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Natural Animal Coloration Can Be Determined by a Non-Fluorescent GFP
Homolog
(Running title: Purple Chromoprotein Homologous to GFP)
Konstantin A. Lukyanov1#, Arkady F. Fradkov1#, Nadya G. Gurskaya1, Mikhail V. Matz1,
Yulii A. Labas2, Aleksandr P. Savitsky3, Mikhail L. Markelov1, Andrey G. Zaraisky1,
Xiaoning Zhao4, Yu Fang4, Wenyan Tan4 and Sergey A. Lukyanov1∗
1Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10,
117871 Moscow, Russia;
2Institute of Ecology and Evolution RAS, Leninsky pr. 33, 117071 Moscow, Russia;
3Institute of Biochemistry RAS, Leninsky pr. 33, 117071 Moscow, Russia;
4CLONTECH Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303-4230, USA
#These authors contributed equally to this work.
∗ To whom correspondence should be addressed: Shemiakin and Ovchinnikov Institute of
Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia. Tel./fax: 7
(095) 330-7056; E-mail: [email protected].
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on June 13, 2000 as Manuscript C000338200 by guest on February 4, 2016
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Summary
It is generally accepted that the colors displayed by living organisms are determined by low-
molecular weight pigments or chromoproteins that require a prosthetic group. The exception
to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a
fluorophore by self-catalyzed protein backbone modification. Here we found a naturally non-
fluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea
anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a
trace amount of red fluorescence (emission λmax = 595 nm). The fluorescence demonstrates
unique behavior: its intensity increases in the presence of green light but is inhibited by blue
light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid
replacement, which probably restores the ancestral fluorescent state of the protein. Other
fluorescent variants of the novel protein have emission peaks which are red-shifted up to 610
nm. They demonstrate that long-wavelength fluorescence is attainable in GFP-like fluorescent
proteins.
Keywords: chromoprotein; GFP; site-directed mutagenesis; red fluorescent protein;
coloration of corals
The nucleotide sequence reported in this paper has been submitted to the GenBank /EBI
Data Bank with accession number AF246709
The abbreviations used are: GFP, green fluorescent protein; FP, fluorescent protein; PKC,
protein kinase C; PMA, phorbol-12-myristate-13-acetate.
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It is generally accepted that the enormous variety of colors and fluorescent hues
displayed by living organisms are determined by chromoproteins and low-molecular weight
pigments. As a rule, chromoproteins typically require a prosthetic group: a small nonpeptide
molecule or metal ion, which binds to the protein and is essential for the chromogenic
properties of the protein (1-6).
The only known exception to this rule is green fluorescent protein (GFP) from
Aequorea victoria (7). In contrast to other naturally occurring fluorescent proteins, GFP’s
fluorescence is entirely due to an internal interaction between amino acids within the protein;
no other cofactors or prosthetic groups are required. GFP owes its intrinsic fluorescence to a
contiguous Ser-Tyr-Gly sequence centrally located within its primary structure. Upon folding
the protein modifies the fluorophore-forming sequence to produce an extended aromatic
system (8-10), which imparts the characteristic green fluorescence to the mature protein. Due
to these distinctive properties, GFP has enjoyed extensive use as a biological marker in vivo
(11, 12). Recently we described six novel GFP-like fluorescent proteins (FP) from non-
bioluminescent Anthozoa species (13). It therefore became clear that GFP-like proteins are not
necessarily components of bioluminescent systems but may simply determine fluorescent
coloration of animals.
In one particular case, we have shown that a GFP-like FP is responsible for the bright
green fluorescence of the tentacle tips in the sea anemone Anemonia majano. However, in
another sea anemone, Anemonia sulcata, we found that, while the tentacle tips do exhibit an
intense purple color they are not significantly fluorescent (Fig. 1). The similarities of the color
localization patterns and the close phylogenetic relationship of these two species led us to
hypothesize that A. sulcata contains a purple non-fluorescent GFP homolog in its tentacles. In
the present work, we describe the isolation of the cDNA for this protein and show that GFP-
like proteins can determine non-fluorescent body coloration.
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EXPERIMENTAL PROCEDURES
Cloning, expression and mutagenesis of the asFP595 cDNATotal RNA from the
tips of tentacles of A. sulcata was isolated as described (14). cDNA synthesis, amplification
of cDNA fragment of interest using degenerate primers, and obtaining the full-length cDNA
were performed as described for other Anthozoa FPs (13). The full-length coding region of
asFP595 was cloned into pQE30 vector (Qiagen). The wild type protein as well as its mutant
variants were expressed in E. coli with 6xHis tag at N-terminus and purified using TALON
metal-affinity resine (Clontech). All preparations of the heterologous expression products
were at least of 95% purity according to electrophoresis. Site-directed mutagenesis was
performed by PCR with primers containing target substitution using the method described in
(15).
SpectroscopyTo calculate the extinction coefficients at 280 nm for new protein
using the average extinction coefficients of tryptophane, tyrosine and cystine, the model
described in (16) was used. This value was then used to determine the concentration of
protein and therefore the molar extinction coefficient in the visible band. Quantum yields for
fluorescent mutants were determined relative to EGFP (Clontech). Perkin-Elmer LS50B
spectrometer was used for quantitative measurements. All samples were excited at 470 nm,
absorbance at this wavelength were 0.02, excitation and emission slits were 5 nm. The spectra
were corrected for photomultiplier response and monochromator transmittance, transformed
to wave number and integrated.
Expression in eukariotic cellsThe expression vector was developed from the
pEGFP-N1 vector (Clontech) as follows. EGFP-coding region was removed and asFP595-
(T70A/A148S) mutant was inserted instead. Then, protein kinase C β1 subunit coding region
was inserted in the multiple cloning site of this vector. As a result, the vector contained the
continuos reading frame that encode the fusion protein PKCβ-asFP595 under the control of
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the cytomegalovirus immediate-early enhancer/promoter region. The construct was expressed
in the human adenovirus 5-transformed embryonic kidney cell line 293 (American Type
Culture Collection [ATCC] CRL 1573). The pictures were taken one day after transfection.
RESULTS AND DISSCUSSION
Using a strategy previously described (13), cDNA for a novel GFP-like protein was
amplified from RNA samples prepared from the tentacle tips of A. sulcata. An alignment
(Fig. 2) of the amino acid sequences of the novel protein (named asFP595 according to
nomenclature we suggested earlier) and other known FPs was manually constructed on the
basis of our previous results (13). It appears that key secondary structure elements observed in
GFP as well as amino acids that form the GFP chromophore (Y66 and G67) or that probably
play a critical role in its formation (R96 and E222), are also present in asFP595. Overall, 20%
of the asFP595 sequence is identical to GFP. When asFP595 is compared with Anthozoa FPs
the identity level is 37—47%. Interestingly, among the Anthozoa species, the closest asFP595
homolog is drFP583 (47% identity)—another red emitter that was cloned from Discosoma
sp.—not amFP485, which derives from the more closely related Anemonia majano (42%
identity).
After expressing asFP595 in E. coli, we measured the absorption and excitation-
emission spectra of the purified protein (Fig. 3). With the exception of a slight shoulder at 530
nm, the protein displays a single absorption wavelength maximum, which occurs at 572 nm
(ε572 = 56,200 M–1cm–1). As is evident from this analysis, asFP595 absorbs efficiently in the
middle range of the visible spectrum but remains translucent when excited with blue or long-
wavelength red light. Consequently, to the observer, the protein appears intensely purple.
Visual inspection of E. coli expressing asFP595 clearly shows that the protein confers a
strong purple hue to its host (Fig. 4A).
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The fluorescence of asFP595 is extremely weak (quantum yield < 0.001);
nevertheless, the purple protein can be detected by spectrofluorimetry (Fig. 3B). When
viewed by fluorescence microscopy, the novel protein shows an unexpected feature: although
the fluorescence at 595 nm is virtually imperceptible at the start of observation, the emission
intensity increases dramatically following a 10–20 sec exposure to green light (Fig. 4B). This
effect is reversible since in the absence of incident green light, the fluorescence capacity
slowly decreases to the basal level. Even more surprisingly, asFP595 fluorescence can be
quenched by a flash of blue light (Fig. 4C). This effect strongly depends on intensity of
irradiation: the brighter light the more pronounced increasing and quenching of the
fluorescence. This phenomenon was observed in both eukariotic and prokariotic hosts
expressing asFP595 as well as in samples of the protein purified from E. coli cells extracts.
The spectral properties of recombinant asFP595 closely resemble those observed in vivo in the
tentacle tips of the corresponding organism, A. sulcata. When examined by fluorescence
microscopy, the purple-colored tentacles display a faint pinkish fluorescence that is enhanced
when viewed under green light and quenched when irradiated with blue light. Collectively,
these data suggest that the purple coloration of A. sulcata tentacles is determined by asFP595
due to differential light absorbency.
Based on their sequence similarities and fluorescent capacities, it is reasonable to
speculate that asFP595 and other Anthozoa GFP-like fluorescent proteins evolved from a
common ancestral fluorescent protein. However, asFP595 has lost the majority of its
fluorescence capability. Using site-directed mutagenesis, we attempted to reconstruct the
fluorescent antecedent of asFP595. The alignment of the nonfluorescent purple protein
sequence with all other known FPs (Fig. 2), revealed a number of interesting sequence
disparities that were evaluated experimentally. We paid particular attention to those residues
surrounding the fluorophore. One of these stood out: residue 148 (numbering based on GFP).
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In GFP and all Anthozoa fluorescent proteins, position 148 is occupied by a polar residue (His
in GFP and Ser in Anthozoa FPs). The equivalent site in asFP595 is occupied by a nonpolar
residue (Ala). Since His-148 is in direct contact with the fluorophore in GFP, it seemed likely
that it played a critical role in the protein’s fluorescence. To test this hypothesis, we generated
an A148S asFP595 point mutant. Indeed, this single substitution increased the quantum yield
of red fluorescence by as much as almost two orders of magnitude (Fig. 3C). The quantum
yield of red fluorescence for A148S was determined to be 0.012. By means of further random
mutagenesis, a brighter variant T70A/A148S was generated (quantum yield = 0.05). It should
be noted that quantum yield determinations in these mutants are hampered by the sensitivity
of the fluorophore to green light. We made these measurements using a spectrometer
equipped with a low power (8 W) light source. Thus, the magnitudes of the quantum yields
reported above probably underestimate the true values and should be used only for
comparison of different asFP595 variants.
Although the asFP595 fluorescent mutant is still rather dim compared with the other
FPs, it has some obvious advantages. First, it is the most red-shifted FP to date (emission λmax
= 595 nm). Second, it matures much faster then another red FP - drFP583. Third, this mutant
is reversibly quenched by blue light in a manner similar to that observed for the wild-type
asFP595 (Fig. 4D). This quenching response can be used for discriminating between target
and background red fluorescence.
Further mutagenesis was performed on the basis of the mutant A148S. Additional
substitutions of amino acids probably adjacent to chromophore (e.g., S68A, W94Y, S165V,
E201A,V,L, and H203S) resulted in appearance of an additional green emission peak at 514
or 523 nm, depending on the mutant (Fig. 3D). In these mutants, the nature of the particular
amino acid substitution affected the ratio between the green and red emission intensities as
well as brightness of the protein but only slightly affected the position of the emission
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maximums. The congruency of these outcomes may indicate that the green and red
fluorescence represent distinct fluorophores in a heterogeneous population of green and red
fluorescent protein molecules. This idea is supported by the observation that the red and green
emissions can be stimulated independently-excitation of the mutants by 338 nm light
produces only red fluorescence, not green. It is tempting to speculate that the red fluorophore
may be produced due to either alternative or additional protein backbone modification that
extents the conjugated π-electron system in comparison with the green fluorophore.
For some of the mutants that displayed dual-color emission spectra (e.g.,
T70A/A148S/S165V), the red emission peak occurred at 610 nm instead of 595 nm. Although
these mutant proteins are of no practical use as fluorescent markers (due to slow maturation
rates, low quantum yields, and dual-color emissions), they demonstrate that long-wavelength
fluorescence is attainable in GFP-like fluorescent proteins.
To evaluate the usefulness of asFP595 for biotechnological applications, we tested its
fluorescent variant in eukaryotic expression system. Fusion of protein kinase C β1 subunit
(PKCβ) and asFP595 fluorescent mutant T70A/A148S was constructed and expressed in a
human cell line. Using fluorescence microscopy, we were able to monitor the real-time
translocation of red fluorescence from the cytoplasm to the plasma membrane upon activation
of the PKC pathway with phorbol-12-myristate-13-acetate (PMA) (Fig. 5).
Vivid coloration is perhaps the most impressive and eye-catching feature of reef-
associated animals – from sponges to fish. However, very little is known about the chemical
identity and function of coloration, especially in invertebrates. For cnidarians, some pigments
were identified as carotenoproteins, proteins containing porphyrin or copper, and low-
molecular pigments (1-6). In many studies, pigments were identified on the basis of indirect
data only (e.g., similarity of spectra). Since carotenoproteins present colors ranging from blue
to red it was generally inferred that they were the agents responsible for the diversity of colors
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in these animals. The idea that an apoprotein—a protein devoid of ligands and prosthetic
groups—could possess chromogenic qualities was not considered. In this work, we show that
coloration can also be determined by a non-fluorescent A. victoria GFP homolog, asFP595.
Existing evidence suggests that GFP-like chromoproteins are widely distributed in
nature. In 1987, Blanquet and Phelan described an unusual blue-colored protein from the
jellyfish Cassiopea xamachana (17). The blue protein contained a covalently bonded
chromophore of unknown chemical composition and appeared not to be associated with any
metal ions. In 1995, Dove and coworkers described similar features for pink, purple, and blue
chromoproteins that were isolated from tissues of reef-building corals (18). The authors
concluded that the unique spectral characteristics of these proteins depended on an
unidentified covalently attached chromophore and not on the presence of any particular
transition metals. In addition to their shared biochemical properties, these proteins have
certain key physical traits in common with asFP595: they possess comparable molecular
weights (~ 28kDa) and display similar absorbency spectra.
Due to its self-contained fluorescence, rapid maturation rate, stability, and negligible
cytotoxicity, GFP has been recruited for use in many research applications. The novel GFP-
homolog described here displays these same desirable properties but also possesses others that
could be particularly beneficial for future research and industrial applications. Those
researchers attempting to resolve two or more fluorescent tags within the same cell may profit
from the use of the T70A/A148S asFP595 fluorescent variant, which extends the emission
range covered by existing FPs to nearly 600 nm. Wild-type asFP595, which displays a highly
distinctive color, could be used to mark an individual organism to distinguish it from others in
its group. Careful monitoring of animals in their natural environments is more conveniently
done with non-fluorescent markers because the label can be easily detected in the field
without the need for special instruments. Chromoproteins such as asFP595 could also serve as
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convenient visual aids for the selection of transgenic organisms. In plant research, for
example, during the early stages of seed germination and prior to the production of
chlorophyll, the expression of a colored protein in developing acrospires should be very
conspicuous. In an industrial setting, transgenic sheep carrying and expressing a particular
chromoprotein could simplify and detoxify the process of producing colored clothing by
eliminating the need for noxious chemical dyes.
AcknowledgmentsWe are grateful to Louis Wollenberger of Clontech for the help in
manuscript preparation and Vera Silina for the image of A. sulcata. Supported by Russian
Foundation for Fundamental Research (grant 99-04-48873) to Y.A.L.
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REFERENCES
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9. Ormö, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y., and Remington, S.J. (1996)
Science 273, 1392–1395
10. Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) Nature Biotechnol. 14, 1246–1251
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12. Kendall, J. M., and Badminton, M. N. (1998) Trends in biotechnol. 16, 216–224
13. Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M.
L., and Lukyanov, S. A. (1999) Nature Biotech. 17, 969-973
14. Chomczynski, P., Sacchi, N. (1987) Anal. Biochem. 162, 156–159
15. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L.R. (1989) Gene 77, 51-59
16. Mach, H., Middaugh, C.R., and Lewis, R.V. (1992) Anal. Biochem. 200, 74-80
17. Blanquet, R. S., and Phelan, M. A. (1987) Marine Biol. 94, 423-430
18. Dove, S. G., Takabayashi M., and Hoegh-Guldberg, O. (1995) Biol. Bull. 189, 288-297
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FIGURE LEGENDS.
Figure 1. A view of the sea anemone Anemonia sulcata. The tips of the tentacles are colored
purple.
Figure 2. Sequence comparison of GFP and GFP-like fluorescent proteins. The protein names
are shaded in colors corresponding to its fluorescence. The numbering is based on Aequorea
victoria GFP. Previously identified fluorescent proteins from Anthozoa species (Matz et al.,
1999) are compared with asFP595: the residues that are identical to the corresponding ones in
asFP595 are represented by dashes. Introduced gaps are represented by dots. The percentage
of sequence identity with asFP595 is given parenthetically to the right of the protein names.
The residues whose side chains form the interior of the β-can are shaded in blue (according to
Yang et al., 1996). In the consensus sequence the amino acids presented in all the proteins are
given. Invariant residues Tyr-66 and Gly-67 that form the GFP fluorophore are shaded in
green. In asFP595 sequence, the residues that were mutated are shaded in magenta.
Figure 3. Spectral characteristics of asFP595 and its fluorescent variants. A, the absorption
spectrum of wild-type asFP595. B and C, the excitation (dotted line) and emission (solid line)
spectra of wild-type asFP595 (B) and its fluorescent mutant A148S (C). D, the excitation-
emission spectra of the asFP595 mutant A148S/S165V. Solid line – double-peak fluorescence
(excitation at 280 nm). The complete emission spectrum (solid line), obtained by exciting the
sample at 280 nm, shows the dual-color (i.e., "green" and "red") fluorescence properties of
this mutant and others in its class (see text). These two fluorophores are energetically distinct
as shown by the excitation spectra for the green (dashed line) and red (dotted line) emissions.
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Figure 4. Colonies of E. coli expressing asFP595 as viewed by fluorescence microscopy. A,
E. coli that express wild-type asFP595 appear purple when illuminated with white light. B,
modulation of asFP595 emission intensity by exposure to green light. The center of an
asFP595-expressing colony was irradiated with green light (TRITC filter set) for 30 sec before
recording the fluorescence. Exposure to green light enhances the fluorescence intensity of
asFP595. The bright spot corresponds to the pre-irradiated area in the center of the colony. C,
quenching of asFP595 fluorescence by exposure to blue light. To generate the maximum
fluorescence intensity, an asFP595-expressing colony was first irradiated with green light
(TRITC filter set) for 30 sec before exposing the central portion of the colony to blue light
(FITC filter set) for 1 sec. The dark spot in the center of the fluorescent colony corresponds to
"quenched" asFP595. D, quenching of fluorescence from the asFP595 mutant T70A/A148S.
The image was recorded as described in (C) except that the colony was irradiated with blue
light for 30 sec. The darkened regions shown in (C) and (D) disappeared approximately 1 min
after re-exposing the colonies to green light.
Figure 5. Monitoring activation of the protein kinase C pathway with PKCβ-asFP595 (mutant
T70A/A148S) fusion. A, rested cells. B, the same cells 30 sec after treatment with 1.5 mg/ml
PMA.
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10 20 30 40 50MSKGEELFTG.VVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT.GKLPVP..W GFP (20%) G GH F G G G G P consensus...MASFLKK.TMPFKTTIEGTVNGHYFKCTGKGEGNPFEGTQEMKIEVI..EGGPLPFAF asFP595MRSSKNVI-E.F-R--VRM-------E-EIE-E---R-Y--HNTV-LK-T..K-------W drFP583 (47%)MSCSK-VI-E.E-LIDLHL---F-----EIK---K-Q-N---NTVTL--T..K------GW dsFP483 (46%)KALTTMGVI-PD-KI-LKM--N----A-VIE-E---K-YD--HTLNL--K..--A----SY cFP484 (45%)MALSNK-IGD.D-KMTYHMD-C------TVK-E-N-K-Y----TSTFK-TMAN----A-S- amFP486 (42%)MAQSKHG-T-.E-TM-YRM--C-D--K-VI--E-I-Y--K-K-AINLC-V..--------E zFP506 (37%)MAHSKHG--E.E-TM-YHM--C----K-VI--E-I-Y--K-K-TINLC--..-------SE zFP540 (37%)
60 70 80 90 100 110PTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGD.. GFP L YG D K P G R D consensusHILSTSCMYGSKTFIKYVSGIP..DYFKQSFPEGFTWERTTTYEDGGFLTAHQDTSLDGD.. asFP595D---PQFQ----VYV-HPAD--..--K-L------K---VMNF----VV-VT--S--QDG.. drFP583---CPQFQ--N-A-VHHPDN-H..--L-L-----Y----SMHF----LCCITN-I--T-N.. dsFP483D---NAFQ--NRALT--PDD-A..----------YS----M-F--K-IVKVKS-I-MEE-.. cFP484D----VFK--NRC-TA-PTSM-..-----A--D-MSY---F------VA--SWEI--K-N.. amFP486D---AAFN--NRV-TE-PQD-V..----N-C-A-Y--D-SFLF---AVCICNA-ITVSVEEN zFP506D---AGFK--DRI-TE-PQD-V..----N-C-A-Y--G-SFLF---AVCICNV-ITVSVKEN zFP540
120 130 140 150 160 170TLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQL GFP G F G G consensusCLVYKVKILGNNFPADGPVM.QNKAGRWEPATEIVYE..VDGVLRGQSLMALKCPGGRHLT asFP595-FI----FI-V---S-----.-K-TMG--AS--RL-P..R----K-EIHK---LKD-G--L drFP583-FY-DI-FT-L---PN---V.-K-TTG---S--RL-P..R----I-DIHH--TVE--G-YA dsFP483SFI-EIRFD-M---PN----.-K-TLK---S---M-V..R----V-DISHS-LLE--G-YR cFP484-FEHKSTFH-V---------.AK-TTG-D-SF-KMTV..C--I-K-DVTAF-MLQ--GNYR amFP486-MYHES-FY-V---------.KKMTDN---SC-KIIPVPKQ-I-K-DVS-Y-LLKD-GR-R zFP506-IYH-SIFN-M---------.KKMTTN--ASC-KIMPVPKQ-I-K-DVS-Y-LLKD-GRYR zFP540
180 190 200 210 220 230 ADHYQQNTPIGDG.PVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK GFP P E consensus CHLHTTYRSKKPASALKMPGFHFEDHRIEIMEEVEKGK.CYKQYEAAVGRYCDAAPSKLGHN asFP595 VEFKSI-MA--...PVQL--YYYV-SKLD-TSHN-DYT.IVE---RTE--HHLFL drFP583 -DIK-V--A--..A------Y-YV-TKLV-WNNDKEFM.KVEEH-I--A-HHPFYEP-KDK dsFP483 -DFKSI-KA--...VV-L-DY--V------LNHDKDYN.KVTL--N--A--SLLPSQA cFP484 -QF--S-KT--...PVT--PN-VVE---ARTDLDKG-N.SVQLT-H--AHITSVF-F amFP486 -QFD-V-KA-S..VPR---DW--IQ-KLTREDRSDAKNQKWHLT-H-IASGSALP zFP506 -QFD-V-KA-S..VPS---EW--IQ-KLLREDRSDAKNQKWQLT-H-IAFPSAL- zFP540
Figure 2.
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Xiaoning Zhao, Yu Fang, Wenyan Tan and Sergey A. LukyanovYulii A. Labas, Aleksandr P Savitsky, Mikhail L. Markelov, Andrey G. Zaraisky,
Konstantin A. Lukyanov, Arkady F. Fradkov, Nadya G. Gurskaya, Mikhail V. Matz,Natural animal coloration can be determined by a non-fluorescent GFP homolog
published online June 13, 2000J. Biol. Chem.
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