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Key words: crystal structure, fluorescent protein, FRET, GFP, mutagenesis
Biophysical and Functional Characterization of asFP504,
a Novel Fluorescent Protein from the Philippines
1Protein Structure and Immunology Laboratory, National Institute of Molecular Biology and Biotechnology,
University of the Philippines Diliman, Quezon City, Philippines
2Laboratory of Molecular and Cellular Biology, National Institute of
Molecular Biology and Biotechnology, University of the Philippines Diliman, Quezon City, Philippines
3Life Science Group, Scientific Research Division,
National Synchrotron Radiation Research Center, Hsinchu, Taiwan
4Department of Physics, National Tsing Hua University, Hsinchu, Taiwan
5Institute of Biotechnology, University Center for Bioscience and
Biotechnology, National Cheng Kung University, Tainan, Taiwan
*Corresponding authors: neilandrew.bascos@mbb.upd.edu.ph
cpsaloma@mbb.upd.edu.ph
cjchen@nsrrc.org.tw
Neil Andrew D. Bascos1,*, Francine Lianne C. Emralino1, Franco Carlos Liu1,
Carla P. Concepcion2, Marvin Altamia2, Yen-Chieh Huang3, Yin-Cheng Hsieh3,
Chun-Jung Chen3,4,5,*, and Cynthia Palmes-Saloma2,*
Fluorescent proteins have proven to be invaluable for a myriad of applications in scientific
research. The discovery and characterization of novel fluorescent proteins promises to expand
this range even further. This report focuses on the biophysical and functional characterization
of a novel green fluorescent protein cloned from a Philippine soft coral species. The asFP504
protein showed peak excitation at 471 nm and at 494 nm (λE1= 471 nm; λE2=494 nm), its emission
maximum from 471 nm excitation was observed at 504 nm. The fluorescence was observed
to be related to its oligomeric state. Both fluorescence and oligomerization were robustly
maintained for a range of temperatures, pH conditions, treatment with chaotropic agents, and
proteolysis. X-ray crystallography documented a molecular packing of three dimers within
each asymmetric unit for the asFP504 protein. The observed absorbance and fluorescence
properties are comparable to that of commercially available fluorescence proteins. Despite its
lower absorbance, asFP504 has higher quantum yield than mCitrine. In addition, the stability
of asFP504 in the presence of multiple denaturants presents the potential of this protein – the
first fluorescent protein from the Philippines – for use in many different research applications.
Philippine Journal of Science
147 (1): 65-74, March 2018
ISSN 0031 - 7683
Date Received: 07 Apr 2017
INTRODUCTION
The green fluorescent proteins or GFPs (Tsien 1998)
comprise a small class of chromoproteins found in
bioluminescent hydrozoan and anthozoan coelenterates.
Among the first of these to be discovered are members
from the Aequorea victoria (avGFP) and Renilla reniformis
(Renilla GFP) species. The GFP family of proteins has now
expanded to include a number of GFP-like fluorescent and
non-fluorescent proteins that have become important tools
in molecular and cell biology. These proteins are used as
protein labels, reporter genes, selection markers, fusion
tags, and biosensors (Lippincott-Schwartz & Patterson
2003). Most of the current information on GFPs is based
65
on the extensive research on the avGFP and Renilla GFP
variants. Continued research on the properties of the
expanding repertoire of GFPs promises the discovery of
new ways for which these proteins may be utilized.
Green fluorescent proteins have been characterized to
have an eleven-stranded β-barrel structure with a coaxial
α-helix. The fluorophore, from which they derive their
function, is buried in the center of the barrel (Ormo et
al. 1996; Yang et al. 1996). Oligomeric states have been
observed to vary between different GFP species. GFP from
Aequoria victoria has been found to exist as a monomer
with tendencies for dimerization, while Renilla GFP has
been observed to be an obligate dimer (Tsien 1998). With
the exception of avGFP, all GFP-like proteins that have
thus far been discovered exist as oligomers, most of which
form tetramers (Matz et al. 1999; Sacchetti et al. 2002;
Weidenmann et al. 2000). Their structure and oligomeric
states have implications on their spectral and biophysical
characteristics.
Oligomeric GFPs often exhibit higher quantum yields
and better stability than their monomeric counterparts
(Campbell et al. 2002; Tasdemir et al. 2008). As a trade-
off, these oligomeric GFPs require longer maturation times
and are often prone to aggregation. Control of protein
oligomerization, maturation, fluorescence, and stability
has been achieved through designed mutations (Baird et
al. 2000; Pedelacq et al. 2005; Sawano & Miyawaki 2000;
Weidenmann et al. 2002; Yanushevich 2001). A recent
publication by Shaner and colleagues (2013) documents
the modification of a tetrameric yellow fluorescent
protein from Branchiostoma lanceolatum (LanYFP) into
its monomeric form (mNeonGreen). The modification
of fluorescent proteins, leading to the availability of
multicolored variants, allows the simultaneous detection
of multiple targets (Chalfie & Kain 2006).
In this study, the researchers report the structural and
functional characterization of a novel green fluorescent
protein, asFP504. This fluorescent protein was cloned
from a soft coral species (Alcyonium sp.) isolated from
Taklong Island, Guimaras, Philippines. The details of
the cloning experiments will be reported in a separate
paper by Concepcion and colleagues (unpublished
results). Biophysical analysis of the recombinantly
expressed asFP504 was conducted to determine its
excitation and emission spectra, its oligomeric states,
its thermostability, and its resistance to denaturation in
acidic and basic conditions. Structural analysis through
X-ray crystallography documented the existence of a
triple dimer oligomeric state, the first recorded for a
GFP-like protein.
MATERIALS AND METHODS
Protein Expression and Purification
Chemically competent Escherichia coli BL21 (DE3) cells
were transformed with expression plasmids (pEXP5-NT/
TOPO®-asFP504) using the heat-shock method (Inoue et
al. 1990). Colonies were allowed to exhibit fluorescence
and mature for one day before being inoculated into starter
cultures. Starter cultures were grown using 5 mL of LB
medium and were incubated at 37°C for 6-8 h, shaking at
220 rpm. These 5 mL starter cultures were then inoculated
into 500 mL LB medium and allowed to grow for 16-18
h at 37°C, shaking at 220 rpm. The bacterial cells were
harvested through centrifugation (15 min; 4,000 x g)
and resuspended in equilibration buffer (50 mM sodium
phosphate; pH 7.0; 300 mM NaCl). The resuspended cell
pellets were treated with 0.75 mg/mL lysozyme at room
temperature for 25 min then lysed with ultrasonication.
Ultrasonication was performed in four 40-second pulses
with 2 min incubation on ice in between. Cell lysate was
centrifuged at 12,000 x g for 15 min and the His-tagged
asFP504 protein was purified from the supernatant
using TalonTM metal affinity resin (BD Biosciences
CLONTECH). The purified proteins were concentrated
and resuspended in 10 mM Tris-Cl, pH 7.5 using a
Microcon-30, a 30-kDa cutoff concentrator from Amicon.
Protein concentration was determined using the Bradford
assay (Bradford 1976). Succeeding protein analysis was
done using variants of these buffer conditions.
Biophysical and Functional Characterization
I. Resistance to Detergents and Chaotropes
The resistance of asFP504 to chemical denaturation was
tested with the addition of detergents and chaotropic
agents. Sodium dodecyl sulfate was added from a 10%
stock solution to a final concentration of 1%. Triton
X-100 was added in 0.5% v/v increments to a final
concentration of 7%. Urea and guanidinium thiocyanate
(6 M stock) were added in 0.5 M increments to the final
concentration of 8 M and 2 M, respectively. The effects of
these treatments on the structure and function of asFP504
were characterized using biophysical techniques including
spectrofluorimetry, circular dichroism (CD) spectroscopy,
and polyacrylamide gel electrophoresis (PAGE).
II. Thermostability
The temperature stability of asFP504 was assessed by
incubating the protein at different temperatures (40, 55, 70,
85, and 90°C) for 5 min each time using a heating block.
Fluorescence was used as a marker of protein function
and was quantified in each case. The ability of denatured
protein samples to renature was tested by checking for
fluorescence after a 5-min recovery period.
Bascos et al.: A Novel Fluorescent Protein
from the Philippines, asFP504
Philippine Journal of Science
Vol. 147 No. 1, March 2018
66
III. pH Stability
The stability of asFP504 in different buffer pH conditions
was assessed by incubating the protein at different pH states
(10 mM Tris, pH 0-14). The effect of these conditions on
the protein’s structure and function were evaluated using
spectrofluorimetry, CD spectroscopy, and PAGE.
IV. Limited Proteolysis
The susceptibility of asFP504 to proteolytic digestion was
tested. Proteolysis was performed with 0.3-0.5 mg/mL
asFP504 (in 10 mM Tris-Cl pH 7.5) at 1:1 ratio (w/w) of
protein to protease. The reactions were analyzed over a
24-h period, with samples taken at different time points.
Trypsin and Proteinase K were used as representative
proteases. Control setups employed BSA as a proteolysis
target. Trypsin reactions were deactivated with the
addition of SDS-PAGE sample buffer (10% w/v SDS;
10 mM dithiothreitol; 20% v/v glycerol; 0.2 M Tris-HCl;
pH 6.8; 0.05% w/v bromophenol blue). Proteinase K was
deactivated with the addition of PMSF to a concentration
of 10 mM (5 mM following the addition of 2x treatment
buffer). Trypsinized samples were analyzed using 8%
and 12% SDS-PAGE.
V. Polyacrylamide Gel Electrophoresis
Protein samples were run in either 8% or 12%
polyacrylamide gels. Denaturing PAGE was performed
by using SDS/PAGE sample buffer. Non-denaturing
PAGE was performed using SDS-PAGE sample buffer
without DTT. Samples were boiled for 10 min prior to
loading onto the gel. The samples used for pseudonative
and native PAGE were not boiled prior to loading.
Pseudonative PAGE was done using SDS-PAGE sample
buffer without boiling. Following electrophoresis the
gels were illuminated with UV light (302 nm) to detect
the fluorescent bands that corresponded to asFP504.
Coomassie blue staining was used to visualize the total
protein content of the samples.
VI. Spectrofluorimetry
Absorbance, fluorescence excitation, and fluorescence
emission profiles were obtained using two fluorimeter
systems: 1) RF-5301 PC spectrofluorometer (Shimadzu)
and 2) Varioskan Flash multimode plate reader (Thermo
Scientific). Wavelengths used for the absorbance,
excitation, and emission scans were varied depending
on the samples tested. Typical absorbance scans
were performed from 230 nm to 600 nm. Observed
absorbance peaks were used as excitation wavelengths
for fluorescence emission scans. Wild-type asFP504 was
excited with 475 nm light and its emission was detected
from 490 nm to 600 nm. Observed emission peaks were
used as the detection wavelengths in the excitation scans.
Wildtype asFP504 emission at 504 nm was detected with
varied excitation from 200nm to 484 nm. Samples were
dissolved in 10 mM Tris-Cl (pH 8.0) at a concentration
of 10 µM per sample. A 100 µl volume of each sample
was then loaded in each of three (3) wells with a fourth
well as the blank. Each well counted as one (1) replicate.
Three (3) replicate runs were performed per sample and
blank. Data was corrected against blank controls.
VII. Circular Dichroism Spectroscopy
Circular Dichroism (CD) spectra were acquired for
the asFP504 protein in different test conditions. CD
experiments were performed on a JASCO J-700
spectropolarimeter. The far-UV spectra (250-190 nm)
were obtained in quartz cuvettes with 1-mm path lengths.
The cuvettes were filled with 0.4 mL of 0.1 mg/mL (3.57
µM) asFP504 in different buffer conditions. Triplicate
readings were taken for each sample. Spectra were taken
at the resolution of 1 nm, scan speed of 50 nm/min,
and response time of 1 s. Blank spectra were acquired
using appropriate buffers and were obtained at identical
conditions. These were subtracted from the test sample
readings to determine the net CD signal.
VIII. Protein Crystallization
Protein crystals were prepared by a hanging-drop vapour
diffusion method. Several protein concentrations and
incubation temperatures were tested to achieve the optimal
crystallization. The best conditions for generating crystals
were achieved with 0.05 M potassium phosphate, 20%
PEG 8000 (Nextal Classic Suite, Qiagen) and a protein
concentration of 22 mg/mL. The hanging drops were
incubated at 4°C, overnight to induce crystallization. The
crystals were allowed to mature at 20°C with different
incubation times, from 1d to 14 d, to maximize their growth.
IX. X-ray Diffraction Analysis
The preliminary X-ray characterization, crystal screening,
and the collection of complete X-ray diffraction data for
asFP504 were performed at the beamline BL13B1 of the
National Synchrotron Radiation Research Center (NSRRC)
in Taiwan. For complete data collection, a total rotation
of 200° with 1.0° oscillation was measured with a CCD
detector (Q315, ADSC) for a X-ray wavelength of 1.00 Å.
Exposure duration of X-ray was set for 30 s at a distance of
300 mm from the crystal to the detector. The temperature
was kept at 110 K using a cryo-system (X-Stream, Rigaku/
MSC). All the data were indexed and processed with the
HKL2000 program suite (Otwinowski & Minor 1997).
The data statistics of X-ray diffraction is given in Table 1.
X. Structure determination and refinement
The asFP504 structure was determined and refined using
CCP4 (Collaborative Computational Project 1994) and
programs supported therein. The initial phasing and model
Bascos et al.: A Novel Fluorescent Protein
from the Philippines, asFP504
Philippine Journal of Science
Vol. 147 No. 1, March 2018
67
building were performed with the molecular replacement
program MOLREP (Murshudov et al. 1997) using the
structure of Azami Green, a monomeric green fluorescent
protein (PDB ID: 3ADF), as a search model. Further
structure refinement was carried out using REFMAC5
(Winn et al. 2001) in the CCP4 suite. After several cyclic
model refinements, a final crystal structure of asFP504
with 2.35 Å resolution was achieved with the final Rwork
and Rfree values of 17.5% and 23.5%, respectively. The
crystal structure contains six molecules per asymmetric
unit as predicted from estimated solvent content 50.8%
(Matthews 1968) and calculations of the self-rotation
function. All graphical works for crystal structure analysis
were performed using the PyMOL program (Delano).
XI. Accession number
The atomic coordinates and structure factor of asFP504
have been deposited in the Protein Data Bank with the
accession code 4JC2.
RESULTS
Fluorescence Spectroscopy
I. Initial Fluorescence Spectra
Initial excitation and emission spectra were determined
for the asFP504 protein using a Shimadzu RF-5301 PC
spectrofluorometer system. The excitation spectrum for
asFP504 was found to have a maximal peak at 494 nm and
a minor peak at 471 nm. Excitation with 471nm revealed
an emission spectrum for the protein with a predominant
maximal peak at 504 nm. This provided the basis for the
name, asFP504 (Figure 1). A minor emission peak was
also observed near 494 nm with 471 nm excitation.
Table 1. Characteristics of cloned fluorescent and chromogenic proteins.
Excitation
maximum (nm)
Emission
maximum (nm) Quantum yield Molar extinction
(M-1cm-1)Brightness In vivo
structure
Fluorescent Proteins
DsRed monomer 556 586 0.10 35,000 3.5 Monomer
mCherry 587 610 0.22 72,000 16 Monomer
avGFP 395/475 507 0.77 21,000 16 Monomer*
asFP504 471/494 504 0.94 29,000 27 Dimer**
EGFP 484 507 0.60 56,000 34 Monomer*
mCitrine 516 529 0.76 77,000 59 Monomer
DsRed 558 583 0.79 75,000 59 Tetramer
*weak dimer
**hexamer in crystal structure
Table 2. Data collection and renement statistics.
Data collection
Wavelength (Å) 1.00
Temperature (K) 110
Space group P212121
Resolution Range (Å) 30.0-2.35 (2.43-2.35)a
Cell dimensions (Å)
a 76.07
b 127.71
c 158.80
Unique reections 65,226 (6,413)a
Completeness (%) 100 (100)a
<I/σI)> 21.0 (5.3)a
Average redundancy 7.2 (7.3)a
Rsymb (%) 10.2 (42.9)a
Mosaicity (°) 0.31
No. of molecules per asymmetric unit 6
Matthews coecient (Å3 Da-1)2.50
Solvent content (%) 50.8
Renement
Resolution range (Å) 30.0-2.35
Rworkc/Rfreed (%) 17.5/23.5
No. of atoms
Protein 10,651
Ligand (QYG) 144
Water molecules 768
B-factors (Å2)
Protein 17.7
Ligand (QYG) 10.1
Water molecules 17.2
R.m.s deviations
Bond lengths (Å) 0.015
Bond angles (°) 1.959
aValues in parentheses are for the highest resolution shell (2.43 - 2.35 Å).
bRsym = Σh Σi [|Ii(h) - <I(h)>|/ Σh Σi Ii(h)], where Ii is the ith measurement and
<I(h)> is the weighted mean of all measurements of I(h).
cRwork = Σh | Fo-Fc |/ Σh Fo, where Fo and Fc are the observed and calculated
structure factor amplitudes of reection h.
dRfree is as Rwork, but calculated with 10% of randomly chosen reections
omitted from renement.
Bascos et al.: A Novel Fluorescent Protein
from the Philippines, asFP504
Philippine Journal of Science
Vol. 147 No. 1, March 2018
68
II. asFP504 Fluorescence Properties
To compare the performance of asFP504 with other
available fluorescent proteins, several of its fluorescence
properties were determined. Figure 2 shows the
absorbance and fluorescence properties of asFP504 in
comparison to fluorescein and mCitrine. Table 1 shows
the comparison of asFP504 properties with other GFPs
in the literature. The tested proteins were suspended in
10 mM Tris-Cl (pH 7.5).
Fluorescence quantum yield measures the ratio of
absorbance to the emitted fluorescence (Sjöback et al.
1995) (Figure 2C). At 0.94, the asFP504 QY is comparable
to that of fluorescein (0.93). A brightness of 27 was
calculated from the asFP504 quantum yield and extinction
coefficient. Interestingly, asFP504 has a higher extinction
coefficient (29,000) and quantum yield than wildtype GFP,
leading to a higher overall brightness (Table 1). Wildtype
GFP has good fluorescence efficiency at 0.77 (Morise
et al. 1974). The asFP504 protein also has a higher QY
than mCitrine (Griesbeck et al. 2001). However, the low
molar absorptivity of asFP504 gives it roughly half the
brightness value of mCitrine. Raw fluorescence values
for the two proteins are similar.
Oligomerization and Fluorescence
Denaturing polyacrylamide gel electrophoresis (SDS/
PAGE) revealed the asFP504 protein to be 28 kDa in
size. This is consistent with the calculated size for the
protein monomer with the addition of the vector-derived
components (i.e., 6XHis tag). Pseudonative (8%) PAGE
showed the majority of the asFP504 protein to be migrating
as trimers (83 kDa). Traces of monomers were observed in
pseudonative gels that contained freshly purified protein
(<7 days). These monomers are suspected to interact with
the protein trimers to form the tetrameric (~115 kDa)
state observed when purified protein is allowed to mature
further for another 4 weeks. These tetrameric bands are
similarly generated with 48 h of incubation of the protein
at 37°C, the temperature at which protein maturation was
found to be optimal. The asFP504 trimers and tetramers
were found to be fluorescent under UV excitation, while
monomers did not exhibit any fluorescence. This suggests
that the formation of oligomers may be necessary for
asFP504 fluorescence (Figure 3). Fluorescence was
observed for all asFP504 oligomers starting from the
trimeric state (Figure 3).
The asFP504 tetramer was not dissociated by treatments
with SDS (3%), Triton X-100 (5%), and urea (8 M) and
showed no change in fluorescence intensity. Treatment
with guanidine thiocyanate exhibited a time-dependent
and concentration dependent loss of fluorescence. This
loss of fluorescence corresponds to a loss in the protein
oligomer form. It is unknown whether the produced
monomer is able to retain its structure or is left unfolded
by guanidine. Fluorescence is completely lost with the
chaotropic agent at the concentration of 3 M. A double
band pattern was observed in the presence of these
Figure 1. The excitation and emission spectra of asFP504.
Figure 2. Absorbance and fluorescence spectra of asFP504 and
commercially available fluorophores (Fluoresceine and
mCitrine). (A) Absorbance spectra; (B) Fluorescence
emission spectra; (C) Quantum yield determination from
the Fluorescence : Absorbance ratio.
Bascos et al.: A Novel Fluorescent Protein
from the Philippines, asFP504
Philippine Journal of Science
Vol. 147 No. 1, March 2018
69
denaturants. The double-banding for the asFP504 protein
is suspected to be a result of a change in protein size due
to partial cleavage of the His-tag (Figure 4).
Thermal Stability
The asFP504 protein was tested for stability within a
given temperature range (25-90°C). For consistency,
tested proteins were kept at similar buffer conditions (10
mM Tris-Cl, pH 7.5). Stability was tested in terms of the
retention of oligomeric states, secondary structure, and
fluorescence. The protein was found to be stable from
25 to 83°C.
Pseudonative PAGE revealed the loss of the trimeric band
at temperatures above 83°C. Instead, non-fluorescent
bands consistent with protein dimers (56 kDa) and
monomers (28 kDa) were detected at all temperatures
above 83°C (Figure 5a). In addition, incubation at 84°C
for ≥10 min showed loss of fluorescence and decreased
oligomerization similar to that of higher temperatures.
Degradation was found to be irreversible with no
observable fluorescence recovery at room temperature
or at 4°C.
Circular Dichroism spectra of heat-treated asFP504
(0.1mg/mL) remained unchanged, indicating the
stability of the protein secondary structure within this
Figure 4. SDS-PAGE analysis of asFP504 treated with detergents and chaotropic agents. Samples were run at 8%
acrylamide gels which were stained with Coomassie blue dye after fluorescence analysis under UV light
(302nm). Lane 1: BenchmarkTM Protein Ladder (Invitrogen); Lane 2: Native asFP504; Lane 3: boiled
asFP504; Lane 4: SDS (3%) treatment; Lane 5: TritonX-100 (5%) treatment; Lane 6: urea (8 M) treatment;
Lane 7: guanidine thiocyanate (2 M) treatment.
Figure 3. SDS-PAGE analysis of native and denatured asFP504. Proteins were run in an 8% acrylamide gel. The
gel was stained with Coomassie blue dye; after fluorescence analysis under UV light (302nm). Lane 1:
Precision Plus Protein Marker (BioRad); Lane 2: boiled asFP504 lysate; Lane 3: native asFP504 lysate;
Lane 4: boiled purified asFP504; Lane 5: native purified asFP504; Lane 6: native purified asFP504 allowed
to mature at 4° C for 4 weeks; Lane 7: native purified asFP504 allowed to mature at 37°C overnight.
Bascos et al.: A Novel Fluorescent Protein
from the Philippines, asFP504
Philippine Journal of Science
Vol. 147 No. 1, March 2018
70
Figure 5. Thermal stability of asFP504. Protein incubated at different temperatures (25-100°C) were run through 8% acrylamide pseudonative
SDS-PAGE and visualized with Coomassie blue staining (a) after undergoing fluorescence analysis under UV light (b). The circular
dichroism (CD) spectra for asFP504 show loss of secondary structures with the loss of fluorescence at temperatures ≥ 84°C.
Figure 6. pH tolerance of asFP504. Pseudonative SDS-PAGE of protein incubated at different pH levels (0-14) run in an 8% acrylamide
gel was stained with Coomassie blue (a) following fluorescence analysis (b). CD spectra obtained for the various pH levels show
minimal loss of secondary structure with pH variance.
Figure 7. Limited proteolysis of asFP504 with trypsin. Protein treated with trypsin (1:1 w/w
ratio) was analyzed through pseudonative SDS-PAGE (8% acrylamide) stained
with Coomassie blue (a) after fluorescence analysis with UV light (b). The same
sample was boiled and run through 12% acrylamide SDS-PAGE and stained
with Coomassie blue (c). Proteolysis of heat-denatured asFP504. Pseudonative
SDS-PAGE (12% acrylamide) of boiled protein samples treated with trypsin (d).
Bascos et al.: A Novel Fluorescent Protein
from the Philippines, asFP504
Philippine Journal of Science
Vol. 147 No. 1, March 2018
71
temperature range (25-83°C). However, this stability
appears to be dependent on the protein concentration.
Greater loss of secondary structure was observed with
the lower concentration samples. CD spectra obtained
at lower concentrations (0.05 mg/mL) showed minor
structural loss beginning at 40°C (Figure 5). The higher
stability observed with higher protein concentrations is
likely associated with the greater probability of oligomer
formation in these conditions.
The appearance of a white precipitate at 85°C was believed
to indicate protein degradation. The protein precipitates
appeared as slow-migrating protein aggregates in
pseudonative PAGE. Heating coupled with treatment of
1% SDS resulted in a decrease in stability and degradation
was observed at a lower temperature (72°C).
CONCLUSIONS
This report focuses on the characterization of the first
GFP-like protein cloned from a soft coral from the
Philippines. Biophysical analysis reveals its existence in
several oligomeric forms. X-ray diffraction analysis of
asFP504 crystals documents the first oligomeric (three-
dimer) molecular packing for a green fluorescent protein.
The oligomeric structure of asFP504 was reported to
influence its resistance to denaturation and proteolysis.
Characterization of asFP504 reveals how its properties
(e.g., quantum yield and stabilities) can compete with
commercially available fluorescent proteins (Heikal et al.
2000; Tomosugi 2009; Tsien 1998; Vrzheshch et al. 2000).
Further modification of these properties are possible
through mutations of the wildtype asFP504 protein and
are the subject of ongoing research projects.
ACKNOWLEDGMENTS
The authors would like to thank the Office of the
Vice Chancellor for Research and Development
and the National Institute of Molecular Biology and
Biotechnology, University of the Philippines Diliman for
their support of this research. The authors acknowledge
the help of Dianne Aster Yunque in the initial cloning
and sequencing of the asFP504 gene and of Dr. Nestor
Yunque in the sample collection from the Taklong Island
National Marine Reserve of UP Visayas. This work was
also supported in part by National Science Council (NSC)
grants NSC 101-2628-B-213-MY4, 102-2627-M-213-
001-MY3, and Ministry of Science and Technology
(MOST) grant MOST 105-2311-B-213-001-MY3
and National Synchrotron Radiation Center (NSRRC)
grants to CJC in Taiwan. The authors are indebted to
the computation facilities at NSRRC and staff at TLS
beamlines BL13B1, BL13C1, BL15A1, and TPS 05A at
NSRRC, as well as BL12B2 and BL44XU at SPring-8.
Figure 8. Structure of the asFP504. Quarternary structure of asFP504 is a loose
hexamer demonstrating the GFP barrel motif. Its central cyclized
chromophore (inset) is stabilized by the coaxial α-helix.
Bascos et al.: A Novel Fluorescent Protein
from the Philippines, asFP504
Philippine Journal of Science
Vol. 147 No. 1, March 2018
72
CONFLICTS OF INTEREST
The authors declare no conflict of interest in this study.
REFERENCES
BAIRD GS, ZACHARIAS DA, TSIEN RY. 2000.
Biochemistry, mutagenesis, and oligomerization of
DsRed, a red fluorescent protein from coral. PNAS
97(22):6.
BRADFORD MM. 1976. Rapid and sensitive method
for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal.
Biochem 72:7.
CAMPBELL RE, TOUR O, PALMER AE, STEINBACH
PA, BAIRD GS, ZACHARIAS DA, TSIEN RY. 2002.
A monomeric red fluorescent protein. PNAS 99(12):6.
CHALFIE M, KAIN S. 2006. Green fluorescent protein:
properties, applications, and protocols. 2nd ed. New
Jersey: John Wiley and Sons.
Collaborative Computational Project, Number 4. 1994.
The CCP4 suite: programs for protein crystallography.
Acta Crystallogr D Biol Crystallogr 50(Pt5):4.
GASTEIGER E, HOOGLAND C, GATTIKER A,
DUVAUD S, WILKINS MR, APPEL RD, BAIROCH
A. 2005. Protein Identification and Analysis Tools
on the ExPASy Server. In The Proteomics Protocols
Handbook, edited by J.M. Walker: Humana Press.
GRIESBECK O, BAIRD G, CAMPBELL R, ZACHARIAS
D, TSIEN R. 2001. Reducing the Environmental
Sensitivity of Yellow Fluorescent Protein. The Journal
of Biol. Chem. 276(31):29188-94.
HEIKAL AA, HESS ST, BAIRD GS, TSIEN RY, WEBB
WW. 2000. Molecular spectroscopy and dynamics of
intrinsically fluorescent proteins: Coral red (dsRed)
and yellow (Citrine). PNAS 97(22):6.
INOUE H, NOJIMA H, OKAYAMA H. 1990. High
efficiency transformation of Escherichia coli with
plasmids. Gene 96:6.
LIPPINCOTT-SCHWARTZ J, PATTERSON G. 2003.
Development and use of fluorescent protein markers
in living cells. Science 200:5.
MASUDA H, TAKENAKA Y, YAMAGUCHI A,
NISHIKAWA S, MIZUNO H. 2006. A novel yellowish-
green fluorescent protein from the marine copepod,
Chiridius poppei, and its use as a reporter protein in
HeLa cells. Gene 372:8.
MATTHEWS BW. 1968. Solvent content of protein
crystals. J. Mol. Biol. 33:8.
MATZ MV, FRADKOV AF, LABAS YA, SAVITSKY AP,
ZARAISKY AG, MARKELOV ML, LUKYANOV SA.
1999. Fluorescent proteins from nonbioluminescent
Anthozoa species. Nature Biotechnology 17(12):1.
MORISE H, SHIMAMURA O, JOHNSON F, WINANT J.
1974. Intermolecular energy transfer in the bioluminescent
system of Aequorea. Biochemistry 13(12):2656-62.
MURSHUDOV GN, VAGIN AA, DODSON E. 1997.
Refinement of macromolecular structures by the
maximum-likelihood method. Acta Crystallogr D Biol
Crystallogr 53:16.
ORMO M, CUBIT AB, KALLIO K, GROSS LA, TSIEN
RY, REMINGTON SJ. 1996. Crystal structure of the
aequorea victoria green fluorescent protein. Science
273:4.
OTWINOWSKI Z, MINOR W. 1997. Processing of
X-ray diffraction data collected in oscillation mode.
In Methods in Enzymology, 307-326. New York:
Academic Press.
PEDELACQ J, CABANTOUS S, TRAN T,
TERWILLIGER T, WALDO G. 2005. Engineering
and characterization of a superfolder green fluorescent
protein. Nature Biotechnology 24:10.
SACCHETTI A, SUBRAMANIAM V, JOVIN TM,
ALBERTI S. 2002. Oligomerization of DsRed
is required for the generation of a functional red
fluorescent chromophore. FEBS Letters 525:7.
SAWANO A, MIYAWAKI A. 2000. Directed evolution
of green fluorescent protein by a new versatile
PCR strategy for site-directed and semi-random
mutagenesis. Nulear Acids Res. 28(16). doi: 10.1093/
nar/28.16.e78.
SHANER NC, LAMBERT GG, CHAMMAS A, NI Y,
CRANFILL PJ, BAIRD MA, SELL BR, ALLEN
JR, DAY RN, ISRAELSSON M, DAVIDSON MW,
WANG J. 2013. A bright monomeric green fluorescent
protein derived from Branchiostoma lanceolatum. Nat
Methods (10)5:407-409.
SJÖBACK R, NYGREN J, KUBISTA M. 1995.
Absorption and fluorescent properties of fluorescein.
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy 51(6):L7-L21.
TASDEMIR A, KHAN F, JOWITT T, LUZZOLINO
L, LOHMER S, CORAZZA S, SCHMIDT T. 2008.
Engineering of a monomeric fluorescent protein
asGFP499 and its applications in a dual translocation
and transcription assay. Protein Engineering, Design
and Selection 21(10):9.
Bascos et al.: A Novel Fluorescent Protein
from the Philippines, asFP504
Philippine Journal of Science
Vol. 147 No. 1, March 2018
73
TOMOSUGI W, MATSUDA T, TANI T, NEMOTO
T, KOTERA I, SAITO K, HORIKAWA K, NAGAI
T. 2009. An ultramarine fluorescent protein with
increased photostability and pH insensitivity. Nature
Methods 6:3.
TSIEN R. 1998. The green fluorescent protein. Annu.
Rev. Biochem 67:35.
VRZHESHCH PV, AKOVBIAN NA, VARFOLOMEYEV
SD, VERKHUSHA VV. 2000. Denaturation and partial
renaturation of a tightly tetramerized DsRed protein
under mildly acidic conditions. FEBS Letters 487:6.
WEIDENMANN J, ELKE C, SPINDLER KD, FUNKE
W. 2000. Cracks in the beta-can: Fluorescent proteins
from Anemonia sulcata (Anthozoa, Actinaria). PNAS
97 (26):6.
Appendix 1. Protein sequence of asFP504.
MSVIKQEMKIKLHMEGNVNGHAFVIEGDGKGKPYDGTQTLNLTVKEGAPLPFSYDILTAAFQYGNRAFTR
YPADIPDYFKQTFPEGYSWERTMSYEDNAICNVRSEISMEGDCFTYKIRFDGKNFPPNGPVMQKKTLKWEP
STEKMYVRDGFLMGDVNMALLLDGGGHHRCDFKTSYKAKKVVQLPDYHFVDHRNEILSHDRDYSKVKL
YENAVARYSLLPSQA
WEIDENMANN J, SCHENK A, ROCKER C, GIROD
A, SPINDLER KD, NIENHAUS GU. 2002. A far-red
fluorescent protein with fast maturation and reduced
oligomerization tendency from Entacmaea quadricolor
(Anthozoa, Actinaria). PNAS 99(18):6.
WINN MD, ISUPOV MN, MURSHUDOV GN.
2001. Use of TLS parameters to model anisotropic
displacements in macromolecular refinement. Acta
Crystallogr D Biol Crystallogr 57:11.
YANG F, MOSS L, PHILLIPS G. 1996. The molecular
structure of green fluorescent protein. Nature
Biotechnology 14:6.
YANUSHEVICH YG. 2001. A strategy for the generation
of non-aggregating mutants of Anthozoa fluorescent
proteins. FEBS Letters 511:4.
Bascos et al.: A Novel Fluorescent Protein
from the Philippines, asFP504
Philippine Journal of Science
Vol. 147 No. 1, March 2018
74
