Biosci. Biotechnol. Biochem., 69 (5), 922–931, 2005
Thermostability of Refolded Ovalbumin and S-Ovalbumin Nobuyuki T AKAHASHI,1; y Maki O NDA,2 Kaori H AYASHI,2 Masayuki YAMASAKI,1 Tomoyoshi M ITA,2 and Masaaki H IROSE1 1
The Division of Applied Life Sciences, The Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan 2 The Department of Environmental Sciences, Faculty of Science, Osaka Women’s University, Daisen-cho 2-1, Sakai, Osaka 590-0035, Japan Received November 1, 2004; Accepted February 15, 2005
Ovalbumin, a member of the serpin superfamily, is transformed into a thermostabilized form, S-ovalbumin, during storage of shell eggs or by an alkaline treatment of the isolated protein (Tm ¼ 8 C). As structural characteristics of S-ovalbumin, three serine residues (Ser164, Ser236 and Ser320) take the D-amino acid residue configuration, while the conformational change from non-thermostabilized native ovalbumin is very small (Yamasaki, M., Takahashi, N., and Hirose, M., J. Biol. Chem., 278, 35524–35530 (2003)). To assess the role of the structural characteristics on protein thermostabilization, ovalbumin and S-ovalbumin were denatured to eliminate the conformational modulation effects and then refolded. The denatured ovalbumin and Sovalbumin were correctly refolded into the original nondenatured forms with the corresponding differential thermostability. There was essentially no difference in the disulfide structures of the native and refolded forms of ovalbumin and S-ovalbumin. These data are consistent with the view that the configuration inversion, which is the only chemical modification directly detected in S-ovalbumin so far, plays a central role in ovalbumin thermostabilization. The rate of refolding of S-ovalbumin was greater than that of ovalbumin, indicating the participation, at least in part, of an increased folding rate for thermodynamic stabilization. Key words:
disulfide bond; S-ovalbumin; ovalbumin; serpin; protein thermostability
It is generally believed that the native state of a globular protein is in the global free energy minimum. However, there are several instances of the native state being a kinetically trapped one with less stability than that in the fully stabilized state.1) Serpin is such a typical example. Upon the expression of inhibitory activity,2) the inhibitors undergo a large conformational change after undergoing the proteolytic cleavage at the P1–P10 y
site. The dynamic conformational change includes insertion of the reactive center loop into central -sheet A2) and accompanies a marked increase in the thermostability.3) The occurrence of a non-proteolyzed loopinserted latent serpin form is also known for 1 antitrypsin4) and plasminogen activator inhibitor-1.5) With respect to antithrombin, there exists a non-proteolyzed locked form with a partially loop-inserted conformation.6) On the basis of the structural homology, the major component of egg white, ovalbumin (OVA), is grouped in a superfamily of the serpins.7,8) The egg white protein has neither the loop-insertion mechanism9–11) nor inhibitor activity,12) and is thus grouped into the noninhibitory serpins.13) Our recent thermodynamic and structural analyses have, however, demonstrated that mutant OVA R339T with replacement of the P14 hinge residue undergoes, following P1–P10 cleavage, structural transition into the fully loop inserted conformer that accompanies marked thermostabilization with a Tm value of 15.8 C.14,15) This implies that native OVA is a metastable serpin conformer as well. The egg white protein is known to be converted into a thermostable form, S-ovalbumin (S-OVA), during the development and storage of the fertilized and nonfertilized eggs or during the incubation of the isolated protein under alkaline conditions.16–19) According to differential scanning calorimetry (DSC), the difference in the denaturation temperature between OVA and SOVA is as large as 8 C.20) During the transformation of OVA to S-OVA, a thermodynamically distinct intermediate (I-OVA) is involved.20) As an analogical situation to the latent and locked forms of the serpins, the transformation of OVA into the thermostabilized SOVA form involves no peptide cleavage.17) The thermostabilization mechanism for S-OVA formation has, therefore, been related to partial insertion of the reactive center loop into central sheet A.21) Our recent crystallo-
To whom correspondence should be addressed. Fax: +81-774-38-3735; E-mail:
[email protected] Abbreviations: OVA, ovalbumin; S-OVA, S-ovalbumin; I-OVA, intermediate of interconversion into S-OVA; DSC, differential scanning calorimetry
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graphic study has, however, unequivocally excluded the occurrence of any loop insertion in S-OVA; the overall structure, including the reactive center loop structure, is almost the same as that of native OVA, except for significant motion of the preceding loop of strand lA away from strand 2A.22) The most striking point is that Ser164, Ser236 and Ser320 take the D-amino acid residue configuration.22) In the present study, to examine whether the configurational inversions are only artifactual or play a central role in the protein thermostabilization, the conformational modulation effects in S-OVA were removed by denaturation, and then the denatured S-OVA was refolded. We report here that S-OVA displayed refolding progress and was consequently distinct from OVA. Either S-OVA or OVA that had been denatured at a high temperature or in an acid-urea condition were refolded into each non-denatured form with the corresponding thermostability. There was essentially no difference in the disulfide structures of the native and refolded forms of OVA and S-OVA. The rate of refolding of S-OVA was greater than that of OVA, indicating the participation, at least in part, of an increased folding rate for thermodynamic stabilization. This increased folding rate was directly related to the lesser involvement of nonnative sulfhydryl/disulfide exchanges in the folding intermediate of S-OVA than in that of OVA. These data support the crucial participation of configurational inversion in OVA thermostabilization.
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by incubating OVA under the same buffer conditions for 4 h. Refolding from the heat-denatured state. Heat denaturation and refolding of OVA and S-OVA were carried out essentially as described elsewhere.26) Briefly, OVA was adjusted to 0.25 mg/ml, and the pH value was adjusted to 7.5 by adding an NaOH solution. OVA was then denatured at 80 C for 10 min. The non-native ‘‘trapped’’ form was obtained by immediately cooling the sample on ice. To achieve native refolding, the heatdenatured protein was first incubated at 55 C for 3 h, and then at 0 C for 30 min. The thermal treatment of S-OVA was carried out in a similar way, except that a higher temperature of 90 C was employed for denaturation. Refolding from the urea-denatured state. The refolding of S-OVA from the urea-denatured state was done in the same way as that for OVA.27) Briefly, S-OVA was denatured at 1.0 mg/ml by incubating in 9 M urea, 0.25 M HCl, and 1 mM Na–EDTA at 37 C for 30 min. The ureadenatured S-OVA was refolded by 20-fold dilution with 52.6 mM Tris–HCl at pH 8.2 and by subsequent incubation for 20 h at 25 C. The refolded S-OVA was concentrated about 15-fold with a membrane concentrator (Centriprep-10, Amicon, Massachusetts, U.S.A.). The denaturation and refolding of the I-OVA-enriched sample were performed in the same way as that for S-OVA.
Materials and Methods Materials. OVA was prepared from fresh egg white by crystallization in an ammonium sulfate solution.23) Diphosphorylated OVA (A1 -OVA) was purified from the crystallized OVA by ion-exchange chromatography,24) using a prepacked column of Hiload 26/10 QSepharose HP (Amersham Biosciences, New Jersey, U.S.A.). The concentration of OVA was calculated from the absorbance at 280 nm based on the molar absorption coefficient of 3:20 104 M1 cm1 .25) Trypsin (EC 3.4.21.4) treated with diphenylcarbamyl chloride (type XI), chymotrypsin (EC 3.4.21.1; type II), and N-iodoacetyl-N0 -(5-sulfo-1-naphtyl) ethylenediamine were purchased from Sigma-Aldrich Fine Chemicals (Missouri, U.S.A.). Achromobacter protease I (EC 3.4.21.50) was obtained from Wako Pure Chemicals (Osaka, Japan). Urea was of a grade for biochemical use (Nacalai Tesque, Kyoto, Japan) and was used without further purification. The other reagents were of guaranteed grade from Nacalai Tesque or Wako Pure Chemicals. Preparation of S-OVA. The preparation of S-OVA involved the alkaline treatment conducted essentially as described by Smith and Back17) with slight modifications; purified OVA was incubated at 1.0 mg/ml in a 0.1 M glycine–NaOH buffer of pH 9.9 at 55 C for 24 h. The OVA sample enriched in I-OVA was prepared
Reduction of the disulfide bond in S-OVA and reoxidization. As described for A1 -OVA,28) S-OVA under non-denaturing conditions was incubated with 15 mM dithiothreitol in a 50 mM Tris–HCl buffer (pH 8.2) and 1 mM Na–EDTA for 2 hours at 37 C. Denaturation under the acid/urea conditions and refolding of disulfide-reduced S-OVA were done in a similar way as that for disulfide-bonded S-OVA, except that the refolding buffer contained 1.0 mM dithiothreitol. Reoxidization of non-denatured and refolded S-OVA was carried out by incubating them at 25 C for 1 h with the reduced (10 mM) and oxidized (1.0 mM) forms of glutathione in a 50 mM Tris–HCl buffer at pH 8.2 containing 1.0 mM Na–EDTA. The refolded and reoxidized OVA was passed through a prepacked Sephadex column that had been equilibrated with a 10 mM sodium phosphate buffer at pH 6.0, and concentrated in the same way. CD measurement. CD spectra were recorded at 25 C with a J720 spectropolarimeter (Jasco, Tokyo, Japan). The data were recorded three times and averaged. FarUV CD spectra were normalized to the mean residue ellipticity by using a mean residue weight of 111. DSC analysis. OVA and S-OVA were analyzed for their thermal stability with an MCS-DSC calorimeter
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(MicroCal, Massachusetts, U.S.A.). The OVA samples were passed through a prepacked Sephadex column (NAP-5, NAP-10 or NAP-25, Amersham Biosciences, New Jersey, U.S.A.) that had been equilibrated with a 10 mM sodium phosphate buffer at pH 6.0. Sample was degassed for 10 minutes under reduced pressure prior to the calorimetric analysis. The temperature was scanned at 1 C/min from 25 C to 100 C. The heat capacity, Cp, was plotted after subtracting the data from a blank experiment without protein. Determination of the disulfide-forming cysteine residues. The cysteine residues involved in the disulfide bond of the OVA derivatives were determined as described previously.27,29) In brief, an OVA sample was denatured in 9 M urea containing 0.25 M HCl and 1 mM Na–EDTA, and the free cysteine sulfhydryls were blocked with 0.1 M iodoacetamide in a 9 M urea solution neutralized with a 1 M Tris base. The alkylated protein was reduced with dithiothreitol, and then the newly generated sulfhydryl groups were modified with the fluorescent reagent, N-iodoacetyl-N0 -(5-sulfo-1-naphtyl) ethylenediamine. The labeled sample was extensively proteolyzed with the combination of trypsin, chymotrypsin, and Achromobacter protease I. The resulting set of peptides was analyzed by reversed-phase HPLC with fluorescence monitoring (excitation at 340 nm, emission at 520 nm). As a standard experiment, OVA was fully reduced with dithiothreitol, and all cysteine residues were labeled with the fluorescent reagent before extensive proteolysis. According to the assignment of the peaks in relation to the individual cysteine residues, the fraction of each cysteine residue participating in the disulfide bond was estimated27) by dividing the peak area in the sample run by that of the corresponding peak in the standard experiment. The disulfide-forming fraction of each cysteine residue was normalized to make their sum within a molecule as 2.0. Intrinsic tryptophan fluorescence. The fluorescence spectrum of OVA was measured with an F-3000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The intrinsic tryptophan residues in OVA were excited at 295 nm, and the emission spectrum was recorded in a wavelength range from 300 to 420 nm. All measurements were carried out at a constant temperature of 25 C. In respect of the spectral measurements at an early refolding time, the time-course characteristics of the fluorescence intensity change were monitored at various emission wavelengths (using an excitation wavelength of 295 nm), and the data at refolding times of 10 sec and 1 min were plotted. The time-course characteristics of refolding after the initial burst phase were monitored by the intrinsic tryptophan fluorescence at 338 nm (using an excitation wavelength of 295 nm). Analysis of disulfide rearrangement during the refolding of S-OVA. S-OVA was refolded by a 20-fold
dilution in the acid and urea-denatured state. The sulfhydryl/disulfide exchange was quenched at various refolding times by mixing the protein sample with 0.24 volume of 2 M HCl, and the disulfide-involved cysteines for the six intrinsic cysteine residues were determined by the peptide mapping method as described.27,29)
Results Refolding from the heat- and urea-denatured states The thermostabilization in the latent forms of 1 antitrypsin and plasminogen activator inhibitor-1 depends on the conformational change by loop insertion without any proteolytic cleavage. No chemical modification is considered to be involved in the formation of the latent form. The latent form of 1 -antitrypsin, which is produced by incubating the native inhibitory form in the presence of 0.7 M sodium citrate at 65–68 C, refolds after denaturation in 6 M guanidine hydrochloride into the non-loop-inserted inhibitory form.4) Latent plasminogen activator inhibitor-1 also refolds after denaturation into the inhibitory form.5) Refolding after denaturation of the latent form into the non-thermostabilized active state is, therefore, a consensus observation. If the conformational modulation observed for the crystal structure of S-OVA is the only underlying structural mechanism for protein thermostabilization as in the latent forms, and if the differential chirality of the serine residues is independent of this thermostabilization, OVA and S-OVA should refold into a common native state with the same thermostability. To investigate this, we conducted refolding experiments on OVA and S-OVA from the heat- and urea-denatured states. Heat-denatured OVA can be refolded into the native form by a two-step procedure:26) OVA denatured at a high temperature (80 C) is first cooled to 55 C and then to 0 C. If the denatured protein is directly cooled to 0 C, OVA is trapped in the non-native form. In that study, however, the question of whether heat-denatured OVA refolds into non-thermostabilized OVA or into thermostabilized S-OVA was not answered. In addition, no attempt was made to investigate the refolding of denatured S-OVA. These omissions prompted us to investigate in greater detail the refolding of OVA and SOVA from the heat-denatured state. Correct refolding by the two-step procedure was confirmed by CD spectroscopyic and DSC analyses. As shown in Fig. 1, the far-UV CD spectrum of the refolded OVA (panel A, dotted line) was almost the same as that of the non-denatured native OVA (panel A, continuous line). Denaturation and refolding of S-OVA were also carried out in a similar way to that for OVA, except that a much higher temperature of 90 C was employed as the denaturation condition. Essentially the same correct refolding profile was observed by the CD spectrum (Fig. 1B, dotted line) for S-OVA. The thermal stability of the refolded protein was also analyzed by DSC. As shown in Fig. 2, both OVA and S-
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Fig. 1. Far-UV CD Spectra of the Refolded OVA and S-OVA. Panels A and B: The CD spectra of the samples refolded from the heat-denatured state (dotted line) were measured in the far UV region for OVA (panel A) and S-OVA (panel B) as described in the text. For comparison, the CD spectrum for each original non-denatured material is plotted (continuous line). Panel C: Far-UV CD spectra for S-OVA denatured in 9 M urea under acidic conditions (broken line) and refolded SOVA from the acid-urea denatured state (dotted line) were recorded as described in the text. The CD spectrum of non-denatured S-OVA (continuous line) is also plotted for comparison.
OVA, when treated by the one-step cooling procedure, showed no endothermic heat flow (profiles b and f). This indicates the absence of a correctly folded conformation in the OVA sample. In contrast, both the refolded OVA and S-OVA displayed distinct endothermic peaks. The most important observation in Fig. 2 is that OVA and SOVA that had been refolded by the two-step procedure showed thermal transition peaks at almost exactly the same temperatures as their original non-denatured forms: the thermal transition temperatures of the nondenatured and refolded forms were, respectively, 77.7 C and 77.2 C for OVA, and 85.5 C and 85.2 C for S-OVA. These data demonstrate the correct refolding of OVA and S-OVA after elimination of the conformational difference by heat denaturation. Heat-denatured OVA, however, assumes a molten globule-like conformation, thereby retaining some residual native conformation.26) The possibility that this residual conformation could determine the subsequent refolding pathway for the original non-denatured state could not be ruled out. We therefore conducted further refolding analyses, using fully denatured OVA as the starting sample. As previously demonstrated, OVA behaves as a random coiled chain in the presence of a high concentration of urea at nearly neutral29) and acidic pH values.30) Although the urea-denatured OVA has been shown to be correctly refolded into a form with the same thermostability as the original non-denatured protein,27,31) the refolding of urea-denatured S-OVA has not been so demonstrated. S-OVA was completely denatured in 9 M urea at pH 2.2 in which practically no sulfhydryl/disulfide exchange occurred30) and then refolded by 20-fold dilution with a nearly neutral refolding buffer and by a
subsequent 20-h incubation in the same way as that for the refolding of OVA.27) Figure 1 (panel C) clearly demonstrates the correct refolding of the urea-denatured S-OVA: the far-UV CD spectrum of the refolded form is almost indistinguishable from that of the original SOVA. As shown in Fig. 2 (profiles g and h), the refolded protein had almost the same endothermic peak (Tm ¼ 85:2 C and 85.4 C, respectively) as the non-denatured S-OVA (Tm ¼ 85:5 C). The refolding into the original non-denatured form was also confirmed for I-OVA. When OVA was incubated under the same alkaline conditions for a shorter incubation time of 4 h, the OVA sample comprised the major component of I-OVA with a Tm value of 82.5 C, together with minor shoulders consisting of OVA and S-OVA (Fig. 2, profile i). Essentially the same DSC profile was obtained after the denaturation and refolding of the OVA sample (profile j). These data indicate that the denatured I-OVA had refolded into the original state. Refolding and reoxidization of disulfide-reduced SOVA The preceding refolding data highlight a clear difference of OVA from 1 -antitrypsin4) and plasminogen activator inhibitor-1.5) OVA, however, has an intrachain disulfide (Cys73–Cys120) along with four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382), while 1 -antitrypsin and plasminogen activator inhibitor-1 comprise no intrachain disulfide bond. To investigate the possibility that the presence of the disulfide bond confines an otherwise (in the absence of the disulfide) thermodynamically or kinetically favored refolding pathway toward a common native state, OVA and SOVA were denatured and refolded under disulfide-
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terparts: the thermal transition temperatures for the disulfide-reduced, refolded OVA and S-OVA were 70.7 C and 79.7 C, respectively. Reoxidation, however, resulted in the proteins regaining almost the same lebel of thermal stability (Tm of 77.5 C for OVA and 85.1 C for S-OVA) as the disulfide-bonded forms. The same was also true for the reoxidized proteins: the transition temperatures were 77.3 C for the refolded, reoxidized OVA and 85.6 C for the refolded, reoxidized S-OVA. These data clearly demonstrate that the denatured OVA and S-OVA were refolded into their original non-denatured forms in the absence of the intramolecular disulfide bond.
Fig. 2. DSC Analyses of Refolded OVA and S-OVA. OVA (profiles b and c) and S-OVA (profiles f and g) were denatured by respective incubation at 80 C and 90 C for 10 min. The samples were directly cooled to 0 C (profiles b and f indicated by one-step), or first incubated at 55 C and then at 0 C (profiles c and g indicated by two-step) as described in the text. In contrast, OVA (profile d) and S-OVA (profile h) were refolded from the ureadenatured state as described in the text (indicated by urea-refold). Likewise, the refolding of I-OVA from the urea-denatured state was confirmed by the DSC analysis (profile j indicated by urea-refold) in comparison with the original I-OVA (profile i). The proteins were subjected to DSC analyses in a 10 mM sodium phosphate buffer at pH 6.0. The heat capacity profiles were recorded at a heating rate at 1 C/min. As control experiments, endothermograms for nondenatured OVA (profile a) and S-OVA (profile e) are shown; for clarity, profiles a–i have been arbitrarily shifted on the ordinate scale. Tm for the sample is shown as its peak temperature on the right side of each thermal transition peak.
reduced conditions27,32) and then reoxidized by glutathione.32) The thermostability of the refolded protein was analyzed by DSC. As shown in Fig. 3, the disulfidereduced OVA (panel A) and S-OVA (panel B) showed less thermostability (Tm values of 70.6 C and 79.3 C, respectively) than their original disulfide-bonded counterparts (Tm of 77.7 C for OVA and 85.5 C for SOVA). The disulfide-reduced, refolded OVA and SOVA were found to have almost the same thermal stability as their disulfide-reduced, non-denatured coun-
Refolding kinetics and disulfide structure The stability of the native conformation depends on the relative ratio of the rate of the folding versus that of unfolding. It was examined whether or not the increased stability of S-OVA was related to an accelerated rate of folding. Previous studies have shown that urea-denatured OVA with the native disulfide Cys73–Cys120 (D[73–120]) formed an initial burst intermediate IN [73– 120] during the refolding with a partially folded conformation that was formed within 4 ms.27,31,33) This intermediate could correctly refold into native form N[73–120] with a first-order rate constant kf of about 0.2 min1 . In the IN state, however, the native cystine, Cys73–Cys120, undergoes extensive sulfhydryl/disulfide exchanges with Cys11, Cys30, Cys367, and Cys382, resulting in the generation of the intermediate with nonnative disulfides IN [mis-SS], hence with a decelerated refolding rate:27,31) kf ¼ 0:2 min1 D[73{120] ! IN [73{120] ! N[73{120]
k1 #" kþ1 IN [mis-SS] where k1 and kþ1 represent the first-order rate constants for the interconversion between IN [73–120] and IN [misSS]. After the initial burst intermediate formation, FN (t), which is defined as the fraction of N[73–120] at refolding time, t, can be expressed as follows: kf þ r2 kf þ r1 FN ðtÞ ¼ 1 þ expðr1 tÞ expðr2 tÞ ðEq. 1Þ r1 r2 r1 r2 where r1 and r2 are related to the rate constants as: pffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffi r1 ¼ þ 2 ; r2 ¼ 2 ; 2 ¼ kþ1 þ k1 þ kf ;
and ¼ kþ1 kf
It was examined whether or not the refolding of SOVA would follow the same process as that of OVA. In a previous study,34) the disulfide structure in S-OVA was analyzed by a classical approach, using the two-dimensional paper chromatography of [14 C]iodoacetatelabeled cysteine peptides, and was concluded to be the same Cys73–Cys120 as the original OVA. As a prerequisite for the disulfide-involved refolding kinetics, we re-examined the disulfide structure by a more
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Fig. 3. Refolding of Disulfide-Reduced OVA and S-OVA. A DSC analysis was carried out for OVA and its derivatives (panel A) and S-OVA and its derivatives (panel B). The protein derivatives were the non-denatured and disulfide-reduced forms of OVA and S-OVA (profile b: Intact hSS formi); the non-denatured, disulfide-regenerated forms of OVA and S-OVA (profile c: Intact hSS regeneratedi); the disulfide-reduced, refolded forms of OVA and S-OVA (profile d: Refolded hSH formi); and the disulfide-reduced, refolded, and then disulfide-regenerated forms of OVA and S-OVA (profile e: hSS regeneratedi). The protein derivatives were produced as described in the text. The DSC profiles for non-denatured, disulfide-intact OVA and S-OVA are shown as the control (profile a). Profiles a–d have been arbitrarily shifted on the ordinate scale for clarity. Tm is shown as a peak temperature on the right side of each peak.
Fig. 4. Identification of the Half Cystine Residues Involved in a Disulfide Bond of OVA and S-OVA Refolded from the UreaDenatured State. In panel A, disulfide-forming cysteine residues in the nondenatured forms of OVA (shaded bars) and S-OVA (unshaded bars) were quantified as described in the text. In panel B, OVA and SOVA were refolded from the urea-denatured state as described in the text. The same disulfide analysis was done for the refolded forms of OVA (shaded bars) and S-OVA (unshaded bars).
quantitative peptide-mapping technique, using reversephase HPLC.29) As shown in Fig. 4, Cys73 and Cys120 were detected as the major disulfide-forming cysteine residues in both OVA and S-OVA (panel A). This reconfirmed the non-involvement of the disulfide rearrangement in the formation of S-OVA. Cys73 and Cys120 were also detected as the major disulfide-
forming cysteines in the refolded OVA and S-OVA (panel B), although the disulfide-forming value was somehow increased, as compared to the non-denatured OVA and S-OVA, for Cys11, Cys30, Cys367, and Cys382. The native and urea-denatured states of OVA can be clearly distinguished by the intrinsic tryptophan fluorescence spectrum.27,31) Figure 5 shows that the same was true for S-OVA; the native and urea-denatured proteins displayed the maximum fluorescence at 338 and 352 nm, respectively, and the maximum fluorescence intensity of the urea-denatured proteins was about 33% of that of the native OVA. Initial burst intermediate IN of OVA displayed the native max , but with much less intensity. In the present study, equivalent intermediate IN was found to have been formed during refolding of the urea-denatured S-OVA. As shown in Fig. 5, at an early refolding time of 10 s, S-OVA displayed an intrinsic fluorescence spectrum with max at 338 nm and 56.9% intensity of that of the native form. After a prolonged refolding time of 20 h, S-OVA had regained almost totally the native fluorescence intensity. These data indicate essentially the same refolding mechanism for OVA and S-OVA. A clear difference between the two OVA forms was, however, observed for the rate of refolding after the formation of IN . As shown in Fig. 6, the rate of refolding of S-OVA, as monitored by the fluorescence intensity at 338 nm, was faster than that of OVA. The first-order rate
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Fig. 5. Intrinsic Tryptophan Fluorescence in the Refolding Processes of OVA (continuous line and shaded symbols) and S-OVA (dotted line and unshaded symbols). The spectra shown are the non-denatured preparation (continuous line), the urea-denatured one (diamonds), and the sample refolded for 10 sec (circles), 1 min (triangles), or 20 h (squares).
Fig. 6. Time-Course Characteristics for the Refolding of OVA and SOVA Traced by Tryptophan Fluorescence. OVA (shaded circle) and S-OVA (unshaded circle) were refolded from their urea-denatured state as described in the text. The fluorescence intensity at 338 nm was recorded (excitation wavelength of 295 nm). A double-exponential least-squares fit for the averaged data is shown as the continuous curve for OVA and as the dotted curve for S-OVA. In the inset, the recorded changes in intrinsic tryptophan fluorescence at 338 nm for OVA (continuous curve) and S-OVA (dotted curve) are shown.
constants obtained by curve-fitting analyses of the data in Fig. 6 for Eq. 1 were kf ¼ 0:24 min1 , kþ1 ¼ 0:015 min1 , and k1 ¼ 0:10 min1 . When these data were compared with the corresponding rate constants for OVA (kf ¼ 0:27 min1 , kþ1 ¼ 0:011 min1 , and k1 ¼ 0:23 min1 ) obtained under the same refolding conditions,27) the accelerated refolding rate in S-OVA was largely accounted for by the 2.3 fold decrease in k1 value.
Fig. 7. Disulfide Rearrangement in the Refolding of S-OVA. S-OVA was refolded by a 20-fold dilution of the acid and ureadenatured state. Sulfhydryl/disulfide exchange was quenched at various refolding times by mixing each protein sample with 0.24 volume of 2 M HCl, and the disulfide-involved cysteine residues were determined by peptide mapping as described in the text. The averaged data from triplicated determinations of the disulfideinvolved cysteins for Cys11, Cys30, Cys73, Cys120, Cys367, and Cys382 are displayed as unshaded circles in panels A, B, C, D, E, and F, respectively. The ordinate represents the ratio of disulfide participation for a cysteine residue, being calculated so that the total ratio for the relevant cysteine residue corresponds to 2.0. The continuous curves are those obtained by smoothing the data. The result for OVA27) is shown by shaded circles for comparison.
The decreased k1 value should result in less formation of non-native disulfide during the refolding of S-OVA. To directly examine this, the extent of the disulfide exchanges during the refolding of OVA and SOVA was monitored by a peptide-mapping analysis.27) As displayed in Fig. 7, all the disulfide-involved cysteines come from the native disulfide bond (Cys73– Cys120) at refolding time 0. The disulfide-involved Cys73 and Cys120, however, were significantly decreased at an early refolding time of 1.0 min (panels C and D); concomitantly at this stage, all the other four cysteines of Cys11, Cys30, Cys367, and Cys382 were detected as disulfide-involved cysteines (panels A, B, E,
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and F). The amounts as disulfide-involved cysteines were minima for Cys73 and Cys120, but maxima for the other cysteines after 15 min of refolding. The disulfideinvolved Cys73 and Cys120, however, both increased gradually after 15 min of refolding, the amounts being about 90% after 20 h of refolding. The respective minimum values for Cys73 and Cys120 at 15 min of refolding were 0.74 and 0.69 in S-OVA, while the corresponding values were 0.59 for Cys73 and 0.63 for Cys120 in OVA (Fig. 7, panels C and D). The maximum values for Cys11 and Cys30 were all lower in SOVA than in OVA (Fig. 7, panels A and B). These data demonstrate that non-native disulfide formation in the IN state was significantly less in S-OVA than in OVA.
The results of the present study demonstrate that denatured OVA and S-OVA were correctly refolded into their original non-denatured forms with the corresponding levels of thermostability (Fig. 2). The redox interconversion of the disulfide-bonded and disulfide-reduced forms are reversible in the native states of OVA and S-OVA (Fig. 3). During refolding, S-OVA forms equivalent early intermediate IN to OVA (Fig. 5). The formation of equivalent early intermediate IN has also been confirmed for the disulfide-reduced forms of OVA27) and S-OVA (unpublished data). These data lead to the overall interconversion scheme of OVA as follows:
Discussion
D(OVA SH )
N(OVA SH)
D(OVA SS )
N(OVA SS)
OVA, a member of the serpin superfamily, is transformed into the thermostabilized form, S-OVA, with a Tm value of about 8 C during the development and storage of fertilized and non-fertilized eggs, or by an alkaline treatment of the isolated protein. The structural mechanism underlying this S-OVA formation has been one of the major unanswered questions in serpin biochemistry and in the food science of egg-white proteins. Previous models can be largely categorized into two mechanisms: covalent modification and conformational change. The deamidation model has been proposed as a covalent modification mechanism.35) This mechanism, however, has not been confirmed, since ammonia released from OVA under alkaline conditions is much less than the stoichiometric amount.36) Studies by far UV CD and Raman spectroscopy have supported the conformational transition by a 2–5% loss of helix21) and 3–4% increase of -sheet content.37) The transformation of OVA into the thermostabilized form of S-OVA involves no peptide cleavage. According to this analogical situation to the latent and rocked forms of serpins, the S-OVA formation mechanism has been related to partial insertion of the reactive center loop into central sheet A. However, our previous observation that P1–P10 was cleaved, and fully loop-inserted OVA mutant R339T was also converted to a further thermostabilized form by an alkaline treatment38) has been inconsistent with the participation of a loop insertion mechanism. More directly, recent crystallographic evidence has demonstrated that thermostabilized S-OVA did not include any loop insertion.22) The overall structure, including the reactive center loop structure, is almost the same as that of native OVA, except for the significant motion of the preceding loop of strand lA away from strand 2A and the changes in the side-chain conformation of Phe99 and Met241.22) The most striking finding was that Ser164, Ser236 and Ser320 took the Damino acid residue configuration.22) The crystallographic evidence, therefore, demonstrates the inclusion of both covalent modification and conformational change in the S-OVA structure.
I N (OVASH )
I N (OVA SS )
I N (S-OVA SS ) D(S-OVA SS )
N(S-OVA SS)
D(S-OVA SH )
N(S-OVA SH) I N (S-OVA SH )
where D(OVA), IN (OVA) and N(OVA) represent the denatured, intermediate and native forms of OVA, respectively, and D(S-OVA), IN (S-OVA) and N(SOVA) are the denatured, intermediate and native forms of S-OVA, respectively. The subscripts SS and SH attached to OVA and S-OVA represent the disulfidebonded and disulfide-reduced states, respectively. One of the most important points in the scheme is that the interconversion between OVA and S-OVA only occurs in the direction from OVA to S-OVA. This is supported by the fact that, despite extensive reports about the conversion from native OVA to S-OVA, no evidence has been shown for the occurrence of the reverse reaction. This irreversible nature is consistent with the view that modification of the covalent structure is directly related to the formation of S-OVA. The refolding scheme is quite different from the refolding data for latent 1 antitrypsin and plasminogen activator inhibitor-1, in which no chemical modification is known to take place during the interconvertion between the inhibitory and latent forms; in these serpins, the latent forms refold after complete denaturation into the non-thermostabilized active form.4,5) The data in the present study also demonstrate that there was essentially no difference in the disulfide structures of non-denatured and refolded OVA and S-
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OVA (Fig. 4). Furthermore, S-OVA after disulfide reduction showed higher thermostability than disulfidereduced OVA (Fig. 3), indicating the existence of disulfide-reduced S-OVA. An altered disulfide structure model for S-OVA formation can therefore be ruled out. The crystal structure of S-OVA clearly shows the presence of the D-amino acid configuration for three serine residues. The occurrence of multiple covalent modifications is consistent with the presence of partially thermostabilized I-OVA and with its refolding into the original non-denatured state (Fig. 2), if partial thermostabilization is related to partial configurational inversion. On the basis of these data, it is very likely that configurational inversion played a central role in OVA thermostabilization. The greater rate of refolding in S-OVA than in OVA indicates the participation (Fig. 6), at least in part, of an increased folding rate for thermodynamic stabilization. The increased folding rate in S-OVA can be directly related to the lesser involvement of non-native sulfhydryl/disulfide exchanges in folding intermediate IN (Fig. 7). Intermediate IN is a molten-globule conformer with a partially folded conformation.27) Unlike the ureainduced denaturation system, where OVA is transformed into a random-coiled, fully denatured state,29) the protein denatured at a high temperature assumes a partially denatured molten-globule conformation.39) These results highlight the importance of the free energy difference between the native and molten-globule states as the determinant for the thermal stability of OVA. The different degree of intrachain sulfhydryl/disulfide exchanges in IN may reflect modulation in the moltenglobule state in S-OVA. A different molten-globule state may be related to the altered stability between OVA and S-OVA. In conclusion, the results from the present refolding studies support the crucial participation of configurational inversion for S-OVA formation. It is, however, not clear whether or not this covalent modification per se works as a direct thermostabilization factor. We speculate that configurational inversion accompanies the conformational changes observed in the crystal structure of S-OVA. It is possible that the modulated conformation is related, at least in part, to the increased thermostability in S-OVA.
References 1) 2)
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12) 13)
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Acknowledgment This work was supported in part by grant-aid for the encouragement of young scientists (no. 08760135 and no. 09760131) from the Ministry of Education, Science, Sports, and Culture of Japan and by grant-aid from the Fujisawa foundation. The authors appreciate the help of Dr. Eizo Tatsumi (Japan International Research Center for Agricultural Sciences) with his participation in a preliminary experiment to determine the disulfide bonds in S-OVA.
16)
17)
18)
Baker, D., Metastable states and folding free energy barriers. Nature Struct. Biol., 5, 1021–1024 (1998). Loebermann, H., Tokuoka, R., Deisenhofer, J., and Huber, R., Human 1 -proteinase inhibitor: Crystal structure analysis of two crystal modifications. The molecular model and preliminary analysis of the implications for function. J. Mol. Biol., 177, 531–556 (1984). Gettins, P., and Harten, B., Properties of thrombin- and elastase-modified human antithrombin III. Biochemistry, 27, 3634–3639 (1988). Lomas, D. A., Elliott, P. R., Chang, W. W., Wardell, M. R., and Carrell, R. W., Preparation and characterization of latent 1 -antitrypsin. J. Biol. Chem., 270, 5282–5288 (1995). Hekman, C. M., and Loskutoff, D. J., Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J. Biol. Chem., 260, 11581–11587 (1985). Carrell, R. W., Evans, D. L. I., and Stein, P. E., Mobile reactive centre of serpins and the control of thrombosis. Nature, 353, 576–578 (1991). Hunt, L. T., and Dayhoff, M. O., A surprising new protein superfamily containing ovalbumin, antithrombinIII, and 1 -proteinase inhibitor. Biochem. Biophys. Res. Commun., 95, 864–871 (1980). Whisstock, J., Skinner, R., and Lesk, A. M., An atlas of serpin conformations. Trends Biochem. Sci., 23, 63–67 (1998). Stein, P. E., Tewkesbury, D. A., and Carrell, R. W., Ovalbumin and angiotensinogen lack serpin S-R conformational change. Biochem. J., 262, 103–107 (1989). Gettins, P., Absence of large-scale conformational change upon limited proteolysis of ovalbumin, the prototypic serpin. J. Biol. Chem., 264, 3781–3785 (1989). Wright, H. T., Qian, H. X., and Huber, R., Crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin. J. Mol. Biol., 213, 513–528 (1990). Wright, H. T., Ovalbumin is an elastase substrate. J. Biol. Chem., 259, 14335–14336 (1984). Huber, R., and Carrell, R. W., Implications of the threedimensional structure of 1 -antitrypsin for structure and function of serpins. Biochemistry, 28, 8951–8966 (1989). Yamasaki, M., Arii, Y., Mikami, B., and Hirose, M., Loop-inserted and thermostabilized structure of P1–P10 cleaved ovalbumin mutant R339T. J. Mol. Biol., 315, 113–120 (2002). Arii, Y., and Hirose, M., Probing the serpin structuraltransition mechanism in ovalbumin mutant R339T by proteolytic-cleavage kinetics of the reactive-center loop. Biochem. J., 363, 403–409 (2002). Smith, M. B., and Back, J. F., Modification of ovalbumin in stored eggs detected by heat denaturation. Nature, 193, 878–879 (1962). Smith, M. B., and Back, J. F., Studies on ovalbumin II. The formation and properties of S-ovalbumin, a more stable form of ovalbumin. Aust. J. Biol. Sci., 18, 365– 377 (1965). Sugimoto, Y., Sanuki, S., Ohsako, S., Higashimoto, Y., Kondo, M., Kurawaki, J., Ibrahim, H. R., Aoki, T., Kusakabe, T., and Koga, K., Ovalbumin in developing
Refolding of Ovalbumin and S-Ovalbumin
19)
20)
21)
22)
23)
24)
25)
26)
27)
28)
29)
chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability. J. Biol. Chem., 274, 11030–11037 (1999). Hatta, H., Nomura, M., Takahashi, N., and Hirose, M., Thermostabilization of ovalbumin in a developing egg by an alkalinity-regulated, two-step process. Biosci. Biotechnol. Biochem., 65, 2021–2027 (2001). Donovan, J. W., and Mapes, C. J., A differential scanning calorimetric study of conversion of ovalbumin to S-ovalbumin in eggs. J. Sci. Food Agric., 27, 197–204 (1976). Huntington, J. A., Patston, P. A., and Gettins, P. G. W., S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins. Protein Sci., 4, 613–621 (1995). Yamasaki, M., Takahashi, N., and Hirose, M., Crystal structure of S-ovalbumin as a non-loop-inserted thermostabilized serpin form. J. Biol. Chem., 278, 35524–35530 (2003). So¨rensen, S. P. L., and Ho¨yrup, M., Studies on proteins. 1. On the preparation of egg-albumin solutions of welldefined composition, and on the analytical methods used. Compt. Rend. Lab. Carlsberg Se´r. Chim., 12, 12–67 (1915). Kitabatake, N., Ishida, A., and Doi, E., Physicochemical and functional properties of hen ovalbumin dephosphorylated by acid phosphatase. Agric. Biol. Chem., 52, 967–973 (1988). Glazer, A. N., Mckenzie, H. A., and Wake, R. G., The denaturation of proteins II. Ultraviolet absorption spectra of bovine serum albumin and ovalbumin in urea and in acid solution. Biochim. Biophys. Acta, 69, 240–248 (1963). Tani, F., Shirai, N., Onishi, T., Venelle, F., Yasumoto, K., and Doi, E., Temperature control for kinetic refolding of heat-denatured ovalbumin. Protein Sci., 6, 1491– 1502 (1997). Onda, M., Tatsumi, E., Takahashi, N., and Hirose, M., Refolding process of ovalbumin from urea-denatured state: Evidence for the involvement of nonproductive side chain interactions in an early intermediate. J. Biol. Chem., 272, 3973–3979 (1997). Takahashi, N., Koseki, T., Doi, E., and Hirose, M., Role of an intrachain disulfide bond in the conformation and stability of ovalbumin. J. Biochem., 109, 846–851 (1991). Tatsumi, E., Takahashi, N., and Hirose, M., Denatured
30)
31)
32)
33)
34)
35)
36)
37)
38)
39)
931
state of ovalbumin in high concentrations of urea as evaluated by disulfide rearrangement analysis. J. Biol. Chem., 269, 28062–28067 (1994). Tatsumi, E., Yoshimatsu, D., and Hirose, M., Conformational state of ovalbumin at acidic pH as evaluated by a novel approach utilizing intrachain sulfhydryl-mixed disulfide exchange reactions. Biochemistry, 37, 12351– 12359 (1998). Onda, M., Tatsumi, E., Takahashi, N., and Hirose, M., Refolding of urea-denatured ovalbumin that comprises non-native disulfide isomers. J. Biochem., 122, 83–89 (1997). Takahashi, N., and Hirose, M., Reversible denaturation of disulfide-reduced ovalbumin and its reoxidation generating the native cystine cross-link. J. Biol. Chem., 267, 11565–11572 (1992). Onda, M., and Hirose, M., Refolding mechanism of ovalbumin: Investigation by using a starting ureadenatured disulfide isomer with mispaired Cys367– Cys382. J. Biol. Chem., 278, 23600–23609 (2003). Webster, D. M., and Thompson, E. O. P., Position of disulfide bond in ovalbumins of differing heat stability. Elimination of thiol-disulfide interchange as a mechanism for the formation of the ovalbumins. Aust. J. Biol. Sci., 33, 269–278 (1980). Nakamura, R., Takemori, Y., and Shitamori, S., Liberation of carboxyl groups on conversion of ovalbumin to S-ovalbumin. Agric. Biol. Chem., 45, 1653–1659 (1981). Johnson, B. A., Shirokawa, J. M., and Aswad, D. W., Deamidation of calmodulin at neutral and alkaline pH: Quantitative relationships between ammonia loss and the susceptibility of calmodulin to modification by protein carboxyl methyltransferase. Arch. Biochem. Biophys., 268, 276–286 (1989). Kint, S., and Tomimatsu, Y., A Raman difference spectroscopic investigation of ovalbumin and S-ovalbumin. Biopolymers, 18, 1073–1079 (1979). Yamamoto, H., Takahashi, N., Yamasaki, M., Arii, Y., and Hirose, M., Thermostabilization of ovalbumin by an alkaline treatment: Examination for the possible implications of an altered serpin loop structure. Biosci. Biotechnol. Biochem., 67, 830–837 (2003). Tani, F., Murata, M., Higasa, T., Goto, M., Kitabatake, N., and Doi, E., Molten globule state of protein molecules in heat-induced transparent food gels. J. Agric. Food Chem., 43, 2325–2331 (1995).
