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Thermal unfolding of eosinophil cationic protein/ribonuclease 3: A nonreversible process

¹ Unitat de Bioquímica, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
² Unitat de Biofísica, Facultat de Medicina, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
³ Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, 17071 Girona, Spain

(RECEIVED March 3, 2006; FINAL REVISION September 1, 2006; ACCEPTED September 3, 2006)

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
Eosinophil cationic protein (ECP)/ribonuclease 3 is a member of the RNase A superfamily involved in inflammatory processes mediated by eosinophils. ECP is bactericidal, helminthotoxic, and cytotoxic to tracheal epithelium cells and to several mammalian cell lines although its RNase activity is low. We studied the thermal stability of ECP by fourth-derivative UV absorbance spectra, circular dichroism, differential scanning calorimetry, and Fourier transform infrared spectroscopy. The T _1/2 values obtained with the different techniques were in very good agreement (T _1/2 72°C), and the stability was maintained in the pH range between 5 and 7. The ECP calorimetric melting curve showed, in addition to the main transition, a pretransitional conformational change with a T _1/2 of 44°C. Both calorimetric transitions disappeared after successive re-heatings, and the ratio H versus H _vH of 2.2 indicated a significant deviation from the two-state model. It was observed that the thermal unfolding was irreversible. The unfolding process gives rise to changes in the environment of aromatic amino acids that are partially maintained in the refolded protein with the loss of secondary structure and the formation of oligomers. From the thermodynamic analysis of ECP variants, the contribution of specific amino acids, such as Trp10 and the region 115–122, to thermal stability was also determined. The high thermal stability of ECP may contribute to its resistance to degradation when the protein is secreted to the extracellular medium during the immune response.

Keywords: eosinophil cationic protein; ribonucleases; thermal stability; protein folding

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
Human eosinophil cationic protein (ECP) (Fig. 1) is a cationic protein found in the large specific granules of eosinophils. ECP is a single polypeptide of 133 residues and a molecular mass of 15.5 kDa, although several glycosylated forms ranging from 16 to 21 kDa have also been identified. ECP, also known as ribonuclease 3, is a member of the ribonuclease A (RNase A) superfamily (EC 3.1.27.5 [EC] ), a vertebrate-specific enzyme family having similar amino acid sequences, enzymatic activity, and a kidney-shaped common folding structure (Boix et al. 1999a). ECP also possesses four disulfide bonds and the catalytic residues known to be involved in the RNase mechanism of action (Findlay et al. 1961). However, its ribonucleolytic activity is rather low and does not appear to be relevant for many of its biological properties that have been described in vitro. ECP is secreted from activated eosinophils, extracellular deposits of ECP are found in tissues undergoing eosinophilic inflammation, and its presence correlates well with tissue damage. ECP possesses bactericidal (Lehrer et al. 1989), antiviral (Domachowske et al. 1998), and helminthotoxic activities (Molina et al. 1988) and inhibits the growth of several mammalian cell lines (Maeda et al. 2002a; Carreras et al. 2005). In addition, eosinophil granules contain other multifunctional proteins such as eosinophil-derived neurotoxin (EDN), also known as RNase 2, which shows a 67% amino acid sequence identity with ECP. Proteins of the RNase A superfamily exhibit diverse expression patterns and biological functions (Beintema et al. 1997). Bovine pancreatic ribonuclease (RNase A) is the archetype of these proteins. Its thermal stability has been extensively characterized as a process following the two-state model, and by means of different techniques, it has been found at a T _1/2 value near to 60°C at pH 6 (Torrent et al. 2001). Eight human proteins that conserve the active-site amino acid residues have been identified by sequence homology. Maeda et al. (2002b) have described, by guanidinium chloride-induced denaturation, the high stability of ECP in comparison with these human proteins. Only the thermal stability of the pancreatic form (HP-RNase), very similar to that of RNase A, is also known (Benito et al. 2002). On the other hand, onconase (ONC), a protein from oocytes of Rana pipiens that belongs to the RNase A superfamily and shows selective cytotoxicity toward specific tumor cell lines, has an unusually high denaturation temperature (T _1/2 = 88.7°C at pH 6.0) (Leland et al. 1998; Notomista et al. 2000).

View larger version (63K): [in this window] [in a new window]   Figure 1. Three-dimensional structure of ECP showing the location of Trp (10 and 35), Tyr (33, 98, 107, and 122), and Phe residues (5, 11, 43, 48, 76, and 106). The picture was drawn using the PyMOL, DeLano Scientific program (PDB code 1QMT).

	Results

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
Fourth-derivative UV spectroscopy
The fourth-derivative spectra give information about the average polarity in the vicinity of aromatic amino acids (Fig. 2). There are two definite regions in the spectrum, the 255–265-nm interval where the bands corresponding to Phe appear and the 270–300-nm interval in which the bands arise from Tyr and Trp residues. In proteins with a Tyr/Trp content ratio value 4, the longest-wavelength minimum (₁) is mainly determined by the nature of the Trp environment (Padrós et al. 1982; Duñach et al. 1983). As the temperature increases, the fourth-derivative UV-absorption spectrum of ECP at pH 5.5 shows changes both in the position of the maxima and in the amplitude of the bands due to changes in the environment of aromatic amino acids. From the structural point of view, these effects are analyzed below in connection with the results obtained for the ECP Trp mutants (see Fig. 9, below). The plot of CDA values versus temperature between 20°C and 90°C (Fig. 3A) shows a slight deviation from a single sigmoidal function at values close to 55°C, and the protein presents a refolding process that is not completely reversible. Taking into account these limitations, we have estimated a T _1/2 for ECP of 72°C. This value indicates a higher thermal stability for ECP with respect to RNase A (T _1/2 60°C). In addition, the thermal stability of ECP is maintained in the pH range between 5 and 7 (Table 1). These experiments were complemented with runs in which the ECP spectra were obtained before and after heating to various intermediate temperatures. The results in which the samples were heated up to 56°C and 76°C are shown as examples in Figure 3, B and C, respectively. The data indicate that the process becomes irreversible when the heating temperature reaches the T _1/2 value. To check whether or not the protein refolding is a slow process, we obtained the ECP spectra at different intervals of time after thermal unfolding; no differences were observed even 24 h later.

View larger version (16K): [in this window] [in a new window]   Figure 2. UV absorbance (dotted line) and fourth-derivative (solid line) spectra of ECP at 20°C. The protein concentration was 1.0 mg/mL in 50 mM sodium acetate buffer (pH 5.5). 1 corresponds to the longest-wavelength minimum of the fourth-derivative UV spectrum, and its value is determined mainly by the nature of the Trp environment.

View larger version (12K): [in this window] [in a new window]   Figure 3. Temperature unfolding and refolding curves for ECP. (A) Spectra were recorded between 20°C and 92°C at 2°C or 5°C intervals. After reaching 92°C, each sample was returned to 20°C following exactly the reverse procedure. The protein concentration was 1 mg/mL in 50 mM sodium acetate buffer (pH 5.5). (B,C) The effect of the heating temperature on the folding reversibility. Spectra were registered between 20°C and 56°C (curve B) or 76°C (curve C). After heating, the sample was returned to 20°C by the reverse procedure. The protein concentration was 1 mg/mL in 50 mM sodium acetate buffer (pH 5.5). CDA was obtained from thermal unfolding (•) and refolding ().

View larger version (20K): [in this window] [in a new window]   Figure 4. UV CD spectra of ECP: Effect of temperature. (A) Far-UV CD spectra; (B) ECP temperature unfolding at 210 nm; (C) near-UV CD spectra. In A and C spectra were recorded between 15°C and 92°C at 2°C or 5°C intervals; after heating to 92°C, the sample was returned to 15°C by the reverse procedure. The protein concentration was 0.2 mg/mL for A and B and 0.6 mg/mL for C in 10 mM sodium cacodylate buffer (pH 5.0).

The effect of the increase of temperature on the near-UV CD spectrum of ECP is presented in Figure 4C. The shape and magnitude of this region of the spectrum depend on several factors such as the number and type of aromatic amino acids and their mobility and microenvironment (Kelly et al. 2005). The increase of temperature gives rise to changes in this spectral region that are partly maintained when the protein returns to 15°C. The changes that were maintained were found in the region of the spectrum (290–305 nm) in which the maximum contribution of the Trp residues is observed. However, no information about changes in their environment was obtained.

Differential scanning calorimetry (DSC)
Figure 5 shows the DSC scans of wt-ECP and the W10K-ECP mutant at pH 5.5 and 7.5. All thermograms present a main transition in the range 55°C–72°C depending on the pH and the variant. The T _1/2, H, H _vH, and the ratio H/H _vH for each pH obtained from the traces in Figure 5 are given in Table 3. The reported enthalpy values were calculated from the first DSC transition in each experimental condition reported in Figure 5. This, together with the fact that our DSC experiments were carried out at a scan rate of 1.5°C/min, results in very low H values, compared to those reported in the literature for the native form of RNase A (carried out at 0.5°C/min) (Robertson and Murphy 1997). Given the irreversible nature of the denaturation process of ECP, a lower denaturation enthalpy would be expected at a higher scan rate. Nevertheless, if all the re-heating scans reported in Figure 5 are taken into account, a H value of 380 kJ/mol for the native form of ECP at pH 5.5 is obtained; this value is very close to the values reported in the literature for the native form of RNase A. In any case, the thermal properties of the different ECP forms were obtained under the same experimental conditions; this allows for the comparison of the total H (first DSC scan plus further re-heating cycles) or just the H calculated from the first scan.

View larger version (12K): [in this window] [in a new window]   Figure 5. DSC thermograms of wt-ECP and the W10K-ECP mutant. (A) ECP in 50 mM sodium acetate buffer (pH 5.5); (B) ECP in 50 mM HEPES buffer (pH 7.5); (C) W10K-ECP in 50 mM sodium acetate buffer (pH 5.5); (D) W10K-ECP in 50 mM HEPES buffer (pH 7.5). The protein concentration was 2 mg/mL. All thermograms were corrected from instrumental and chemical baselines. The heating rate was 1.5°C/min. At each pH, two consecutive re-heatings (dashed and dotted lines) are shown.

View this table: [in this window] [in a new window]   Table 3. T1/2 and H of the temperature-induced denaturation of wt-ECP and the W10K-ECP mutant determined by DSC

Fourier transformed infrared spectroscopy (FTIR)
Figure 6A shows the absorption infrared spectra of wt-ECP and of the W10K-ECP mutant in 50 mM sodium acetate buffer (pD 5.5). Both spectra showed a broad and asymmetric spectrum with a maximum centered at 1639 cm^–1 with two shoulders at 1652 cm^–1 and 1673 cm^–1. The spectral features were manifest after Fourier deconvolution, as shown in Figure 6B. The deconvoluted spectra of ECP and the W10K-ECP mutant, obtained by using a full width at half height (FWHH) of 15 cm^–1 and a k factor of 1.8, were very similar, showing four main peaks. According to the well-established assignments found in the literature (Byler and Susi 1986; Cladera et al. 1992a; Goormaghtigh et al. 1994a,b), the peak at 1673 cm^–1 can be assigned to turns, the peak at 1652 cm^–1 to -helix, and the peaks at 1639 cm^–1 and 1628 cm^–1 to -sheet structure. In order to quantify the relative amount of secondary structure, a curve-fitting of the deconvoluted spectra was carried out as shown in Figure 6C. The values obtained for the different types of secondary structures (31% turns, 24% helical structure, and 45% -sheet structure) are in good agreement with the values obtained from the crystallographic structure of ECP (PDB 1QMT) (Boix et al. 1999a).

View larger version (15K): [in this window] [in a new window]   Figure 6. (A) Absorption infrared spectra of ECP (trace a) and W10K-ECP mutant (trace b) in sodium acetate buffer (pD 5.5). (B) Spectra of ECP (trace a) and W10K-ECP (trace b) deconvoluted using FWHH = 15, k = 1.8. (C) Curve-fitted deconvoluted spectrum of ECP.

View larger version (19K): [in this window] [in a new window]   Figure 7. Thermal denaturation of ECP. (A) Absorption infrared spectra of ECP in 50 mM sodium acetate buffer (pD 5.5) at temperatures between 20°C and 92°C. The inset shows the deconvoluted spectra of ECP at 20°C (thick line) and after cooling down from 92°C (thin line) using the same parameters as in Fig. 6B. (B) Difference spectra of ECP calculated from A: A 92°C – An °C, where A 92°C is the absorbance spectrum of ECP recorded at 92°C, and An °C are the absorbance spectra at the corresponding temperatures between 20°C and 92°C. (C) Temperature dependence of the peak intensity at 1663 cm–1 () and 1629 cm–1 () for ECP.

Aggregation is a frequent feature in irreversible unfolding processes. In our case, the FTIR spectra do not show any of the characteristic bands associated to the presence of -sheet aggregates (1616 cm^–1 and 1683 cm^–1). The possibility that the thermal aggregates of ECP have amyloid-like properties was also analyzed by the binding of the thioflavin-T dye (Le Vine 1993; Pallarès et al. 2004); the binding of this dye to ordered crossed -sheet aggregates results in an increase in the fluorescence emission spectrum that is not observed in the ECP samples. However, a nondenaturing electrophoresis analysis of the temperature-dependent denaturation of ECP showed the formation of oligomeric structures (Fig. 8).

View larger version (139K): [in this window] [in a new window]   Figure 8. Nondenaturating polyacrylamide gel electrophoresis in 15% polyacrylamide (pH 4). ECP samples were submitted to the thermal process at the same conditions as those described for the UV absorption spectroscopy methods and were analyzed by nondenaturing 15% polyacrylamide gel electrophoresis (pH 4.0) and Coomassie Blue staining. (Lane 1) Heating up to 20°C; (lane 2) heating up to 76°C; (lane 3) heating up to 92°C; and (lane 4) after heating to 92°C the sample was returned to 20°C by the reverse procedure.

Spectral properties of wt-ECP and the W10K-ECP and W35A/R36A-ECP mutants
In order to obtain information about the specific environmental changes that take place around the regions of the two Trp residues of ECP as a consequence of the thermal unfolding, we analyzed the fourth-derivative UV spectra of ECP and two variants modified in either one of these amino acids (positions 10 and 35, respectively) (Fig. 9). The three-dimensional structure of ECP (Figs. 1, 10A,B) shows that the side chain of W35 is solvent-exposed at the molecule surface, whereas W10 is partly buried and close to the active site (Boix et al. 1999a). The contribution of each Trp residue, their specific environments, and the effect of the thermal unfolding on the fourth-derivative UV spectrum were analyzed, taking into account the results obtained for two ECP mutants, W10K-ECP and W35A/R36A-ECP. Substitution of Trp by Lys at position 10 was designed with the aim of mimicking the corresponding region of RNase A, which constitutes a noncatalytic phosphate binding subsite (p₂) that plays an indirect role in the catalysis (Boix et al. 1994) and that contributes to the endonucleolytic activity of RNase A (Cuchillo et al. 2002). In the other mutant, the specific region of W35 and R36 involved in the cytotoxic properties of ECP (Carreras et al. 2003, 2005) was substituted by Ala residues. Upon unfolding, all three proteins showed an increased exposure to the solvent of Tyr side chains, as indicated by a blue shift of the derivative band located in the region between 275 and 280 nm (Fig. 9). However, for the three proteins, differences in the band corresponding to Trp residues, due to changes in the environment of these residues, were also observed. Duñach et al. (1983) indicated that the values of the long-wavelength minimum of the fourth-derivative UV spectrum, ₁ (Fig. 2), are mainly dependent on the Trp environment for a Tyr/Trp molar ratio of 4 or smaller. This is the case for the ECP sequence, which contains four tyrosines and two tryptophans (Fig. 1), and also for both mutants. Blue or red shifts of the ₁ band may be associated with an increase of the polar or nonpolar environment, respectively. For the W35A/R36A-ECP mutant (Fig. 9B), the ₁ value of 294.8 nm corresponds to W10 in a nonpolar environment (Fig. 10), and the thermal unfolding results in a blue shift associated with an increase of the polarity due to the exposure of the side chain to the solvent. An opposite effect has been observed for the W10K-ECP mutant (Fig. 9C): at the initial conditions, the low ₁ value is assigned to W35 located at the protein surface in a polar environment, and in the heat unfolding state this band shows a red shift. This shift indicates a decrease in the polarity of the surrounding medium that may be explained by hydrophobic contacts with nonpolar amino acid side chains of the same molecule, such as, for example, the proximity of Tyr 33, or with nonpolar regions of other protein molecules. In the case of ECP (Fig. 9A), the observed result is a consequence of the opposite effects on both Trp residues and, therefore, an intermediate behavior is observed.

View larger version (28K): [in this window] [in a new window]   Figure 9. Thermal unfolding of ECP and variants monitored by fourth-derivative UV spectroscopy. Spectra were recorded between 20°C and 92°C at intervals of 2°C or 5°C. After heating to 92°C, each sample was returned to 20°C by the reverse procedure. The protein concentration was 1 mg/mL in 50 mM sodium acetate (pH 5.5). (A) ECP; (B) W35A/R36A-ECP; (C) W10K-ECP.

View larger version (32K): [in this window] [in a new window]   Figure 10. Three-dimensional structure of the regions around the ECP Trp amino acids. (A) Trp 35 region; (B) Trp 10 region; (C) molecular modeling of the mutation of Trp 10 to Lys. Molecular modeling was carried out with the program Deep View/Swiss PDB Viewer, and the picture was drawn using PyMOL, DeLano Scientific program (PDB code 1QMT).

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
We have studied the thermal unfolding and refolding of ECP by means of fourth-derivative UV spectroscopy, UV-CD, FTIR, and DSC (Figs. 3,4,5,7). The T _1/2 values obtained with each technique were in good agreement and indicate that ECP is very stable to an increase in temperature (T _1/2 72°C) within the range of pH 5–7 (Tables 1,3). ECP is, therefore, much more thermostable than either RNase A and HP-RNase (T _1/2 60°C at pH 6) (Torrent et al. 2001; Benito et al. 2002). These techniques have shown that the thermal denaturation of ECP is an irreversible process that modifies the microenvironment of aromatic amino acids. These structural effects may contribute to the formation of protein aggregates that, according to our results, should be of the non-amyloid type.

The fourth-derivative UV spectroscopy studies (Fig. 9) demonstrate that irreversible changes in the environment of aromatic amino acids take place as a consequence of raising the temperature; the CD (Fig. 4) and FTIR spectra (Fig. 7) show that the temperature alters the protein structure, and the DSC experiments (Fig. 5) allow us to observe the increase in the amount of denatured protein due to the successive re-heatings. Moreover, the DSC data indicate that the thermal denaturation does not follow the two-state model (Fig. 5; Table 3), that the process shows a pretransition before the main transition takes place, and that the H/H _vH ratio has a value higher than 1. The thermal denaturation curves obtained from the fourth-derivative UV (Fig. 3) and FTIR spectra (Fig. 7) confirm this phenomenon, although not so clearly. All these results indicate that the thermal denaturation of ECP does not follow the two-state model that has been described for the other members of the RNase A superfamily for which these sort of studies have already been carried out. Nevertheless, even in the case of RNase A—one of the archetypes of the two-state model of folding/refolding—Stelea et al. (2001) have shown that, depending on the analytical procedure and on the conditions used, subtle variations associated with the thermal denaturation of RNase A can be observed. They have also noticed the appearance of structural changes previous to the main transition. These changes modify the protein secondary structure in a not completely reversible process that is strongly influenced by the presence of phosphate and by the time at which the protein is kept at high temperature. In addition, as observed with ECP (Fig. 8), RNase A aggregation associated to the thermal unfolding was also detected by nondenaturing gel electrophoresis (Kita and Arakawa 2002). Recent studies suggest that specific regions in the proteins act driving the aggregation, a fact that should be especially relevant for the unfolded states of globular proteins. The analysis of the aggregation prediction of ECP and RNase A according to the procedure described by Sánchez de Groot et al. (2005) (http://bioinf.uab.es/aap/) shows in both proteins an aggregation-prone sequence located at the C-terminal sequence, which has been proposed as a hydrophobic core for RNase A. The analysis of the ECP aggregation profile shows the presence of a specific additional aggregation-prone sequence that corresponds to the amino acid sequence 45–55; this sequence forms part of the 1-sheet and 3-helix and is poorly exposed to the solvent in the native structure (Boix et al. 1999a).

Using guanidinium chloride-induced denaturation, and based on the assumption of a two-state model for the unfolding of ECP, Maeda et al. (2002b) showed that ECP is more stable than RNase A and some human RNases (HP-RNase, EDN, RNase 4, and angiogenin), and suggested that its conformational stability contributes to the resistance to proteolytic degradation and is partly responsible for its cytotoxic properties.

The thermal denaturation of ECP results in changes in the environment of the Trp residues that are partially maintained in the protein after refolding. Recently, Torrent et al. (2005) using fourth-derivative analysis of the pressure-assisted thermal unfolding/refolding of the Y150W variant of the cellular prion protein, have observed a similar effect with changes in the polarity of this Trp residue's environment that are maintained after refolding.

ECP is nine residues longer than RNase A and shares 32% sequence identity and a similar three-dimensional structure (Boix et al. 1999a). The major differences between both structures occur at the L2 loop (residues 17–21), which separates helices 1 and 2, that is truncated in ECP and at the L7 loop (residues 115–122) between strands 4 and 5, which contains a large insertion and is packed against the N terminus (Fig. 1). In addition, ECP contains two Trp residues (positions 10 and 35, respectively), and because of the larger number of Arg residues located at the protein's surface, the overall surface charge for ECP is 14, instead of 3 as in RNase A (Boix et al. 1999a). The contributions of the two Trp residues, of some Arg residues in the surface, and the sequence of the 115–122 loop to the thermal stability of ECP have been analyzed using ECP variants obtained by site-directed mutagenesis (Table 3). The W10K-ECP variant, with only one amino acid substitution, decreases T _1/2 to a value close to that of RNase A. The location of this residue—partly buried in the protein structure—and the interaction of the substituting Lys 10 with amino acid side chains close to it (Fig. 10C) can explain the contribution of Trp 10 to the stabilization of the N-terminal region and the interaction of two -helical structures in ECP (1 and 2). Conversely, in the case of Trp 35, its side chain is exposed to the solvent and, hence, it contributes little to the overall stability of the protein. The variant with a deletion of the 115–122 region shows the highest decrease in thermal stability (T _1/2 = 58°C), very likely as a consequence of the structural strain that appears in the connecting region between the 4- and 5-sheets.

The thermal stability of a protein is an indicator of the flexibility of the protein structure that is maintained by a multitude of weak forces. Protein flexibility is essential to many biochemical functions in which conformational changes in the protein are involved (Petsko and Ringe 2004). In the case of ECP its stability might explain, at least in part, the low RNase activity shown in a way similar to that proposed for onconase in the same RNase A superfamily (Notomista et al. 2000); in both cases, the higher intrinsic rigidity can limit the conformational changes that are associated with the interaction of the enzyme with the substrate and/or the transition state.

Another aspect that it is important to point out is that a higher rigidity in the protein structure has been related to the higher resistance shown by the extracellular proteins to degradation (Petsko and Ringe 2004). In the case of ECP, the high thermal stability described here (and also in front of guanidinium chloride) (Maeda et al. 2002b) could confer a higher resistance to degradation when it is released from the eosinophyl granules to the extracellular medium during the immune response in which this protein is involved. Future studies will be directed to characterize the molecular basis of ECP stability; such a knowledge should be useful in the design of new RNases as therapeutic agents.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
Expression and purification of ECP and mutants
A human ECP synthetic gene was used to obtain the recombinant ECP as well as the corresponding mutants (Boix et al. 1999b). Mutants were constructed following the procedure described by Carreras et al. (2003). The recombinant proteins were expressed in the Escherichia coli BL21 (DE3) strain (Novagen) using the pET1 1c expression vector and were purified from inclusion bodies as described previously (Boix et al. 1999b). The homogeneity of the purified proteins was checked first with 15% SDS-PAGE and Coomassie staining and then by MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry on a Brucker Biflex mass spectrometer.

Fourth-derivative UV spectroscopy
UV absorption spectra were recorded using a Cary spectrophotometer (Cary 400Bio, Varian) equipped with a thermostatted cell holder. The proteins were dissolved at concentrations ranging between 0.5 and 1 mg/mL in different buffers according to the pH value of each experiment. In all cases, the buffer concentration was 50 mM (see tables and figure legends for specific buffers). A 1-cm optical pathlength quartz cell with lid was used to record absorption spectra between 250 and 310 nm. All spectra were recorded in steps of 0.1 nm, and the time of data acquisition per step of 1 sec in the temperature range between 20°C and 92°C. Following each temperature increment, typically in steps of 2°C–5°C, the protein absorption was observed to equilibrate well within a 3 min pause before each measurement. The same procedure was applied to follow the effect of the decrease of temperature. Cary Win UV software was used to control the system and to obtain the fourth-derivative spectra. The temperature-induced transitions were determined using a method that exploits the whole spectral region giving the parameter named cumulative difference amplitude (CDA) described in Torrent et al. (1999). CDA corresponds to the area of the difference spectrum at each temperature with respect to the initial spectrum, at 20°C. T _1/2 values were estimated from unfolding transition curves obtained from CDA analysis as previously described (Torrent et al. 1999).

Circular dichroism spectroscopy (CD)
CD spectra were recorded using a Jasco-J715 spectropolarimeter equipped with a thermostatted cell holder. ECP was dissolved in 10 mM sodium cacodylate (pH 5.0) at 0.2 mg/mL and 0.6 mg/mL for the far- and near-UV CD spectra, respectively. Quartz cells with 0.1- and 1-cm optical pathlengths were used to record the spectra both in the far-UV (180–260 nm) and in the near-UV (255–310 nm), respectively. CD spectra were recorded at a scan speed of 20 nm/min, a 2-nm bandwidth, and a response time of 1 sec in the temperature range between 15°C and 90°C. The sample chamber was purged with pure dry nitrogen. Spectra were signal-averaged over four scans. The contribution of the solvent to the spectra was subtracted using the Jasco software. The ECP temperature unfolding curve at 210 nm was obtained operating at a heating rate of 1°C/min within the range 15°C–90°C.

Differential scanning calorimetry (DSC)
DSC experiments were carried out on a MicroCal MC2 instrument (MicroCal Inc.) operating at a heating rate of 1.5°C/min within the range 25°C–100°C. A nitrogen pressure of 1.7 atm was kept during scans to avoid sample evaporation at high temperatures. 1.33 mL of solution was introduced into the sample cell at a final protein concentration of 2 mg/mL. The reversibility of the thermal transitions was checked by re-heating the samples immediately after cooling at 20°C. Data were processed with the Origin software supplied by MicroCal Inc. Each thermogram was corrected by subtraction of buffer thermograms acquired in the same conditions as the sample and by subtraction of the chemical baseline using the method of Takahashi and Sturtevant (1981). T _1/2 was defined as the temperature where the Cp^ex value is maximum, and the calorimetric enthalpy (H) was determined by direct integration of the area under the curve (Cladera et al. 1992b). The Van't Hoff enthalpy (H _vH) was calculated according to Krishnan et al. (1978) as:

where T _1/2 is the temperature at which Cp^ex value is maximum and Cp^ex is the heat capacity at this temperature.

Fourier transformed infrared spectroscopy (FTIR)
For the FTIR measurements, lyophilized proteins were dissolved in deuterated buffers according to the pH value of each experiment. In all cases, the buffer concentration was 50 mM (see tables and figure legends for specific buffers). Samples were stored overnight in the corresponding deuterated buffer in order to ensure a complete H/D exchange. The samples were sandwiched between two CaF₂ windows separated by a 50-µm Teflon spacer at a final concentration of 15 mg/mL. The temperature was controlled with a Julabo circulating bath, and FTIR spectra were recorded as a function of temperature. For each spectrum, 200 scans at a nominal resolution of 2 cm^–1 were averaged with a sample shuttle device using a FTIR-Mattson Polaris spectrometer. The spectrometer was equipped with a cooled nitrogen mercury-cadmium-telluride (MCT) detector, and it was continuously purged with dry air (dew point lower than –60°C). To obtain the pure spectrum of the protein, spectra of the solvent were recorded under identical conditions. The criterion for a good subtraction was to obtain a flat line between 1800 cm^–1 and 2000 cm^–1. All spectra were also corrected for atmospheric water. The spectra were deconvoluted using an FHHH of 15 cm^–1 and a k factor of 1.8. In order to measure the relative areas of the amide I' band components, deconvoluted spectra were curve-fitted by means of a least-squares iterative program.

Determination of the oxidation state of disulfide bonds
The presence of free cysteines in ECP samples at different steps of the thermal unfolding and refolding was determined by a derivatization reaction with 4-vinylpyridine and MALDI-TOF mass spectrometry. Protein samples were submitted to the thermal process at the conditions described under the fourth-derivative UV-spectroscopy methods, and aliquots of 40 µL were collected during the thermal process and freeze-dried. Each sample was dissolved in 50 µL of 0.1 M vinylpyridine in 0.1 M Tris-HCl (pH 8.4) and maintained for 1.5 h at room temperature. The reaction was quenched by 1/10 dilution of the sample in water containing 0.1% TFA. Each free cysteine residue modified by vinylpyridine increases the molecular mass of the protein by 105 Da. Mass determination of each sample was carried out by MALDI-TOF mass spectrometry.

Nondenaturing gel electrophoresis
ECP samples submitted to the thermal process at the conditions described under the fourth-derivative UV spectroscopy methods were analyzed by nondenaturing 15% polyacrylamide gel electrophoresis (pH 4.0) and Coomassie Blue staining.

Dye binding assay
A thioflavin-T binding assay directed to determine highly ordered amyloid structures (Le Vine 1993; Pallarès et al. 2004) was carried out in ECP samples at the conditions described under the fourth-derivative UV spectroscopy. Aliquots of 100 µL were diluted into buffer (10 mM sodium phosphate at pH 7.4, 150 mM NaCl) containing 65 µM thioflavin-T, and adjusted to a final volume of 1 mL. Fluorescence data were collected after 5 min to ensure that thermal equilibrium had been achieved. Fluorescence emission spectra were recorded using an excitation wavelength fixed at 440 nm.

	Footnotes

 
Reprint requests to: M. Victòria Nogués, Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; e-mail: victoria.nogues{at}uab.es; fax: 34-93-5811264.

Abbreviations: ECP, eosinophil cationic protein; RNase A, bovine pancreatic ribonuclease; EDN, eosinophil-derived neurotoxin; HP-RNase, human pancreatic ribonuclease; ONC, onconase; CD, circular dichroism; FTIR, Fourier transform infrared; DSC, differential scanning calorimetry; MALDI-TOF, matrix assisted laser desorption ionization-time of flight; CDA, cumulative difference amplitude; Cp^ex, heat capacity; H, calorimetric enthalpy; H _vH, Van't Hoff enthalpy; MCT detector, mercury-cadmium-telluride detector; FWHH, full width at half-height.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062196406.

	Acknowledgments

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
We thank Esteve Padrós (Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona) for helpful suggestions; Elodia Serrano (Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona) for her helpful assistance with DSC data acquisition; Ester Boix, Marc Torrent, and Elisabet Cuyàs (Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona) for molecular modeling and helpful discussions; Virtudes Villegas (Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona) for helpful suggestions about CD results; and Antoni Benito and Josep Font (Departament de Biologia, Universitat de Girona) for technical support. This work was supported by Grants 2001SGR-00196 from the Direcció General de Recerca of the Generalitat de Catalunya, BMC2003-08485-C02 from the Dirección General de Investigación of the Ministerio de Educación y Ciencia (Spain), and from the funds FEDER of the European Union.

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