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International Journal of Biological Macromolecules 74 (2015) 29–35

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

stable and functional single peptide phycoerythrin (15.45 kDa) fromyngbya sp. A09DM

avi Raghav Sonani, Rajesh Prasad Rastogi, Meghna Joshi, Datta Madamwar ∗

RD School of Biosciences, Sardar Patel University, Vadtal Road, Satellite Campus, Post Box No. 39, Vallabh Vidyanagar 388120, Gujarat, India

r t i c l e i n f o

rticle history:eceived 22 September 2014eceived in revised form5 November 2014ccepted 27 November 2014vailable online 5 December 2014

eywords:hycoerythrin

a b s t r a c t

A functional and stable truncated-phycoerythrin (T-PE) was found as a result of spontaneous in vitro trun-cation. Truncation was noticed to occur during storage of purified native-phycoerythrin (N-PE) isolatedfrom Lyngbya sp. A09DM. SDS and native-PAGE analysis revealed the truncation of N-PE, containing �(19.0 kDa) – and � (21.5 kDa) – subunits to the only single peptide of ∼15.45 kDa (T-PE). The peptide massfingerprinting (PMF) and MS/MS analysis indicated that T-PE is the part of �-subunit of N-PE. UV–visibleabsorption peak of N-PE was found to split into two peaks (540 and 565 nm) after truncation, suggestingthe alterations in its folded state. The emission spectra of both N-PE and T-PE show the emission bandcentered at 581 nm (upon excitation at 559 nm) suggested the maintenance of fluorescence even after

◦

n vitro truncationtructural stability

significant truncation. Urea-induced denaturation and Gibbs-free energy (�GD ) calculations suggestedthat the folding and structural stability of T-PE was almost similar to that of N-PE. Presented bunch ofevidences revealed the truncation in N-PE without perturbing its folding, structural stability and function-ality (fluorescence), and thereby suggested its applicability in fluorescence based biomedical techniqueswhere smaller fluorescence molecules are more preferable.

. Introduction

Cyanobacteria are the most primitive among oxygenic pho-oautotrophs and well known for their characteristic antennaystem called phycobilisome (PBS) [1,2]. PBS is a giant lightarvesting complex made up of phycobiliproteins (PBPs) andssociated linker peptides. Morphologically, PBS contains twoajor sub-structures: core consists of 2/3/5 cylinders situated

orizontally on the outer surface of thylakoid membrane and/8 rods radiating out from the core [3–5]. Rods always containhycocyanin (PC, �max ∼610–620 nm), and often also phycoery-hrin (PE, �max ∼540–570 nm); whereas core invariantly containsllophycocyanin (APC, �max ∼653 nm) [1]. The chromophores likehycoerythrobilin, phycocyanobilin, phycourobilin and phycovi-lobilin attach covalently to the PBP-apoproteins and therebyirect the spectral properties of particular PBP [1,6]. Unique

bsorption and emission properties of PBP enable PBS to capturend funnel the sunlight energy in a unidirectional manner fromE → PC → APC → chlorophyll a for photosynthesis [7]. Moreover,

∗ Corresponding author. Tel.: +91 02692 229380; fax: +91 02692 231042/236475.E-mail addresses: ravi123sonani@gmail.com (R.R. Sonani),

aj rastogi@rediffmail.com (R.P. Rastogi), datta madamwar@yahoo.comD. Madamwar).

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.11.030141-8130/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

protein microenvironment, optimum geometry (distance and ori-entation) and the appropriate docking of adjacent sub-complexeswithin PBS govern the expression of overall spectral propertiesand energy transfer efficiency of PBS supra-molecule [1].

PE, the blue/green light absorbing PBP, is basically made up oftwo types of polypeptides called �- (∼164 amino acids) and �-subunit (∼171 amino acids) with 65–70% sequence identity amongdifferent species [8,9]. The � and �-subunits, contain covalentlyattached chromophore/s, associate to construct �–� dimer, thebuilding block of PBP assembly [9]. In vitro proteolytic cleavagestudies [10] and structural analysis [8] has shown the impor-tant role of N-terminal domain in the formation of stable �–�dimer as well as in prevention of �–�/�–� homodimerization. The�–� dimers form ring shaped trimmer with threefold symmetry.Trimmers congregate further in a face-to-face manner to form hex-americ disk, serving as a structural unit in the peripheral part of PBSrod [3,11].

It is believed that PE cannot be functional without charac-teristic folding of chromophore/s appended �- and �-subunits.Many reports [12–16] have described the purification and char-acterization of PE made up of two native folded subunits from

both cyanobacteria and red algae. Moreover, available structuralinformation also suggested the important role of molecular inter-actions between �- and �-subunits of PE to associate productivelywithin PBS [17–19]. Only few reports are available, which have

3 of Biol

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escribed the alternative forms of PE. Huber [20] described the PE,ade up of three different peptides such as �-, �- and �-subunits.n the contrary, Thomas and Passaquet [21] reported PE, com-osed of only �-subunit from red algae. Steglich et al. [22] andiethaus et al. [23] have also described degenerated form of PE

made up of only �-subunit) as remnants of PBS upon high-lightxposure. Furthermore, Soni et al. [8] and Parmar et al. [24] haveeported fragmented-PE, composed of only truncated �-subunit. Inhe present study, we report one more variant of PE, obtained viaruncation in native-PE (N-PE) (with �- and �-subunits) of Lyngbyap. A09DM during in vitro storage condition. We have purified andharacterize the folding, stability and functionality (fluorescence)f truncated-PE (T-PE) obtained as a part of N-PE.

. Materials and methods

.1. Culture, growth conditions and protein purification

Lyngbya sp. A09DM, a filamentous cyanobacterium was isolatedrom the rocky shores of Okha, Gujarat, India. It was grown in ASN III

edium [25,26] and subjected for PE extraction and purification asescribed earlier [26,27]. Purified PE was concentrated and storedt 4 ◦C under dark condition, and labeled as native-PE (N-PE).

After fifty days of storage, N-PE truncated spontaneously to giveingle and smaller peptide. This single peptide was purified by pass-ng the fifty days stored N-PE through gel permeation (Sephadex-100) column. Fractions with high purity were collected, concen-

rated and labeled as truncated-PE (T-PE).

.2. Characterization of T-PE and N-PE

.2.1. Gel electrophoresis analysisN-PE and T-PE were resolved on native- and SDS-PAGE as

escribed earlier [26,28]. Proteins on resolved gels were visualizedy silver and zinc-acetate staining as described earlier [29–31].

.2.2. Protease assayThe presence of protease in PE – solution was traced by pro-

ease assay as described in Sarath et al. [32]. Casein was used as aubstrate. Trypsin and Prteinase K were used as positive control.

.2.3. Spectroscopic analysisThe absorbance spectra of purified T-PE and N-PE were recorded

ver 250–750 nm wavelength range using cuvette of 1 cm pathength on UV–visible spectrophotometer (Specord 210, Analytikena AG, Jena, Germany). The purity of protein was probed as ‘purityatio’, calculated by the formula A565/A280 (Ax stands for absorbancet x nm wavelength) [26]. The fluorescence emissions of T-PE and-PE (∼0.5 mg/ml) were recorded to verify their functionality uponxcitation at 559 nm by fluorescence spectrophotometer (F-7000,itachi High Technologies Corp.).

.2.4. Matrix-assisted laser desorption ionization time-of-flightass spectrometry analysis (MALDI-TOF-MS)

MALDI-TOF of T-PE was performed as described by Gupta et al.33] with slight modification. In brief, the purified T-PE was dilutedo 10–15 pmol �l−1 in potassium phosphate buffer (20 mM, pH 7.0)

nd 2 �l of diluted T-PE was mixed with sinapinic acid matrix (pre-ared in trifluoroacetic acid). This mixture was spotted on a targetALDI plate, allowed to dry at room temperature and analyzed by

he MASSLYNX program.

ogical Macromolecules 74 (2015) 29–35

2.3. Peptide mass fingerprinting (PMF)

2.3.1. Gel elution and trypsin digestion of T-PE and N-PE subunitsPurified T-PE and N-PE (50 �g of each) were loaded and resolved

on 15% PAGE under denaturing condition. Proteins bands on theresolving gel were made visual by coomassie brilliant blue G250staining. Stained bands were cut and digested individually byTrypsin-Gold (Promega Corp., Madison, WI, USA) according tomanufacturer’s protocol. Digested samples were dried and re-solubilized in 0.1% trifluoroacetic acid (TFA). Digested solution waspurified by passing through Millipore® ZipTips (Sigma–Aldrich,USA) with TA buffer (made up of 1:1 ratio of 0.1% TFA and ace-tonitrile) [34].

2.3.2. Mass spectrometryA matrix solution was prepared by mixing �-cyano-4-

hydroxycinnamic acid (5 mg ml−1) in TA buffer containing purifiedpeptide mixture. Matrix solution (2 �l) was applied to the MALDItarget plate and allowed to dry before MS analysis. PMF spectraof �-N-PE, �-N-PE and T-PE were obtained in the positive reflec-tion mode by the MALDI-TOF/TOF mass spectrometer (ULTRAFLEXIII, Bruker Daltonics, USA). Spectra were recorded over the rangeof 0–4000 Da with an accumulation of 1000 shots for each sample[35]. MS/MS spectra of selected peptides were generated at 1 keVacceleration voltage by gas/air-collision induced dissociation under10−6 Torr pressure [34].

2.3.3. Bioinformatics analysisPMF spectrums were analyzed by Flex analysis software to

extract peak list. Mascot (Matrix Science, UK) was used to findmatches against the UniProtKB database. Mascot analysis was donewith trypsin as the enzyme, carbamidomethylation (C) as a fixedmodification, oxidation (M) as variable modifications, maximummissed cleavages as 1, peptide mass tolerance (MS) of ±100 ppmand fragment mass tolerance (MS/MS) of ±1 Da.

2.4. Chemical denaturation and renaturation study

2.4.1. Sample preparationUrea was used to study chemical-induced denaturation of T-

PE and N-PE. A solution of urea was prepared freshly in 20 mmpotassium phosphate buffer (pH 7.2) as described in Pace [36].The proteins were extensively dialyzed against the potassiumphosphate buffer (20 mM, pH 7.2) to remove residual salts. Dia-lyzed proteins were quantified spectrophotometrically by using themolar absorption coefficient value (265,000 M−1 cm−1) for 565 nm[37]. Increasing amount of urea (0.2–9.0 M) was mixed with N-PE/T-PE and allowed to incubate for 45 min at 25 ◦C as describedearlier [38]. Renaturation was also performed by following thesame procedure, but the concentration of urea was decreased grad-ually by diluting the 9.0 M urea in the reaction mixture.

2.4.2. Measurement of UV–visible absorbance spectrumAbsorption spectra of denatured/renatured T-PE and N-PE were

measured as described in Section 2.2.3. The protein concentrationwas used in the range of 0.2–0.4 mg ml−1. All spectral measure-ments were done in triplicates.

2.4.3. Data analysisThe sigmoidal chemical-induced denaturation curve (change in

molar extinction coefficient, �ε vs. [urea], M) was plotted and ana-lyzed for the deduction of �GD

◦ (Gibbs free energy change for

denaturation of protein), m (slope of the plot of �GD

◦, the Gibbsfree energy change vs. [urea], i.e., ∂�GD/∂[urea]) and Cm (midpointof denaturation curve, i.e., [urea] at which �GD

◦ = 0) [9]. The least-squares method was used to fit the data of the denaturation curve

f Biological Macromolecules 74 (2015) 29–35 31

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R.R. Sonani et al. / International Journal o

y assuming the linear relationship between �GD and [urea], whichas expressed by

= yN + yD × exp[−(�GD◦ − m[Urea])/RT]

1 + (exp[−(�GD◦ − m[Urea])/RT]

(1)

here yN and yD are absorbance properties of the native (N) andenatured (D) protein molecules under identical experimental con-ition except [urea], R is the gas constant and T is the temperature

n Kelvin (K).

. Results and discussion

.1. Truncation of native PE (N-PE)

We found spontaneous truncation in purified N-PE during longerm in vitro storage at 4 ◦C. While monitoring the pattern ofruncation, we noticed that N-PE containing � (19.0 kDa) and �

21.5 kDa) subunit truncates to give a single peptide of ∼15.5 kDaT-PE) (Fig. 1A) after around 50 days of storage. However, no reduc-ion in the size and intensity of T-PE band (∼15.5 kDa) (Fig. 1B) upon

Marker N-PE T-PEkDa

43.0

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ig. 1. (A) Silver stained (S) and zinc acetate (Z) stained 15% SDS-PAGE of proteinolecular mass standard (Marker), N-PE and T-PE. (B) Silver stained (S) and zinc

cetate (Z) stained 15% SDS-PAGE of T-PE at every 25 days of interval. (C) Proteaseontent in terms of unit activity and color development (inset) in control (A), trypsinB), proteinase K (C), N-PE (D) and T-PE (E). N-PE and T-PE showing non-significantrotease activity as well as color development indicated the complete absence ofrotease in PE – preparations.

0

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3000

4000

5000

6000

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Fig. 2. UV–visible and fluorescence emission spectrum of N-PE (A) and T-PE (B)(excitation was provided at 559 nm in emission spectrum measurement).

further storage (of 200 days) revealed its stability over truncationphenomenon.

3.2. Purification and characterization of N-PE and T-PE

Freezing and thawing at −25 ◦C and 4 ◦C, respectively, wasfound optimum to cause cell wall breakage and thus leaching outintracellular content. Brick red color supernatant after removal ofcell debris was named as ‘crude extract’. Crude extract was fur-ther subjected to 20–70% ammonium sulfate fractionation, whichresulted in the retention of all PE in the pellet upon centrifuga-tion. The pellet containing PE was passed through gel permeationresin sephadex G-150 using potassium phosphate buffer (20 mM,pH 7.0) as the mobile phase. Elutes from gel permeation matrixwas further purified by using diethyl amino ethyl-cellulose (DEAE-cellulose) matrix. PE was eluted by mobile phase containing 0.15 MNaCl in potassium phosphate buffer (20 mM, pH 7.0). Purified N-PEand T-PE did not show significant protease activity as well as colordevelopment during protease assay (Fig. 1C). These results signif-icantly ruled out the possibility of presence of any protease in PE– preparations. Furthermore, the purity assessment by SDS-PAGE(Fig. 1A) and UV–visible spectroscopy (Fig. 2 and Table 1) affirmedthat N-PE and T-PE were very pure. The controlled truncation ofN-PE in its pure form (without any external factor) suggested thatthe N-PE is might be truncated via some self-catalyzed and auto-governed mechanisms.

Zinc acetate stained SDS-PAGE showing the identical band pat-tern to that of silver stained gel revealed the presence of the

chromophore/s with each subunit of N-PE and T-PE (Fig. 1A). How-ever, the intensity of N-PE and T-PE band differed in zinc acetatestained native-PAGE under UV light illumination (Fig. 3). T-PEshowed higher fluorescence intensity than that of the N-PE for

32 R.R. Sonani et al. / International Journal of Biological Macromolecules 74 (2015) 29–35

Table 1Purification parameters in terms of purity ratios, protein contents and % impurities of purified N-PE and T-PE.

Absorbance at 280 nmA280

Absorbance at 565 nmA565

Purity ratioA565/A280

Total proteincontent (mg)

N-PE/T-PE content (mg) Impurities (%)

Purified N-PE 0.0509 0.3205 6.29 20.15 19.90 1.49Purified T-PE 0.0163 0.0603 3.70 12.45 12.31 1.12

N-PE T-PE

S

Z

FT

iPaaceci

ig. 3. Silver stained (S) and zinc acetate stained (Z) 12% native-PAGE of N-PE and-PE.

dentical protein load (Fig. 3). Variation in band intensity in native-AGE is justified as the manifestation of altered chromophore/sccessibility and reactivity (related with its conformation) to zinccetate in the altered folding of T-PE than that of N-PE. Trun-ation might alter the native folding of T-PE in a manner that either

xposes chromophore/s on the outer solvent accessible surface orhanges the chromophore/s conformation toward more reactivesoform.

Fig. 5. Peptide mass fingerprinting (PMF) spectra of T

Fig. 4. Matrix-assisted laser-desorption ionization time-of-flight mass spectrome-try (MALDI-TOF-MS) spectrum of intact T-PE.

The absorbance peak of N-PE was found to be split into two peaks

(540 nm and 565 nm) upon truncation, which is similar to that ofrecombinant �-N-PE [39]. Appearance of slight fall at ∼550 nmin absorption of T-PE reflected the occurrence of conformational

-PE, �-subunit of N-PE and �-subunit of N-PE.

R.R. Sonani et al. / International Journal of Biological Macromolecules 74 (2015) 29–35 33

F igestes

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ig. 6. MS/MS spectrum of two major peptides 2431 (A) and 1777 Da (B) of trypsin dimilarities with that of PE-�-subunit (accession no. P05098, UniProtKB).

hanges in its native folding to fulfill the absenteeism of lost aminocid residues after truncation (Fig. 2). However, T-PE showed theuccessive emission band centered at 581 nm when excited at59 nm (Fig. 2B) similar to that of N-PE (Fig. 2A) and only �-N-PE40]. Pattern of alteration occurring in absorption and fluorescence

mission properties of PE upon truncation revealed the mainte-ance of its functionality even in perturbed folding.

MALDI-TOF spectrum of T-PE shows a significant peak cor-esponds to 15.45 kDa (Fig. 4) suggested the presence of single

d T-PE. (C) Deduced amino acid sequences of these peptides shows 100% sequence

substantial forms of T-PE in solution. Another small peak corre-sponds to 14.87 kDa in MALDI-TOF is might be of chromophore/sdetached PE-apoproteins, generated due to laser desorption.

3.3. T-PE is a part of ˛-subunit of N-PE

PMF of N-PE subunits and T-PE was performed to identify theorigin of truncated subunit. PMF spectrum of truncated subunitshowed high similarities with that of �-subunit as compared to

34 R.R. Sonani et al. / International Journal of Biological Macromolecules 74 (2015) 29–35

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ig. 7. Urea-induced denaturation study of native and truncated PE. Changes in abso 9.0 M. Denaturation curves of N-PE (C) and T-PE (D) constructed by following churea] during denaturation and renaturation experiments, respectively.

-subunit of N-PE (Fig. 5). Amino acid sequences of two major pep-ides (1777 and 2431 Da) of trypsin digested T-PE were deducedrom their MS/MS spectrums as shown in Fig. 6A and B. Moreover,

ascot similarity search has revealed 100% sequence similaritiesf these peptides with PE-�-subunit (accession no. P05098) inhe UniProtKB database (Fig. 6C). Furthermore, we found signif-cant loss of some peaks (1402, 1913, 2252, 3121, 3381 m/z) inhe PMF spectra of T-PE while comparing it with that of �-N-PE.he peaks at ∼2252 and ∼3381 m/z correspond to 18thArg–37thArgnd 1stMet–33thArg oligopeptides, respectively, possibly producedrom N-terminus of �-N-PE as a result of trypsine digestion. Theesults of PMF and MALDI-TOF analysis collectively suggested thathe T-PE is produced from �-subunit of N-PE via truncation of5–30 amino acids from N-terminus. Since �-subunit of PE has beeneported to contain two chromophores attached with two con-erved cysteine situated on 84/82 and 140/139 position [18,19,41],-PE is assumed to still contain both chromophores according tohe site of cleavage. However, this assumption has been alreadyonfirmed by chromophore specific Zn-acetate staining as wells an absorption and fluorescence emission spectroscopy of T-PEFigs. 1 and 2B).

This result reflected the pattern of N-PE truncation toward T-E. Absence of any matches between PMF spectrum of �-subunitof N-PE) and T-PE (Fig. 5) indicated complete degradation of N-PE

-subunit. Whereas, significant matches between PMF spectrumf �-subunit (of N-PE) and T-PE (Fig. 5) indicated the presence ofny sequence/structural motifs in �-subunits as a stopping point ofegradation. Altogether, this result inferred that T-PE is made up of

n spectra of N-PE (A) and T-PE (B) at pH 7.0 and 25 C, as [urea] increases from 0.0 in �ε565 as a function of [urea]. Circles and triangles showed the plot of �ε565 vs.

a single peptide belonging to �-subunit family and hypothesized toobtain from N-PE by complete degradation of �-subunit and limitedtruncation of �-subunit, spontaneously.

3.4. Role of truncated part in folding and stability

The aim of this experiment was to understand the role oftruncated residue in the folding and structural stability of N-PE.For this purpose, we have performed in vitro denaturation andrenaturation study using urea as denaturing agent. Due to owingsimilar absorption maxima, folding/unfolding of T-PE and N-PE canbe followed by recording the changes in their absorption maximaat 565 nm. We thus measured the absorption spectrum of bothproteins in the range of 250–800 nm as a function of [urea] to checkthe difference in their structural stability. Fig. 7A and B showsthe absorbance spectra of N-PE and T-PE as a function of [urea].Successive reduction in the absorption property (ε565) of both pro-teins was observed upon an increase in [urea]. The characteristicabsorption at 565 nm of PE is because of the phycoerythrobilin(PEB)–apoprotein interaction, spatial arrangement and the sur-rounding microenvironment of PEB [42]. Reduction in ε565 ismainly due to the loss of PEB–apoprotein interaction and tertiaryand quaternary level protein-folding [42]. A significant decrease inε565 of both N-PE and T-PE was observed in the presence of 4–6 M

[urea]. However, above 6 M [urea] no significant decrease wasobserved. This may be due to the decrease in the stringency causedby disruption in balanced hydrophilic–hydrophobic environment[43]. Fig. 7C and D shows the denaturation curves, plots of �ε565

R.R. Sonani et al. / International Journal of Biol

Table 2Stability parameters of the F-PE and T-PE associated with the urea-induced dena-turation at pH 7.0 and 25 ◦C.a

Protein �GD◦ (kcal mol−1 M−1) m (kcal mol−1) Cm (M)

F-PE 9.76 ± 0.31 2.02 ± 0.31 4.83 ± 0.12

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a A ± mean of errors from the average of three independent measurements.

difference in molar absorption coefficients at 565 nm) of N-PEnd T-PE as a function of [urea]. These curves were analyzed forhe �GD

◦, m and Cm values of both proteins. Transition curvesnalysis according to Eq. (1) was based on a two basic assumptions.irst is that the urea-induced denaturation of N-PE and T-PE iseversible. Reversibility of denaturation was checked by dilutinghe denatured proteins with the same buffer and absorption spec-ra were recorded after sufficient incubation to allow completeenaturation. The spectrum of denaturation and renaturation wasound to be indistinguishable, which intended us to conclude thathe urea-induced denaturation of N-PE and T-PE is reversible. Asecond assumption, urea-induced transition (native to denaturedtate) is a two-state process for PE, that have been described in ourrevious study showing the overlapping values of �GD

◦, obtainedrom different probes used to monitor the denaturation [10,44].

The resultant values of �GD◦, m and Cm are summarized in

able 2. The structural stability and folding of T-PE and N-PEhowed no significant difference as the value of their respectiveGD

◦, 8.42 ± 0.24 kcal mol−1 M−1 and 9.76 ± 0.31 kcal mol−1 M−1,ere not very different. The results of this experiment suggested

hat the truncation phenomenon may affect the overall geometryf N-PE, but does not significantly perturb folding and structuraltability because �GD

◦ for both N-PE and T-PE has very small differ-nce of ∼1 kcal mol−1 M−1. This negligible difference in �GD

◦ ruledut the role of the truncated portion in stability and folding, andhus rationally justified the retention of structural and functionalntegrity of T-PE even after the truncation.

The presented bunch of evidences reflected the spontaneousize reduction in natural PE to produce truncated but still functionalE. Moreover, on the basis of results obtained, we also hypothesizedhat this phenomenon might be self-catalyzed and governed by thenformation present inside inherent primary and/or tertiary struc-ure of PE. However, the exact molecular phenomenon of truncationn PE is not reflected by the present investigation. Nevertheless, theroperties of T-PE including smaller size, structural stability, char-cteristics fluorescence and absorption as reported in this studyuggested the prospective use of T-PE as a potential fluorescenceag; however, more extensive study is needed to explore its fluo-escence half-life time and other spectral properties.

. Conclusion

The macromolecule, T-PE was reported as stable and func-ional end product of N-PE via spontaneous in vitro truncation.he results obtained are of great importance elucidating the com-lete degradation of �-subunit and partial truncation of �-subunitf N-PE to generate T-PE. Chemical denaturation kinetics show-ng the difference of only ∼1 kcal mol−1 M−1 in �GD

◦ of T-PE tohat of N-PE, suggested very negligible difference between theirolding and stability. Moreover, the function of T-PE, probed byuorescence emission and UV–visible spectroscopy suggested thereservation of functionality (fluorescence) even after significant

runcation. Altogether, the results showed that truncation did noterturb the folding and thus the stability and functionality of the T-E, suggested its prospective use as a tag in spectroscopic detectionechniques where small molecule are more desirable.

[

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ogical Macromolecules 74 (2015) 29–35 35

Acknowledgements

Ravi R. Sonani gratefully acknowledges the Department of Sci-ence and Technology (DST), New Delhi, India for financial help inthe form of the INSPIRE (IF120712) fellowship. Rajesh P. Rastogiis thankful to the University Grant Commission (UGC), New Delhi,India for Dr. D.S. Kothari Postdoctoral Research Grant.

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