Pressure perturbation calorimetic studies of the solvation properties and the thermal unfolding of staphylococcal nuclease

A rather new technique, pressure perturbation calorimetry (PPC), was applied to study volumetric and solvation properties of staphylococcal nuclease (Snase) in its native and unfolded state with high precision.

Furthermore, the effects of various chaotropic and cosmotropic co-solvents on the solvation and unfolding behaviour of Snase was investigated in detail.

In PPC, the apparent coefficient of thermal expansion of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent at the protein–solvent interface.

In the native state, the protein shows a very strong thermal expansion of 1.0 × 10−3 K−1 at 10 °C, which decreases steeply to 0.65 × 10−3 K−1 at 40 °C.

This behaviour is discussed in terms of a continuous release of condensed water from the protein surface.

Upon unfolding, the volume decreases by about 19 mL × mol−1.

Solutions of the cosmotropic and chaotropic compounds glycerol, sorbitol, K2SO4 and urea, respectively, show characteristic deviations from the thermal expansion and volumetric properties of the pure buffer solution.

The solvent contribution to the apparent coefficient of thermal expansion of the protein, α, is enhanced considerably when the protein is immersed in a solvent known to be more structured than H2O (even the more structured D2O has a drastic effect) and nearly eliminated in a solvent in which “normal” water is largely absent (e.g., in 1.5 M urea).

Similarly to D2O, a continuous increase in solvation was observed with increase in glycerol or sorbitol content in the buffer, which leads to an increase in protein stability, as is verified by the increasing Tm and ΔH values obtained by microcalorimetric measurements (DSC).

In this regard, sorbitol is the more efficient agent.

The reduction of ΔV in the presence of these stabilisers can in part be attributed to the formation of a partial unfolded state of the protein, in part it is due to the temperature dependence of ΔV.

The preferential binding of urea reduces the hydration level, also in the native state, causing the protein to approach a more disordered state at high urea concentration.

The increase in ΔV and the decrease in ΔH with increasing urea concentration support these conclusions.

A stabilising effect, even though there is a reduction in solvation around the protein is observed for 0.5 M K2SO4 as co-solvent.

In this case, surprisingly, ΔV is found to be positive which is an indication of the formation of a swollen, molten globule kind of unfolded state at the transition.

Finally, ΔV values for the temperature-induced unfolding are compared with corresponding data for the pressure-induced unfolding of Snase.


The in depth knowledge of various thermodynamical properties of proteins in solution has been drawing attention of many researchers, as these form the basis for understanding their physiological functions, but also their use in drug design and formulations.

The stability of globular proteins depends on temperature,1–4 solvent properties,5–8 and its hydration capacity.9,10

Another important factor contributing to the stability of proteins is their relative affinity towards a particular reagent (in the present context, a co-solvent) in comparison to water or buffer solution.

The use of co-solvents such as glycerol and sucrose have been used widely, primarily because of their ability to stabilize the folded proteins through a mechanism that does not involve direct contact, but rather their preferential exclusion from the protein surface.11

Brandts12 and Pohl13,14 proposed that the driving force for stabilizing the protein folded conformation is a non-specific solvation effect in which the preferential exclusion of the co-solvents from the protein surface arises from enhanced water ordering (structure makers).

In contrast, when denaturating co-solvents bind to proteins, water–protein and water–co-solvent interactions, that occur in the unbound state, are replaced by relatively stronger co-solvent–protein interactions in the bound state with concomitant release of water molecules into the bulk phase.

Furthermore, these compounds tend to reduce the solvent ordering (structure breakers).

Such ligand interactions can be studied based on determining the solvent accessible surface area (SASA).15,16

The structure-making and structure-breaking tendencies of these compounds on protein-bound water have been studied extensively by Lin et al.17

Their results indicate that when hydrophilic groups, such as charged or polar side chains of proteins, are exposed or come in contact with the surrounding water, they show a pattern characteristic of structure breakers, with a large positive apparent expansion coefficient of the protein, in particular at low temperature, which decreases drastically with increasing temperature.

On the contrary, apolar, hydrophobic amino acid side groups act in the reverse manner, as structure makers, by enhancing the space consuming hydrogen-bonded network structure of water, particularly at low temperature.

This is accompanied by a decrease in solvent density around the hydrophobic groups.

Aliphatic side chains, for example, are known to have even negative apparent thermal expansion coefficients, α, at low temperatures (near 0 °C) with a large positive temperature coefficient.

Increasing thermal energy, however, allows the water molecules to free themselves from this expanded ordered solvation state.

Hence, in general, for proteins in dilute aqueous solutions, the temperature dependent thermal expansion coefficient at the protein–solvent interface is dominated by protein–water interactions, which arise mainly from interactions between the protein groups with the surrounding hydration layer.

Irrespective of whether co-solvents directly interact with or are preferentially excluded from the protein surface, they induce definite changes in the quantity of bound water and its associated properties, such as hydrogen-bonded structure, and chemical potential.18–22

Densimetric studies are often not of sufficient sensitivity to reveal these changes in solvation properties or volume changes upon unfolding of proteins.22–25

In the present paper, we examine these perturbations in the hydration layer around a protein caused by addition of co-solvents using a relatively new and efficient technique called pressure perturbation calorimetry (PPC).17,26–28

Applying this method, determination of the thermal expansion coefficient and relative volume changes, ΔV/V, upon unfolding of proteins in solution has advantages over densimetric measurements since its sensitivity is higher by more than one order of magnitude.17,29–31

This high sensitivity is a prerequisite for such studies, as the expansivity and the volume change upon unfolding, which can be either positive of negative, are usually very small (ΔV/V < 0.3%).

The observed volume and expansivity changes are correlated with further thermodynamic properties obtained from differential scanning calorimetry (DSC).

Taken together, these data lead to a deeper understanding of the solvation process of proteins in different co-solvents in their native and unfolded states.

We have chosen to study the solvation properties and stability of a well characterized monomeric protein, staphylococcal nuclease (Snase), in H2O and D2O buffer solutions, as well as in the presence of various types of co-solvents, such as glycerol, sorbitol, urea and potassium sulfate.

Their influence on the expansivity as well as the denaturation temperature, relative volume and enthalpy changes upon unfolding were obtained with high accuracy.

Snase is a small protein of about 17.5 kDa containing 149 amino acid residues and no disulfide bonds.

It has an extraordinarily high fraction of ionisable groups.

The X-ray diffraction pattern of native crystalline Snase32 reveals that in the crystalline state the protein contains 26.2% helices, 24.8% β-sheets (barrel), 7.4% extended chains, 24.8% turns and loops, and 8.7% unordered chains, and the remaining 8.1% is uncertain.

Volume and expansivity changes of Snase upon pressure and acid induced denaturation were given elsewhere33–37 and are compared with these data.

The present study aims to obtain more insight into the basic thermodynamic properties of protein solvation and volume effects accompanying unfolding scenarios on one hand, and on the other hand, to initiate further potential applications of pressure perturbation calorimetry in studies of biomolecular systems in general.

Materials and methods

Protein preparation

Recombinant Snase with the sequence of nuclease A from the V8 strain of Staphylococcus aureus was obtained using the λ expression system in the Escherichia coli strain Arλ9 as described by Shortle and Lin.38

The cells were grown according to the procedure described by Shortle et al39. except that SB rather than MOPS media was employed.

The protein purification was carried out according to the method described by Shortle and Meeker40 with modifications described by Frye et al.41

Potassium sulfate (K2SO4), deuterium oxide (D2O) were obtained from Aldrich, urea and D-sorbitol from Fluka, and glycerol from Merck.

The chemicals were used without further purification.

10 mM of phosphate buffer (di-sodium hydrogen phosphate, anhydrous, from Merck) was used for all experiments.

Differential scanning and pressure perturbation calorimetry (PPC)

The thermal unfolding of Snase was measured by means of a high precision VP DSC micro-calorimeter from MicroCal, Northamption, MA, USA.

The cell volume is 0.51 mL.

The reference cell was filled with matching buffer.

Both buffer and protein solutions were degassed before being injected into the respective cells.

Degassing was performed at a slightly reduced atmospheric pressure and for a short time only, so that no concentration changes occurred.

The instrument was operated in the high gain mode at a rate of 40 °C h−1 for all experiments.

The instrument is also equipped with a pressuring cap that allows application of ca.

1.8 bar to the cells in order to avoid air bubbles at elevated temperatures.

Sample concentration of Snase in 10 mM phosphate buffer was 0.2 wt% with a known quantity of co-solvent.

Baseline subtraction (pure buffer) and normalization with respect to protein concentration were performed by the instrument software, yielding the temperature-dependent apparent molar heat capacity of the protein, Cp.

The pressure perturbation (PPC) experiments were performed in the DSC calorimeter using the MicroCal PPC accessory.

The reference and sample cell volume are identical (0.51 mL) and they open to a common pressure chamber containing a sensor.

An equal pressure of 5 bar was applied to both cells in a programmed manner using nitrogen gas.

The pressure effect on the sample volume is negligibly small (0.02%).

The protein concentration used for the PPC studies was ca.

4 mg mL−1.

Each pressure jump starts with equilibration at 5 bar pressure of the calorimeter in the isothermal high gain and low noise modes at the desired temperature with 0.02 °C tolerance.

The software then initiates a pressure release to ambient pressure.

The temperature of the cell is kept constant by active compensation of the heat change caused by the pressure jump.

The compensation power returns to the baseline typically within one minute and integration of the supplied power vs. time yields the heat consumed or released by the sample.

After complete equilibration, automatically checked by an adjustable slope criterion, an upward pressure jump is applied when the PPC controller reconnects the PPC cells with nitrogen gas.

The heat peak of compressed and decompressed pressures should agree in absolute values; they are of opposite sign, however.

For both compression and decompression experiments, temperature, pressure, and heat flow are recorded as a function of time.

The calorimeter is then automatically heated or cooled to the next desired temperature and the next two pressure jumps are applied.

Various pH variation studies from 2 to 9 on Snase using DSC indicated that it is most stable at pH 5.5 in 10 mM phosphate buffer.

As the technique has been introduced quite recently only,17,26–28,31 we briefly summarize the thermodynamics on which it is based.

From the second law of thermodynamics we know that an entropy change for a reversible process carried out at temperature T, whose heat change is dQrev, can be written as dS = dQrev/T.

Differentiation with respect to pressure, at constant T, gives (∂Qrev/∂p)T = T (∂S/∂p)T.

Using the Maxwell relation (∂S/∂p)T = −(∂V/∂T)p, we obtain eqn. (1): where V is the volume and α is the coefficient of thermal expansion, (1/V)(∂V/∂T)p.

Here it is assumed that V and α are nearly invariant within small pressure changes (here 5 bar), which is a very good approximation for all liquids.

Integration of eqn. (1), at constant temperature, over a small pressure range, Δp, gives the working equation for calculating α The coefficient of thermal expansion, α, can thus be determined from an isothermal measurement of the heat consumed or released upon small pressure changes.

Here, ΔQrev is the heat difference between the sample cell that contains the dissolved sample, and the reference cell, which contains the same solvent, upon the pressure changes Δp.

Moreover, the relative volume changes ΔV/V at the unfolding transition, taking place in the temperature range from To to Te, can be obtained by eqn. (3):

In extending eqn. (2) to protein solute (in buffer solvent) and solvent (buffer only) binary component systems, we obtain eqn. (4): where α and αsolv are the thermal expansion coefficients of the protein and solvent, respectively, and Vcell is the active cell volume.17,26

The experiment thus yields the difference in expansivity between the protein solution (sample cell) and the buffer (reference cell).

The apparent expansion coefficient of the protein is measured, which includes changes in hydrational properties at the protein surface with regard to the pure solvent system.

The uncertainties in α determined from at least three measurements using different sample preparations is ±0.05 × 10−3 K−1.

Results and discussion

Pure solvents

The values of the expansion coefficients of the solvents, αsolv, measured for the phosphate buffer containing 0.5 M co-solvents are shown in Fig. 1.

The solutions with 0.5 M glycerol, sorbitol, urea, and K2SO4 exhibit a greater thermal expansion compared to pure water (shown for comparison), in particular at low temperature.

In the case of K2SO4, above 80 °C, however, its expansivity becomes smaller than that of pure water, which is likely due to the “structure making” effect of the salt.


Fig. 2a represents PPC curves of the apparent expansion coefficient, α(T), of 0.4 wt% Snase dissolved in 10 mM phosphate buffer at pH 5.5, from which α at different temperatures, the midpoint of thermal unfolding (Tm) and the relative volume change upon unfolding, ΔV/V, are obtained.

Tm and the enthalpy change of the transition (ΔH) are obtained from the DSC experiments (Fig. 2b).

The protein begins to unfold (onset temperature) at ca.

43 °C and the temperature of the unfolding midpoint, Tm, was found to be 51.5 °C.

The enthalpy change obtained by integration over the DSC peak amounts to 178 ± 5 kJ mol−1.

We find an increase in Cp of 5.7 ± 0.2 kJ mol−1 K−1 between the unfolded and native state.

Between 10 and 40 °C, the apparent thermal expansion coefficient of the protein, α, decreases almost linearly from 1.0 × 10−3 K−1 to a value near 0.65 × 10−3 K−1.

At approximately 51.7 °C, where the DSC curve Cp(T) exhibits its maximum, a distinct dip is observed.

Above that temperature, α increases again to a relatively constant level in the post-transition temperature range.

Above 70 °C, α then decreases again.

High α values at lower temperatures (1.0 × 10−3 K−1 at 10 °C) and their steep decrease up to 40 °C (dα/dT)10–40 = −1.17 × 10−5 K−2) are indicative of the presence of a large number of structure-breaking hydrophilic side chains on the surface of Snase.17

As the temperature increases, water molecules are released from the surface and the value of the thermal expansivity is reduced.

Since Snase has an extraordinarily large number of ionisable residues (39%), it exhibits very large values of α and |(dα/dT)10–40|.

The relative volume change upon unfolding, ΔV/V, as obtained from the area under the transitional peak, is calculated by integrating the α(T) transition curve (after baseline subtraction); ΔV/V = −1.5 × 10−3.

The absolute volume change upon unfolding, ΔV, can be calculated from ΔV/V using the molar mass of Snase (16 812 Da) and its partial specific volume (0.754 mL g−1)33 which yields ΔV = −19 mL mol−1, which is of the order of −0.15% of the whole system.

In agreement with the DSC data, the transition has been found to be reversible up to temperatures of 80 °C.

Snase–H2O, D2O

The effect of solvation strength on the temperature dependence of α, which is largely controlled by protein–solvent interactions, should be expected to change significantly upon replacing H2O for D2O as solvent.

Fig. 3a shows PPC results of Snase in dilute buffer prepared from D2O (Snase – D2O) in comparison to those of Snase in H2O (Snase – H2O).

From this figure it is clear that in the lower temperature region, the negative slope of α(T) is considerably enhanced in D2O: (dα/dT)10–40 = −2.2 × 10−5 K−2 in Snase–D2O, whereas (dα/dT)10–40 = −1.17 × 10−5 K−2 for Snase in dilute buffer prepared from H2O. The larger absolute (dα/dT)10–40 value in the case of Snase–D2O points to a higher degree of solvation than in the Snase–H2O system.17

D2O has a stronger structuring tendency which, in contact with structure-breaking protein groups results in a relatively larger solvation layer around the protein surface.15

The decrease in thermal expansivity with temperature is indeed largely controlled by the net structure-breaking tendencies of the polar protein groups interacting with water.

This is not to say that the intrinsic expansivity of the protein is negligible, but simply that the protein interior does not appear to contribute significantly to the decrease in expansivity with increasing temperature (see below).

The Tm value shifts from 51.5 °C in H2O to 56 °C in D2O. Furthermore, there is a ca.

33% reduction in ΔVV = −12.7 mL mol−1 for Snase–D2O).

All the results are listed in Table 1.

Concerning Tm values, similar results as from PPC were obtained from the DSC traces of Snase in D2O and H2O, respectively.

From Fig. 3b it is clear that there is a shift in Tm from 51.7 °C in H2O to 56.3 °C in D2O. We observe also a considerable increase in the enthalpy of unfolding in D2O. The transition enthalpy change of the system Snase–D2O (ΔH ≈ 227 kJ mol−1) is ca.

25% greater than that of the system Snase–H2O (ΔH ≈ 178 kJ mol−1), which is, again, an indication that the protein is more stable in D2O. Such an enhancement of the structure of a solvation layer contributing to the protein stability was also reported elsewhere43,44.


Glycerol and other polyolic co-solvents have been shown to lead to a “preferential hydration” of the protein, i.e. the exclusion of the co-solvent molecules from the protein surface.

Hence, in their presence proteins are preferentially hydrated.18,45–51

For sugars (e.g., sucrose), glycerol and polyhydric alcohols (e.g., ethylene glycerol, sorbitol), the preferential hydration was found to induce also protein stabilization.

Glycerol, being a strongly hydrophilic co-solvent, interacts strongly with H2O and has a weaker affinity for the polar residues on the protein surface, thus leading to preferential hydration of the protein.

Furthermore, the steric exclusion principle should make the distance of closest approach of a co-solvent with a larger molecular volume greater than that of water.

As a result, the volume fraction occupied by the co-solvent at the surface of the protein should be less than that in the bulk solvent.

Thermodynamically, this thus also manifests itself as preferential hydration.

Upon denaturation, the surface of the protein increases, which results in an enhanced preferential hydration of the denatured protein relative to the native protein.51,52

The PPC results of Snase dissolved at various molar concentrations of glycerol in 10 mM phosphate buffer at pH 5.5 are shown in Fig. 4a.

The glycerol concentration in the buffer was varied from 0.5 M (ca.

5 wt%) to 3.5 M (ca.

32 wt%).

From the figure it can be clearly seen that there is a continuous increase in α, |(dα/dT)10–40|, and Tm values with increasing glycerol concentration.

The increase in Tm value is clearly an evidence of the stabilization of the folded state, arising from the energetically unfavourable increase in preferential hydration due to the increase in exposed surface area upon unfolding.

Similar results were obtained for the Snase–D2O system (data not shown).

The increase in glycerol concentration leads to a drastic increase of the thermal expansivity at 10 °C due to the preferential hydration, even though the total amount of water in the system is reduced with increasing glycerol concentration.

The comparison of the (dα/dT)10–40 value of Snase–3.5 M glycerol (−2.4 × 10−5 K−2) with that of Snase–H2O (−1.17 × 10−5 K−2) in buffer solution indicates that the addition of glycerol contributes to a ca.

100% increase in solvation, assuming that (dα/dT)10–40 is proportional to the degree of solvation.

The possibility of extensive glycerol binding to the protein, even at high glycerol concentration, might be ruled out because in such a case part of the water should be expelled from the protein surface to accommodate glycerol which would lead to a decrease in α and hence |(dα/dT)10–40|.

The values of Tm from DSC (Fig. 4b) and PPC are in good agreement.

The steady increment of Tm and ΔH in the glycerol containing buffer solutions, up to 56 °C and ΔH ≈ ∼260 kJ mol−1 for the 3.5 M glycerol containing solution (Table 1), confirms the continuous increase in protein stability with increasing glycerol concentration.

The volume change of unfolding is drastically reduced for the glycerol solvent systems (e.g., ca.

100% for the 3.5 M glycerol solution), which might be due to a less disordered unfolded state.

This would be in agreement with the smaller ΔCp values as obtained by DSC (Table 1).


Several reports state that stabilisation of proteins in the presence of sorbitol, which is about twice as large as glycerol, is also mainly due to it being excluded from the surface of the protein,47,51i.e., in their presence, proteins are preferentially hydrated.

To quantify the relative hydration changes, PPC experiments were also conducted using 0.5 M (ca.

9 wt%) and 1.5 M (ca.

27 wt%) sorbitol as co-solvents in 10 mM phosphate buffer.

The PPC results are shown in Fig. 5a and indicate that the hydration changes and stabilisation due to sorbitol are even higher than those for the corresponding concentrations of glycerol (Table 1).

The comparison of (dα/dT)10–40 values of Snase–1.5 M sorbitol (−1.7 × 10−5 K−2) and Snase–1.5 M glycerol (−1.5 × 10−5 K−2) indicates that there is an ca.

12% increase in solvation of the native protein in the sorbitol containing solvent, which also acts as a more effective stabilising agent.

The shift in Tm value, e.g., from 51.5 to 58.6 °C in 1.5 M sorbitol (53.7 °C in 1.5 M glycerol) indicates the more effective stabilising effect of sorbitol compared to glycerol.

The volume change upon unfolding is much smaller, −5 mL mol−1 in 1.5 M sorbitol as compared to −16 mL mol−1 in 1.5 M glycerol and −19 mL mol−1 in the pure phosphate buffer system.

The high ΔH (ca.

208 kJ mol−1) and Tm (59 °C) values obtained from the DSC data, which are shown in Fig. 5b, are in good agreement with the conclusions drawn from the PPC results.


It is well known that the destabilising nature of the chaotropic agent urea on protein stability is caused by binding efficiently to the unfolded state of the protein, compared to the folded state, due to exposure of more binding sites upon unfolding.52,53

The PPC and DSC results on Snase–urea solutions displayed in Fig. 6 are in agreement with these general findings.

As expected, the Tm value decreases with increasing urea concentration, for example Tm(1.5 M urea) = 39.1 °C, Tm(2.5 M urea) = 32.5 °C (Table 1).

The α values at low temperature are much smaller than those of the pure Snase–H2O system, and the absolute value of dα/dT, taken between 10 °C and the onset temperature of the denaturation peak, also decreases with increasing urea concentration (Fig. 6a).

These effects are related to specific binding of urea to the polypeptide, thereby replacing water molecules and releasing them into the bulk phase, such that the hydration of the protein is largely diminished in the presence of urea, prior to denaturation.

Interestingly, the ΔV values of Snase–0.5 M urea and Snase–1.5 M urea buffer solutions with −32 and −56 mL mol−1, respectively, are much greater than those of Snase in the pure buffer solution.

Beyond a concentration of 1.5 M of urea, it is not possible to calculate ΔV accurately as the protein does not denaturate in a cooperative manner anymore.

This is supported by the reduction in the enthalpy values of denaturation, ΔH, as calculated from the DSC data, which are shown in Fig. 6b.

ΔH values are 168 and 108 kJ mol−1 for the 0.5 and 1.5 M urea containing solutions, i.e., they decreased by ca.

6% and ca.

39%, respectively, when compared to Snase–H2O (buffer).

The Tm values as obtained by DSC also decrease with increasing urea concentration and are in good agreement with the values obtained by the PPC method.

Above a concentration of 2.5 M urea, no clear transition can be observed anymore.

All experimental data are again listed in Table 1.

In the case of urea, an increase in absolute ΔV values is observed at the unfolding transition, which is an indication that the protein tends to form a rather extended structure, and the solvation properties at the protein surface are probably largely determined by binding of urea.

As a consequence, the α values in the denatured state are rather small.


Some specific anions are known to induce the formation of molten globule forms in proteins, for example from acid-unfolded proteins.54

The mechanism of stabilisation by anions is due to a reduction of the net positive charge on the protein through anion binding.

Stabilisation of proteins due to the presence of such salts are thus more effective at lower pH than at neutral pH.55

At reduced protein surface charge and at higher anion concentrations, the Hofmeister effects46,55 prevail, however.

Hofmeister anions interact favourably with the peptide groups whereas they interact unfavourably with the nonpolar peptide side chains.

Sulfate, for example, is known to be a good protein stabilizer, because sulfate strongly salts out nonpolar compounds and only weakly interacts with and thus salts in the peptide group.55

Here, we looked at the role of the cosmotropic salt K2SO4 in a solvent environment where the net charge of the protein is still small.

Under these conditions, the K2SO4 specific binding tendency is reduced.

The PPC and DSC results for Snase dissolved in 0.5 M K2SO4 buffer solution are presented in Figs. 7a and 7b, respectively.

From the PPC experiments it is clear that the shift in the Tm value from 51.5 to 59.5 °C is an indication that the salt acts as a strong stabilising agent.

In contrast to the osmolytes glycerol and sorbitol, the apparent expansion coefficient of the protein in the native state is smaller than that of the protein in pure buffer solution, however.

The value of (dα/dT)10–40 = −0.77 × 10−5 K−2 obtained for the K2SO4 system is ca.

30% less than that of the Snase–H2O (buffer) system.

In the present case, even though there is a net reduction in solvation, similar to urea, the K2SO4–buffer system increases the stability of the protein.

The high ΔH value of ca.

295 kJ mol−1 obtained from the DSC results indicates the drastically increased stability of the protein in the K2SO4 buffer solution.

Surprisingly, in this and only in this case, ΔV becomes positive (ΔV = 19 mL mol−1).

This volume increase at the unfolding transition could be due to the formation of a protein conformation which is not fully unfolded but rather achieves a flexible, expanded state only, which might be a molten globule kind of structure.

In the unfolded state at ca.

80 °C, α is similar to that of the pure buffer system, again.

The thermal unfolding scenario

The interpretation of the present results must be made in the context of previously published work on the volumetric properties of Snase (and proteins in general) as a function of temperature and pressure.

Direct densimetric measurements on Snase as a function of temperature and pressure36 revealed that below the unfolding transition temperature at atmospheric pressure, the slope of the change in specific volume with temperature exhibits a distinct downward curvature, indicating that the apparent thermal expansivity of the native state of the solvated protein decreases with increasing temperature.

The present more accurate studies are in good agreement with these earlier observations, as they demonstrate a marked decrease in α between 4 and 40 °C, below the transition temperature.

At higher temperatures, between 55 and 65 °C, our earlier studies show that the increase in specific volume with temperature was nearly linear, i.e. the thermal expansivity appeared nearly constant over this temperature range.

In the present study, it is seen that this temperature range corresponds to the tail end of the transition, and thus to the region of rollover in which dα/dT ≈ 0 before changing sign.

Thus, the present determination of α as a function of temperature using PPC is in good agreement with the previous observations based on a determination of the specific volume by densimetry, which had to be carried out at a much higher (20×) protein concentration, however.

The estimation, based on the present PPC results, of the change in α upon unfolding, Δα, at the transition temperature yields a positive value of about 1 × 10−4 K−1, and is in the range (0.5–1.5 × 10−4 K−1) of the values obtained by Lin et al17. on a number of proteins.

Our earlier densimetric measurements also revealed an increase in the apparent value of the expansivity between 45 and 65 °C, consistent with a positive value for Δα.

Intrinsic protein volume, including the effects of protonation and deprotonation of amino acids upon unfolding, volume changes due to thermal motions and changes in water structure at the protein surface are the three major factors contributing to the apparent thermal expansivity of a protein.42,56

The thermal volume at the protein solvent interface and the intrinsic volume of the protein increase with temperature leading to a positive contribution to α.

The thermal expansivity of the protein interior has been measured over a limited temperature range, essentially using fluorescence and X-ray diffraction (on single crystals),57–60 and the changes observed are rather small.

This is in accord with our SAXS measurements of the radius of gyration, Rg, of Snase in solution; within the accuracy of our data (±1–2 Å), the Rgvalue is unchanged up to ca.

40 °C.34,61

The solvation effect also represents a positive contribution to the thermal expansivity, but is expected to decrease with increasing temperature.

This behaviour is due to the fact that the protein surface binds adjacent water.

Vicinal water molecules may be oriented to the surface charges leading to a more or less layered structure.17,62,63

This layering is probably substantial at low temperature, and thermal activation leads to a continuous release of this “condensed” water from the protein surface.

Once the water is released, it no longer contributes to the partial volume of the protein and hence to α.

Thus at low temperatures, the solvation effect (in particular, of the polar/charged peptide groups) leads to large positive contributions to α and will decrease steeply with increasing temperature.

Lin et al17. proposed that the dα/dT value of a protein in its native state is a direct measure of the solvation contribution to the volume expansivity.

The volume change that accompanies the unfolding transition of a protein (in this case Snase) clearly will depend upon the temperature at which the transition occurs.

The value of ΔV at the transition temperature of 51.5 °C obtained in this study (−19 mL mol−1) is smaller than those reported in denaturation studies by pressure36 or acid induced33 denaturation.

However, the acid denaturation studies may include significant electrostriction effects.

More to the point, the pressure-induced unfolding was carried out over a temperature range of 4 to 45 °C, below the unfolding transition temperature.34

These pressure studies demonstrated that the absolute value of the volume change upon unfolding was strongly temperature dependent, decreasing significantly in magnitude with increasing temperature.

At 40 °C, the ΔV was near −65 ± 10 mL mol−1, whereas at 45 °C it had decreased in absolute value to −37 mL mol−1.

Our previous data were not of high enough quality to determine whether this temperature dependence of ΔV was linear or non-linear.

However, one can assume that at 52.5 °C it would have decreased to at least −25 mL mol−1, a value within experimental error of that determined in the present studies.

It is possible, however, that differences in ΔV values may also arise from differences in the structure of the denatured states at high temperature as compared to high pressure.

Studies involving the application of hydrostatic pressure to solutions of native proteins have demonstrated that at temperatures below that corresponding to the unfolding transition, the volume change upon unfolding of proteins is negative, i.e. the partial specific volume of the unfolded state is smaller than that of the folded state and therefore pressure induces unfolding.64,65

Early work on metmyoglobin66 and ribonuclease67 and later on Snase as noted above34 has also shown that the pressure effect decreases in magnitude with increasing temperature, and may even change sign, i.e., the partial specific volume of the unfolded state becomes larger than that of the folded state at high temperature and pressure can induce folding, rather than unfolding under these conditions.

Moreover, even though it is negative at low temperatures, the absolute value of the volume change upon unfolding is quite small.

Thus, clearly the value and sign of the volume change upon unfolding at higher temperature will depend upon how the thermal expansivities of folded and unfolded states change with temperature.

Unfortunately, the temperature dependence of the thermal expansivity of the unfolded state cannot be determined at temperatures below the transition temperature, while that of the folded state cannot be determined at temperatures above the transition temperature.

Nonetheless, since Δα is positive at the transition temperature, the expansivity of the unfolded state is higher than that of the folded state at the transition.

As the temperature is lowered, it is likely that α of the unfolded state increases, but probably to a lesser degree than that of the folded state.

This might be due to the fact that relatively more aliphatic side chains of the protein are exposed to the solvent in the unfolded state, and the temperature dependence of the thermal expansivity of aliphatic side chains is opposite to that of the polar side chains and peptide bonds, given the fact that the aliphatic groups tend to promote a higher degree of structure in the water of solvation than exists in bulk water.17

Thus, as temperature is lowered this ordering significantly reduces the expansivity.

Aromatic groups as well are exposed to the solvent in the unfolded state, whereas they are significantly less exposed in the folded state.

The temperature dependence of the thermal expansivity of these groups is very small.

Thus overall, while the thermal expansivity of the unfolded state is likely to be higher than that of the folded state (at least near the transition temperature), its temperature dependence is probably reduced in comparison.

Given that the specific volume of the unfolded state is smaller at low temperature than that of the folded state, the temperature evolution of the specific volume of the two states may be similar to that schematized in Fig. 8.

Such a scheme of the individual contributing factors to the volume change upon unfolding changes as a function of temperature.

At low temperature, the volume change upon unfolding, ΔV, is negative because elimination of internal cavities and electrostrictive effects around exposed polar groups upon unfolding offset the positive contributions of exposure of hydrophobic groups and the larger thermal volume of the unfolded state.

At higher temperature, the decreased solvation effect can no longer offset the larger thermal volume of the unfolded state compared to the unfolded state, and the ΔV changes sign.

The Δα and ΔCp values for Snase in phosphate buffer solution at pH 5.5 are 0.12 × 10−3 K−1 and 5.7 kJ mol−1 K−1, respectively.

These values are taken as a reference to compare relative SASAs in the presence of the various co-solvents.

The larger absolute (dα/dT)10–40 value in the case of Snase–D2O points to a higher solvation than that of the Snase–H2O system.

D2O has a stronger structuring tendency which, in contact with structure-breaking protein groups results in a relatively larger solvation layer around the protein surface.

For the Snase in glycerol, the changes in expansivity and heat capacity upon unfolding indicate a decrease compared to those measured in pure buffer, indicating a compaction of the unfolded state as the glycerol concentration increases.

This would be in agreement with the reduced ΔV values observed.

Indeed, glycerol has been found to decrease the specific volume and the adiabatic compressibility of the protein interior, suggesting that the mechanism involves the collapse of voids in the protein core following a glycerol-induced elimination of flexibility-associated (“lubricant”) water.45

However, in pressure unfolding experiments on Snase at 21 °C as a function of increasing osmolyte concentration (in that case xylose up to ca.

0.5 M), we observed that while the apparent free energy of unfolding increased with increasing xylose concentration as would be expected, the volume change of unfolding remained essentially constant.68

One explanation for this discrepancy between previous studies and the present can be found in the temperature dependence of the volume change, which is quite large.

Indeed, addition of osmolyte stabilizes the folded state, such that the Tm (the temperature at which ΔV is measured in the present experiments) increases with increasing glycerol concentration.

Since the volume change of unfolding decreases strongly with increasing temperature, we can conclude that at least part of the observed effect of glycerol on the ΔV is due to the increase in temperature.

A similar but inverse explanation may also hold for the observed increase in the absolute value of ΔV as a function of increasing urea concentration.

Indeed, urea destabilizes the folded state of the protein leading to a decrease in the Tm as a function of increasing urea concentration.

Since the ΔV is of larger absolute value as the temperature decreases, increasing urea concentration results automatically in a larger absolute value of ΔV.

Another contribution may be that increasing urea concentration leads to a more completely unfolded state.

We observed that there is a continuous decrease in Δα which finally approaches zero with increasing urea concentration.

This could arise from significant binding of urea to the protein in the pre- and post-transitional temperature range.

The excess area available after protein denaturation would be occupied by the remaining free urea molecules, such that electrostatic or hydrophobic hydration effects could be largely reduced.

We note in contrast to this explanation, that another effective denaturant, guanidinium hydrochloride, had no effect on the value of the volume change of unfolding obtained in the pressure-induced unfolding of Snase at 21 °C,69 indicating that such solvation effects do not play a role in the value of the volume change under those high pressure conditions.

Moreover, the radius of gyration observed for urea denatured Snase (35 Å)70,71 is smaller than that which we observed by temperature denaturation (45 Å),34 indicating that, on the contrary, urea leads to a more ordered unfolded state than does temperature.

However, it is altogether possible that the combination of both temperature and urea significantly destabilizes residual interactions in the unfolded state compared to either perturbation alone.

Very interesting observations are made when the protein is stabilised in the presence of the salt K2SO4.

Firstly, ΔV being positive is probably an indication of the dominance of the thermal volume expansion due to an increase in the dynamics of backbone and side chains of the protein over the internal dead volume being exposed during denaturation.

The state might be characterized by a swollen, molten globule kind of state.

The experimental Δα and ΔCp values of −0.03 × 10−3 K−1 and ca. −9.5 kJ mol−1 K−1 (Table 1), respectively, point to no drastic increase in SASA in the denatured state, again indicative of a less unfolded and hence less hydrophobically hydrated structure in the denatured state.

This state can be achieved only, if K2SO4–H2O interactions dominate over Snase–K2SO4–H2O interactions and specific anion binding is largely missing.

Concluding remarks

Pressure perturbation calorimetry has been found to be an effective tool for measuring the thermal expansion coefficient and volume changes of biomolecular systems with very high precision.

Accurate information related to solvent binding, ligand binding and volume changes during unfolding and denaturation of proteins can be obtained by this technique.

The observed thermodynamic data obtained drastically depend on the interfacial solvent conditions, which drastically influence the structural and dynamic behaviour of the biomolecular system.

We clearly demonstrate that solvation properties contribute extensively towards the stability of proteins.

The apparent expansion coefficient of the protein, α, is strongly dependent on the type and concentration of co-solvent and its interactions with functional groups of the polypeptide.

This effect is already clearly observed when the thermodynamic data obtained using water as solvent are compared with those of a chemically structurally similar solvent, D2O. The increase in thermal stability of the protein in the presence of D2O can be understood to be due to an increase in solvation around the protein as indicated by the much larger α value in the native state of the protein.

The solvation effects leading to the strong temperature dependence of α are controlled by two contributions.

Firstly, as shown in the seminal paper by Lin et al.,17 hydrophobic groups known to act as structure-makers in water have a large positive slope and a negative curvature in α(T) plots, while structure-breaking hydrophilic groups show the opposite behaviour.

For charged and polar groups, α(T) curves exhibit a large negative slope and a positive curvature.

Since the solvation effects of a protein are dominated by hydrophilic groups, both the native and unfolded protein exhibit a net structure-breaking profile for α(T).

Upon unfolding of the Snase, the internal dead volume becomes accessible to water (leading to a decrease in system volume) and the dynamics of the protein backbone, and amino acid side chains increases thus leading to an increase of the thermal volume.

Moreover, the water-accessible surface area becomes larger upon unfolding, thus increasing the number of water molecules being affected.

The surface term may reflect two contributions: Firstly, since charged or polar surface-affected water is supposed to be condensed, this will give rise to a negative contribution to ΔV.

Secondly, upon unfolding of the surface, the protein becomes more hydrophobic because the native structure tends to bury more of the hydrophobic side chains in the interior.

It can be supposed that this effect counteracts the first one to some extent.

The negative volume change upon unfolding observed under most conditions implies that at the transition temperature, the opening of void volume and the increase in accessible surface area with more charged and polar groups upon unfolding overcompensate the positive effects of thermal volume and the decrease in the hydrophilic to hydrophobic balance of the surface area.

Similarly to D2O, a continuous increase in solvation was observed with increase in glycerol or sorbitol content in the buffer, hence indicating that they act as protein stabilisers, as is indeed verified by the increasing Tm and ΔH values.

The values of ΔV and ΔCp are also smaller for these co-solvents.

The observed reduction in absolute value of ΔV in the presence of these stabilisers may be in part attributed to the formation of a partially unfolded state of the protein (which is consistent with a smaller change in heat capacity upon unfolding) and in part directly due to the temperature dependence of ΔV; as Tm increases, ΔV decreases (in absolute value).

On the contrary, the chaotropic agent urea destabilizes the protein by direct ligand binding and restricts direct water contact with the protein surface.

This preferential binding of urea to the protein reduces the hydration level causing the protein to approach a disordered, random coil-like state at high urea concentration.

The increase in ΔV and the decrease in ΔH with increasing urea concentration support these conclusions, although a significant contribution to the increase in ΔV arises as well from its temperature dependence; as Tm decreases ΔV increases in absolute value.

The Δα and ΔCp values are less affected by changes in hydrophilic and hydrophobic hydration, which is attributed to the strong ligand binding of urea with the protein.

A stabilising effect, even though there is a reduction in solvation around the protein (|ΔαT10–40| is ca.

30% lower compared to the pure buffer system) is also observed for 0.5 M K2SO4 as co-solvent.

Near pH 5.5, K2SO4 is still left with little chance to bind strongly to the protein.

Yet, the sulfate anions are still able to decrease the hydration layer around the protein.

In this case, ΔV is found to be positive which is an indication of the formation of a swollen, molten globule kind of unfolded state at the transition.

Indeed, the negative values for Δα and ΔCp in presence of this salt point to no marked increase in SASA upon unfolding.

These results for the various chaotropic and cosmotropic co-solvents clearly show that the co-solvent not only markedly changes the stability of Snase, but also its solvation, and perhaps also the structures of the denatured state.

More generally, the present results on Snase combined with the few previously reported studies on other small globular proteins17 demonstrate that the PPC technique provides an exquisitely sensitive handle on the hydration properties of proteins in solution, the understanding of which is prerequisite to a global comprehension of protein stability and dynamics.