From the Shemyakin and Ovchinnikov Institute of
Bioorganic Chemistry, Russian Academy of Science and
§ Evrogen, Joint Stock Company, Miklukho-Maklaya 16/10,
117997 Moscow, Russia
Received for publication, November 25, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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asCP, the unique green fluorescent
protein-like nonfluorescent chromoprotein from the sea anemone
Anemonia sulcata, becomes fluorescent ("kindles") upon
green light irradiation, with maximum emission at 595 nm. The kindled
protein then relaxes to a nonfluorescent state or can be "quenched"
instantly by blue light irradiation. In this work, we used asCP mutants
to investigate the mechanism underlying kindling. Using site-directed
mutagenesis we showed that amino acids spatially surrounding
Tyr66 in the chromophore are crucial for kindling.
We propose a model of the kindling mechanism, in which the key event is
chromophore turning or cis-trans isomerization. Using site-directed
mutagenesis we also managed to transfer the kindling property to the
two other coral chromoproteins. Remarkably, most kindling mutants were
capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.
Recently a GFP-like1
chromoprotein from the sea anemone Anemonia sulcata was
discovered (1). This protein, named asCP, absorbs light effectively,
with a maximum at 568 nm, and causes purple coloration of anemone
tentacle tips. Initially nonfluorescent, asCP becomes fluorescent
(kindles) in response to intense green light irradiation, with an
excitation maximum at 575 nm and an emission maximum at 595 nm. The
protein then relaxes back to its initial nonfluorescent state, or it
can be quenched instantly by short blue light irradiation. Both
kindling in green light and quenching in blue light are reversible
processes for the wild-type protein. The nature of these striking
changes remains unclear; here we propose an explanation based on the
wild-type and mutant asCP properties.
Cloning, Expression, and Mutagenesis--
For heterologous
expression of proteins, full-length coding regions were cloned into the
pQE30 vector (Qiagen). Proteins fused to an N-terminal 6xHis tag were
expressed in Escherichia coli and purified using the Talon
metal affinity resin (Clontech). Site-directed
mutagenesis was performed by overlap extension PCR with primers
containing the appropriate target substitutions (2).
Screening--
Screening of E. coli colonies
expressing mutant proteins was performed using a Nikon Optiphot
fluorescent microscope and an Olympus US SZX12 fluorescent stereo
microscope. Photographs were made using an Olympus DP50 camera.
Spectroscopy--
Absorption spectra were recorded on a Beckman
DU520 UV/VIS spectrophotometer. A Varian Cary eclipse fluorescence
spectrophotometer was used to measure excitation-emission spectra and
as a light source to determine the action spectrum for the quenching of
asCP-A148G mutant. The following external light sources were used: 458 nm (Ar-ion laser line), 430-490 nm (fluorescent microscope filter), 460-490 nm (fluorescent microscope filter), 514 nm (Ar-ion laser line), 532 nm (Nd laser line), 543 nm (HeNe laser line), and 546 (±14
nm, fluorescent microscope filter) as the light sources for estimating
asCP kindling and quenching optima; 430-490 nm (fluorescent microscope
filter) and 460-490 nm (fluorescent microscope filter) as the
quenching light sources for asCP and its mutants and as the kindling
light sources for hcCP-N165A,G mutants; 532 nm (Nd laser line) and 546 (±14) nm (fluorescent microscope filter) as the kindling light sources
for the asCP, its mutants and for those cgCP and hcCP mutants that
kindle in green light.
asCP Kindling and Relaxation--
asCP kindles in response to
green light and quenches in blue light. As the kindling light intensity
is relatively high, it was difficult to measure the optimum kindling
wavelength precisely. We roughly estimated kindling and quenching
wavelength optima using different light sources. asCP irradiation at
532, 543, or 546 nm caused bright kindling (hereinafter referred to as
kindling green light). However, the 514 nm light caused only low level kindling, but it quenched previously kindled asCP considerably, indicating a probable intersection with a blue quenching light. The
blue light irradiation at 458, 430-490, or 460-490 nm caused no
kindling and quenched previously kindled asCP. It is likely that the
kindling optimum corresponds to the absorption peak at 568 nm (Fig.
1A). (Extinction
coefficient = ~120,000 M
The intensity and duration of the green light illumination needed for
kindling depend on several parameters: the protein sample thickness and
transparency, protein concentration, and wavelength used. In the
protein sample, kindling is observed when kindling speed (which depends
both on irradiation power and duration) is higher than the kindled asCP
relaxation speed. For example, irradiation of asCP expressing E. coli colony in fluorescent microscope through 10× objective
(TRITC filter set, 100-W lamp) causes quick kindling, which
reaches its maximum in ~10-20 s. Irradiation through a 5× objective
causes very slow kindling, which reaches its maximum in about 10 min.
A quarter of 5× objective intensity causes no visible kindling.
We also measured the relaxation kinetics of the kindled asCP at room
temperature, which was shown to have a 7-s half-life. However, kindled
asCP stayed fluorescent for a much longer period of time, for more than
24 h, upon immediate cooling with liquid nitrogen and subsequent
placement at Chromophore Environment in GFP-like Proteins--
Currently, the
x-ray structures for two GFP-like proteins are known, i.e.
GFP itself and coral DsRed protein (3-6). These data can be used as
three-dimensional models and are suitable for the analysis of asCP and
other GFP-like fluorescent proteins (FPs) and nonfluorescent
chromoproteins (CPs). According to the x-ray structure,
Tyr66 of the GFP chromophore is oriented to
Thr203 and His148, forming a hydrogen bond with
the latter (3, 4). Our previous studies also showed the importance of
the amino acids at positions 148, 203, and, interestingly, 165 for the
fluorescent properties of coral GFP-like proteins (1, 7, 8) (to
facilitate comparisons, we have denoted all amino acids according to
their alignment with GFP).
Remarkably, position 148 is clearly different between coral FPs and CPs
cloned to date. The known coral FPs (about 20) contain Ser148 (Table I). Similar to
GFP, the DsRed crystal structure shows that the chromophore is
stabilized through position 148 (5, 6). It is highly probable that in
most coral FPs the conserved Ser148 is engaged in
chromophore stabilization in the fluorescent state. In CPs (except
asCP), position 148 is Cis or Asn (Table I), whereas asCP contains the
uncommon Ala148. At the same time, asCP is an exceptional
protein, capable of being in both the nonfluorescent or "chromo"
state and the fluorescent state. (The chromo state indicates
that the protein has a high extinction coefficient but a low quantum
yield, whereas in the fluorescent state the protein is characterized by
a high quantum yield.) Noticeably, a single X148S mutation
makes coral CPs (including asCP) fluorescent, with the
excitation/emission characteristics corresponding to their absorption
peaks (1, 8). It is likely that in coral CP X148S mutants, the
chromophore state is similar to that in FPs.
Like Ala148, Ser165 in asCP is also
exceptional. Typically this position is occupied by Val or Ile in FPs
or by Asn in CPs (Table I). Position 203 does not differ so definitely
between coral FPs and CPs, although most FPs contain His203.
Properties of asCP Mutants--
We examined the kindling
properties of asCP with mutations at amino acid position 148, in
combination with wild-type Ser165 and mutated S165V.
His203 was also mutated. Table
II lists the properties of the mutant proteins. Random mutagenesis of Ala148 in asCP gave mutants
with Gly, Ser, Cys, Asn and Thr148, which folded in
E. coli at 37 °C. All of these mutants were intensely
colored except for the poorly folded A148T. Mutants A148C and
A148N lost the kindling property and stayed nonfluorescent, the
A148G kept the wild-type protein properties in general (see detailed
study below), and mutants A148S and A148T became fluorescent and
kept the kindling property (although A148T lost the quenching property). It is reasonable to assume that changes to essential position 148 affected asCP fluorescent and kindling properties because
this position is important for FPs chromophore stabilization. However,
interestingly, our results demonstrated that changes in position 148 caused no significant alteration in protein coloration.
It is noteworthy that the asCP-S165V mutant was fluorescent and
completely lost all its kindling/quenching properties. Moreover, this
mutation made asCP fluorescent independently of position 148. Thus,
asCP mutant S165V with mutation at position 148 (A148G, A148S,
A148C, or A148N) were fluorescent and incapable of quenching or
additional kindling (A148T/S165V mutant folding was poor). At the same
time, all S165V mutants were relatively weakly colored compared with
wild-type asCP and A148X mutants.
Mutation of His203 also influenced asCP kindling. The
asCP-A148S/H203S mutant was fluorescent with an emission maximum at 590 nm and, in contrast to the A148S mutant, was incapable of kindling or quenching.
Model of the Kindling Mechanism--
Two plausible explanations
can be proposed for a protein switching from a chromo to fluorescent
form: either a change in chromophore charge or a change in chromophore
environment. Neutral and anionic forms of the GFP chromophore have
clearly different excitation spectra, peaking at 396 and 476 nm,
respectively, but have close emission maxima at 508 and 503 nm (9). A
simultaneous decrease of the 396 nm and increase of the 476 nm
excitation peaks was observed in the course of irreversible GFP
photoactivation (10, 11). However, the excitation maximum wavelength of
the kindled asCP was very close to its absorption maximum before
kindling (Fig. 1A). This argues for the same chromophore
charge of both initial nonfluorescent and kindled fluorescent asCP,
suggesting that some other changes put the chromophore into a new
state. The influence of temperature on kindled asCP relaxation time
(see above) indicates that protein conformation changes are involved.
On the basis of our findings, we propose a model whereby kindling is
related to cis-trans isomerization (or turning) of the excited
chromophore from a chromo to a fluorescent state. As
Ser148, characteristic of coral FPs, makes asCP
fluorescent, we presume that the asCP chromophore fluorescent (kindled)
state is similar to the fluorescent state of coral FPs and GFP.
Therefore, if kindling is tied with the chromophore cis-trans
isomerization, asCP Tyr66 should contact Ser165
before kindling. (The latter follows from the DsRed crystal map. In
DsRed, Ile165 spatially blocks this conformation (Fig.
2A).) Therefore, we suggest
that the exceptional wild-type Ser165 in asCP stabilizes
the chromophore in the chromo state. However, the excited chromophore
has a chance to isomerize to the fluorescent state, close to that of
GFP and DsRed (Fig. 2, B and C). Notably, the
S165V change makes asCP fluorescent. This mutant loses all of the
kindling/quenching properties of the wild-type protein, suggesting that
the chromo state is blocked by Val165.
As in GFP Thr203 is known to take part in chromophore
stabilization, and asCP contains His203, which is present
in most coral FPs, it is possible that His203 takes part in
the temporary asCP chromophore stabilization in the kindled state. The
proposed model correlates well with asCP mutants properties (see Table
II).
For example, previously mentioned asCP-A148S mutant is fluorescent and
is capable of additional kindling in green light and quenching in blue
light. Within several minutes after kindling or quenching irradiation
is stopped, the protein is restored to its initial fluorescent
brightness. Within the proposed model, these properties of the A148S
mutant may be explained as such; Ser148 functions to
stabilize the chromophore in the fluorescent state as it does in coral
FPs. Nevertheless, some portion of the chromophore rests stabilized by
Ser165 in the chromo state, making the protein capable of
additional kindling. In turn, the fluorescent chromophore fraction may
be quenched, as the chromo state is not prohibited.
Mutants asCP-A148C and A148N (both variants are typical for
coral CPs) lose kindling ability and stay nonfluorescent, although intensely colored, with an absorption maximum close to wild-type asCP.
The A148C or A148N mutation probably makes the fluorescent state
noncompetitive, so that no fluorescence or kindling is observed. However, mutants asCP-A148C/S165V and asCP-A148N/S165V were fluorescent and incapable of kindling or quenching, indicating that all
chromophores were in the fluorescent state, which was noncompetitive in
the presence of Ser165.
Remarkably, cis-trans chromophore photoisomerization was
proposed earlier to explain GFP switching from the fluorescent
to the long-lived "dark" state. Quantum chemical calculations
showed a high probability of excited GFP chromophore cis-trans
isomerization (12). Extending our model, we may also surmise that two
chromophore conformations are common for the chromo, fluorescent, and
kindling GFP-like proteins. We have summarized this hypothesis in Table III, taking the initial GFP chromophore
state as cis and the dark state as trans.
AsCP-A148G Mutant Study--
The A148G mutant behaved similar to
wild-type asCP, with very low initial fluorescence and an ability to
kindle in green light and quench in blue light. However, the kindled
state half-life of the A148G mutant was 50 s, which is severalfold
longer than the wild-type protein. We used the A148G mutant, the
closest to wild-type asCP, as a model because its prolonged kindled
state half-life allowed us to measure and compare the absorption
spectra before and after reversible kindling.
We found that absorption at 568 nm fell considerably in the course of
A148G kindling (Fig. 3A), and
simultaneous absorption growth at 445 nm was observed (Fig.
1B). (We observed the same effect on the wild-type protein,
but the fast relaxation hampered accurate measurement.) The
fluorescence excitation maximum of kindled A148G and asCP was 575 nm,
making it evident that the kindled chromophore still absorbs at this
wavelength. Therefore, we suggest that there are at least two
equilibrium forms of the kindled chromophore. The first is fluorescent,
with the excitation peaked at 575 nm, whereas the second absorbs at 445 nm and is nonfluorescent. It is likely that Tyr66 is
protonated in this second state, as a close absorption maximum (430 nm)
was registered for acid-denatured asCP (13). It is also very likely
that this equilibrium state is responsible for kindled protein
quenching, which happens upon blue light irradiation. We determined the
action spectrum for the quenching using asCP-A148G mutant by measuring
the decrease of its weak initial fluorescence upon different wavelength
light irradiation (Fig. 1C). The quenching wavelength
optimum was shown to be close to the absorption peak, which appears
after asCP-A148G kindling.
Transfer of the Kindling Property to Nonkindling
Chromoproteins--
To confirm the crucial role of positions 165, 148, and 203 in kindling and to obtain novel kindling variants, we made
several point mutations in two other coral chromoproteins, hcCP (from Heteractis crispa (7)) and cgCP (from Condilactis
gigantea (7)). As a result, we managed to transfer the kindling
property to both proteins and also identified a novel type of kindling upon blue light irradiation.
Wild-type cgCP contains Cys148, Asn165, and
Leu203. A single C148S mutation made this protein
fluorescent with an emission maximum at 620 nm, also making it
capable of additional kindling in green light. The cgCP-C148S plus
N165S mutant was weakly fluorescent and could be brightly kindled in
green light, similar to asCP.
For hcCP we started with a mutant containing a number of folding
substitutions (A5S, T39A, L181H, P208L, K211E), because wild-type protein folding is very poor in E. coli at 37 °C. We made
several changes of Cys148, Asn165, and
Ile203. Among the mutants obtained, hcCP-C148S/N165S/I203H
and hcCP-C148A/N165G/I203H clearly showed the kindling property. Both
of these proteins were weakly fluorescent and could be brightly kindled
in green light.
The only mutant protein obtained on the basis of hcCP and cgCP that
demonstrated quenching under blue light was the cgCP and hcCP hybrid
protein: cgCP-C148S before Ser165 and hcCP after
Ser165. This mutant protein was fluorescent and
could be additionally kindled in green light and considerably quenched
in blue light, similar to the asCP-A148S mutant.
Novel Type of Kindling--
A novel type of kindling was
found in two hcCP mutants containing a single change at position 165, N165A and N165G (with no additional random mutations). These
nonfluorescent proteins kindled quickly upon blue light irradiation. In
the kindled form they were fluorescent with an emission peak at 620 nm
and excitation at 590 nm. The hcCP-N165A mutant fluorescence was
observed for about 1 min after kindling, whereas the N165G mutant was
fluorescent for 10 s.
In contrast to other kindling variants, the kindling wavelength of
these mutants was different than the fluorescent excitation wavelength,
giving advantages for biotechnology applications, as there is no
background kindling in the course of object tracking. Also, dual red
fluorescent labeling becomes possible (Fig. 3B).
Irreversible Kindling--
Surprisingly, for most mutant kindling
proteins (but not for wild-type asCP) the irradiation with kindling
light of greater intensity caused irreversible kindling. For example,
irreversibly kindled A148G mutant gave stable red fluorescence with an
intensity up to 40 times brighter than the unkindled protein, reaching
about 50% of reversibly kindled protein brightness. The fluorescence of the irreversibly kindled A148G mutant protein did not fade for at
least a year after kindling. Further studies are needed to understand
the nature of this irreversible effect.
Concluding Comments--
The kindling effect was relatively easy
to transfer to nonkindling, nonfluorescent, GFP-like Anthozoa
chromoproteins. This was achieved by performing amino acid
substitutions at positions 148, 165, and 203 around chromophore
Tyr66, confirming the key role of these positions in kindling.
asCP Ala148 and Ser165 positions mutagenesis
gave proteins with essentially modified or blocked kindling and
quenching. The properties of these mutants enabled us to propose a
model that implies that asCP kindling is linked to chromophore
cis-trans isomerization. Although Ser165 takes part in
chromophore stabilization in the initial chromo state,
His203 takes part in kindled chromophore temporary
stabilization in the fluorescent (kindled) state, which is similar to
the fluorescent chromophore state in GFP and DsRed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1
cm
1 according to our refined data. Previous studies
showed a lower extinction coefficient, probably due to the incomplete
protein maturation (1)). The action spectrum for the quenching was determined precisely using an asCP mutant (see "asCP-A148G
Mutant Study").
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Fig. 1.
Changes in excitation and absorption spectra
during kindling. A, normalized asCP absorption spectra
before kindling (solid line) and fluorescence excitation
spectra after kindling in green light (dotted line).
B, difference between kindled and initial asCP-A148G mutant
protein absorption spectra. The main absorption peak at 568 nm falls,
but absorption growth is observed with a peak at 445 nm.
C, relative decrease of the fluorescent brightness for the
asCP-A148G mutant after quenching using different wavelengths of
light irradiation. Fluorescent emission brightness at 610 nm was
measured using 570 nm excitation light.
70 °C.
Amino acids in positions 148, 165, and 203 in Aequorea victoria GFP and
Anthozoa chromoproteins and fluoroproteins
asCP mutant
properties
View larger version (49K):
[in a new window]
Fig. 2.
DsRed chromophore and asCP kindling
model. A, schematic stereo outline of the DsRed
chromophore and selected neighboring residues in a "space-filled"
representation. Carbon atoms are shown in gray, nitrogen
atoms in blue, and oxygen atoms in red. This
image was generated using RasMol 2.6 software based on PDB file 1G7K
(6). B and C, model of the asCP chromophore
cis-trans isomerization, which leads to asCP kindling. Schematic
outline of the chromophore (known for DsRed) and selected neighboring
residues in chromo (B, before kindling) and fluorescent
(C, after kindling) asCP states. Carbon atoms are shown in
gray, nitrogen atoms in blue, and oxygen atoms in
red. Images were generated using RasMol 2.6 software.
Computer modeling for asCP was performed using Swiss-PdbViewer and
HyperChem 5.01 software based on DsRed crystal structure.
Possible chromophore conformations in GFP-like proteins
View larger version (31K):
[in a new window]
Fig. 3.
Imaging of kindling proteins in E. coli colonies. A, chromo-fluorescent
switching of asCP-A148G mutant. The round-shaped part of the E. coli colony expressing the A148G mutant was kindled with intense
green light for 1 min using a fluorescent microscope (TRITC filter set,
100-W lamp, 10× magnification). This part fluoresces brightly, but
absorption of the irradiated region is low. After several minutes, the
kindled protein relaxed to the nonfluorescent state while its
absorption recovered. B, dual red fluorescent labeling using
two kindling proteins. E. coli colonies expressing
hcCP-N165A and asCP-A148G mutants were grown on the same Petri dish.
Colonies were irradiated by blue light (460-490 nm) for several
seconds using a fluorescent microscope. Immediately after blue light
irradiation, colonies expressing hcCP mutant were kindled and
fluoresced brightly. After 2 min of green light irradiation (TRITC
filter set), the hcCP mutant relaxed to the nonfluorescent state,
whereas asCP mutant was kindled and fluoresced brightly. This switching
could be repeated many times.
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FOOTNOTES |
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* This work was supported by grants from the Russian Foundation for Basic Research (01-04-49037) and the Russian Foundation for Support of Domestic Science (to S. L.).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: Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia. Tel.: 7-095-429-80-20; E-mail: kluk@ibch.ru.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M211988200
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ABBREVIATIONS |
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The abbreviations used are: GFP, green fluorescent protein; FP, fluorescent protein; CP, chromoprotein; TRITC, tetramethylrhodamine isothiocyanate.
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