White Wine Proteins: How Does the pH Affect Their Conformation at

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White Wine Proteins: How Does the pH Affect Their Conformation at Room Temperature? Marie Dufrechou,†,‡,§ Aude Vernhet,†,‡,§ Pierre Roblin,∥,⊥ François-Xavier Sauvage,†,‡,§ and Céline Poncet-Legrand*,†,‡,§ †

INRA, UMR1083 SPO, F-34060 Montpellier, France Montpellier SupAgro, UMR1083 SPO, F-34060 Montpellier, France § Université Montpellier 1, UMR1083 SPO, F-34060 Montpellier, France ∥ Synchrotron SOLEIL, Gif Sur Yvette, France ⊥ URBIA-Nantes, INRA Centre de Nantes, 60 rue de la Géraudière, 44316 Nantes, France ‡

S Supporting Information *

ABSTRACT: Our studies focused on the determination of aggregation mechanisms of proteins occurring in wine at room temperature. Even if the wine pH range is narrow (2.8 to 3.7), some proteins are affected by this parameter. At low pH, the formation of aggregates and the development of a haze due to proteins sometimes occur. The objective of this work was to determine if the pH impacted the conformational stability of wine proteins. Different techniques were used: circular dichroism and fluorescence spectroscopy to investigate the modification of their secondary and tertiary structure and also SAXS to determine their global shape. Four pure proteins were used, two considered to be stable (invertase and thaumatin-like proteins) and two considered to be unstable (two chitinase isoforms). Two pH values were tested to emphasize their behavior (pH 2.5 and 4.0). The present work highlighted the fact that the conformational stability of some wine proteins (chitinases) was impacted by partial modifications, related to the exposure of some hydrophobic sites. These modifications were enough to destabilize the native state of the protein. These modifications were not observed on wine proteins determined to be stable (invertase and thaumatin-like proteins).

1. INTRODUCTION Protein aggregation is of importance in many different fields (biology, enzymology, medicine, food science, etc.). It can take place via different mechanisms: it may be triggered by protein unfolding, involve other cosolutes, and lead to the formation of stable or unstable complexes.1,2 Different environmental parameters such as the pH, the ionic strength, and the addition of chaotropic compounds can impact the conformational stability of proteins and then their aggregative behavior. Depending on their size and structure, the formation of protein aggregates can lead to the development of a haze, or these aggregates can grow and sediment to form deposits. In beverages such as beers, clear juices, or white wines,3,4 such hazes or deposits constitute a visual defect for the consumer. In white wines, this haze can develop even if the protein concentration is low (15 to 330 mg·L−1).5,6 This defect does not impact wine sensory properties but leads to a poor image of the products and results in sale losses.7,8 This instability is generally related to thermal protein unfolding, occurring during wine transport or storage, as a result of exposition to high temperatures. It may also develop under correct storage conditions, but following slower aggregation kinetics (12 months or longer).9−11 One of the objectives of our studies was to identify the mechanism of aggregation occurring in white wines at room temperature. © XXXX American Chemical Society

Because of the complexity and the variability of the wine matrix, the identification of aggregation mechanisms remains difficult. Four different classes of proteins comprising different isoforms are present in wine: thaumatin-like proteins (TLPs), chitinases, β-glucanases, and invertases.9,12−16 Until now, the mechanism of protein aggregation induced by high-temperature heat treatment has been thoroughly studied. β-Glucanases and chitinases were characterized as the most heat-sensitive proteins, whereas invertase and some thaumatin-like proteins did not aggregate.12,14,17 Differential scanning calorimetry experiments performed on purified proteins gave access to the melting temperature of some chitinases (55 °C), thaumatinlike protein isoforms (55, 61, and 62 °C), and invertase (81 °C).12 Our previous work reported the strong impact of the protein thermosensitivity on the mechanisms of aggregation, depending on the treatment used.17 The impact of pH (wine range 2.8−3.7) on the protein melting temperature was also demonstrated: decreasing the pH in the wine range decreased the melting temperature of wine proteins and modified the reversible unfolding of TLPs.18 Temperature and pH are thus important to consider for the study of the mechanisms of Received: April 22, 2013 Revised: July 19, 2013

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dx.doi.org/10.1021/la401524w | Langmuir XXXX, XXX, XXX−XXX

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aggregation of wine proteins. However, a few studies have investigated the mechanism of protein aggregation that can occur at room or lower temperatures. In previous work, we highlighted that wine protein aggregation at 25 °C was induced when the pH was lower than 3.5.18 Aggregation kinetics were strongly enhanced when the pH was lowered to 2.5. In this case, we hypothesized that aggregation was triggered by conformational changes. At low pH, because of their isoelectric point (pI mainly around 4 to 5), wine proteins are more charged. These modification can induce a destabilization of the native state of the protein, leading to a conformational modification and then to their aggregation. Our objective was to validate the hypothesis of the pHinduced conformational changes of some wine proteins. Four pure wine proteins were used: two identified as stable at room temperature and low pH (an invertase and a thaumatin-like protein) and two unstable (chitinases).18 The effect of the pH on their conformational stability was studied using complementary and classical methods: circular dichroism (determination of secondary structures), fluorescence spectroscopy (exposure of hydrophobic sites due to a modification of the tertiary structure), and small-angle X-ray scattering (global tertiary structure of the proteins).

Figure 1. One-dimensional SDS-PAGE analysis of the three proteins (A, C, and D) purified from Sauvignon white wine (molecular weight (MW) standards on left). The protein in lane B was provided by Dr. Matteo Marangon (AWRI, Adelaide, Australia). highlighted that chitinases with molecular weights of about 27 to 28 kDa were affected by pH variations. These proteins are not present in high quantity in our wine; therefore, we used a chitinase at 27.5 kDa (lane B). This latter was kindly provided by Dr. Matteo Marangon (AWRI, Adelaide, Australia). Purification steps used for this protein purification are detailed in Marangon et al.20 The obtained bands were excised for their identification by mass spectrometry. Analyses of 1D bands were performed at the BIBS platform (INRA, Nantes, France) according to the procedure described by Sauvage et al.14 Briefly, proteins were digested with trypsin, and the resulting peptides were analyzed by LC−MS/MS. The Vitis vinifera protein database was queried locally using the mascot search engine (v. 2.2.04; Matrix Science, London, U.K.) with the following parameters: all entries for the taxonomy, trypsin as enzyme, one missed cleavage allowed, carbamidomethylation of cystein as a fixed modification, oxidation of methionine as a variable modification, and 0.6 Da mass accuracy in both MS and MS/MS. Under these conditions, individual ions scoring above 36 indicated identity or extensive homology (p < 0.05), and proteins were validated once they showed at least one peptide over this threshold. 2.2. Circular Dichroism Spectroscopy. Circular dichroism experiments were performed on a Chirascan spectrometer (Applied Photophysics, United Kingdom) at the IBISA platform of the CBS Institute (Centre de Biochimie Structurale, Montpellier, France). Experiments were performed with protein concentrations from 0.3 to 0.5 g·L−1 in model solutions at pH 2.5 or 4.0 (12% ethanol, 0.013 M malic acid)12 and at an ionic strength of 0.02 M adjusted with NaCl. The temperature was set at 25 °C. Experiments were conducted in a short-path-length demountable cuvette (80 μL, 0.2 mm). CD spectra were recorded in the far-UV region, between 190 and 260 nm, and Pro-data suite software (Applied Photophysics, United Kingdom) was used to set up the parameters. Each spectrum was an average of two accumulations (scan rate 30 nm·min−1, bandwidth 1 nm, response time 2s). The percentage of helices, antiparallel β-sheets, parallel βsheets, β-turns, or random coils, contributing to the secondary structure of the proteins, was calculated by the CDNN CD spectra deconvolution software. 2.3. Small Angle X-ray Scattering. 2.3.1. Data Acquisition. Small angle X-ray scattering (SAXS) experiments were performed on the SWING beamline at Synchrotron SOLEIL (Gif-sur-Yvette, France). The incident beam energy was 12 keV (λ = 1.03 Å), and the distance from the sample to the Aviex CCD detector was 1.793 m. The corresponding scattering vector q = (4π)/λ sin θ ranged from 0.005 to 0.530 Å−1, with 2θ being the scattering angle and λ being the incident wavelength. Experiments were conducted at 25 °C. Invertase was studied in a model solution containing 0.013 M tartaric acid/0.15 M NaCl (pH 4.0 and 2.5 adjusted with NaOH) and was processed in autosampler mode, with the same acquisition and delay times (500 ms/500 ms). The two chitinases and VVTL1 were studied in a solution designed to mimic the white wine medium (12% ethanol, 7 g· L−1 glycerol, 0.013 M tartaric acid), adjusted to pH 2.5 and 4.0 at an ionic strength of 0.02 M. The coupling of size exclusion

2. MATERIALS AND METHODS 2.1. Protein Purification and Identification. A first purification step was performed on a Sauvignon white wine by cation-exchange chromatography. A 1.6-cm-diameter column was filled with 125 mL of SP Sepharose high performance gel (GE Healthcare). The method used was adapted from the one described by van Sluyter et al.19 The column was first equilibrated with a 13 mM tartrate buffer at pH 3.0 (buffer A) for 15 min. The flow rate was set up at 18 mL·min−1. Nine liters of Sauvignon wine were loaded, followed by a washing step with buffer A. Protein elution was performed using successive mixtures of buffers A and B (30 mM MES/1 M NaCl, pH 6.0): 0−70 min, 5% buffer B to 30% buffer B; 70−95 min, 30% buffer B to 100% buffer B, and 95−105 min, 100% buffer B. Protein elution was monitored by UV detection (280 nm), and fractions of 12 mL were collected. Proteins in the fractions were analyzed by 1D SDS-PAGE analysis, according to the method described previously.18 Results of these analyses (Supporting Information S1) were used to pool the fractions according to their protein composition. Three fractions of interest were selected and kept for further purification: fraction 1 (volume of 120 mL) containing a band with a high molecular weight (∼71 kDa) but also two bands with a molecular weight lower than 25 kDa, fraction 2 (volume of 120 mL) mainly composed of a band of about 25 kDa and a smaller-intensity band at 19 kDa, and fraction 3 (volume of 145 mL) containing mostly a band of about 19 kDa. These fractions were concentrated to 10 mL using a 200 mL ultrafiltration stirred cell equipped with a 5 kDa membrane (Amicon, Millipore). The applied trans-membrane pressure was 4 bar. Concentrated samples were then stored at −20 °C before further use. A second purification step was performed on the three selected fractions by size exclusion chromatography (Superdex 75, 120 mL, hiload 16/60, Amersham Biosciences). The column was first equilibrated with a pH 4 tartrate buffer 13 mM/10 mM NaCl at a flow rate of 1 mL·min−1 for 2 h. A 2 mL loop was used for sample injection, and 1 mL fractions were collected. Absorbance measurements at 280 nm and SEC-HPLC analyses (Agilent Zorbax GF-250 column on Agilent 1100 series system, same buffer) were then performed to pool protein fractions according to concentration and purity criteria. Purified proteins were analyzed first by SDS-PAGE. Before analysis, the protein concentration in the samples was adjusted to 0.2 g·L−1on the basis of UV absorbance. Three pure proteins were selected (Figure 1): lane A at 71.7 kDa, lane C at 24.2 kDa, and lane D at 19.6 kDa. In previous work performed with Sauvignon white wine proteins,18 we B


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Table 1. Protein Identification by Mass Spectrometry, Where A−D Are the Same as in Figure 1

a

lane

peptide number

scorea

accession number (uniprot KB)

theoretical mass (Da)

measured mass (Da)b

name

A B C D

16 17 8 18

158 134 151 451

Q9S944_VITVI Q7XAU6_VITVI Q7XAU6_VITVI O04708_VITVI

71 746 27 528 27 528 24 866

71 000 27 500 24 200 19 600

vacuolar invertase 1 class IV - chitinase class IV - chitinase VVTL1

Identification score that matched with peptides of the Uniprot database. bObtained from 1D SDS-PAGE electrophoresis with image analysis.

Table 2. Proteins with Highly Similar Structure in the PDB as Identified by the I-Tasser Server (TM-align Score) with Invertase, Chitinase, and VVTL1 UniprotkB

TM-align score

6-fructosyltransferase class I chitinase

E3PQS3 Q7DNA1

0.814 0.860

chitinase

P23951

0.724

zeamatin (member of the thaumatin-like protein family) thaumatin-like protein

P33679

0.849

O22322

0.850

protein invertase chitinase

VVTL1

chromatography and SAXS was used to get a better accuracy, especially for buffer subtraction, but also to remove possible aggregates. A size exclusion column (Biosec3, Agilent) was used with an Agilent 1200 HPLC system: 40 μL of proteins (thaumatin-like protein and chitinases) was injected. The system was equilibrated with the same buffer as the buffer used for protein dissolution (flow rate 0.2 mL·min−1 for 1 h). The acquisition and delay times (respectively 500 ms/500 ms) were chosen to minimize protein degradation. Two hundred frames were recorded before (blank) and during protein elution.21 Because of the partial unfolding of proteins under X-ray treatment, only the 12−20 frames of the scattering intensity of the protein at higher intensity were chosen. The blank was subtracted from the average of the chosen frames using the Foxtrot program. The forward scattering angle (I0) and the radius of gyration (RG) of proteins were determined first using the Guinier approximation and PRIMUS software. The pair-distance distribution function P(r) was then calculated with GNOM software.22 It provides the estimated radius of gyration (Rg), the maximum diameter (Dmax), the forward scattering angle (I0) and gives an indication of the global shape of the protein in solution.23 2.3.2. Structure and Function Prediction. SAXS data were used to build the final 3D structure of the proteins at the two tested pH values by comparison with crystallized proteins having sequence similarities. To this end, the sequence of the studied protein, determined by mass spectrometry, was processed via the I-TASSER online program (http://zhanglab.ccmb.med.umich.edu/I-TASSER/).24,25 The theoretical structures obtained were matched against proteins of known structure and function in the protein data bank (PDB). The experimental scattering intensity curve determined by SAXS was compared to the solution scattering calculated from the model using the CRYSOL program.26 A better fit was achieved by the modification of protein structure (loop modification) from the PDB file with COOT software and the DADIMODO program.27 2.4. Fluorescence Spectroscopy. Measurements were performed with an RF 5301 PC fluorescence spectrophotometer (Shimadzu, Japan) and were used to evidence modifications of the tertiary structure of the proteins. Hydrophobic fluorescent probe ANS (1anilinonaphthalene-8-sulfonate, Sigma) was used because of its capacity to bind specifically to hydrophobic sites of the protein.28,29 Experiments were performed in model solutions (12% ethanol, 7 g·L−1 glycerol, 0.013 mM tartaric acid) at pH values of 2.5 and 4.0 and an ionic strength of 0.02 M. The pH was adjusted with 5 M NaOH, and the ionic strength, with 2 M NaCl. The protein concentration was set to a value of about 0.1 g·L−1. The ANS probe was added to a final concentration of 0.1 mM. Experiments were performed at 25 °C. The

organism

function

publication

Pachysandra terminalis Oryza sativa Japonica group (rice) Hordeum vulgare (barley seeds) Zea mays (maize)

transferase hydrolase and antifungal protein antifungal protein

31 32

antifungal protein

34

Musa acuminata (banana fruit)

antifungal protein

35

33

excitation wavelength was 350 nm, and the emission wavelength ranged from 400 to 600 nm. Data were recorded and analyzed with the RFPC software (Shimadzu, Japan).

3. RESULTS AND DISCUSSION As stated before, our aim was to investigate the impact of the pH (at 2.5 and 4.0) on the conformation of four selected wine proteins. To this end, experimental data along with structure prediction using I-TASSER were used. Indeed, there is only a little information about the structure of the wine proteins. To our knowledge, only some thaumatin-like protein isoforms have been crystallized,19 and diffraction data are now available (PDB code 4l5h). 3.1. Identification of Purified Proteins. The identity of the purified proteins was assessed by mass spectrometry (Table 1). These proteins were an invertase, a thaumatin-like protein, and two chitinases. It is important to note that the two chitinases (Figure 1, lanes B and C) correspond to the same accession number. The difference between their molecular weights could be related to partial hydrolysis during wine making or aging.30 The sequence of the proteins, obtained by mass spectrometry, was computed using the I-TASSER server.24,25 Software analysis designated proteins with highly similar structure in the PDB (identified by TM-align). For each protein, homologies with the structure of plant protein (Pachysandra, rice, barley, maize, or banana) were found. They belong to the same class of proteins and have the same functions: transferase for the invertase31 and mainly antifungal activities for the class IV chitinase and the VVTL1.34,35 Results for proteins having the best score are summarized Table 2. At low pH, because of their isoelectric point (pI mainly around 4 to 5), wine proteins get more and more charged when the pH decreases from 4 to 2.5. These modifications may induce a destabilization of the native state of the protein, leading to conformational modification and then to their aggregation. In the following text, the impact of pH changes on the conformation of wine proteins are discussed. 3.2. pH Impact on the Secondary Structure of Proteins. The secondary structure of the four proteins was studied by circular dichroism.36 Model solutions were C


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in helices (as already described by Falconer et al.12). The VVTL1 was mainly made up of helices and random coils. These results were consistent with the 3D structure of the plant proteins previously mentioned (Table 2). Circular dichroism spectra of the proteins at pH 4.0 and 2.5 were recorded between 190 and 260 nm (data not shown). A modification of the protein spectrum can indicate a loss of the native state, and high signal intensity is related to good-quality CD spectra. Whatever the studied protein, CD spectra at the two pH values overlapped. Small changes were observed but were not considered to be significant. In particular, decreasing the pH did not induce changes in the ellipticity of the chitinases whereas the pH decrease was previously shown to induce their aggregation in wine and model winelike protein solutions. Falconer et al.12 studied the impact of temperature on the secondary structure of a class IV chitinase (25.6 kDa). At 60 °C, α-helices unwound, leading to protein unfolding and a reduction in chitinase ellipticity. The temperature-induced conformational changes thus differ from the pH-induced ones. However, local changes involving movements in external loops may occur when the pH decreases. Such changes could be detected by SAXS or fluorescence spectroscopy. 3.3. Global Structure of the Proteins by SAXS. SAXS experiments were performed to determine the structure of the

simplified in comparison to small-angle X-ray scattering and fluorescence spectroscopy experiments. Indeed, the use of a buffer with 12% ethanol, 7 g·L−1 glycerol, and 0.013 M tartaric acid induced a large amount of noise related to the symmetry of the tartaric acid structure. Therefore, tartaric acid was replaced by malic acid. It was determined by fluorescence spectroscopy that choosing malic acid instead of tartaric acid does not impact the protein structure. The contribution of the different components was calculated with the CDNN program. Results obtained at pH 4.0 are shown in Table 3. They indicate that Table 3. Percentage of Contributions of the Various Components to the Protein Secondary Structure Predicted by CDNN Software

helix antiparallel β-sheet parallel β-sheet β-turn random coil

invertase

chitinase 27.5 kDa

chitinase 24.2 kDa

VVTL1

19 23 9 17 32

51 2 6 15 25

65 2 4 16 13

31 11 9 18 31

invertase exhibits a structure mainly composed of random coils and antiparallel β-sheets. The two class IV chitinases were rich

Figure 2. SAXS curve analysis of (A) VVTL1, (B) invertase, (C) 27.5 kDa chitinase, and (D) 24.2 kDa chitinase. The measurements were performed at pH 4 (curves in red) and pH 2.5 (curves in blue) and superimposed for comparison. The normalized Kratky plot is used to compare the SAXS curves at the two pH values, with normalized scattering vector qRg on the horizontal axis and normalized intensity (qRg)2I(q)/I(0) on the vertical axis. From each SAXS curve at the two pH values, the corresponding autocorrelation function P(r) was calculated and superimposed (with the same color as above). D


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the 27.5 kDa chitinase appears more compact than at pH 4.0, with reductions in Rg (22.1 to 20.5 Å) and Dmax (90 to 71 Å) values. This behavior was also observed with the 24.2 kDa chitinase (from 65 to 55 Å) but with less importance. To understand the structural changes induced by the pH, a starting model that takes every atom of the 27.5 kDa chitinase was used. Several models for the protein structure were generated by I-TASSER from the protein sequence of the chitinase identified by mass spectrometry. The best model selected corresponded to a class I chitinase from Oryza sativa L. japonica (PDB code 2DKV) containing an N-terminal chitinbinding domain (ChBD) and a C-terminal catalytic domain (CatD), which are connected by a short linker peptide.32 This full model of the 27.5 kDa chitinase was refined with the Dadimodo program against SAXS data at pH 4.0. Results are shown in Figure 4. Good agreement was observed between the experimental SAXS curve at pH 4.0 and the modeled structure as shown by the residual. If the model is confronted with data obtained at pH 2.5, then the quality of the fit is significantly worse. In this case, we can highlight that the change in pH locally affects the structure of the chitinase. However, SAXS cannot determine which regions are affected. 3.4. Study of the Tertiary Structure by Fluorescence Spectroscopy. To confirm the results of SAXS and CD, which highlight local rather than global changes, we performed fluorescence spectroscopy experiments. The modification of the protein structure can lead to the exposure of some buried sites, such as hydrophobic ones. Fluorescence spectroscopy in the presence of the ANS probe was used. This probe fluoresces differently in an apolar microenvironment: an increase in

four proteins but also to monitor possible changes with decreasing pH. The scattering patterns collected at pH 2.5 and 4.0 are displayed in Figure 2. The radius of gyration Rg for each protein was determined by the Guinier approximation and summarized in Table 4. The Table 4. Radii of Gyration of the Different Tested Proteins at pH 2.5 and 4.0 Determined by the Guinier Equation and Estimated Using GNOM Program Rg (Guinier, Å)

Rg (GNOM, Å)

Dmax (GNOM, Å)

proteins

2.5

4.0

2.5

4.0

2.5

4.0

invertase chitinase 27.5 kDa chitinase 24.2 kDa VVTL1

28.0 ± 0.03 20.0 ± 0.03

27.8 ± 0.03 21.6 ± 0.02

27.9 20.5

28 22.1

89 71

89 90

17.2 ± 0.07

18.1 ± 0.03

17.3

17.6

55

65

17.0 ± 0.03

16.9 ± 0.02

16.7

16.8

49

51

distance distribution functions P(r) were also calculated (Figure 3) using the GNOM program to estimate the radius of gyration, the maximum internal distance Dmax (Table 4), and the shape of the studied proteins. The Gaussian shape of the P(r) function is typical of globular proteins37 and remained similar at pH 4.0 and 2.5. The results show that there are no measurable differences between the two pH values, except for the 27.5 kDa chitinase. In that case, a slight but significant change in the form factor is observed at small angles, as confirmed by the calculation of the P(r) function. At pH 2.5,

Figure 3. Molecular modeling of the 27.5 kDa chitinase from the I-TASSER program. The different domainsthe N-terminal helix (blue), the Nterminal chitin-binding domain (yellow), and the C-terminal catalytic domain CatD (red)are displayed in different colors for more clarity in model A. Comparison of experimental SAXS data at pH 4.0 (red curve, B) and pH 2.5 (blue curve, C) with one model of the 27.5 kDa chitinase. The experimental curves and the curves calculated from the model (in black) are displayed on a log scale on the horizontal and vertical axes to enhance the small-angle regions. To illustrate the difference between both curves, the corresponding residual plot is also shown. E


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Figure 4. Emission spectra of (A) VVTL1, (B) invertase, (C) 27.5 kDa chitinase, and (D) 24.2 kDa chitinase in model solutions (0.02 M ionic strength) at pH 2.5 and 4.0 and in the presence of ANS. The excitation wavelength was 350 nm. A shift of the emission length and an increase in fluorescence intensity indicate a modification of the protein tertiary structure.

expected that the exposure of hydrophobic sites results in a loss of solubility and then in the formation of aggregates. Previous papers suggested that protein hydrophobicity plays an important part in the formation of aggregates.39 These experiments also pointed out time effects: conformational changes induced by pH are not instantaneous, and this is consistent with what is observed for wine haze formation, which is triggered after a few months. Last, reversibility experiments were performed on the 27.5 kDa chitinase: increasing the pH from 2.5 to 4.0 after 1 week led to the observation of the same spectra as the initial one at pH 4.0. The conformational changes are reversible, at least after 1 week.

intensity and a shift of the maximum wavelength of emission are observed. Sample analyses were performed at room temperature, and spectra obtained at pH 4.0 and 2.5 for the four proteins are shown in Figure 4. The spectra of the VVTL1 (Figure 4A) at pH 4.0 and 2.5 are very similar, indicating that the tertiary structure of this protein was not affected by the pH. Likewise, only a very minor shift of the emission wavelength (1 nm) between pH 4.0 and 2.5, along with a small increase in the fluorescence intensity (20 au), was observed for invertase (Figure 4B). By contrast, important modifications were evidenced for the two class IV chitinases (Figure 4C,D), the different molecular weights of which are likely related to the partial hydrolysis of the 27.5 kDa isoform during wine making. Indeed, Monteiro et al.38 showed that these two isoforms were already present in the grape berry. At pH 4.0, the maximum emission intensity was obtained at wavelengths of 510 and 514 nm for the 24.2 and 27.5 kDa chitinases, respectively. It is worth noting that the proteins were stored at pH 4 and that their fluorescence spectra did not change with time. These maxima were shifted to 500 and 506 nm at pH 2.5, and a significant increase in the fluorescence intensity was observed. This increase was much more pronounced for the chitinase at 24.2 kDa (around 50%) than for the one at 27.5 (16%). The differences observed for the 27.5 kDa chitinase were emphasized after 2 days at pH 2.5. These results indicated a strong exposure of some hydrophobic sites previously buried inside the protein backbone. It is

4. CONCLUSIONS The aim of the present work was to assess that the aggregation of some of the wine proteins at pH below 3.0 observed in a previous study18 at room temperature is triggered by conformational changes. To this end we studied the conformation of four different proteins at pH 4.0 (where no aggregation was observed in wine or model solution) and 2.5 (formation of protein aggregates in wine and in model solutions): a thaumatin-like protein (VVTL1) and an invertase, shown not to be involved in haze formation, and two chitinases (class IV chitinases at 27.5 and 24.2 kDa) involved in aggregates. Circular dichroism experiments demonstrated that the secondary structure of the four proteins was the same for both pH values. This indicated that their secondary structure F


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was not dramatically impacted by this parameter. In contrast, some local changes in the structure of the unstable chitinases were identified by means of SAXS and fluorescence spectroscopy. Decreasing the pH affected the protein shape and led to the exposure of some buried hydrophobic sites. Hydrophobic interactions between theses patches then likely favor protein aggregation or coaggregation with other macromolecules. These modifications were not observed on the stable invertase and 19.6 kDa VVTL1. The hypothesis of pH-induced conformational changes triggering the aggregation of class IV chitinases in wine and at room temperature was thus validated. In addition, it was demonstrated that the instability of these proteins is related only to minor and local conformational changes. The time dependence is also an important parameter to consider when studying the stability of wine proteins: as demonstrated previously, aggregation rates are increased when the pH is lowered between 3.2 and 2.5.18 Though not studied here, it is likely that these different aggregation kinetics are related to the time dependence of pH-induced conformational changes. This information contributes to a better understanding of the mechanisms involved in the aggregation of wine proteins. Four important protein isoforms, belonging to the three main protein classes, were thoroughly studied here. However, purification steps (Figure S1), in accordance with other works,19,40 evidence the presence of a wide diversity of isoforms with different hydrophobicities and charges, especially among thaumatin-like proteins and chitinases. It has been shown that different thaumatin-like protein isoforms have different aggregative behaviors.41 Experiments of fluorescence spectroscopy performed on these different isoforms could complement these results.

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ASSOCIATED CONTENT

S Supporting Information *

Protein elution profile after the first chromatographic separation and corresponding SDS-PAGE. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 33 (0)4 99 61 20 23. Fax: 33 (0)4 99 61 28 57. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. Matteo Marangon for providing the chitinase protein and to Dr. Christian Dumas (CBS, Montpellier, France) for helpful discussions about circular dichroism. We thank the Pech Rouge Experimental Unit (INRA, Gruissan, France) for providing the Sauvignon white wine.

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ABBREVIATIONS CD, circular dichroism; SEC, size exclusion chromatography; SAXS, small-angle X-ray scattering; VVTL1, Vitis vinifera thaumatin-like 1

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REFERENCES

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White Wine Proteins: How Does the pH Affect Their Conformation at

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