45 JBSD Ubaid

PROOF COVER SHEETJournal acronym: TBSD

Author(s): Faizan Ahmad

Article title: Effect of sequential deletion of extra N-terminal residues on the structure and stability of yeast iso-1-

cytochrome-c

Article no: 848826

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Effect of sequential deletion of extra N-terminal residues on the structure and stability of yeast

iso-1-cytochrome-c

Shah Ubaid-ullaha, Md. Anzarul Haquea, Sobia Zaidia, Md. Imtaiyaz Hassana, Asimul Islama, Janendra K. Batrab,5 Tej P. Singhc and Faizan Ahmada*

aCentre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India;bImmunochemistry Lab, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India; cDepartment ofBiophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

(Received 19 July 2013; accepted 23 September 2013)

10 A sequence alignment of yeast cytochrome-c (y-cyt-c) with mammalian cyts-c shows that the yeast protein has afive residue long N-terminal extension. A question arises: Does this N-terminal extension play any roles in the stability,structure, and folding of the yeast protein? To answer this question, in silico and in vitro studies were carried out on thewild type (WT) protein and its five deletants (Δ(−5/−5), Δ(−5/−4), Δ(−5/−3), Δ(−5/−2), and Δ(−5/−1) where Δ denotesthe deletion and the numbers refer to the residues deleted, e.g. Δ(−5/−1) denotes the deletion of residues numbered from

15 −5 to −1 (TEFKA), while Δ(−5/−2) denotes the deletion of resides numbered from −5 to −2 (TEFK) and so on). Themain conclusion of the in silico study is that the order of stability of deletants and WT protein is Δ(−5/−4) > WT > Δ(−5/−3) > Δ(−5/−5) > Δ(−5/−1) ~ Δ(−5/−2). In vitro studies involved (i) measurements of thermody-namic stability of all proteins by differential scanning calorimetry and from sigmoidal curves of two different structuralproperties ([θ]222, a probe for detecting change in secondary structure, and Δε405, a probe for detecting alteration in the

20 heme environment), and (ii) characterization of all proteins by various spectral properties. The main conclusions of thein vitro studies are as follows: (i) The order of thermodynamic stability of all proteins is in excellent agreement with thatpredicted by in silico studies, and (ii) A sequential deletion of the N-terminal extension has no effects on proteinstructure and folding.

Keywords: yeast iso-1-cytochrome-c; protein stability; two-state denaturation; differential scanning calorimetry

25 Introduction

Cytochrome-c (cyt-c) is a membrane-bound electrontransport protein found in wide varieties of prokaryoticand eukaryotic organisms, shuttling electrons betweendifferent components of the respiratory chain (Bertini,

30 Cavallaro, & Rosato, 2006). It also plays a pivotal rolein the activation of apoptotic process in cells (Gao, Ren,Liou, Bao, & Zhang, 2013; Jiang & Wang, 2004). Sinceits discovery, cyt-c has served as a model system tostudy a wide range of biological phenomena, including

35 protein folding, stability, electron transport, and evolutionof proteins. Because of its ease in expression, purifica-tion, high stability, and the presence of heme, cyt-c hasbeen extensively used for various biophysical studies.Moreover, this small soluble protein exhibits reversible

40 temperature-, pH-, and chemical-induced denaturation.Therefore, it is considered as an appreciable experimentalsystem to relate primary sequences to its structure, func-tion, and stability (Agueci et al., 2007). Furthermore, thepresence of red-colored prosthetic group heme has

45 allowed cyt-c to be studied through a variety of spectro-scopic techniques (Bertini et al., 2006). This wealth of

information available makes it a suitable candidate tostudy the protein structure, folding, and function (Zaidi,Hassan, Islam, & Ahmad, 2013).

50We have been interested in understanding the role ofvarious conserved residues in structure, stability, andfolding of mammalian heart cyt-c from horse, cow, andgoat (Alam Khan et al., 2009; Alam Khan et al., 2010;Khan, Rahaman, & Ahmad, 2011; Moza, Qureshi, &

55Ahmad, 2003; Moza et al., 2006; Qureshi, Moza, Yadav,& Ahmad, 2003; Rahaman et al., 2008; Rahaman et al.,2013). One main conclusion of these studies is that thereexist two thermodynamically stable intermediates,namely molten and premolten globule states on the fold-

60ing/unfolding pathway of the protein. When amino acidsequences of mammalian cyts-c are aligned with thesequence of the yeast iso-1-cyt-c (y-cyt-c), it is observedthat the yeast protein contains a five-residue N-terminalextension (Lett, Rosu-Myles, Frey, & Guillemette, 1999).

65These amino acids residues are generally referred to asresidues −5 to −1, when using the eukaryotic numberingsystem for cyts-c (Figure S1). Presently, we areinterested in understanding the role of these five extra

*Corresponding author. Email: [email protected] Ubaid-Ullah, Md. ImtaiyazHassan and SobiaZaidi contributed equally to this work.

© 2013 Taylor & Francis

Journal of Biomolecular Structure and Dynamics, 2013http://dx.doi.org/10.1080/07391102.2013.848826

TBSD 848826 CE: LS QA: CL23 October 2013 Initial

mailto:[email protected]

http://dx.doi.org/10.1080/07391102.2013.848826

residues at the N-terminus in the structure, stability and5 folding of y-cyt-c. In this communication, we present in

silico and in vitro data to answer the question whetherone or more of these residues at the N-terminal extensionis (are) required for the structure and stability of theprotein.

10 Three-dimensional crystallographic structure of thewild-type (WT) y-cyt-c is known (Berghuis & Brayer,1992; Louie & Brayer, 1990). From this study, we deter-mined the interactions of each extra residue at the N-ter-minus with rest of the protein and predicted the stability

15 of the WT protein and its five deletants, Δ(−5/−5),Δ(−5/−4), Δ(−5/−3), Δ(−5/−2), and Δ(−5/−1). The orderof stability in terms of ΔGD°, the Gibbs free energychange associated with the equilibrium, native (N) state↔ denatured (D) state at 25 °C, that we observed is

20 Δ(−5/−4) > WT > Δ(−5/−3) > Δ(−5/−5) > Δ(−5/−1) ~Δ(−5/−2). Our in vitro experiments on the measurementsof thermal stability using differential scanning calorime-try (DSC) and conformational techniques, support the in

silico studies.

25 Materials and methods

Ampicillin, SDS, glycine, ammonium persulfate, tris-base, dNTPs were obtained from Amersham PharmaciaBiotech (Sweden). Oligonucleotide primers were suppliedby Eurofins Genomics (MWG) India Pvt. Ltd. Agarose,

30 bacto-tryptone and yeast extract were purchased from Hi-media Laboratories (India). Potassium ferricyanide waspurchased from Loba Chemical Company, India.Millipore syringe filters (0.22 μm) were purchased fromMillipore Corporation (USA). Chemicals like NaCl,

35 EDTA, and (NH4)2SO4 were bought from Merck (India).Sodium cacodylate trihydrate, SP-Sepharose, and dialysistubing were obtained from Sigma Chemical Co.(St. Louis, USA). All the other chemicals and reagentsused were of analytical-grade purchased from local

40 supplier.

Nomenclature scheme

The direct comparison of the sequences of y-cyt-c andh-cyt-c shows five additional residues at the N-terminalof the yeast protein. Using the vertebrate numbering sys-

45 tem these residues, Thr-Glu-Phe-Lys-Ala, are numberedfrom −5 to −1. In our study, we have deleted these resi-dues sequentially from the N-terminal. These deletantsare referred as; Δ(−5/−5), Δ(−5/−4), Δ(−5/−3), Δ(−5/−2), and Δ(−5/−1) where Δ denotes the deletion and the

50 numbers refer to the residues deleted, e.g. Δ(−5/−1)denotes the deletion of residues numbered from −5 to−1 (i.e. TEFKA), while Δ(−5/−2) denotes the deletionof resides numbered from −5 to −2 (i.e. TEFK) and soon (Lett et al., 1999).

55In silico structural analysis

The ribbon diagram of y-cyt-c was drawn in PyMOL(DeLano, 2002) using the atomic coordinates of crystalstructure of y-iso-1-cyt-c (PDB code: 1YCC). Contactsbetween the atoms of the five extra N-terminal residues

60to the atoms of other residues were determined using thecontact program from the CCP4 package (Emsley &Cowtan, 2004).

Preparation of the deletants

The expression plasmid pBTR1 (WT) was a kind gift65from Dr. Bruce E. Bowler, University of Montana, USA.

pBTR1(WT) contains a CYC1 gene encoding y-cyt-c andCYC3 gene encoding heme-lyase, essential for the cova-lent attachment of the heme to the polypeptide. Since theWT protein has free cysteine at position 102 that causes

70dimerization upon denaturation (Cohen & Pielak, 1994;Cutler, Pielak, Mauk, & Smith, 1987), Cys102 was,therefore, replaced by serine (Cys102Ser) to prevent theformation of intermolecular disulfide bond which maycomplicate interpretation of physical data. The CYC1

75gene is cloned using BamHI restriction sites that lieupstream and downstream of the start and stop codonsof the CYC1 gene as shown in vector map (Figure S2).

To delete each residue from the N-terminal, we haveincorporated restriction sites MluI at the start and XhoI at

80the end of the gene by site directed mutagenesis usingthe earlier procedure (Ho, Hunt, Horton, Pullen, &Pease, 1989). The desired restriction sites were incorpo-rated at the ends of the CYC1 gene by PCR amplifyingthe DNA encoding CYC1 in two fragments AB and CD

85using two sets of primers Ub-1 and Ub-3, and Ub-2 andUb-4, respectively (Figure S3(A)). The primer Ub-1annealed to CYC1 gene upstream to the 5’ BamHI site.The primers Ub-2 and Ub-3 were reverse and comple-mentary of each other and carried the site MluI, while

90Ub-4 carried sites XhoI and BamHI as shown in the pri-mer sequences in lower case (Table S1). With primerUb-4, we also shifted BamHI site 16 bases upstream.The amplified fragments AB and CD were purified byQaigen kit for PCR purification. The fragments AB and

95CD were fused by denaturing and used as template inthe subsequent primer extension reaction. The overlapallowed one strand from each fragment to act as a primeron the other, and extension of this overlap resulted in thedesired mutant product (mutant fusion product). The

100fusion product was further amplified using primers Ub-1and Ub-4 as illustrated in the cloning strategy (Figure S3(A)). The amplified product and the vector were digestedwith BamHI and ligated. The ligation mixture was trans-formed into Escherichia coli, DH5α cells. The transfor-

105mants were screened, and the insertion of restriction siteswas confirmed by restriction digestion and subsequently

2 S. Ubaid-ullah et al.


by DNA sequencing. The modified vector thus generatedwas named as pBTR1SU and was further used to deletethe N-terminal residues from WT y-cyt-c (Figure S3(B)).

5 To sequentially delete the N-terminal residues, wedesigned primers Ub-9 to Ub-5. Each primer had MluIrestriction site and was 3 nucleotides shorter than thepreceding one (Table S2). Amplification was carried outwith each primer separately and using primer Ub-4 as

10 reverse primer. The amplified product was purified andinserted into the vector pBTR1SU using MluI and XhoIsites. The deletions were later confirmed by sequencing.

Expression and purification

Specific clone of each deletant and pBTR1 (WT) was15 transformed into BL21 (λe3) strain of E. coli

(Stratagene) for protein expression. The proteins werepurified by the protocol described earlier (Patel, Lind,& Pielak, 2001) with slight modifications. Fractioncontaining y-cyt-c was allowed to bind to SP-Sephar-

20 ose cation-exchange resin and was eluted with highsalt buffer (Na-phosphate buffer (pH 7.2) containing1 M NaCl). Fractions with absorbance ratio (A410/A280) greater than 4.0, considered as rich in cyt-c,were pooled. The purity of proteins was further con-

25 firmed by SDS-PAGE (see Figure S4).

Preparation of protein solutions

Heme iron in cyt-c can exist in both reduced form(Fe2+) and oxidized form (Fe3+). The reduced form hasa tendency to auto-oxidize upon denaturation, making

30 the process irreversible (Bixler, Bakker, & McLendon,1992). For these reasons, the oxidized form is bestsuited for equilibrium denaturation studies. All thepurified proteins (WT proteins and its deletants) wereoxidized by adding 0.1% potassium ferricyanide (Goto,

35 Takahashi, & Fink, 1990). The concentration of eachprotein was measured using molar absorption coefficient(ε) of 106.1 × 103 M−1 cm−1 at 410 nm (Margoliash &Frohwirt, 1959). Solutions for all measurements wereprepared in 30 mM sodium cacodylate buffer (pH 6.0)

40 containing 0.1 M NaCl.

Absorbance measurements

Absorption spectra were measured in Jasco-660 spec-trophotometer equipped with a Peltier-type temperaturecontroller (ETCS761). The protein concentrations used

45 were 5–7 and 80 μM for spectral measurements inthe range 700–300 and 800–600 nm, respectively. Acell of path length of 1.0 cm was used. Baseline cor-rections were always carried out with respectiveblank.

50Circular dichroism (CD) measurements

CD spectra were recorded in Jasco-715 spectropolarimeterequipped with Peltier-type temperature controller(PTC348WI). The far-UV CD spectra (250–200 nm) wererecorded using a 1 mm path length cell, whereas 1 cm

55path length cell was used for the near-UV (300–270 nm)and Soret (450–370 nm) CD spectral measurements. Eachspectrum was corrected for contribution due to the blanksolution. The protein concentration used was in the range14–18 μM. The raw CD data were expressed in terms of

60mean residue ellipticity, [θ]λ (deg cm2 dmol−1) using therelation,

½h�k¼ Mohk=10lc (1)

where θλ is the observed ellipticity in millidegrees, Mo is65the mean residue weight of the protein, c is concentra-

tion in mg ml−1, and l is the path length of the cell incentimeters.

Fluorescence spectra measurements

Trp fluorescence spectra of proteins in native conditions70were measured in Jasco spectrofluorimeter (Model FP-

6200) using 3 mm quartz cell at 25 ± 0.1 °C. The tem-perature of the cell was maintained by circulating waterfrom an external thermostated water bath. The proteinconcentration used was 7 μM. Excitation wavelength was

75280 nm, and the emission spectra were recorded in thewavelength range 300–400 nm.

Thermal denaturation measurements

Heat-induced denaturations were carried out in Jascospectropolarimeter (J-715) and Jasco UV–Vis Spectro-

80photometer (J-660) at a heating rate of 1 °C min−1.This heating rate was found adequate to reach equilib-rium. Changes in CD signal at 222 nm and absorp-tion at 405 nm were recorded as a function oftemperature in the range 20–80 °C. At the end of

85each experiment, reversibility was checked by coolingthe denatured protein and then matching the spectrumof the protein with the spectrum taken before heatingthe protein solution. The raw CD data were convertedinto [θ]222 using Equation (1), and the raw absorption

90data were converted into Δε405 (ε405 at any tempera-ture T−ε405 at 25 °C). In order to obtain values ofTm (midpoint of denaturation), ΔHm (van’t Hoffenthalpy change at Tm) and ΔCp (constant pressureheat capacity change), entire transition curve of each

95protein was fitted according to the relation (Swint &Robertson, 1993),

Stability of yeast cytochrome-c 3


Where yobs is the experimentally observed optical prop-erty of the protein at temperature T (K), yn and yd arethe optical properties of the native (N) and denatured

5 (D) molecules at same temperature, and R is universalgas constant. It should be noted that Equation (2)assumes that the heat-induced denaturation is a two-stateprocess.

DSC measurements

10 DSC measurements were performed in a MicroCal VPDSC having a cell volume of 0.5 ml. The scanning ratewas 1 °C min−1 in the temperature range 20–80 °C. Theprotein and buffer solutions were degassed for 15 minprior to loading into DSC cells. First, buffer was filled in

15 both the sample and reference cells, and a series of scanswere made to determine the buffer-buffer baseline. Afterthis, protein solution was filled in the sample cell andsubjected to the upward scan from 20 to 80 °C. Eachsample was scanned in triplicate at a protein concentra-

20 tion of 1.5 mg ml−1. Data from scanning calorimeterwere analyzed using the Origin software supplied by

MicroCal, Inc. The buffer-buffer baseline was subtractedfrom the sample data, and the data were normalized fromcalories per degree Celsius to calories per mole per

25Kelvin. The baselines were created with the softwarebaseline function.

Results and discussion

In-silico studies

Crystal structures of y-iso-1-cyt-c are known at resolu-30tions of 2.8, 1.9, and 1.23 Å (Berghuis & Brayer, 1992;

Louie & Brayer, 1990; Louie, Hutcheon, & Brayer,1988). For our studies, we have used the co-ordinates ofthe protein structure refined at 1.23 Å resolution. It isbecause these coordinates are more accurate as this struc-

35ture is refined at a higher resolution and also the fiveextra N-terminal residues were defined more clearly inthe electron density. These extra residues were foundcontributing to the stability of the structure significantly.As seen from Figure 1, several interactions are formed

40within these five extra residues as well as they alsointeract with the amino acid residues of the rest of the

Monofor

print

colour

online

Figure 1. Structure of y-cyt-c showing interaction of the extra five N-terminal residues with other proteins atoms. The overall struc-ture of y-cyt-c is shown in cartoon model. Five N-terminal residues (yellow), other protein atoms (green), and heme (cyan) are shownin ball and stick. Structure was drawn in PyMol using the atomic coordinates of 1YCC (Louie & Brayer, 1990).

Yobs ¼ðyn þ mnTÞ þ ðyd þ mdTÞ expfð½DCpððTm=T � 1Þ þ lnðT=TmÞÞ� � ½DHmðTm=T � 1Þ�Þ=Rg

1þ expfð½DCpððTm=T � 1Þ þ lnðT=TmÞÞ� � ½DHmðTm=T � 1Þ�Þ=Rg(2)



protein extensively. Table 1 shows all the observed inter-actions involving the extra five N-terminal residues. Asseen from this Table 1 and Figure 1, it is evident that

5the N-terminal extension (residues at positions −5 to −1)appears to contribute appreciably to the stability of WTprotein. The main interactions of the N-terminal extrafive residues in the WT protein with the rest of theprotein atoms are provided by Thr-5 through one H-bond

10between Thr-5(Oγ1) and Asn62(Oε1) (Thr-5 Oγ1(H)–Asn62 Oδ1 = 3.04 Å) and another with Glu61(Oε1) (Thr-5 Oγ1(H) – Glu61 Oε1 = 3.13 Å) as well as several vander Waals contacts involving atoms of Asn62(Cγ, Oδ1,Nε2) and Glu61(Oε1) with Thr-5(Cβ,Oγ1, Cγ2). All these

15interactions help in further stabilizing the structure. Theintrasegment H bonds are formed between Thr-5(Oγ1H)and Glu-4(NH) and Phe-3(NH) that stabilize the structureof the N-terminal pentapeptide. Thus, the removal of res-idue Thr-5 that gives deletant Δ(−5/−5) causes a consid-

20erable loss to the stability of protein structure as well asto the stability of the N-terminal pentapeptide.

On removal of Thr-5, the new negatively chargedN-terminal residue, Glu-4 may destabilize the structurebecause of its proximity to the protruding negatively

25charged residues, Asp60, Glu62, Glu66, and Glu88 fromthe neighbouring regions of the protein. However, whenfirst two residues from the N-terminus, Thr-5 and Glu-4are removed (deletant Δ(−5/−4)), the effect of thenegative charge from the N-terminus is fully removed.

30As a result, the new N-terminus consisting of residues,Phe-3–Lys-2–Ala-1 will be attracted toward negativelycharged face of the neighboring α-helix, the neighboringα-helix, thus stabilizing the protein structure substan-

Table 1. List of interactions offered by extra five N-terminalresidues.

Source atoms Target atoms Distance (Å)a

−5(Thr)N −4(Glu)–N 3.39C −4(Glu)–Cα 3.83

−4(Glu)–Cβ 3.31−4(Glu)–Cγ 3.31

O −4(Glu)–Cγ 3.24−4(Glu)–Cα 2.92

Cβ−4(Glu)–N 2.8162(Asn)–Cγ 3.4862(Asn)–Oδ1 2.8962(Asn)–Nδ2 3.25

Oγ1−4(Glu)–N 2.90−3(Phe)–N 3.01−3(Phe)–Cα 3.36−3(Phe)–Cβ 3.10−3(Phe)–C 3.3561(Glu)–Oε1 3.1362(Asn)–Oδ1 3.04

Cγ2 61(Glu)–Oε1 2.9462(Asn)–Oδ1 3.2462(Asn)–Nδ2 3.49

−4(Glu)N −5(Thr)–N 3.39

−5(Thr)–Cβ 2.81−5(Thr)–Oγ1 2.90

Cα−5(Thr)–O 2.92

C −3(Phe)–Cα 2.52−3(Phe)–Cβ 3.47

O −3(Phe)–Cα 2.99Cβ

−5(Thr)–C 3.31Cγ

−5(Thr)–C 3.31−5(Thr)–O 3.24

−3(Phe)N −2(Lys)–N 3.49

−5(Thr)–Oγ1 3.01Cα

−4(Glu)–C 2.52−4(Glu)–O 2.99−5(Thr)–Oγ1 3.36

C −2(Lys)–Cβ 3.44O −2(Lys–Cα 2.87Cβ

−4(Glu)–C 3.47−2(Lys)–N 3.41−5(Thr)–Oγ1 3.1062(Asn)–Oδ1 3.32

Cγ−2(Lys)–N 3.22

Cδ1−2(Lys)–N 3.4892(Asn)–Oδ1 3.18

Cδ2 61(Glu)–Oε1 3.0961(Glu)–Cδ 3.1761(Glu)–Oε2 3.47

Cε1 92(Asn)–Oδ1 3.46Cε2 61(Glu)–Oε2 3.44−2(Lys)N −3(Phe) N 3.49

−3(Phe)–Cβ 3.41−3(Phe)–Cγ 3.22−3(Phe)–Cδ1 3.48

Cα−3(Phe)–O 2.87

C −1(Ala)–C 3.05

(Continued)

Table 1. (Continued).

Source atoms Target atoms Distance (Å)a

−1(Ala)–O 3.26O 92(Asn)–Cβ 3.14

92(Asn)–Cγ 3.1792(Asn)–Nε2 3.57−1(Ala)–Cα 2.87−1(Ala)–C 3.06−1(Ala)–O 3.39

Cβ−3(Phe)–C 3.44−1(Ala)–N 3.06

−1(Ala)Cα

−2(Lys)–O 2.87C −2(Lys)–C 3.05

−2(Lys)–O 3.061(Gly)–C 3.47

O −2(Lys)–C 3.26−2(Lys)–O 3.391(Gly)–Cα 2.89

Cβ 1(Gly)–N 3.08N −2(Lys)–Cβ 3.06

aDistance was calculated using the contact program of the CCP4 pack-age (Emsley & Cowtan, 2004).



tially. That is, the deletant Δ(−5/−4) will be more stable5 than WT protein.

Phe-3 forms several van der Waals interactionsthrough its side chain with the side chains of Glu61,Asn62, Asn92, Ile95, and Thr96 (Table 1). There appearsto be a binding pocket-like structure for the side chain of

10 Phe-3, giving rise to strong stabilizing effect of this resi-due. Thus, when Thr-5–Glu-4–Phe-3 segment is removedin the deletant Δ(−5/−3), the main stabilizing effects ofThr-5 and Phe-3 are removed leading to the considerable

reduction in stability. However, the new N-terminal resi-15due, Lys-2, might still interact with the negatively

charged surface of the neighboring helix. This electro-static attraction will contribute to stability, but there maystill be an overall loss, and hence, this deletant will beless stable than the WT protein.

20The H-bond between Lys-2(O) and Asn92(Nδ2) andvan der Waals interactions between Lys-2(NH) andAsn92(Nδ2) and other van der Waals forces may also con-tribute to the stability of protein structure (see Table 1).

(A) (B)

(C) (D)

(E) (F)

Wavelength, nm

400 500 600 700-2

0

2

4

6

8

10

12

WT

Δ(−5/−5)

Δ(−5/−4)

Δ(−5/−3)

Δ(−5/−2)

Δ(−5/−1)

Wavelength, nm

600 650 700 750 8000

2

4

6

8

10

12

14

WT

Δ

(-5/-5)

Δ

(-5/-4)Δ (-5/-3)Δ

(-5/-2)

Δ

(-5/-1)

Wavelenth, nm

200 210 220 230 240 250

-14

-12

-10

-8

-6

-4

-2

0

2

4

6

8

WT

Δ

(-5/-5)

Δ

(-5/-4)Δ (-5/-3)Δ

(-5/-2)

Δ

(-5/-1)

Wavelenth, nm

270 275 280 285 290 295 300-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

WT

Δ

(-5/-5)

Δ

(-5/-4)Δ (-5/-3)Δ

(-5/-2)

Δ

(5/-1)

Wavelength, nm

390 400 410 420 430 440 450-5

-4

-3

-2

-1

0

1

2

3

WT

Δ

(-5/-5)

Δ

(-5/-4)Δ (-5/-3)Δ

(-5/-2)

Δ

(-5/-1)

Wavelength, nm

300 320 340 360 380 400

Flu

ore

scen

ce I

nte

nsi

ty

-2

0

2

4

6

WT

Δ

(-5/-5)

Δ

(-5/-4)Δ (-5/-3)Δ

(-5/-2)

Δ

(-5/-1)

Monofor

print

colour o

nline

Figure 2. Structural characterization of WT y-cyt-c and its deletants at pH 6.0 and 25 °C. (A) UV–Vis absorbance spectra, (B) Visi-ble spectra, (C) Far-UV CD spectra, (D) Near-UV spectra, (E) Soret CD spectra, and (F) Trp fluoresence spectra.



Thus, the deletant Δ(−5/−2) will be less stable than the5 deletant Δ(−5/−3).

As seen from Table 1, Ala-1 forms several van derWaals interactions with Lys-2 and Gly1. Thus, it isexpected that the stability of the deletant Δ(−5/−1) willnot be significantly different from that of the Δ(−5/−2),

10 because Lys-2 that was involved in making van der Wa-als interactions with Ala-1, was removed in this deletant.Therefore, when all the five residues are removed, thestabilizing contributions of N-terminal segment are lostthus making the protein molecule less stable than the

15 WT.

Structural characterization

From the analysis of the three-dimensional structure ofthe protein, it is concluded that the order of the stabilityof WT protein and its deletants is as follows: Δ(−5/

20 −4) > WT > Δ(−5/−3) > Δ(−5/−5) > Δ(−5/−2) ~ Δ(−5/−1). It should, however, be noted that this conclusion isbased on the assumption that deletions of these residuesfrom N-terminal do not perturb the overall conformationof the protein molecule. In order to validate our assump-

25 tion, we prepared deletants by sequentially removingthese extra residues. We successfully purified andcharacterized each deletant and compared their structuralproperties using various spectroscopic techniques. Thefar-UV CD spectrum provides information about second-

30 ary structure of a protein. Figure 2(C) shows the far-UVCD spectra of WT y-cyt-c and its deletants. The far-UVCD spectrum of y-cyt-c, revealed negative Cotton effectsat 208 and 222 nm, as expected for an α-protein(Figure 2(C)). The far-UV CD spectrum obtained for

35 WT protein is in agreement with the one previouslyreported (Herrmann & Bowler, 1997). A far-UV CDspectrum can be analyzed to determine secondarystructure content of the protein with various softwareavailable (Kelly, Jess, & Price, 2005; Louis-Jeune, And-

40 rade-Navarro, & Perez-Iratxeta, 2011). Reliability ofanalysis using these software, indeed, depends on theaccuracy of data up to 190 nm. Due to experimental

constraints, we could obtain reliable data up to 200 nmonly, which are shown in Figure 2(C). However, one

45may determine α-helical content of a protein from theCD value at 222 nm (Correa & Ramos, 2009). We haveused the procedure of Morrisett, David, Pownall, andGotto (1973) to determine the amount of α-helix in eachprotein from its observed [θ]222 value, which is given in

50Table 2. To check whether the estimates of α-helix inthese proteins are correct, we have used the procedure ofGreenfield and Fasman which estimates α-helical contentfrom the measured value of [θ]208 (Greenfield & Fasman,1969). Values of α-helix in all proteins, thus obtained,

55are shown in Table 2. An agreement between α-helicalcontent using two different methods provides confidencein our estimation of the secondary structure of pro-teins (Correa & Ramos, 2009). Most importantly, valuesof α-helical content in WT protein is in excellent agree-

60ment with that (40%) reported from the studies of thecrystal structure (Berghuis & Brayer, 1992; Louie &Brayer, 1990; Louie et al., 1988). Since no crystal struc-ture studies were carried out for deletants, a comparison,such as in the case of WT protein, is therefore, not pos-

65sible. Work on the crystallization of all deletants is inprogress in our laboratory. It is seen in Figure 2(C) thatthe far-UV CD spectra of the deletants are, within exper-imental errors, comparable with CD spectrum of WTprotein suggesting negligible change in the secondary

70structure on the subsequent deletion of these extraresidues. Thus, it can be concluded that these residuesdo not have any effect on the formation of secondarystructure of WT y-cyt-c.

After examining the effect of these extra N-terminal75residues on secondary structure of y-cyt-c, we investi-

gated their effect in maintenance of the tertiary structure.Four probes were used to scrutinize different perspec-tives of the tertiary structure. Absorption spectrum in thevisible region of cyt-c is a highly sensitive probe for

80detecting any alteration in heme co-ordination or hemeenvironment (Margoliash & Schejter, 1966; Schejter &George, 1964). Owing its origin to porphyrin chromo-phore, the visible spectrum of the native ferri-cyt-c

Table 2. α-helical content of WT iso-1-cyt c and its deletants.

Proteinsa [θ]222 deg cm2 dmol−1% age ofα-helixb [θ]208 deg cm2 dmol−1

% age ofα-helixc

WT −12,230 ± 205 39.0 ± 0.4 −10,164 ± 150 38.3 ± 0.1Δ(−5/−1) −11,445 ± 192 36.3 ± 0.3 −9260 ± 137 38.2 ± 0.1Δ(−5/−2) −10,940 ± 178 35.7 ± 0.3 −8945 ± 157 35.2 ± 0.2Δ(−5/−3) −10,882 ± 165 35.6 ± 0.3 −9055 ± 152 35.3 ± 0.1Δ(−5/−4) −11,200 ± 170 36.4 ± 0.3 −9025 ± 127 35.1 ± 0.3Δ(−5/−5) −11,690 ± 180 37.6 ± 0.3 −10,129 ± 110 35.8 ± 0.1

aWT and its deletants all have C102S modification.bα-helical content at 222 nm was calculated using equation given by Morrisett et al. (1973).cα-helical content at 208 nm was estimated using equation given by Greenfield & Fasman (1969).



displays the characteristic Soret band at 409 nm and a5 weaker Q band at 528 nm (Dixon, Hill, & Keilin, 1931;

Silke Oellerich & Hildebrandt, 2002). Another band inthe absorption spectrum at 695 nm is diagnostic forMet80-Fe ligation, and weakening or disappearance ofthis band suggests conformational or structural perturba-

10 tion in the protein molecule (Schejter & George, 1964;Stellwagen & Cass, 1974). We observed almost overlap-ping absorbance spectra in the wavelength range 800 –

300 nm (highly sensitive to oxidation and ligation stateand heme environment (Margoliash & Schejter, 1966;

15 Schejter & George, 1964; Stellwagen & Cass, 1974) ofthe WT and deletants (Figure 2(A) and (B)), suggestingthat a sequential deletion of the N-terminal extensiondoes not have any effect on heme co-ordination, includ-ing Met80-Fe interaction, or heme environment in WT

20 protein.Aromatic residues (one Trp, four Phe and five Tyr

residues) and two thioether bonds present in y-cyt-ccontribute to the near UV-CD spectrum (320–270 nm)of the protein (Davies et al., 1993). The near-UV CD

25 spectrum of cyt-c shows negative cotton effects at 282and 289 nm, attributed to the interaction of Trp59 withone heme propionate, a signature of natively foldedcyts-c (Davies et al., 1993; Moore & Pettigrew, 1990).Figure 2(D) shows the effect of deletion on the envi-

30 ronment of aromatic amino acid residues with respectto the WT. As seen in this figure, no considerablechange in the CD spectrum of WT protein wasobserved on deletion of the five N-terminal residues.This observation suggests that the N-terminal extension

35 does not have any effect on the environment of Trp/Tyr/ Phe residues in cyt-c.

To further verify the effect of deletion on tertiarystructure, we measured Soret CD spectra (450–390 nm)of WT protein and its deletants, and the results are shown

40 in Figure 2(E). Soret CD spectrum of y-cyt-c has beenproposed to arise from the interaction of heme ring withnearby polarizable groups and from protein-induceddeformation of heme (Blauer, Sreerama, & Woody, 1993;Hsu & Woody, 1971). CD band at 416 nm is highly

45 sensitive to the strength of heme-Met80 and Phe82-hemeinteraction (Pielak, Oikawa, Mauk, Smith, & Kay, 1986;Santucci & Ascoli, 1997), and any alteration in this bandsuggests structural transition (Santucci & Ascoli, 1997;Takeda, Takahashi, & Batra, 1985). Soret CD spectra

50 (Figure 2(E)) of the deletants are almost coincident(overlapping within experimental error) on thespectrum obtained for the WT protein suggesting nochange in heme structure and heme-axial ligandsinteraction.

55 Another probe used for measuring the change in ter-tiary structure was Trp fluorescence which in cyt-c isexceedingly sensitive for overall conformation of theprotein. In the native y-cyt-c, fluorescence of single

Trp (at position 59) is strongly quenched by the heme,60situated at a distance of 10 Å, attributable to fluores-

cence resonance energy transfer to heme group (Das,Mazumdar, & Mitra, 1998; Tsong, 1974). Thus, Trp fluo-rescence quenching is insightful to heme pocket distor-tion and thereby monitors tertiary structure. Trp

65fluorescence spectra of the WT protein and its mutantsare shown in Figure 2(F). It is seen in this figure that thefluorescence intensity of all the deletants is almost negli-gible as observed in the case of the WT cyt-c, suggestingthat the Trp-heme distance remained unperturbed on

70deletions. Thus, this probe also substantiates that tertiarystructure is unaffected due to the deletion of extra N-ter-minal residues.

Measurements of secondary and tertiary structuralchanges using all the four probes led us to conclude that

75the extra N-terminal residues of y-cyt-c are not contribut-ing in maintenance of secondary and tertiary structure ofthe protein. Thus, these observations support ourassumption that the sequential deletion of the N-terminalextension does not lead to any change in conformation

80of the y-cyt-c.

Measurements of thermodynamic stability

The in silico studies have predicted that the order ofstability of the WT protein and its deletants isΔ(−5/−4) > WT > Δ(−5/−3) > Δ(−5/−5) > Δ(−5/

85−2) ~ Δ(−5/−1). In order to confirm this order of stabil-ity, we measured the stability of all proteins. Heat-induced transition curves of all the proteins (WT proteinand its deletants) were measured at pH 6.0. It should benoted that each denaturation curve was checked for

90reversibility and found to be reversible. Each denatur-ation curve was analyzed to estimate ΔGD° associatedwith the transition, N state ↔ D state at 25 °C and pH6.0. It should be noted that this analysis assumes thetransition between N and D states to be a two-state

95process. One of the methods to validate if the processfollows a two-state mechanism is to compare the thermo-dynamic parameters obtained for the same transition bytwo different probes (Dill & Shortle, 1991). We carriedout heat-induced denaturation and monitored changes in

100Δε405 and [θ]222 in the temperature range 20–80 °C (Fig-ure 3). To estimate Tm, ΔHm, and ΔCp, each transitioncurve was fitted to Equation (2) (Swint & Robertson,1993). The values of Tm, ΔHm, and ΔCp thus obtainedare given in Table 3. The Gibbs-free energy change at

10525 °C (ΔGDo) was estimated using values of Tm, ΔHm,

and ΔCp in Gibbs–Helmholtz equation,

DGDðTÞ ¼ DHmTm � T

Tm

� �

� DCp ðTm � TÞ þ T lnT

Tm

� ��

(3)



Values of ΔGDo of all proteins thus obtained are

5 given in the Table 3. As it can be seen in Table 3, allthermodynamic parameters obtained for each protein byboth optical probes are in excellent agreement witheach other, supporting our assumption of two-statemechanism.

10 To validate our thermodynamic parameters obtainedfrom spectroscopic methods discussed in the precedingparagraph (see Table 3), DSC measurements were alsocarried out at pH 6.0. DSC studies of WT protein and itsdeletants were carried out under the same experimental

15 conditions that were used during the measurements ofheat-induced denaturation of these proteins using opticalprobes (Figure 3). Another purpose for the DSC studieswas to validate our assumption of the two-state mecha-nism which was used in the analysis of heat-induced

20 denaturation curves shown in Figure 3. Figure 4 showsthe results of DSC measurements of the WT proteinand its deletants. These endotherms were analyzed forΔHcal (calorimetric enthalpy change on thermal

denaturation), Tm and ΔCP. These values from calorimet-25ric measurements are given in Table 4. It is seen in this

table that there is an excellent agreement between calori-metric values and those obtained from the analysis ofdenaturation curves monitored by spectroscopic tech-niques. It is noteworthy that the values of the thermody-

30namic parameters of the WT protein, obtained here, arein excellent agreement with the values reported earlier(Liggins, Sherman, Mathews, & Nall, 1994; Pollock,Rosell, Twitchett, Dumont, & Mauk, 1998). This agree-ment led us to conclude that (a) Thermodynamic parame-

35ters obtained from the analysis of results shown inFigure 3 are accurate and valid and (b) Our assumptionof two-state mechanism for the heat induced denaturationof the WT protein and its deletants seems to be valid.

The endotherms shown in Figure 4 can also be40used to determine ΔHvH. The values of ΔHvH of all

the proteins are obtained with an assumption that eachendotherm shown in this figure represents a two-stateprocess. Values of ΔHvH thus obtained are given in

Temperature, oC

20 30 40 50 60 70 80

405

x 1

03, M

-1cm

-1

-4

-2

0

2

4

6

8

10

12

14

16

WT

(-5/-5)

(-5/-4)

(-5/-3)

(-5/-2)

(-5/-1)

Temperature, oC

20 30 40 50 60 70 80

222

x 1

0-3

, d

eg c

m2 d

mol-1

-14

-12

-10

-8

-6

-4

WT

(-5/-5)

(-5/-4)

(-5/-3)

(-5/-2)

(-5/-1)

A B

Monofor

print

colour

online

Figure 3. Thermal denaturation curves of WT protein and its deletants monitored by following changes in Δε405 (A) and [θ]222 (B)at pH 6.0. In order to maintain clarity, data points are not shown on the curve.

Table 3. Thermodynamic parameters obtained from thermal denaturation of WT protein and its deletants at pH 6.0.

Probe used

Thermodynamicparameters Proteinsa,b,c

[θ]222 (deg cm2 dmol−1) Δε405 (M−1 cm−1)

Tm (oC)ΔHm

(kcal mol−1)ΔCp

(kcal mol−1 K−1)ΔGD

o

(kcal mol−1) Tm (oC)ΔHm

(kcal mol−1)ΔCp


o

(kcal mol−1)

Δ(−5/−1) 52.3 ± 0.3 69 ± 2 1.52 ± 0.15 3.98 ± 0.10 51.5 ± 0.2 68 ± 3 1.52 ± 0.22 3.96 ± 0.12Δ(−5/−2) 53.2 ± 0.4 65 ± 3 1.29 ± 0.12 3.99 ± 0.11 52.8 ± 0.3 65 ± 2 1.42 ± 0.23 3.81 ± 0.10Δ(−5/−3) 54.5 ± 0.3 72 ± 2 1.51 ± 0.14 4.41 ± 0.16 54.8 ± 0.2 73 ± 2 1.61 ± 0.22 4.38 ± 0.12Δ(−5/−4) 58.2 ± 0.2 82 ± 3 1.25 ± 0.12 5.94 ± 0.12 58.5 ± 0.3 81 ± 2 1.35 ± 0.20 5.81 ± 0.12Δ(−5/−5) 53.6 ± 0.4 69 ± 2 1.40 ± 0.08 4.23 ± 0.10 53.2 ± 0.3 70 ± 3 1.25 ± 0.18 4.48 ± 0.14Wild type 56.3 ± 0.4 75 ± 2 1.51 ± 0.10 4.90 ± 0.11 56.8 ± 0.2 76 ± 3 1.61 ± 0.20 4.77 ± 0.10

a± With each parameter represents an error from the mean of errors from the triplicate measurementsbMeasurements were performed in 30 mM sodium cacodylate buffer (pH 6.0) containing 0.1 M NaClcWT and its deletants all have C102S modification.



Table 4. The ratio of ΔHvH–ΔHcal (ΔHvH/(ΔHcal) is

5 important, as it provides an accurate thermodynamictest of the mechanistic complexity of folding. Theratio (ΔHvH/(ΔHcal) when comes out to be unity sug-gest an ideal two-state transition. A value less than ormore than unity indicates the presence of intermediates

10 or reversible intermolecular association, respectively(Sturtevant, 1987). It is seen in Table 4 that this ratiois very close to unity, for the deviation is within 1–5%. Such small deviations, as suggested by Privalovand Khechinashvili (1974), can be neglected and the

15 denaturation may be considered as a co-operative two-state transition between the N and D states, thusverifying our two-state mechanism. That is, the heat-induced denaturation of WT protein and its deletants,indeed, follows a two-state mechanism.

20 As discussed above (see paragraphs 1–4 under thesection “Results and Discussion”), it is evident from thecrystal structure of y-cyt-c that the five-residue long

N-terminal extension must contribute to the stability ofWT protein. From our in silico studies, we have pre-

25dicted that the order of stability of WT protein and itsdeletants should be Δ(−5/−4) > WT > Δ(−5/−3) > Δ(−5/−5) > Δ(−5/−1) ~ Δ(−5/−2). It is seen in Tables 3 and 4that stability of all proteins in terms of ΔGD°, obtainedfrom in vitro studies, confirms the in silico prediction. It

30is also seen in these tables that stability of WT proteinand its deletants in terms of Tm, follows the same orderas predicted by in silico studies as well. It must, how-ever, be noted that it is dangerous to draw conclusionsabout protein stability, defined as ΔGD° for the reaction

35N state ↔ D state, based solely on Tm values. Forinstance, DSC measurements gave values of 78.5, 90.0,and 100.0 °C for Tm, of hens’ egg lysozyme, carp par-valbumin and bovine trypsin inhibitor, respectively,whereas the ΔGD° values are identical, within experi-

40mental error, for all these proteins (Oobatake & Ooi,1993).

Temperature, oC

20 30 40 50 60 70 80

Cp, kcal m

ol-1

oC

-1

4

6

8

10

12

14WT

(-5/-5)

(-5/-4)

(-5/-3)

(-5/-2)

(-5/-1)

30 40 50 60 70 80

5

10

Cp

(kca

l/m

ole

/oC

)

Temperature (oC)

(-5/-1)

Cp

Monofor

print

colour o

nline

Figure 4. DSC endotherms of WT y-cyt-c and its deletants in the native buffer at pH 6.0. In order to maintain clarity, baselines arenot drawn. The inset is a representative endotherm showing the pre- and post-transition base lines and the value of ΔCP.

Table 4. Thermodynamic parameters of unfolding transition of WT and its deletants by DSC at pH 6.0.

Thermodynamic parametersProteinsa,b,c Tm (oC)

ΔHcal

(kcal mol−1)ΔHvH

(kcal mol−1)ΔHvH/ΔHcal

ΔCp


o

(kcal mol−1)

Δ(−5/−1) 52.0 ± 0.2 69 ± 2 70 ± 3 1.01 1.45 ± 0.10 4.06 ± 0.12Δ(−5/−2) 52.4 ± 0.1 68 ± 2 69 ± 3 1.01 1.47 ± 0.12 3.98 ± 0.10Δ(−5/−3) 53.7 ± 0.2 73 ± 3 77 ± 3 1.05 1.40 ± 0.08 4.59 ± 0.11Δ(−5/−4) 57.6 ± 0.2 86 ± 3 87 ± 3 1.01 1.45 ± 0.09 6.06 ± 0.14Δ(−5/−5) 52.4 ± 0.1 69± 2 73 ± 2 1.05 1.38 ± 0.08 4.17 ± 0.12Wild type 56.2 ± 0.2 74 ± 3 75 ± 3 1.01 1.33 ± 0.07 4.95 ± 0.12

a± With each parameter represents an error from the mean of errors from the triplicate measurementsbMeasurements were performed in 30 mM sodium cacodylate buffer (pH 6.0) containing 0.1 M NaClcWT and its deletants all have C102S modification.



Conclusions

From our in silico and in vitro studies, we are sure ofthe three things. (a) The five-residue N-terminal exten-

5 sion of the WT protein does not have any effect on thesecondary and tertiary structures of the WT protein. (b)The N-terminal extension contributes significantly to thethermodynamic stability of the protein. (c) The N-termi-nal extension does not have effect on the folding mecha-

10 nism of the protein, for the denaturation of the WTprotein and its deletant follows a two-state mechanism.

Acknowledgments

SU, MAH, and SZ are thankful to DBT, UGC, andICMR, respectively, for their fellowships. FA and MIH

15 gratefully acknowledge the financial support from DST(SB/SO/BB-71/2010 (G)). We thank Professor Bruce E.Bowler, University of Montana (USA) for providing usthe plasmid pBTR1(WT). We also thank Professor RajivBhat, Jawaharlal Nehru University (India) for his help

20 with the DSC measurements.

Supplementary material

The supplementary material for this paper is availableonline at http://dx.doi.10.1080/07391102.2013.848826.

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100doi:10.1007/s00018-013-1341-1

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