Báo cáo khoa học: The thermodynamic analysis of protein stabilization by sucrose and glycerol against pressure-induced unfolding The typical example of the 33-kDa protein from spinach photosystem II docx

The thermodynamic analysis of protein stabilization by sucrose and glycerol against pressure-induced unfolding The typical example of the 33-kDa protein from spinach photosystem II Kangcheng Ruan 1 , Chunhe Xu 2 , Tingting Li 1 , Jiong Li 1 , Reinhard Lange 3 and Claude Balny 3 1 Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Science, Chinese Academy of Sciences, Shanghai, China; 2 Institute of Plant Physiology, Shanghai Institute for Biological Science, Chinese Academy of Sciences, Shanghai, China; 3 Institut National de la Sante ´ et de la Recherche Me ´ dicale, INSERM U 128, IFR 24, CNRS, Montpellier, France We have studied the reaction native « denatured for the 33-kDa protein isolated from photosystem II. Sucrose and glycerol have profound effects on pressure-induced unfolding. The additives shift the equilibrium to the left; they also cause a significant decrease in the standard volume change (DV). The change in DV was related to the sucrose and glycerol concentrations. The decrease in DV varied with the additive: sucrose caused the largest effect, glycerol the smallest. The theoretical shift of the half-unfolding pressure (P 1/2 )calculatedfromthenet increase in free energy by addition of sucrose and glycerol was lower than that obtained from experimental mea- surements. This indicates that the free energy change caused by preferential hydration of the protein is not the unique factor involved in the protein stabilization. The reduction in DV showed a large contribution to the theoretical P 1/2 shift, suggesting that the DV change, caused by the sucrose or glycerol was associated with the protein stabilization. The origin of the DV change is discussed. The rate of pressure-induced unfolding in the presence of sucrose or glycerol was slower than the refolding rate although both were significantly slower than that observed without any stabilizers. Keywords: conformational changes; hydrostatic pressure; spinach particle; protein denaturation. Understanding protein folding mechanisms is one of the big challenges in protein science. For example, an unusual property of prion protein unfolding in neutral salt solution has recently been shown [1]. However, the prion protein is not easy to work with and to go further, convenient models must be used. The 33-kDa protein from spinach photosys- tem II is a good system with which to explore the role of additives in protein folding and unfolding; their effects on the chemical denaturation of this protein have been described previously. This protein has a very low free energy of unfolding and it is easy to modulate its unfolding transition [2]. Most protein denaturation studies use chemicals (such as urea or guanidine hydrochloride) or thermal perturbation to influence the folding pattern. Reversibility is frequently a problem. For many years, various chemicals like ÔneutralÕ salts, glycerol, sucrose have been known as protein stabi- lizers. Initially it was thought that these molecules could form coating shells around the proteins. Subsequently, other studies on sucrose and glycerol indicated that these substances do not usually bind to protein; their presence changes the water surface tension around protein. They are preferentially depleted from the protein surface layer [3–5]. In other words, the proteins are preferentially hydrated around the surface in the presence of these stabilizers. This leads to an increase in free energy and consequently protection against denaturation [5]. An increasing number of researchers are using high- pressure as a denaturing agent. Compared to other meth- ods, pressure denaturation is often rapidly reversible [6]. High hydrostatic pressure has been used extensively to denature single chain proteins and oligomeric proteins [6–17]. Generally, single chain proteins such as trypsin, chymotrypsinogen, phospholipase, etc. can be unfolded in the pressure range 300–600 MPa; the 33-kDa protein and staphylococcal nuclease unfold at lower pressures [10,11]. High pressure induces a system volume decrease which governs the protein unfolding equilibrium; it has been shown that this volume change can be modulated by various factors. Different workers have studied this phenomenon [10,12,18] 1 . For example, Royer and coworkers found that Correspondence to K. Ruan, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China. Fax: + 86 21 64338357, Tel.: + 86 21 64740532, E-mail: kcruan@sunm.shcnc.ac.cn or C. Balny, INSERM U128, 1919 route de Mende, 34293 Montpellier Cedex 5, France. Fax: +33 47523681, Tel.: +33 467613360, E-mail: balny@montp.inserm.fr Abbreviations: GdmCl, guanidinium chloride; 4thD, fourth derivative absorbance spectra; CSM, centre of spectral mass; P 1/2 , experimental half pressure of denaturation; P 1/2 *, value of half pressure denaturation obtained from calculation for the net increase in free energy; P 1/2 **, value of half pressure denaturation obtained from calculation for the net reduction of the standard volume change. (Received 7 October 2002, revised 9 December 2002, accepted 27 January 2003) Eur. J. Biochem. 270, 1654–1661 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03485.x xylose stabilizes staphylococcal nuclease mainly by increas- ing the protein free energy (DG) of denaturation, while the standard volume change (DV) seems to be independent of the xylose concentration [10,18]. In earlier work we explored the pressure-induced dena- turation of the 33-kDa protein isolated from spinach photosystem II; the equilibrium is a two-state reversible transition which is influenced by NaCl [11]. P 1/2 (the half- pressure of denaturation) was shifted from 118 MPa to 127 MPa and 195 MPa in the absence and in the presence of 0.5 M and 1.0 M NaCl, respectively. It was also observed that the volume change, DV, decreases from )120.0 (without salt) to )108.1 (0.5 M NaCl) and to )80.0 mLmol )1 (1.0 M NaCl). DV and DG both contribute to the folding–unfolding shift. Some questions are still without clear answers: (a) is the reduction in DV also found with stabilizing agents such as glycerol and sucrose? (b) with these agents, is the same stabilization mechanism involved when the denaturation is induced either by chemical denaturants or by hydrostatic pressure? To answer these questions the effect of sucrose and glycerol on the pressure-induced unfolding of the 33-kDa protein has been studied in the present work. We chose the 33-kDa protein as a model because of its very low DG of unfolding at pH 6.0 and 20 °C()3.5 kcalÆmol )1 ) and because of its large standard DV ()120 mLÆmol )1 ). The response of the 33-kDa protein to pressure is completely reversible [11]. Moreover, the protein molecule contains only one tryptophan residue (Trp241) buried in a very strong hydrophobic region. This allows for easy fluorescence detection when this residue is exposed to the solvent. In the native form, the fluorescence emission, k max , is at 317 nm, shifting to 352 nm when unfolded. In this report we show that sucrose, glycerol and NaCl protect the 33 kDa protein against denaturation by either hydrostatic pressure or guanidine hydrochloride. Materials and methods Purification of the 33-kDa protein The 33-kDa protein was isolated and purified from spinach chloroplast photosystem II as described in our previous report [11]. The purified protein was dialysed against 10 m M NH 4 HCO 3 and then lyophilized. The protein concentrations were determined as described by Xu and Briker [19]. In most experiments, the protein was dissolvedin0.05 M pH 6.0 Mes buffer. All other reagents were of A. R. grade. Distilled water was further purified by a Millipore system to a resistance of 18 MW. Fluorescence measurements The fluorescence measurements were carried out either on an Aminco Bowman Series 2 (AB2) fluorospectrophoto- meter (SLM Co.) or on a SLM 48000 fluorospectrophoto- meter (SLM Co.). These have been modified thereby allowing us to measure fluorescence in a pressure range from 0.1 MPa to 600 MPa at temperatures between )20 °C and 100 °C. The fluorescence spectra were quantified by specifying the centre of spectral mass (CSM) <m>as introduced in our previous and related papers [20,21]. The excitation wavelength for the intrinsic fluorescence was 295 nm, which excited only the tryptophan residue. To measure the unfolding–refolding kinetics of the protein, the fluorescence spectrophotometer was further modified to adapt a pressure jump device designed in the INSERM laboratory [22]. Positive or negative pressure- jumps up to 150 MPa were possible in a pressure range from 0.1 to 600 MPa, with a dead time of 5 ms. Fourth derivative UV absorbance spectra Absorption spectra of the protein between 260 and 300 nm were recorded at 20 °C using a modified Cary3 (Varian) absorption spectrophotometer as described elsewhere; this instrument allows experiments in a pressure range from atmospheric pressure to 500 MPa at temperatures between )20 °Cand100°C [23]. The 4th derivative (4 th D) absorb- ance spectra were calculated from the corresponding absorption spectra as described previously [23,24]. Unfolding degree calculations The basic scheme for a denaturation reaction is N « D where N and D are the native and the denatured forms, respectively. The method for determining the degree of unfolding of the protein (a) was the same as reported previously and was calculated either from the centre of spectral mass (CSM) <m> for fluorescence measurement or from the amplitude of the change at 293 nm in the 4thD spectra [11]. The degree of unfolding (a) was plotted against pressure to draw the unfolding curve and to determine the half-denaturation pressure, P 1/2 . The free energy and standard volume change were calculated from the unfolding curve according to the method of Li et al. [13]. The values of DG were also estimated from half-denaturation pressure, P 1/2 , according to: DG ¼ 0:234 Â DV Â P 1=2 where P 1/2 is in MPa, DV in mLÆmol )1 and DGincalÆmol )1 , respectively [11]. The free energy of unfolding due to guanidine hydro- chloride was calculated according to the Tanford method [2,25]. Results Sucrose stabilization of the 33-kDa protein pressure-induced unfolding Fig. 1 shows the degree of unfolding, a,ofthe33-kDa protein plotted against pressure. The curves are shifted to the higher pressures as the sucrose concentration is increased. Consequently, P 1/2 is shifted from a minimum of 118 MPa to 320 MPa at 1.47 M sucrose. This indicates that in the presence of sucrose, the 33-kDa protein is more stable and is protected from pressure-induced denaturation. DG of unfolding is listed in Table 1. It increases as the sucrose concentration increases. This observation is in good agreement with the Timasheff model and with the results reported by Frye and Royer for the xylose study on Ó FEBS 2003 Stabilization of 33-kDa protein of spinach PS II against pressure unfolding (Eur. J. Biochem. 270) 1655 staphylococcal nuclease [18]. In contrast to the free energy, DV is found to decrease with the sucrose addition. In the absence of sucrose, DV is )120 mLÆmol )1 ; it decreases to 53.7 mLÆmol )1 at 1.47 M sucrose. The decrease in DV is obviously dependent on the sucrose concentration. Fig. 2 shows that the change in DV reduction is a linear function of the sucrose concentration (in osmolarity). However, the linearity is not followed when the concentration is rather high (1.47 M ). DGandDV of unfolding in the presence of sucrose have been also determined using 4thD spectra. The unfolding curve of the protein in the presence of 0.83 M sucrose obtained from the 4thD spectra (n)asshowninFig.1is very close to that obtained from the tryptophan fluorescence measurement (m). The free energy and the standard volume change are 3.94 kcalÆmol )1 and )66.1 mLÆmol )1 , in good agreement with that obtained from fluorescence experi- ments (3.9 kcalÆmol )1 and )72 mLÆmol )1 ). Stabilization effect of glycerol Fig. 3 shows the effect of glycerol on pressure-induced unfolding of the 33-kDa protein. When the glycerol concentration is increased from 0 to 40%, the unfolding curves shift to the higher pressures. P 1/2 increases from 118 MPa to 280 MPa (see Table 1). In 40% glycerol the 33-kDa protein is totally unfolded at about 400 MPa, a value much higher than that observed in the absence of glycerol (180 MPa). These results indicate that the glycerol, like sucrose, stabilizes the protein against pressure denatur- ation. DG of unfolding in the presence of glycerol increases from 3.5 to 5.3 kcalÆmol )1 (see Table 1); DG is dependent on glycerol concentration. All of these results also provide evidence supporting the Timasheff model [5]. DV of unfolding decreases with increasing glycerol concentration (see Table 1). It goes from )120 mLÆmol )1 (without glycerol) to )80.4 mLÆmol )1 (in 40%), giving results similar to those obtained from experiments using sucrose. Import- antly the decrease in DV seems to be associated with the stabilization effect. It should be noticed that the 10% glycerol concentration is an exception. Under this condition the DV has a small increase (of % 8mLÆmol )1 ), which is similar to that observed in the staphylococcal nuclease study [18]. They found a small increase in DV when xylose was added. However, they found that the increase in DV is independent of the xylose concentration. The linearity between the reduction in DV and the glycerol concentration isshowninFig.2(d). Denaturation of the 33-kDa protein by guanidine hydrochloride To understand the sucrose and glycerol effects on the stabilization of this protein against pressure-induced denaturation, guanidinium chloride (GdmCl)-induced protein denaturation has been studied. Tryptophan fluor- escence was used as a probe. The unfolding curves are plottedinFigs4and5forsucroseandglyceroleffects, respectively. The unfolding curves are obviously shifted to higher GdmCl concentrations when sucrose or glycerol concentrations are increased. The free energy values are listed in Table 1 and show a significant increase with the sucrose or glycerol concentrations. This indicates that Fig. 1. The unfolding of the 33-kDa protein induced by hydrostatic pressure in the presence of different sucrose concentrations. Curves from left to right: 0.0, 0.1, 0.2, 0.41, 0.83 and 1.47 M sucrose, respectively. The unfolding degrees (a) were calculated from the fluorescence spectra of the protein excited at 295 nm or from the 4 th Dspectrum. Protein concentration for fluorescence and 4 th D measurements: 0.1 mgÆmL )1 and 0.7 mgÆmL )1 , respectively, in 0.05 M Mes buffer, pH 6.0, 20 °C. Table 1. Thermodynamic parameters for the 33-kDa protein unfolding. DG, obtained from pressure-induced unfolding; DG*, obtained from GdmCl- induced unfolding; TP 1/2 *, obtained from calculation for the net increase in free energy; TP 1/2 ** obtained from calculation for the net reduction of the standard volume change. Reactions were performed at pH 6.0 and 20 °C. DG (kcalÆmol )1 ) DV (mLÆmol )1 ) P 1/2 (MPa) DG* (kcalÆmol )1 ) TP 1/2 * (MPa) TP 1/2 ** (MPa) No sucrose 3.5 )120.0 118 )2.6 118 118 0.10 M sucrose 3.4 )118.9 118 – 115 119 0.20 M sucrose 3.2 )103.5 130 )3.1 109 136 0.41 M sucrose 3.6 )92.7 163 )3.3 121 152 0.83 M sucrose 3.9 )72.0 234 )3.8 132 197 1.47 M sucrose 4.0 )53.7 320 )4.6 135 264 10% glycerol 4.0 )128.3 132 )3.3 135 110 20% glycerol 4.3 )101.5 177 )4.7 144 140 30% glycerol 5.2 )89.0 248 )5.2 175 159 40% glycerol 5.3 )80.4 280 )5.7 178 176 1656 K. Ruan et al.(Eur. J. Biochem. 270) Ó FEBS 2003 both sucrose or glycerol can inhibit the chemical dena- turation of the 33-kDa protein by the GdmCl, according to the preferential hydration around protein surface in the presence of the stabilizers. The values of DG obtained from either pressure- or GdmCl-induced unfold- ing are very similar. Some differences in quantitative values are observed (but they remain within a reasonable range for the results collected from various experimental methods). DGforthenative« denatured transition in the absence of protectants is 2.6 kcalÆmol )1 , a value in good agreement with those reported by Tanaka et al. (2.8 kcalÆmol )1 )[2]. The change in P 1/2 is caused by effects on DG and D V P 1/2 , the pressure at which 50% of the protein is unfolded, is a parameter often used to evaluate protein stability. The higher is P 1/2 , the more stable is the protein to pressure- induced denaturation. P 1/2 is related both to DGandDV according to: P 1=2 ¼ DG=DV or ln Kp ¼ DG þ P Ã DV=RT From the above formulae, the change in P 1/2 caused by the net variation in DG or by the net standard change alone (termed theoretical half unfolding pressure, TP 1/2 )canbe obtained. The TP 1/2 caused both by the net increase in DG and the net reduction of DV upon either sucrose or glycerol addition were calculated and listed in Table 1. It was found that the TP 1/2 * caused by the net increases in DG were lower than the experimental P 1/2 . Typically, the difference between P 1/2 and TP 1/2 *isaslargeas% 185 MPa in the presence of 1.47 M sucrose and 102 MPa in 40% glycerol (see Table 1). Even when the rather large values of DG obtained in GdmCl denaturation experiments were used for calculation, TP 1/2 *(153MPafor1.47 M sucrose and 192 MPa for 40% glycerol) were still much lower than those obtained from Fig. 2. The effect of sucrose (m), glycerol (j)andsalt(d)onthe standard volume change of the protein. The concentrations of the additives are expressed in osmolarity. Fig. 3. The unfolding of the 33-kDa protein induced by hydrostatic pressure in the presence of different glycerol concentrations (in volume). Curves from left to right: 0%, 10%, 20%, 30% and 40%, respectively. Other conditions as in Fig. 1. Fig. 4. The unfolding of the 33-kDa protein induced by GdmCl in the presence of different sucrose concentrations. Other conditions as in Fig. 1. Fig. 5. The unfolding of the 33-kDa protein induced by GdmCl in the presence of different glycerol concentrations (in volume). Other condi- tionsasinFig.1. Ó FEBS 2003 Stabilization of 33-kDa protein of spinach PS II against pressure unfolding (Eur. J. Biochem. 270) 1657 measurements (320 MPa and 280 MPa, respectively). This indicates that the net increase in free energy upon sucrose or glycerol addition cannot explain totally the protection effect—other factors are certainly involved. Meanwhile it was found that the theoretical changes in TP 1/2 ** calculated from the net reduction of DV were larger. In thecaseofsucrose,theTP 1/2 ** values calculated from the DV measurements were larger than the values calculated from the net increase in DG. Meanwhile, in the case of glycerol the TP 1/2 ** values calculated from both DV and DG were close to each other. These observations indicate that the reduction in DV probably plays an important role in the sucrose or glycerol protein protection against pressure denaturation. Effects of sucrose and glycerol on the kinetics of the pressure induced protein unfolding and refolding The unfolding and refolding kinetics in the presence of either 0.83 M sucrose or 30% glycerol have been investi- gated using positive and negative pressure jumps (Figs 6 and 7). Fluorescence intensity was monitored at 350 nm [11]. A single exponential was fit to the data (solid lines); the curves showed a rather slow, two-state transition processes. The corresponding relaxation times for the protein unfolding and refolding in the presence of both 0.83 M sucrose and 30% glycerol are listed in Table 2. Compared with the kinetics of the protein unfolding–refolding measured with- out additive where both are about 100 s [11], sucrose and glycerol slow both the folding and unfolding rates. It was also found that the presence of sucrose or glycerol induced an unfolding relaxation time significantly longer than that for the refolding reaction. For sucrose, the unfolding relaxation time is 2.5 times longer than refolding, and, for glycerol it is 1.5 times longer. The actual data suggest that the rather slow unfolding rate could be associated with the sucrose and glycerol stabilization effect. Discussion Our goal is to understand how high hydrostatic pressure induces the refolding of proteins and how sugars and salts influence this refolding. To this end, we used the effects of both hydrostatic pressure and osmotic pressure as probes [26]. As mentioned in the introduction, the stabilization mechanism of these agents has been attributed to a protein preferential hydration mechanism as proposed by Timasheff [5] or by an osmotic stress [27] where, mathematically, the two mechanisms cannot be distinguished [28]. In a very well documented paper, Parsegian et al.[28] indicated that there has been much confusion about the relative merits of different approaches, osmotic stress, preferential interaction (i.e. preferential hydration), and crowding, to describe the indirect effect of solutes on macromolecular conformations and reactions. The two first mechanisms (and crowding) cannot be distinguished as they are derived from the same solution theory. In the prefer- ential hydration model proposed by Timasheff, both the chemical nature and the size of the solute determine water exclusion from the protein surfaces [5]. Osmotic stress emphasizes the role of the water that is necessarily included if solutes are excluded [28], dealing also with the movement of water molecules [27]. Upon addition of solutes (in the present case, stabilizers), surface tension around the protein changes because of water exclusion. Consequently, the protein free energy increases, resulting in protein stabilization. However, the question is Table 2. Relaxation time of unfolding and refolding of the protein induced by pressure (times obtained from the data in Figs 6 and 7). Sample condition Relaxation time (s) Unfolding Folding 0% Glycerol, 0 M sucrose 125 100 30% Glycerol 360 145 0.8 M Sucrose 276 175 Fig. 6. Kinetics of the pressure-induced unfolding and refolding of the 33-kDa protein in the presence of 0.83 M sucrose. Curve A for a pressure jump from 100 MPa to 180 MPa. Curve B for a pressure jump from 180 MPa to 100 MPa. Solid lines are the fitted curves. Protein con- centration: 0.1 mgÆmL )1 in 0.05 M Mesbuffer,pH6.0,20°C. Exci- tation wavelength, 295 nm; emission wavelength, 350 nm. Fig. 7. Kinetics of the pressure-induced unfolding and refolding of the 33-kDa protein in the presence of 30% glycerol. Otherconditionsasin Fig. 6. 1658 K. Ruan et al.(Eur. J. Biochem. 270) Ó FEBS 2003 still open: how do additives protect proteins from pressure denaturation and what is the best model with which to interpret these effects? Frye and Royer reported that xylose can protect staphylococcal nuclease from pressure-induced unfolding mainly by increasing the protein free energy [18]. The present results show that both sucrose and glycerol increase the protein free energy, resulting in a stabilization effect. However, we also found that the contribution from theincreaseinDG is only a part of whole stabilization effect. The stabilization effect is actually stronger than that expected according to the preferential hydration model, implying that the model is insufficient to fully interpret the observations. It is more in keeping with the osmotic formulations [26,27]. The calculated standard volume changes decreasing (in absolute value) with both the sucrose and glycerol concentrations show an important contribu- tion to stabilization. Most studies concerning protein stabilization effects are not associated with the hydrostatic pressure denaturation. Consequently, the protein volume change (DV) and its variation is not usually taken into consideration. This parameter is usually inaccessible when the experiments have been achieved using only chemical denaturation. However, as pointed out above, for pressure- induced protein unfolding, DV is an important variable which governs the folding–unfolding equilibrium when pressure is applied (for a recent review see [6]). The variation in DV influences the folding–unfolding equilibrium accord- ing to Le Chatelier’s principle. The reduction in DV (in absolute value), favoured by folding, will shift the equilib- rium to the native state, there by resulting in a protective effect for the pressure-induced protein unfolding. On the other hand, the formula P 1/2 ¼ DG/DV indicates, from a mathematical point of view, that either an increase in DGor a decrease in DV will increase P 1/2 and, consequently, protect the protein. It is the cooperative effects of the increase in DG and the reduction in DV that protect the 33-kDa protein from pressure-induced unfolding by addi- tion of sucrose and glycerol. A reduction in DV, resulting from the addition of either sucrose or glycerol has been observed for other systems. In 1989, Ruan and Weber reported that glycerol can protect glyceraldehydephosphate dehydrogenase from pressure- induced dissociation mainly by the reduction in DV [29]. They found that 10% and 25% glycerol could reduce the DV from 230 mLÆmol )1 to 143 mLÆmol )1 and 86 mLÆmol )1 , respectively. Oliviera et al. also reported that glycerol reduces the DV of Arc repressor resulting in a protein stabilization [30]. With the actual results and data from the literature, it is difficult to make a generalization. However, we suggest that the stabilizing effects of sucrose and glycerol should be attributed to the effects on both DV and DG. The reduction in DV caused by these stabilizers is not the same for all proteins. For staphylococcal nuclease, the standard volume change shows a small increase upon xylose addition (smaller than 10%), suggesting that the protein is stabilized mainly by increasing DG[18]. Concerning the origin of the DV reduction, we think that many factors could be involved. First of all, as pointed out in a recent review by Taulier and Chalikian [31], the compressibility of protein transitions must be taken into consideration. In their paper they analyzed the compres- sibility changes accompanying conformational transitions of globular proteins in conjunction with the role of hydration. In the present work, the pressure-induced unfolding of the 33-kDa protein is a two-state equilibrium [11] where the detection of any transient intermediates (such as molten-globule like intermediates) can be excluded. It is possible to calculate changes in isothermal compressibility (Dk T ) associated with the pressure-induced denaturation [11]. Assuming that the change in DV depends only very slightly on both sucrose and glycerol, a specific change in compressibility of Dk T ¼ )1 · 10 )6 cm 3 Æg )1 Æbar )1 2 can be calculated. This value is within the range of compressibility changes accompanying protein denaturation as listed in [31]. However, Dk T for most proteins studied so far is positive; we have no evidence to show that Dk T for the 33 k-Da protein is negative. On the other hand, the assumption that DV depends only slightly on adducts is not fully support by experiments as the DV, in absence of any adduct is )120 mLÆmol )1 , a value which can be modulated (higher or lower) depending on the nature of the adduct (this work, and [11]). Even with a negative Dk T , the question of why the isothermal compressibility of the protein in the absence of adducts becomes negative, is still open. At this stage, further pressure-related studies on a larger set of globular proteins are required to determine the validity of each of the possible explanations [31]. The contraction of the protein–solvent interface caused by the increase in chemical potential is accomplished by water release from inside the protein, which consequently increases the core density [32]. The additives could significantly reduce the volume of the protein interior [33]. The preferential hydration of the protein when compounds are present leads to some protein conformational changes associated with variations in the apparent volume. Additionally, the reduc- tion in DV might be the result of osmotic stress which could be, for the present data, the result of the exclusion of the adducts from the protein core [27]. Sucrose and glycerol as osmolytes can induce an osmotic stress between the bulk solvent and the water in the cleft or core of the protein [26,34,35]. Consequently, the water can be removed from the cleft (or cavity). In any event, it is necessary to consider effects not on the protein alone, but on the complete system. In Fig. 2, the reductions in DV are plotted against osmolarity of the added agents (data from previous work [11] is included). In both conditions, it was found that the DV reductions are linearly proportional to the osmolarity. Moreover, the slopes of the curves are obviously different, meaning that the ability to reduce the DV by the additives is different: sucrose has the strongest effect, glycerol the weakest. This may be related to the extent to which the additives have access to the protein interior. The strength of osmotic stress close to the cleft or cavity in protein is dependent on both the molecular size and on the concentration of the osmolyte, but alsodepends on the so-called Ôsemipermeable membraneÕ (or ÔchannelsÕ as defined by Parsegian et al. [27]) which is determined by the protein itself [35]. The sucrose molecular size is larger than that of sodium and chloride ions; it will be more excluded on the basis of size. Sucrose has a stronger effect compared to NaCl at the same osmolarity, because the Ôsemipermeable mem- braneÕ of the protein surface might be more effective at excluding sucrose. For glycerol, the situation is more complicated because glycerol can also bind to the protein molecule. Ó FEBS 2003 Stabilization of 33-kDa protein of spinach PS II against pressure unfolding (Eur. J. Biochem. 270) 1659 In Fig. 8, the standard volume change has been plotted as a function of the reciprocal osmolarity values of NaCl and sucrose. For the sucrose curve, the extrapolation up to zero (a value at which the osmolarity is infinite), the standard volume change of the protein is )43 mLÆmol )1 which might be the DV minimum of the protein (or of the system: protein, solvent and sucrose) in the presence of sucrose. From this value, the P 1/2 of the protein was estimated to be % 400 MPa according to the formula reported above. DG was supposed to be 4.0 kcalÆmol )1 (estimated from the free energy of the protein in presence of either 0.87 M or 1.43 M sucrose: 3.9 and 3.95 kcalÆmol )1 , respectively). The behaviour in the presence of NaCl is qualitatively similar (see Fig. 8). Another interesting phenomenon concerning the signifi- cant decrease in DV is the observation that the presence of either sucrose or glycerol did not cause any protein conformational changes. In the fluorescence measurements, as well as in the CD and in the 4thD absorption, the protein spectra were almost the same in presence or in absence of additives. A recent report of Twist et al. about the effect of sucrose and glycerol on the environment of two tryptophan residues in apomyoglobin provides a clue to explain this phenomenon [36]. They found that the environment of Trp7 was obviously affected by these compounds, whereas the other one nearby Trp14 was not influenced. There is only a single tryptophan residue in the 33-kDa protein. We speculate that this behaves like those of Trp14 in apo- myoglobin. Conclusion In conclusion, we would like to stress that the fundamental principles from in vitro folding experiments have practical application in understanding the pathology of diseases of protein misfolding. High pressure, associated with the action of either denaturant and/or other chemical adducts, is an interesting tool to denature, aggregate, or disaggregate proteins, offering a number of unique advantages [6]. To this end, we must mention the first very recent publications which appear in the field of prion proteins where high pressure is used as a new approach to identify several conformers [37–40]. Acknowledgements This work was supported by a grant from National Natural Science Foundation of China and a grant from INSERM/Academia China (K. R. and C. B.). R. L. thanks the Gis-Prion and the HSFP for financial assistance. The authors warmly thank Prof J. Kornblatt (Concordia University, Canada) for fruitful discussions and critical comments. 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The unfolding of the 33-kDa protein induced by. role in the sucrose or glycerol protein protection against pressure denaturation. Effects of sucrose and glycerol on the kinetics of the pressure induced protein unfolding and refolding The unfolding