Accepted Manuscript

Folding and stability studies on C-PE and its natural N-terminal truncant

Khalid Anwer, Asha Parmar, Safikur Rahman, Avani Kaushal, DattaMadamwar, Asimul Islam, Md. Imtaiyaz Hassan, Faizan Ahmad

PII: S0003-9861(14)00008-3DOI: http://dx.doi.org/10.1016/j.abb.2014.01.005Reference: YABBI 6602

To appear in: Archives of Biochemistry and Biophysics

Received Date: 18 November 2013Revised Date: 3 January 2014

Please cite this article as: K. Anwer, A. Parmar, S. Rahman, A. Kaushal, D. Madamwar, A. Islam, Md. ImtaiyazHassan, F. Ahmad, Folding and stability studies on C-PE and its natural N-terminal truncant, Archives of

Biochemistry and Biophysics (2014), doi: http://dx.doi.org/10.1016/j.abb.2014.01.005

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Running head: Folding and stability measure of C-Phycoerythrin

Folding and stability studies on C-PE and its natural N-

terminal truncant

Khalid Anwer1, Asha Parmar2, Safikur Rahman1, Avani Kaushal2, Datta Madamwar2, Asimul

Islam1, Md. Imtaiyaz Hassan1* and Faizan Ahmad1

1Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (Central

University), Jamia Nagar, New Delhi 110 025, India

2BRD School of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat 388 120,

India

*To whom all correspondence should be addressed,

Dr. Md. Imtaiyaz Hassan

(Assistant Professor)

Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India E-mail: mihassan@jmi.ac.in

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ABSTRACT

The conformational and functional state of biliproteins can be determined by optical properties

of the covalently linked chromophores. �-Subunit of most of the phycoerythrin contains 164

residues. Recently determined crystal structure of the naturally truncated form of �-subunit of

cyanobacterial phycoerythrin (Tr-�C-PE) lacks 31 N-terminal residues present in its full length

form (FL-�C-PE). This provides an opportunity to investigate the structure-function relationship

between these two natural forms. We measured guanidinium chloride (GdmCl)-induced

denaturation curves of FL-�C-PE and Tr-�C-PE proteins, followed by observing changes in

absorbance at 565 nm, fluorescence at 350 and 573 nm, and circular dichroism at 222 nm. The

denaturation curve of each protein was analyzed for �GD0, the value of Gibbs free energy change

on denaturation (�GD) in the absence of GdmCl. The main conclusions of the this study are: (i)

GdmCl-induced denaturation (native state � denatured state) of FL-�C-PE and Tr-�C-PE is

reversible and follows a two-state mechanism, (ii) FL-�C-PE is 1.4 kcal mol-1 more stable than

Tr-�C-PE, (iii) truncation of 31-residue long fragment that contains two �-helices, does not alter

the 3-D structure of the remaining protein polypeptide chain, protein-chromophore interaction,

and (iv) amino acid sequence of Tr-�C-PE determines the functional structure of the

phycoerythrin.

Keywords: Biliproteins; C-phycoerythrin; Chromophore; Protein denaturation; Folding and

stability

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Introduction

Cyanobacteria are the most primordial among oxygenic photosynthetic organisms, and

their antenna system consists of characteristic phycobiliproteins (PBPs) which are categorized as

phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC) that form a supramolecular

assembly called phycobilisomes (PBS) [1-3]. PBS is a light harvesting antennae of photosystem

II in cyanobacteria, red algae and glaucophytes. These protein complexes are anchored to

thylakoid membranes which are made of stacks of PBPs and their associated linker polypeptides

[4]. Each PBS consists of a core made of APC from which several outwardly oriented rods made

of stacked disks of PC and (if present) PE or phycoerythrocyanin [3, 5]. The energy transfer in

the organism proceeds successively in the direction PE →PC →APC → chlorophyll a, with an

overall efficiency of almost 100% [1, 2, 6]. The lights absorbed by PBP are in the regions of

visible spectrum where chlorophyll a has lower efficiency. The spectral properties of PBPs are

mainly governed by their prosthetic groups which are linear tetrapyrroles known as phycobilin

including phycocyanobilin, phycoerythrobilin, phycourobilin and phycoviolobilin. Interestingly,

spectral properties of phycobilins are highly influenced by the protein environment [1, 2, 7].

PE is one of the unique and special accessory light harvesting pigment and exists in the

form of hexamer (�6�6), dimers (�2�2) or dimers of trimers (�3�3)2 in light harvesting system of

blue-green algae, cryptophytes and red algae [1]. PE of blue-green algae (C-PE) contains 6� and

6� subunits arranged in ring-like assemblies where the 3 monomers are present in the threefold

symmetry [8, 9]. Each �-subunit of PE consists of 164 amino acid residues while each �-subunit

is 171-residue long [1, 9]. The structure of C-PE resembles that of the globin protein which

contains two open-chain linear tetrapyrrole chromophores known as phycoerythrobilins (PEBs)

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covalently attached to each �-subunit at Cys82 and Cys139. While �-subunit of C-PE contains

three chromophores where two are covalently attached to the Cys84 and Cys155, and third

chromophore is doubly linked to Cys50 and Cys61 [1, 8, 9]. The interactions between PEB with

protein atoms and protein-chromophore environments are critically responsible for the photon

absorption properties of C-PE.

Recently, we identified and determined the crystal structure of �-subunit of C-PE from P.

tenue, which is devoid of 31 residues from its amino terminal side [9] as compared to the full

length �C-PE containing complete 164 residues [8]. It is interesting to note that truncation of 31

N-terminal residues occurs naturally when the organism is grown or kept in prolonged starved

condition. However, the truncated �C-PE maintains its light absorbing capabilities [8, 9]. The

full length �C-PE (FL-�C-PE) and truncated �C-PE (Tr-�C-PE) can be used as a model system

to study protein folding problems in order to establish the role of 31 N-terminal residues. We,

therefore, studied folding/unfolding of the full length and truncated �C-PEs using absorption,

fluorescence and circular dichroism (CD) techniques. Apart from determination of role of 31 N-

terminal residues in protein folding and stability, our findings may be helpful to understand the

mechanism of survival of this cyanobacterium and other marine algae under stress conditions. As

under these conditions including starvation the organism maintains its pigment proteins and

absorption properties for food preparation and survival. Furthermore, our study may also provide

an insight to grow higher photosynthetic plants under this and similar types of stress

environmental conditions.

This report on structure, folding and stability of FL-�C-PE and naturally truncated Tr-�C-

PE will be helpful for better understanding of mechanism of energy transfer in photosynthetic

5

cyanobacteria, even under stress conditions like starvation and salinity. Furthermore, we

established the role of 31 N-terminal residues in the folding and stability of C-PE.

Materials and methods

Materials

Guanidinium chloride (GdmCl) of ultra pure grade was purchased from MP Biomedicals,

LLC (Illkirch, France). Sodium cacodylate trihydrate and Sephadex G-150 matrix were obtained

from Sigma-Aldrich (St Louis, USA) and GE Healthcare UK Limited, respectively. Other

chemicals were purchased from local suppliers. All chemicals used were of molecular biology

research grade.

Isolation and purification

Protein was isolated and purified from P. tenue sp. A27DM collected from rocky shores of

Bet Dwarka, Okha and estuarine mouth of river Daman Ganga (India). The enrichment,

provision of growth conditions, extraction and purification of C-PE were carried out using our

well optimized protocol described elsewhere [10-12]. Briefly, the cyanobacterium was harvested

in artificial sea water nutrients, and the cell mass was washed with 1.0 M Tris-HCl buffer (pH

8.1) containing 3.0 mM sodium azide. For the purification of FL-�C-PE and Tr-�C-PE, cultures

were harvested when they were young (20 days) and old (90–200 days), respectively. The

procedures for getting both types of proteins have already been published [9-11]. Washed cell

mass was resuspended in the same buffer, and repeated freeze–thaw cycles (−30 °C and 4 °C)

were applied for C-PE release. This was followed by two-step (20% and 70%) ammonium

sulfate precipitation. The precipitate was dissolved in 10 mM Tris buffer and subjected to gel (G-

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150) permeation chromatography column pre-equilibrated with 10 mM Tris buffer. The purity of

the purified protein was checked by the native-PAGE and SDS-PAGE using silver staining [11,

13, 14]. The entire purification procedure was carried out in dark at 4 °C. All buffer solutions

required above during isolation and purification were prepared in mili-Q water supplemented

with 0.01% sodium azide. Stock solutions of both proteins were stored in the Tris-HCl buffer

(pH 8.1) containing 0.01% sodium azide at 4 °C.

To remove buffer constituents and sodium azide, stock solutions were dialyzed against

several changes with 0.1 M KCl (pH 7.0). The concentrations of the dialyzed and filtered stock

solutions of FL-�C-PE and Tr-�C-PE were determined using a value of 265000 M-1 cm-1 molar

absorption coefficient at 565 nm [15].

Sample preparation for denaturation and renaturation experiments

For the study of GdmCl-induced denaturation, 8 M stock solution of the denaturant was

prepared in 50 mM sodium cacodylate buffer (pH 7.0) containing 0.1 M KCl. Following the

procedure reported earlier [16], protein solutions for denaturation and renaturation experiments

were prepared. Briefly, for each denaturation experiment known amounts of the protein stock

solution, buffer and concentrated denaturant solution were mixed and incubated for a time long

enough to ensure the complete denaturation. This was cross-checked by taking absorbance. A

similar procedure was employed in preparing the solution for renaturation experiments with the

exception that the protein was first denatured by adding denaturant solution and then dilated with

the buffer. The GdmCl-induced denaturation was studied at pH 7.0. We have always checked the

pH of the protein solutions thus prepared.

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Measurements of absorption spectra

Absorption spectra of FL-�C-PE and Tr-�C-PE were measured in Jasco UV/visible

spectrophotometer (Jasco V-660, Model B 028661152) equipped with peltier-type temperature

controller (ETCS-761). All measurements were carried out in the wavelength range 700-350 nm

using 1-cm path length cuvettes. The protein concentration used was in the range of 0.1-0.3 mg

ml-1. All spectral measurements were done at least three times at 25 ± 0.1 oC.

Measurements of fluorescence spectra

Fluorescence measurements were carried out in Jasco spectrofluorimeter (Model FP-6200)

using 5-mm quartz cuvette. All experiments were carried out at 25 ± 0.1 oC which is maintained

by an external thermostated water circulator. FL-�C-PE and Tr-�C-PE have intrinsic (Trp77) and

extrinsic (PEB) fluorophores. To measure Trp77 fluorescence the protein was excited at 287 nm

and emission spectra was recorded in the range 300-400 nm. To measure PEB’s fluorescence,

protein solution was excited at 479 nm, and emission spectra were recorded in the range 500-600

nm [17]. The protein concentration used was 0.1-0.2 mg ml-1.

CD measurements

The far-UV and Soret CD measurements of FL-�C-PE and Tr-�C-PE were carried out in

Jasco spectropolarimeter (J-715) equipped with peltier type of temperature controller (PTC-

348WI). CD spectra of FL-�C-PE and Tr-�C-PE were collected with a response time of 1 s and a

scan speed of 100 nm min-1. Each spectrum was an average of at least five scans. All

measurements were carried out at 25 ± 0.1 °C. The far-UV spectra were collected in the

wavelength range 250-200 nm using 0.1-cm path length cuvette. The protein concentration used

8

was 0.2-0.4 mg ml-1. Soret CD spectra measurements of FL-�C-PE and Tr-�C-PE were carried

out in the wavelength range 600-400 nm using protein concentration of 0.3-0.5 mg ml-1 in a

cuvette of a path length of 1.0-cm. The raw data at a given wavelength, � (nm) were converted

into concentration-independent parameter [�]� (deg cm2 dmol-1), the mean residue ellipticity at

wavelength � using the relation,

[�]� = M0 �� / 10lc (1)

where �� is t he observed ellipticity in millidegrees at wave lengt h � , M0 is the mean

residue weight of the protein, l is the path length of the cell in centimeters, and c is the protein

concentration in mg ml-1.

Analysis of data

Sigmoidal GdmCl-induced denaturation curve (plot of a physical property (y) versus

[GdmCl], the molar concentration of the denaturant) were analyzed for stability parameters

(�GD0, the value of �GD (Gibbs free energy change) in the absence of the GdmCl; m, the slope (=

��GD/�[GdmCl]); and Cm, the midpoint of the denaturation curve, i.e., [GdmCl] at which �GD =

0. This analysis involves a least-squares method to fit the entire denaturation curve, obtained at

constant pH and temperature T (K), according to the relation,

y = yN + yD × Exp [−−−−(∆GD0 −−−− m [GdmCl]) / RT] / 1+ Exp [−−−−(∆GD

0 −−−− m [GdmCl]) / RT] (2)

where yN and yD are the properties of the native (N) and denatured (D) protein molecules under

the same experimental condition in which y has been measured, R is gas constant, and T is

temperature (K). It should be noted that equation 2 assumes that a plot of ∆GD versus [GdmCl] is

9

linear, and the dependencies of yN and yD on [GdmCl] are also linear (i.e., yN = aN + bN [GdmCl],

yD = aD + bD [GdmCl], where aN, bN, aD and bD are [GdmCl]-independent parameters, and

subscripts N and D signify that parameters are for the native and denatured states, respectively).

The denatured fraction of the protein, fD at a given [GdmCl] was determined using the relation,

fD = (y −−−− yN) / (yD −−−− yN) = y −−−− (aN + bN [GdmCl]) / (aD −−−− aN) + (bD −−−− bN) [GdmCl] (3)

In silico structural analysis

The crystal structure of Tr-�C-PE from P. tenue is already (PDB code 3MWN). However,

the crystal structure of FL-�C-PE is still not determined. Hence, we modeled the structure of FL-

�C-PE using modeler [18]. The energy minimized coordinate [19] was validated using online

Structural Analysis and Verification Server (http://nihserver.mbi.ucla.edu/SAVES/), showing

most of the residues in allowed region of Ramachandran plot (Figure S1). A superimposed

ribbon diagrams of full length and truncated �C-PE were drawn using PyMOL (The PyMOL

Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC) [20]. Contacts offered by

various residues were measured by using the contact program from the CCP4 suite [21].

Results

Polyacrylamide gel electrophoresis

The purified FL-�C-PE and Tr-�C-PE showed single band on both the native PAGE and SDS-

PAGE (results not shown) [10]. This observation led us to conclude that both full length and

truncated proteins exist as monomer.

Spectral characterization

10

Insets in Fig. 1 show visible absorbance spectra of the native FL-�C-PE and Tr-�C-PE at

pH 7.0 (50 mM sodium cacodylate containing 0.1 M KCl) and 25 ± 0.1 °C. It has been argued

that this absorption of both proteins is due to the interaction between the folded polypeptide

backbone and the two thioether bonded PEBs [10, 11]. This argument was based on the fact that

the apoprotein does not absorb in the visible region, and PEB has a very low absorbance [1, 2,

22]. These findings suggest that the folding/unfolding reactions of FL-�C-PE and Tr-�C-PE can

be followed by observing changes in the visible absorption at 565 nm. To see the effect of

GdmCl on the structural stabilty of FL-�C-PE and Tr-�C-PE, we measured the absorption

spectra of both proteins in the presence of different concentrations of GdmCl in the range 700-

350 nm. Results of these measurements are shown in Fig. S2 (also see insets in Fig. 1). Figs. 1A

and 1B respectively show plots of ��565 (difference in molar absorption coefficient at 565 nm in

the presence of GdmCl and that in the absence of GdmCl) of FL-�C-PE and Tr-�C-PE as a

function of [GdmCl]. These plots were analyzed for �GD0, m and Cm values for each protein

according to equation 2. Results of this analysis are shown in Table 1.

Both the FL- and Tr-�C-PE proteins have a lone tryptophan at position 77 (numbering is

according to the full length protein). This intrinsic fluorophore is buried and is at a distance of

~17 Å from the second chromophore (PEB2) attached at Cys139 [9]. After excitation at 287 nm,

we measured tryptophan emission spectra of both proteins in the native (in 50 mM cacodylate

buffer) and denatured (GdmCl solution) states at pH 7.0 and 25 ± 0.1 °C (see insets in Fig. 2). To

make corrections for the effect of solvent (GdmCl solution) on the D state of the protein,

following procedure was used. Values of fluorescent intensity at different wavelengths were

determined from the observed emission spectra of a protein in the presence of [GdmCl] > 2.5 M

i.e., the post transition region (Figure S3 and S4). At a given wavelength, values of fluorescent

11

intensity were plotted against [GdmCl]. To obtain the solvent independent value, an extrapolated

value at [GdmCl] = 0 M was determined from this plot. These extrapolated values of fluorescent

intensity values at different wavelengths were used to obtain the fluorescence spectrum of the

denatured state which does not depend on the denaturant concentration. It has been shown that

�max of tryptophan fluorescence spectrum shifts to longer wavelengths and the intensity at �max

decreases on transferring this fluorophore from non-polar medium to polar medium [23, 24]. As

can be seen in Fig. 2, when a protein is transferred from the native buffer to the denaturant

solution, there is an expected red shift of the emission spectrum from 350 to 360 nm. However,

contrary to this expectation, the emission intensity of the proteins is increased on denaturation.

This increase can be explained by invoking quenching phenomenon due to the presence of PEB2

near Trp77, which will decrease when the protein unfolds [22]. We exploited this phenomenon to

see the effect of GdmCl on emission spectra of FL- and Tr-�C-PEs. These spectra are shown in

Fig. S3. Values of emission intensity at 350 nm (F350) of both proteins are read from these

spectra and plotted as a function of [GdmCl] in Figs. 2A and 2B. To obtain stability parameters

(�GD0, m and Cm) each denaturation curve shown in Fig. 2 was fitted to equation 2. Results of

this analysis for both proteins are shown in Table 1.

PEB has fluorescence property [25-27]. It has been shown that the covalently linked PEBs

bonded to the folded protein polypeptide chain via thioether bonds have a strong emission

spectrum in the wavelength region 500-600 nm, which vanishes on protein denaturation [26, 28].

After exciting FL-�C-PE and Tr-�C-PE at 479 nm, we measured emission spectra of both

proteins in the presence of different concentrations of GdmCl at pH 7.0 and 25 ± 0.1 °C. These

spectra are shown in Fig. S4. Figs. 3A and 3B show the change in F573, the fluorescence intensity

of FL-�C-PE and Tr-�C-PE at 573 nm as a function of [GdmCl], respectively. For each protein,

12

GdmCl-induced denaturation curves were analyzed for �GD0, m and Cm values by fitting the

entire denaturation curve according to equation 2. The results of this analysis are shown in Table

1.

To see the effect of GdmCl on the stability of FL-�C-PE and Tr-�C-PE, we measured the

far-UV CD spectra of both proteins in the presence of different concentrations of GdmCl. These

measurements are shown in Fig. S5. Values of [�]222 (probe for the secondary structure) in the

presence of different concentrations of GdmCl were determined from these CD spectra shown in

this figure. Figs. 4A and 4B show plots of [�]222 versus [GdmCl] for FL-�C-PE and Tr-�C-PE,

respectively. Each of these plots (denaturation curves) was analyzed for values of �GD0, m and

Cm by fitting the entire denaturation curve according to equation 2. These stability parameters

thus obtained are given in Table 1.

The insets in Fig. 4 show the far-UV CD spectra of FL-�C-PE and Tr-�C-PE. We have

used k2d program (http://dichroweb.cryst.bbk.ac.uk/html/process.shtml) to estimate the

percentage of elements of secondary structure of those proteins. Analysis of the far-UV CD

spectrum of the native FL-�C-PE (see inset in Fig. 4A) for the contents of �-helix and �-sheet

yielded values of 75 and 2%, respectively. Analysis of the far-UV CD spectrum of Tr-�C-PE

(see inset in Fig. 4B) for the secondary structure gives 60 and 2% for the �-helix and �-structure,

respectively, which is in excellent agreement with that determined by the X-ray diffraction

studies [9].

Refolding Experiments

In order to check the reversibility of denaturation of FL-�C-PE and Tr-�C-PE, proteins

were exposed to concentrated GdmCl solution ([GdmCl] ≥ 2.5M) for a time long enough to

13

ensure complete denaturation. The denatured protein was diluted with the native buffer to check

the reversibility of denaturation at 3.0, 2.5, 2.0 and 0.5 M GdmCl. Figs. 5 A-H show

representative denaturation (curve 1) and renaturation (curve 2) spectra. It has been observed that

for each protein in a given denaturatnt concentration, spectra obtained from denaturation and

renaturation experiments are indistinguishable (also see Figs. 1-4).

In silico structure analysis

A structural comparison of FL-�C-PE and Tr-�C-PE suggests a difference of 31 residues

from N-terminal side, which is comprised of two �-helices. This segment is completely away

from the whole compact polypeptide chain (Fig. 6). It is seen in Fig. 6 that there are only 6

hydrogen bonds between residues Lys2 and Gly105, Gly29 and Arg33, Gln28 and Gln32, Asn30

and Gln32, Gln28 and Gln32 (through side chain) and Ile31 and Gln32 (see Table 2). However,

for the sake of clarity the first three hydrogen bonds are shown in the Fig. 6. Furthermore, this N-

terminal segment is involved in 25 van der Waals interactions (see Table 2). Since these 31

residues of FL-�C-PE are completely away from the packed polypeptide core, these are expected

to be highly exposed to water, if the protein exists in the form of a monomer.

The crystal structure of Tr-�C-PE was recently determined [9]. It was observed that this

protein belongs to a class of all �-protein. Likewise, our modeled structure of FL-�C-PE shows

the presence of 75% �-helical content. Fig. 6 shows overlapping 3-D structures of Tr-�C-PE and

FL-�C-PE. The 31-residue long N-terminal sequence of FL-�C-PE contains 12% �-helix.

Discussion

14

The analysis of transition curves (Figs. 1-4) for the stability parameters, �GD0, m and Cm

(Table 1) according to equation 2 is based on a few assumptions. A few comments are therefore

necessary. First, it was assumed that the GdmCl-induced denaturation of the full length and

truncated �C-PE proteins are reversible. To check the reversibility of denaturation, we denatured

each protein by adding different concentrations of the denaturant. The GdmCl-denatured protein

was diluted with the native buffer. After incubation of the denatured protein in the buffer for a

time enough for the completion of the renaturation process, optical spectra (absorption,

fluorescence and CD) were recorded. A spectrum of the renatured protein was then compared

with that obtained during denaturation experiments. It was observed that the spectral properties

of renatured protein in a given [GdmCl] are identical to those obtained during denaturation

experiments. Fig. 5 shows the representative data which were obtained on denaturing the protein

by 2.5 M GdmCl (curve 1), and renaturing this denatured protein by diluting with the buffer to

bring the denaturant concentration to 0.5 M (curve 2). It has been observed that the spectra of the

protein in a given [GdmCl] obtained from the denaturation and renaturation experiments were

indistinguishable. The spectra of the denatured and renatured proteins are also compared with

those of the native proteins (curve 3; Fig. 5). The coincidence of optical properties of the proteins

obtained from denaturation and renaturation experiments (Fig. 5 and also see Fig. 1-4) led us to

conclude that the GdmCl-induced denaturation of FL-�C-PE and Tr-�C-PE is reversible.

The second assumption was that the GdmCl-induced transition between N and D states is a

two-state process. The most authentic criterion for a two-state (native and denatured) behavior of

the protein denaturation is to show that the calorimetric and van’t Hoff enthalpy changes on

denaturation are identical. We could not succeed to make differential scanning calorimetry

measurements on Fl-�C-PE and Tr-�C-PE proteins due to the fact that the heat denatured

15

proteins formed aggregates. However, another test for the two-state behavior of the protein

denaturation is to show coincidence of the normalized sigmoidal curves of different physical

property induced by GdmCl [29]. Figs. 7A and 7B show normalized denaturation curves (plots

of fD versus [GdmCl]) of the full length and truncated proteins, respectively. It is seen in these

figures that for a protein, the normalization of each transition curve of Figs. 1-4, using equation 3

yields a coincidence of curves of different physical properties. As pointed out earlier [29], this is

a necessary but not sufficient criterion for a two-state behavior of the protein denaturation.

Customarily, the observation of non-coincidence of normalized transition curves of different

physical properties constitutes a proof of existence of stable intermediate(s) on the folding �

unfolding pathways [29]. On the contrary, basing their arguments on theoretical grounds, Dill

and Shortle [30] have shown that the non-coincidence of sigmoidal curves of two different

physical properties is also observed for a two-state protein denaturation. Recently, Rahaman et al

[31] provided experimental evidence that this non-coincidence is consistent with two-state

denaturation behavior.

In another approach for testing the two-state behavior of GdmCl-induced denaturation,

�GD0 values of the protein are measured from sigmoidal curves of different structural proteins,

and it is shown that this thermodynamic property is independent of techniques (probes) used to

monitor the denaturation [32]. Table 1 shows that values of �GD0 of each protein, measured by

four different structural properties are, within experimental errors, identical. This overlap in

�GD0 values suggest that GdmCl-induced denaturation of Fl-�C-PE and Tr-�C-PE is a two-state

process.

16

It is known that if absorption spectra of two non-interacting species separately intersect

exactly at one or more wavelengths, then each intersecting point (isosbestic point) of absorption

no longer depends on the concentration ratios of these species in their mixtures. The presence of

isosbestic points on the absorption spectra of the native and denatured proteins alone and in their

equilibrium mixtures is therefore taken as an evidence for a two-state denaturation [22, 29]. Fig.

S2 shows absorption spectra of the FL- and Tr- �C-PEs in the presence of different concentration

of GdmCl in which a protein exists either in the N state (pre-transition region) or as an

equilibrium mixture of N and D states (transition region) or in the D state (post-transition

region). It is seen in this figure that there exist isosbestic points at wavelengths 460 and ~594 nm

in the case of FL-�C-PE and at wavelengths 440 and ~602 nm in case of Tr-�C-PE. These

observations therefore support an assumption that GdmCl-induced denaturation of both proteins

follows a two-state mechanism.

Estimates of �GD0 from the denaturation curves depend not only on the mechanism of

denaturation but also on the models describing the dependencies of the observed �GD0 on

[GdmCl]. There are three models to estimate �GD0 from the extrapolation of �GD to 0 [M]

GdmCl concentrations [33, 34]. One model assumes that �GD value varies linearly with

[denaturant]. The other one (binding model) assumes the presence of ‘specific’ binding sites for

the chemical denaturant on the native and denatured protein molecules. In this model dependence

of �GD on the [denaturant] is non-linear. The third model known as transfer-free energy model,

describes the dependence of �GD on the transfer free energy of small model compounds for the

protein groups from water to different concentrations of the chemical denaturants. It has been

observed that the analysis of the same set of denaturation data using different models give

different values of �GD0 of a protein. However, we used the linear free energy model for the

17

analysis of denaturation curves of both FL-�C-PE and Tr-�C-PE (Figs. 1-4), for it has supports

of both theory [35, 36] and experiments [37-41].

It is seen in Table 1 that the full length protein is only ~1.4 kcal mol-1 more stable than the

truncated protein, or in other words, the contribution of 31 N-terminal residues to protein

stability (�GD0) of FL-�C-PE is equivalent to the stability provided by only one buried hydrogen

bond (1.5 ± 1 kcal mol-1) [42]. To understand this difference in stability between FL-�C-PE and

Tr-�C-PE, we performed in silico study. Since the sequence of FL-�C-PE is not determined so

far we have taken a conserved sequence of 31 N-terminal residues from cyanobacteria. We

assumed that our model is quite similar to the crystal structure of FL-�C-PE. This assumption is

supported by following observations. (i) We aligned the sequences of all �-PEs of cyanobacteria

and red algae available in the UniProt database using multiple sequence alignment protcol on

ClustalW2 [43]. Out of 31 N-terminal residues, only 5 positions are not fully conserved; 5A/I/A,

8V/A, 9I/V, 10A/S/G/T and 21 T/A/Q/S (see Fig. 8). This comparison indicates that there is 97%

sequence homology as far as 31 N-terminal residues are concerned. (ii) A sequence alignment of

Tr-�C-PE with that of 32-164 residues shows 94% homology (Fig. 8). (iii) An overlaying of 3-D

structure of Tr-�C-PE on that of FL-�C-PE shows almost identical structural homology (Fig. 6).

(iv) Analysis of the far-UV CD spectra for the elements of secondary structures in FL-�C-PE and

Tr-�C-PE (Figs. 4A and 4B), and comparison of these elements of secondary structure with

those of Tr-�C-PE obtained from the X-ray diffraction studies (Fig. 6) led to the conclusion that

31 N-terminal residues of the full length intact protein contain 12% �-helix. This observation is

identical to that observed for the same segment in the FL-�C-PE (see Fig. 6).

18

Table 1 shows m-values of FL-�C-PE and Tr-�C-PE. The average values of this parameter

(m) obtained from measurements of GdmCl-induced denaturation using different optical

properties are 5.99 kcal mol-1 M-1 for the full length protein and 5.65 kcal mol-1 M-1 for the

truncated protein. Myer et al [44] have developed a method to predict m-value of a protein from

its known change in the accessible surface area (�ASA) on denaturation. Using their equation

that relates m-value with �ASA (i.e., m = 953 + 0.23 �ASA), we estimated m-values of 5.65 and

5.71 kcal mol-1 M-1 for FL-�C-PE and Tr-�C-PE, respectively. This predicted value of m for each

protein is in excellent agreement with the average value determined experimentally (also see

Table 1). It should be noted that for the determination of total �ASA of the protein, we have

used DSSP method [45] for the estimation of ASA of the folded protein and the procedure of

Creamer et al [46] for estimation of the lower bound value of ASA of the unfolded protein. The

�-subunit of each PBP contains 164 residues and consists of 9-helices packed in globular shape

to form main core of the protein molecule [9]. The N-terminal segment (1-31 residues) contains

only 2-helices X and Y which are antiparallel to each other and perturbed away from the main

core (32-164 residues). Structural analysis suggests that residues of the N-terminal segment form

a total of 30 hydrogen bonds and many van der Waals interactions among themselves (see Table

3). In addition to these non-covalent interactions this segment forms six inter-segment hydrogen

bonds: Lys2 and Gly105, Gly29 and Arg33, Gln28 and Gln32, Asn30 and Gln32, Gln28 and

Gln32 (through side chain) and Ile32 and Gln32) and 25 van der Waals interactions (see Table

2). It seems that among these six hydrogen bonds, only one hydrogen bond, 2LysO---105GlyN

may have a significant role holding the N-terminal segment to the main core, for other hydrogen

bonds are formed between neighboring atoms. Both types of non-covalent interactions (hydrogen

bonding and van der Waals interactions) are known to provide stability to folded proteins [47-

19

49]. It is then expected that the full length protein (FL-�C-PE) should be more stable than

truncated protein (Tr-�C-PE) which lacks 31-residues long N-terminal segment of the full length

protein. This is indeed true; FL-�C-PE is 1.4 kcal mol-1 more stable than the Tr-�C-PE (see

Table 1).

A buried hydrogen bond in the protein core (dielectric constant 4-6) provides a stability of

about 2 kcal mol-1 [42]. Thus an observed stability difference of 1.4 kcal mol-1 between the full

length and truncated proteins appears to be too small, for 31-residue long N-terminal is involved

in six hydrogen bonds formation [20]. A possible reason for this small difference in stability

could be due to full or partial exposure of amino acid residues involved in non-covalent

interactions, to the highly polar solvent water (dielectric constant 80). The reason for saying this

is that both hydrogen bonds and van der Waals interactions are electrostatic in nature and

inversely proportional to the dielectric constant of the medium. For instance, hydrogen bonds in

water do not have a net stabilizing effect on protein structure due to the fact that in order to form

a D-H���A (where A and D are N and O atoms) bond one needs to disrupt another set of very

favorable hydrogen bonds, those the protein groups (D-A and A) can make with water. Using

software RVP-Net (version 1), we estimated exposure of all residues involved in non-covalent

interactions. Interestingly, it has been observed that almost all groups involved in such

interactions are fully or partially exposed.

Conclusions

(a) The 31-residue long N-terminal segment of the full length phycoerythrin (i) does not

contribute significantly to the overall stability of the protein, for the full length protein is only 1.4

kcal mol-1 more stable than the truncated protein, (ii) has 12% �-helix, and (iii) has no effect on

20

the mechanism of folding of the protein fragment (32-164 residues), for both proteins undergo a

two-state transition between N and D states. (b) The non-covalent interactions due to 31 N-

terminal residues in the FL-�C-PE monomeric protein provide structural rigidity, not significant

stability. (c) Deletion of the N-terminal segment does not perturb the protein-PEBs interaction,

thus both FL-�C-PE and Tr-�C-PE have same absorbance capability.

Acknowledgements

MIH and FA are thankful to the Department of Science and Technology (DST) for

financial support (Project No. SR/SO/BB-0124/2010A). KA is thankful to University Grant

Commission (UGC) for providing Maulana Azad National Fellowship (MANF).

21

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24

Figure Legends

Fig. 1. GdmCl-induced denaturation of FL-�C-PE and Tr-�C-PE at pH 7.0 and 25 °C. A,

Denaturation curve of FL-�C-PE followed by observing changes in ��565 as a function of

[GdmCl]. B, Plots of ��565 of Tr-�C-PE versus [GdmCl]. Open and filled symbols show the

results obtained from denaturation and renaturation experiments. The insets in A and B show the

absorption spectra of FL-�C-PE and Tr- �C-PE of the native and GdmCl-induced denatured

states, respectively.

Fig. 2. GdmCl-induced denaturation of FL-�C-PE and Tr-�C-PE followed by observing

changes in F350 at pH 7.0 and 25 °C. A and B show denaturation curves of FL-�C-PE and Tr-

�C-PE, respectively. Open and filled symbols have same meaning as in Fig. 1. Fluorescence

spectra of FL-�C-PE (inset in A) and Tr-�C-PE (inset in B) in their native and denatured states,

respectively. The excitation wavelength was 287 nm. D state of each protein was corrected for

the solvent effect.

Fig. 3. Denaturation curves of FL-�C-PE and Tr-�C-PE induced by GdmCl at pH 7.0 and

25 °C. A, Plot of F573 of FL-�C-PE versus [GdmCl]. B, Plot of F573 of Tr-�C-PE versus

[GdmCl]. Open and filled symbols have the same meaning as in Fig. 1. Insets in A and B show

the fluorescence spectra of the native and denatured FL-�C-PE and Tr-�C-PE, respectively.

Spectra of the denatured proteins were corrected for the solvent effect. For both proteins

excitation wavelength was 479 nm.

Fig. 4. GdmCl-induced denaturation curves of FL-�C-PE and Tr-�C-PE at pH 7.0 and 25

°C. For the full length and truncated proteins A and B show the plots of [θ]222 versus [GdmCl],

25

respectively. Open and filled symbols have the same meaning as in Fig. 1. The insets in A and B

show the far-UV CD spectra of the native and denatured states of FL-�C-PE and Tr-�C-PE,

respectively.

Fig. 5. Renaturation of the GdmCl-denatured FL-�C-PE and Tr-�C-PE at pH 7.0 and 25

°C. Absorption spectra of A, FL-�C-PE and B, Tr-�C-PE: (1) denatured protein in 2.5 M

GdmCl, (2) renatured protein in 0.5 M GdmCl, and (3) native protein. C and D show Trp

fluorescence spectra, E and F show PEB fluorescence spectra, and G and H the far-UV CD

spectra of FL-�C-PE and Tr-�C-PE, respectively. Spectra meaning has the same meaning as in

A.

Fig. 6: Overlaid structure of Tr-�C-PE (light green) and FL-�C-PE illustrated in cartoon

model. The chromophores, PEBs are shown in ball and stick model. 31 N-terminal residues

forming hydrogen bonded interactions with other part (32-164) of molecule are shown in ball

and stick. Structure is drawn in PyMol using the atomic coordinates of FL-�C-PE and �C-PE

from the Phormidium tenue (PDB code: 3MWN).

Fig. 7. Normalized GdmCl-induced denaturation curves of A, FL-�C-PE and B, Tr-�C-PE

at pH 7.0 and 25 °C. Results shown in Figs. 1-4 were used to construct plots of fD versus

[GdmCl].

Fig. 8. Multiple sequence alignment of �-subunit of PBPs. Names of sequences is given

according to the sequence identifier on the UniProt (www.uniprot.org). Non-conserved residues

from N-terminal side (1-31) are shaded in yellow. Secondary structures of PBPs are illustrated

on the top of sequence.

26

Figure Legends (Supplementary figures)

Fig. S1. Ramachandran Plot of FL-�C-PE modeled structure.

Fig. S2. Absorption spectra of FL-�C-PE and Tr-�C-PE at pH 7.0 and 25 °C. A and B,

absorption spectra of FL-�C-PE and Tr-�C-PE in increasing concentration of GdmCl (0.0 M to

4.5 M), respectively.

Fig. S3. Fluorescence spectra of FL-�C-PE and Tr-�C-PE at pH 7.0 and 25 °C. Trp

fluorescence spectra of A, FL-�C-PE and B, Tr-�C-PE in the presence of different

concentrations of GdmCl used in absorption measurements (Fig. S2). The wavelength of

excitation was 287 nm.

Fig. S4. Fluorescence spectra of FL-�C-PE and Tr-�C-PE at pH 7.0 and 25 °C. PEB

fluorescence spectra of A, FL-�C-PE and (B) Tr-�C-PE in the presence of different

concentrations of GdmCl used in absorption measurements (Fig. S2). The wavelength of

excitation was 573 nm.

Fig. S5. Far-UV CD measurement of FL-�C-PE and Tr-�C-PE at pH 7.0 and 25 ± 0.1 °C:

Far-UV CD spectra of A, FL-�C-PE and B, Tr-�C-PE in the presence of different concentrations

of GdmCl used in absorption measurements (Fig. S2).

27

Fig. 1

[GdmCl], M

0.0 0.5 1 .0 1 .5 2.0 2.5 3.0 3 .5 4 .0 4.5

∆∆ ∆∆εε εε

55 556

5 x

10

-3,

M-1

cm

-1

-3

-2

-1

0

1

A

Wavelength, nm

350 400 450 500 550 600 650 700

εε εελλ λλ x

10-4

, M

-1 c

m-1

0

5

10

15

20

25

30

Native FL- ααααC-PE

Denatured FL-ααααC-PE

[GdmCl], M

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

∆∆ ∆∆εε εε55 5565 x

10-3

, M

-1cm

-1

-3

-2

-1

0

1

Wavelength, nm350 400 450 500 550 600 650 700

εε εελλ λλ x

10-4

, M

-1cm

-1

0

5

10

15

20

25

30

Native Tr-Native Tr-Native Tr-Native Tr- ααααC-PE C-PE C-PE C-PE

Denatured Tr-Denatured Tr-Denatured Tr-Denatured Tr- ααααC-PEC-PEC-PEC-PE

B

28

29

Fig. 3

[GdmCl], M

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

F57

3 (

a. u

.)

0

20

40

60

0

60

20

40

520 600540 560 580

Native FL-�C-PE

Denatured FL-�C-PE

Wavelength, nm

Flu

ores

cenc

e (a

. u.)

A

[GdmCl], M

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

F5

73

(a.

u.)

0

20

40

60

0

60

20

40

520 600540 560 580

Wavelength, nm

Flu

ores

cenc

e (a

. u.)

Native Tr-�C-PE

Denatured Tr-�C-PE

B

30

Fig. 4

[GdmCl], M

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

[ θθ θθ] 2

22 x

10-3

, d

eg c

m2 d

mol-1

-25

-20

-15

-10

-5

0

5

A

Wavelength, nm

200 205 210 215 220 225 230 235 240 245 250

[ θθ θθ] λλ λλ

x 1

03, d

eg c

m2 d

mol-1

-25

-20

-15

-10

-5

0

5

10

Native FL-ααααC-PE

Denatured FL-ααααC-PE

[GdmCl], M

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

[ θθ θθ] 2

22x

10-3

, d

eg c

m2

dm

ol-1

-25

-20

-15

-10

-5

0

5

B

Wavelength, nm

200 205 210 215 220 225 230 235 240 245 250

[ θθ θθ] λλ λλ

x 1

03,

deg

cm

2 d

mol-1

-25

-20

-15

-10

-5

0

5

10

Native Tr- ααααC-PE

Denatured Tr- ααααC-PE

31

Fig. 5

0

15

5

10

300 400320 340 360 380

Wavelength, nm

Flu

ores

cenc

e (a

. u.)

C

1

3

2

0

15

5

10

300 400320 340 360 380

Wavelength, nm

Flu

ores

cenc

e (a

. u.)

D

0

60

20

40

520 600540 560 580

Wavelength, nm

Flu

ores

cen

ce

(a. u

.)

E

0

60

20

40

520 600540 560 580Wavelength, nm

Flu

ores

cen

ce

(a. u

.)

F

1

3

2

1

2

3

1

2

3

Wavelength, nm

350 400 450 500 550 600 650 700

εε εελλ λλ x

10-4

, M

-1cm

-1

0

5

10

15

20

25

30

32

1

A

Wavelength, nm

350 400 450 500 550 600 650 700

εε εελλ λλ x

10-4

, M

-1 c

m-1

0

5

10

15

20

25

30

3

1

2

B

Wavelength, nm

200 205 210 215 220 225 230 235 240 245 250

[ θθ θθ] λλ λλ

x 1

0-3

, d

eg c

m2

dm

ol-1

-25

-20

-15

-10

-5

0

5

10

3

2

1

G

Wavelength, nm

200 205 210 215 220 225 230 235 240 245 250

[ θθ θθ] λλ λλ

x 1

0-3

, d

eg c

m2 d

mol-1

-25

-20

-15

-10

-5

0

5

10

3

2

1

H

32

Fig. 6

Lys2

Gly105

Gly29

Asn30

Gln28

Gln32

Arg33

Ile31

33

Fig. 7

[GdmCl], M

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

f D

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

F350

F573

∆ε565

[θ]222

[GdmCl], M

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

f D

0.0

0.2

0.4

0.6

0.8

1.0

1.2

F350

F573

∆ε565

[θ]222

A

B

N4_9CYAN(Tr-αC-PE)-------------------------------QRAAARLEAAEKLGSNLEAVTKEAGDACF 29 PHEA_POLUR(αR-PE) MKSVITTTISAADAAGRYPSTSDLQSVQGNIQRAAARLEAAEKLGSNHEAVVKEAGDACF 60 PHEA_FREDI MKSVVTTVIAAADAAGRFPSTSDLESVQGSIQRAAARLEAAEKLANNIDAVATEAYNACI 60

PHEA_PSEA9 MKSVVTTVISAADAAGRFPSTSDLESVQGSIQRAAARLEAAEKLANNIDAVAKEAYNAAI 60

PHEA_SYNY1 MKSVITTVVAAADAAGRFPSTSDLESVQGSIQRAAARLEAAEKLAANLDAVAKEAYDAAI 60

PHEA2_SYNPX MKSVITTVVGAADSASRFPSASDMESVQGSIQRAAARLEAAEKLAGNYDQVAQEAVDAVY 60

PHEA1_SYNPW MKSVVTTVVTAADAAGRFPSQNDLEAVQGNIQRAAARLEAAEKLAAGLDAVTKEAGDACF 60

PHEA2_SYNPY MKSVITTVVGAADSASRFPSASDMESVQGSIQRAAARLEAAEKLSANYDAIAQRAVDAVY 60

PHEA2_SYNPZ MKSVITTVVGAADSASRFPSASDMESVQGSIQRAAARLEAAEKLAGNYDQVAQEAVDAVY 60

Q7U4Q1_SYNPX MKSVVTTVVTAADAAGRFPSQNDLEAVQGNIQRAAARLEAAEKLAAGLDNVTREAGDACF 60

Q0ICU1_SYNS3 MKSVITTVVGAADSASRFPSASDMESVQGSIQRAAARLEAAEKLSANYDAIAQRAVDAVY 60

Q0ICU9_SYNS3 MKSVVTTVVTAADAAGRFPSQNDLEAVQGNIQRAAARLEAADKLAAGLDAVTREAGDACF 60

A5GJ03_SYNPW MKSVLTTVIGSADSGSRFPTSSDLESVQGSLQRAAARLEAAEKIAQNYDAIAQRAVDAVY 60

A5GVQ1_SYNR3 MKSVVTTVVTAADAAGRFPSQNDLEAVQGNIQRAAARLEAAEKLAAGLDAVTREAGDACF 60

A5GVP3_SYNR3 MKSVITTVIGAADSASRFPSASDMESVQGSIQRAAARLEAAEKLAGNYDAIAQRAVDAVY 60

Q3AWB3_SYNS9 MKSVVTTVVTAADAAGRFPSQNDLEAVQGNIQRAAARLEAAEKLAAGLDAVTREAGDACF 60

Q3AWC1_SYNS9 MKSVITTVVGAADSASRFPSSSDMESVQGSIQRAAARLEAAEKLSQNYDAIAQRAVDAVY 60

Q3AMH2_SYNSC MKSVITTVVGAADSASRFPTASDMESVQGSIQRASARLEAAEKLASNYDQVAQEAVDAVY 60

Q3AMH9_SYNSC MKSVVTTVVTAADAAGRFPSQNDLEAVQGNIQRAAARLEAAEKLAAGLDNVTREAGDACF 60

I4IE78_9CHRO MKSVITTAVASADAAGRFPSTSDLESVQGSIQRATARLEAAEKLAGNLDAVAKEAYDACI 60

D0CKC3_9SYNE MKSVITTVVGAADSASRFPTASDMESVQGSIQRASARLEAAEKLASNYDQVAQEAVDAVY 60

D0CKD5_9SYNE MKSVVTTVVTAADAAGRFPSQNDLEAVQGNIQRAAARLEAAEKLAAGLDNVTREAGDACF 60

Q05Q58_9SYNE MKSVVTTVVTAADAAGRFPSQNDLEAVQGNIQRAAARLEAAEKLAAGLDKVTREAGDACF 60

Q05Q50_9SYNE MKSVITTVVGAADSASRFPSASDMESVQGSIQRAAARLEAAEKLASNYDGIAQRAVDAVY 60

A4CSG2_SYNPV MKSVATTVVTAADAAGRFPSQNDLEAVQGNIQRAAARLEAAEKLAAGLDAVTKEAGDACF 60

Q05Z96_9SYNE MKSVITTVVGAADSASRFPSSSDMESVQGSIQRAAARLEAAEKLSQNYDGIAQRAVDAVY 60

Q05ZA4_9SYNE MKSVVTTVVTAADAAGRFPSQNDLEAVQGNIQRAAARLEAAEKLASGLDAVTKEAGDACF 60

N4_9CYAN SKYSYNKNPGEAGENQEK-VDKCYRDILHYMRLINYTLVVGGTGPLDEWGIAGAREVYRT 88

PHEA_POLUR SKYGYNKNPGEAGENQEK-INKCYRDIDHYMRLINYTLVVGGTGPLDEWGIAGAREVYRT 120

PHEA_FREDI KKYPYLNNSGEANSTDTF-KAKCARDIKHYLRLIQYSLVVGGTGPLDEWGIAGQREVYRA 119

PHEA_PSEA9 KKYPYLNNAGEANSTDTF-KAKCARDIKHYLRLIQYSLVVGGTGPLDEWGIAGQREVYRS 119

PHEA_SYNY1 KKYSYLNNAGEANSTDTF-KAKCLRDIKHYLRLINYSLVVGGTGPLDEWGIAGQREVYRT 119

PHEA2_SYNPX NQYPNGATGRQPRKCATEGKEKCKRDFVHYLRLINYCLVTGGTGPLDELAINGQKEVYKA 120

PHEA1_SYNPW NKYPYLKQPGEAGENQTK-VDKCYRDLGHYLRLINYCLVVGGTGPLDEWGIAGAREVYRT 119

PHEA2_SYNPY AQYPNGATGRQPRQCATEGKEKCKRDFVHYLRLINYCLVTGGTGPLDELAINGQKEVYKA 120

PHEA2_SYNPZ NQYPNGATGRQPRKCATEGKEKCKRDFVHYLRLINYCLVTGGTGPLDELAINGQKEVYKA 120

Q7U4Q1_SYNPX NKYAYLKQPGEAGDSQVK-IDKCYRDLGHYLRLINYCLIVGGTGPLDEWGIAGAREVYRT 119

Q0ICU1_SYNS3 AQYPNGATGRQPRQCATEGKEKCKRDFVHYLRLINYCLVTGGTGPLDELAINGQKEVYKA 120

Q0ICU9_SYNS3 SKYSYLKQAGEAGDSQVK-VDKCYRDLGHYLRLINYCLVVGGTGPLDEWGIAGAREVYRS 119

A5GJ03_SYNPW TQYPNGATGRQPRQCATEGKEKCKRDFVHYLRLINYSLVVGGTGPLDELAINGQREVYKA 120

A5GVQ1_SYNR3 NKYSYLKQPGEAGDSQVK-VDKCYRDLGHYLRLINYCLVVGGTGPLDEWGIAGAREVYRS 119

A5GVP3_SYNR3 SQYPNGATGRQPRQCATEGKEKCKRDFVHYLRLINYCLVTGGTGPLDELAINGQKEVYKA 120

Q3AWB3_SYNS9 NKYAYLKQPGEAGENQVK-VDKCYRDLGHYLRLINYCLVVGGTGPLDEWGIAGAREVYRS 119

Q3AWC1_SYNS9 AQYPNGATGRQPRQCATEGKEKCKRDFVHYLRLINYCLVTGGTGPLDELAINGQKEVYKA 120

Q3AMH2_SYNSC AQYPNGATGRQPRKCATEGKEKCKRDFVHYLRLINYCLVTGGTGPLDELAINGQKEVYKA 120

Q3AMH9_SYNSC NKYAYLRQPGEAGDSQVK-VDKCYRDLGHYLRLINYCLIVGGTGPLDEWGIAGAREVYRT 119

I4IE78_9CHRO KKYPYLNEAGQANSTNTF-KEKCLRDIKHYLRLVNYCLVVGGTGPLDEWGIAGQREVYRA 119

D0CKC3_9SYNE AQYPNGATGRQPRKCATEGKEKCKRDFVHYLRLINYCLVTGGTGPLDELAINGQKEVYKA 120

D0CKD5_9SYNE NKYAYLRQPGEAGDSQVK-VDKCYRDLGHYLRLINYCLIVGGTGPLDEWGIAGAREVYRT 119

Q05Q58_9SYNE NKYAYLKQPGEAGDSQIK-VDKCYRDLGHYLRLINYCLVVGGTGPLDEWGIAGAREVYRS 119

Q05Q50_9SYNE AQYPNGATGRQPRKCATEGKEKCKRDFVHYLRLINYCLVTGGTGPLDELAINGQKEVYKA 120

A4CSG2_SYNPV NKYPYLKQPGEAGENQTK-VDKCYRDLGHYLRLINYCLVVGGTGPLDEWGIAGAREVYRT 119

Q05Z96_9SYNE AQYPNGATGRQPRQCATEGKEKCKRDFVHYLRLINYCLVAGGTGPLDELAINGQKEVYKA 120

Q05ZA4_9SYNE NKYAYLKQPGEAGDTQVK-VDKCYRDLGHYLRLINYCLVVGGTGPLDEWGIAGAREVYRS 119

N4_9CYAN LNLPSAAYIAAFTFTRDRLCAPRDMSAQAGTEFSTALDYLINSLS 133

PHEA_POLUR LNLPSAAYIAAFVFTRDRLCIPRDMSAQAGVEFCTALDYLINSLS 164

PHEA_FREDI LGLPTAPYVEALSFARNRGCAPRDMSAQALTEYNALLDYAINSLS 164

PHEA_PSEA9 LGLPTAPYVEALSYARNRGCSPRDLSPQALTEYNALLDYAINSLS 164

PHEA_SYNY1 LGLPTAPYVEALSFARNRGCSPRDLSAQALTEYNSLLDYVINSLS 164

PHEA2_SYNPX LSIDAGTYVAGFSHLRSRGCAPRDMSAQALTAYNQLLDYVINSLG 165

PHEA1_SYNPW LGLPTGPYVEALTYTRDRACAPRDMSPQALNEFKSYLDYVINALS 164

PHEA2_SYNPY LSIDAGTYVAGFSNMRNDGCSPRDMSAQALTAYNTLLDYVINSLG 165

PHEA2_SYNPZ LSIDAGTYVAGFSHLRSRGCAPRDMSAQALTAYNQLLDYVINSLG 165

Q7U4Q1_SYNPX LGLPTNAYIEALTYTRDRACAPRDMSAQALNEFKSYLDYAINALS 164

Q0ICU1_SYNS3 LSIDAGTYVAGFSNMRNDGCSPRDMSAQALTAYNTLLDYVINSLG 165

Q0ICU9_SYNS3 LSLPTGPYVEALTYTRDRACAPRDMSPQALNEFKSYLDYVINALS 164

A5GJ03_SYNPW LSIDPGTYVAGFTQMRNDGCAPRDLSPQALTEYNAALDYVINSLA 165

A5GVQ1_SYNR3 LGLPTGPYVEALTYTRDRACAPRDMSPQALNEFKSYLDYVINALS 164

A5GVP3_SYNR3 LSIDAGTYVAGFSNMRNDGCAPRDMSPQALTAYNTLLDYVINSLG 165

Q3AWB3_SYNS9 LGLPTGPYVEALTYTRDRACAPRDMGPQALNEFKSYLDYVINALS 164

Q3AWC1_SYNS9 LSIDAGTYVAGFSQMRNDGCAPRDMSPQALTSYNTLLDYVINSLG 165

Q3AMH2_SYNSC LSIDAGTYVAGFSHLRSRGCAPRDMSAQALTAYNQLLDYVINSLG 165

Q3AMH9_SYNSC LGLPTNAYIEALTYTRDRACAPRDMSPQALNEFKSYLDYAINALS 164

I4IE78_9CHRO LGLPTAPYVEALSFARNRGCAPRDMSAQALTEYNALLDYVINSLS 164

D0CKC3_9SYNE LSIDAGTYVAGFSHLRSRGCAPRDMSAQALTAYNQLLDYVINSLG 165

D0CKD5_9SYNE LGLPTNAYIEALTYTRDRACAPRDMSPQALNEFKSYLDYAINALS 164

Q05Q58_9SYNE LGLPTGPYVEALIYTRDRACAPRDMSPQALNEYKSYIDYLINALS 164

Q05Q50_9SYNE LSIDAGTYVAGFANMRNDGCSPRDMSPQALTAYNTLLDYVINSLG 165

A4CSG2_SYNPV LRLPTAPYVEALTYTRDRACAPRDMSPQALNEFKAYLDYAINALS 164

Q05Z96_9SYNE LSIDAGTYVAGFSQMRNDGCAPRDMSPQALTSYNQLLDYVINSLG 165

Q05ZA4_9SYNE LSLPTGPYVEALTYTRDRACAPRDMSPQALNEFKSYLDYLINALS 164 α-Helix Blank

Fig. 8

Table 1

Stability parameters of FL-αC-PE and Tr-αC-PE associated with the GdmCl-induced denaturation at pH 7.0 and 25 ± 0.1 oCa

Probes (Fit parameter)

∆GD° (kcal mol-1 M-1) m (kcal mol-1 M-1) Cm (M)

FL-αC-PE Tr-αC-PE FL-αC-PE Tr-αC-PE FL-αC-PE Tr-αC-PE

Δε565 11.45 ± 0.34 10.11 ± 0.40 5.67 ± 0.44 5.77 ± 0.36 2.02 ± 0.43 1.75 ± 0.29

F350 11.77 ± 0.38 10.42 ± 0.66 6.13 ± 0.35 6.35 ± 0.22 1.92 ± 0.24 1.68 ± 0.22

F573 11.56 ± 0.23 10.16 ± 0.27 5.99 ± 0.56 5.83 ± 0.39 1.93 ± 0.35 1.74 ± 0.34

[θ]222 11.82 ± 0.21 10.47 ± 0.56 6.16 ± 0.49 6.41 ± 0.57 2.03 ± 0.27 1.77 ± 0.30

a A ± with a value is a mean of errors from the average of three independent measurements

Table 2

Hydrogen bonds and van der Waals Interactions that the N-terminal segment (1-31 residues) forms with atoms of the remaining polypeptide chain (32-164) of αR-PEa

a Residues forming hydrogen bonds are shown in bold letters

Residues involved in non-covalent interactions

Bond Length

1MetC 109GluO 3.30

1MetS 106ProC 3.43

1MetC 109GluC 3.47

109GluO 3.42

2LysN 105GlyC 3.48

2LysO 105GlyN 3.27

103GlyC 3.12

103GlyC 3.18

103GlyO 3.48

28GlnO 32GlnN 2.84

32GlnC 3.31

32GlnN 3.18

28GlnC 32GlnN 3.10

28GlnN 32GlnN 3.22

29GlyO 33ArgN 3.08

30Asn N 32GlnN 3.41

30AsnC 34AlaN 2.81

34AlaC 3.42

30AsnO 33ArgN 3.23

37ArgN 3.05

30AsnC 37ArgN 2.86

30AsnO 37ArgN 2.89

30AsnN 32GlnN 2.88

31IleN 32GlnC 3.00

31IleC 33ArgN 3.23

31IleC 34AlaN 3.29

32GlnC 2.62

31IleO 32GlnO 3.43

32GlnC 2.79

33ArgN 3.15

32GlnN 3.38

Table 3

Residues of the N-terminal segment (1-31 residues) involved

in the formation of hydrogen bonds

Serial number

Redidues involved in the hydrogen bond formation

1

6ThrN 3SerOγ1 2

7ThrN 3SerO 3

7ThrOγ1 3SerO 4

8ThrN 4ValO 5

8ThrOγ1 4ValO 6

8ThrOγ1 5IleO 7

9IleN 5IleO 8

10SerN 6ThrO 9

11AlaN 8ThrO 10

11AlaN 9IleO 11

12AlaN 8ThrO 12

12AlaN 9IleO 13

13GluN 9IleO 14

14AlaN 10SerO 15

15AlaN 12AlaO 16

15AlaO 13GluO 17

16GlyN 12AlaO 18

16GlyN 13GluO 19

17ArgN 12AlaO 20

17ArgN 15AlaO 21

20SerN 23AspOδ2 22

23GluN 20SerO 23

23GluN 21ThrO 24

24LeuN 20SerO 25

24LeaN 21hrO 26

25GlnN 21ThrO 27

25GlnO 21ThrOγ1 28

26SerN 22SerO 29

29GlyN 26SerO 30

30AsnN 27ValO

34

Lys2

Gly105

Gly29

Asn30

Gln28

Gln32

Arg33

Ile31

Wavelength, nm

350 400 450 500 550 600 650 700

εε εελ

λ

λ

λ

X 1

04,

M-1

Cm

-1

0

5

10

15

20

25

30FL(0 M GdmCl & pH 7.0)

Tr (0 M GdmCl & pH 7.0)

FL(6 M GdmCl & pH 7.0)

Tr (6 M GdmCl & pH 7.0)

Table of Content

Graphical abstract

35

Highlights

• Two isoform of αC-PE were compared for structure and stability.

• Denaturation of both FL-�C-PE and Tr-�C-PE is follows a two-state mechanism.

• FL-�C-PE is 1.4 kcal mol-1 more stable than the Tr-�C-PE.

• Truncation of 31-residue does not alter the structure of the remaining polypeptide.