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INTRODUCTION |
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
-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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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
-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
-NH2 group, although intrachain
-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.
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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
( max 430 nm) at pH 3.0 (dashed line); red
( max 535 nm) at pH 8.0 (solid line); and
colorless ( 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.
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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
electrons, it
is reasonable to conclude that the molecule stabilizes the cation
radical by delocalizing the charge over the entire
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
-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
-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.
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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 (
max 430 nm) to red
(
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.
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DISCUSSION |
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
-carboxyl group (30).
Base-catalyzed opening of the chromophore heterocycle confirms the
above data, showing that methionine
-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.