RAS, Leninsky Pr. 33, 117071 Moscow, Russia

Received for publication, January 18, 2001, and in revised form, March 16, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Anemonia sulcata purple protein (asFP595) belongs to a family of green fluorescent protein (GFP)-like proteins from the Anthozoa species. Similar to GFP, asFP595 apparently forms its chromophore by modifying amino acids within its polypeptide chain. Until now, the GFP-like proteins from Anthozoa were thought to contain chromophores with the same imidazolidinone core as GFP. Mass spectral analysis of a chromophore-containing tryptic pentapeptide from asFP595 demonstrates that chromophore formation in asFP595 is stoichiometrically the same as that in GFP: one H2O and two H+ are released while a Schiff base and dehydrotyrosine are formed. However, structural studies of this asFP595 chromopeptide show that in contrast to GFP, the other peptide bond nitrogen and carbonyl carbon are required for chromophore cyclization, a reaction that yields the six-membered heterocycle 2-(4-hydroxybenzylidene)-6-hydroxy-2,5-dihydropyrazine. Spectrophotometric titration reveals three pH-dependent forms of the asFP595 chromopeptide: yellow (absorption maximum = 430 nm) at pH 3.0; red (absorption maximum = 535 nm) at pH 8.0; and colorless (absorption maximum = 380 nm) at pH 14.0. The pKa values for these spectral transitions (6.8 and 10.9) are consistent with the ionization of the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation in the chromophore heterocycle, respectively. The amidinium group in asFP595 accounts for the unique absorption spectrum of the protein, which is substantially red-shifted relative to that of GFP. When the asFP595 chromophore cyclizes, the Cys-Met bond adjacent to the chromophore hydrolyzes, splitting the chromoprotein into 8- and 20-kDa fragments. High performance liquid chromatography analysis of a tryptic digest of denatured asFP595 shows that a pentapeptide with the cleaved Cys-Met bond is the only fragment associated with the red-shifted absorbance. These results imply that fragmentation of asFP595 is a critical step in protein maturation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The green fluorescent protein (GFP)1 from jellyfish Aequorea victoria is a well known light-emitting protein that is widely used for visualizing gene expression in living cells and organisms (1-6). The most outstanding feature of GFP is the creation of its chromophore, a self-catalyzed reaction consisting of two main steps: cyclization of the protein backbone at positions 65-67 (Ser-Tyr-Gly) followed by dehydrogenation of the Tyr66 side chain (7-10). In its final form, the GFP chromophore comprises two cyclic structures. One cycle originates from Tyr66; the other, a five-membered heterocycle, forms when the nitrogen of Gly67 bonds with the carbonyl carbon of Ser65.

Recently, a number of GFP homologues were cloned from the Anthozoa species (11-13). As a group, these GFP-like proteins display colors spanning the visible spectrum. Sequence comparisons of all known fluorescent proteins (FPs) reveal that Tyr66 and Gly67 (numbering in accordance with GFP) are invariant. The most impressive feature of some of these proteins is a dramatic difference in their absorption maxima. Presumably, there are three plausible explanations for the structural basis of color differences. First, spectral variability could arise from different noncovalent interactions of the chromophore with its environment. Second, the GFP-like imidazolidinone structure might be extended by an additional reaction. Third, chromophore formation could occur via an alternative pathway yielding a different heterocyclic structure. Apparently, all three possibilities might be embodied among diverse FPs. Among a number of GFP mutants, for example, spectral diversity results from differences in the way a chromophore interacts with its protein environment (for review see Ref. 14). Gross et al. (15) have demonstrated that the red emitter drFP583 (DsRed) derives its spectral quality from an additional autocatalytic dehydrogenation, which extends the chromophore's system of conjugated pi -bonds.

Recently, we reported the discovery of a GFP-like purple chromoprotein, asFP595, from the sea anemone Anemonia sulcata (13). This protein is naturally nonfluorescent and is responsible for the purple coloration of the anemone tentacles. In vitro, asFP595 absorbs light maximally at 572 nm and displays extremely weak red fluorescence at 595 nm (quantum yield < 0.001). We have found that the spectral and biophysical properties of both the native and denatured forms of asFP595 differ markedly from GFP and drFP583. These observations prompted us to investigate the chemical nature of the asFP595 chromophore to identify the structural basis for the outlined differences. Our studies show that the asFP595 chromophore forms via an alternative pathway, one that requires protein cleavage in addition to amino acid modification.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Trypsin Digestion of asFP595 and Purification of the Chromopeptide-- After dialysis against 10 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA, asFP595 was denatured by acidification to pH 2.3 with dilute HCl. The solution with denatured protein was immediately adjusted to pH 7.8 with dilute NaOH. After the addition of trypsin (20% by weight) the suspension was stirred continuously at room temperature for 4 h. Finally, the clarified digest was adjusted to pH 4.0 and applied to an HPLC column (Beckman Ultrasphere ODS, 4.5 × 250 mm). The column was equilibrated with a starting buffer of 10 mM sodium phosphate, pH 4.0, at a flow rate of 0.4 ml/min. Protein was eluted with a linear gradient of 10 mM sodium phosphate, pH 4.0, containing 60% acetonitrile. The eluent was monitored at 210 and 430 nm.

The protein concentration was determined with a Bio-Rad protein assay using bovine serum albumin as a standard. SDS-polyacrylamide gel electrophoresis analyses routinely employed 15% polyacrylamide gels (16) calibrated with low molecular weight standards from Amersham Pharmacia Biotech. The proteins were transferred to Immobilon-P membrane using a semi-dry Hoefer transfer unit following the protocol provided by PerkinElmer Life Sciences (17). Automated Edman degradation was performed with an Applied Biosystems 491 sequenator. Amino acid analyses were done with a Biotronik amino acid analyzer LC 3000.

Mass Spectra-- Matrix-assisted laser desorption ionization (MALDI) mass spectra were acquired using a Vision 2000 (Thermo Bio Analyses) mass spectrometer operating in the linear mode. Samples of peptides (10-20 pmol each) prepared by HPLC were deposited directly in the sample well of the MALDI plate using 2,5-dihydroxybenzoic acid as a matrix.

Spectroscopy-- Absorption spectra were recorded on a Beckman DU520 UV/VIS spectrophotometer. A PerkinElmer Life Sciences LS50B fluorescence spectrophotometer was used for registration of the excitation-emission spectra.

Carboxypeptidase A Treatment-- The reaction was carried out in 0.2 M N-ethylmorpholine acetate buffer, pH 8.5, for 4 h at 37 °C. The blotted fragments of asFP595 (30 µg of protein) were excised from the Immobilon membrane and incubated with carboxypeptidase A. Finally, the liberated amino acids were identified as dansyl derivatives on thin-layer silica gel plates or on polyamide sheets.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fragmentation of asFP595-- Based on its cDNA sequence (13), monomeric asFP595 should have a mass of 27.2 kDa. However, when we analyzed recombinant asFP595 (expressed by Escherichia coli and purified using metal affinity chromatography) by SDS-polyacrylamide gel electrophoresis, we detected very little protein at 28 kDa. Instead, most of the protein distributed between two major bands at 20 and 8 kDa (Fig. 1A). After blotting onto polyvinylidene difluoride membrane, these two bands were analyzed directly by Edman degradation and carboxypeptidase digestion. Amino acid sequencing of the 8-kDa band revealed the Met-Arg-Gly-Ser-His(6)-Gly-Ser-Ala sequence, which corresponds to the N-terminal part of the expressed protein (13). Edman degradation of the 20-kDa band provided no sequence information. The 20-kDa band was further analyzed by reaction with dansyl chloride. We reasoned that although the amino acid participating in cyclization of the chromophore may contain a free alpha -NH2 group, the adjoining peptide bond might be somehow modified and therefore resistant to Edman degradation. However, dansylation of the 20-kDa fragment followed by acid hydrolysis failed to reveal an accessible alpha -NH2 group, although intrachain epsilon -Lys and o-Tyr dansyl derivatives were observed.


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Fig. 1.   Fragmentation of asFP595. A, after expression in E. coli, purified asFP595 was analyzed on a 15% SDS-polyacrylamide gel. The 28-kDa band corresponds to the full-length protein. The 8- and 20-kDa bands are the N- and C-terminal fragments of the protein, respectively. Protein bands were visualized by staining the gel with Coomassie Blue. Molecular mass standards are shown on the left of the gel. B, scheme outlining how the asFP595 polypeptide chain splits during maturation. The position of the cleavage is indicated by an arrow. (Residues corresponding to the expression vector backbone are in lowercase.) The amino acid sequence of the chromopeptide is shaded.

Carboxypeptidase A treatment of the blotted 20-kDa band liberated Asn and His as expected for the C terminus of the protein. Similar analysis of the 8-kDa band showed the presence of Cys, Ser, and Thr. Taking into account the molecular mass of 8 kDa, we have found the characteristic Thr-Ser-Cys sequence at positions 62-64 just before the predicted chromophore motive Met-Tyr-Gly.

The above results indicate that the mature asFP595 undergoes fragmentation by splitting at the Cys-Met bond, yielding two of the three bands observed on SDS-polyacrylamide gel electrophoresis. The minor 28-kDa band corresponds to the full-length protein, whereas the major 8- and 20-kDa bands correspond to the N- and C-terminal fragments of the protein, respectively (Fig. 1B).

Spectral Characteristics of Denatured asFP595-- Under acidic conditions (pH 2.5), the absorption peak of asFP595 occurs at 430 nm (data not shown). Under alkaline conditions (pH 14.0), the absorption maximum of the protein shifts to an even shorter wavelength, with the peak in this case occurring at 380 nm. The pH-dependent spectral shift for asFP595 is the opposite of that observed for GFP. Under acidic conditions, GFP absorbs maximally at 380 nm; under basic conditions, it absorbs maximally at 445 nm. Prolonged incubation of asFP595 at pH 14.0 led to a decrease in the absorbance at 380 nm, implying that the asFP595 chromophore is unstable and undergoes degradation under alkaline conditions. In contrast, incubation of asFP595 at pH 3.0 at room temperature for several hours did not alter the characteristic 430-nm absorbance.

Spectral Behavior of asFP595 Trypsin-derived Chromopeptide-- The absorption spectra (Fig. 2A) of an HPLC-purified chromopeptide (a peptide produced by digesting asFP595 with trypsin) were similar to those for the denatured protein, asFP595. For example, at pH 3.0 the peptide showed an absorption maximum at 430 nm. This peak shifted to 380 nm when the pH was adjusted to 14.0. As the pH increased from 3.0 to 9.0, the peak, formerly at 430 nm, shifted to 535 nm (Fig. 2A), and the color of the peptide solution changed from yellow to red. This pH-dependent transition showed an isosbestic point at 466 nm with a pKa of 6.8 (Fig. 3). The yellow to red color shift is reversible; titrating back to pH 3.0 restored the absorbance at 430 nm. For GFP, the pH-dependent shift from 380 to 445 nm is known to be caused by the ionization of a phenolic group (18), which has a pKa of 7.9-8.1 (7, 19). Presumably, the pH-dependent shift from 430 to 535 nm for the asFP595 chromopeptide also corresponds to the ionization of a phenolic group. But the relatively lower pKa of the asFP595 implies the presence of a stronger electron-withdrawing group in the asFP595 chromophore. As the chromopeptide-containing solution was titrated to pH 14.0, the 535-nm absorbance peak shifted to 380 nm, as was seen for the denatured asFP595. The pKa of this transition was 10.9 (Fig. 3), suggesting that a protonated base is crucial for the structure of the chromophore. This transition is only partially reversible, because at pH 14.0 the chromophore gradually falls apart as discussed above.


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Fig. 2.   Absorption and emission spectra of the purified asFP595 tryptic chromopeptide. A, the chromopeptide exists in three pH-dependant forms: yellow (lambda max 430 nm) at pH 3.0 (dashed line); red (lambda max 535 nm) at pH 8.0 (solid line); and colorless (lambda max 380 nm) at pH 14.0 (dotted line). The yellow and colorless forms exhibit the same maxima as the acid- and alkali-denatured asFP595. B, excitation (dotted line) and emission (solid line) spectra of the HPLC-purified chromopeptide of asFP595 in 20 mM Tris-HCl buffer containing 150 mM NaCl, pH 8.5.


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Fig. 3.   Spectrophotometric titration of the purified asFP595 chromopeptide. Purified chromopeptide in 10 mM sodium phosphate buffer, pH 3.0, was titrated by the addition of microliter quantities of dilute NaOH solution. Absorbance of the chromopeptide was monitored at 430 (circles), 535 (squares), and 380 nm (triangles). The measured pKa of 6.8 corresponds to conversion of the chromopeptide from yellow to red, and a pKa of 10.9 corresponds to the conversion from red to colorless.

The red form of the chromopeptide exhibits emission spectra similar to those of asFP595 (Fig. 2B) with the emission peak at 595 nm and excitation maximum at 535 nm. Because the 430-nm (yellow) and 380-nm (colorless) forms of the chromopeptide do not show an appreciable emission at 595 nm, it is reasonable to assume that the structure of the chromophore in the 535-nm-absorbing peptide represents the structure of the chromophore in mature asFP595.

Structural Studies of the Chromopeptide-- Acid hydrolysis of the chromopeptide yielded 1.8 mol of glycine, 1 mol of lysine, and 0.6 mol of serine. Edman degradation or reaction with dansyl chloride failed to reveal any accessible N-terminal NH2 group of the chromopeptide. Carboxypeptidase A treatment of the purified peptide liberated Ser and Lys. Collectively, these results lead us to conclude that the isolated chromopeptide corresponds to the N-terminal pentapeptide from the 20-kDa fragment Met-Tyr-Gly-Ser-Lys. (Fig. 1B).

With MALDI/time-of-flight mass spectrometry, the chromopeptide was detected as a molecular ion at m/z = 564.6 (the molecular mass for the precursor peptide Met-Tyr-Gly-Ser-Lys was calculated to be 584.6 Da). To confirm that the mass of 564.6 Da corresponded to the cation radical and not to the protonated molecular ion [M+H]+, MALDI/time-of-flight mass spectrometry experiments were carried out in both positive and negative ion detection modes and included the synthetic hexapeptide Thr-Gly-Glu-Asn-His-Lys as an internal standard (Fig. 4, A and B). The masses of the peptide standard, detected by MALDI/time-of-flight mass spectrometry in positive and negative ion modes, differed by 2 atomic mass units, a value that corresponds to the difference between the [M+H]+ and [M-H]- molecular ions. In contrast, the masses of the chromopeptide differed by only 1 atomic mass unit, corresponding to the difference between the M and [M-H]- ions. Because the chromophore contains an extended system of conjugated pi  electrons, it is reasonable to conclude that the molecule stabilizes the cation radical by delocalizing the charge over the entire pi  framework. Resonance effects such as these have also been used to explain the stability of charged radicals formed from other polyunsaturated compounds including carotenoids (20), glucuronide metabolites of certain aromatic drug molecules such as indomethacine (21), and highly conjugated textile dyes (22). Comparing the mass of the molecular ion at m/z = 564.6 Da with the calculated mass of the corresponding unmodified peptide (584.6 Da), one can conclude that the cyclization reaction of the asFP595 chromophore is stoichiometrically the same as that for the GFP chromophore: one H2O and two H+ are released while a Schiff base and dehydrotyrosine are formed. Because the N-terminal methionine of the chromopeptide does not contain an accessible alpha -NH2 group, a covalent bond to this nitrogen is probably formed during cyclization. Based on the results of the carboxypeptidase A treatment of the chromopeptide, the carbonyl carbons of Ser and Gly remain unmodified. Therefore, the tyrosine carbonyl is the only reasonable candidate for the nucleophilic attack by the methionine NH2 group (Fig. 5, a and b).


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Fig. 4.   MALDI/time-of-flight mass spectra of the chromopeptide. Mass spectra of the purified chromopeptide were acquired in positive (A) and negative (B) ion detection modes with the inclusion of a synthetic hexapeptide Thr-Gly-Glu-Asn-His-Lys (calculated molecular mass = 684.7 Da) as an internal standard. The mass of 587.4 Da in A corresponds to the sodium form of the chromopeptide.


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Fig. 5.   A complete scheme of the asFP595 chromophore formation, pH-dependant conversions, and degradation. The first step in asFP595 chromophore formation is cyclization, which is the result of a nucleophilic reaction between the alpha -nitrogen of Met65 and carbonyl carbon of Tyr66 catalyzed by the protein. After cyclization, the N-acylamidine bond (the former peptide bond between Cys64 and Met65) hydrolyses resulting in the splitting of the protein into 8 and 20-kDa fragments as shown in Fig. 1. The chromopeptide of asFP595 exists in the three pH-dependant forms (absorption maxima are indicated). The determined pKa values of 6.8 and 10.9 are consistent with ionization of the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation, respectively. The red form of the chromopeptide exhibits the same emission maximum as the native protein (595 nm), suggesting that this form is a major structure of the chromophore in mature asFP595. The colorless form of the chromophore is unstable and undergoes degradation by hydrolysis at two C=N bonds, yielding free methionine and the shortened polypeptide.

The results presented above can be summarized as follows (Fig. 5). After the Schiff base forms, the N-acylamidine bond undergoes hydrolysis (c), cleaving the Cys-Met peptide bond and fragmenting the protein. Cyclization is followed by oxidation of the tyrosine side chain by molecular oxygen (similar to the reaction in GFP (23, 24)), yielding dehydrotyrosine. N,N'-disubstituted amidines usually exist in the two resonance forms shown. Protonation of the imino nitrogen of form c leads to resonance form d and a C=N double bond within the heterocycle. Amidines are strong bases (25). The amidinium cation is a strong electron-withdrawing substituent, which shifts the absorbance of asFP595 to longer wavelengths as compared with GFP. Ionization of the phenolic group of dehydrotyrosine shown in e accounts for the yellow (lambda max 430 nm) to red (lambda max 535 nm) transition of the chromopeptide. Deprotonation of the amidinium cation abolishes the long-wavelength (red) form of the chromophore, yielding the product shown in f, which absorbs at 380 nm.

Base-catalyzed Opening of the Chromophore Heterocycle-- As discussed above, under alkaline conditions asFP595 and the chromopeptide each display a 380-nm absorption, which declines over time as the chromophore degrades. We realized that if we could identify the degradation products we could reconstruct the chromophore structure. To this end, the purified chromopeptide was incubated in 1 N NaOH in the dark at room temperature for 24 h, conditions that normally do not hydrolyze ordinary peptide bonds. Dansylation of the hydrolysis products revealed the presence of methionine, apparently the only amino acid liberated upon decomposition of the chromopeptide (Fig. 5g). Dansylation followed by hydrolysis in 6 N HCl for 8 h at 105 °C showed that tyrosine is the N-terminal amino acid of a newly formed tetrapeptide (Fig. 5g), although a much lower amount of glycine was also detected. Extensive hydrolysis in 6 N HCl for 24 h followed by dansylation yielded dansyl derivatives of Gly, Ser, and Lys. It should be mentioned that extensive acid-catalyzed hydrolysis of dehydrotyrosine yields p-hydroxybenzaldehyde and glycine (7, 8). The conditions we routinely employ for liberating N-terminal residues labeled with dansyl chloride (hydrolysis for 8 h) might explain why tyrosine, although predominant, is not the only hydrolysis product; the treatment also liberates a small amount of glycine. Also, it should be noted that we were unable to distinguish dehydro-Tyr from Tyr-dansyl derivatives using thin-layer chromatography.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chromophore formation in asFP595 is unlike that in GFP or any other GFP-like protein studied to date. asFP595 is unique because after the autocatalyzed cyclization of its chromogenic amino acids, the polypeptide chain splits into two fragments with molecular masses of 8 and 20 kDa. Our results suggest that this fragmentation is a critical step in the maturation of asFP595.

The purple protein splits when the Cys64-Met65 bond adjacent to the chromophore is cleaved in an autocatalyzed reaction, the final step in protein maturation. Because all GFP-like proteins, including asFP595, possess an extraordinarily rigid structure, harsh denaturing conditions must be applied to study this phenomenon. Therefore, it was initially unclear to us whether the splitting is an inevitable consequence of inadequate treatment of the protein or whether fragmentation of asFP595 is a necessary step in protein maturation. To resolve these uncertainties, we optimized the conditions for denaturation and analyzed the chromophore-bearing peptides after proteolytic digestion. We found that alkaline conditions, sufficient for denaturation, lead to irreversible degradation of the asFP595 chromophore. In a mildly acidic solution, the protein denatures but the chromophore is chemically stable. Therefore, acid-induced denaturation followed by a short digestion with trypsin (at a high trypsin/protein ratio) at room temperature was selected as the optimal method for isolating the chromophore-bearing peptide of asFP595.

Recently, Gross et al. (15) showed that the long-wavelength fluorescence of drFP583 can be explained by an additional autocatalytic dehydrogenation, a reaction that extends the system of overlapping p orbitals in the GFP-like chromophore. The dehydrogenation forms an acylimine at the 2nd position of the 4-(p-hydroxybenzylidene)-5-imidazolone. The acylimine bond exists only in the undenatured protein; harsh denaturation of drFP583 leads to hydrolysis of the acylamine, which is located at the Phe64-Gln65 junction (numbering based on GFP), the same position as the Cys64-Met65 cleavage in asFP595. The hydrolysis splits drFP583 into two fragments and causes the red chromophore to revert to its nascent green form. If the red-shifted absorbance of asFP595 had been a property of the full-length protein (as in drFP583), then the characteristic 430-nm absorbance must have been fully associated with the peptide with an uncleaved Cys-Met bond. However, fractionation of a trypsin digest of asFP595 by HPLC showed that the 430-nm absorbance distributes with the pentapeptide Met-Tyr-Gly-Ser-Lys. Our structural studies show that this sequence is located at the N terminus of a 20-kDa fragment produced when the Cys64-Met65 peptide bond breaks in the full-length protein. Thus, these results favor a model in which full-length asFP595 represents an immature form of the purple protein. In this model, fragmentation of asFP595 is a critical step in protein maturation.

The chromophore structure presented here illustrates why fragmentation is crucial for the development of the mature asFP595 chromophore. The pKa values of the N-acyl-N,N'-disubstituted amidines, similar to those in Fig. 5b, are considerably decreased in comparison with unacylated amidines (26). This means the protonated amidinium cation is much more stable in mature (fragmented) asFP595 in which the N-acylamidine bond has been cleaved (Fig. 5c), which explains the bathochromic shift upon fragmentation. It should be noted that because the amidines have resonance forms, both nitrogens of the amidinium cation should lie in the plane of the conjugated system.

According to our data, the chromopeptide from asFP595 does not contain an acylimine bond. (An acylimine bond is responsible for the red-shifted fluorescence of drFP583 (15, 27, 28).) Hydrolysis of the acylimine bond would yield an amide of cysteine at the C terminus of the 8-kDa fragment. Carboxypeptidase A treatment of the 8-kDa fragment liberated cysteine but not cysteine amide. Carboxypeptidase A does not display amidase activity (29), and the absolute requirement for this enzyme is the presence of an amino acid in the terminal position with a free alpha -carboxyl group (30). Base-catalyzed opening of the chromophore heterocycle confirms the above data, showing that methionine alpha -nitrogen does not exist in the form of a cysteine amide-leaving group, but it is indeed located within the chromophore heterocycle.

We interpret these discrepancies to mean that drFP583 and asFP595 mature by different pathways. Indeed, even after prolonged maturation, drFP583 still contains a substantial amount of GFP-like green chromophores (15, 31). asFP595, on the other hand, exhibits no green fluorescence (or GFP-like absorbance) at any time during its maturation. Thus, it is likely that the green GFP-like chromophore in drFP583 is an intermediate in the maturation of red fluorescent protein. And although this intermediate is a prerequisite for forming the red chromophore, it does not seem to be a part of the pathway that leads to formation of the purple (asFP595) chromophore. This idea is supported by the spectra of the drFP583 and asFP595 denaturation products. When drFP583 is denatured at an alkaline pH, it displays a UV-visible absorbance spectrum similar to that of denatured GFP, demonstrating that the drFP583 chromophore is a derivative of imidazolidinone (15). Under the same denaturing conditions, asFP595 yields a product absorbing at 380 nm.

Shortly before this report was published, an unusually small GFP-like chromoprotein, asCP562 from A. sulcata, was described (32). In comparing the predicted amino acid sequence of asCP562 with the known sequence of asFP595 (13), we find that these proteins have an identical stretch of 131 amino acids at their N termini. Their cDNAs differ by four bases and one frameshift at the position corresponding to the 132nd amino acid residue (CCCC in asFP595 cDNA versus CCCCC in asCP562 cDNA).

Armed with this information, we decided to study the maturation of truncated asFP595. We generated a pQE30-based clone expressing cDNA identical to the cDNA sequence reported for asCP562 (32). The shortened protein, however, was completely colorless. In light of this result, we believe that Wiedenmann et al. (32) have misinterpreted the asCP562 length because of a sequencing mistake. Consequently, the observed 19.1-kDa band for asCP562 does not comprise the N-terminal sequence of asFP595 but presumably corresponds to the C-terminal 20-kDa fragment of mature asFP595 (see Fig. 1). Interpreted this way, the results reported by Wiedenmann et al. support our finding that fragmentation plays a critical role in the development of asFP595 red shift. They have shown that only the 19-kDa band exhibits red-shifted fluorescence associated with the native protein.

    ACKNOWLEDGEMENTS

We thank Natalya I. Khoroshilova for the perfect experiments with dansyl derivatives of amino acids, Arkady F. Fradkov for cloning of the truncated asFP595 cDNA, and Alexander S. Arseniev for fruitful discussion. We are grateful to Louis Wollenberger (CLONTECH) and Maria E. Bulina for the help with the manuscript preparation.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117997 Moscow, Russia. Tel.: 7-095-330-7056; Fax: 7-095-330-7056; E-mail: luk@ibch.ru.

Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M100500200

    ABBREVIATIONS

The abbreviations used are: GFP, green fluorescent protein; FP, fluorescent protein; drFP583, red fluorescent protein from Discosoma sp. (DsRed); asFP595, purple chromoprotein from A. sulcata; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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