Thermodynamics of denaturation of mutants of barnase with disulfide crosslinks1

J. Mol. Biol. (1997) 268, 198±208

Thermodynamics of Denaturation of Mutants of Barnase with Disulfide Crosslinks Christopher M. Johnson, Mikael Oliveberg, Jane Clarke and Alan R. Fersht* Cambridge Centre for Protein Engineering, Hills Road Cambridge CB2 2QH, UK

We have measured the effects of disul®de crosslinks on the thermodynamics of denaturation of three mutants of barnase that contain cystine and the corresponding single and double cysteine mutants. At ®rst sight, the data are consistent with the hypothesis that disul®de crosslinks stabilise proteins through entropic destabilisation of the denatured state, but the decreases in the entropy of denaturation are larger than predicted and are accompanied by decreases in the enthalpy of denaturation. These effects are not a unique feature of the disul®de crosslink and are observed in a range of non-crosslinked mutants of barnase as part of a general enthalpy-entropy compensation phenomenon. Similarly, effects on the heat capacity change for denaturation (
Cdp), determined from the slope of the enthalpy of denaturation versus temperature, are not con®ned to mutants with disul®de crosslinks. The value of
Cdp is lower in four stabilised mutants than in wild-type barnase, irrespective of the presence of a disul®de crosslink, while the
Cdp remains unchanged in a destabilised mutant containing a disul®de. The variation in
Cdp may result from an inherent temperature-dependence of
Cdp, since it is measured for each mutant over a different temperature range. The thermodynamics of denaturation of the disul®de mutant with a crosslink between positions 70 and 92 change anomalously with pH but in a similar way to that of the D93N mutant of barnase, which lacks the D93 ±R69 salt-bridge present in the wild-type. This ®nding con®rms initial observations in the X-ray structure of this disul®de mutant that the saltbridge has been disrupted by the introduced crosslink. # 1997 Academic Press Limited

*Corresponding author

Keywords: thermodynamics; denaturation; folding; enthalpy; entropy

Introduction Naturally occurring disul®de bonds generally increase the stability of proteins. It is widely believed that this results from a reduction in con®gurational Abbreviations used:
Cdp, heat capacity change of denaturation; 43-80, 85-102 and 70-92, mutants of barnase with disul®de crosslinks between residues 43 and 80, 85 and 102, and 70 and 92, respectively; 43-80SH, 85-102SH and 70-92SH, double cysteine mutants under reducing conditions; DSC, differential scanning calorimetry; CD, circular dichroism; Tm, midpoint of thermal denaturation;
Hvh, van't Hoff enthalpy of denaturation;
Hcal, calorimetric enthalpy of denaturation;

GD-N,

HD-N and

SD-N change on mutation in free energy, enthalpy and entropy of denaturation, respectively (
GD-N wild-type, ÿ
GD-N mutant, etc.);
v, net change in protonation on denaturation. 0022±2836/97/160198±11 $25.00/0/mb970928

entropy (
Sconf) of the denatured state (Flory, 1956; Poland & Scheraga, 1965). Changes in stability on removal of naturally occurring disul®des are often consistent with the changes in
Sconf, the con®gurational entropy, in the denatured protein calculated according to the equation: ÿ

G ˆ T

Sconf ˆ T…ÿ2:1 ÿ f1:5 R ln ng† cal molÿ1

…1†

where R is the gas constant and n is the number of residues enclosed by the disul®de (Pace et al., 1988). But, Doig & Williams (1991) concluded from a survey of published data that disul®de bonds increase the
S and
H of denaturation while decreasing the heat capacity change of denaturation (
Cdp). They rationalised this by proposing that the denatured state becomes more compact on the introduction of crosslinks, so decreasing its exposure # 1997 Academic Press Limited

199

Thermodynamics of Denaturation of Barnase Mutants

to solvent. This change in solvation increases the
S and
H of denaturation to a greater extent than the corresponding decreases in
S and
H of denaturation expected from the residual intramolecular interactions. At ®rst sight, it would appear that a detailed thermodynamic characterisation of disul®de-containing proteins should resolve the mechanism of stabilisation. The chain entropy model predicts decreases in the
S of denaturation on introduction of a disul®de while the model proposed by Doig & Williams (1991) predicts increases in the
H and
S of denaturation. There are two ways to approach the problem of understanding the thermodynamics of disul®de formation. The ®rst employs proteins that contain novel engineered disul®des, while the second uses proteins where native disul®des are removed by mutation or by covalent modi®cation of the cysteine side-chain (Betz, 1993 and references therein). These studies have shown that effects on the
S of denaturation can also be accompanied by changes in the
H and
Cdp of denaturation. It is dif®cult to attribute these varied changes solely to effects on the denatured state. It seems likely, therefore, that disul®des also perturb the native state of proteins. This idea has received some support from both experimental and theoretical studies introducing the concept of a potentially destabilising ``strain'' energy in the native state (Katz & Kossiakoff, 1986; Tidor & Karplus, 1993). This destabilisation, which may be larger than the chain entropy effects on the denatured state, explains why the success rate in stabilising proteins by introducing disul®des is not high (Wetzel, 1987). There are important differences between the two experimental strategies. Introducing a disul®de into a protein may cause some disruption to the native state, however carefully the mutations are designed. Control experiments should be performed that measure the thermodynamics of the single and double cysteine mutations. On the other hand, removing a disul®de from a protein may produce different changes in the folded state because the crosslink has some evolutionary role in the structure and dynamics of the system. Thiol blocking generally introduces bulky chemical groups that may also perturb the system in an idiosyncratic manner. It may not be surprising, therefore, if the different approaches do not always produce consistent results. Three disul®de mutants of barnase have been generated to probe the folding pathway of this protein. These mutants have been characterised by a range of biophysical techniques including measurements of equilibrium and kinetic stability, structural analysis by X-ray crystallography and structural and dynamic studies using NMR and H-D exchange (Clarke & Fersht, 1993; Clarke et al., 1993, 1995a,b). Two of the mutants, in which a disul®de has been engineered between residues 43 and 80 (43-80) and between 85 and 102 (85-102),

are more stable than wild-type barnase or their corresponding reduced states. The third mutant, with a disul®de between residues 70 and 92 (70-92), is less stable than wild-type barnase or its reduced state, suggesting that the crosslink has additional effects on the protein. A possible source of this destabilisation has been identi®ed in the X-ray structure of this mutant where a salt-bridge between R69 and D93 appears to be disrupted (Clarke et al., 1995a). Equilibrium unfolding at 298 K using urea or GdmCl has indicated that the stabilities of the disul®de mutants are not consistent with the predictions of equation 1 (Clarke et al., 1995a). The 85-102 mutant is 4.8 kcal molÿ1 more stable than its corresponding dithiol in comparison with the 3.1 kcal molÿ1 predicted from the 17 residues enclosed by the disul®de. The 43-80 mutant is 3.2 kcal molÿ1 more stable than the dithiol, which is close to the 3.8 kcal molÿ1 expected from the 37 residue loop. On the other hand, the 70-92 mutant is 0.6 kcal molÿ1 less stable than its dithiol, while the 22 residues in the loop are expected to increase the stability 3.4 kcal molÿ1. These discrepancies make this group of mutants an interesting system in which to study the thermodynamic effects of disul®des. Barnase is also suited to such studies since its thermal unfolding is fully reversible and characterised by a two-state transition with a large enthalpy change. Several studies have used differential scanning calorimetry (DSC) to examine the thermodynamics of the wild-type protein and a limited number of mutants (Griko et al., 1994; Martinez et al., 1994; Matouschek et al., 1994; Johnson & Fersht, 1995). Here, we report the thermodynamic characterisation of mutants of barnase with disul®de crosslinks as well as the corresponding single and double cysteine mutants obtained under reducing conditions. We have examined a range of other stabilised and destabilised mutants of barnase to determine whether the thermodynamic changes are speci®c to the introduction of the crosslinks.

Results Thermal unfolding of barnase DSC and circular dichroism (CD) data for the thermal denaturation of barnase are of a similar or higher quality to that published from our group earlier (Matouschek et al., 1994; Oliveberg et al., 1994; Johnson & Fersht, 1995). The unfolding of wild-type barnase and the mutants examined here is reversible under a range of conditions as judged from the repeatability of DSC endotherms or CD transitions on rescanning. The Tm and van't Hoff enthalpy of unfolding (
Hvh) are essentially identical by either technique and the ratio of
Hvh to the calorimetric enthalpy of unfolding (
Hcal) obtained from the analysis of DSC endotherms is close to 1.0 over the pH range examined (average 1.01  0.04). The values of Tm,
Hvh and
Hcal by

200

Thermodynamics of Denaturation of Barnase Mutants

Figure 1. DSC and CD data for the thermal denaturation of barnase. Values of
Hcal (*) and
Hvh (*) measured at low ionic strength are plotted at the Tm of unfolding. Values of
Cdp were determined using equation (2) by linear regression of the
Hvh data using weighted errors, as indicated by the continuous lines, A, Wild-type barnase,
Cdpˆ 1.70 (0.06) kcal Kÿ1 molÿ1 (R ˆ 0.997); B, 85-102,
Cdpˆ 1.47 (0.06) kcal Kÿ1 molÿ1 (R ˆ 0.992); C, 43-80,
Cdp ˆ 1.54 (0.07) kcal Kÿ1 molÿ1 (R ˆ 0.993); D, 70-92,
Cdp ˆ 1.72 (0.10) kcal Kÿ1 molÿ1 (R ˆ 0.992). The variation of each value of
Hvh measured at pH 2.8 predicted by the temperature-dependent
Cdp function for wild-type barnase (Martinez et al., 1994) is indicated by the dotted lines.

DSC are independent of the protein concentration (tenfold range) or instrumental scan rate (threefold range), indicating that the thermal unfolding of barnase is a true two-state equilibrium between native and denatured states with no signi®cant accumulation of other conformations detectable on the time-scale of measurement. This observation is supported elsewhere for wild-type barnase and a number of mutants (Griko et al., 1994; Martinez et al., 1994; Matouschek et al., 1994; Johnson & Fersht, 1995).

Changes in the heat capacity of denaturation Values of
Hvh are plotted against the Tm of unfolding for wild-type barnase, 85-102, 43-80 and 70-92 in Figure 1. The values of
Cdp in kcal Kÿ1 molÿ1 determined from these plots is 1.70 for wildtype barnase, 1.47 for 85-102, 1.54 for 43-80 and 1.72 for 70-92. The values of
Hcal obtained from DSC measurements are included for comparison and similar values of
Cdp are obtained by ®tting these data. The values of
Cdp observed for the disul®de mutants correlate with the stabilities of the proteins such that the most stable 85-102 mutant has the smallest
Cdp and the destabilised 70-92 mutant the largest. These differences may re¯ect speci®c physico-chemical effects of the mutations on the native and denatured states (and thus the value of
Cdp) which are correlated to the relative stabilising effects of the disul®de. The enthalpy data for each mutant are recorded over a different temperature range, however, so the possibility of

an effect of temperature on the value of
Cdp should also be considered. It was originally accepted that the value of
Cdp was constant and independent of the temperature of measurement (Privalov, 1979). Recent work has indicated, however, that the
Cdp may decrease at both high and low temperatures but reaches a maximal and fairly constant value over a temperature range encompassing the Tm values typical for the thermal unfolding of proteins (Privalov & Makhatadze, 1990; Gomez et al., 1995). As a result of this, any deviation from linearity in plots of enthalpy versus temperature is dif®cult to detect experimentally and a temperature-independent
Cdp is a good approximation to the temperature-dependent function. The expected variation in
Cdp with temperature for barnase can be calculated using the difference between the observed heat capacity of the folded state and a calculated pro®le for the unfolded polypeptide based on heat capacities of model amino acids (Martinez et al., 1994). We have used this empirical variation in
Cdp to calculate the predicted temperature dependence of the enthalpy using the experimental value of
Hvh at pH 2.8 for each protein studied here. The Tm at this pH is the median value of the range observed. This variation in
Hvh, indicated by the dotted lines in Figure 1, ®ts the measured enthalpies almost as well as the linear regression. Nevertheless, if the
Cdp for each mutant is plotted at the middle of the temperature range over which values of
Hvh were measured, the values follow the expected variation in
Cdp with temperature (Figure 2). Data obtained at an ionic strength of 5 to 20 mM for 85-

201

Thermodynamics of Denaturation of Barnase Mutants

servation rather than a consequence of the introduced crosslink. Changes in the free energy of denaturation

Figure 2. Variation of
Cdp for barnase on mutation. Values of
Cdp determined from equation (2) are plotted at the middle of the temperature range over which
Hvh values were measured. For wild-type barnase ( & ), 85-102 (*), 43-80 (~) and 70-92 (!) the values were obtained from Figure 1. The values of
Cdp of 85-102 (*), 43-80 (~) and 43-80/85-102 ( &) at I < 20 mM and the
Cdp for 70-92 at I ˆ 600 mM (!) were obtained from similar plots (unpublished data). Similarly, the values of
Cdp of the triple mutant Q15I, T16R, K19R (^) and the multiple mutant Q15I, T16R, K19R, G65S, K66A, K108R (^) of barnase were determined from data in the I ˆ 125 to 150 mM buffer system (unpublished data). The temperature-dependent
Cdp function for wild-type barnase (Martinez et al., 1994) is indicated by the continuous line.

102, 43-80 and the double disul®de mutant combining these crosslinks (43-80/85-102) are included as well as values obtained for two multiple mutants of barnase that are signi®cantly stabilised relative to wild-type without the introduction of disul®de bonds (C. M. J. and A. R. F. unpublished results). The latter two mutants suggest that the variation in
Cdp is a function of temperature of ob-

The thermal unfolding of the disul®de mutants and the corresponding single and double cysteine mutants under reducing conditions was studied using DSC at either pH 4.4 and/or 3.4. The changes in the free energy of denaturation for these mutations (

GD-N ˆ
GD-N wildtype ÿ
GD-N mutant) are given in Table 1. These changes in stability have been determined by urea or GdmCl equilibrium unfolding and from NMR hydrogen ± deuterium exchange rates as indicated in Table 1 (Clarke & Fersht, 1993; Clarke et al., 1993, 1995a,b). The agreement between the various estimates of

GD-N is good. The dithiol mutants are destabilised by the sum of the destabilisation energies of the corresponding single cysteine mutants, within the errors of measurement. The additivity observed in these mutant cycles indicates that there is no interaction energy between the free cysteine residues in the dithiol form. The energetic changes on formation of the disul®de can, therefore, be attributed to the crosslink. The thermal unfolding data in Table 1 also con®rm that the 70-92 mutant is less stable than wild-type barnase or its dithiol despite the introduction of the disul®de. The values of

GD-N obtained from DSC measurements on a variety of other mutants of barnase examined in this study and elsewhere (Matouschek et al., 1994) are also close to estimates from urea or GdmCl unfolding (as shown in Figure 3). The correlation between the two values is strong and the slope of the plot is close to 1. Equivalent or better correlations are observed for data recorded at pH 4.4 or 3.5 using other temperatures of comparison or with the simpli®ed relation given in equation (7) (data not shown). The close agreement between values of

GD-N obtained from thermal and chemical denaturation has been demonstrated elsewhere (Matouschek et al., 1994)

Table 1. Changes in free energy on mutation Protein

na

85-102 85-102SH S85C H102C 43-80 43-80SH A43C S80C 70-92 70-92SH T70C S92C

3 2 1 1 2 1 3 2 2 1 2 2

a

b

G298 D-N

b

G320 D-N

ÿ3.3  0.3 0.1  0.3 0.4  0.3 ÿ0.1  0.3 ÿ0.9  0.2 1.1  0.3 0.9  0.3 0.2  0.3 3.8  0.2 3.1  0.3 1.2  0.3 2.3  0.3

ÿ4.2  0.2 0.1  0.3 0.3  0.3 ÿ0.3  0.3 ÿ1.8  0.2 0.9  0.3 1.0  0.2 0.1  0.3 3.1  0.2 2.2  0.3 0.8  0.3 1.1  0.3


Tm 
S c

GDÿN

d

GD50% D-N

e

GH-D D-N

ÿ4.5 0.1 0.3 ÿ0.3 ÿ2.0 1.0 1.0 0.1 3.5 2.8 0.8 1.3

ÿ4.3  0.2 ÿ0.2  0.1 0.6  0.1 0.0  0.1 ÿ2.1  0.2 1.1  0.1 1.1  0.1 0.1  0.1 2.9  0.2 2.3  0.3 1.0  0.1 1.3  0.2

ÿ3.9 ± ± ± ÿ2.0 ± ± ± ± ± ± ±

n is the number of measurements at pH 3.4 and/or 4.4 that are averaged. Values of

G in kcal molÿ1 calculated relative to wild-type barnase using a
Cdp of 1.7 kcal molÿ1 Kÿ1. c m 
S

G
T calculated from equation (7). DÿN d

GD50% D-N calculated at the denaturant mid-point from urea or GdmCl equilibrium data at 298 K (Clarke & Fersht, 1993; 1995a). e

GH-D D-N calculated from NMR hydrogen-deuterium exchange rates (Clarke et al., 1993, 1995b). b

202

Thermodynamics of Denaturation of Barnase Mutants

Figure 3. Values of

GD-N from thermal unfolding and urea or GdmCl equilibrium measurements for 35 mutants of barnase. The

GD-N at 324 K obtained from thermal unfolding data at pH 4.4 was calculated using equation (4) and a
Cdp of 1.7 kcal Kÿ1 molÿ1. Values of

GD-N determined by urea or GdmCl equilibrium unfolding at the denaturant mid-point have been published elsewhere. Linear regression of the data gives D50% the line indicated,

G324 D-N ˆ ÿ0.02 ‡ 0.96

GD-N kcal molÿ1 (Rˆ 0.990).

but here we extend the observation and include a number of stabilised mutants. Changes in the enthalpy and entropy of denaturation Values of

HD-N and

SD-N (

HD-N ˆ
HD-N wild-type ÿ
HD-N mutant, etc.) of the various mutants of barnase were evaluated at 320 K. This is the mean of the Tm range (307.6 to 336.8 K) of all the mutants examined at pH 4.4 and/or pH 3.4. The single cysteine and dithiol mutants can be compared as mutant cycles as well as with the corresponding oxidised disul®de, as shown in Figure 4. For the 43-80 and 85102 disul®des, it appears that the single cysteine and dithiol mutations do not affect signi®cantly the values of
HD-N or T
SD-N. This is consistent with the relatively small effects of these mutations on
GD-N (Table 1). On oxidation of the protein to form the disul®de bonds there is a large decrease in
HD-N and T
SD-N in both mutants relative to wild-type barnase and the dithiol. The changes in entropy are consistent with the classical stabilising effect expected from entropic limitation of the unfolded state by the crosslink, although the decrease in T
SD-N is approximately three times the values of 4.1 kcal molÿ1 and 3.4 kcal molÿ1 predicted from the loop size in 43-80 and 85-102, respectively (equation (1)). The two single cysteine and the dithiol mutants at position 70 and 92 produce large decreases in both
HD-N and T
SD-N, despite the changes in
GD-N being similar to those in the 43-80 and 85-102 cycles. The 70-92 disul®de crosslink does not cause any additional change in the values of
HD-N and T
SD-N relative to these mutants.

Figure 4. Changes in
HD-N and T
SD-N in the disul®de mutant cycles. Values of

HD-N (open bars) and
(T
SD-N) (shaded bars) relative to wild-type barnase were calculated at 320 K from data recorded at pH 3.4 and/or 4.4 using equation (4) with a
Cdp of 1.7 kcal Kÿ1 molÿ1. Data are averaged from the number of measurements as indicated in Table 1.

Signi®cant changes in
HD-N and
SD-N are not a unique feature of the mutations in the 70-92 disul®de cycle but are observed in a wide range of other barnase mutants. The values of
HD-N and
SD-N for each mutant (or the values of

HD-N and

SD-N) are strongly correlated, as shown in Figure 5, and over half of the 41 mutants examined have signi®cantly smaller values of
HD-N and
SD-N than wild-type barnase. The decreases in both
HD-N and
SD-N on mutation have compensating effects on the value of

GD-N such that the ®nal change in stability is relatively small. This is typical enthalpy-entropy compensation behaviour

Thermodynamics of Denaturation of Barnase Mutants

203

Figure 5. Values of
HD-N and
SD-N for the thermal unfolding of wild-type barnase (*) and 41 different mutants (*). The values of
HD-N and
SD-N were calculated at 320 K from all data obtained at pH 3.4 and/or 4.4 using equation (4) and a
Cdp of 1.7 molÿ1. Duplicated kcal Kÿ1 measurements of the same mutant were averaged and the errors indicated were calculated as described in Materials and Methods. The continuous line uses the wild-type values and has a slope of 320 indicating the variation of
HD-N and
SD-N corresponding to a

GD-N of 0.

as observed in a range of biological systems (Lumry & Rajender, 1970). Effects of pH on the free energy of denaturation The values of
GD-N for wild-type barnase and the 85-102, 43-80 and 70-92 mutants are plotted as a function of pH in Figure 6. For wild-type barnase, the derivative of a smoothed ®t to the values of
GD-N has a maximum around pH 3, corresponding to the uptake of about four protons on unfolding (data not shown). This pro®le and the magnitude of the protonation changes are in excellent agreement with earlier work (Oliveberg et al., 1994). Similarly, the value of
GD-N of 10.2 kcal molÿ1 at 298 K for wild-type barnase at near neutral pH is close to estimates obtained in other studies (Griko et al., 1994; Martinez et al., 1994; Oliveberg et al., 1994; Johnson & Fersht, 1995). The value obtained from urea equilibrium unfolding of barnase at this temperature is somewhat lower and the reasons for this discrepancy have been examined elsewhere (Johnson & Fersht, 1995). The titration properties of the disul®de mutants are similar to that of wild-type barnase and, therefore, the value of

GD-N is fairly constant with pH. Closer examination of Figure 6, however, reveals some minor differences between the pro®les. The stability of 70-92 is increasing relative to wildtype barnase with decreasing pH, i.e. the pro®les are converging. These differences in the stability pro®le at low pH are more obvious at high ionic strength because lower pH values can be achieved in the HCl buffer system. For 70-92, this extends the pH range of comparison down to pH 1.25. Values of
GD-N at high ionic strength for wildtype barnase and the D93N mutant (Oliveberg

et al., 1995) and for the 70-92 disul®de are shown in Figure 7A. The derivative of these stability pro®les and the

GD-N for the mutants are plotted in Figure 7B and 7C, respectively. Clearly, the 70-92 and D93N mutants exhibit similar anomalous titration behaviour over this extended pH range. Both the derivative of
GD-N with pH, which indicates the proton ¯ux during unfolding (
v; equation (8)), and

GD-N decrease below pH 4. In D93N these changes have been attributed to the removal of the salt-bridge between D93 and R69 by the mutation with the effects occurring between the pKa of D93 in the denatured state (4) and the signi®cantly shifted pKa of about 1.5 for this group in the folded state (Oliveberg et al., 1995). The R69D93 salt bridge in the X-ray structure of the 70-92

Figure 6. The stability of barnase as a function of pH at low ionic strength. Values of
GD-N at 298 K for wildtype barnase (*), 85-102 (*), 43-80 (~) and 70-92 (~) were calculated using equation (4) and a
Cdp of 1.7 kcal Kÿ1 molÿ1. The lines represent a smoothed ®t to each data set using Kaleidagraph2 (Abelbeck Software).

204

Thermodynamics of Denaturation of Barnase Mutants

titration properties of this mutant and D93N provides independent evidence that con®rms that the R69-D93 salt-bridge is disrupted or signi®cantly weakened in solution. There may also be a slight decrease in the stability of the 85-102 mutant above pH 5 relative to wild-type barnase (Figure 6). This change would be consistent with the removal of H102 in the mutant, since in wild-type barnase the pKa of this group is 0.2 to 0.4 unit lower in the folded state than the unfolded state (Sali et al., 1988; Loewenthal et al., 1992). This residue will therefore contribute a positive
v in this pH region, so that its removal by mutation will reduce
v and thus decrease the derivative of the
GD-N function and the value of

GD-N.

Discussion
Cdp for barnase

Figure 7. The stability of barnase as a function of pH at high ionic strength. A, Values of
GD-N at 298 K for wild-type barnase (*), D93N (*) and 70-92 (~) were calculated using equation (4) and a
Cdp of 1.7 kcal Kÿ1 molÿ1. The lines represent a smoothed ®t to each data set using Kaleidagraph2 (Abelbeck Software). B, Derivative of the smoothed ®ts in A for wild-type barnase ( ± ± ± ) , D93N (




) and 70-92 ( ÐÐ ). C,

GD-N at 298 K calculated from the smoothed ®ts in A for the D93N (




) and 70-92 ( ÐÐ ) mutants.

mutant is disrupted by the introduction of the disul®de, because of a displacement of the loop between residues 65 and 70 (Clarke et al., 1995a). This observation has been interpreted with caution, since this loop is involved in some crystal packing interactions. The similarities in the

The large difference between the heat capacity of the folded and unfolded states of proteins in aqueous solution results from the exposure of buried groups to the solvent along with minor contributions from changes in conformational entropy (Sturtevant, 1977). The value measured for wildtype barnase of 1.70 kcal Kÿ1 molÿ1 is comparable with estimates obtained elsewhere (Griko et al., 1994; Martinez et al., 1994; Matouschek et al., 1994; Johnson & Fersht, 1995). The
Cdp of proteins is expected to be slightly temperature-dependent because the folded and unfolded states expose a different proportion of polar and non-polar groups and the heat capacity of these components show different temperature dependencies (Privalov & Makhatadze, 1990). It is generally dif®cult to demonstrate any temperature-dependence from plots of enthalpy versus Tm (equation (2)), however, since the thermal stability of most systems limits the temperature range over which unfolding can be measured. The stabilised disul®de mutants of barnase have smaller values of
Cdp than wild-type, while the
Cdp is essentially unchanged for the destabilised mutant. This variation may be part of the overall temperature-dependence of
Cdp as indicated in Figure 2 because
Cdp is measured over different temperature ranges in each mutant. It has been suggested, however, that the introduction of a disul®de should be accompanied by a decrease in
Cdp of about 0.2 kcal Kÿ1 molÿ1 for each disul®de bond because of restrictions to the hydration of the unfolded state (Doig & Williams, 1991). The values of
Cdp for 43-80 and 85-102 are decreased by this amount but that for 70-92 remains unchanged. Similar arguments would not explain the decreases in the value of
Cdp for the two stabilised mutants that do not contain disul®des, since their unfolded states should be similar to that of wild-type barnase. There is also some evidence that thermally unfolded proteins containing disul®des are surpris-

205

Thermodynamics of Denaturation of Barnase Mutants

ingly similar to their corresponding reduced state, so that the conformational effects on
Cdp may be rather small. For example, the heat capacity and the CD spectra of these states are similar (Privalov et al., 1989). The NMR hydrogen-deuterium exchange rate is also similar, suggesting that the thermally unfolded state has equal solvent exposure (Roder et al., 1985; Robertson & Baldwin, 1991). It is also possible that changes in the solvation of the folded state could contribute to the changes in
Cdp of the disul®de mutants. The X-ray structures of the mutants are essentially identical with wildtype barnase, however, with the exception of some localised rearrangements in 70-92 (Clarke et al., 1995a). The effects of disul®de bonds on
Cdp has been examined experimentally using protein engineering techniques and by thiol blocking. There are few studies, however, where
Cdp has been determined accurately. Both increases and decreases (Kuroki et al., 1992) as well as no signi®cant change in the value of
Cdp (Cooper et al., 1992) have been reported when disul®de bonds are removed. It seems, therefore, that the effects of disul®des on
Cdp cannot be distinguished from other mutational effects or from a dependence on the temperature of measurement. Changes in the thermodynamics of denaturation The values of

GD-N on mutation estimated from the thermal unfolding data are similar to those determined by other techniques (Table 1, Figure 3). The changes in
GD-N for the single cysteine and dithiol mutants form a series of additive thermodynamic cycles indicating that there is no interaction free energy between the original residues as expected (Table 1, Clarke & Fersht, 1995a). The changes in
HD-N and T
SD-N are consistent with an absence of interaction between residues 85 and 102 and between 43 and 80, although the relatively large errors in these values may mask out smaller levels of non-additivity (Figure 4A and B). In contrast, the decreases in the values of
HD_N and T
SD-N for mutations at positions 70 and 92 are signi®cant and do not form an additive cycle suggesting large interaction energies (Figure 4C). It has been known for many years that the changes in enthalpy and entropy of a process occurring in solution are extremely dif®cult to interpret in terms of a detailed physico-chemical mechanism (Jencks, 1969). This is because changes in the structure of a reactant, for example on mutation, lead to changes in its solvation and, therefore, the observed

HD-N and

SD-N can contain large and misleading contributions from the thermodynamics of the solvent rearrangement, i.e. H2 O reactant

HD-N ˆ

HD -N ‡

HD-N

and H2 O

SD-N ˆ

Sreactant ‡

SD D-N -N ;

where the superscripts reactant and H2O refer to the changes in the thermodynamics of the reactant and solvent, respectively. Fortunately, the solvation terms tend to cancel out when combined in the free energy function because H2 O H2 O

HD -N  T

SD-N :

The

GD-N for the change in structure is, therefore, generally correct and much smaller than the component changes in

HD-N or T

SD-N. The large apparent interaction enthalpy and entropy between residues 70 and 92 is probably the result of non-additive changes in the solvation of the folded and/or unfolded states of these proteins, which tend to cancel when combined in

GD-N. These changes could include a similar or smaller reorganisation of the 65-70 loop, which is observed in the X-ray structure of the 70-92 disul®de mutant. The mutation of S92 to cysteine also results in the loss of a hydrogen bond between the hydroxyl oxygen atom of S92 and the amide nitrogen atom of T70, which may be disruptive. H2 O The approximate equivalence of

HD -N H2 O and T

SD-N is a fundamental component of the enthalpy-entropy compensation behaviour of biological systems in solution (Lumry & Rajender, 1970). Dunitz (1995) has presented a semi-quantitative analysis, which demonstrated that enthalpyentropy compensation may be a feature of weak intermolecular interactions in general. Compensating changes in
H D-N and
SD-N can be introduced, however, from errors in the measurement of
HD-N, since this value and the relatively accurate Tm are used to calculate
SD-N. Underestimating the value of
HD-N therefore causes a compensating error in
SD-N. This may represent a signi®cant component in smaller changes to these parameters. We have observed strong enthalpy-entropy compensation in the thermodynamics of denaturation in a variety of mutants of barnase (Figure 5). Further, it is clear that few, if any, mutants have a larger
HD-N or
SD-N than the wild-type protein. This may indicate that the intramolecular protein interactions and intermolecular solvent interactions are optimised in the structure of the wild-type protein so that any mutation causes a decrease in
HD-N or
SD-N. It has already been noted that
HD-N for barnase is unusually large in comparison with other proteins (Martinez et al., 1994). Further, the nature of the mutants of barnase that have been produced in our group generally involve the removal of speci®c moieties and/or interactions for application of the protein engineering approach to protein folding. This may also tend to bias the data set that we have obtained. Changes in the thermodynamics of unfolding of the three disul®de mutants of barnase are not fully

206 consistent with either of the models of disul®de bond formation. Firstly, the stability of the 70-92 mutant is decreased on formation of the disul®de relative to wild-type barnase and its corresponding dithiol despite the entropic effects on its unfolded state. The destabilisation probably originates from the signi®cant local reorganisation in structure suggested by the X-ray structure of this mutant. Here we have con®rmed that the electrostatic properties of this mutant are consistent with this change and the associated disruption of the R69D93 salt-bridge by examining the thermodynamics of unfolding as a function of pH. Secondly, the changes in
HD-N,
SD-N and
Cdp of the disul®de mutants relative to wild-type barnase or the corresponding dithiols do not form a pattern consistent with the simple chain entropy effects of equation (1) or the contrasting model proposed by Doig & Williams (1991). It is likely that each individual disul®de contributes effects from both intramolecular and solvation contributions in both the native and denatured states and it is the subtle balance of these that leads to the variety of unpredictable thermodynamic changes observed.

Materials and Methods Materials Acetate, formate, glycine and HCl-based buffers were prepared from reagents of Analar quality using water deionised to 18 M resistance in an Elgastat UHP system. The heats of ionisation of all the buffers used are negligible, so that the enthalpies of unfolding do not contain signi®cant contributions from the associated protonation changes. Buffers used for calorimetric measurements contained 50 mM acetate, formate or glycine with 100 mM KCl. This system has a variable ionic strength (I) between 110 and 130 mM for the pH range and buffers used. This buffer was used for some CD measurements. Alternatively, a constant ionic strength buffer system, with I maintained at 50 or 600 mM using KCl, was used for the CD studies as previously (Oliveberg et al., 1994). Measurements on mutants containing free thiol groups were performed in the same buffers as above but containing 10 mM DTT and 1 mM EDTA. These samples were all examined for the presence of dimers or higher-order aggregates using SDS-PAGE before use. The mutagenesis, expression and puri®cation of the proteins used in this study are described elsewhere (Clarke & Fersht, 1993, 1995a; Horovitz et al., 1990; Serrano et al., 1990).

Thermodynamics of Denaturation of Barnase Mutants to 50 or 1 mM, respectively. Data were ®tted as described using a
Cdp of 1.7 kcal Kÿ1 molÿ1 (Oliveberg et al., 1994). Within the error of measurement, the data obtained from DSC and CD measurements made in the same buffer were identical. Measurements at a ®xed I ˆ 50 mM or variable I ˆ 110 to 130 mM at the same pH were also comparable. While some effect of ionic strength on the stability of barnase has been observed, the largest effects occur between 200 and 600 mM (Oliveberg et al., 1994). The data recorded at I ˆ 50 mM and I ˆ 110 to 130 mM were, therefore, combined and analysed as one set, which we term the low ionic strength data. The data set recorded at I ˆ 600 mM (high ionic strength data) were obtained solely from CD measurements at 230 nm because barnase tends to aggregate upon unfolding at high and low pH using the protein concentrations required for DSC. DSC studies on barnase can be complicated by additional association events that occur at very low ionic strength and between pH 2 and 4 (Sanz et al., 1994) and so here, as in earlier work (Johnson & Fersht, 1995), we used higher ionic strength 550 mM, which eliminates these processes. The source and magnitude of the errors in the values of the enthalpy for unfolding (
Hcal ˆ 4 kcal molÿ1;
Hvh ˆ 2 kcal molÿ1 from DSC measurement;
Hvh ˆ 10 kcal molÿ1 from CD measurement) and the midpoint for unfolding (Tm ˆ 0.1 K) have been discussed elsewhere (Matouschek et al., 1994; Johnson & Fersht, 1995). In proteins such as barnase, where the unfolding appears to follow very closely a two-state mechanism and
Hcal and
Hvh have the same value (as we have found over a wide range of measurements (Matouschek et al., 1994; Johnson & Fersht, 1995)), we use
Hvh in all analyses, since it is independent of errors of measurement of protein concentration. This also allows values of
Hvh obtained from other techniques to be combined with DSC data. The
Cdp can be measured directly from individual DSC endotherms but it is generally more reliable, however, to determine the value from the temperature dependence of the enthalpy of unfolding according to the relation:
Cdp ˆ d
Hm =dTm

…2†

where
Hm is the enthalpy of unfolding at the Tm. For measurements other than by calorimetry this is essentially the only method of determining
Cdp, since alternatives require taking the derivative of a non-linear ®t to van't Hoff plots. The value of
Cdp can normally be determined with an error of 5% or less (e.g. Figure 1). Despite this level of precision, we assume a larger error of 10%, since this provides some tolerance for additional changes in the value arising from mutation or extrapolations over a range of temperatures.

Thermal denaturation

Extrapolation and comparison of thermodynamic data

DSC measurements were performed with a Microcal MC-2D instrument using a nominal scan rate of 60 K hÿ1. The protein concentration was 30 to 50 mM. The preparation of samples, operation of the DSC and data analysis were as described earlier (Johnson & Fersht, 1995). Thermal unfolding was monitored by CD in a Jasco J-720 spectropolarimeter at 230 nm using 0.1 or 2 cm pathlength cells. The protein concentration was 30

DSC and CD measurements give the Tm of thermal unfolding and the value of
HD-N at this temperature.
GD-N is zero at the Tm and so the value of
SD-N ˆ
HD-N/Tm. These values are obtained with reasonable certainty. To compare the thermodynamics of unfolding for different proteins, however, the values obtained by measurement at the Tm of unfolding must be extrapolated to a common temperature. This extrapolation uses

207

Thermodynamics of Denaturation of Barnase Mutants the value of
Cdp and standard thermodynamic relationships: T T
GTD-N ˆ
HD -N ÿ T
SD-N Tm d
GTD-N ˆ …
HD -N ‡
Cp …T ÿ Tm †† d ÿ T…
STm DÿN ‡
Cp ln…T=Tm ††

…3†

…4†

where T is the temperature of comparison. The errors in
HTD-N and
STD-N can be quanti®ed and are dependent on the length of the temperature extrapolation (T ÿ Tm) d T and errors in the values of
HTm D-N,
Cp. For
HD-N this simpli®es to: T Tm 2 d 2 1=2 d
HD -N  ‰…d
HD-N † ‡ …‰T ÿ Tm Š  d
Cp † Š

…5†

and for
STD-N to: 2 Tm d
STD-N  ‰…
HD -N =Tm †

‡ …d
Cdp  ln…T=Tm ††2 Š1=2

…6†

Both errors and changes in the value of
Cdp on mutation or with temperature act to compound the exand isting errors in the measurement of
HTm D-N
STm D-N. Fortunately, the errors in the extrapolated values of
HTD-N and
STD-N tend to cancel out when combined in
GTD-N, however, such that its value is obtained with a relatively small uncertainty (Matouschek et al., 1994). Differences in the thermodynamic parameters on mutation can be evaluated at any temperature but clearly the errors are minimised if a temperature between the Tm values of the two proteins is used. The value of

GD-N can also be estimated using the simpli®ed equation:

GD-N ˆ
Tm

STm D- N

…7†

where
Tm is the difference in unfolding temperature (Tm wild-type ÿ Tm mutant) and
STm D-N is value of the wild-type protein (Becktel & Schellman, 1987). Quite accurate estimates of

GD-N can be obtained from this relation (Matouschek et al., 1994). Nevertheless, it is clear from equation (4) that the free energy of each protein is dependent on the relative values of d
HTm D-N, Tm and
Cp and the temperature of comparison. If these parameters are signi®cantly different then the value of

GD-N will be temperature dependent.

Effects of pH on protein stability The stability pro®les of a protein with pH gives information about the titration of ionisable groups in the folded and unfolded states of these proteins since the protonation equilibrium and the pH are linked functions. Speci®cally, the derivative of the
GD-N pro®les indicate the changes in protonation according to: d
GD-N =dpH ˆ 2:303 RT
v

…8†

where
v represents the net proton ¯ux during unfolding (Wyman, 1964). This relation has been used to characterise the electrostatic properties of barnase in earlier work (Oliveberg et al., 1994, 1995).

References Becktel, W. J. & Schellman, J. A. (1987). Protein stability curves. Biopolymers, 26, 1859± 1877. Betz, S. F. (1993). Disul®de bonds and the stability of globular proteins. Protein Sci. 2, 1551± 1558. Clarke, J. & Fersht, A. R. (1993). Engineering disul®de bonds as probes of the folding pathway of barnase: increasing the stability of proteins against the rate of denaturation. Biochemistry, 32, 4322 ±4329. Clarke, J., Hounslow, A. M., Bycroft, M. & Fersht, A. R. (1993). Local breathing and global unfolding in hydrogen exchange of barnase and its relationship to protein folding pathways. Proc. Natl Acad. Sci. USA, 90, 9837± 9841. Clarke, J., Henrick, K. & Fersht, A. R. (1995a). Disul®de mutants of barnase I: changes in stability and structure assessed by biophysical methods and X-ray crystallography. J. Mol. Biol. 253, 493± 504. Clarke, J., Hounslow, A. M. & Fersht, A. R. (1995b). Disul®de mutants of barnase II: changes in structure and local stability identi®ed by hydrogen exchange. J. Mol. Biol. 253, 505± 513. Cooper, A., Eyles, S. J., Radford, S. E. & Dobson, C. M. (1992). Thermodynamic consequences of the removal of a disulphide bridge from hen lysozyme. J. Mol. Biol. 225, 939± 943. Doig, A. J. & Williams, D. H. (1991). Is the hydrophobic effect stabilizing or destabilizing in proteins? The contribution of disulphide bonds to protein stability. J. Mol. Biol. 217, 389± 398. Dunitz, J. D. (1995). Win some, lose some: enthalpyentropy compensation in weak intermolecular interactions. Chem. Biol. 2, 709± 712. Flory, P. J. (1956). Theory of elastic mechanisms in ®brous proteins. J. Am. Chem. Soc. 78, 5222± 5235. Gomez, J., Hilser, V., Xie, D. & Freire, E. (1995). The heat capacity of proteins. Proteins: Struct. Funct. Genet. 22, 404± 412. Griko, Y. V., Makhatadze, G. I., Privalov, P. L. & Hartley, R. W. (1994). Thermodynamics of barnase unfolding. Protein Sci. 3, 669± 676. Horovitz, A., Serrano, L., Avron, B., Bycroft, M. & Fersht, A. R. (1990). Strength and cooperativity of contributions of surface salt bridges to protein stability. J. Mol. Biol. 216, 1031± 1044. Jencks, W. (1969). Catalysis in Chemistry and Enzymology, McGraw-Hill, London. Johnson, C. M. & Fersht, A. R. (1995). Protein stability as a function of denaturant concentration: the thermal stability of barnase in the presence of urea. Biochemistry, 34, 6795± 6804. Katz, B. A. & Kossiakoff, A. (1986). The crystallographically determined structures of atypical strained disul®des engineered into subtilisin. J. Biol. Chem. 261, 15480± 15485. Kuroki, R., Inaka, K., Taniyama, Y., Kidokoro, S., Matsushima, M., Kikuchi, M. & Yutani, K. (1992). Enthalpic stabilization of a mutant human lysozyme lacking a disulphide bridge between cysteine-77 and cysteine-95. Biochemistry, 31, 8323± 8328. Loewenthal, R., Sancho, J. & Fersht, A. R. (1992). Histidine-aromatic interactions in barnase: elevation of histidine pKa and contribution to protein stability. J. Mol. Biol. 224, 759± 770. Lumry, R. & Rajender, S. (1970). Enthalpy-entropy compensation phenomena in water solutions of proteins and small molecules: a ubiquitous property of water. Biopolymers, 9, 1125± 1227.

208 Martinez, J. C., El Harrous, M., Filimonov, V. V., Mateo, P. L. & Fersht, A. R. (1994). A calorimetric study of the thermal stability of barnase and its interaction with 30 GMP. Biochemistry, 33, 3919± 3926. Matouschek, A., Matthews, J. A., Johnson, C. M. & Fersht, A. R. (1994). Extrapolation to water of kinetic and equilibrium data for the unfolding of barnase in urea solutions. Protein. Eng. 7, 1089± 1095. Oliveberg, M., Vuilleumier, S. & Fersht, A. R. (1994). Thermodynamic study of the acid denaturation of barnase and its dependence on ionic strength: evidence for residual electrostatic interactions in the acid/thermally denatured state. Biochemistry, 33, 8826± 8832. Oliveberg, M., Arcus, V. & Fersht, A. R. (1995). pKa values of carboxyl groups in the native and denatured states of barnase: the pKa values of the denatured state are on average 0.4 units lower than those of model compounds. Biochemistry, 34, 9424± 9433. Pace, C. N., Grimsley, G. R., Thomson, J. A. & Barnett, B. J. (1988). Conformational stability and activity of ribonuclease T1 with zero, one and two intact disulphide bonds. J. Biol. Chem. 263, 11820± 11825. Poland, D. C. & Scheraga, H. A. (1965). Statistical mechanics of noncovalent bonds in polyamino acids. VIII. Covalent loops in proteins. Biopolymers, 3, 379± 399. Privalov, P. L. (1979). Stability of proteins. Advan. Protein Chem. 33, 167 ± 236. Privalov, P. L. & Makhatadze, G. I. (1990). Heat capacity of proteins II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects. J. Mol. Biol. 213, 385±391. Privalov, P. L., Tiktopulo, E. I., Venyaminov, S. Y., Griko, Y. V., Makhatadze, G. I. & Khechinashvili,

Thermodynamics of Denaturation of Barnase Mutants N. N. (1989). Heat capacity and conformation of proteins in the denatured state. J. Mol. Biol. 205, 737 ± 750. Robertson, A. D. & Baldwin, R. L. (1991). Hydrogen exchange in thermally denatured ribonuclease A. Biochemistry, 30, 9907± 9914. Roder, H., Wagner, G. & WuÈtrich, K. (1985). Individual amide proton exchange rates in thermally unfolded basic pancreatic trypsin inhibitor. Biochemistry, 24, 7407± 7411. Sali, D., Bycroft, M. & Fersht, A. R. (1988). Stabilization of protein structure by interaction of an alpha-helix dipole with a charged side chain. Nature, 335, 496 ± 500. Sanz, J. M., Johnson, C. M. & Fersht, A. R. (1994). The A-state of barnase. Biochemistry, 33, 11189± 11199. Serrano, L., Horovitz, A., Avron, B., Bycroft, M. & Fersht, A. R. (1990). Estimating the contribution of engineered surface electrostatic interactions to protein stability using double-mutant cycles. Biochemistry, 29, 9343± 9352. Sturtevant, J. M. (1977). Heat capacity and entropy changes in processes involving proteins. Proc. Natl Acad. Sci. USA, 74, 2236± 2240. Tidor, B. & Karplus, M. (1993). The contribution of cross-links to protein stability: a normal mode analysis of the con®gurational entropy of the native state. Proteins: Struct. Funct. Genet. 15, 71± 79. Wetzel, R. (1987). Harnessing disulphide bonds using protein engineering. Trends Biochem. Sci. 12, 478 ± 482. Wyman, J. (1964). Linked functions and reciprocal effects in hemoglobin: a second look. Advan. Protein Chem. 19, 223 ±286.

Edited by J. Karn (Received 5 November 1996; accepted 27 January 1997)