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Heat capacity and conformation of proteins in the denatured state
J. Mol. Biol. (1989) 205, 731-750
Heat Capacity and Conformation of Proteins in the Denatured State P. L. Privalovl, E. I. Tiktopulol, S. Yu. Venyaminovl, Yu. V. Grikol, G. I. Makhatadzel and N. N. Khechinashvili’ ‘Institute of Protein Research and 21nstitute of Biological Physics, Academy of Sciences of the U.S.S.R., 142292 Pushchino, Moscow Region, U.S.S.R. (Received 20 July 1988, and in revised form 14 October 1988) Heat capacity, intrinsic viscosity and ellipticity of a number of globular proteins (pancreatic ribonuclease A, staphylococcal nuclease, hen egg-white lysozyme, myoglobin and cytochrome c) and a fibrillar protein (collagen) in various states (native, denatured, with and without disulfide crosslinks or a heme) have been studied experimentally over a broad range of temperatures. It is shown that the partial heat capacity of denatured protein significantly exceeds the heat capacity of native protein, especially in the case of globular proteins, and is close to the value calculated for an extended polypeptide chain from the known heat capacities of individual amino acid residues. The significant residual structure that appears at room temperature in the denatured states of some globular proteins (e.g. myoglobin and lysozyme) at neutral pH results in a slight decrease of the heat capacity, probably due to partial screening of the protein non-polar groups from water. The heat capacity of the unfolded state increases asymptotically, approaching a constant value at about 100°C. The temperature dependence of the heat capacity of the native state, which can be determined over a much shorter range of temperature than that of the denatured state and, correspondingly, is less certain, appears to be linear up to 80°C. Therefore, the denaturational heat capacity increment seems to be temperature-dependent and is likely to decrease to zero at about 140°C.
guanidinium hydrochloride (Gu * HCl) or urea, while a heat or acid-denatured protein contains too much residual structure to be regarded as a completely unfolded, unstructured state (see Tanford, 1968). This conventional view was shaken first when it was shown, by direct calorimetric measurements, that the enthalpies of lysozyme denaturation by heat and GuoHCl are identical if the denaturant solvation is correctly taken into account (Pfeil & Privalov, 1976a). It was found also that the partial heat capacities of the heat and GueHCl denatured lysozyme are indistinguishable. The importance of the partial heat capacity in specifying the denatured state of proteins follows from the fact that this parameter is a sensitive index of the completeness of protein unfolding. The exposure of the internal non-polar groups to water should result in a heat capacity increment, since the transfer of non-polar compounds to water is associated with a significant increase of the heat capacity (Kauzmann, 1959). On the other hand, the heat capacity is the temperature derivative of a basic thermodynamic function, the enthalpy Therefore, the denaturational heat capacity increment
1. Introduction The denatured states of proteins have attracted increasing attention. This interest arises from the realization that the denatured state is the only experimentally achievable state of a protein that can be taken as an initial reference in considering the mechanism of folding and stabilizing the native protein structure, However, a denatured state can play this role only if it can be associated with a completely unfolded, random coil conformation of polypeptide chain. The latter, as is clear, is an idealized model that is never realized in practice, since real polypeptides have too many groups interacting in very different ways with each other and with the solvent. However, it is unclear how far the real situation is from the ideal, and how large an error arises from equating a random coil with the denatured state, particularly when we estimate thermodynamic parameters for folding of the native protein structure. For many years it has been accepted that the best approximation of a random coil polypeptide is a denatured protein in concentrated solutions of 0022-2836/89/040737-14
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P. L. Privalov et al determines the temperature dependence of the enthalpy and, hence, of the entropy of denaturation, i.e. the parameters that determine the native state stability. The first more or less circumstantial study of the denaturational heat capacity increment of globular proteins was carried out by Privalov & Khechinashvili (1974), who showed that its value is likely to be specific for a given protein and does not depend on temperature. However, in that study, the heat capacity was measured over a limited temperature range from 20 to 90°C and calorimetric experiments were not accompanied by studies of the conformation of the denatured proteins. Therefore, one cannot exclude the possibility that heat capacity increment is caused by gradual melting of the residual structure upon increasing temperature and that, over a broader range of temperature, there will not appear some temperature dependence of the denaturational heat capacity increment on temperature. To clarify these points, it was necessary to make calorimetric measurements in a much broader range of temperature than had been done before, and to accompany the measurements with studies of protein conformation. Here, we report the results of a detailed calorimetric study of the heat capacity of a number of globular proteins in a temperature range from -5 to 13O”C, and the results of viscosimetric and spectropolarimetric studies of these proteins. Such an extension of the temperature range of calorimetric measurements became possible due to a new -model of the precision scanning microcalorimeter with capillary cells, which permits measurements under considerable excess pressure at temperatures above 100°C and scanning down the temperature scale to measure aqueous solutions in a supercooled state. We chose five globular proteins that are most popular among physical chemists; pancreatic ribonuclease A, staphylococcal nuclease, hen egg-white lysozyme, sperm whale myoglobin and horse heart cytochrome c. These proteins were examined with the disulfide crosslinks intact or disrupted and with the heme groups in place or removed (apo-form). The sixth globular protein used in this study was catalase from thermophilic micro-organisms, which was included for its extreme thermostability, in order to clarify aspects of the heat capacity of the native state. The seventh example, collagen, was chosen to represent a quite different class of fibrillar proteins.
2. Materials and Methods Metmyoglobin (Mb?) was isolated from frozen sperm whale muscle according to Hapner et al. (1968). The procedure for its further purification was as described by Privalov et al. (1986). Apomyoglobin (aMb) was obtained from sperm whale metmyoglobin by precipitation with acetone according to Rossi Fanelli et al. (1958). Cytochrome c (Cyt) was isolated from bovine heart as described by Margoliash & Walashek (1967).
Apocytochrome c (aCyt) was obtained according to Fisher et al. (1973). Ribonuclease A (RNase) was chromatographically purified from the commercial preparation of bovine pancreatic ribonuclease (“Reanal”, Hungary) using the procedure described by Crestfield et al. (1962). Hen egg-white lysozyme (Lys), a commercial preparation from “Reanal” (Hungary), was recrystallized 3 times before use. Staphylococcal nuclease (Nase) was isolated from a homogenate of Escherichia coli cells that had been transformed with a recombinant plasmid containing the gene for the enzyme by Dr Robert Fox (Yale University) and kindly provided for this study by Professor J. Sturtevant (Yale University). This construction results in the synthesis of a modified nuclease in which the heptapeptide Met-Asp-Pro-Thr-Val-Tyr-Ser is appended to the amino-terminal end of the nuclease; i.e. its molecular mass is 17,600 and not 16,807 Da (for a detailed description of this preparation, see Calderon et al., 1985). Catalase from Thermus therwwphilus (CTT) was a kind gift from Dr Barynin from the Institute of Crystallography of the USSR Academy of Sciences, who isolated it from micro-organisms and purified as described by Barynin & Grebenko (1986). Collagen from pike skin (Coll) was purified from an acid-soluble extract according to Glimcher et al. (1964). Glu-Lys-Lys-Leu-Glu-Gln-Ala heptapeptide The (Lys = lysine) was synthesized by Potekhin et al. (1988) in our Institute and kindly provided for our experiments. The purity of each preparation was checked electrophoretically in denaturing (Laemmli, 1970) and nondenaturing (Ornstein, 1964) conditions, which showed that the amount of impurities was less than 2%. Disulfide crosslinks in RNase and Lys were disrupted by reducing cysteine with thioglycolic acid in the presence of 8.5 M-urea at 20°C. The SH groups of cysteine residues were then carboxamidomethylated by iodoacetamide according to the method of Sela et al. (1959). The
t Abbreviations used: Mb, metmyoglobin; aMb, apomyoglobin; aMb”, apomyoglobin previously heated; Cyt, cytochrome c; aCyt, apocytochrome c; RNase, ribonuclease A; RNase-“, ribonuclease A with disrupted S-S crosslinks; Lys, hen egg-white lysozyme; Lys-“, hen egg-white lysozyme with disrupted S-S crosslinks; Nase, staphylococcal n&ease; C!l!T,
cat&se from Thrmua them$iZus;
Coil, collagen from pike
skin; c.d., circular dichroism; EH, 3ro helix; Gu, guanidinium. recorded apparent difference AC’“s(T) pl,aol,~lv, calorimetrically in the heat capacity of the protein solution and solvent. C (Thzo, spe cific heat capacity of water at temperature T. C$T) partial specific heat capacity of native protein. C$T)z: partial sp cific heat capacity of denatured protein. AC&, = q,af - Cv,s, denaturational increment of protein heat capacity. A$f(T) = HD(T) - HN(T), the enthalpy difference of the denatured and native states of protein. A$S(T)=SD(T)-SN(T), the entropy difference of the denatured and native states of protein. V(T),,, partial specific volume of the protein in solution at temperature T. V(T),,,, specific volume of water at temperature T. w(T),,, protein concentration in the solution at temperature
T.
intrinsic viscosity at temperature T. molecular elliptioity at the wavelength 2. molecular mass of an amino acid residue of a m.l, mean ’ protein. A, number of residues in the polypeptide chain of protein. $yd.ptein
739
Heat Capacity of Proteins amount of reduced SH groups was tested &a described by Boyer (1954). All the measurements were carried out in 10 mM-saltfree glycine or phosphate buffer solutions of acidic pH since, under these conditions, the proteins studied are most soluble and no aggregation is observed upon heating. The presence of aggregation was tested by highpressure liquid chromatography at various temperatures. After heating experiments, the preparation was checked electrophoretically under reducing conditions to ensure the absence of hydrolysis of the polypeptide chain at elevated temperatures. Prior to measurements, solutions were carefully dialyzed against solvent. Several replacements of the solvent were used to achieve complete equilibration of low molecular weight compounds in the solvent and solution used in differential measurements. After dialysis, solutions were centrifuged for 40 min at 24,000 g. Protein concentrations in solution were determined spectrophotometrically with correction for light-scattering (Winder & Gent, 1971). The extinction coefficients were found, in separate experiments, by measuring the nitrogen content in stock solutions according to Jaenicke (1974). For solutions of pH 2.0 to 5.0, the following extinction coefficients were used: Er&,., = 176 for Mb, 9.0 for aMb, 26.9 for intact Lys, 25.25 for Lys with reduced disulfides, 10.0 for CTT, 9.39 for Nase, 9.2 for aCyt, E:%,,snm = 7.32 for intact RNase, 7.25 for RNase with reduced disulfides, and Eaon, = 9-06 for Cyt. Calorimetric measurements were peformed using a DASM-4A capillary scanning microcalorimeter equipped with gold cells of 1.0 ml volume (Bureau of Biological Instrumentation of the Academy of Sciences of the USSR). To extend the heating range to 13O”C, all measurements were performed under an excess pressure of 506,625 Pa. For measurements below O”C, the solutions were supercooled as described earlier (Privalov et al., 1986). The heating rate was from 1.0 to 2.0 K min- ‘. The protein concentration in the solutions used for the experiments varied from I.0 to 5.0 mgml-‘. The partial specific heat capacity of the protein in the solution was determined as described by Privalov &, Potekhin (1986) from the calorimetrically recorded apparent difference in the heat capacity of the protein solution and solvent, ACBpPP(T)pr.sollsol, and the apparent difference in the heat capacities of the solvent and water, ACPpPpV)so,v,u~o:
where v(T) is the operational volume of the calorimetric cell at temperature T, w(T),, is the protein concentration in the cell at this temperature in gml-i, V(T)H20 and C,(T),,, are, respectively, the specific volume and specific heat capacity of water at this temperature and is the partial specific volume of protein at this UT),, temperature. For the specific volume and specific heat capacity of water, data tabulated in standard handbooks (Kell, 1971) were used. The partial specific volumes of proteins were determined from the density of the protein solution, P(T)~~.~~, and that of the solvent, P(T)~~~“, measured by a vibrational densimeter DMA-2 (Anton Paar, Austria), using the equation (Masterton, 1954):
WY,, = PICwm,rIH~ - ~P~~~,,.,,,l/~P~~~~O,“l (2) + b4m/wh,,4. It should be noted that, at the protein solution concentrations (20) studied, the values determined by eqns (1) and (2) do not show any concentration dependence and can be considered as corresponding to infinitely dilute solutions; i.e. Cp,pr = CEpr and VP, = VE (Fig. 1). The partial specific volume of the protein was determined over a temperature range of 2 to 8O”C, where it exhibited a linear temperature dependence (Fig. 2). For heat capacity determinations at higher temperatures, we used the extrapolated values of the partial specific volume. At the accuracy with which partial specific volumes could be determined in these experiments (*0403 cm3g-‘), no difference was noticed between the partial specific volumes of the native, denatured and completely unfolded forms of a protein at a given temperature. The solution viscosity was measured in an Ostwald viscosimeter with a water flow-time of about 200 s at 25°C and in a VBA-1 automatic rotational viscosimeter (Bureau of Biological Instrumentation of the USSR Academy of Sciences). A great advantage of the second instrument is that the protein solution does not contact air and is under an excess pressure. Therefore, the protein solution does not foam during experiments, as occurs in the usual capillary instruments at elevated temperatures. The protein intrinsic viscosity [q]r was determined by extrapolating the reduced viscosity determined from the solution flow-time (or period of rotation) t to zero concentration:
tvl~ = lim [@--WM W-+0
where the protein
T m 7 Y 2
20
-A+.--.-
& 1.5G -
d,-
concentration
(3)
w (in gml-‘)
and the
and LYS-‘~ (0)
in 10 mM-
7-----*---R-----+ #
-o-e-
AL?
o--o----o
I.O1 I.0
I
I 3.0
1
Concentration
Figure 1. Concentrational glycine (pH 2.5) at 25°C.
dependence of the partial
I 5.0
1
I 7.0
I
I 9.0
(mg mPl
specific heat capacity
of Lys (0)
740
P. L. Privalov et al.
0.65’
1 0
I
I 20
I
I 40
I 60
I
Temperature
I
I 80
PC)
Figure 2. Temperature dependence of the partial specific volume of Lys (O), RNase (n), Mb (A) and Cyt (0) in 10 mru-glycine (pH 2.5). Filled symbols correspond to proteins with disrupted S-S crosslinks and apo-forms. solvent flow-time to are measured at the same temperature, T. The extrapolation was done using at least 5 measurements on solutions with different concentrations over the range of 1 to 5 mg/ml-’ (Fig. 3). The circular dichroism spectra in the range of 183 to 320nm were measured with a Jasco-41A spectropolarimeter (Japan) in thermostated cells with a pathlength from 0.15 to 0.9 mm using solutions with -protein concentration varying from 0.2 to 2.0 mg ml . The molar ellipticity was determined as [e]pw = (4%+ ~aA/W) (4) where w is the protein concentration (in g ml-‘), 1 is the light pathlength in the cell (in mm), e1 is the measured ellipticity (in degrees) at a wavelength 1, and g,, is the mean molecular mass (in daltons) of an amino acid residue of a protein determined from the known sequence of the given protein. For the proteins studied, this quantity varied from 93 for collagen to 116 for apomyoglobin (for details of the experimental procedure, see Venyaminov & Gogia, 1982; Brzeska et al., 1983).
3. Results (a) Viscosimetric study As seen in Figure 3 for lysozyme with disrupted S-S crosslinks (Lys-““), the reduced viscosity of the unfolded polypeptide does not depend significantly on the protein concentration in solution at pH 2.2 at any temperature studied. This shows that, under the chosen solvent conditions, the interaction between protein molecules is rather low even in the
I
I
I.0
2.0 Concentration
Figure 3. Concentrational temperatures (“C).
dependence
unfolded conformation. This is a very important circumstance for further interpretation of all the experimental results obtained in this study. The intrinsic viscosity of the native protein at 10°C is low and is almost independent of pH throughout the pH region in which this protein is in the native state (Fig. 4). For Lys and RN&se with intact
S-S crosslinks,
the native
state
is realized
throughout the entire acidic pH range at 10°C. However, in the case of much less stable proteins that denature at acidic pH values, the intrinsic viscosity
increases
considerably
at decreasing
pH.
The observed denaturational increment of intrinsic viscosity is especially large in cases where the polypeptide chain is not crosslinked by disulfide bonds, as occurs for Mb. Upon disruption of S-S crosslinks, intrinsic viscosities of Lys and RNase increase at low pH to the same extent as that of Nase, Mb and Cyt. It is notable that with increasing pH, the intrinsic viscosities of these polypeptide chains decrease, though not to the level of the native compact proteins. A similar situation is observed with previously heated apomyoglobin @Mb”), which does not regain its native-like compact structure (Griko et aZ., 1988), but its intrinsic viscosity drops to a remarkably low level at neutral pH values. It appears that heating of aMb to 100°C leads to such a chemical modification of some of its amino acid residues that its
polypeptide structure 1986).
I
3.0 (mg
of the reduced viscosity
chain (on this
cannot
fold into a native see Zale & Klibanov,
aspect,
I
I
4.0
5.0
ml-‘)
of Lys e-S’ in 10 mM-glycine
(pH 25) at various
741
Heat Capacity of Proteins
Table 1
-I
(a)
Intrinsic viscosity of unfolded proteins at 25°C (RNase and Lys without S-S crosslinks and Mb and Cyt without the heme in solutions with pH 2.2 and 6 M-Cu. HCI) [11
rtl1t
11,702 104 13,683 124 14,396 129
15.0 15.6 17.0
16.1 17.6
17,170
20.0
20.1
W’c
pH 2.2 6 M-Gu.HCI for random coil 12 (cm’ g-l)
Protein acyt RNase-‘”
153
149 16.6 17.0 19.1
M, molecular mass; 12,number of amino acid residues; [q], intrinsic viscosity; [q]c”c, values for the random coiled polypeptide calculated according to Tanford (1968) by eqn (5). t Values from Ahmad & Salahuddin (1974).
last column of Table 1. As is seen, the experimental values
1
0'
,
I
I
I
I
4.0
2.0
6.0
PH
Figure 4. pH dependence of the intrinsic viscosity of proteins at 10°C. (a) Lys and RNaae with intact and disrupted S-S crosslinks and aCyt; (b) Mb with the heme groups in place and removed. Symbols: Lys (O), Lys-” (01, R&se (Cl), RNase-w (ml, Cyt (01, aCyt (+), Mb (AL aMb (A), aMbh (A).
With decreasing pH, the intrinsic viscosity of a non-crosslinked protein rises to some constant level, which is specific for a given protein at a given
temperature and ionic strength of the solution. These maximal values of intrinsic viscosity, which are achieved at pH below 3-O at 25”C, are summarized in Table 1 for RNase and Lys without SS crosslinks and for the apo-forms of Mb and Cyt. The Table presents values for the intrinsic viscosity of the same proteins in 6 M-Gu.HCl solution obtained by Ahmad & Salahuddin (1974). As can be seen, they
are in rather
good correspondence
of
intrinsic
viscosity
of
the
considered
polypeptide chains without S-S crosslinks or a heme are rather close to the value calculated for a random coiled polypeptide. Thus, one can suppose that in solution at pH 2.0, the polypeptide chains of all the proteins studied are in a random coil conformation at 25°C if they are not fixed by S-S crosslinks. An increase of temperature leads to a significant decrease of the intrinsic viscosity of the unfolded polypeptide chain (Fig. 5). The effect of temperature on the intrinsic viscosity of unfolded polypeptide chains was observed by Ahmad & Salahuddin (1974) for proteins in concentrated solutions of Gu.HCl; but, in that case, the temperature influence is even more pronounced than in the case where unfolding of the polypeptide chain is caused by extreme pH, i.e. by the charges on the polypeptide chain. The temperature influence on the intrinsic viscosity of the unfolded polypeptide chain can be
with
our values obtained in acidic solutions without a denaturant. There is a clear correlation of intrinsic viscosity values with the molecular weight of the corresponding protein or with the number of amino acid residues in the polypeptide chain. According to Tanford (1968), the intrinsic viscosity of the polypeptide chain in a random coil conformation at 25°C can be expressed as: [q] = 77.0 n”.666/iiZ5.r,
(5) where iii,, is the mean molecular mass of an amino acid residue and n is the number of residues in the polypeptide chain. The values of intrinsic viscosity calculated bv eauation (5) frnlcslc) are given in the Y
1
T
I
\L
,J
I
0
Figure 5. Temperature viscosity pH 4.0; and of pH 3.0: symbols
dependence of the intrinsic of proteins: (1) RNase, pH 3.0; (2) RNase, (3) Lys, pH 3.0; (4) Mb, pH 5.0; (5) Lys, pH 4.0; unfolded polypeptide chains: (6) aMb and Mb, (7) Lys-““, pH 2.2; (8) RNase-=, pH 2.2. The are the same as for Fig. 4.
742
P. L. Privalov
explained by the increase of flexibility of the chain caused by the increase of rotational freedom of backbones at increasing temperature. Clearly, increased flexibility of the chain should lead to a decrease in the hydrodynamic volume occupied by this chain (Ahmad & Salahuddin, 1974). The decrease of the hydrodynamic volume of the coil might be caused also by an increase of the hydrophobic pressure; i.e. the random hydrophobic interactions between the non-polar groups of a chain, since the hydrophobic interactions are known to increase at increasing temperature (Privalov et al., 1986).
It follows from the above that the conformation of a heat-denatured protein does not differ greatly from that of a random coil, as it is supposed to be when the intrinsic viscosity of a heat-denatured protein as measured at 80°C is compared with that of Gu.HCl-denatured protein measured at 25°C. Judging by Figure 5, the intrinsic viscosity of a random coil polypeptide chain at 80°C is only twice as large as that of the corresponding heat-denatured protein with intact S-S crosslinks, while for proteins without S-S crosslinks, such as Mb, the intrinsic viscosity of a
-15
’ ,‘.j,
et al.
heat-denatured state is almost the same as that of the random coil state. (b) Circular
dichroism study
Figure 6 presents c.d. spectra of the studied proteins in various states; native, acid-denatured, heat-denatured and denatured by GuaHCl. The spectra for all proteins in the concentrated solution of GueHCl are similar to those found by Dearborn & Wetlaufer (1970), Tiffany 6 Krimm (1973) and Cortijo et al. (1973). The spectra of heat-denatured proteins are rather close to the spectra of unfolded polypeptide chains without disulfide crosslinks or heme at acidic pH, but deviate significantly from the spectra of proteins in 6 M-Gu.HCl. This deviation is especially clear at 222 nm, lO”C, but with a temperature increase it decreases and is likely to disappear above 80°C (Fig. 7). It is notable that the deviation decreases due to a considerable temperature dependence of the ellipticity of protein in the presence of Gu. HCl. The ellipticity of polypeptide chain at 222 nm is usually considered as an index of its secondary
i 200
, Cyt., pH , 3.0 , 220 240
,
, 200
I
Collagen, , ( pH, 3.5 ( 220 240
Wavelength km)
Figure 6. Circular dichroism spectra of proteins in the native state at 10°C (continuous line) and temperaturedenatured state at 80°C (dot-and-dash line), both at the pH indicated in the boxes; in the acid-unfolded state with disrupted S-S crosslinks or removed heme, at 10% pH 2.5 (dashed line), and in the presence of 6 M-GU . HCl at 10°C (dotted line).
743
Heat Capacity of Proteins
o
,+ 2.0
Figure 7. Dependence of the ellipticity
(
, 4.0 PH
,
, 6.0
I
,
40 Temperature
60
1 43 . . . . . . . ,...‘..’ 20
,
,
/ 60
PC)
of proteins at 222 nm on (a) pH at 10°C and (b) on temperature
at various pH
values. The symbols are the same as for Fig. 4. The crossed symbol corresponds to the results in the presence of 6 MGu.HCl. structure (Chen et al., 1972). If one accepts that the polypeptide chain is in a completely random coiled conformation in a concentrated solution of Gu . HCl (see Tanford, 1968), then it follows from Figures 6 and 7 that heat-denatured protein has considerable but its content residual secondary structure decreases upon a temperature increase and practically disappears at about 100°C. However, the question is whether one can use the cd. spectra of proteins in concentrated solutions of Gu.HCl as a reference for the random coil conformation of the polypeptide chain? Our experiments with short synthetic polypeptides that could hardly have any helical conformation in aqueous solutions showed that the presence of Gu . HCl induces significant changes in ellipticity (Fig. 8). This is not unexpected, as it is known that Gu * HCI interacts with peptide groups with a large negative enthalopy (Lee & Timashelf), 1974; Pfeil & Privalov, 1976a). According to Robinson & Jencks (1965), one molecule of Gu . HCl links with two peptide bonds; therefore, one should expect that it can affect the conformation of the polypeptide group. According to Tiffany & Krim (1972, 1973), the high concentration of Gu . HCl and urea favors the locally extended 3,,-helix conformation (EH) of polypeptides. If so, the
effect of temperature on the ellipticity of proteins in concentrated Gu * HCl solutions could be explained by the decrease of the content of the EH conformation at a temperature increase. This seems quite likely, as increasing temperature should lead to desolvation of Gu * HCl bound to polypeptide if its binding enthalpy is negative. It follows then, that proteins in concentrated solutions of Gu * HCI attain a random coil conformation only at a rather high temperature. Another possibility that should not be excluded is that GuaHCl can influence the intrinsic optical properties of the individual amino acid residues, and this influence is temperature-dependent (see Hvidt et al., 1985). In this case, the observed optical effect cannot be interpreted in conformation terms. The above leads us to conclude that the conformation of protein denatured by Gu. HCI or acid is certainly close to a random coil only at temperatures about 100°C. At lower temperatures, there is some difference in the ellipticities of the Gu*HCl and acid-unfolded polypeptides, which in some cases amounts to 5666 deg cm2 dmol-‘; that is, to about 15% of the value specific for the 166% a-helix. However, this difference is hardly interpretable with certainty as evidence for the residual helicity in the acid-unfolded polypeptide chain,
744
P. L. Privalov et al.
0
20
40 60 80 Temperature PC)
10°C v
,
200
(
,
220 Wovelength
,
240
(nm)
Figure 8. Ellipticity of the heptapeptide Glu-Lys-Lys-Leu-Glu-Gln-Ala (Lys = lysine) in 100 mM-sodium phosphate, pH 7-O (continuous line) and in the presence of 6 M-GU. HCl (dotted line) at different temperatures.
since the nature of this systematic difference in the ellipticities is unclear. Therefore, in considering the conformation of a polypeptide chain we can only neglect this difference (especially when it is small) and suppose that the conformation of the acid and GueHCl-unfolded polypeptide chains are both close to a random coil. For some of polypeptide chains, such as RNase-“” and aCyt, the random-coil conformation is likely to persist at higher pH up to 7.0 (Fig. 7). In the case of Nase, whose unfolding and folding is a perfectly reversible process, an increase of pH leads to folding of its polypeptide chain into the native structure. The increase of pH leads to an increase of negative ellipticity at 222 nm of Lys-“’ and aMb. If lOOo/o a-helicity disposes the ellipticity of 34,500 deg cm2 dmol- ’ at 222 nm, as reported by Chen et al. (1972), then one can suppose that at pH 5.0 the content of u-helicity reaches 8% in Lys-“’ and 25% in preliminarily heated aMb’. This increase of the a-helicity is accompanied by a decrease of the intrinsic viscosity of these polypeptide chains (see Fig. 4). It is remarkable with increasing that, temperature, the high ellipticity of Lys-“” drops to the initial value within a temperature range from 30°C to 70°C (Fig. 7). In contrast to Lys-“, the ellipticity of the preliminarily heated aMb6 is not very sensitive to temperature. Thus, it does not
seem to be associated operative structure.
with
(c) Calorimetric
an extended
co-
study
Figure 9 represents the partial specific heat capacity of the proteins studied here in solutions with various pH values as determined calorimetrically over the temperature range -5°C to 130°C. To expand the y-axis, we have omitted the tops of the denaturational heat absorption peaks as, in this paper, we are not interested in the denaturational process itself, but in the heat capacities of the native and denatured states. As can be seen, increasing pH shifts the denaturation temperatures to higher values, but does not noticeably change either the heat capacity of the native state or the heat capacity of the denatured state. These two quantities are equal to within the experimental error of kO.04 J K-r g- 1 with only two exceptions (considered below). However, the most remarkable observation is that RNase and Lys without S-S crosslinks in solutions of pH below 3.0, in which they are in completely unfolded, random coil conformations, have the same heat capacity as the denatured states of these proteins with intact S-S crosslinks. A similar situation is observed with Mb and Cyt; in solutions with pH below 3.0, the denatured states of
745
Heat Capacity of Proteins
RNase
Mh
2Jz#Y-I-’
14
0
I
20
I
Nase I
40
60
Temperature
I
80
I
too
/
120
PC)
Figure 9. Temperature dependence of the partial specific heat capacity of proteins with intact and disrupted disulfide crosslinks and with or without the heme, in solutions with different pH values (indicated on the curves).
these molecules have the same heat capacities as the apo-forms of these proteins, which, according to viscosimetric and c.d. studies, are in the unfolded conformation close to a random coil. The influence of pH on the heat capacity of the denatured state is observed only for Lys-“” and aMbh. As shown above, with increasing pH above 4.0, both these polypeptides, and especially aMbh, form structures that are characterized by considerable helicity. Upon heating Lys-“” in pH 5.0 solution, its additional helicity disappears over the temperature range 30°C to 70°C (Fig. 7) and, in the same temperature range, an extra heat absorption is observed that appears as a broad and flat peak, shown by a dotted line in Figure 8. The area of this peak is about 5 Jg-’ (70 kJ mol-‘), which is less of the enthalpy of lysozyme than 20% denaturation. The van? Hoff enthalpy, determined from the breadth of this peak, is of the same order. This shows that the structure formed by Lys-‘” at pH 5.0 is co-operative. From this point of view, this structure resembles the native structure of lysozyme with intact crosslinks, the more so because they are also comparable in ellipticity. However, these two structures differ essentially in the enthalpy of formation and in heat capacity. The heat capacity of Lys-” at pH 5-O and 25”C, where temperature-induced melting of its structure does not occur yet judging by its ellipticity, is almost the same as that of Lys-” at pH 2.5, which is in a
completely unfolded state. Therefore, one can suppose that the structure formed by Lys-” at pH values close to neutral is quite open. This is confirmed by viscosimetric studies, which show that the intrinsic viscosity of Lys-“” at pH 5-O is about 10. In contrast to the structure formed by Lys-“‘, the structure formed by the preliminarily heated aMbh at pH above 4.0, as mentioned in the previous does not change significantly with section, increasing temperature (Fig. 7). Correspondingly, we do not observe a clear heat effect that can be interpreted as additional heat absorption, although there is some difference between the heat capacity functions found at pH 5.0 and pH 2.5 (Fig. 9). This difference amounts to O-1 J K-‘g-’ at a low temperature (i.e. is about a 5th of the heat capacity difference between the native and denatured states) and gradually disappears upon heating from 40°C to 70°C. In all cases, the heat capacities of the native protein are much lower than that of the denatured protein, and they differ considerably, providing a large variation between the values of the denaturaE;nal heat capacity increment A$?,,,, = for the proteins studied here (Table 2). P-PI - CF,PI The A% N p,pr values obtained at 25°C agree well with previous findings (Privalov & Khechinashvili, 1974). It is remarkable that the temperature dependences of the heat capacity of all the proteins Table 2 Partial speci$c capacities of proteins in native (N) and denatured (D) states at 25°C measured calorimetrically (exp) and calculated (talc) for the unfolded polypeptide chain (U) from the heat capacities of constituting amino acid residues Protein, condition
Cr.,, (exp)t
Nase, pH 7.0 pH 2.0-3.0 RNaN!, pH 4.0 RNaWZ-““, pH 2.fS5.0 LYS, pH 2Xk5.0 LysC”, pH 2.0-4.0 Mb, pH 4+6.0 pH 2.C3.0 aMb, pH 5.Ck6.0 pH 2.0-4.0 Cyt, pH 3.0-6.0 aCyt, pH 2G6.0 Coll, pH 3.0 f c,.,r in JK-‘g-l.
CLpr (exp)t
c;,r
(calcH
1.45 1.90
2.15
1.90
1.85
1.88
1.96
1.52
1.40
1.36 1.98 1.60
1.90 2.02
-
1.42
1.35
2.28
2.03
2.22
1.55
1.58
746
P. L. Privalov et al.
40
60 80 Temperature PC)
100
120
Figure 10. Temperature dependence of the heat capacity of RNase (pH 4.0), Lys (pH 3.5), Mb (pH 4.4), C!l!T (pH 8.5) and RNaseWsS,Lys-“, aMb ai pH 2.5. studied here in the completely unfolded state are represented by rather similar functions. Over the broad temperature range of - 5°C to 13O”C, this function does not appear linear, as it appeared when examined over the smaller temperature range from 20°C to 90°C (Privalov t Khechinashvili, 1974). For the native protein, one cannot draw the same conclusion, since the temperature range over which the native state can exist is much smaller. Even for such a thermostable protein as catalase from thermophilic micro-organisms (CTT), the pure native state can be observed with certainty only up to 80°C (Fig. 10).
4. Discussion As has been shown above, the polypeptide chain of protein without S-S crosslinks and the heme in acidic solutions with pH below 3-O appears to be in an unfolded state with little, if any, residual structure. An increase in temperature leads to some squeezing of the hydrodynamic volume of an unfolded polypeptide chain, but this does not at all imply the formation of a compact structure. On the contrary, temperature-induced squeezing appears to be a general property of all polymers and, at high temperature, the conformation of a polypeptide chain is likely to be even closer to a random coil judging by its ellipticity. With increasing pH, polypeptide chains fold into a structure that is characterized by some helicity (Fig. 7). In some cases this is a compact, cooperative native structure (as in the cases of Nase and aMb); in other instances, this is a not quite compact, but co-operative structure (as in the case of Lys-““); and in some cases it is neither compact nor co-operative, as with the preliminary heated apomyoglobin, aMb”. In the latter case, the enthalpy of the structure formation is found to be close to zero (Griko et al., 1988). Therefore, temperature does not affect this structure greatly.
Crosslinking of a polypeptide chain by disulfide bonds results in a significant decrease of its intrinsic viscosity, i.e. of the hydrodynamic volume occupied by this chain, as expected. However, this does not mean in itself that a crosslinked polypeptide chain has a significant amount of residual structure. Indeed, the ellipticity of heat-denatured Lys and RNase with intact S-S crosslinks is very close to the ellipticity of these polypeptide chains without S-S crosslinks, which are in a completely unfolded state, and, upon temperature increase, these two values of ellipticity become closer (Fig. 7). Thus, it appears that, at a sufficiently high temperature (above SO’%), the conformation of the heatdenatured protein in acidic solutions is rather close to a random coil (i.e. crosslinked random coil). Before discussing our experimental results on the heat capacity of proteins, it is interesting to consider what should be expected for the heat capacity of an extended polypeptide chain in which all the groups are in perfect contact with water, and in which no residual structure is supposed to be present. Let us assume, in the first approximation, that each amino acid residue contributes additively to the integral heat capacity of such a chain as if these residues have an identical peptide group (CHCONH) and differ in the side-chains (R), and there are two terminal groups (NH,) and (CHCOOH) in the chain. The integral heat capacity of the extended polypeptide can be represented as: _ c C ca1c p,NH2 + (n~~‘P,CHCONH P>PI + f
i=l
‘p,r
+
cp,C”COOH.
(6)
The partial heat capacities of all these groups were calculated from the known partial heat capacities of model compounds in dilute aqueous solutions, and for some of the groups they were determined by direct calorimetric measurements of the heat capacity of tripeptide Gly-X-Gly in solution (Makhatadze, Medvedkin & Privalov, unpublished results). The heat capacity values used are listed in
Heat Capacity of Proteins Table 3 Partial heat capacities C,,i of polypeptide groups
Group NH, CHCOOH CHCONH R WY) R (Ala) R (Val) R (He) R (Phe) R (TY~) R (Ser) R (Thr) R (Asp) R (Am) R (Glu) R (Gln) R (LYs) R (I-) R (Pro) R (HYP) R IMet) ~ R (His) R (Trp) R (Ax) R (CY#I
Procedure for calculating 6,
Analog t
ql,CH+ c, co + C,,,,
CH,CONHf Hydrogen?’ Methane5 Propane§ 1-Butane§ Methylhenzene§ 4-MethylphenoQ Methanol5 Ethanols Acetic acid5 Acetamide$ Propionic acids Propionamide§ n-Butylamine§
Calorimetric(I
Gly-X-Gig G&X-Glj Glv-X-Glv GI;-X-G6 Gly-X-Gly n-Propylguanidinefr (:p,t.,e,- q,.cH
Calorimetric~~ CalorimetricI] CalorimetricI/ Calorimetric/l CalorimetricI\
c -c CT;::: - c;::: CPA - ql,” %A-c,,, GA- C&H C&A - C,,” GA-c,., %A- C,.” CPA -c&H %A- C pH (I,,* - c,:, ~P.A -(Jim G.“,, + %,H
%4
-
(kH
+ %co + CF.,,”
Heat capacities of groups CH,. CH, NH,, were taken from Gutrie (1977). t Gutrie (1977). $ Jolicoeur & Boileau (1978). 4 Cabani et al. (1981). 1)Makhatadze et al., unpublished. 7 Carboxymethylated.
-5 36 19 75 168 293 395 355 309 83 183 90 85 178 179 347 377 175 168 173 176 332 266 17
CO, OH, 0 and H
Table 3, which gives also the model compounds together with corresponding references to their heat capacity values and to the procedure used to derive the heat capacity of a group from the heat capacity of an analog. The values of the integral heat capacity of extended polypeptide chains, calculated per gram of protein, are given in Table 2. As can be seen, they agree well with the experimental values of the heat capacity of the denatured protein, but are somewhat larger in all cases. The deviation, which does not exceed lo%, is not surprising, as the polypeptide chain, even in the random coil conformation, can hardly be considered as a dilute solution of the constituent groups. It was expected that a heat-denatured protein, which was supposed to be not as unfolded as the Gu.HCl-denatured protein, should have a lesser heat capacity than the protein in a concentrated solution of Gu. HCl (Tanford & Aune, 1970). However, direct calorimetric measurements carried out on lysozyme, showed that, if the GueHCl solvation effects are correctly taken into account, no difference between the Gu.HCl and heatdenatured protein heat capacities can be observed (Pfeil & Privalov, 1976a). Now we see that even a very extended residual structure, which persists in the preliminarily heated aMbh, at pH values close
747
to neutral, does not affect significantly the heat capacity function. Its deviation from the function for a fully unfolded polypeptide does not exceed 7% of the value of the specific heat capacity of the protein, i.e. is less than 20% of the heat capacity difference between the native and denatured states of a protein. It follows then, that the denaturational heat capacity increment cannot be explained by a gradual melting of the residual structure in a denatured protein. It cannot be explained either by the increase of the configurational freedom of the polypeptide chain upon the “disruption” of the According to structure. protein rigid native Sturtevant (1977) and Velicelebi & Sturtevant (1979), the contribution of this effect to the observed denaturational increment of the protein heat capacity cannot be large. This was shown experimentally by measuring the heat capacity change upon melting of membranes, which can be considered as a model of protein denaturation without penetration of water inside the molecule, i.e. without unfolding of the hydrophobic core, but with a significant increase of the configurational freedom (Bendzko et al., 1988). The inconsistency of the explanation of the protein denaturation heat capacity increment by the decrease of the configurational freedom becomes especially evident in considering the cold denaturation phenomenon, which induces a similar heat capacity increase as heat denaturation, notwithstanding the fact that it is induced by decreasing temperature (Privalov et al., 1986). Therefore, the only reasonable explanation for the observed denaturational heat capacity increment is the assumption that it is caused mainly by hydration of the non-polar groups, which are exposed to water upon unfolding of the compact protein structure. This was first suggested by Kauzmann (1959), on the basis of the well-known fact that the transfer of non-polar molecules to water is accompanied by a significant heat capacity increase (Edsall, 1935; for a review, see Franks & Reid, 1973). One of the arguments confirming this assumption is that A$, is found to be proportional to the number of contacts between the non-polar groups in native protein, i.e. actually to the surface area of the non-polar groups that are exposed to water upon folding of a compact protein structure (Privalov & Khechinashvili, 1974; Privalov, 1979). One of the most important facts established by these calorimetric studies of protein performed over a broader temperature range than that examined previously is that the partial heat capacity of a denatured protein is not a linear function of temperature. At temperatures above 6O”C, the slope of this function asymptotically decreases and almost disappears at about 100°C. It is tempting to explain the observed curvature of the heat capacity function by extended heat absorption caused by some gradual process (i.e. the melting of the residual structure), which is superimposed on the trivial temperature-induced increase of the heat
748
i? L. Privalov et al. for the enthalpy
and entropy functions
we have:
T
A;H(T)
= A;H(T,)
+ 1 A:C,dT T,
(7)
@W’) = C~~HV’,W,+ j N$&M’l dT, T, where T, is transition A;H(T,) I
,
100
50 Temperature
150
PC)
Figure 11. Denaturational increment of the partial heat capacity of RNase, Lys and Mb. The dashed lines represent parts of these functions that were obtained by a linear extrapolation of the heat capacity of the native state. The dot-and-dash lines show the behavior when the values measured at 50°C are assumed to be temperature-
independent.
A$,
= (aAEH)/aT and (A$Y,)/T
= (aA$S)/aT,
at which:
- T,A$‘(T,)
= 0.
If A$?, does not depend on temperature, the enthalpy and entropy of protein denaturation will be increasing functions of temperature; namely, the enthalpy will be a linearly increasing function (see Privalov, 1979). However, if A$?, is temperature dependent and decreases to zero at some temperature T,, then AEH and A$S will be functions that are asymptotically approaching some constant value at To. It is most interesting that, for the specific enthalpy and entropy, this constant value appears
capacity of the polypeptide chain. However, the universality of this effect, and the fact that it is observed under conditions when the unfolded polypeptide chain hardly possesses any residual structure, urge us to consider the shape observed for the heat capacity function as an intrinsic property of the unfolded polypeptide chain in water, as we consider the effect of squeezing the hydrodynamic volume of a random coil with increasing temperature. The situation with the heat capacity function of the native state is much less clear, since it can be experimentally determined only to 80°C even for the most thermostable protein, such as CTT (Fig. 10). Within the region 0°C to SOY!, this function appears to be linear but it is not improbable that this linearity is only apparent, being caused by a low-temperature tail of the denaturation heat effect. Assuming this function to be linear above 80°C we would conclude that the denaturational heat capacity increment should be a decreasing function of temperature, as shown in Figure 11. The main argument for the correctness of such an extrapolation is the fact that the heat capacity increment of transfer of non-polar solutes to water behaves in a similar way, asymptotically decreasing with increasing temperature (Gill et al., 1985; Makhatadze & Privalov, 1988). Then, such a behavior of the difference heat capacity function seems to be much more natural since, if it is caused by the influence of protein non-polar groups on the state of surrounding water, one can expect that, at increasing temperature, this influence should diminish and even disappear at a sufficiently high temperature. This is a very important conclusion for evaluating the temperature dependence of the enthalpy and entropy of protein denaturation. Since :
temperature
(8)
to be universal
for
all compact
globular
proteins. This is shown in Figures 12 and 13 for two proteins (RNase and Mb) that differ greatly in their denaturational heat capacity increments. As seen from these Figures, these asymptotic values are very close in magnitude to the values at the intersection of the enthalpy and entropy functions derived on the assumption that AEC, is constant, which takes place at about 110°C. However, while the values at the intersection of these functions at 110°C do not have a direct physical sense (as upon a further extrapolation above 110 “C these functions
, 100
I 50 Temperature
(‘73
Figure 12. Temperature dependence of the specific enthalpy of denaturation of Mb and RNase (per mol of amino acid residues) in solutions with pH and buffer providing maximal stability of these proteins and compensation of heat effect of ionization (see Privalov & Khechinashvili, 1974). The broken extension of the continuous lines represents a region that is less certain due to uncertainty in the A$?, function (see Fig. 10). The dot-and-dash lines represent the functions calculated with the assumption that the denaturation heat capacity increment is temperature-independent.
Heat Capacity
r
749
of Proteins
We are grateful to Drs S. J. Gill and B. G. Bar&s for helpful discussion of the manuscript in its preparation.
References 15 ? 2 T x 3 2
a
IO i 5 4 t 0 t -5 / 1 0 Temperature PC)
Figure 13. Temperature dependence of the specific entropy of denaturation of Mb and RNase (per mol of amino acid residues) under the same conditions as indicated for Fig. 12. The dot-and-dash lines represent the functions calculated on the assumption that the denatured heat capacity increment is temperatureindependent.
are diverging and can increase infinitely), the universal asymptotic values reached above 140 “C are quite interpretable. They seem to correspond to the enthalpy and entropy of protein unfolding in the absence of hydration effects. The final point we would like to call attention to is the following: the only argument for using Gu *HCl and urea denaturation in thermodynamic studies of unfolding (and, thus, folding) of protein structure is that the protein, denatured in this way, is supposed to be in a completely unfolded state. On the other hand, up to the present, we do not have a reliable procedure for evaluating the thermodynamic parameters of conformational transition caused by denaturants. We do not know how to take into account the denaturant solvation effect and, even more importantly, we do not know what kind of reaction we are analyzing and usually only assume for simplicity that it is a two-state transition. The main advantage of studying temperature/pH-induced conformational transitions in proteins is that, in this case, we can measure directly all the thermodynamic parameters required for a complete description of the process, even if it is complicated and proceeds in several discrete steps (see Privalov, 1979, 1982; Privalov & Potekhin, 1986). We can also properly deal with all ionization effects (Pfeil & Privalov, 1976b). But, in such cases, there has always been some suspicion that the results obtained do not adequately describe the protein folding process, since we do not take into account the contributions of residual structure. Now we see that this contribution is, in fact, so small that it can be neglected in many cases.
Ahmad, F. Rt Salahuddin, A. (1974). Biochemistry, 13, 2465249. Barynin, V. V. & Grebenko, A. I. (1986). Dokl. Akad. Nauk SSSR, 286, 461464. Bendzko, P. I., Pfeil, W. A., Privalov, P. L. & Tiktopulo, E. I. (1988). Biophylys. Chem. 29, 301-307. Boyer, P. D. (1954). J. Amer. Chem. Sot. 76, 43314337. Brzeska, H., Venyaminov, S. Yu., Grabarek, Z. & Drabikowski, W. (1983). FEBS Letters, 153, 169 173. Cabani, S., Gianni, P., Mollica, V. & Lepori, L. (1981). Canadian J. Chem. 56, 2707-2713. Calderon, R. O., Stolowich, N. J., Gerlt, J. A. BE Sturtevant, J. M. (1985). Biochemistry, 24, 6044 6049. Chen, Y.-H., Yang, J. T. & Martinez, H. M. (1972). Biochemistry, 11, 412M131. Cortijo, M., Panijpan, B. & Gratzer, W. B. (1973). Int. J. Pept. Protein Res. 5, 179-186. Crestfield, A., Stein, W. H. & Moore, S. (1962). Arch. Biochem. Biophys. (Suppl.), 1, 217-222. Dearborn, D. G. t Wetlaufer, D. B. (1970). B&hem. Biophys. Res. Commun. 39, 314-320. Edsall, J. (1935). J. Amer. Chem. Sot. 57, 1506-1507. Fisher, W. R., Taniuchi, H. & Anfinsen, C. B. (1973). J. Biol. Chem. 248, 3188-3195. Franks, F. & Reid, D. S. (1973). In Water. A Comprehensive Treatise (Franks, F., ed.), vol. 2, pp. 323-380, Plenum Press, New York and London. Gill, S. J., Dee, S. F., Olofsson, G. & Wadso, I. (1985). J. Phys. Chem. 89, 3758-3761. Glimcher, M. K., Francois, C. J., Richards, L. & Krane, S. M. (1964). Biochim. Biophys. Acta. 93, 585-602. Griko, Yu. V., Privalov, P. L., Venyaminov, S. Yu. & Kutyshenko, V. P. (1988). BioJizika (USSR), 33, 18-26. Gutrie, J. P. (1977). Canadian J. C&m. 55, 3700-3701. Hapner, K. D., Bradshaw, R. A., Hartzell, C. R. & Gurd, F. R. N. (1968). J. Biol. Chem. 243, 683-689. Hvidt, S., Rodgers, M. E. & Harrington, W. F. (1985). Biopolymers, 24, 1647-1662. Jaenicke, L. (1974). And. B&hem. 61, 623-627. Jolicoeur, C. & Boileau, J. (1978). Canad. J. Chem. 56, 2707-2713. Kauzmann, W. (1959). Advan. Protein Chem. 14, l-63. Kell, G. S. (1971). In Handbook of Chemistry and Physics (Weast, R. C., ed.), p. F-5, Chemical Rubber Co., Cranwood Parkway, Cleveland, OH. Laemmli, U. K. (1970). Nature (Lo&m), 227, 680-685. Lee, B. & Timasheff, S. N. (1974). Biochemistry, 13, 257265. Makhatadze, G. I. & Privalov, P. L. (1988). J. Chem. Themnodynam. 20,405-412. Margoliash, E. & Walashek, 0. F. (1967). Methods Enzymol. 10, 33S348. Masterton, W. L. (1954). J. Chem. Phys. 22, 1833. Ornstein, L. (1964). Ann. N. Y. Acud. Sci. 121, 312-349. Pfeil, W. & Privalov, P. L. (1976a). Biophys. Chem. 4, 33-40. Pfeil, W. & Privalov, P. L. (19766). Biophys. Chem. 4, 41-50. Potekhin, S. A., Medvedkin, V. N. & Yatsuk, 0. I. (1988). Biojzika (USSR), in the press. Privalov, P. L. (1979). Advan. Protein Chem. 33, 167-241.
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Privalov, P. L. (1982). Adwan. Protein Chem. 35, l-104. Privalov, P. L. & Khechinashvili, N. N. (1974). J. Mol. Biol. 86, 665-684. Privalov, P. L. & Potekhin, S. A. (1986). Methods Enzymol. 131, 4-51. Privalov, P. L., Griko, Yu. V., Venyaminov, S. Yu. & Kutyshenko, V. P. (1986). J. Mol. Biol. 190, 487498. Robinson, D. R. & Jencks, W. P. (1965). J. Amer. C&m. Sot. 87, 2462-2470. Rossi Fanelli, A., Antonini, E. & Caputo, A. (1958). B&him. Biophys. Acta, 30, 608-615. Sela, M., White, F. H., Jr t Anfinsen, C. B. (1959). Biochim. Biophys. Acta, 31, 417426. Sturtevant, J. M. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 2236-2240. Edited
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by R. Huber
Heat Capacity and Conformation of Proteins in the Denatured State P. L. Privalovl, E. I. Tiktopulol, S. Yu. Venyaminovl, Yu. V. Grikol, G. I. Makhatadzel and N. N. Khechinashvili’ ‘Institute of Protein Research and 21nstitute of Biological Physics, Academy of Sciences of the U.S.S.R., 142292 Pushchino, Moscow Region, U.S.S.R. (Received 20 July 1988, and in revised form 14 October 1988) Heat capacity, intrinsic viscosity and ellipticity of a number of globular proteins (pancreatic ribonuclease A, staphylococcal nuclease, hen egg-white lysozyme, myoglobin and cytochrome c) and a fibrillar protein (collagen) in various states (native, denatured, with and without disulfide crosslinks or a heme) have been studied experimentally over a broad range of temperatures. It is shown that the partial heat capacity of denatured protein significantly exceeds the heat capacity of native protein, especially in the case of globular proteins, and is close to the value calculated for an extended polypeptide chain from the known heat capacities of individual amino acid residues. The significant residual structure that appears at room temperature in the denatured states of some globular proteins (e.g. myoglobin and lysozyme) at neutral pH results in a slight decrease of the heat capacity, probably due to partial screening of the protein non-polar groups from water. The heat capacity of the unfolded state increases asymptotically, approaching a constant value at about 100°C. The temperature dependence of the heat capacity of the native state, which can be determined over a much shorter range of temperature than that of the denatured state and, correspondingly, is less certain, appears to be linear up to 80°C. Therefore, the denaturational heat capacity increment seems to be temperature-dependent and is likely to decrease to zero at about 140°C.
guanidinium hydrochloride (Gu * HCl) or urea, while a heat or acid-denatured protein contains too much residual structure to be regarded as a completely unfolded, unstructured state (see Tanford, 1968). This conventional view was shaken first when it was shown, by direct calorimetric measurements, that the enthalpies of lysozyme denaturation by heat and GuoHCl are identical if the denaturant solvation is correctly taken into account (Pfeil & Privalov, 1976a). It was found also that the partial heat capacities of the heat and GueHCl denatured lysozyme are indistinguishable. The importance of the partial heat capacity in specifying the denatured state of proteins follows from the fact that this parameter is a sensitive index of the completeness of protein unfolding. The exposure of the internal non-polar groups to water should result in a heat capacity increment, since the transfer of non-polar compounds to water is associated with a significant increase of the heat capacity (Kauzmann, 1959). On the other hand, the heat capacity is the temperature derivative of a basic thermodynamic function, the enthalpy Therefore, the denaturational heat capacity increment
1. Introduction The denatured states of proteins have attracted increasing attention. This interest arises from the realization that the denatured state is the only experimentally achievable state of a protein that can be taken as an initial reference in considering the mechanism of folding and stabilizing the native protein structure, However, a denatured state can play this role only if it can be associated with a completely unfolded, random coil conformation of polypeptide chain. The latter, as is clear, is an idealized model that is never realized in practice, since real polypeptides have too many groups interacting in very different ways with each other and with the solvent. However, it is unclear how far the real situation is from the ideal, and how large an error arises from equating a random coil with the denatured state, particularly when we estimate thermodynamic parameters for folding of the native protein structure. For many years it has been accepted that the best approximation of a random coil polypeptide is a denatured protein in concentrated solutions of 0022-2836/89/040737-14
$03.00/0
737
0 1989 Academic Press Limited
P. L. Privalov et al determines the temperature dependence of the enthalpy and, hence, of the entropy of denaturation, i.e. the parameters that determine the native state stability. The first more or less circumstantial study of the denaturational heat capacity increment of globular proteins was carried out by Privalov & Khechinashvili (1974), who showed that its value is likely to be specific for a given protein and does not depend on temperature. However, in that study, the heat capacity was measured over a limited temperature range from 20 to 90°C and calorimetric experiments were not accompanied by studies of the conformation of the denatured proteins. Therefore, one cannot exclude the possibility that heat capacity increment is caused by gradual melting of the residual structure upon increasing temperature and that, over a broader range of temperature, there will not appear some temperature dependence of the denaturational heat capacity increment on temperature. To clarify these points, it was necessary to make calorimetric measurements in a much broader range of temperature than had been done before, and to accompany the measurements with studies of protein conformation. Here, we report the results of a detailed calorimetric study of the heat capacity of a number of globular proteins in a temperature range from -5 to 13O”C, and the results of viscosimetric and spectropolarimetric studies of these proteins. Such an extension of the temperature range of calorimetric measurements became possible due to a new -model of the precision scanning microcalorimeter with capillary cells, which permits measurements under considerable excess pressure at temperatures above 100°C and scanning down the temperature scale to measure aqueous solutions in a supercooled state. We chose five globular proteins that are most popular among physical chemists; pancreatic ribonuclease A, staphylococcal nuclease, hen egg-white lysozyme, sperm whale myoglobin and horse heart cytochrome c. These proteins were examined with the disulfide crosslinks intact or disrupted and with the heme groups in place or removed (apo-form). The sixth globular protein used in this study was catalase from thermophilic micro-organisms, which was included for its extreme thermostability, in order to clarify aspects of the heat capacity of the native state. The seventh example, collagen, was chosen to represent a quite different class of fibrillar proteins.
2. Materials and Methods Metmyoglobin (Mb?) was isolated from frozen sperm whale muscle according to Hapner et al. (1968). The procedure for its further purification was as described by Privalov et al. (1986). Apomyoglobin (aMb) was obtained from sperm whale metmyoglobin by precipitation with acetone according to Rossi Fanelli et al. (1958). Cytochrome c (Cyt) was isolated from bovine heart as described by Margoliash & Walashek (1967).
Apocytochrome c (aCyt) was obtained according to Fisher et al. (1973). Ribonuclease A (RNase) was chromatographically purified from the commercial preparation of bovine pancreatic ribonuclease (“Reanal”, Hungary) using the procedure described by Crestfield et al. (1962). Hen egg-white lysozyme (Lys), a commercial preparation from “Reanal” (Hungary), was recrystallized 3 times before use. Staphylococcal nuclease (Nase) was isolated from a homogenate of Escherichia coli cells that had been transformed with a recombinant plasmid containing the gene for the enzyme by Dr Robert Fox (Yale University) and kindly provided for this study by Professor J. Sturtevant (Yale University). This construction results in the synthesis of a modified nuclease in which the heptapeptide Met-Asp-Pro-Thr-Val-Tyr-Ser is appended to the amino-terminal end of the nuclease; i.e. its molecular mass is 17,600 and not 16,807 Da (for a detailed description of this preparation, see Calderon et al., 1985). Catalase from Thermus therwwphilus (CTT) was a kind gift from Dr Barynin from the Institute of Crystallography of the USSR Academy of Sciences, who isolated it from micro-organisms and purified as described by Barynin & Grebenko (1986). Collagen from pike skin (Coll) was purified from an acid-soluble extract according to Glimcher et al. (1964). Glu-Lys-Lys-Leu-Glu-Gln-Ala heptapeptide The (Lys = lysine) was synthesized by Potekhin et al. (1988) in our Institute and kindly provided for our experiments. The purity of each preparation was checked electrophoretically in denaturing (Laemmli, 1970) and nondenaturing (Ornstein, 1964) conditions, which showed that the amount of impurities was less than 2%. Disulfide crosslinks in RNase and Lys were disrupted by reducing cysteine with thioglycolic acid in the presence of 8.5 M-urea at 20°C. The SH groups of cysteine residues were then carboxamidomethylated by iodoacetamide according to the method of Sela et al. (1959). The
t Abbreviations used: Mb, metmyoglobin; aMb, apomyoglobin; aMb”, apomyoglobin previously heated; Cyt, cytochrome c; aCyt, apocytochrome c; RNase, ribonuclease A; RNase-“, ribonuclease A with disrupted S-S crosslinks; Lys, hen egg-white lysozyme; Lys-“, hen egg-white lysozyme with disrupted S-S crosslinks; Nase, staphylococcal n&ease; C!l!T,
cat&se from Thrmua them$iZus;
Coil, collagen from pike
skin; c.d., circular dichroism; EH, 3ro helix; Gu, guanidinium. recorded apparent difference AC’“s(T) pl,aol,~lv, calorimetrically in the heat capacity of the protein solution and solvent. C (Thzo, spe cific heat capacity of water at temperature T. C$T) partial specific heat capacity of native protein. C$T)z: partial sp cific heat capacity of denatured protein. AC&, = q,af - Cv,s, denaturational increment of protein heat capacity. A$f(T) = HD(T) - HN(T), the enthalpy difference of the denatured and native states of protein. A$S(T)=SD(T)-SN(T), the entropy difference of the denatured and native states of protein. V(T),,, partial specific volume of the protein in solution at temperature T. V(T),,,, specific volume of water at temperature T. w(T),,, protein concentration in the solution at temperature
T.
intrinsic viscosity at temperature T. molecular elliptioity at the wavelength 2. molecular mass of an amino acid residue of a m.l, mean ’ protein. A, number of residues in the polypeptide chain of protein. $yd.ptein
739
Heat Capacity of Proteins amount of reduced SH groups was tested &a described by Boyer (1954). All the measurements were carried out in 10 mM-saltfree glycine or phosphate buffer solutions of acidic pH since, under these conditions, the proteins studied are most soluble and no aggregation is observed upon heating. The presence of aggregation was tested by highpressure liquid chromatography at various temperatures. After heating experiments, the preparation was checked electrophoretically under reducing conditions to ensure the absence of hydrolysis of the polypeptide chain at elevated temperatures. Prior to measurements, solutions were carefully dialyzed against solvent. Several replacements of the solvent were used to achieve complete equilibration of low molecular weight compounds in the solvent and solution used in differential measurements. After dialysis, solutions were centrifuged for 40 min at 24,000 g. Protein concentrations in solution were determined spectrophotometrically with correction for light-scattering (Winder & Gent, 1971). The extinction coefficients were found, in separate experiments, by measuring the nitrogen content in stock solutions according to Jaenicke (1974). For solutions of pH 2.0 to 5.0, the following extinction coefficients were used: Er&,., = 176 for Mb, 9.0 for aMb, 26.9 for intact Lys, 25.25 for Lys with reduced disulfides, 10.0 for CTT, 9.39 for Nase, 9.2 for aCyt, E:%,,snm = 7.32 for intact RNase, 7.25 for RNase with reduced disulfides, and Eaon, = 9-06 for Cyt. Calorimetric measurements were peformed using a DASM-4A capillary scanning microcalorimeter equipped with gold cells of 1.0 ml volume (Bureau of Biological Instrumentation of the Academy of Sciences of the USSR). To extend the heating range to 13O”C, all measurements were performed under an excess pressure of 506,625 Pa. For measurements below O”C, the solutions were supercooled as described earlier (Privalov et al., 1986). The heating rate was from 1.0 to 2.0 K min- ‘. The protein concentration in the solutions used for the experiments varied from I.0 to 5.0 mgml-‘. The partial specific heat capacity of the protein in the solution was determined as described by Privalov &, Potekhin (1986) from the calorimetrically recorded apparent difference in the heat capacity of the protein solution and solvent, ACBpPP(T)pr.sollsol, and the apparent difference in the heat capacities of the solvent and water, ACPpPpV)so,v,u~o:
where v(T) is the operational volume of the calorimetric cell at temperature T, w(T),, is the protein concentration in the cell at this temperature in gml-i, V(T)H20 and C,(T),,, are, respectively, the specific volume and specific heat capacity of water at this temperature and is the partial specific volume of protein at this UT),, temperature. For the specific volume and specific heat capacity of water, data tabulated in standard handbooks (Kell, 1971) were used. The partial specific volumes of proteins were determined from the density of the protein solution, P(T)~~.~~, and that of the solvent, P(T)~~~“, measured by a vibrational densimeter DMA-2 (Anton Paar, Austria), using the equation (Masterton, 1954):
WY,, = PICwm,rIH~ - ~P~~~,,.,,,l/~P~~~~O,“l (2) + b4m/wh,,4. It should be noted that, at the protein solution concentrations (20) studied, the values determined by eqns (1) and (2) do not show any concentration dependence and can be considered as corresponding to infinitely dilute solutions; i.e. Cp,pr = CEpr and VP, = VE (Fig. 1). The partial specific volume of the protein was determined over a temperature range of 2 to 8O”C, where it exhibited a linear temperature dependence (Fig. 2). For heat capacity determinations at higher temperatures, we used the extrapolated values of the partial specific volume. At the accuracy with which partial specific volumes could be determined in these experiments (*0403 cm3g-‘), no difference was noticed between the partial specific volumes of the native, denatured and completely unfolded forms of a protein at a given temperature. The solution viscosity was measured in an Ostwald viscosimeter with a water flow-time of about 200 s at 25°C and in a VBA-1 automatic rotational viscosimeter (Bureau of Biological Instrumentation of the USSR Academy of Sciences). A great advantage of the second instrument is that the protein solution does not contact air and is under an excess pressure. Therefore, the protein solution does not foam during experiments, as occurs in the usual capillary instruments at elevated temperatures. The protein intrinsic viscosity [q]r was determined by extrapolating the reduced viscosity determined from the solution flow-time (or period of rotation) t to zero concentration:
tvl~ = lim [@--WM W-+0
where the protein
T m 7 Y 2
20
-A+.--.-
& 1.5G -
d,-
concentration
(3)
w (in gml-‘)
and the
and LYS-‘~ (0)
in 10 mM-
7-----*---R-----+ #
-o-e-
AL?
o--o----o
I.O1 I.0
I
I 3.0
1
Concentration
Figure 1. Concentrational glycine (pH 2.5) at 25°C.
dependence of the partial
I 5.0
1
I 7.0
I
I 9.0
(mg mPl
specific heat capacity
of Lys (0)
740
P. L. Privalov et al.
0.65’
1 0
I
I 20
I
I 40
I 60
I
Temperature
I
I 80
PC)
Figure 2. Temperature dependence of the partial specific volume of Lys (O), RNase (n), Mb (A) and Cyt (0) in 10 mru-glycine (pH 2.5). Filled symbols correspond to proteins with disrupted S-S crosslinks and apo-forms. solvent flow-time to are measured at the same temperature, T. The extrapolation was done using at least 5 measurements on solutions with different concentrations over the range of 1 to 5 mg/ml-’ (Fig. 3). The circular dichroism spectra in the range of 183 to 320nm were measured with a Jasco-41A spectropolarimeter (Japan) in thermostated cells with a pathlength from 0.15 to 0.9 mm using solutions with -protein concentration varying from 0.2 to 2.0 mg ml . The molar ellipticity was determined as [e]pw = (4%+ ~aA/W) (4) where w is the protein concentration (in g ml-‘), 1 is the light pathlength in the cell (in mm), e1 is the measured ellipticity (in degrees) at a wavelength 1, and g,, is the mean molecular mass (in daltons) of an amino acid residue of a protein determined from the known sequence of the given protein. For the proteins studied, this quantity varied from 93 for collagen to 116 for apomyoglobin (for details of the experimental procedure, see Venyaminov & Gogia, 1982; Brzeska et al., 1983).
3. Results (a) Viscosimetric study As seen in Figure 3 for lysozyme with disrupted S-S crosslinks (Lys-““), the reduced viscosity of the unfolded polypeptide does not depend significantly on the protein concentration in solution at pH 2.2 at any temperature studied. This shows that, under the chosen solvent conditions, the interaction between protein molecules is rather low even in the
I
I
I.0
2.0 Concentration
Figure 3. Concentrational temperatures (“C).
dependence
unfolded conformation. This is a very important circumstance for further interpretation of all the experimental results obtained in this study. The intrinsic viscosity of the native protein at 10°C is low and is almost independent of pH throughout the pH region in which this protein is in the native state (Fig. 4). For Lys and RN&se with intact
S-S crosslinks,
the native
state
is realized
throughout the entire acidic pH range at 10°C. However, in the case of much less stable proteins that denature at acidic pH values, the intrinsic viscosity
increases
considerably
at decreasing
pH.
The observed denaturational increment of intrinsic viscosity is especially large in cases where the polypeptide chain is not crosslinked by disulfide bonds, as occurs for Mb. Upon disruption of S-S crosslinks, intrinsic viscosities of Lys and RNase increase at low pH to the same extent as that of Nase, Mb and Cyt. It is notable that with increasing pH, the intrinsic viscosities of these polypeptide chains decrease, though not to the level of the native compact proteins. A similar situation is observed with previously heated apomyoglobin @Mb”), which does not regain its native-like compact structure (Griko et aZ., 1988), but its intrinsic viscosity drops to a remarkably low level at neutral pH values. It appears that heating of aMb to 100°C leads to such a chemical modification of some of its amino acid residues that its
polypeptide structure 1986).
I
3.0 (mg
of the reduced viscosity
chain (on this
cannot
fold into a native see Zale & Klibanov,
aspect,
I
I
4.0
5.0
ml-‘)
of Lys e-S’ in 10 mM-glycine
(pH 25) at various
741
Heat Capacity of Proteins
Table 1
-I
(a)
Intrinsic viscosity of unfolded proteins at 25°C (RNase and Lys without S-S crosslinks and Mb and Cyt without the heme in solutions with pH 2.2 and 6 M-Cu. HCI) [11
rtl1t
11,702 104 13,683 124 14,396 129
15.0 15.6 17.0
16.1 17.6
17,170
20.0
20.1
W’c
pH 2.2 6 M-Gu.HCI for random coil 12 (cm’ g-l)
Protein acyt RNase-‘”
153
149 16.6 17.0 19.1
M, molecular mass; 12,number of amino acid residues; [q], intrinsic viscosity; [q]c”c, values for the random coiled polypeptide calculated according to Tanford (1968) by eqn (5). t Values from Ahmad & Salahuddin (1974).
last column of Table 1. As is seen, the experimental values
1
0'
,
I
I
I
I
4.0
2.0
6.0
PH
Figure 4. pH dependence of the intrinsic viscosity of proteins at 10°C. (a) Lys and RNaae with intact and disrupted S-S crosslinks and aCyt; (b) Mb with the heme groups in place and removed. Symbols: Lys (O), Lys-” (01, R&se (Cl), RNase-w (ml, Cyt (01, aCyt (+), Mb (AL aMb (A), aMbh (A).
With decreasing pH, the intrinsic viscosity of a non-crosslinked protein rises to some constant level, which is specific for a given protein at a given
temperature and ionic strength of the solution. These maximal values of intrinsic viscosity, which are achieved at pH below 3-O at 25”C, are summarized in Table 1 for RNase and Lys without SS crosslinks and for the apo-forms of Mb and Cyt. The Table presents values for the intrinsic viscosity of the same proteins in 6 M-Gu.HCl solution obtained by Ahmad & Salahuddin (1974). As can be seen, they
are in rather
good correspondence
of
intrinsic
viscosity
of
the
considered
polypeptide chains without S-S crosslinks or a heme are rather close to the value calculated for a random coiled polypeptide. Thus, one can suppose that in solution at pH 2.0, the polypeptide chains of all the proteins studied are in a random coil conformation at 25°C if they are not fixed by S-S crosslinks. An increase of temperature leads to a significant decrease of the intrinsic viscosity of the unfolded polypeptide chain (Fig. 5). The effect of temperature on the intrinsic viscosity of unfolded polypeptide chains was observed by Ahmad & Salahuddin (1974) for proteins in concentrated solutions of Gu.HCl; but, in that case, the temperature influence is even more pronounced than in the case where unfolding of the polypeptide chain is caused by extreme pH, i.e. by the charges on the polypeptide chain. The temperature influence on the intrinsic viscosity of the unfolded polypeptide chain can be
with
our values obtained in acidic solutions without a denaturant. There is a clear correlation of intrinsic viscosity values with the molecular weight of the corresponding protein or with the number of amino acid residues in the polypeptide chain. According to Tanford (1968), the intrinsic viscosity of the polypeptide chain in a random coil conformation at 25°C can be expressed as: [q] = 77.0 n”.666/iiZ5.r,
(5) where iii,, is the mean molecular mass of an amino acid residue and n is the number of residues in the polypeptide chain. The values of intrinsic viscosity calculated bv eauation (5) frnlcslc) are given in the Y
1
T
I
\L
,J
I
0
Figure 5. Temperature viscosity pH 4.0; and of pH 3.0: symbols
dependence of the intrinsic of proteins: (1) RNase, pH 3.0; (2) RNase, (3) Lys, pH 3.0; (4) Mb, pH 5.0; (5) Lys, pH 4.0; unfolded polypeptide chains: (6) aMb and Mb, (7) Lys-““, pH 2.2; (8) RNase-=, pH 2.2. The are the same as for Fig. 4.
742
P. L. Privalov
explained by the increase of flexibility of the chain caused by the increase of rotational freedom of backbones at increasing temperature. Clearly, increased flexibility of the chain should lead to a decrease in the hydrodynamic volume occupied by this chain (Ahmad & Salahuddin, 1974). The decrease of the hydrodynamic volume of the coil might be caused also by an increase of the hydrophobic pressure; i.e. the random hydrophobic interactions between the non-polar groups of a chain, since the hydrophobic interactions are known to increase at increasing temperature (Privalov et al., 1986).
It follows from the above that the conformation of a heat-denatured protein does not differ greatly from that of a random coil, as it is supposed to be when the intrinsic viscosity of a heat-denatured protein as measured at 80°C is compared with that of Gu.HCl-denatured protein measured at 25°C. Judging by Figure 5, the intrinsic viscosity of a random coil polypeptide chain at 80°C is only twice as large as that of the corresponding heat-denatured protein with intact S-S crosslinks, while for proteins without S-S crosslinks, such as Mb, the intrinsic viscosity of a
-15
’ ,‘.j,
et al.
heat-denatured state is almost the same as that of the random coil state. (b) Circular
dichroism study
Figure 6 presents c.d. spectra of the studied proteins in various states; native, acid-denatured, heat-denatured and denatured by GuaHCl. The spectra for all proteins in the concentrated solution of GueHCl are similar to those found by Dearborn & Wetlaufer (1970), Tiffany 6 Krimm (1973) and Cortijo et al. (1973). The spectra of heat-denatured proteins are rather close to the spectra of unfolded polypeptide chains without disulfide crosslinks or heme at acidic pH, but deviate significantly from the spectra of proteins in 6 M-Gu.HCl. This deviation is especially clear at 222 nm, lO”C, but with a temperature increase it decreases and is likely to disappear above 80°C (Fig. 7). It is notable that the deviation decreases due to a considerable temperature dependence of the ellipticity of protein in the presence of Gu. HCl. The ellipticity of polypeptide chain at 222 nm is usually considered as an index of its secondary
i 200
, Cyt., pH , 3.0 , 220 240
,
, 200
I
Collagen, , ( pH, 3.5 ( 220 240
Wavelength km)
Figure 6. Circular dichroism spectra of proteins in the native state at 10°C (continuous line) and temperaturedenatured state at 80°C (dot-and-dash line), both at the pH indicated in the boxes; in the acid-unfolded state with disrupted S-S crosslinks or removed heme, at 10% pH 2.5 (dashed line), and in the presence of 6 M-GU . HCl at 10°C (dotted line).
743
Heat Capacity of Proteins
o
,+ 2.0
Figure 7. Dependence of the ellipticity
(
, 4.0 PH
,
, 6.0
I
,
40 Temperature
60
1 43 . . . . . . . ,...‘..’ 20
,
,
/ 60
PC)
of proteins at 222 nm on (a) pH at 10°C and (b) on temperature
at various pH
values. The symbols are the same as for Fig. 4. The crossed symbol corresponds to the results in the presence of 6 MGu.HCl. structure (Chen et al., 1972). If one accepts that the polypeptide chain is in a completely random coiled conformation in a concentrated solution of Gu . HCl (see Tanford, 1968), then it follows from Figures 6 and 7 that heat-denatured protein has considerable but its content residual secondary structure decreases upon a temperature increase and practically disappears at about 100°C. However, the question is whether one can use the cd. spectra of proteins in concentrated solutions of Gu.HCl as a reference for the random coil conformation of the polypeptide chain? Our experiments with short synthetic polypeptides that could hardly have any helical conformation in aqueous solutions showed that the presence of Gu . HCl induces significant changes in ellipticity (Fig. 8). This is not unexpected, as it is known that Gu * HCI interacts with peptide groups with a large negative enthalopy (Lee & Timashelf), 1974; Pfeil & Privalov, 1976a). According to Robinson & Jencks (1965), one molecule of Gu . HCl links with two peptide bonds; therefore, one should expect that it can affect the conformation of the polypeptide group. According to Tiffany & Krim (1972, 1973), the high concentration of Gu . HCl and urea favors the locally extended 3,,-helix conformation (EH) of polypeptides. If so, the
effect of temperature on the ellipticity of proteins in concentrated Gu * HCl solutions could be explained by the decrease of the content of the EH conformation at a temperature increase. This seems quite likely, as increasing temperature should lead to desolvation of Gu * HCl bound to polypeptide if its binding enthalpy is negative. It follows then, that proteins in concentrated solutions of Gu * HCI attain a random coil conformation only at a rather high temperature. Another possibility that should not be excluded is that GuaHCl can influence the intrinsic optical properties of the individual amino acid residues, and this influence is temperature-dependent (see Hvidt et al., 1985). In this case, the observed optical effect cannot be interpreted in conformation terms. The above leads us to conclude that the conformation of protein denatured by Gu. HCI or acid is certainly close to a random coil only at temperatures about 100°C. At lower temperatures, there is some difference in the ellipticities of the Gu*HCl and acid-unfolded polypeptides, which in some cases amounts to 5666 deg cm2 dmol-‘; that is, to about 15% of the value specific for the 166% a-helix. However, this difference is hardly interpretable with certainty as evidence for the residual helicity in the acid-unfolded polypeptide chain,
744
P. L. Privalov et al.
0
20
40 60 80 Temperature PC)
10°C v
,
200
(
,
220 Wovelength
,
240
(nm)
Figure 8. Ellipticity of the heptapeptide Glu-Lys-Lys-Leu-Glu-Gln-Ala (Lys = lysine) in 100 mM-sodium phosphate, pH 7-O (continuous line) and in the presence of 6 M-GU. HCl (dotted line) at different temperatures.
since the nature of this systematic difference in the ellipticities is unclear. Therefore, in considering the conformation of a polypeptide chain we can only neglect this difference (especially when it is small) and suppose that the conformation of the acid and GueHCl-unfolded polypeptide chains are both close to a random coil. For some of polypeptide chains, such as RNase-“” and aCyt, the random-coil conformation is likely to persist at higher pH up to 7.0 (Fig. 7). In the case of Nase, whose unfolding and folding is a perfectly reversible process, an increase of pH leads to folding of its polypeptide chain into the native structure. The increase of pH leads to an increase of negative ellipticity at 222 nm of Lys-“’ and aMb. If lOOo/o a-helicity disposes the ellipticity of 34,500 deg cm2 dmol- ’ at 222 nm, as reported by Chen et al. (1972), then one can suppose that at pH 5.0 the content of u-helicity reaches 8% in Lys-“’ and 25% in preliminarily heated aMb’. This increase of the a-helicity is accompanied by a decrease of the intrinsic viscosity of these polypeptide chains (see Fig. 4). It is remarkable with increasing that, temperature, the high ellipticity of Lys-“” drops to the initial value within a temperature range from 30°C to 70°C (Fig. 7). In contrast to Lys-“, the ellipticity of the preliminarily heated aMb6 is not very sensitive to temperature. Thus, it does not
seem to be associated operative structure.
with
(c) Calorimetric
an extended
co-
study
Figure 9 represents the partial specific heat capacity of the proteins studied here in solutions with various pH values as determined calorimetrically over the temperature range -5°C to 130°C. To expand the y-axis, we have omitted the tops of the denaturational heat absorption peaks as, in this paper, we are not interested in the denaturational process itself, but in the heat capacities of the native and denatured states. As can be seen, increasing pH shifts the denaturation temperatures to higher values, but does not noticeably change either the heat capacity of the native state or the heat capacity of the denatured state. These two quantities are equal to within the experimental error of kO.04 J K-r g- 1 with only two exceptions (considered below). However, the most remarkable observation is that RNase and Lys without S-S crosslinks in solutions of pH below 3.0, in which they are in completely unfolded, random coil conformations, have the same heat capacity as the denatured states of these proteins with intact S-S crosslinks. A similar situation is observed with Mb and Cyt; in solutions with pH below 3.0, the denatured states of
745
Heat Capacity of Proteins
RNase
Mh
2Jz#Y-I-’
14
0
I
20
I
Nase I
40
60
Temperature
I
80
I
too
/
120
PC)
Figure 9. Temperature dependence of the partial specific heat capacity of proteins with intact and disrupted disulfide crosslinks and with or without the heme, in solutions with different pH values (indicated on the curves).
these molecules have the same heat capacities as the apo-forms of these proteins, which, according to viscosimetric and c.d. studies, are in the unfolded conformation close to a random coil. The influence of pH on the heat capacity of the denatured state is observed only for Lys-“” and aMbh. As shown above, with increasing pH above 4.0, both these polypeptides, and especially aMbh, form structures that are characterized by considerable helicity. Upon heating Lys-“” in pH 5.0 solution, its additional helicity disappears over the temperature range 30°C to 70°C (Fig. 7) and, in the same temperature range, an extra heat absorption is observed that appears as a broad and flat peak, shown by a dotted line in Figure 8. The area of this peak is about 5 Jg-’ (70 kJ mol-‘), which is less of the enthalpy of lysozyme than 20% denaturation. The van? Hoff enthalpy, determined from the breadth of this peak, is of the same order. This shows that the structure formed by Lys-‘” at pH 5.0 is co-operative. From this point of view, this structure resembles the native structure of lysozyme with intact crosslinks, the more so because they are also comparable in ellipticity. However, these two structures differ essentially in the enthalpy of formation and in heat capacity. The heat capacity of Lys-” at pH 5-O and 25”C, where temperature-induced melting of its structure does not occur yet judging by its ellipticity, is almost the same as that of Lys-” at pH 2.5, which is in a
completely unfolded state. Therefore, one can suppose that the structure formed by Lys-” at pH values close to neutral is quite open. This is confirmed by viscosimetric studies, which show that the intrinsic viscosity of Lys-“” at pH 5-O is about 10. In contrast to the structure formed by Lys-“‘, the structure formed by the preliminarily heated aMbh at pH above 4.0, as mentioned in the previous does not change significantly with section, increasing temperature (Fig. 7). Correspondingly, we do not observe a clear heat effect that can be interpreted as additional heat absorption, although there is some difference between the heat capacity functions found at pH 5.0 and pH 2.5 (Fig. 9). This difference amounts to O-1 J K-‘g-’ at a low temperature (i.e. is about a 5th of the heat capacity difference between the native and denatured states) and gradually disappears upon heating from 40°C to 70°C. In all cases, the heat capacities of the native protein are much lower than that of the denatured protein, and they differ considerably, providing a large variation between the values of the denaturaE;nal heat capacity increment A$?,,,, = for the proteins studied here (Table 2). P-PI - CF,PI The A% N p,pr values obtained at 25°C agree well with previous findings (Privalov & Khechinashvili, 1974). It is remarkable that the temperature dependences of the heat capacity of all the proteins Table 2 Partial speci$c capacities of proteins in native (N) and denatured (D) states at 25°C measured calorimetrically (exp) and calculated (talc) for the unfolded polypeptide chain (U) from the heat capacities of constituting amino acid residues Protein, condition
Cr.,, (exp)t
Nase, pH 7.0 pH 2.0-3.0 RNaN!, pH 4.0 RNaWZ-““, pH 2.fS5.0 LYS, pH 2Xk5.0 LysC”, pH 2.0-4.0 Mb, pH 4+6.0 pH 2.C3.0 aMb, pH 5.Ck6.0 pH 2.0-4.0 Cyt, pH 3.0-6.0 aCyt, pH 2G6.0 Coll, pH 3.0 f c,.,r in JK-‘g-l.
CLpr (exp)t
c;,r
(calcH
1.45 1.90
2.15
1.90
1.85
1.88
1.96
1.52
1.40
1.36 1.98 1.60
1.90 2.02
-
1.42
1.35
2.28
2.03
2.22
1.55
1.58
746
P. L. Privalov et al.
40
60 80 Temperature PC)
100
120
Figure 10. Temperature dependence of the heat capacity of RNase (pH 4.0), Lys (pH 3.5), Mb (pH 4.4), C!l!T (pH 8.5) and RNaseWsS,Lys-“, aMb ai pH 2.5. studied here in the completely unfolded state are represented by rather similar functions. Over the broad temperature range of - 5°C to 13O”C, this function does not appear linear, as it appeared when examined over the smaller temperature range from 20°C to 90°C (Privalov t Khechinashvili, 1974). For the native protein, one cannot draw the same conclusion, since the temperature range over which the native state can exist is much smaller. Even for such a thermostable protein as catalase from thermophilic micro-organisms (CTT), the pure native state can be observed with certainty only up to 80°C (Fig. 10).
4. Discussion As has been shown above, the polypeptide chain of protein without S-S crosslinks and the heme in acidic solutions with pH below 3-O appears to be in an unfolded state with little, if any, residual structure. An increase in temperature leads to some squeezing of the hydrodynamic volume of an unfolded polypeptide chain, but this does not at all imply the formation of a compact structure. On the contrary, temperature-induced squeezing appears to be a general property of all polymers and, at high temperature, the conformation of a polypeptide chain is likely to be even closer to a random coil judging by its ellipticity. With increasing pH, polypeptide chains fold into a structure that is characterized by some helicity (Fig. 7). In some cases this is a compact, cooperative native structure (as in the cases of Nase and aMb); in other instances, this is a not quite compact, but co-operative structure (as in the case of Lys-““); and in some cases it is neither compact nor co-operative, as with the preliminary heated apomyoglobin, aMb”. In the latter case, the enthalpy of the structure formation is found to be close to zero (Griko et al., 1988). Therefore, temperature does not affect this structure greatly.
Crosslinking of a polypeptide chain by disulfide bonds results in a significant decrease of its intrinsic viscosity, i.e. of the hydrodynamic volume occupied by this chain, as expected. However, this does not mean in itself that a crosslinked polypeptide chain has a significant amount of residual structure. Indeed, the ellipticity of heat-denatured Lys and RNase with intact S-S crosslinks is very close to the ellipticity of these polypeptide chains without S-S crosslinks, which are in a completely unfolded state, and, upon temperature increase, these two values of ellipticity become closer (Fig. 7). Thus, it appears that, at a sufficiently high temperature (above SO’%), the conformation of the heatdenatured protein in acidic solutions is rather close to a random coil (i.e. crosslinked random coil). Before discussing our experimental results on the heat capacity of proteins, it is interesting to consider what should be expected for the heat capacity of an extended polypeptide chain in which all the groups are in perfect contact with water, and in which no residual structure is supposed to be present. Let us assume, in the first approximation, that each amino acid residue contributes additively to the integral heat capacity of such a chain as if these residues have an identical peptide group (CHCONH) and differ in the side-chains (R), and there are two terminal groups (NH,) and (CHCOOH) in the chain. The integral heat capacity of the extended polypeptide can be represented as: _ c C ca1c p,NH2 + (n~~‘P,CHCONH P>PI + f
i=l
‘p,r
+
cp,C”COOH.
(6)
The partial heat capacities of all these groups were calculated from the known partial heat capacities of model compounds in dilute aqueous solutions, and for some of the groups they were determined by direct calorimetric measurements of the heat capacity of tripeptide Gly-X-Gly in solution (Makhatadze, Medvedkin & Privalov, unpublished results). The heat capacity values used are listed in
Heat Capacity of Proteins Table 3 Partial heat capacities C,,i of polypeptide groups
Group NH, CHCOOH CHCONH R WY) R (Ala) R (Val) R (He) R (Phe) R (TY~) R (Ser) R (Thr) R (Asp) R (Am) R (Glu) R (Gln) R (LYs) R (I-) R (Pro) R (HYP) R IMet) ~ R (His) R (Trp) R (Ax) R (CY#I
Procedure for calculating 6,
Analog t
ql,CH+ c, co + C,,,,
CH,CONHf Hydrogen?’ Methane5 Propane§ 1-Butane§ Methylhenzene§ 4-MethylphenoQ Methanol5 Ethanols Acetic acid5 Acetamide$ Propionic acids Propionamide§ n-Butylamine§
Calorimetric(I
Gly-X-Gig G&X-Glj Glv-X-Glv GI;-X-G6 Gly-X-Gly n-Propylguanidinefr (:p,t.,e,- q,.cH
Calorimetric~~ CalorimetricI] CalorimetricI/ Calorimetric/l CalorimetricI\
c -c CT;::: - c;::: CPA - ql,” %A-c,,, GA- C&H C&A - C,,” GA-c,., %A- C,.” CPA -c&H %A- C pH (I,,* - c,:, ~P.A -(Jim G.“,, + %,H
%4
-
(kH
+ %co + CF.,,”
Heat capacities of groups CH,. CH, NH,, were taken from Gutrie (1977). t Gutrie (1977). $ Jolicoeur & Boileau (1978). 4 Cabani et al. (1981). 1)Makhatadze et al., unpublished. 7 Carboxymethylated.
-5 36 19 75 168 293 395 355 309 83 183 90 85 178 179 347 377 175 168 173 176 332 266 17
CO, OH, 0 and H
Table 3, which gives also the model compounds together with corresponding references to their heat capacity values and to the procedure used to derive the heat capacity of a group from the heat capacity of an analog. The values of the integral heat capacity of extended polypeptide chains, calculated per gram of protein, are given in Table 2. As can be seen, they agree well with the experimental values of the heat capacity of the denatured protein, but are somewhat larger in all cases. The deviation, which does not exceed lo%, is not surprising, as the polypeptide chain, even in the random coil conformation, can hardly be considered as a dilute solution of the constituent groups. It was expected that a heat-denatured protein, which was supposed to be not as unfolded as the Gu.HCl-denatured protein, should have a lesser heat capacity than the protein in a concentrated solution of Gu. HCl (Tanford & Aune, 1970). However, direct calorimetric measurements carried out on lysozyme, showed that, if the GueHCl solvation effects are correctly taken into account, no difference between the Gu.HCl and heatdenatured protein heat capacities can be observed (Pfeil & Privalov, 1976a). Now we see that even a very extended residual structure, which persists in the preliminarily heated aMbh, at pH values close
747
to neutral, does not affect significantly the heat capacity function. Its deviation from the function for a fully unfolded polypeptide does not exceed 7% of the value of the specific heat capacity of the protein, i.e. is less than 20% of the heat capacity difference between the native and denatured states of a protein. It follows then, that the denaturational heat capacity increment cannot be explained by a gradual melting of the residual structure in a denatured protein. It cannot be explained either by the increase of the configurational freedom of the polypeptide chain upon the “disruption” of the According to structure. protein rigid native Sturtevant (1977) and Velicelebi & Sturtevant (1979), the contribution of this effect to the observed denaturational increment of the protein heat capacity cannot be large. This was shown experimentally by measuring the heat capacity change upon melting of membranes, which can be considered as a model of protein denaturation without penetration of water inside the molecule, i.e. without unfolding of the hydrophobic core, but with a significant increase of the configurational freedom (Bendzko et al., 1988). The inconsistency of the explanation of the protein denaturation heat capacity increment by the decrease of the configurational freedom becomes especially evident in considering the cold denaturation phenomenon, which induces a similar heat capacity increase as heat denaturation, notwithstanding the fact that it is induced by decreasing temperature (Privalov et al., 1986). Therefore, the only reasonable explanation for the observed denaturational heat capacity increment is the assumption that it is caused mainly by hydration of the non-polar groups, which are exposed to water upon unfolding of the compact protein structure. This was first suggested by Kauzmann (1959), on the basis of the well-known fact that the transfer of non-polar molecules to water is accompanied by a significant heat capacity increase (Edsall, 1935; for a review, see Franks & Reid, 1973). One of the arguments confirming this assumption is that A$, is found to be proportional to the number of contacts between the non-polar groups in native protein, i.e. actually to the surface area of the non-polar groups that are exposed to water upon folding of a compact protein structure (Privalov & Khechinashvili, 1974; Privalov, 1979). One of the most important facts established by these calorimetric studies of protein performed over a broader temperature range than that examined previously is that the partial heat capacity of a denatured protein is not a linear function of temperature. At temperatures above 6O”C, the slope of this function asymptotically decreases and almost disappears at about 100°C. It is tempting to explain the observed curvature of the heat capacity function by extended heat absorption caused by some gradual process (i.e. the melting of the residual structure), which is superimposed on the trivial temperature-induced increase of the heat
748
i? L. Privalov et al. for the enthalpy
and entropy functions
we have:
T
A;H(T)
= A;H(T,)
+ 1 A:C,dT T,
(7)
@W’) = C~~HV’,W,+ j N$&M’l dT, T, where T, is transition A;H(T,) I
,
100
50 Temperature
150
PC)
Figure 11. Denaturational increment of the partial heat capacity of RNase, Lys and Mb. The dashed lines represent parts of these functions that were obtained by a linear extrapolation of the heat capacity of the native state. The dot-and-dash lines show the behavior when the values measured at 50°C are assumed to be temperature-
independent.
A$,
= (aAEH)/aT and (A$Y,)/T
= (aA$S)/aT,
at which:
- T,A$‘(T,)
= 0.
If A$?, does not depend on temperature, the enthalpy and entropy of protein denaturation will be increasing functions of temperature; namely, the enthalpy will be a linearly increasing function (see Privalov, 1979). However, if A$?, is temperature dependent and decreases to zero at some temperature T,, then AEH and A$S will be functions that are asymptotically approaching some constant value at To. It is most interesting that, for the specific enthalpy and entropy, this constant value appears
capacity of the polypeptide chain. However, the universality of this effect, and the fact that it is observed under conditions when the unfolded polypeptide chain hardly possesses any residual structure, urge us to consider the shape observed for the heat capacity function as an intrinsic property of the unfolded polypeptide chain in water, as we consider the effect of squeezing the hydrodynamic volume of a random coil with increasing temperature. The situation with the heat capacity function of the native state is much less clear, since it can be experimentally determined only to 80°C even for the most thermostable protein, such as CTT (Fig. 10). Within the region 0°C to SOY!, this function appears to be linear but it is not improbable that this linearity is only apparent, being caused by a low-temperature tail of the denaturation heat effect. Assuming this function to be linear above 80°C we would conclude that the denaturational heat capacity increment should be a decreasing function of temperature, as shown in Figure 11. The main argument for the correctness of such an extrapolation is the fact that the heat capacity increment of transfer of non-polar solutes to water behaves in a similar way, asymptotically decreasing with increasing temperature (Gill et al., 1985; Makhatadze & Privalov, 1988). Then, such a behavior of the difference heat capacity function seems to be much more natural since, if it is caused by the influence of protein non-polar groups on the state of surrounding water, one can expect that, at increasing temperature, this influence should diminish and even disappear at a sufficiently high temperature. This is a very important conclusion for evaluating the temperature dependence of the enthalpy and entropy of protein denaturation. Since :
temperature
(8)
to be universal
for
all compact
globular
proteins. This is shown in Figures 12 and 13 for two proteins (RNase and Mb) that differ greatly in their denaturational heat capacity increments. As seen from these Figures, these asymptotic values are very close in magnitude to the values at the intersection of the enthalpy and entropy functions derived on the assumption that AEC, is constant, which takes place at about 110°C. However, while the values at the intersection of these functions at 110°C do not have a direct physical sense (as upon a further extrapolation above 110 “C these functions
, 100
I 50 Temperature
(‘73
Figure 12. Temperature dependence of the specific enthalpy of denaturation of Mb and RNase (per mol of amino acid residues) in solutions with pH and buffer providing maximal stability of these proteins and compensation of heat effect of ionization (see Privalov & Khechinashvili, 1974). The broken extension of the continuous lines represents a region that is less certain due to uncertainty in the A$?, function (see Fig. 10). The dot-and-dash lines represent the functions calculated with the assumption that the denaturation heat capacity increment is temperature-independent.
Heat Capacity
r
749
of Proteins
We are grateful to Drs S. J. Gill and B. G. Bar&s for helpful discussion of the manuscript in its preparation.
References 15 ? 2 T x 3 2
a
IO i 5 4 t 0 t -5 / 1 0 Temperature PC)
Figure 13. Temperature dependence of the specific entropy of denaturation of Mb and RNase (per mol of amino acid residues) under the same conditions as indicated for Fig. 12. The dot-and-dash lines represent the functions calculated on the assumption that the denatured heat capacity increment is temperatureindependent.
are diverging and can increase infinitely), the universal asymptotic values reached above 140 “C are quite interpretable. They seem to correspond to the enthalpy and entropy of protein unfolding in the absence of hydration effects. The final point we would like to call attention to is the following: the only argument for using Gu *HCl and urea denaturation in thermodynamic studies of unfolding (and, thus, folding) of protein structure is that the protein, denatured in this way, is supposed to be in a completely unfolded state. On the other hand, up to the present, we do not have a reliable procedure for evaluating the thermodynamic parameters of conformational transition caused by denaturants. We do not know how to take into account the denaturant solvation effect and, even more importantly, we do not know what kind of reaction we are analyzing and usually only assume for simplicity that it is a two-state transition. The main advantage of studying temperature/pH-induced conformational transitions in proteins is that, in this case, we can measure directly all the thermodynamic parameters required for a complete description of the process, even if it is complicated and proceeds in several discrete steps (see Privalov, 1979, 1982; Privalov & Potekhin, 1986). We can also properly deal with all ionization effects (Pfeil & Privalov, 1976b). But, in such cases, there has always been some suspicion that the results obtained do not adequately describe the protein folding process, since we do not take into account the contributions of residual structure. Now we see that this contribution is, in fact, so small that it can be neglected in many cases.
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by R. Huber