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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
International Journal of Biological Macromolecules 49 (2011) 616– 621
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journa l h o me pag e: www.elsev ier .com/ locate / i jb iomac
Acidic residue modifications restore chaperone activity of �-caseininteracting with lysozyme
A.A. Moosavi-Movahedia,b,∗, H. Rajabzadehc, M. Amanid, D. Nourouziane, K. Zarec,H. Hadia, A. Sharifzadeha, N. Poursasana, F. Ahmadf, N. Sheibanig
a Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iranb Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iranc Chemistry Department, Science and Research Branch, Islamic Azad University, Tehran, Irand Faculty of Medicine, Ardabil University of Medical Sciences, Ardabil, Irane Pasteur Institute of Iran, Karaj Highway, Tehran, Iranf Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, Indiag Department of Ophthalmology and Visual Sciences, and Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792-4673, USA
a r t i c l e i n f o
Article history:Received 3 April 2011Received in revised form 15 June 2011Accepted 19 June 2011Available online 22 July 2011
Keywords:Modified �-caseinLysozymeHydrophobicityWoodward’s Reagent KElectrostatic potentialAccessible surface area
a b s t r a c t
An important factor in medicine and related industries is the use of chaperones to reduce protein aggre-gation. Here we show that chaperone ability is induced in �-casein by modification of its acidic residuesusing Woodward’s Reagent K (WRK). Lysozyme at pH 7.2 was used as a target protein to study �-caseinchaperone activities. The mechanism for chaperone activity of the modified �-casein was determinedusing UV–vis absorbencies, fluorescence spectroscopy, differential scanning calorimetry and theoreticalcalculations. Our results indicated that the �-casein destabilizes the lysozyme and increases its aggre-gation rate. However, WRK-ring sulfonate anion modifications enhanced the hydrophobicity of �-caseinresulting in its altered net negative charge upon interactions with lysozyme. The reversible stability oflysozyme increased in the presence of WRK-modified �-casein, and hence its aggregation rate decreased.These results demonstrate the enhanced chaperone activity of modified �-casein and its protective effectson lysozyme refolding.
Casein belongs to a family of phosphoproteins, which are suit-able for use in a variety of food products with protective roles inmilk and in the mammary gland [1–3]. Caseins exhibit unique struc-tural characteristics. They are neither globular nor fibrous proteinsin nature and, they don’t have well-defined secondary and tertiarystructures [4]. Caseins are extremely flexible and are essentiallyunfolded [5]. There are specific differences among the four maincaseins including charge, hydrophobicity, and calcium sensitivity[6]. �-casein and �s-casein have molecular chaperone-like prop-erties [7,8]. Caseins are also shown to decrease turbidity of wheyproteins under stress conditions [9]. �s-casein exhibits a consid-erable anti-aggregation activity [4,7,8]. However, the mechanisms
involved, and more specifically the contribution of charge–chargeinteractions, in chaperone activity needs further investigation. Theprimary structure of �-casein has a highly amphiphilic character[10], which is crucial for its function in aggregation and micel-lization processes [11]. �-casein also acts as a surfactant moleculein solution [3] and is considered a natural detergent [3]. There isa short N-terminal hydrophilic polar domain in �-casein chain,which carries most of the protein’s net charge (mainly negative),and a prominent C-terminal hydrophobic domain [12,13]. Recently,a novel function for �-casein has been reported, namely a molecularchaperone that protects many proteins against heat and chem-ical induced aggregation [14–17]. The �-casein hydrophilic andhydrophobic domains enhance solubility in aqueous medium andallow binding of hydrophilic molecules, respectively [18,19].
The approach applied throughout this study was that of thechemical modification of �-casein, especially that of carboxylresidues (aspartate and glutamate residues on the surface), whichwere specifically selected for their effects on diminishing lysozymeaggregation. The carboxylic side chains of glutamate and aspartateresidues on the �-casein surface were modified using Woodward’sReagent K (WRK), an isoxosolium salt [20–28]. The reaction of WRKwith a carboxylate is outlined in Scheme 1 [29].
A.A. Moosavi-Movahedi et al. / International Journal of Biological Macromolecules 49 (2011) 616– 621 617
Scheme 1. The reaction mechanism for WRK.
Recent studies have shown that �-casein acts as a molecu-lar chaperone when it interacts with proteins that have negativecharge on their surface, including insulin [20,30] and alcoholdehydrogenase [17,31–33]. The presence of negative charge on�-casein surface, which results in electrostatic and hydrophobicfactors to act in the same direction, promotes �-casein molecu-lar chaperone property. In this report we chose lysozyme at pH7.2 with 8 positive charges and pI 11[34] as a target-protein inorder to evaluate the role of electrostatic and hydrophobic forcesin chaperone ability of native or WRK (aspartate and glutamateresidues)-modified �-casein. For this purpose, we measured thestability and aggregation rate of lysozyme using different meth-ods including differential scanning calorimetry (DSC). DSC is widelyused for the study of thermal protein denaturation, and is the solemethod for direct determination of the thermodynamic parame-ters of proteins [35–39]. Here we demonstrate how �-casein withnegative charge on its surface can change its chaperone activitytoward a target protein with positive charge.
2. Materials and methods
2.1. Materials
Bovine �-casein, hen-egg white lysozyme, dithiothreitol(DTT), 2-ethyl-5-phenylisoxazolium-3-sulfonate (Woodward’sreagent K), 8-anilino-1-naphthalensulfonic acid (ANS) were fromSigma–Aldrich (St. Louis, MO), and other chemicals used were ofanalytical grade obtained from Merck (Germany).
2.2. Methods
2.2.1. Chemical modification of ˇ-caseinSelective modification of aspartic and glutamic acid residues on
�-casein’s surface was carried out with WRK. �-casein (0.4 mg/mL)was incubated in 150 mM phosphate buffer (pH 7.2) with WRK(1 mM) at 37 ◦C for up to 1 h. In order to remove excess WRK, thereaction mixture was dialyzed for 72 h. The above sample treatedwith WRK had an absorption band with a �max within 340–350 nm,which was absent in the untreated sample. The stability of WRK isa function of pH, and it is rapidly destroyed at pH values above 3.0[40]. Thus, large excess of reagent over �-casein was used in orderto compensate for the instability of the reagent at pH 7.2 [20].
2.2.2. Chaperone–like activity assay and analysis of the kineticsof aggregation
The aggregation of DTT-induced lysozyme (0.177 mg/ml) in150 mM sodium phosphate buffer (pH 7.2) was monitored at360 nm at 37 ◦C for 80 min using a Cary Varian UV-Vis spectropho-tometer model 100 Bio. In order to reduce lysozyme aggregation,200 �l of normal- or modified-�-casein (0.4 mg/ml) in 150 mMsodium phosphate buffer (pH 7.2) was added to 300 �l reaction
solution (lysozyme 0.177 mg/ml and 10 mM DTT) and transferredto a 600 �l cuvette. Analysis of the kinetics of aggregation wasbased on the assumption that absorbance is proportional to theamount of the aggregated protein [41]. Given the aggregation pro-cess as the first order kinetics, the following equations was used forquantitative description of the kinetic curves [42,43]:
A = Alim
{1 − exp[−k1(t − t0)]
}(1)
where Alim and A are the limiting value (maximum value) ofabsorbance at t → ∞ and absorbance at given time, respectively,k represents the first-order rate constant for aggregation reaction,t indicates time and to represents aggregation starting time (orminimum value of A).
2.2.3. ANS fluorescence spectroscopy(a) The ANS fluorescence of lysozyme was recorded using
Cary Eclipse Varian fluorescence spectrophotometery. The fol-lowing solutions were prepared lysozyme with a concentration0.177 mg/ml in 150 mM phosphate buffer at pH 7.2, (b) lysozymeconcentration 0.177 mg/ml in the presence of 0.4 mg/ml modi-fied �-casein in 150 mM sodium phosphate buffer (pH 7.2), (c)lysozyme with a concentration 0.177 mg/ml in the presence of0.4 mg/ml normal �-casein in 150 mM sodium phosphate buffer(pH 7.2). All above solutions were transferred to a 0.4 ml cuvetteand their fluorescence was measured in the presence of 10 mM ANSat �excitation 365 nm at 37 ◦C with a 10 nm band width.
2.2.4. Circular dichroism spectropolarimetryCircular dichroism (CD) spectra in the far-UV regions
(190–260 nm) were obtained in J-810 Jasco spectropolarime-ter using 1 mm path cell at 25 ◦C. �-casein concentration was0.4 mg/ml and WRK-modified �-casein 0.4 mg/ml [WRK (1 mM)] inthe presence of 150 mM sodium phosphate buffer, pH 7.2 and 25 ◦C.Protein secondary structure was determined by CDNN program,version 2.1.0.223.
2.2.5. Theoretical approachesThe �-casein 3D model was generated using the MODELER
9V7 software [44] based on homology modeling procedures andloop refinement implemented in modeler software. The templateuses for homology modeling is cytosolic iron–sulphur assemblyprotein-1 with 2HES pdb accession code. The overall identity is28%. Multiple sequence alignment shows 2HES as a good templatefor casein also. In aligned region there are about 80 residues withsimilarity or identity.
To study the action of modified casein on lysozyme, a WRKwas attached to an accessible aspartate of casein, and subse-quently docked to the lysozyme via the HEX6 software [45]. Theeffects of WRK modification on global protein structure neglects.The docking energy would represent an estimation of protein-protein interaction affinity and the docking results sort by the
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618 A.A. Moosavi-Movahedi et al. / International Journal of Biological Macromolecules 49 (2011) 616– 621
overall energy. The final structures of the docking results refinewith newton-like energy minimization after the docking process.The same procedure was performed for native casein. To calculatethe protein–protein interface properties, the InterPro SurfPro-tein server was used. The provided images were generated usingthe Swiss PDB viewer software. To estimate in vacuum electro-static potential of the proteins the Swiss PDB viewer 4 used withPoisson–Boltzman computation method.
2.2.6. Calorimetric studiesCalorimetric studies were carried out by Nano-DSC differen-
tial scanning microcalorimeter (Setaram, France) equipped with0.348 ml cells. All DSC experiments were done under 2 atm pres-sure. The concentrations of native and modified �-casein were0.4 mg/ml, lysozyme 0.177 mg/ml in 150 mM sodium phosphatebuffer (pH 7.2) and the experiments were performed at scan rateof 2 ◦C/min. To check the reversibility of denaturation, the samplewas heated 1–2 ◦C above the Tm then cooled, and is reheated. If theoriginal curve completely or partially reproduced, it is evident thatdenaturation is fully or partially reversible; otherwise it is an irre-versible process. The extent of reversibility depends on a numberof factors including temperature to which the protein was heatedin the first scan [38].
3. Results and discussion
Molecular chaperones are group of proteins that act not onlyin protection and folding correction of inappropriately foldedproteins, but also participate in refolding, targeting, and the degra-dation of defective proteins or their undesired forms [14,46,47].This is accomplished, at least in part, by their binding to theexposed hydrophobic patches of the denatured and/or unfoldedproteins. It has been demonstrated that �-casein has remarkableanti-aggregation properties [7,8]. Our study shows that the �-casein does not have the ability to prevent aggregation of lysozyme,and failed to show chaperone ability under our experimental con-ditions (pH 7.2).
In order to revive chaperone ability via hydrophobic moieties,aspartate and glutamate residues on the �-casein surface weremodified using WRK. We showed modification of the �-caseinacidic residues induced its chaperone properties toward lysozymeat pH7.2, a condition of net positive charge. The UV-Vis absorbancespectra of the native and WRK-modified �-casein are shown inFig. 1. The modification of the �-casein by WRK showed a newabsorption peak in the wavelength range 340–350 nm, establish-ing the modification of aspartate and glutamate residues [33,48].The first step in WRK reaction is the formation of ketoketenimine,which then reacts with carboxylate groups to produce an enolester [49] with an absorbance peak at 340–350 nm. CD spectra of
Fig. 1. UV–vis absorbance spectra of native and modified �-casein. (–) native �-casein and (�) �-casein + WRK (1 mM). The �-casein concentration was 0.4 mg/mlat pH 7.2 at 25 ◦C.
- 70
- 60
- 50
- 40
- 30
- 20
- 10
0
10
190 210 230 250 270
mol
ar e
lip�c
ity x
10-5
(m
deg
.Mol
-1Cm
-1)
Wavelenght (nm)
Fig. 2. The Molar ellipicity of 0.4 mg �-casein (thin line) and WRK-modified �-casein(thick line) at pH 7.2 and 25 ◦C.
0.00
0.50
1.00
1.50
2.00
0 20 40 60 80 100
Abs
orba
nce(
360n
m)
Time(min)
Fig. 3. Chemical aggregation assay of the lysozyme using DTT in the presence ofnative and modified �-casein as a function of time at 360 nm and 37 ◦C. Lysozyme0.1% (- - - -), lysozyme 0.1% in the presence of native �-casein 0.4 mg/ml (–),lysozyme 0.1% in the presence of modified �-casein 0.4 mg/ml (�), and lysozyme0.1% in the presence of W RK 30 �M (�).
WRK-modified �-casein showed more negative ellipticity relativeto native �-casein in the range of 250–204 nm (Fig. 2). Fig. 3 showsthe status of DTT induced aggregation of lysozyme in the absenceand presence of �-casein, WRK, and WRK-modified �-casein at37 ◦C.
In Eq. (1), rate constant and Alim are estimations of aggrega-tion rate and amount of aggregated protein, respectively. Fittingthe obtained data to Eq. (1) showed these values diminished in thepresence of modified �-casein (Table 1). Decreasing Alim might bedue to the precipitation of aggregated form of the protein. Native�-casein had no considerable effect on rate constant and Alim.
To further investigate the effect of native and modified �-caseinon the refolding of lysozyme, we conducted a calorimetric studyusing DSC. Lysozyme showed a peak with Tm at 72.2 ◦C. In the pres-ence of native �-casein, lysozyme showed a lower Tm and enthalpyindicating its destabilization. There were no significant differencesin the DSC profile of lysozyme in the presence or absence of modi-fied �-casein (Fig. 4A).
The reversibility of protein refolding depends on various con-ditions such as the nature of the protein, heating rate, coolingrate and the maximum temperature in the first scan [38]. Whenlysozyme was heated up to 80 ◦C, its denaturation was reversible.
Table 1The fitting parameters of DTT induced aggregation of lysozyme in the absence orpresence of �-casein (�CN) and WRK-modified �CN.
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Fig. 4. (A) The DSC profiles of lysozyme (dashed line), lysozyme in the presence ofnative �-casein (thin line), and lysozyme in the presence of modified �-casein (thickline). (B) The DSC profile of lysozyme (thick line) and rescan of lysozyme heated upto 85 ◦C in the absence (dashed line) and presence of modified �-casein (continuesline).
However, increasing the temperature over 80 ◦C in the first scanresulted in decreased amount of reversibility. Lysozyme unfoldedirreversibly as temperature reached 85 ◦C in the absence of �-casein(Fig. 4B), while in the presence of WRK-modified �-casein it showedreversible stability for such interaction. These results indicate thechaperone activity of modified �-casein and its protective effect onlysozyme refolding.
The �-casein amino acid sequence indicates that there are 5glutamate and 2 aspartate residues on its surface. The modifica-tion of acidic residues did not change the net negative charge dueto the presence of WRK sulfonate anions (see Scheme 1) [29]. Wenext determined the reason for refolding and diminished aggrega-tion of lysozyme in the presence of modified �-casein. In orderto illustrate the changes in hydrophobic parts of lysozyme, weused ANS for monitoring the effect of native and modified �-caseinon hydrophobic parts of lysozyme. Fig. 5 shows that fluorescence
0
50
100
150
200
400 500 600 700
Fluo
resc
ence
Inte
nsity
(a
rbitr
ary
unit)
Wavelength (nm)
Fig. 5. ANS fluorescence spectra of lysozyme (0.177 mg/ml) in the presence of nativeor modified �-casein (0.4 mg/ml) and DTT. Phosphate buffer pH 7.2 (. . .), native�-Casein (—), modified �-casein(�), Lysozyme (- - -), lysozyme + WRK (thick line),lysozyme + normal �-casein (�), and lysozyme + modified �-casein (�) at 37 ◦C.
intensity was increased in the presence of modified �-casein butdecreased in the presence of native �-casein. The results obtainedfrom ANS fluorescence of lysozyme indicated that there wereinteractions between the exposed hydrophobic patches of the mod-ified �-casein and exposed hydrophobic patches of the lysozyme.Thus, modified �-casein prevented the direct interactions betweenlysozyme hydrophobic patches, which cause lysozyme aggregation.
Based on ANS fluorescence spectra results, the chemical mod-ification of �-casein carboxyl residues caused conformationalchanges. Therefore, the hydrophobic and electrostatic interactionsmay play an important role in protein–chaperone interactions,which appear to compete here. It is important to note thatFigs. 3 and 5 shows that WRK alone has no effect on lysozyme dis-aggregation and its tertiary structure. It appears that WRK alonedid not cause any changes in lysozyme.
To understand the mechanisms responsible for diminishedaggregation of lysozyme in the presence of modified �-casein, wecalculated the polar and non-polar accessible surface area (ASA)of native and modified �-casein in lysozyme-�-casein complex.The protein calculations demonstrated that the polar ASA of mod-ified �-casein was decreased by 13%, while its non-polar ASA wasincreased by 3%, in lysozyme-modified �-casein complex com-pared with native �-casein (Table 2).
Figs. 6 and 7A indicate the remaining 10% of non-polar ASAwas relative to buried residues at the interface between modi-fied �-casein and lysozyme in the complex. Fig. 6A shows that themodified �-casein electrostatic potential (EP) did not penetrate tothe lysozyme core, but that of the native �-casein did and fit to itspatially (Fig. 6A).
Fig. 6. The �-casein-lysozyme-WRK. (A) The green color (in the online system and dark in the print version) SWISS pdb viewer produced electrostatic potential surfacesindicate modified �-casein and the yellow (in the online system and the light in the print version) is lysozyme. (B) The tube structures indicate �-casein protein and theribbon one shows the lysozyme protein. The ball structures were used to indicate aspartate 152 and added WRK.
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620 A.A. Moosavi-Movahedi et al. / International Journal of Biological Macromolecules 49 (2011) 616– 621
Fig. 7. The �-casein-lysozyme. (A) The green color (in the online system and the dark in the print version) SWISS pdb viewer produced electrostatic potential surfaces indicatenative casein and the yellow (in the online system and the light in the print version) one is lysozyme. (B) The tube structures indicate �-casein protein and the ribbon oneshows the lysozyme protein. The ball structure was used to indicate aspartate 152 on native �-casein surface.
Table 2ASA values for native and modified �CN with lysozyme. Delta ASA is defined as:the differences in polar and non-polar accessible surface areas of native or modified�CN in casein-lysozyme complex, in percent value.
The results obtained from additional calculations (data notshown) revealed that aspartate 152 was more accessible than othercarboxyl residues (glutamate and aspartate) on the �-casein sur-face.
Figs. 6 and 7 show the different interactions site betweenaspartate 152 on the surface of native and modified �-casein in �-casein-lysozyme complex. Figs. 6B and 7B indicate lysozyme bindsto native and modified �-casein at different binding sites. Theseresults confirmed that the alterations of charge position, reduc-tion of ASA polarity and enhancement of hydrophobicity inducedthe chaperone ability of modified �-casein upon interactions withlysozyme at pH 7.2. Thus, the change of position and reductionof positive–negative charge interactions revitalized the chaperoneability of �-casein. Therefore, this study demonstrates a significantrole for charge-charge interactions in promotion or inhibition ofchaperone activity, and emphasizes the importance of these inter-actions in chaperone ability.
4. Conclusions
It is possible to enhance chaperone ability through increasedhydrophobic moiety and changing the charge positions upon inter-action of modified �-casein and lysozyme. In this study, we showedhow changes in charge positions without alteration of net charge,caused an increase in the hydrophobic interactions between �-casein and lysozyme, and consequently revived chaperone activityof �-casein. Our data clearly indicate that the modified �-casein haschaperone ability and protects lysozyme against chemical aggrega-tion. This helps to refold and induces the reversibility of the thermaldenatured state. Thus, the changes in charge position may play animportant role in increasing the refolding and diminishing aggre-gation of the lysozyme.
Acknowledgements
The support of Research Council of University of Tehran, IranNational Science Foundation (INSF), Pasteur Institute of Iran andIAU are gratefully acknowledged.
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