- Email: [email protected]
Contribution of hydration and non-covalent interactions to the heat capacity effect on protein unfolding
J. Mol. Biol. (1992) 224, 715-723
Contribution of Hydration and Non-covalent Interactions the Heat Capacity Effect on Protein Unfolding
to
Peter L. Privalov and George I. Makhatadze Department and Institute
of Biology, The Johns Hopkins University Baltimore, MD 21218, U.S.A. of Protein Research of the USSR Academy of Sciences Puschino, USSR
(Received 25 July
1991; accepted 29 November 1991)
The heat capacity change upon protein unfolding has been analysed using the heat capacity data for the model compounds’ transfer into water, corrected for volume effects. It has been shown that in the unfolding, the heat capacity increment is contributed to by the effect of hydration of the non-polar groups, which is positive and decreases with temperature increase, and by the effect of hydration of the polar groups, which is negative and decreases in magnitude as temperature increases. The sum of these two effects is very close to the total heat capacity increment of protein unfolding at room temperature but is likely to deviat,e from it at higher temperatures. Therefore, the expected heat capacity effect caused by the increase of configurational freedom of the polypeptide chain upon unfolding seems to be compensated for by some other effect, perhaps associated with fluctuation of the native protein structure. Keywords: protein; unfolding;
heat capacity; hydration;
1. Introduction One of the most general specificities of globular protein unfolding is the significant heat capacity increase accompanying this process (for reviews, see Privalov, 1979, 1989). This heat capacity increment determines the temperature dependencies of all the thermodynamic characteristics of protein unfolding and, consequently, the stability of their native states. Therefore, an understanding of this effect is necessary for an understanding of the mechanism of the native protein structure stabilization. The heat capacity increment was thought to be caused by the hydration and the increase of configurational freedom of internal protein groups upon unfolding (Sturtevant, 1977; Privalov & Gill, 1989). According to indirect estimates, the increase of configurational freedom upon unfolding contributes about 30% of the observed heat capacity increment (Velicelebi & Sturtevant, 1979). We have shown (Privalov & Makhatadze, 1990) that hydration of groups that are exposed to water upon unfolding of the polypeptide chain comprises more than 70% of the observed heat capacity increment. This was done by summing all the heat capacity effects of the hydration of the protein interior groups, which were determined for model compounds (Makhatadze & Privalov, 1990). By heat capacity effect of 715 0022-2836/92/070715-09
$03.00/O
calorimetry
hydration, we meant a difference between the partial heat capacity of the molecule in aqueous solution and its heat capacity in the gaseous state, as it is conventionally accepted (Gill & Wads& 1976). Later, however, we realized that the gaseous state is not the appropriate standard state when considering the effect of hydration, because of the volume expansion effect, which is significant in gases. The liquid and solid states, which have been used in determining the hydration effects (Spolar et al., 1989; Livingstone et aZ., 1991; Murphy 6 Gill, 1990) are even less appropriate as standards of anhydrous non-polar solutes. The heat capacities of these condensed phases include considerable effects associated with fluctuation of interactions between the molecules, which cannot be taken into account without referring to the gaseous phase. The most appropriate standard state for the analysis of hydration effects in proteins appears to be a “compact” state of the unfolded polypeptide chain and, correspondingly, a compact phase of model compounds, which was introduced to analyse the hydrophobic interactions (Privalov & Gill, 1989). The compact state assumes the hypothetical state of a macroscopic system that has the same density as the condensed phase of this system, but all noncovalent interactions between its components are switched off. These components are the small 0
1992 Academic
Press Limited
716
P. L. Privalov
and G. I. Makhatadze
molecules (e.g. the non-polar molecules of benzene) if we are considering the system of these molecules, or the amino acid residues if we are considering a single protein molecule as a macroscopic system. According to the definition, the molecules of the surrounding medium (e.g. water molecules in the case of an aqueous solution of protein) do not penetrate into a compact system and are in contact with the same groups that are on the surface of protein in the native state. This is one of the principal features distinguishing the compact state of protein from the “molten globule” state. which has been widely discussed in the literature (for reviews, see Ptitsyn. 1987; Kuwajima. 1989). The other principal feature is t,hat. in the molten globule state, which is assumed to be maintained by the hydrophobic interactions maintains the elements of and secondary structure, not all internal non-covalent interactions are switched off. One should bear in mind that, while the molten globule is supposed to be a t’hermodynamically stable state of protein. the compact state is a hypothetical transient state, which has been suggested to take into account the volume effects and effects of non-covalent interactions in evaluating hydration upon protein unfolding. Makhatadze & Privalov (1990) and Privalov & Makhatadze (1990) showed that the heat, capacity effects of hydration of non-polar and of polar groups have different signs; while the hydration of nonpolar groups increases the partial heat capacity of proteins in aqueous solutions, the hydration of polar groups decreases it. Unfortunately, we did not separate these two opposite effects in considering the overall heat capacity change upon protein unfolding and, further, this point of paramount importance for understanding the thermodynamics of protein molecules has escaped the attention of many of our readers. Here, we consider all three components of the complex heat capacity effect of protein unfolding: the effects of hydration of non-polar groups; the hydration of polar groups; and disruption of the internal bonds that maintain the native structure; recalculated in accordance with the definition of hydration given above. For a demonstration, we have used four globular proteins for which the denaturation has been studied in detail (Privalov et al., 1989).
2. Method Privalov & Makhatadze (1990) and Makhatadze & Privalov (1990) determined the heat capacity effect of hydration according to the conventional definition as the difference between the partial heat capacity of the solute molecule in an aqueous solution and the heat capacity of this molecule in the gaseous state?: dchyd = c+ - CP (1) P P P’ Now we determine it as the difference between the partial heat capacity of a solute molecule in an aqueous solution and the heat capacity of this molecule in the compact
state.
(A. which
we take as a standard S(‘y
st,ate:
= ‘7; - p.
(4
The compact state differs from the gaseous state by the molar volume, which is the same for the considered substance as it is in the condensed, liquid-like state of this substance. On the other hand. the compact state differs from the condensed state by the absence of int,erac%iorr between the molecules (Privalov & Gill. 1989). Therefore, t’he heat capacity of the molecule in the cotnpac+ st)ate should differ from its heat capacity in the condrnsrd state by the thermal effects associated with the fiuctuation of molecules. and from that of’ interactions between molecules in the gaseous state by the term associated with volume expansion at constant pressure. To determine the heat c>apaGty of a molrculr in the compact state, let us consider the process of vapourization of a liquid. The enthalpy of this process can be presented in 2 terms, one representing the energy of int,eractiott between molecules. and the other representing the work on expansion of a substame from the condensed state to the gaseous state at normal pressure: A;H
= A;E + /‘A,p I’.
0)
Since the volume of a gas at normal pressure considerably exceeds that of the condensed state. one can neglect thr volume of a condensed state and. assuming that thr considered gas is close to ideal in the first approximation. i.e. PI’ = RT, write: AgI H = ACE I + R7’. Differentiating
this expression
(4)
by temperature
we get:
AY’ 1 P = A;(‘,+ R ()“a- (71 = (” - ( 1’ + fi P P P P or: (‘C = (‘g--R. P P Correspondingly, we will have:
for
the hydration
(5) heat
SChYd P = cg - q f R.
ca1)acit.v
effeclt (6)
It is known that the hydration
heat capacity effect for non-polar substances is proportional to the water-accesset al., ible surface area of the substance, ASA (Naghibi 1986, 1987). The ratio:
is the hydration heat capacity effect of the non-polar group of type i normalized to a surface area. It is similar for various types of non-polar groups, differing slightly for aliphatic and ring compounds (Makhatadze & Privalov. 1989). In the case of a molecule consisting of polar and nont AfH = Ha-H’, the enthalpy change upon transition from the liquid state to the gaseous state; A,FE = EC-E’, the energy change upon transition from the liquid state to the compact state; AfC, = CE-Cb, the heat capacity change upon transition from the liquid state to the gaseous state; Cg, the partial heat capacity of substance in solution; Ck, the heat capacity of substance in the liquid state; C$ the heat capacity of substance in the gaseous state; Cz, the heat capacity of substance in the compact state; &Zy, the heat capacity effect of hydration; ASA,, the water-accessible surface area of the group of type i.
Heat Capacity
Effects on Protein
Unfolding
717
Table 1 Hydration
heat capacity effect, @2,,ilhyd, per unit of surface-accessible various constituent groups of protein
Croup or side-chain of amino acid residue Non-polar: Aliphatic Aromatic Heme Polar parts of:
‘W -(:ONHp Met QS His Ser AM Asp Gin Glu Lys Tyr A% Thr
Temperature
area for
(“C)
5
25
50
75
190
125
2.30 1.36 507
2.22 1.23 475
2.10 1.14 440
1.98 1.05 406
1.88 @98 375
1.74 0.91 346
3.68 - 1.92 - 3.53 -353 -0.86 - 1.52 - 1.22 - 1.66 -634 -0-68 - 1.34 602 -637 - 1.03
3.78 - 1.64 -388 -388 - 1.28 - 1.30 -0.96 - 1.35 -019 - 0.52 - 1.57 608 -020 - 1.25
358 - 1.39 -4.10 -410 - 1.32 - 1.10 -962 - 1.01 - 0.03 - 0.32 - 1.62 0.05 -912 - 1.16
3.40 - 1.35 -408 - 4.08 - 1.26 -0.85 - 0.36 -065 611 -914 - 1.39 0.16 - 0.04 -083
3.15 - 1.30 - 3.95 -395 -1.13 -0.61 -0.10 -034 0.21 -001 - 1.19 0.35 901 -0.24
2.95 - 1.36 - 3.80 - 3%0 - 0.97 -637 0.15 - 0.05 633 612 - 0.99 0.50 ow 059
t The temperature dependence of the -NH group of the Trp side-chain Therefore its contribution was summed with non-polar groups. All heat capacity values are given in J K-’ molF’ A-‘.
polar parts, the normalized hydration effect of the polar part is determined by excluding the effect of the non-polar part from the total partial heat capacity of the molecule and dividing the difference by the surface area of the polar part. The values of partial heat capacities of all the groups constituting the polypeptide chain, the heat capacities of these groups in the gaseous state, and their ASA values have been reported (Makhatadze & Privalov, 1990). The normalized hydration heat capacity effects. corrected for thermal expansion, of all these groups are given in Table 1. It’ should be noted that experimental error in determination of the partial heat capacities of model compounds and proteins in aqueous solutions by scanning microcalorimeter is about 3%. The uncertainty in ASA values is rather large because of the uncertainty of van der Waals radii of atoms and the imperfection of computer programs (see Livingstone et aZ., 1991). However, since we are using the same radii and program in calculating the ASA values of model compounds and proteins, this uncertainty in ASA values is eliminated in the evaluated hydration heat capacity effects in proteins. Therefore, the overall uncertainty in these effects does not exceed 5%. Here, we consider the same 4 proteins that we analysed in our previous work. Native pancreatic ribonuclease A (Rns(N)t) and its unfolded polypeptide chain with t Abbreviations used: Rns(N), native pancreatic ribonuclease A; Lys(N), native hen egg-white lysozyme; Mb(N), native sperm-whale myoglobin; Cyt(N), native horse-heart cytochrome c; Rns(U, -ss), unfolded pancreatic ribonuclease A with reduced and carboxymethylated cysteine residues; Lys(U, -ss), unfolded hen egg-white lysozyme with reduced and carboxymethylated cysteine residues; Mb(U, apo), unfolded sperm-whale apo myoglobin; Cyt(U, ape), unfolded horse-heart apo cytochrome c.
behaves as non-polar
reduced and carboxymethylated cysteine residues (Rns(U, -ss)), native hen egg-white lysozyme (Lys(N)) and its unfolded polypeptide chain with reduced and carboxymethylated cysteine residues (Lys(U, ss)), native spermwhale myoglobin (Mb(N)) and its unfolded polypeptide chain without heme (Mb(U, ape)), and native horse heart cytochrome c (Cyt(N)) and its unfolded polypeptide chain without heme (Cyt(U, apo)).
3. Results and Discussion (a) The heat capacity
of proteins
denatured
in the unfolded
and
states
Privalov & Makhatadze (1990) compared the calorimetrically measured partial heat capacities of four proteins in the denatured state (Rns(U, -ss), Lys(U, -ss), Mb(U, apo) and Cyt(U, apo)) with the partial heat capacities of completely unfolded polypeptide chains of these proteins calculated from the partial heat capacities of individual amino acid residues, which we purposely measured (Makhatadze & Privalov, 1990). The close correspondence of these two sets of values for all proteins considered in a broad temperature range (from 5 to 125°C) showed that the denatured proteins in acidic solutions can be regarded as completely unfolded from a thermodynamic point of view. (b) Hydration effect of the unfolded polypeptide chain If the areas of the surfaces exposed to water of the non-polar, polar and charged groups of the unfolded polypeptide chains are known, and the heat capacity effects of hydration of these groups
P. L. Privalov
718
and G. I. Makhatadze
Table 2 Contribution
of the hydration
to the heat capacity unfolded states
of proteins
in the native and
Hydration heat capacity
increment (kJ/K mol)
Temperature ASA (AZ)
5
25
50 Cytochrome
(“C) 75
100
125
Polar Non-polar Total
2360 3580 5940
-3.5 7.7 4.2
-3.2 7.4 42
-2.8 7.0 4.1
c (12.3 kDa) -2.4 6.5 42
-21 6.2 4.1
Polar Non-polart Total?
6880 10,890 17,770 4520 7310 11,830
- 10.6 23.3 12.7
-95 22.2 12.7
- 8.3 21.0 12.6
- 7.4 19.7 123
-6.8 18.6 11.9
- 1.9 5.7 3.9 -6.3 17.3 11.0
-7.1 15.6 8.5
-6.3 149 8.5
-5.5 14.0 %5
-51 13.2 8.1
- 4.6 12.4 7.8
-44 11.5 7.1
Polar Non-polar Total
3360 3760 7120
-42 8.1 39
-3.7 7.8 40
-31 7.3 4.2
(13.6 kDa) -25 6.9 44
-21 65 45
Polar Non-polar Total
9330 11,120 20,450
- 141 235 9.4
- 12.6 22.5 9.9
- Il.0 21.2 l@O
-98 20.0 10.0
-8.7 18.9 10.0
Polar Non-polar Total
5970 7350 13,330
- 9.9 15.4 55
-8.9 147 54
-7.9 13.9 60
- 7.3 13.1 58
- 6.6 12.4 58
- 1.6 61 4.4 - 7.9 17.5 9.6 -6.2 11.5 52
Polar Non-polar Total
3530 3160 6690
-43 6.8 2.4
-3.6 6.5 2.9
Lysozyme - 3.0 6.1 3.2
(14.3 kDa) - 2.4 58 3.4
-21 55 34
-1.7 -.1 i.4
Polar Non-polar Total
9050 11,730 20,780
- 13.4 247 11.3
-11.6 23.7 12.0
-99 22.3 12.4
- 8.9 21.0 12.1
-8.0 19.9 11.9
- 7.4 18.4 11.0
Polar Non-polar Total
5520 8750 14,090
-9.0 17.9 8.9
- 8.0 17.1 92
-7.0 16.2 92 Myoglobin
-6.5 152 88
-6.0 14.4 8.4
-57 13.3 7.6
Polar Non-polar Total
3250 4620 7870
-40 9.6 56
- 3.5 9.0 5.5
-3.0 8.5 55
-pfj
-2.3 7.5 52
Polar Non-polart Total?
9910 16,210 26,120
-4.2 l@O 57 - 147 346 19.9
- 13.2 33.1 199
-11.4 31.2 19.8
10.3 29.3 19.1
- 9.4 278 18.4
Polar Non-polar? Total?
6660 11,590 18,250
-1@5 246 142
-9.2 23.5 14.3
-7.9 22.2 143
- 7.3 20.8 13.6
-6.8 197 13.0
Polar Non-polar? Total?
Ribonuclease
t Includes the heme group. All heat capacity values are precise within
(17.8 kDa) 8.0
54
-8.9 25.7 16.8 - 6.6 l&3 Il.7
5%
are known, one can determine the contribution of their hydration to the total partial heat capacity of the polypeptide chain. The corrected values of the normalized hydration heat capacity effects at different temperatures for all the groups constituting the polypeptide chain are given in Table 1. The surface area of the various groups of considered proteins in the native and unfolded states and the calculated values of the heat capacity effect associated with hydration of the non-polar and polar groups of the considered unfolded polypeptide chains are presented in Table 2. Figure 1 illustrates the temperature dependencies of these effects recalculated per gram of protein (the specific heat
capacity effects) to facilitate comparison of proteins that differ in size and in content of prosthetic groups. The values are corrected for the effect of carboxymethylation of cysteine residues in the cases of Rns(U, -ss) and Lys(U, -ss), and the absence of hemes in the cases of Mb(U, apo) and Cyt(U, ape). As seen, the positive heat capacity effect is provided only by the hydration of the non-polar groups, while the heat capacity effect of hydration of polar groups is negative at all temperatures. The remarkable feature of the hydration heat capacity effects of these two sets of groups is that, on temperature increase they both decrease in absolute value. The decrease of hydration effects
Heat Capacity IZfjects on Protein Unfolding
I .6
719
1
__ T
-”
- 0.8
-0.8
-1.2 I -20
6 20
60
100
Temperature
140
180
1
-I.:, Temperature
PC)
Figure 1. Specific heat capacity effects of hydration of the unfolded polypeptide chains. Continuous line, the total hydration effect; long broken line, the hydration effect of non-polar groups; broken line, the hydration effect of polar groups; short broken line, the extrapolation. The bars show the experimental error in heat
1°C)
Figure 2. Specific heat capacity effects of hydration of the native proteins. The meaning of the lines is the same as in Fig. 1. A, native myoglobin; B, native cytochrome c;
C, native lysozyme; D, native ribonuclease A.
capacity determination. A, unfolded apo myoglobin; B, unfolded apo cytochrome c; C, unfolded lysozyme with reduced and carboxymethylated cysteine residues; D, unfolded ribonuclease A with reduced and carboxymethylated cysteine residues.
with temperature proceeds monotonously and rather slowly. It appears that the hydration effect of the non-polar groups disappears much above 125”C, up to which temperature they were experimentally determined, perhaps, somewhere above 450°C as judged by extrapolations of experimentally determined functions. However, since the hydration effects of polar and non-polar groups have different signs, and they depend on temperature in different ways, their sum, which corresponds to the total hydration heat capacity effect, is represented by a curved function with a maximum at about 50°C. This shape of the hydration heat capacity effect seems to be the main cause of the calorimetrically observed curved shape of the partial heat capacity function of denatured proteins (Privalov et al., 1989).
(c) Hydration
(d) Hydration
effect of protein unfolding
Most important for understanding the thermodynamics of protein unfolding/refolding is the difference hydration effect of the native and unfolded states: KY(U)-SC?(N)
= A;GC~d.
(8) All the specific difference functions have a maximum at about 50°C (Table 2, Fig. 3). At higher temperatures they decrease and, as can be
1.64 1.2- ; 0,80.4-
6 c D
effect of the native protein
Using the surface areas of polar and non-polar groups exposed in the native structure (Table 1 of Privalov & Makhatadze, 1990), we calculated the hydration heat capacity effects for the native proteins (Table 2, Fig. 2). As might be expected, these effects are smaller than those for the unfolded polypeptide chains, because of the smaller exposed surfaces, especially of the non-polar groups, which are mostly buried in the protein interior in the native protein.
-1.21 -2.0
,
, 20
,
, 60
,
, 100
Temperature
I 140
,
, 180
PC)
Figure 3. The differences of the specific heat capacity effects of proteins in the unfolded and folded states, which correspond to the hydration effects of unfolding. The meaning of the lines is the same as in Fig. 1. A, myoglobin; B. cytochrome c; C, lysozyme; D, ribonuclease A. In the case of myoglobin and cytochrome c. the heme contribution is taken into account.
P. L. Privalov
720
and G. I. Makhatadze
Table 3 The partial heat capacities of the unfolded, C,(U), C,,(C),
native, C,(N), and compact, difference between the compact and the native states, AC,(N = C)
states and the heat capacity
Temperature 5
25
22.7 158 149 -@9
24.3 17.5 16.4 -1.1
25.8 19.7 17.9 - 1.8
241 195 18.0 -1.5
26.0 208 19.5 -1.3
27.8 225 20.9 -1.6
(“C)
50
75
Cytochrome
loo
125
c 26.3 21.9* 18.8 -3.1
26-8 240* 19.5 -4.5
268 2fi2* 202 -6.0
293 258* 22.4 -3.4
29.5 27.5* 23.0 -4.5
31.8 24.4* 22.0 -2.4
32.4 26.7* 22.8 -3.9
325 28.9* 237 -5.2
4@0 30.3* 27.0 -3.3
405 33.4* 28.1 -5-3
403 365* 29.2 -7.3
ttibonuclease 285 242* 21.7 -2.5
Lysozyme 26.7 18.2 172 -1.0
29.1 20.0 19.2 -0.8
31.1 2%2 21.0 -1.2
356 21.8 22.0 @2
37.6 242 23.9 -1.3
39.5 27.3 258 - 1.5
Myoglobin
C,(U)
C,(N) c&m AC&N-C)
* Extrapolated values. All heat capacity values
are in kJ K-’
per
mol
imagined, the total hydration effect falls to zero at about 25O”C, although the hydration effect of nonpolar groups decreases to zero at much higher temperatures. A remarkable feature of these specific functions is that their maximal height is very different for different proteins: it is the largest for Mb and the smallest for Rns. This difference is caused by the different contributions of non-polar groups to hydration effects, and by the different contributions of polar groups. It is notable that the larger the positive contribution of non-polar groups, the smaller the negative contribution of polar groups. This is understandable, because the total number of these groups in the unit volume of a protein molecule is almost constant, so that an increase in content of one type occurs at the expense of the other. The fact that non-polar and polar groups contribute in opposite ways to the heat capacity increment of protein unfolding was shown also for 25°C by Murphy & Gill, (1990), who measured the heat of dissolution of cyclic dipeptide crystals in water. (e) The heat capacity
of protein
in the compact
state
Excluding the estimated hydration heat capacity effect of protein unfolding from the observed partial heat capacity of unfolded protein, we will get the heat capacity of protein without internal bonds but not hydrated, i.e. the heat capacity of protein in the compact state: C,,(C) = C,(U) - A;&$? (9)
of protein.
The deviation of these values from the heat capacity values of the native protein should correspond, clearly, to the heat capacity effect of disruption of the internal bonds that maintain native protein structure and the increase of configurational freedom of the polypeptide chain. Therefore, the comparison of these two heat capacity functions, that of the compact state and the native state, is a matter of great interest, (Table 3). The heat capacity of the compact state is very similar to that of the native state. The difference between them at room temperature does not exceed 10% of the denaturation heat capacity increment and seems to be negative. Since this difference is on the limit of experimental accuracy, one can state only that the heat capacity of the native state is very close to, if not higher than, the heat capacity of the compact state. This is rather surprising, because it was always assumed that disruption of the internal non-covalent bonds that maintain the rigid native protein structure should lead to a significant 1977; of heat capacity (Sturtevant, increase Velicelebi & Sturtevant, 1979). The expectation that the heat capacity of protein in the compact state should be higher than in the native state was based on the assumption that the only result of disruption of internal bonds is an increase in the configurational freedom of protein groups. However, it is known that the heat capacities of many substances in the solid or liquid states are significantly higher than the heat capacities of these substances in the gaseous state (see e.g. Table
Heat Capacity
@fects on Protein
2.2-
c
i
1.8.
I.O-
O-6j 0
25
50 75 100 Temperature PC)
125
Figure 4. The partial specific heat capacity of proteins in the native (continuous line) and compact (broken line) states.
The bars show experimental
H, cytochrome
721
judging by the divergence values for the native protein
2’6/ T~
Uqfolding
error.
A, myoglobin;
c; C. lysozyme; D, ribonuclease A. In the
case of myoglobin and cytochrome tion is taken into account.
c, the heme contribu-
III of Privalov & Gill, 1989, or Table II of Livingstone et al., 1991). Their heat capacities are higher because of interactions between molecules, the fluctuation of which is intensified as temperature increases and thermal energy is accumulated. One can expect then that the heat capacity of a protein in the native state should also be higher than that in the compact state. Unlike the native protein, there is no fluctuation of interactions in the system of non-interacting components and, correspondingly, no energy consumption by the rise of fluctuations upon temperature increase, i.e. there vibrational modes in such a system are no “soft” that are excited at ordinary temperatures. The increase of thermal fluctuations on heating, the breathing of the native structure, manifests itself in many properties of protein, e.g. in the increase of hydrogen exchange rate and the decrease in the resolution of X-ray crystallographs with increasing temperature. The accumulation of thermal energy in fluctuations should certainly appear in the excess heat capacity of the native structure, and this excess heat capacity should increase with intensification of fluctuations at increasing temperature. However, disruption of the rigid native structure should also lead to an increase of configurational freedom and, consequently, to an increase of heat capacity. Since the heat capacity at room temperature of protein in the compact state is not larger than that in the native state, one can conclude that at this temperature the excess heat capacity associated with the configurational freedom increase upon protein unfolding is of the same magnitude as the effect of fluctuation of the native structure. At higher temperatures, the latter is likely to prevail,
of the extrapolated heat capacity (Fig. 4).
(f) Temperature dependence of the heat capacity effect of internal interactions in protein The heat capacity of protein in the native state can be measured in only a rather limited temperature range, in which the native state exists in the liquid aqueous solution. Even for proteins from extreme thermophiles, the upper limit of this range does not exceed 8O”C, but for the majority of globular proteins it is much lower, about 60°C. In this temperature range, the heat capacity of t,he native protein appears to be an almost linear function of temperature (Privalov et al., 1989). It is easiest to suppose that the heat’ capacity of native protein should be close to linear at higher temperatures as well. The main argument for this is that the absolute heat capacities of the anhydrous proteins, according to calorimetric measurements, are almost linear functions of temperature up to 100°C (Hutchens et al., 1969; Haly & Snaith, 1971; Roles & Wunderlich, 1991). At higher temperatures one can expect, according to the general theory of the heat capacity of solids, that it should have positive curvature because of the excit,ement of increased frequency modes. This positive curvature should be more pronounced in the presence of water, which labilizes the protein structure. On the other hand, the hydration heat capacity effect of the native protein is an increasing function of temperature with a slightly negative curvature (Fig. 2). These two curvatures could compensate for each other to some extent. In any case, one can hardly expect that the heat capacity function of the native protein would have significant negative curvature at high temperatures. Consequently, one can expect that the difference between heat capacities of the native and compact states would increase with increasing temperature. In other words, the heat capacity effect of native structure fluctuations is likely to increase faster with increasing temperature than the effect of configurational freedom increase. (g) Convergence
of the heat capacity to zero
increments
If the heat capacity of the native protein is a linear function of temperature, the overall heat capacity increment of protein unfolding should decrease to zero at much lower temperatures than the heat capacity effect associated with the hydration of groups that are exposed to water upon protein unfolding and, more so, the hydration of the non-polar groups (Fig. 5). Since the denaturation heat capacity increment was usually associated only with the hydration of non-polar groups, a temperature of about 150°C was regarded as the temperature above which non-polar groups do not affect the state of water (Privalov & Gill, 1989). We see now that the real situation is much more complex: the influence of non-polar groups on water persists to
722
P. L. Privalov
and G. I. Makhatadxe hydration of polar groups is significant in value and negative, then why was the heat capacity of protein unfolding found to be in such good correspondence with the effect of transfer of non-polar molecules from the liquid phase into water (Livingstone et al., 1991)? The reason is that the heat capacity of nonpolar liquid, which has been used as a standard state in this study, includes the effect of fluctuation of interactions between the molecules. This excess heat capacity effect significantly decreases the hydration effect, and brings the apparent hydration effect closer to the calorimetrically observed heat capacity effect of protein unfolding when normalized to corresponding surface area.
0
25
50
75
Temperature
100
125
PC)
Figure 5. The overall change of partial specific heat capacity of proteins upon unfolding in aqueous solution. The broken line corresponds to the part of function calculated by linear extrapolation of the native protein heat capacity. A, myoglobin; B, cytochrome c; C, lysozyme; D; ribonuclease A.
much higher temperatures and, at 15O”C, we have a situation where four different contributors to the heat capacity effect of protein unfolding (hydration of non-polar groups, hydration of polar groups, fluctuation of the native structure and increase of configurational freedom) compensate for each other. (h) Conclusion It follows from the above, that the denaturation heat capacity increment is due, to a great extent, to hydration of the groups that are exposed to water upon protein unfolding. However, both the nonpolar groups, as was supposed before, and the polar groups contribute to this effect. The contribution of these two types of groups is of opposite sign: while the hydration of non-polar groups increases the heat capacity, the hydration of polar groups decreases the heat capacity. The contribution of polar groups is smaller in absolute value than that of non-polar groups and, because of that, the overall heat capacity change upon unfolding is positive. This heat capacity effect correlates with the exposed surface area of non-polar groups in proteins (Privalov, 1979; Livingstone et al., 1991) but only because the relative surface area of polar groups that are exposed upon protein unfolding also correlates with the relative surface area of the non-polar groups, and because two other expected contributors to the heat capacity effect of protein unfolding (disruption of internal non-covalent bonds and increase of configurational freedom) are likely to compensate for each other to a great extent. However, if the contribution of these two effects to protein unfolding heat capacity is close to zero at and the contribution of room temperature,
We thank Drs R. Baldwin, J. A. Shellman and J. M. Sturtevant for helpful discussions of the manuscript in its preparation.
References Gill, S. J. & Wads& I. (1976). An equation of state describing hydrophobic interactions. Proc. Nat. Acad. Sci., U.S.A.
73, 2955-2958.
Haly, A. R. & Snaith, J. W. (1971). Calorimetry of rat tail tendon collagen before and after denaturation. Biopolymers, 10, 1681-1689. Hutchens, J. O., Cole, A. G. & Stout, J. W. (1969). Heat capacities from 11 to 305 K and entropies of hydrated and anhydrous bovine zinc insulin and chymotrypsinogen A. J. Biol. Chem. 244, 25-32. Kuwajima, K. (1989). The molten globule state as a clue for understanding the folding and cooperativity of globular protein structure. Proteins: Struck Funct. Genet. 6, 877103. Livingstone, J. R., Spolar, R. S. & Record, M. T. (1991). Contribution to the thermodynamics of protein folding from the reduction in water-accessible nonpolar surface area. Biochemistry, 30, 4237-4244. Makhatadze, G. I. & Privalov, P. L. (1989). Heat capacity of alcohols in aqueous solutions in the temperature range from 5 to 125°C. J. Sol. Chem. 18, 927-937. Makhatadze, G. I. & Privalov, P. L. (1990). Heat capacity of proteins. 1. Partial molar heat’capacity of individual amino acid residues in aqueous solution. J. Mol. Biol. 213, 375-384. Murphy, K. P. & Gill, S. J. (1990). Group additivity thermodynamics for dissolution of solid cyclic dipeptides into water. Thermochim. Acta, 172, 11-20. Naghibi, H., Dee, S. F. & Gill, S. J. (1986). Heats of solution of methane in water from 0 to 50°C. J. Phys. Chem. 90, 4621-4623. Naghibi, H., Dee, S. F. & Gill, S. J. (1987). Heats of solution of ethane and propane in water from 0 to 50°C. J. Phys. Chem. 91, 245-248. Privalov, P. I,. (1979). Protein stability. 1. Small globular proteins. Advan. Protein Chem. 33, 167-241. Privalov. P. L. (1989). Thermodynamic problems of protein structure. Annu. Rev. Biophys. Biophys. Chem. 18, 47-69. Privalov, P. L. & Gill, S. J. (1989). Stability of protein structure and hydrophobic interaction. Advan. Protein Chem. 39, 191-234. Privalov, P. L. & Makhatadze, G. I. (1990). Heat capacity of proteins. 11. Partial molar heat capacity of the unfolded polypeptide chain of proteins. J. Mol. Biol. 213, 385-391.
Heat Capacity Efects on Protein Unfolding Privalov, P. L., Venyaminov, S. Yu., Tiktopulo, E. I., Griko, Yu. V., Makhatadze, G. I. & Khechinashvili, N. N. (1989). Heat capacity and conformation of proteins in the denatured state. J. Mol. Biol. 205, 131-750. Ptitsyn, 0. B. (1987). Protein folding: hypotheses and experiments. J. Protein Chem. 6, 272-293. Roles K. A. & Wunderlich, B. (1991). Heat capacities of solid poly amino acids. Polyglycine, polyalanine and polyvaline. Biopolymers, 31, 477-481.
723
Spolar, R. S., Ha, J. & Record, M. T. (1989). Hydrophobic effect in protein folding and other noncovalent processes involving proteins. Proc. ,Vat. Acnd. Sci., U.S.A. 86, 8382-8385. Sturtevant, J. M. (1977). Heat capacity and entropy changes in processes involving proteins. Proc. Nat. Acad. Sci., U.S.A. 74, 2236-2240. Velicelebi, G. & Sturtevant, J. M. (1979). Thermodynamics of the denaturation of lysozyme in alcohol-water mixture. Biochemistry, 18, 1180-l 186.
Edited by R. Huber
Contribution of Hydration and Non-covalent Interactions the Heat Capacity Effect on Protein Unfolding
to
Peter L. Privalov and George I. Makhatadze Department and Institute
of Biology, The Johns Hopkins University Baltimore, MD 21218, U.S.A. of Protein Research of the USSR Academy of Sciences Puschino, USSR
(Received 25 July
1991; accepted 29 November 1991)
The heat capacity change upon protein unfolding has been analysed using the heat capacity data for the model compounds’ transfer into water, corrected for volume effects. It has been shown that in the unfolding, the heat capacity increment is contributed to by the effect of hydration of the non-polar groups, which is positive and decreases with temperature increase, and by the effect of hydration of the polar groups, which is negative and decreases in magnitude as temperature increases. The sum of these two effects is very close to the total heat capacity increment of protein unfolding at room temperature but is likely to deviat,e from it at higher temperatures. Therefore, the expected heat capacity effect caused by the increase of configurational freedom of the polypeptide chain upon unfolding seems to be compensated for by some other effect, perhaps associated with fluctuation of the native protein structure. Keywords: protein; unfolding;
heat capacity; hydration;
1. Introduction One of the most general specificities of globular protein unfolding is the significant heat capacity increase accompanying this process (for reviews, see Privalov, 1979, 1989). This heat capacity increment determines the temperature dependencies of all the thermodynamic characteristics of protein unfolding and, consequently, the stability of their native states. Therefore, an understanding of this effect is necessary for an understanding of the mechanism of the native protein structure stabilization. The heat capacity increment was thought to be caused by the hydration and the increase of configurational freedom of internal protein groups upon unfolding (Sturtevant, 1977; Privalov & Gill, 1989). According to indirect estimates, the increase of configurational freedom upon unfolding contributes about 30% of the observed heat capacity increment (Velicelebi & Sturtevant, 1979). We have shown (Privalov & Makhatadze, 1990) that hydration of groups that are exposed to water upon unfolding of the polypeptide chain comprises more than 70% of the observed heat capacity increment. This was done by summing all the heat capacity effects of the hydration of the protein interior groups, which were determined for model compounds (Makhatadze & Privalov, 1990). By heat capacity effect of 715 0022-2836/92/070715-09
$03.00/O
calorimetry
hydration, we meant a difference between the partial heat capacity of the molecule in aqueous solution and its heat capacity in the gaseous state, as it is conventionally accepted (Gill & Wads& 1976). Later, however, we realized that the gaseous state is not the appropriate standard state when considering the effect of hydration, because of the volume expansion effect, which is significant in gases. The liquid and solid states, which have been used in determining the hydration effects (Spolar et al., 1989; Livingstone et aZ., 1991; Murphy 6 Gill, 1990) are even less appropriate as standards of anhydrous non-polar solutes. The heat capacities of these condensed phases include considerable effects associated with fluctuation of interactions between the molecules, which cannot be taken into account without referring to the gaseous phase. The most appropriate standard state for the analysis of hydration effects in proteins appears to be a “compact” state of the unfolded polypeptide chain and, correspondingly, a compact phase of model compounds, which was introduced to analyse the hydrophobic interactions (Privalov & Gill, 1989). The compact state assumes the hypothetical state of a macroscopic system that has the same density as the condensed phase of this system, but all noncovalent interactions between its components are switched off. These components are the small 0
1992 Academic
Press Limited
716
P. L. Privalov
and G. I. Makhatadze
molecules (e.g. the non-polar molecules of benzene) if we are considering the system of these molecules, or the amino acid residues if we are considering a single protein molecule as a macroscopic system. According to the definition, the molecules of the surrounding medium (e.g. water molecules in the case of an aqueous solution of protein) do not penetrate into a compact system and are in contact with the same groups that are on the surface of protein in the native state. This is one of the principal features distinguishing the compact state of protein from the “molten globule” state. which has been widely discussed in the literature (for reviews, see Ptitsyn. 1987; Kuwajima. 1989). The other principal feature is t,hat. in the molten globule state, which is assumed to be maintained by the hydrophobic interactions maintains the elements of and secondary structure, not all internal non-covalent interactions are switched off. One should bear in mind that, while the molten globule is supposed to be a t’hermodynamically stable state of protein. the compact state is a hypothetical transient state, which has been suggested to take into account the volume effects and effects of non-covalent interactions in evaluating hydration upon protein unfolding. Makhatadze & Privalov (1990) and Privalov & Makhatadze (1990) showed that the heat, capacity effects of hydration of non-polar and of polar groups have different signs; while the hydration of nonpolar groups increases the partial heat capacity of proteins in aqueous solutions, the hydration of polar groups decreases it. Unfortunately, we did not separate these two opposite effects in considering the overall heat capacity change upon protein unfolding and, further, this point of paramount importance for understanding the thermodynamics of protein molecules has escaped the attention of many of our readers. Here, we consider all three components of the complex heat capacity effect of protein unfolding: the effects of hydration of non-polar groups; the hydration of polar groups; and disruption of the internal bonds that maintain the native structure; recalculated in accordance with the definition of hydration given above. For a demonstration, we have used four globular proteins for which the denaturation has been studied in detail (Privalov et al., 1989).
2. Method Privalov & Makhatadze (1990) and Makhatadze & Privalov (1990) determined the heat capacity effect of hydration according to the conventional definition as the difference between the partial heat capacity of the solute molecule in an aqueous solution and the heat capacity of this molecule in the gaseous state?: dchyd = c+ - CP (1) P P P’ Now we determine it as the difference between the partial heat capacity of a solute molecule in an aqueous solution and the heat capacity of this molecule in the compact
state.
(A. which
we take as a standard S(‘y
st,ate:
= ‘7; - p.
(4
The compact state differs from the gaseous state by the molar volume, which is the same for the considered substance as it is in the condensed, liquid-like state of this substance. On the other hand. the compact state differs from the condensed state by the absence of int,erac%iorr between the molecules (Privalov & Gill. 1989). Therefore, t’he heat capacity of the molecule in the cotnpac+ st)ate should differ from its heat capacity in the condrnsrd state by the thermal effects associated with the fiuctuation of molecules. and from that of’ interactions between molecules in the gaseous state by the term associated with volume expansion at constant pressure. To determine the heat c>apaGty of a molrculr in the compact state, let us consider the process of vapourization of a liquid. The enthalpy of this process can be presented in 2 terms, one representing the energy of int,eractiott between molecules. and the other representing the work on expansion of a substame from the condensed state to the gaseous state at normal pressure: A;H
= A;E + /‘A,p I’.
0)
Since the volume of a gas at normal pressure considerably exceeds that of the condensed state. one can neglect thr volume of a condensed state and. assuming that thr considered gas is close to ideal in the first approximation. i.e. PI’ = RT, write: AgI H = ACE I + R7’. Differentiating
this expression
(4)
by temperature
we get:
AY’ 1 P = A;(‘,+ R ()“a- (71 = (” - ( 1’ + fi P P P P or: (‘C = (‘g--R. P P Correspondingly, we will have:
for
the hydration
(5) heat
SChYd P = cg - q f R.
ca1)acit.v
effeclt (6)
It is known that the hydration
heat capacity effect for non-polar substances is proportional to the water-accesset al., ible surface area of the substance, ASA (Naghibi 1986, 1987). The ratio:
is the hydration heat capacity effect of the non-polar group of type i normalized to a surface area. It is similar for various types of non-polar groups, differing slightly for aliphatic and ring compounds (Makhatadze & Privalov. 1989). In the case of a molecule consisting of polar and nont AfH = Ha-H’, the enthalpy change upon transition from the liquid state to the gaseous state; A,FE = EC-E’, the energy change upon transition from the liquid state to the compact state; AfC, = CE-Cb, the heat capacity change upon transition from the liquid state to the gaseous state; Cg, the partial heat capacity of substance in solution; Ck, the heat capacity of substance in the liquid state; C$ the heat capacity of substance in the gaseous state; Cz, the heat capacity of substance in the compact state; &Zy, the heat capacity effect of hydration; ASA,, the water-accessible surface area of the group of type i.
Heat Capacity
Effects on Protein
Unfolding
717
Table 1 Hydration
heat capacity effect, @2,,ilhyd, per unit of surface-accessible various constituent groups of protein
Croup or side-chain of amino acid residue Non-polar: Aliphatic Aromatic Heme Polar parts of:
‘W -(:ONHp Met QS His Ser AM Asp Gin Glu Lys Tyr A% Thr
Temperature
area for
(“C)
5
25
50
75
190
125
2.30 1.36 507
2.22 1.23 475
2.10 1.14 440
1.98 1.05 406
1.88 @98 375
1.74 0.91 346
3.68 - 1.92 - 3.53 -353 -0.86 - 1.52 - 1.22 - 1.66 -634 -0-68 - 1.34 602 -637 - 1.03
3.78 - 1.64 -388 -388 - 1.28 - 1.30 -0.96 - 1.35 -019 - 0.52 - 1.57 608 -020 - 1.25
358 - 1.39 -4.10 -410 - 1.32 - 1.10 -962 - 1.01 - 0.03 - 0.32 - 1.62 0.05 -912 - 1.16
3.40 - 1.35 -408 - 4.08 - 1.26 -0.85 - 0.36 -065 611 -914 - 1.39 0.16 - 0.04 -083
3.15 - 1.30 - 3.95 -395 -1.13 -0.61 -0.10 -034 0.21 -001 - 1.19 0.35 901 -0.24
2.95 - 1.36 - 3.80 - 3%0 - 0.97 -637 0.15 - 0.05 633 612 - 0.99 0.50 ow 059
t The temperature dependence of the -NH group of the Trp side-chain Therefore its contribution was summed with non-polar groups. All heat capacity values are given in J K-’ molF’ A-‘.
polar parts, the normalized hydration effect of the polar part is determined by excluding the effect of the non-polar part from the total partial heat capacity of the molecule and dividing the difference by the surface area of the polar part. The values of partial heat capacities of all the groups constituting the polypeptide chain, the heat capacities of these groups in the gaseous state, and their ASA values have been reported (Makhatadze & Privalov, 1990). The normalized hydration heat capacity effects. corrected for thermal expansion, of all these groups are given in Table 1. It’ should be noted that experimental error in determination of the partial heat capacities of model compounds and proteins in aqueous solutions by scanning microcalorimeter is about 3%. The uncertainty in ASA values is rather large because of the uncertainty of van der Waals radii of atoms and the imperfection of computer programs (see Livingstone et aZ., 1991). However, since we are using the same radii and program in calculating the ASA values of model compounds and proteins, this uncertainty in ASA values is eliminated in the evaluated hydration heat capacity effects in proteins. Therefore, the overall uncertainty in these effects does not exceed 5%. Here, we consider the same 4 proteins that we analysed in our previous work. Native pancreatic ribonuclease A (Rns(N)t) and its unfolded polypeptide chain with t Abbreviations used: Rns(N), native pancreatic ribonuclease A; Lys(N), native hen egg-white lysozyme; Mb(N), native sperm-whale myoglobin; Cyt(N), native horse-heart cytochrome c; Rns(U, -ss), unfolded pancreatic ribonuclease A with reduced and carboxymethylated cysteine residues; Lys(U, -ss), unfolded hen egg-white lysozyme with reduced and carboxymethylated cysteine residues; Mb(U, apo), unfolded sperm-whale apo myoglobin; Cyt(U, ape), unfolded horse-heart apo cytochrome c.
behaves as non-polar
reduced and carboxymethylated cysteine residues (Rns(U, -ss)), native hen egg-white lysozyme (Lys(N)) and its unfolded polypeptide chain with reduced and carboxymethylated cysteine residues (Lys(U, ss)), native spermwhale myoglobin (Mb(N)) and its unfolded polypeptide chain without heme (Mb(U, ape)), and native horse heart cytochrome c (Cyt(N)) and its unfolded polypeptide chain without heme (Cyt(U, apo)).
3. Results and Discussion (a) The heat capacity
of proteins
denatured
in the unfolded
and
states
Privalov & Makhatadze (1990) compared the calorimetrically measured partial heat capacities of four proteins in the denatured state (Rns(U, -ss), Lys(U, -ss), Mb(U, apo) and Cyt(U, apo)) with the partial heat capacities of completely unfolded polypeptide chains of these proteins calculated from the partial heat capacities of individual amino acid residues, which we purposely measured (Makhatadze & Privalov, 1990). The close correspondence of these two sets of values for all proteins considered in a broad temperature range (from 5 to 125°C) showed that the denatured proteins in acidic solutions can be regarded as completely unfolded from a thermodynamic point of view. (b) Hydration effect of the unfolded polypeptide chain If the areas of the surfaces exposed to water of the non-polar, polar and charged groups of the unfolded polypeptide chains are known, and the heat capacity effects of hydration of these groups
P. L. Privalov
718
and G. I. Makhatadze
Table 2 Contribution
of the hydration
to the heat capacity unfolded states
of proteins
in the native and
Hydration heat capacity
increment (kJ/K mol)
Temperature ASA (AZ)
5
25
50 Cytochrome
(“C) 75
100
125
Polar Non-polar Total
2360 3580 5940
-3.5 7.7 4.2
-3.2 7.4 42
-2.8 7.0 4.1
c (12.3 kDa) -2.4 6.5 42
-21 6.2 4.1
Polar Non-polart Total?
6880 10,890 17,770 4520 7310 11,830
- 10.6 23.3 12.7
-95 22.2 12.7
- 8.3 21.0 12.6
- 7.4 19.7 123
-6.8 18.6 11.9
- 1.9 5.7 3.9 -6.3 17.3 11.0
-7.1 15.6 8.5
-6.3 149 8.5
-5.5 14.0 %5
-51 13.2 8.1
- 4.6 12.4 7.8
-44 11.5 7.1
Polar Non-polar Total
3360 3760 7120
-42 8.1 39
-3.7 7.8 40
-31 7.3 4.2
(13.6 kDa) -25 6.9 44
-21 65 45
Polar Non-polar Total
9330 11,120 20,450
- 141 235 9.4
- 12.6 22.5 9.9
- Il.0 21.2 l@O
-98 20.0 10.0
-8.7 18.9 10.0
Polar Non-polar Total
5970 7350 13,330
- 9.9 15.4 55
-8.9 147 54
-7.9 13.9 60
- 7.3 13.1 58
- 6.6 12.4 58
- 1.6 61 4.4 - 7.9 17.5 9.6 -6.2 11.5 52
Polar Non-polar Total
3530 3160 6690
-43 6.8 2.4
-3.6 6.5 2.9
Lysozyme - 3.0 6.1 3.2
(14.3 kDa) - 2.4 58 3.4
-21 55 34
-1.7 -.1 i.4
Polar Non-polar Total
9050 11,730 20,780
- 13.4 247 11.3
-11.6 23.7 12.0
-99 22.3 12.4
- 8.9 21.0 12.1
-8.0 19.9 11.9
- 7.4 18.4 11.0
Polar Non-polar Total
5520 8750 14,090
-9.0 17.9 8.9
- 8.0 17.1 92
-7.0 16.2 92 Myoglobin
-6.5 152 88
-6.0 14.4 8.4
-57 13.3 7.6
Polar Non-polar Total
3250 4620 7870
-40 9.6 56
- 3.5 9.0 5.5
-3.0 8.5 55
-pfj
-2.3 7.5 52
Polar Non-polart Total?
9910 16,210 26,120
-4.2 l@O 57 - 147 346 19.9
- 13.2 33.1 199
-11.4 31.2 19.8
10.3 29.3 19.1
- 9.4 278 18.4
Polar Non-polar? Total?
6660 11,590 18,250
-1@5 246 142
-9.2 23.5 14.3
-7.9 22.2 143
- 7.3 20.8 13.6
-6.8 197 13.0
Polar Non-polar? Total?
Ribonuclease
t Includes the heme group. All heat capacity values are precise within
(17.8 kDa) 8.0
54
-8.9 25.7 16.8 - 6.6 l&3 Il.7
5%
are known, one can determine the contribution of their hydration to the total partial heat capacity of the polypeptide chain. The corrected values of the normalized hydration heat capacity effects at different temperatures for all the groups constituting the polypeptide chain are given in Table 1. The surface area of the various groups of considered proteins in the native and unfolded states and the calculated values of the heat capacity effect associated with hydration of the non-polar and polar groups of the considered unfolded polypeptide chains are presented in Table 2. Figure 1 illustrates the temperature dependencies of these effects recalculated per gram of protein (the specific heat
capacity effects) to facilitate comparison of proteins that differ in size and in content of prosthetic groups. The values are corrected for the effect of carboxymethylation of cysteine residues in the cases of Rns(U, -ss) and Lys(U, -ss), and the absence of hemes in the cases of Mb(U, apo) and Cyt(U, ape). As seen, the positive heat capacity effect is provided only by the hydration of the non-polar groups, while the heat capacity effect of hydration of polar groups is negative at all temperatures. The remarkable feature of the hydration heat capacity effects of these two sets of groups is that, on temperature increase they both decrease in absolute value. The decrease of hydration effects
Heat Capacity IZfjects on Protein Unfolding
I .6
719
1
__ T
-”
- 0.8
-0.8
-1.2 I -20
6 20
60
100
Temperature
140
180
1
-I.:, Temperature
PC)
Figure 1. Specific heat capacity effects of hydration of the unfolded polypeptide chains. Continuous line, the total hydration effect; long broken line, the hydration effect of non-polar groups; broken line, the hydration effect of polar groups; short broken line, the extrapolation. The bars show the experimental error in heat
1°C)
Figure 2. Specific heat capacity effects of hydration of the native proteins. The meaning of the lines is the same as in Fig. 1. A, native myoglobin; B, native cytochrome c;
C, native lysozyme; D, native ribonuclease A.
capacity determination. A, unfolded apo myoglobin; B, unfolded apo cytochrome c; C, unfolded lysozyme with reduced and carboxymethylated cysteine residues; D, unfolded ribonuclease A with reduced and carboxymethylated cysteine residues.
with temperature proceeds monotonously and rather slowly. It appears that the hydration effect of the non-polar groups disappears much above 125”C, up to which temperature they were experimentally determined, perhaps, somewhere above 450°C as judged by extrapolations of experimentally determined functions. However, since the hydration effects of polar and non-polar groups have different signs, and they depend on temperature in different ways, their sum, which corresponds to the total hydration heat capacity effect, is represented by a curved function with a maximum at about 50°C. This shape of the hydration heat capacity effect seems to be the main cause of the calorimetrically observed curved shape of the partial heat capacity function of denatured proteins (Privalov et al., 1989).
(c) Hydration
(d) Hydration
effect of protein unfolding
Most important for understanding the thermodynamics of protein unfolding/refolding is the difference hydration effect of the native and unfolded states: KY(U)-SC?(N)
= A;GC~d.
(8) All the specific difference functions have a maximum at about 50°C (Table 2, Fig. 3). At higher temperatures they decrease and, as can be
1.64 1.2- ; 0,80.4-
6 c D
effect of the native protein
Using the surface areas of polar and non-polar groups exposed in the native structure (Table 1 of Privalov & Makhatadze, 1990), we calculated the hydration heat capacity effects for the native proteins (Table 2, Fig. 2). As might be expected, these effects are smaller than those for the unfolded polypeptide chains, because of the smaller exposed surfaces, especially of the non-polar groups, which are mostly buried in the protein interior in the native protein.
-1.21 -2.0
,
, 20
,
, 60
,
, 100
Temperature
I 140
,
, 180
PC)
Figure 3. The differences of the specific heat capacity effects of proteins in the unfolded and folded states, which correspond to the hydration effects of unfolding. The meaning of the lines is the same as in Fig. 1. A, myoglobin; B. cytochrome c; C, lysozyme; D, ribonuclease A. In the case of myoglobin and cytochrome c. the heme contribution is taken into account.
P. L. Privalov
720
and G. I. Makhatadze
Table 3 The partial heat capacities of the unfolded, C,(U), C,,(C),
native, C,(N), and compact, difference between the compact and the native states, AC,(N = C)
states and the heat capacity
Temperature 5
25
22.7 158 149 -@9
24.3 17.5 16.4 -1.1
25.8 19.7 17.9 - 1.8
241 195 18.0 -1.5
26.0 208 19.5 -1.3
27.8 225 20.9 -1.6
(“C)
50
75
Cytochrome
loo
125
c 26.3 21.9* 18.8 -3.1
26-8 240* 19.5 -4.5
268 2fi2* 202 -6.0
293 258* 22.4 -3.4
29.5 27.5* 23.0 -4.5
31.8 24.4* 22.0 -2.4
32.4 26.7* 22.8 -3.9
325 28.9* 237 -5.2
4@0 30.3* 27.0 -3.3
405 33.4* 28.1 -5-3
403 365* 29.2 -7.3
ttibonuclease 285 242* 21.7 -2.5
Lysozyme 26.7 18.2 172 -1.0
29.1 20.0 19.2 -0.8
31.1 2%2 21.0 -1.2
356 21.8 22.0 @2
37.6 242 23.9 -1.3
39.5 27.3 258 - 1.5
Myoglobin
C,(U)
C,(N) c&m AC&N-C)
* Extrapolated values. All heat capacity values
are in kJ K-’
per
mol
imagined, the total hydration effect falls to zero at about 25O”C, although the hydration effect of nonpolar groups decreases to zero at much higher temperatures. A remarkable feature of these specific functions is that their maximal height is very different for different proteins: it is the largest for Mb and the smallest for Rns. This difference is caused by the different contributions of non-polar groups to hydration effects, and by the different contributions of polar groups. It is notable that the larger the positive contribution of non-polar groups, the smaller the negative contribution of polar groups. This is understandable, because the total number of these groups in the unit volume of a protein molecule is almost constant, so that an increase in content of one type occurs at the expense of the other. The fact that non-polar and polar groups contribute in opposite ways to the heat capacity increment of protein unfolding was shown also for 25°C by Murphy & Gill, (1990), who measured the heat of dissolution of cyclic dipeptide crystals in water. (e) The heat capacity
of protein
in the compact
state
Excluding the estimated hydration heat capacity effect of protein unfolding from the observed partial heat capacity of unfolded protein, we will get the heat capacity of protein without internal bonds but not hydrated, i.e. the heat capacity of protein in the compact state: C,,(C) = C,(U) - A;&$? (9)
of protein.
The deviation of these values from the heat capacity values of the native protein should correspond, clearly, to the heat capacity effect of disruption of the internal bonds that maintain native protein structure and the increase of configurational freedom of the polypeptide chain. Therefore, the comparison of these two heat capacity functions, that of the compact state and the native state, is a matter of great interest, (Table 3). The heat capacity of the compact state is very similar to that of the native state. The difference between them at room temperature does not exceed 10% of the denaturation heat capacity increment and seems to be negative. Since this difference is on the limit of experimental accuracy, one can state only that the heat capacity of the native state is very close to, if not higher than, the heat capacity of the compact state. This is rather surprising, because it was always assumed that disruption of the internal non-covalent bonds that maintain the rigid native protein structure should lead to a significant 1977; of heat capacity (Sturtevant, increase Velicelebi & Sturtevant, 1979). The expectation that the heat capacity of protein in the compact state should be higher than in the native state was based on the assumption that the only result of disruption of internal bonds is an increase in the configurational freedom of protein groups. However, it is known that the heat capacities of many substances in the solid or liquid states are significantly higher than the heat capacities of these substances in the gaseous state (see e.g. Table
Heat Capacity
@fects on Protein
2.2-
c
i
1.8.
I.O-
O-6j 0
25
50 75 100 Temperature PC)
125
Figure 4. The partial specific heat capacity of proteins in the native (continuous line) and compact (broken line) states.
The bars show experimental
H, cytochrome
721
judging by the divergence values for the native protein
2’6/ T~
Uqfolding
error.
A, myoglobin;
c; C. lysozyme; D, ribonuclease A. In the
case of myoglobin and cytochrome tion is taken into account.
c, the heme contribu-
III of Privalov & Gill, 1989, or Table II of Livingstone et al., 1991). Their heat capacities are higher because of interactions between molecules, the fluctuation of which is intensified as temperature increases and thermal energy is accumulated. One can expect then that the heat capacity of a protein in the native state should also be higher than that in the compact state. Unlike the native protein, there is no fluctuation of interactions in the system of non-interacting components and, correspondingly, no energy consumption by the rise of fluctuations upon temperature increase, i.e. there vibrational modes in such a system are no “soft” that are excited at ordinary temperatures. The increase of thermal fluctuations on heating, the breathing of the native structure, manifests itself in many properties of protein, e.g. in the increase of hydrogen exchange rate and the decrease in the resolution of X-ray crystallographs with increasing temperature. The accumulation of thermal energy in fluctuations should certainly appear in the excess heat capacity of the native structure, and this excess heat capacity should increase with intensification of fluctuations at increasing temperature. However, disruption of the rigid native structure should also lead to an increase of configurational freedom and, consequently, to an increase of heat capacity. Since the heat capacity at room temperature of protein in the compact state is not larger than that in the native state, one can conclude that at this temperature the excess heat capacity associated with the configurational freedom increase upon protein unfolding is of the same magnitude as the effect of fluctuation of the native structure. At higher temperatures, the latter is likely to prevail,
of the extrapolated heat capacity (Fig. 4).
(f) Temperature dependence of the heat capacity effect of internal interactions in protein The heat capacity of protein in the native state can be measured in only a rather limited temperature range, in which the native state exists in the liquid aqueous solution. Even for proteins from extreme thermophiles, the upper limit of this range does not exceed 8O”C, but for the majority of globular proteins it is much lower, about 60°C. In this temperature range, the heat capacity of t,he native protein appears to be an almost linear function of temperature (Privalov et al., 1989). It is easiest to suppose that the heat’ capacity of native protein should be close to linear at higher temperatures as well. The main argument for this is that the absolute heat capacities of the anhydrous proteins, according to calorimetric measurements, are almost linear functions of temperature up to 100°C (Hutchens et al., 1969; Haly & Snaith, 1971; Roles & Wunderlich, 1991). At higher temperatures one can expect, according to the general theory of the heat capacity of solids, that it should have positive curvature because of the excit,ement of increased frequency modes. This positive curvature should be more pronounced in the presence of water, which labilizes the protein structure. On the other hand, the hydration heat capacity effect of the native protein is an increasing function of temperature with a slightly negative curvature (Fig. 2). These two curvatures could compensate for each other to some extent. In any case, one can hardly expect that the heat capacity function of the native protein would have significant negative curvature at high temperatures. Consequently, one can expect that the difference between heat capacities of the native and compact states would increase with increasing temperature. In other words, the heat capacity effect of native structure fluctuations is likely to increase faster with increasing temperature than the effect of configurational freedom increase. (g) Convergence
of the heat capacity to zero
increments
If the heat capacity of the native protein is a linear function of temperature, the overall heat capacity increment of protein unfolding should decrease to zero at much lower temperatures than the heat capacity effect associated with the hydration of groups that are exposed to water upon protein unfolding and, more so, the hydration of the non-polar groups (Fig. 5). Since the denaturation heat capacity increment was usually associated only with the hydration of non-polar groups, a temperature of about 150°C was regarded as the temperature above which non-polar groups do not affect the state of water (Privalov & Gill, 1989). We see now that the real situation is much more complex: the influence of non-polar groups on water persists to
722
P. L. Privalov
and G. I. Makhatadxe hydration of polar groups is significant in value and negative, then why was the heat capacity of protein unfolding found to be in such good correspondence with the effect of transfer of non-polar molecules from the liquid phase into water (Livingstone et al., 1991)? The reason is that the heat capacity of nonpolar liquid, which has been used as a standard state in this study, includes the effect of fluctuation of interactions between the molecules. This excess heat capacity effect significantly decreases the hydration effect, and brings the apparent hydration effect closer to the calorimetrically observed heat capacity effect of protein unfolding when normalized to corresponding surface area.
0
25
50
75
Temperature
100
125
PC)
Figure 5. The overall change of partial specific heat capacity of proteins upon unfolding in aqueous solution. The broken line corresponds to the part of function calculated by linear extrapolation of the native protein heat capacity. A, myoglobin; B, cytochrome c; C, lysozyme; D; ribonuclease A.
much higher temperatures and, at 15O”C, we have a situation where four different contributors to the heat capacity effect of protein unfolding (hydration of non-polar groups, hydration of polar groups, fluctuation of the native structure and increase of configurational freedom) compensate for each other. (h) Conclusion It follows from the above, that the denaturation heat capacity increment is due, to a great extent, to hydration of the groups that are exposed to water upon protein unfolding. However, both the nonpolar groups, as was supposed before, and the polar groups contribute to this effect. The contribution of these two types of groups is of opposite sign: while the hydration of non-polar groups increases the heat capacity, the hydration of polar groups decreases the heat capacity. The contribution of polar groups is smaller in absolute value than that of non-polar groups and, because of that, the overall heat capacity change upon unfolding is positive. This heat capacity effect correlates with the exposed surface area of non-polar groups in proteins (Privalov, 1979; Livingstone et al., 1991) but only because the relative surface area of polar groups that are exposed upon protein unfolding also correlates with the relative surface area of the non-polar groups, and because two other expected contributors to the heat capacity effect of protein unfolding (disruption of internal non-covalent bonds and increase of configurational freedom) are likely to compensate for each other to a great extent. However, if the contribution of these two effects to protein unfolding heat capacity is close to zero at and the contribution of room temperature,
We thank Drs R. Baldwin, J. A. Shellman and J. M. Sturtevant for helpful discussions of the manuscript in its preparation.
References Gill, S. J. & Wads& I. (1976). An equation of state describing hydrophobic interactions. Proc. Nat. Acad. Sci., U.S.A.
73, 2955-2958.
Haly, A. R. & Snaith, J. W. (1971). Calorimetry of rat tail tendon collagen before and after denaturation. Biopolymers, 10, 1681-1689. Hutchens, J. O., Cole, A. G. & Stout, J. W. (1969). Heat capacities from 11 to 305 K and entropies of hydrated and anhydrous bovine zinc insulin and chymotrypsinogen A. J. Biol. Chem. 244, 25-32. Kuwajima, K. (1989). The molten globule state as a clue for understanding the folding and cooperativity of globular protein structure. Proteins: Struck Funct. Genet. 6, 877103. Livingstone, J. R., Spolar, R. S. & Record, M. T. (1991). Contribution to the thermodynamics of protein folding from the reduction in water-accessible nonpolar surface area. Biochemistry, 30, 4237-4244. Makhatadze, G. I. & Privalov, P. L. (1989). Heat capacity of alcohols in aqueous solutions in the temperature range from 5 to 125°C. J. Sol. Chem. 18, 927-937. Makhatadze, G. I. & Privalov, P. L. (1990). Heat capacity of proteins. 1. Partial molar heat’capacity of individual amino acid residues in aqueous solution. J. Mol. Biol. 213, 375-384. Murphy, K. P. & Gill, S. J. (1990). Group additivity thermodynamics for dissolution of solid cyclic dipeptides into water. Thermochim. Acta, 172, 11-20. Naghibi, H., Dee, S. F. & Gill, S. J. (1986). Heats of solution of methane in water from 0 to 50°C. J. Phys. Chem. 90, 4621-4623. Naghibi, H., Dee, S. F. & Gill, S. J. (1987). Heats of solution of ethane and propane in water from 0 to 50°C. J. Phys. Chem. 91, 245-248. Privalov, P. I,. (1979). Protein stability. 1. Small globular proteins. Advan. Protein Chem. 33, 167-241. Privalov. P. L. (1989). Thermodynamic problems of protein structure. Annu. Rev. Biophys. Biophys. Chem. 18, 47-69. Privalov, P. L. & Gill, S. J. (1989). Stability of protein structure and hydrophobic interaction. Advan. Protein Chem. 39, 191-234. Privalov, P. L. & Makhatadze, G. I. (1990). Heat capacity of proteins. 11. Partial molar heat capacity of the unfolded polypeptide chain of proteins. J. Mol. Biol. 213, 385-391.
Heat Capacity Efects on Protein Unfolding Privalov, P. L., Venyaminov, S. Yu., Tiktopulo, E. I., Griko, Yu. V., Makhatadze, G. I. & Khechinashvili, N. N. (1989). Heat capacity and conformation of proteins in the denatured state. J. Mol. Biol. 205, 131-750. Ptitsyn, 0. B. (1987). Protein folding: hypotheses and experiments. J. Protein Chem. 6, 272-293. Roles K. A. & Wunderlich, B. (1991). Heat capacities of solid poly amino acids. Polyglycine, polyalanine and polyvaline. Biopolymers, 31, 477-481.
723
Spolar, R. S., Ha, J. & Record, M. T. (1989). Hydrophobic effect in protein folding and other noncovalent processes involving proteins. Proc. ,Vat. Acnd. Sci., U.S.A. 86, 8382-8385. Sturtevant, J. M. (1977). Heat capacity and entropy changes in processes involving proteins. Proc. Nat. Acad. Sci., U.S.A. 74, 2236-2240. Velicelebi, G. & Sturtevant, J. M. (1979). Thermodynamics of the denaturation of lysozyme in alcohol-water mixture. Biochemistry, 18, 1180-l 186.
Edited by R. Huber