Occurrence of a functionally stable photoharvesting single peptide allophycocyanin α-subunit (16.4 kDa) in the cyanobacterium Nostoc sp. R76DM

Abstract

Allophycocyanin (APC) is a primary photoreceptor, usually composed of α- and β-polypeptide subunits. Herein, we report the occurrence of a functionally stable single peptide APC α-subunit in cyanobacterium Nostoc sp. R76DM. APC was purified successfully by ammonium sulfate fractionation. A series of biochemical characterizations like SDS-PAGE, native-PAGE, UV-visible spectroscopy, fluorescence spectroscopy and circular dichroism were performed to ensure the purity, integrity and functionality of the purified APC. Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of intact PBP revealed a ∼16.4 kDa protein. The MS/MS spectrum of five major peptides 1051, 1431, 936, 2291 and 2163 Da of trypsin digested purified PBP, followed by amino acid sequences of these peptides, shows a high degree (100%) of sequence similarities with that of the APC α-subunit (accession no. P16570, UniProtKB). The absorption as well as the fluorescence spectra of a single peptide APC α-subunit was shifted from normal absorption at 652 nm to 613 nm, and fluorescence at 663 nm to 645 nm. Urea-induced denaturation based Gibbs-free energy (ΔG^°_D) calculations suggested that the folding and structural stability of the APC α-subunit is almost similar to that of the standard APC (αβ) heterodimer from Lyngbya sp. Moreover, due to its conserved structural and functional integrity, the APC α-subunit may be widely used as a relatively low molecular weight fluorescent tag for fluorescence detection techniques.

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1. Introduction

Cyanobacteria are ubiquitous in nature and are major contributors to the evolution of atmospheric oxygen. They are an immense source of several high value compounds¹ in connection with the most vital life supporting biological phenomenon, photosynthesis.² Phycobiliproteins (PBPs) such as phycorerythrin (PE), phycocyanin (PC) and allophycocyanin (APC) are core photoharvesting pigment biomolecules which are crucial for photosynthesis in cyanobacteria and red algae. These biliproteins are associated with the light-harvesting complex (or antenna complex) of a photosystem called phycobilisome (PBS). Morphologically, PBS consists of a core located near the photosynthetic reaction center, most proximal to the outer surface of the thylakoid membrane, from which some rod-like structures are projected outwardly. The rod element of a PBS mainly contains PC (λ_max: ∼610 and 620 nm) and/or PE (λ_max: ∼540 and 570 nm) and linker proteins; whereas the core contains the PBP APC (λ_max: ∼600 and 680 nm). During photosynthesis, solar energy traverses unidirectionally down the rods of the PBS where light energy can be shifted from the PE via the PC to the APC in the core, and subsequently, some other core biliproteins permit this absorbed light energy to shift to chlorophyll within the thylakoid membrane. The unique spectral properties of a particular PBP depend on the presence of some chromophores such as phycocyanobilin, phycoerythrobilin, phycourobilin and phycoviolobilin which attach covalently to the PBP-apoproteins.³

PBPs are considered major metabolic products of cyanobacteria as almost 20% of the total dry weight of cyanobacteria is composed of PBPs.⁴ The unique color, non-toxic protein nature, strong antioxidant capacity and the exclusive absorption and fluorescence emission property of the PBPs makes them ecologically as well as economically very important. In recent decades, PBPs have been extensively used in food, cosmetic and pharmaceutical industries. Furthermore, some imperative properties of PBPs like being hepato-protective,⁵ anti-oxidative,^6,7 anti-aging^8,9 and showing anti-inflammatory activity¹⁰ make them highly promising macromolecules for therapeutic, diagnostic and pharmacological applications.

Structurally, most biliproteins generally exist in heterodimeric forms composed of alpha (α) and beta (β) subunits.¹¹ The amino acid sequence of APC from Mastigocladus laminosus revealed the occurrence of 160 and 161 amino acid residues for α- and β-subunits, respectively, exhibiting a high affinity for one another with 37% homology.¹² Moreover, the self-assembly of all PBP is initiated by the docking of α- and β-subunits that are only impartially homologous at the amino acid sequence level (25–40%) but are highly homologous at the three-dimensional structural level.^13,14 Native APC is a trimeric protein, consisting of three (αβ) monomers.¹⁵ Each α- and β-subunit of APC contains a single covalently attached chromophore phycocyanobilin (PCB), which assists in constructing the functional α–β dimer, the building block of PBP assembly.^16,17 It has been reported that the stability and functionality of the APC (αβ)₃ trimer are mainly due to the polar enhanced hydrophobicity of the PCB binding pocket.¹⁸

Some reports have described the alternative forms of PBP from cyanobacteria and red algae. Thomas and Passaquet (1999) have reported a PE composed of only β-subunits from unicellular red algae.¹⁹ A degenerated form of PE made up of only β-subunits has been reported from a marine cyanobacterium Prochlorococcus sp. growing under intense light conditions.^20,21 In the previous study, our group have also reported some fragmented-PE, composed of only truncated α-subunits from the marine cyanobacterium Phormidium sp.^22,23 and Lyngbya sp. A09DM.²⁴ Contrary to PC and PE, a few works have been conducted on the general physiology and biochemistry of APC from cyanobacteria or red algae. In the study presented here, we have reported the occurrence of a functionally stable APC composed of a single peptide α-subunit from the fresh water cyanobacterium Nostoc sp. R76DM. To the best of our knowledge there are no reports on the occurrence and in vivo biosynthesis of a single peptide APC from any cyanobacterial species/strains studied so far.

2. Materials and methods

2.1. Cyanobacterium and growth conditions

The cyanobacterium Nostoc sp. R76DM was routinely grown under axenic conditions in a BG11 liquid culture medium²⁵ in a culture room at 27 ± 2 °C with 12 : 12 h light : dark cycles and an illumination of 12 W m⁻² with cool white fluorescent lamps. The cyanobacterium was identified on the basis of 16S-rRNA gene sequence homology (accession number KJ994254).

2.2. Extraction and purification of allophycocyanin (APC)

The grown cell mass was subjected to ultra-sonication using a metal-probe to homogenize the cyanobacterial cell aggregates (VC505, Vibra Cell, Sonics and Material Inc., USA). The homogenized cell mass was frozen (at −25 °C) and thawed (at 4 °C) to achieve cell lysis and the maximum extraction of intracellular content with phycobiliproteins (PBPs). The cell extract obtained in this way was subjected to ammonium sulfate precipitation to separate the PBP from other impurities as described earlier.²⁶ The separated PBPs were further passed through a DEAE-cellulose anionic exchange column to yield pure APC. The fractions containing pure APC were concentrated by ultra-filtration using a Macrosep® (10 kDa MWCO centrifugal device, Pall Corporation). Purification was carried out under dark conditions at 4 °C unless specified. The purity of the APC was recorded as the ‘purity ratio’ calculated by the formula A₆₁₃/A₂₈₀, where A_x stands for the absorbance at x nm wavelength.

2.3. Characterization of APC

2.3.1. Gel electrophoresis analysis. Analysis of the purified APCs (5 μg of each) was carried out using native- and SDS-PAGE as described earlier.²⁷ Proteins on resolved gels were visualized by silver and zinc-acetate staining as described earlier.²⁸

2.3.2. Spectroscopic analysis. Spectroscopic analysis of the purified APCs (0.4 mg ml⁻¹) was performed using a UV-Vis spectrophotometer (Specord 210, AnalytikJena AG, Jena, Germany). The data were recorded over a 250–750 nm wavelength range using a cuvette with a 1 cm path length. The purity of the standard dimeric APC (isolated from Lyngbya sp.) as well as the single peptide APC (form Nostoc sp.) was verified as the ‘purity ratio’ calculated by the formulas A₆₅₃/A₂₈₀ and A₆₁₃/A₂₈₀, respectively.

The fluorescence emission of the APC was measured at room temperature by fluorescence spectrophotometry (F-7000, Hitachi High Technologies Corp.) to verify the functionality upon excitation at 589 nm. The raw data were transferred to a microcomputer and both absorption and emission peaks were analyzed with the respective software provided by the manufacturer.

2.3.3. Circular dichroism (CD) measurement. The far-UV CD measurement of APC was performed using a Jasco spectropolarimeter (J-810). The instrument was equipped with a Peltier type of temperature controller (PTC-348WI). CD spectra of the APC samples were collected in the wavelength range of 250–200 nm with a response time of 1 s and a scan speed of 100 nm min⁻¹. All measurements were carried out at 25 ± 0.1 °C. Molar ellipticity at 222 nm ([θ]₂₂₂) was used as a probe to investigate the secondary structure of the protein.

2.3.4. High performance liquid chromatography (HPLC). HPLC was performed in non-denaturing and denaturing conditions to determine the molecular weights of intact and monomerized Nostoc APC. A Bio-Sil SEC 125-5 gel filtration column was used with ultra-fast liquid chromatography (UFLC, Shimazdu) systems. The results were interpreted with HP Chemstation software.

Operating parameters: mobile phase – 50 mM potassium phosphate buffer (pH 8.0), column back pressure – 55 kg cm⁻², column temperature: 25 °C, protein detection: PDA detector (at 280 nm).

2.4. Gel elution, trypsin digestion of intact APC

The purified APC α-subunit (0.06 mg) was electrophoresed on sodium dodecyl sulfate (SDS)–polyacrylamide (15%) gels. The desired protein on the resolved gel was stained with Coomassie Brilliant Blue G250 dye and a portion of the envisioned stained band of APC was cut carefully using a sterile razor blade and subjected to in-gel trypsin digestion by Trypsin-Gold (Promega Corp., Madison, WI, USA) according to the manufacturer’s protocol. The digested protein sample was dried and re-solubilized in trifluoroacetic acid (TFA; 0.1%). Finally, the solution was purified by passing through Millipore®ZipTips (Sigma-Aldrich, USA) with a TA buffer (0.1% TFA + acetonitrile; 1 : 1 v/v) as described earlier.²⁹

2.4.1. MALDI-TOF-MS analysis. To evaluate the molecular weight of the pure intact peptide, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed using a AB Sciex TOF/TOF™ 2046 system as described by Benedetti et al. (2006)³⁰ with slight modifications. Briefly, 2 μl of the purified APC (15–20 pmol μl⁻¹) dissolved in potassium phosphate buffer (20 mM, pH 7.0) was mixed with a sinapinic acid matrix (in TFA). The sample was eluted directly onto the MALDI target, allowed to dry at room temperature and analyzed by the MASSLYNX program.

2.4.2. MS/MS analysis. Tryptic digested proteins were mixed with an α-cyano-4-hydroxycinnamic acid (5 mg ml⁻¹) TA buffer and a small quantity of the solution (2 μl) was allowed to dry on the MALDI plate before MS analysis. Peptide mass fingerprinting was obtained in the positive ion mode by the MALDI-TOF/TOF mass spectrometer (ULTRAFLEXIII, Bruker Daltonics, USA). Mass spectra of selected peptides were recorded over 4000 m/z using the ionization conditions as described earlier.^29,30

2.5. Bioinformatics analysis

Peptide mass fingerprints were analyzed by Flex analysis software to produce a peak list. Selected peptide identification was performed by searching in a non-redundant protein sequence database (NCBInr) using the Mascot program (http://www.matrixscience.com). The Mascot search was performed using parameters such as the significance threshold: p < 0.05, enzyme: trypsin, fixed modifications: carbamidomethylation (C), variable modifications: oxidation (M), peptide mass tolerance: ±80 ppm, maximum missed cleavages: 2 and fragment mass tolerance: ±1 Da.

The secondary and tertiary structures of the APC α-chain (accession no. P16570, UniProtKB) were predicted using the RaptorX server (http://raptorx.uchicago.edu/).³¹ An image of the tertiary structure was generated using PyMOL software (http://www.pymol.org/).³²

2.6. In vivo and in vitro stability experiments

To observe the in vivo stability and nullify the hypothesis of truncation, the PBPs were extracted and purified from cyanobacterium grown for 10, 20, 30, 40, 50 and 60 days. The purified PBPs from the log-phase culture were stored at 4 °C for 180 days to observe the possibility of truncation during a long term storage period. All of the biliproteins isolated from cultures grown for a different number of days, as well as the long term (20 to 180 days) storage samples, were analyzed by SDS-PAGE for their in vivo and in vitro stability, respectively.

Furthermore, the in vitro stability of PBPs was also investigated under three different physico-chemical stressors such as temperature, pH and a strong oxidizing agent. The thermal stability of the purified APC α-subunit was investigated by exposing the APC solutions to temperatures of 20, 40, 60 and 80 ± 2 °C for 60 min in an incubator (Innova 42, New Brunswick Scientific Co., New Jersey). To observe the effects of pH and an oxidizing agent (H₂O₂) the freeze-dried samples (1 mg) of the APC α-subunit were re-dissolved in 20 mM potassium phosphate buffers (100 μl) of different pHs (i.e., 2, 4, 6, 7, 8, 10 and 12) and a different percentage of H₂O₂ (i.e., 0.2, 0.4, 0.8 and 1.0%), respectively, and incubated for an hour under dark conditions.

2.7. Chemical denaturation and renaturation study

Chemical-induced denaturation of the purified APC was studied using an organic compound urea (CH₄N₂O) as described earlier (Sonani et al., 2015). In brief, an increasing amount of urea was mixed with the protein and denaturation was allowed for 45 min at 25 °C. Whereas, renaturation was performed by diluting a reaction mixture containing 9.0 M urea. Absorption spectra of denatured/renatured APC (0.2–0.4 mg ml⁻¹) were measured as described in Section 2.3.2 with a scan range of 200–300 nm. All spectral measurements were done in triplicate.

2.7.1. Data analysis. The plot of the change in molar extinction coefficient, Δε vs. [urea] was generated using the UV-visible results. This plot was used for the calculation of ΔG^°_D (the Gibbs free energy change for denaturation of protein), m (the slope of the plot of ΔG^°_D, the Gibbs free energy change vs. [urea], i.e., ∂ΔG_D/∂ [urea]) and C_m (the midpoint of the denaturation curve, i.e., [urea] at which ΔG^°_D = 0). The linear relationship between ΔG_D and [urea] was assumed and expressed by

y = y_N + y_D × Exp[−(ΔG^°_D − m[denaturant])/RT]/(1 + (Exp[−(ΔG^°_D − m[denaturant])/RT]). (1)

where, y_N and y_D are the spectral properties of the native (N) and denatured (D) state of protein, R is the gas constant and T is the temperature in Kelvin (K).

3. Results and discussion

3.1. Extraction, fractionation and purification of PBP

The sequential freeze-thaw actions of the cyanobacterial cell mass caused the release of intracellular content with photo-harvesting PBP pigments. All of the biliproteins released out of the cells were purified by precipitation using different concentrations of ammonium sulfate, followed by chromatographic techniques. The purity ratios of the APC from Nostoc sp. established after different fractionations was found to be up to 3.12 (Table 1). The APC obtained from Lyngbya sp.⁷ was used as a standard (hereafter S-APC) against the APC from Nostoc sp. (hereafter N-APC).

Table 1 Allophycocyanin content, purity and yield at each stage of purification

Organism	Purification	Total protein content (mg)	APC content (mg)	Purity ratio Amax/A280	Yield (%)
Nostoc APC	Crude	40.18	5.18	0.29	100.00
	Purified APC	3.15	3.08	3.12	59.45
Lyngbya APC	Crude	54.16	7.12	0.23	100.00
	Purified APC	5.38	5.17	3.09	72.61

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Furthermore, the purity of the APC was affirmed by SDS-PAGE. Contrary to S-APC, only one band of the N-APC corresponding to a smaller subunit of PBP was observed without detectable contamination in the cyanobacterium Nostoc sp. (Fig. 1A). The UV-visible spectrum of N-APC (λ_max: 613 nm), as well as S-APC (λ_max: 652 nm), shows the high purity of the APCs as the peak for N-APC and S-APC is highly prominent over the peak at 280 nm (Fig. 1B). Ammonium sulfate precipitation and aqueous two phase separation (ATPS) are well established methods for PBP separation and purification.^4,33 Several methods have been described to extract and purify the specific biliproteins from different cyanobacteria;³⁴ however, our group has recently reported an efficient technique for concurrent purification of all three major PBPs from Lyngbya sp. A09DM.⁷ Contrary to phycoerythrin (PE) and phycocyanin (PC), the purification of allophycocyanin (APC) is very arduous due to its availability in very low quantities and/or a lower resolution to discriminate between their surface hydrophobic properties; however, in the present study, a high concentration (59.45% of crude extract) of a single peptide APC was purified from the cyanobacterium Nostoc sp. R76DM.

Fig. 1 (A) Silver (left panel) and zinc acetate (right panel) stained 15% SDS-PAGE of protein molecular mass standard (Marker), APC from Lyngbya sp. (AP-L) and Nostoc sp. R76DM (AP-N). (B) UV-visible absorption spectra of purified APC from Nostoc sp. (a) and Lyngbya sp. (b) with absorption maximums at 613 nm and 652 nm, respectively.

3.2. Characterization of APC

The purified APC from Nostoc sp. R76DM was chemically as well as structurally and functionally characterized using a series of biochemical characterizations like SDS-PAGE, native-PAGE, UV-visible spectroscopy, fluorescence spectroscopy, circular dichroism and MALDI-TOF-MS.

The silver stained SDS-PAGE (Fig. 1A) of the purified N-APC along with the high range protein markers revealed a single band with molecular weight of about 16.4 to 17.5 kDa. The zinc acetate stained SDS-PAGE of the purified N-APC also showed a single distinct fluorescent band under UV light, and this band co-migrated with the ∼16.4 to 17.5 kDa silver stained band, indicating the presence of chromophore/s linked to the single peptide APC from Nostoc sp. (Fig. 1A). The MALDI-TOF-MS spectrum of the intact N-APC also showed a significant peak for a major component of m/z 16.4 kDa (Fig. 2). Moreover, the results obtained by electrophoresis (Fig. 1A) and mass spectrometry (Fig. 2) confirm the existence of a single peptide PBP without any contaminating proteins. Moreover, the appearance of a similar peak (with ∼17.50 RT) in the HPLC-chromatograms of the Nostoc APC under non-denaturing and denaturing conditions indicated the existence of APC in a non-oligomeric form (ESI Fig. S1†).

Fig. 2 Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) spectrum of the intact APC from Nostoc sp. R76DM.

The reported molecular mass (i.e., 16.4 kDa) is close to those of other single peptide PBPs isolated from different cyanobacteria. Recently, Sonani et al. (2015)²⁴ characterized a single peptide phycoerythrin (15.45 kDa) from Lyngbya sp. A09DM. Parmar et al. (2011)²³ characterized a 14 kDa functional α-subunit PE from marine cyanobacterium Phormidium sp. A27DM. Moreover, all of the PBPs characterized as single peptide α-and/or β-subunits from different cyanobacteria were the result of truncation by means of different abiotic factors.^22,23 However, herein we report the biosynthesis of a single peptide α-subunit APC from the cyanobacterium Nostoc sp. growing under natural conditions without any external abiotic influence.

3.2.1. Characterization of APC subunit composition. Pure fractions of the single peptide PBP (from Nostoc sp.), as determined by their electrophoretic and mass composition, were used to obtain a matrix-assisted laser desorption ionization (MALDI) peptide mass fingerprint (PMF) to identify the subunit composition of the single peptide APC. The mass spectrum showed several protonated ions [M + H]⁺ of peptide fragments as shown in Fig. 3. The amino acid sequences of five major peptides i.e., 1051 (Fig. 3A), 1431 (Fig. 3B), 936 (Fig. 3C), 2291 (Fig. 3D) and 2163 Da (Fig. 3E), deduced from the MS/MS analysis of the trypsin digested single peptide APC, was a match in the NCBInr protein sequencing database, using the Mascot peptide fingerprint search engine, with the full APC alpha chain of Microchaete diplosiphon with top protein scores >92 and p < 0.05. Moreover, the Mascot similarity search has revealed 100% sequence similarities of these peptides with the APC α-subunit (accession no. P16570) (Fig. 3F). Furthermore, the results obtained from MALDI-TOF and PMF analysis clearly identify the purified PBP as a single peptide APC α-subunit (ESI Fig. S2†).

Fig. 3 MS/MS spectra of five major peptides 1051 (A), 1431 (B), 936 (C), 2291 (D) and 2163 Da (E) of trypsin digested APC. (F) Deduced amino acid sequences of these peptides show 100% sequence similarity with that of the APC α-subunit (accession no. P16570, UniProtKB).

Recently, some studies have been conducted to synthesize the stable single peptide β-subunit in metabolically engineered Escherichia coli cells.^35,36 The biosynthesis of a recombinant APC β-subunit from a cyanobacterium was successfully reconstituted in E. coli which had spectroscopic properties that were similar to native APC.³⁶ Moreover, the present study would be helpful in understanding the uniqueness of the photosynthetic machinery of cyanobacteria and the promising use of a single peptide APC as a fluorescent tag as a substitute for native APC.

The APC alpha chain structure was predicted using a sequence from Microchaete diplosiphon. The three-state secondary structure of Microchaete APC was predicted and represented by helix, beta-sheet and loop sequences, with 74%, 3% and 21% of sequences, respectively (Fig. 4). The predicted 3D structure showed eight alpha helices (modelled residues-161) (Fig. 4). The p-value of the predicted structure was 1.13 × 10⁻⁵, which indicated a good quality structure. MS/MS derived five different peptides (i.e., 1051 Da, 1431 Da, 936 Da, 2291 Da and 2163 Da) having sequence similarities with that of the APC α-subunit (accession no. P16570, UniProtKB), shown in red (A), blue (B), brown (C), purple (D) and orange (E) colors, respectively (Fig. 4).

Fig. 4 The predicted secondary structure of the allophycocyanin alpha chain (with helices, beta strands and coils) of Microchaete diplosiphon and the amino acid sequence similarities of the five major peptides 1051 (A), 1431 (B), 936 (C), 2291 (D) and 2163 Da (E) of the trypsin digested single peptide APC α-subunit from Nostoc sp.

3.2.2. Functional characterization of APC. The functionality of the single peptide APC pigment was further analyzed with regard to its spectral characteristics using far-UV CD, absorption and emission. The data clearly showed the functionality of the APC α-subunit bearing bilin chromophores (as revealed by Zn-acetate staining) with an absorption maximum at 613 nm and fluorescence emission at 645 nm (Fig. 5A). However, the absorption as well as the fluorescence of a single peptide APC α-subunit were shifted from the normal absorption and fluorescence emission of a heterodimeric (αβ) standard APC (from Lyngbya sp.) to 652 nm and 663 nm, respectively (Fig. 5B). The far-UV CD spectrum of the native APC α-subunit exhibited double minima at 208 and 222 nm (Fig. 6), indicating its preserved substantial secondary structure. These values revealing the functionality of the purified APC α-subunit are consistent with those in previous reports.³⁷

Fig. 5 Absorption and fluorescence emission spectra of the APC α-subunit from Nostoc sp. R76DM (A) and dimeric (αβ) APC from Lyngbya sp. (B). The excitation wavelength for emission measurements was 589 nm.

Fig. 6 Far-UV CD spectrum of the APC α-subunit in 20 mM phosphate buffer (pH 7.0) at 25 °C.

3.3. Folding and stability dynamics of single peptide α-subunit APC

The in vitro denaturation and renaturation capability of the α-subunit of APC was studied using urea (CH₄N₂O) as a denaturing agent. Folding/unfolding of N-APC and S-APC was performed by recording the changes in their UV-visible absorption maxima at 613 (Fig. 7A) and 652 nm (Fig. 7B), respectively. Absorption spectra of both biliproteins were recorded in the range of 250–750 nm to check the difference in their structural stability as a function of variable urea concentrations. The absorbance of both APC α-subunits (Fig. 7A) as well as the standard heterodimeric APC (Fig. 7B) was found to decrease successively with the increase in urea concentration, which is in agreement with those determined previously for other PBPs.^24,38 It has been determined that the unique absorption characteristics of APC are due to the presence or interaction between a chromophore PCB and a respective apoprotein.^16,17 Moreover, the decrease in the specific absorbance of a biliprotein might be due to the loss/alteration of chromophore–apoprotein interactions or a specific level of protein folding.^39–41 A significant loss of APC absorbance was observed up to 4 M urea; however, no significant difference in the absorbance properties was observed above 5–6 M urea, probably due to the decrease in the rigidity of chromophore configurations in the native protein caused by a balanced hydrophilic/hydrophobic network distraction.⁴² Fig. 7C and D shows the denaturation curves of a single peptide APC α-subunit and standard heterodimeric APC plotted against the difference in molar absorption coefficients (ε) at 613 nm (Δε₆₁₃) and 652 nm (Δε₆₅₂), respectively, as a function of the presence or absence of urea. The trend of the denaturation–renaturation phenomena for the single peptide APC α-subunit was similar to that of the standard heterodimeric APC, suggesting the basic structural integrity of the novel APC α-subunit from Nostoc sp. The plots of Δε₆₁₃ and Δε₆₅₂ as a function of urea were analyzed to obtain ΔG^°_D, m, and C_m values for each APC protein in relation to eqn (1), and are depicted in Table 2. The data obtained from the denaturation and/or renaturation processes distinctly revealed that denaturation of APC is a two-state reversible process as described in our previous study using other PBPs.^24,38 It has been stated that two-state mechanisms of denaturation–renaturation for a particular protein depend on the coincidence of the normalized sigmoidal curves of several physical properties induced by certain denaturing agents such as urea.³⁸ However, non-coincidence of sigmoidal curves of different physical properties of a protein may also regulate two-state protein folding/unfolding characteristics.^43,44 Furthermore, the two-state folding/unfolding behavior of a protein as a function of urea can be monitored by the observed values of ΔG^°_D.⁴⁵ In the present study, the values of ΔG^°_D (protein stability) of a single peptide APC α-subunit (3.98 ± 0.18 kcal mol⁻¹) from Nostoc sp. and standard heterodimeric APC (4.12 ± 0.21 kcal mol⁻¹) from Lyngbya sp. were almost similar (Table 2), indicating the conserved geometry of the APC α-subunit.

Fig. 7 Urea-induced denaturation study of the single peptide APC α-subunit (A and C) and dimeric (αβ) APC (B and D). The changes in absorption spectra of the APC α-subunit (A) and dimeric (αβ) APC (B) at pH 7.0 and 25 °C, as urea increases from 0.0 to 6.0 M. Denaturation curves of the APC α-subunit (C) and dimeric (αβ) APC (D) were constructed by following changes in Δε₅₆₅ as a function of urea. The down arrow (↓) denotes absorption spectra of APCs with increasing [urea] from 0.0 to 6.0 M. Circles and triangles show the plot of Δε₅₆₅ vs. urea during denaturation and renaturation experiments, respectively.

Table 2 Denaturation kinetics of allophycocyanin from Nostoc and Lyngbya sp

Protein	ΔG^°D (kcal mol^−1)	M (kcal mol^−1 M^−1)	Cm (M)
Nostoc APC	3.98 ± 0.18	2.23 ± 0.12	1.78 ± 0.11
Lyngbya APC	4.12 ± 0.21	2.17 ± 0.09	1.89 ± 0.09

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3.3.1. Thermal and chemical stability of α-subunit APC. The stability of a protein against different physico-chemical factors is an utmost requirement for their use in food, pharmaceuticals, cosmeceuticals and other biomedical research. We have inspected the thermal and chemical stability of the APC α-subunit under different temperatures, pHs and with an oxidizing agent.

Fig. 8A shows the UV-visible absorption spectrum of the purified APC α-subunit before and after 1 h of heat exposure. The single peptide APC α-subunit showed considerably good stability towards temperatures up to 40 °C; however, the degradation rate increased rapidly over 60 °C (Fig. 8A). Moreover, PBPs isolated from different taxonomic groups differ in their stability under different physicochemical factors including thermostability.^46–51 Similar to the APC α-subunit, a loss of UV-visible spectral properties for the heterodimeric form of APC isolated from Lyngbya was also observed at 60–80 °C.⁵⁰ It has been found that the functional property of APC is also lost at high temperatures.⁵⁰ It has been suggested that the changes in the thermal stability of a PBP might be due to a number of sequence-dependent structural changes.⁵²

Fig. 8 UV-visible spectra of the single peptide PBP APC α-subunit after exposure to various physico-chemical stressors such as temperature (A), pH (B) and an oxidizing agent (C). The down arrow (↓) denotes the absorption spectra of heat (4 °C: control, 20 °C, 40 °C, 60 °C and 80 °C), pH (7: control, 6, 4, 8, 2, 10 and 12) and H₂O₂ (control, 0.2, 0.4, 0.6, 0.8 and 1.0%) treated samples from top to bottom, respectively.

The stability of the purified APC α-subunit was also investigated in a wide range of pHs. Fig. 8B shows the effects of different pHs on its UV-visible spectral properties. The APC α-subunit showed functional stability in the pH range 4.0–7.0; however, a relatively low stability was observed at pH 8.0 (Fig. 8B). The residual fraction of APC was severely decreased under a range of acidic (pH 2.0) and alkaline (pH 10.0–12.0) conditions (Fig. 8B). The PBP (B-PE) obtained from the red algae Porphyridium cruentum showed stronger functional stability in the pH range 4.0–10.0.⁴⁹ Recently, Rastogi et al. (2015) have observed the structural and functional stability of PBPs under different physico-chemical stressors.⁵⁰ The percentage decrease in dimeric APC concentration after 1 h storage at pH 2.0, 4.0, 10.0 and 12.0 was 56.8 ± 3.1, 48.58 ± 2.8, 72.47 ± 1.6 and 87.98 ± 1.9, respectively. Moreover, both single peptide and/or dimeric APC may differ in their sensitivity towards different pH conditions.

The incubation of the purified APC α-subunit with the oxidizing agent H₂O₂ led to a successive decrease in their absorption (Fig. 8C), accompanied with the disappearance of their respective color (data not shown), with an increase in H₂O₂ concentration. Moreover, contrary to high temperature and pH change, the spectral properties of APC were relatively maintained under oxidative stress, indicating the potential of PBP as free radical scavengers.

3.3.2. In vivo and In vitro stability of α-subunit APC. Both the in vivo and in vitro stability of the purified single peptide APC α-subunit were performed. No additional reduction in peptide size or intensity was observed upon storage for 180 days (Fig. 9A). To clarify the possibility of in vivo truncation in the culture growing for a long term, we isolated and purified the biliproteins from the Nostoc cultures growing at different time intervals. Fig. 9B shows the existence of similar biliproteins isolated from cyanobacterium cultures grown for 10 to 60 days, indicating the in vivo stability of the single peptide APC α-subunit. The results obtained from SDS-PAGE analysis for the purified PBP (Fig. 9) directly support the synthesis of a single peptide APC α-subunit and discard or nullify the possibilities of an in vivo or in vitro PBP truncation process in the cyanobacterium Nostoc sp. R76DM during long-term culture storage or in vitro storage of purified PBP at 4 °C. Moreover, the occurrence of single peptide PBPs has also been reported as a result of in vivo or in vitro truncation of PBPs in the cyanobacterium Phormidium tenue²² and Lyngbya sp. A09DM,²⁴ respectively.

Fig. 9 SDS-PAGE of the purified APC α-subunit every 20 days up to 180 days (A) of storage at 4 °C. SDS-PAGE of the APC α-subunit isolated and purified from cyanobacterium cultures grown for 10 to 60 days. Z: zinc acetate and S: silver stained.

4. Conclusions

Very few works have reported the occurrence of APC, particularly on the occurrence of a single peptide APC subunit. We have purified and characterized the occurrence of a single peptide APC α-subunit (16.4 kDa) in the cyanobacterium Nostoc sp. R76DM. Urea-induced denaturation and Gibbs-free energy (ΔG^°_D) calculations suggested the folding and structural stability of the APC α-subunit is almost similar to that of the standard APC (αβ) heterodimer from Lyngbya sp. The PBP APC α-subunit showed both in vivo and in vitro stability against long-term culture storage or storage of purified PBP at 4 °C, respectively. The occurrence of APC composed of a single subunit may reveal some changing aspects of photosynthesis in photoautotrophs. Moreover, due to conserved structural and functional integrity, the APC α-subunit may be used as a low molecular weight fluorescent tag as a constituent of a relatively higher molecular weight native APC in fluorescence detection techniques for different diagnostic purposes.

Acknowledgements

Rajesh P. Rastogi is thankful to the University Grant Commission (UGC), New Delhi, India for the Dr D. S. Kothari Postdoctoral Research Grant. Ravi R. Sonani thanks the Department of Science and Technology (DST), New Delhi, India for financial help in the form of the INSPIRE (IF120712) fellowship.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra14508b

‡ Contributed equally.

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