Biophysical and Functional Characterization of asFP504, a ...philjournalsci.dost.gov.ph/images/pdf_upload/pjs... · were characterized using biophysical techniques including spectrofluorimetry,

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, Taiwan5Institute of Biotechnology, University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan

*Corresponding authors: [email protected] [email protected] [email protected]

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 Science147 (1): 65-74, March 2018ISSN 0031 - 7683Date Received: 07 Apr 2017

INTRODUCTIONThe 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

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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 PurificationChemically 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 ScienceVol. 147 No. 1, March 2018

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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



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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 SpectraInitial 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 refinement statistics.Data collectionWavelength (Å) 1.00Temperature (K) 110Space 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 reflections 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 (°) 0.31No. of molecules per asymmetric unit 6Matthews coefficient (Å3 Da-1) 2.50Solvent content (%) 50.8RefinementResolution range (Å) 30.0-2.35Rwork

c/Rfreed (%) 17.5/23.5

No. of atoms Protein 10,651 Ligand (QYG) 144 Water molecules 768B-factors (Å2) Protein 17.7 Ligand (QYG) 10.1 Water molecules 17.2R.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 reflection h.dRfree is as Rwork, but calculated with 10% of randomly chosen reflections

omitted from refinement.



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II. asFP504 Fluorescence PropertiesTo 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 FluorescenceDenaturing 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.



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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 StabilityThe 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.



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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).



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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).

CONCLUSIONSThis 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.

ACKNOWLEDGMENTSThe 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.



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CONFLICTS OF INTERESTThe authors declare no conflict of interest in this study.

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Appendix 1. Protein sequence of asFP504. MSVIKQEMKIKLHMEGNVNGHAFVIEGDGKGKPYDGTQTLNLTVKEGAPLPFSYDILTAAFQYGNRAFTR YPADIPDYFKQTFPEGYSWERTMSYEDNAICNVRSEISMEGDCFTYKIRFDGKNFPPNGPVMQKKTLKWEP STEKMYVRDGFLMGDVNMALLLDGGGHHRCDFKTSYKAKKVVQLPDYHFVDHRNEILSHDRDYSKVKL YENAVARYSLLPSQA

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Biophysical and Functional Characterization of asFP504, a ...philjournalsci.dost.gov.ph/images/pdf_upload/pjs... · were characterized using biophysical techniques including spectrofluorimetry,