The thermodynamic analysis of protein stabilization by sucroseand glycerol against pressure-induced unfolding The typical example of the 33-kDa protein from spinach photosystem II Kangch
Trang 1The 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 Ruan1, Chunhe Xu2, Tingting Li1, Jiong Li1, Reinhard Lange3and Claude Balny3
1 Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Science,
Chinese Academy of Sciences, Shanghai, China;2Institute of Plant Physiology, Shanghai Institute for Biological Science,
Chinese Academy of Sciences, Shanghai, China;3Institut 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 (P1/2) calculated from the net
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 P1/2 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 sysphotosys-tem 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 neutral 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)
Trang 2xylose 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] P1/2(the
half-pressure of denaturation) was shifted from 118 MPa to
127 MPa and 195 MPa in the absence and in the presence of
0.5Mand 1.0MNaCl, respectively It was also observed that
the volume change, DV, decreases from)120.0 (without salt)
to )108.1 (0.5M NaCl) and to )80.0 mLmol)1 (1.0M
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 20C ()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, kmax, 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 mM NH4HCO3 and then lyophilized The
protein concentrations were determined as described by
Xu and Briker [19] In most experiments, the protein was
dissolved in 0.05MpH 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)
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 100C 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 20C 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 C and 100 C [23] The 4th derivative (4thD) 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, P1/2 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,
P1/2, according to:
DG ¼ 0:234 DV P1=2 where P1/2is in MPa, DV in mLÆmol)1and DG in calÆmol)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, of the 33-kDa protein plotted against pressure The curves are shifted to the higher pressures as the sucrose concentration is increased Consequently, P1/2 is shifted from a minimum
of 118 MPa to 320 MPa at 1.47Msucrose 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
Trang 3staphylococcal 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 1.47M 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 (1.47M)
DG and DV of unfolding in the presence of sucrose have
been also determined using 4thD spectra The unfolding
curve of the protein in the presence of 0.83M sucrose
obtained from the 4thD spectra (n) as shown in Fig 1 is
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)1and)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 P1/2 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 8 mLÆmol)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
is shown in Fig 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 plotted in Figs 4 and 5 for sucrose and glycerol effects, 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 4thD spectrum.
Protein concentration for fluorescence and 4thD 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)
Trang 4both 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) DG for the native« 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 inP1/2is caused by effects on DG and DV
P1/2, the pressure at which 50% of the protein is unfolded, is
a parameter often used to evaluate protein stability The
higher is P , the more stable is the protein to
pressure-induced denaturation P1/2is related both to DG and DV according to:
P1=2 ¼ DG=DV or ln Kp ¼ DG þ P DV=RT From the above formulae, the change in P1/2caused by the net variation in DG or by the net standard change alone (termed theoretical half unfolding pressure, TP1/2) can be obtained The TP1/2caused 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 TP1/2* caused by the net increases in DG were lower than the experimental P1/2 Typically, the difference between
P1/2and TP1/2* is as large as 185 MPa in the presence of 1.47Msucrose 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,
TP1/2* (153 MPa for 1.47Msucrose and 192 MPa for 40% glycerol) were still much lower than those obtained from
Fig 2 The effect of sucrose (m), glycerol (j) and salt (d) on the
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-tions as in Fig 1.
Trang 5measurements (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 TP1/2**
calculated from the net reduction of DV were larger In
the case of sucrose, the TP1/2** 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 TP1/2** 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 0.83M 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 0.83Msucrose and
30% glycerol are listed in Table 2 Compared with the
kinetics of the protein unfolding–refolding measured
with-out additive where both are abwith-out 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
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)1in 0.05 M Mes buffer, pH 6.0, 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 Other conditions as in Fig 6.
Trang 6still 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
the increase in DG 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 stabilizacontribu-tion 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 P1/2¼ DG/DV indicates, from a
mathematical point of view, that either an increase in DG or
a decrease in DV will increase P1/2 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)1to 143 mLÆmol)1and 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 (DkT) 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 DkT¼)1 · 10)6cm3Æg)1Æbar)1
calculated This value is within the range of compressibility changes accompanying protein denaturation as listed in [31] However, DkT for most proteins studied so far is positive; we have no evidence to show that DkT 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 DkT, 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 also depends on the so-called semipermeable membrane (or channels 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 semipermeable mem-brane 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
Trang 7In 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)1which 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 P1/2of 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 0.87Mor 1.43Msucrose: 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 We thank also one referee who pointed out the problem of relationship between denaturation and compressibility.
References
1 Nandi, P.K., Leclerc, E & Marc, D (2002) Unusual property of prion protein unfolding in neutral salt solution Biochemistry 41, 11017–11024.
2 Tanaka, S., Kuwada, Y., Wada, K & Hamaguchi, K (1989) Extrinsic 33-kDa protein of spinach oxygen-evolving complexes: kinetic studies of folding and disulfide reduction Biochemistry 28, 7188–7193.
3 Gekko, K & Timasheff, S.N (1981) Mechanism of protein sta-bilization by glycerol: preferential hydration in glycerol-water mixtures Biochemistry 20, 4667–4676.
4 Lee, J.C & Timasheff, S.N (1981) The stabilization of proteins by sucrose J Biol Chem 256, 7193–7201.
5 Timasheff, S.N (1993) The control of protein stability and association by interactions with water: how do solvent affect these processes? Annu Rev Biophys Biomol Structure 22, 67–97.
6 Balny, C., Heremans, K & Masson, P., eds (2002) Frontiers in High Pressure Biochemistry and Biophysics Elsevier, Amsterdam.
7 Ruan, K., Lange, R., Bec, N & Balny, C (1997) A stable partly denatured state of trypsin induced by high hydrostatic pressure Biochem Biophys Res Commun 239, 150–154.
8 Ruan, K., Lange, R., Meersman, F., Heremans, K & Balny, C (1999) Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin Eur J Biochem 265, 79–85.
9 Dumoulin, M., Ueno, H., Hayashi, R & Balny, C (1999) Con-tribution of the carbohydrate moiety to conformational stability
of the carboxypeptidase Y High pressure study Eur J Biochem.
262, 475–483.
10 Vidugiris, G.J.A., Truckses, D.M., Markley, J & Royer, C (1996) High-pressure denaturation of staphylococcal nuclease proline-to-glycine substitution mutants Biochemistry 35, 3857–3854.
11 Ruan, K & Xu, C., YuY., Li, J., Lange, R., Bec, N & Balny, C (2001) Pressure-exploration of the 33-kDa protein from spinach photosystem II particle Eur J Biochem 268, 2742–2750.
12 Saad-Nehme, J., Silva, J.L & Meyer-Fernandes, J.R (2001) Osmolytes protect mitochondrial F 0 F 1 -ATPase complex against pressure inactivation Biochim Biophys Acta 1546, 164–170.
13 Li, T., Hook, J., Drickamer, H.K & Weber, G (1976) Plurality of pressure-denatured forms in chymotrypsinogen and lysozyme Biochemistry 15, 5571–5580.
14 Torrent, J., Connelly, J., Coll, M.G., Robo, M., Lange, R & Vilanova, M (1999) Pressure versus heat-induced unfolding of ribonuclease A: The case of hydrophobic interactions within a chain-folding initiation site Biochemistry 38, 15952–15961.
15 Ruan, K., Lange, R., Zhou, Y & Balny, C (1998) Unusual effect
of high hydrostatic pressure on basic phospholipase A2 from venom of Agkistrodon halys pallas Biochem Biophys Res Com-mun 249, 844–848.
16 Zhou, J.-M., Zhu, L., Balny, C., Inactivation of creatine kinase by high pressure may precede dimer dissociation E ur J Biochem.
267, 1247–1253.
Fig 8 Plot of the standard volume change of the 33-kDa protein in the
presence of different concentration of NaCl (d) and sucrose (m) vs the
reciprocal of the sucrose osmolarity.
Trang 817 Zhang, J., Peng, X., Jonas, A & Jonas, J (1995) NMR study of
the cold, heat, and pressure unfolding of ribonuclease A.
Biochemistry 34, 8631–8641.
18 Frye, K.J & Royer, C.A (1997) The kinetic basis for the
stabili-zation of staphylococcal nuclease by xylose Protein Sci 6, 789–
793.
19 Xu, Q & Briker, T.M (1992) Structural organization of proteins
on the oxidizing side of photosystem II J Biol Chem 267, 25816–
25821.
20 King, L & Weber, G (1986) Conformational drift and
cryo-inactivation of lactate dehydrogenase Biochemistry 25, 3637–
3640.
21 Silva, J.L., Moles, E.W & Weber, G (1986) Pressure dissociation
and conformational drift of the b-dimer of tryptophan synthase.
Biochemistry 25, 5781–5786.
22 Mombelli, E (1999) Stabilite´ conformationnelle et me´canisme de
de´naturation de prote´ines thermostables The`se, Universite´ de
Montpellier II, Montpellier, France.
23 Lange, R., Frank, J., Saldana, J.-L & Balny, C (1996) Fourth
derivative UV-spectroscopy of proteins under high pressure I.
Factors affecting the fourth derivative spectrum of the aromatic
amino acids Eur Biophys J 24, 277–283.
24 Lange, R., Bec, N., Mozhaev, V.V & Balny, C (1996) Fourth
derivative UV-spectroscopy of proteins under high pressure II.
Application to reversible structural changes Eur Biophys J 24,
284–292.
25 Tanford, C (1970) Protein denaturation Adv Prot Chem 24,
1–95.
26 Kornblatt, J.A & Kornblatt, M.J (2002) The effects of osmotic
and hydrostatic pressures on macromolecular systems Biochim.
Biophys Acta 1595, 30–47.
27 Parsegian, V.A., Rand, R.P & Rau, D (1995) Macromolecules
and water: probing with osmotic stress Methods Enzymol 259,
43–94.
28 Parsegian, V.A., Rand, R.P & rau, D.C (2000) Osmotic
stress, crowding, preferential hydration and binding: a
com-parison of perspectives Proc Natl Acad Sci USA 97, 3987–
3992.
29 Ruan, K & Weber, G (1989) Hysteresis and conformational drift
of pressure-dissociated glyceraldehydephosphate dehydrogenase Biochemistry 28, 2144–2153.
30 Oliveira, A.C., Gaspar, L.P., Da Poian, A.T & Silva, J.L (1994) Arc repressor will not denature under pressure in the absence of water J Mol Biol 240, 184–187.
31 Taulier, N & Chalikian, T.V (2002) Compressibility of protein transitions Biochim Biophys Acta 1595, 48–70.
32 Boonyaratanakornkit, B.B., Park, C.B & Clark, D.S (2002) Pressure effects on intra– and intermolecular interactions within proteins Biochim Biophys Acta 1595, 235–249.
33 Priev, A., Almagor, A., Yedgar, S & Gavish, B (1996) Glycerol decreases the Volume and compressibility of protein interior Biochemistry 35, 2061–2066.
34 Robinson, C.R & Sligar, S.G (1994) Hydrostatic pressure reverses osmotic pressure effect on the specificity of EcoRI–DNA interactions Biochemistry 33, 3787–3793.
35 Robinson, C.R & Sligar, S.G (1995) Hydrostatic pressure and osmotic pressure as tools to study macromolecular recognition Methods Enzymol 259, 359–427.
36 Twist, C., Royer, C.A & Alpert, B (2002) Effect of solvent dif-fusion on the apomyoglobin–water interface Biochemistry 41, 10343–10350.
37 Zhou, J.M., Zhu, L., Balny, C & Perrett, S (2001) Pressure denaturation of the yeast prion protein Ure2 Biochem Biophys Res Comm 287, 147–152.
38 Kuwata, K., Li, H., Yamada, H., Legname, G., Prusiner, S.B., Akasaka, K & James, T.L (2002) Locally disorded conformer of the hamster prion protein: a crutial intermediate to PrP Sc ? Biochemistry 41, 12277–11283.
39 Alvarez-Martinez, M.T., Torrent, J., Lange, R., Verdier, J.M., Balny, C & Liautard, J.P (2003) Optimized overproduction, purification, characterization and high-pressure sensitivity of the prion protein in the native (PrPC-like) or amyloid (PrPSc-like) conformation Biochim Biophys Acta 1645, 228–240.
40 Torrent, J., Alvarez-Martinez, M.T., Heitz, F., Liautard, J.P., Balny, C & Lange, R (2003) Alternative prion structural changes revealed by high pressure Biochemistry 42, 1318–1325.