The Unfolding Thermodynamics of c-Type Lysozymes: A Calorimetric Study of the Heat Denaturation of Equine Lysozyme

J. Mol. Biol. (1995) 252, 447–459

The Unfolding Thermodynamics of c-Type Lysozymes: A Calorimetric Study of the Heat Denaturation of Equine Lysozyme Yuri V. Griko1, Ernesto Freire1, George Privalov1, Herman Van Dael2 and Peter L. Privalov1* 1

Department of Biology and Biocalorimetry Center, The Johns Hopkins University Baltimore, MD 21218, USA 2 Interdisciplinary Research Center, K.U. Leuven Campus Kortrijk, B-8500 Kortrijk Belgium

The energetics of the temperature-induced unfolding of equine lysozyme was studied calorimetrically and compared with that of two structurally homologous proteins: hen egg white lysozyme and a-lactalbumin. The structure of each of these proteins is characterized by the presence of a deep cleft that divides the molecule into two regions called the a and b domains. In equine lysozyme and a-lactalbumin the latter domain specifically binds Ca2+. It is shown that, in contrast to hen egg white lysozyme in which the a and b domains unfold as a single cooperative unit, in equine lysozyme the two domains unfold in two separate cooperative stages even in the presence of excess Ca2+. The calcium binding b-domain unfolds at a lower temperature and with more extensive heat absorption than the a-domain. Binding of Ca2+ increases the stability of the b-domain, but even in the holo form it is less stable than the a-domain. The thermodynamic characteristics of Ca2+ binding have been determined, and indicate that it is an entropically driven process. The unfolding of equine lysozyme largely resembles the unfolding of a-lactalbumin, which also unfolds in two stages, but in the latter case the second stage is much less cooperative and proceeds with a smaller and diffuse heat absorption. As a result, the total enthalpy of unfolding of equine lysozyme is significantly larger than that of a-lactalbumin, being almost of the same magnitude as the enthalpy of egg white lysozyme unfolding, which proceeds as a single two-state transition. Analyses of the unfolding enthalpy function of various lysozymes, which bind or do not bind Ca2+, and unfold in one or two stages, have led us to the conclusion that the main reason for the loss of interdomain cooperativity in equine lysozyme is not the cluster of negative charges forming the calcium binding site, but the difference in atomic packing in the interior and at the interface between the a and b domains. 7 1995 Academic Press Limited

*Corresponding author

Keywords: equine lysozyme; calcium binding; unfolding; molten globule; thermodynamics

Introduction Equine lysozyme (EL) is a close relative of hen egg white lysozyme (HEWL). Both belong to the same family of c-type lysozymes and have the same function, similar sequence, and very similar tertiary structure characterized by the presence of a deep Abbreviations used: EL, equine lysozyme; HEWL, hen egg white lysozyme; HL, human lysozyme; LA, a-lactalbumin; Tt , transition temperature; DH, enthalpy change; DS, entropy change; DG, Gibbs free energy change; k, equilibrium constant; Cp , heat capacity; CD, circular dichroism. 0022–2836/95/390447–13 $12.00/0

cleft that divides the molecule into two domains (Figure 1). One of the domains is formed by the central part of the polypeptide chain and contains the only b-sheet in the molecule and some a helical structure (b-domain) while the other one is formed by the two peripheral parts and is rich in a-helical structure (a-domain). The thermodynamic properties of these two molecules, however, are very different: Upon increasing temperature or denaturant concentration, HEWL unfolds in a single two-state transition under all experimental conditions that have been examined, i.e. it behaves as a single cooperative unit (Khechinashvili et al., 1973; 7 1995 Academic Press Limited

448

Equine Lysozyme Unfolding

Figure 1. Schematic representation of the three-dimensional structure of EL according to Tsuge et al. (1992). The b-domain is shown on the right side together with the atoms that form the calcium binding site. The a-domain is shown on the left side of the Figure.

Privalov & Khechinashvili, 1974; Pfeil & Privalov, 1976). For EL, on the contrary, unfolding is complex and proceeds in two separate stages occurring at different temperatures or denaturant concentrations (Morozova et al., 1991). Previously, the heat effect associated with the temperature-induced unfolding was interpreted as including three overlapping transitions (Van Dael et al., 1993). Another property that distinguishes these two proteins qualitatively is their calcium binding ability: HEWL does not specifically bind Ca2+, while EL binds it tightly (Nitta et al., 1988). The Ca2+ binding site in EL, rich in negative charges, is formed by three Asp side-chains and two main-chain carbonyls located in the b-domain (Tsuge et al., 1992). In HEWL the Asp residues are not present, and thus it lacks the ability to bind the positively charged calcium ion. However, a similar calcium binding site is present in a-lactalbumin (LA), a distant relative of the c-lysozymes, with a quite different function but with a very similar three-dimensional structure (Figure 1) (Stuart et al., 1986). LA is also composed of an a and b domain divided by a cleft and its Ca2+ binding site is also formed by three Asp residues located on the continuous domain (Acharya et al., 1991). It is intriguing that this protein also unfolds in two stages upon increasing temperature or denaturant concentration, although with a much more diffuse second stage than EL (Griko et al., 1994a). In the presence of excess Ca2+, however, LA unfolds as a single cooperative unit. Thus, in its thermodynamic properties EL lies somewhere between HEWL and LA. Based on NMR studies of EL, it was proposed that the intermediate state of this protein corresponds in many respects to the molten globule (Van Dael et al., 1993), that appears to be the intermediate state in LA unfolding (Kuwajima, 1989). In that case the second transition should represent the unfolding of the molten globule state of these proteins. On the other hand, there are major differences in the cooperativity of this stage in these two proteins. It is also unclear

Figure 2. Temperature dependencies of the partial heat capacity of EL in 10 mM Na-acetate and Glycine/HCl solutions without calcium at various pH: a, pH 4,5; b, pH 3.1; c, pH 2.5. The broken line represents the heat capacity function calculated for the completely unfolded polypeptide chain of EL. The dot-and-dashed line represents the heat capacity function of the native state of EL obtained by linear extrapolation with a slope specific for the native state of globular proteins.

whether the complex unfolding behavior of these two proteins is somehow associated with the domain organization of their structure and if so why the unfolding of HEWL proceeds as a simple two-state transition. To answer these questions, we performed a detailed calorimetric study of the thermodynamics of EL unfolding and complemented these studies with a systematic analysis of the structural and thermodynamic differences between LA, EL and HEWL.

Results Denaturation of EL in the absence of calcium Figure 2 shows the temperature dependence of the partial heat capacity of EL in acidic solutions without calcium. Under these conditions EL is in the apo-form. In all the solutions at low pH, two partly overlapping heat absorption peaks were observed, indicating that the temperature induced denaturation of EL proceeds in at least two distinct stages. The temperature at which both heat absorption peaks reach their maximum decreases at lower pH values in almost the same manner. The similarity in the pH dependence suggests that the same number of groups become protonated at lower pH for both transitions. At pH 2.5 (curve c) the denaturation of EL starts below 5°C, which accounts for the larger initial partial heat capacity of the protein observed under these conditions. At room temperature, the partial

Equine Lysozyme Unfolding

heat capacity of the native protein can be determined only in solutions at pH > 4.5. At 20°C it equals 21(21) kJ K−1 mol−1 which is close to the value obtained for native LA, 20(21) kJ K−1 mol−1, (Griko et al., 1994a) and native HEWL, 19(21) kJ K−1 mol−1 (Privalov & Khechinashvili, 1974; Pfeil & Privalov, 1976). It is known that the temperature dependence of the heat capacity of native proteins is approximated well by a linear function of temperature with a rather universal slope of about 6.1 × 10−3 JK−1 g−1 (Makhatadze & Privalov, 1990, 1992). This function is represented in Figure 2 by the dot-and-dashed line. The broken line in Figure 2 gives the partial heat capacity expected for the completely unfolded EL, which has been calculated from the known sequence assuming that all amino acid residues are exposed to water, using the known partial heat capacities of amino acid residues in aqueous solutions (Makhatadze & Privalov, 1990). It appears that, upon heating, the heat capacity of EL approaches this expected value after the second heat absorption is complete. Because the partial heat capacity of a protein is a very sensitive indicator of the exposure of protein groups to water (Makhatadze & Privalov, 1992), we can conclude that upon completion of the second transition EL is completely unfolded. At pH 4.5 this occurs above 100°C. Therefore it is not surprising that in the NMR spectra of EL some deviation from the random-coil spectrum has been observed at 82°C (Van Dael et al., 1993). As follows from Figure 2, the calculated difference between the heat capacities of apo-EL in the native and denatured state, is temperature dependent. At 25°C it equals 7.2(20.7) kJ K−1 mol−1, it increases to 7.6(20.7) kJ K−1 mol−1 at 50°C and drops to 6.07 kJ K−1mol−1 at 80° C. For HEWL it was found to be 7.3(20.8) kJ K−1 mol−1 at 50°C (Makhatadze & Privalov, 1992), and for LA it is 7.5(20.8) kJ K−1 mol−1 at this temperature (Griko et al., 1994a). Thus, the total heat capacity increments upon unfolding for these three proteins are quite similar.

449

Figure 3. Temperature dependencies of the partial heat capacity of EL in 10 mM Na-acetate (pH 4.5) in the presence of various concentration of CaCl2 : 1, 0.00 mM; 2, 0.10 mM; 3, 0.75 mM and 4, 1.50 mM CaCl2 . The broken line represents the heat capacity function calculated for the completely unfolded polypeptide chain of EL. The dot-and-dashed line represents the heat capacity function of the native state of EL obtained by linear extrapolation with a slope specific for the native state of globular proteins.

measured heat capacity function of EL does not depend noticeably on the heating rate and its reproducibility upon repeated heating is maximal at low pH provided that the protein is not kept for a long time at high temperature. At pH values close to neutral, the protein aggregates immediately after unfolding, releasing heat in an irreversible fashion (data not shown). Thus, under all conditions studied except at high temperatures and neutral pH the observed process of EL unfolding is close to equilibrium. Therefore, the measured heat effects can be treated by using rigorous thermodynamic methods.

Denaturation of EL in the presence of calcium The temperature dependence of the partial heat capacity of EL in 10 mM Na-acetate buffer (pH 4.5) in the presence of various concentrations of CaCl2 is shown in Figure 3. The increase in calcium concentration leads to a shift of the first heat absorption peak to higher temperatures without noticeably affecting the second peak. This result indicates that the first peak of heat absorption is associated with the disruption of the calcium binding domain and, correspondingly, results in the loss of the ability of EL to specifically bind Ca2+. The second heat absorption peak should be associated with the cooperative disruption of the rest of the molecule, i.e. with the a-domain. The stability of this domain is not affected by the stability of the Ca2+ binding b-domain. It should be noted that the calorimetrically

Analysis of the excess heat capacity function Thermodynamic analysis of the observed excess heat absorption is straightforward in the case of a homogeneous protein solution, i.e. when all protein molecules in the solution are identical and are initially in the same macroscopic state. In the case of a calcium binding protein in which the kinetics of binding is slow, this situation exists only in solutions which do not contain calcium, in which all protein molecules are in the apo form, or in solutions in which the concentrations of calcium are so high that all protein molecules are in the holo form. According to Nitta et al. (1988) the calcium binding constant of EL in neutral solutions, pH 7.0, is 2.5 × 106 M−1. Therefore, one can expect that in the presence of 0.1 mM free CaCl2 , EL will be completely in the holo form. At pH 4.5 the Ca2+ binding constant of EL

450

Equine Lysozyme Unfolding

should be lower. For LA at this pH, it is approximately 105 M−1 (Bratcher & Kronman, 1984). If EL behaves in a similar manner, we can expect that complete saturation with Ca2+ should occur at concentrations of free calcium on the order of 1 mM or higher. From a rigorous statistical thermodynamic point of view, the partial molar heat capacity of a macromolecule, Cp is given by the following equation: Cp = Cp,0 +

0

1 N s P ·DHi 1T i = 1 i

1

(1)

where Cp,0 is the partial molar heat capacity of the reference state, which we take as the native state. The second term accounts for any temperature-induced transition and the possibility that the transition involves an arbitrary number of states, N. In equation (1), Pi represents the population of molecules in the ith state and DHi its relative enthalpy, DHi = Hi − H0 . Expanding equation (1), one obtains N

i=1

(2)

i=1

0

N

Pi = exp(−DGi /RT )/ s exp(−DGi /RT ) i=0

1

= exp(−DGi /RT )/Q

(3a) (3b)

$

N

s DHi2 ·exp(−DGi /RT )/Q i=0

N

i=0

%7

%

DH(T ) = DH(TR ) +

g

T

DCp dT

(6a)

DH(T ) = DH(TR ) + a × (T − TR ) + (b/2) ×(T 2 − T R2 ) + (c/3) × (T 3 − T R3 ) DS(T ) = DS(TR ) +

g

(6b)

T

DCp d ln T

(7a)

DS(T ) = DS(TR ) + a × ln(T/TR ) + b × (T − TR ) + (c/2) × (T 2 − T R2 )

/RT 2

N

i=0

=Cp,0 + 4DH 2 − DH25/RT 2 N

(4)

i=0

The first term on the right-hand side of equation (4) is simply the heat capacity of the reference state; the second term is the transition excess heat capacity term that gives rise to the observed transition peaks; and the third term accounts for the change in the heat capacity ‘‘baseline’’ due to the existence of a heat

(7b)

where TR is the reference temperature. In the analysis of the calorimetric curves, the temperature at which the Gibbs free energy is zero, Tt , is taken as the reference temperature (TR0Tt ). At this temperature, DS(Tt ) = DH(Tt )/Tt . For convenience, equation (5) can be written as: DCp (T ) = DCp (Tt ) + b × (T − Tt ) + c × (T 2 − T t2 )

2

+ s Pi ·DCp,i

+ s Pi ·DCp,i

the relative enthalpies and entropies are written as:

TR

where Q, the sum over all the states is the conformational partition function of the macromolecule (Freire & Biltonen, 1978). Equation (3) allows expansion of equation (2) as (Freire, 1995)

− s DHi ·exp(−DGi /RT )/Q

(5)

TR

where DCp,i is the relative heat capacity of state i. The population of each state is a function of the relative free energy of each state, DGi ,

6$

DCp (T ) = a + b × T + c × T 2

N

Cp = Cp,0 + s DHi ·(1Pi /1T ) + s Pi ·DCp,i

Cp = Cp,0 +

capacity difference between states. The formalism described above and summarized in equation (4) is completely general and independent of the character or mechanism of a transition (Freire & Biltonen, 1978). It does not assume a sequential ordering of states. It is valid for any monomeric macromolecular system which exists in equilibrium between an arbitrary number of states. In the past, equation (4) could not be applied directly, primarily because differential scanning calorimeters lacked the sensitivity and baseline stability for accurate routine determinations of the partial molar heat capacities. It must be noted, that a direct application of equation (4) does not require arbitrary baseline subtractions in the analysis. Equation (4) was used to analyze the heat capacity function of EL in calcium-free solutions and in excess calcium. Since DCp is temperature-dependent and obeys a quadratic function of the form (Griko et al., 1994a; Xie et al., 1995; Gomez et al., 1995)

(8)

where DCp (Tt ) is the heat capacity change at the transition temperature. Equations (4) to (8) are used for deconvolution of the experimental heat capacity function by means of a non-linear least squares procedure. Analysis of the data in the absence of calcium, in which the protein is in the apo form, shows that the temperature-induced changes take place in two distinct temperature ranges (Figure 4). The situation is similar for EL in the presence of high concentration of CaCl2 (Figure 5) except that in this case the first transition is shifted to higher temperatures. Most notably, the second transition occurs at the same temperature and with the same enthalpy in the apo and in the holo form of the protein. Table 1 summarizes the thermodynamic parameters obtained from the deconvolution of the heat

Equine Lysozyme Unfolding

451

Figure 4. Deconvolution of the excess heat absorption of EL in solutions of 10 mM Na-acetate (pH 4.5) without CaCl2 . Top, The experimental and calculated heat capacity curves. The continuous line is the theoretical curves calculated with the deconvolution parameters summarized in Table 1. Also shown by broken lines are the heat capacities of the native and unfolded states. Middle, Temperature dependence of the population of the three states that become populated during the transition, the native (N), intermediate (I) and unfolded (U) states. Bottom, The contributions of the individual N : I and I : U transitions to the heat capacity function.

Figure 5. Deconvolution of the excess heat absorption of EL in solutions of 10 mM Na-acetate pH 4.5 in the presence of 1.5 mM CaCl2 . Top, The experimental and calculated heat capacity curves. The continuous line is the theoretical curve calculated with the deconvolution parameters summarized in Table 1. Also shown by broken lines are the heat capacities of the native and unfolded states. Middle, Temperature dependence of the population of the three states that become populated during the transition, the native (N), intermediate (I) and unfolded (U) states. Bottom, The contributions of the individual N : I and I : U transitions to the heat capacity function.

capacity functions obtained at pH 4.5 for the apo and holo forms. In Figures 4 and 5; the continuous lines represent the theoretical curves calculated with the deconvolution parameters summarized in Table 1. The enthalpy of the first transition is significantly larger than that of the second one. Also the increment in heat capacity is almost twice as large for the first transition. Figure 6 shows the temperature dependence of the enthalpy and entropy changes associated with the first and second transitions for the apo and holo forms of the protein. The enthalpy of the second transition is identical for both the apo and holo forms, indicating that within the experimental error Ca2+ does not affect the character of this transition. The enthalpy of the first transition is also very similar for the apo and holo forms; however, it appears that the

enthalpy of the apo form is slightly larger (010 kJ/mol at 25°C) than that of the holo form suggesting that the binding enthalpy of Ca2+ is slightly positive. Also, the entropy change of the apo form is slightly larger, suggesting that the binding entropy of Ca2+ is positive. In all cases, the enthalpy change is not a linear function of temperature due to the fact that DCp is not constant as illustrated in Figures 2, 4 and 5. The analysis of the heat capacity function of the apo form obtained at different pH values gave results consistent with those summarized in Table 1 except for the shift in transition temperatures. In particular, the heat capacity change estimated from the temperature dependence of the enthalpy changes obtained at different pH were similar to those obtained from individual calorimetric scans.

452

Equine Lysozyme Unfolding

Table 1. Thermodynamic parameters from the deconvolution of the heat capacity function of apo and holo EL at pH 4.5 DH1 (Tt1 ) (kJ/mol) Tt1 DS1 (Tt1 ) (J/K mol) DCp,1 (Tt1 ) (kJ/K mol b1 (J/K2 mol) c1 (J/K3 mol) DH2 (Tt2 ) (kJ/mol) Tt2 DS2 (Tt2 ) (J/K mol) DCp,2 (Tt2 ) (kJ/K mol) b2 (J/K2 mol) c2 (J/K3 mol)

Apo pH 4.5

Holo pH 4.5

153.7 41.5 488.5 5.04 315.8 −0.464 123.8 66.44 364.6 2.56 299.6 −0.46

204.7 54.73 624.3 4.90 315.8 −0.464 133.3 66.2 392.8 2.50 299.6 −0.46

For each transition Tt is the temperature at which DG = 0. The heat capacity change is a quadratic function of temperature: DCp (T ) = DCp (Tt ) + b × (T − Tt ) + c × (T 2 − T t2 ), where the temperature is given in degrees K.

Analysis of a mixture of apo and holo forms In solutions containing less than 1.0 mM free Ca2+, the excess heat absorption function of EL could not be deconvoluted into two transitions. In this case, it was observed that the excess heat absorption curve was represented best by a weighted combination of the heat capacity curves corresponding to the apo and holo forms: Cp = (1 − X(a)) × Cp,apo + X(a) × Cp,holo

(9)

where X(a) is the population of molecules in the holo form and Cp,apo and Cp,holo the characteristic heat capacity functions of the apo and holo forms, respectively. X(a) is a function of the free calcium concentration, a. Since the functional forms for Cp,apo and Cp,holo are known from the deconvolution

Figure 7. The heat absorption peaks in heterogeneous solution of EL in the presence of intermediate concentration of Ca2+. A, 0.1 mM; B, 0.50 mM CaCl2 (10 mM Na-acetate, pH 4.5). The broken lines are the individual contributions to the total heat absorption peaks.

analysis of the homogeneous solutions, it is possible to perform a non-linear least squares analysis of the heat capacity function using equation (9). Strictly speaking X(a) is a function of temperature, however since the binding enthalpy of calcium is small it was assumed that X(a) is constant within the transition region. This procedure was applied to the data obtained at Ca2+ concentrations lower than 1 mM in order to estimate X(a). This behavior results in the presence of three heat absorption peaks (Figure 7), as observed before by Van Dael et al. (1993). Upon increasing the calcium concentration the area of the low temperature peak (peak I, corresponding to the first peak in the apo form) decreases while that of the second peak (peak I', corresponding to the first peak in the holo form) increases at the expense of peak I. As expected the transition temperature of I' increases with Ca2+ concentration. The third or highest temperature peak remains invariant since its thermodynamic characteristics are similar in both the apo and holo forms. This type of behavior is indicative of a very slow binding process that does not equilibrate during the time frame of the calorimetric scan. Determination of Ca2+ binding parameters

Figure 6. The enthalpy (top) and entropy (bottom) changes for the first and second transition of EL in solutions of 10 mM Na-acetate (pH 4.5) in the absence of CaCl2 (apo) and in the presence of 1.5 mM CaCl2 (holo).

As discussed above, there are only two temperature-induced transitions in homogeneous solutions of apo or holo EL and only the first of these transitions is affected by the presence of calcium. Binding of calcium results in a significant increase in the temperature of this transition reflecting a

453

Equine Lysozyme Unfolding

Table 2. Relative population of holo form as a function of the Ca2+ concentration at pH 4.5 a 0 0.10 0.50 0.75 1.50

X(a) 0 0.3920.07 0.720.04 0.8020.06 1.00

k — 6 × 103 5 × 103 5 × 103 —

a, concentration of free calcium ions in mM; X(a), relative population of the ligated (holo) form; k, Ca2+ binding constant in M−1.

Figure 8. Temperature dependence of the Gibbs energy of first and second transition of EL in the apo form and in the holo form obtained in the presence of 1.5 mM Ca2+. The difference between the curves corresponding to the first transition in the apo and holo form is proportional to the Gibbs energy of Ca2+ binding at the given free Ca2+ concentration and can be used to estimate the binding constant as a function of temperature. Continuous line: first transition in apo form; dotted line: first transition in holo form; broken line: second transition in both forms.

heterogeneous solutions in which the heat capacity of EL is represented as a mixture of the apo and holo functions. Since the first transition in the holo form takes place at a higher temperature than in the apo form, the appearance of transition I' in solutions containing intermediate calcium concentrations can serve as an index of the appearance of the holo form. As discussed above, the population of molecules in the holo form, X(a), can be estimated by non-linear least squares analysis of the heat capacity function of EL obtained at intermediate calcium concentrations. Knowing X(a) and the free calcium concentration, a, it is possible to calculate the Ca2+ binding constant k as: k = X(a)/[(1 − X(a))a]

significant stabilization of the corresponding structure. Using calorimetric data we can estimate the Gibbs energy associated with each transition: DG = DH(T ) − T × DS(T ) DG = DH(Tt ) +

g

T

(10a)

DCp dT − T

Tt

0

× DS(Tt ) +

g

T

DCp d ln T

Tt

1

(10b)

The Gibbs energy functions for the first and second transitions in the apo and holo forms of EL are presented in Figure 8. The difference of these functions can be used to calculate the Gibbs energy of Ca2+ binding: DG(a) − DGapo = RT ln(1 + k × a)

(11)

where DG(a) is the Gibbs energy obtained at a free calcium concentration a, and k is the calcium binding constant. According to this analysis, the calcium binding constant equals 6.5 × 103 M−1 at 25°C and increases with temperature as expected for a binding process characterized by a positive enthalpy change. The intrinsic Gibbs energy of calcium binding by EL is given by the equation DGCa = −RT ln(k)

(12)

and equals 22 kJ/mol at 25°C. The calcium binding constant can also be estimated by using the results obtained for the

(13)

2+

It appears that at pH 4.5 the Ca binding constant of EL is between 5 × 103 and 6 × 103 M−1 (Table 2) and, correspondingly, the Gibbs energy of binding is close to 21 kJ mol−1. The perfect correspondence of these values with those obtained from the direct calorimetric estimate of the intrinsic Gibbs energy of binding provides strong support to the assumption that at intermediate Ca2+ concentrations the population of molecules is heterogeneous. This result also indicates that the apo and holo forms of EL exhibit an extremely slow rate of interconversion.

Discussion Thermodynamics of Ca2+ binding According to our results, the binding of Ca2+ to EL is characterized by a binding enthalpy close to 10 kJ/mol and a binding entropy of 105 JK−1 mol−1 at 25°C. Thus, it appears that the entropy, as well as the enthalpy of Ca2+ binding by EL are positive and therefore the binding process is entropically controlled. This conclusion contrasts with previous findings: according to Desmet et al. (1989) the enthalpy of Ca2+ binding by EL is −76 kJ mol−1, and the entropy of binding is −131 JK−1 mol−1. According to Kuroki et al. (1992a), the enthalpy of binding is −40 kJ mol−1 and the entropy of binding is −19 JK−1 mol−1. If both of these parameters are negative this means that Ca2+ binding by EL is an enthalpically driven process. This is in conflict with what is known about Ca2+ binding by chelate compounds such as EDTA and EGTA, and also by other calcium binding proteins. Therefore, if the

454 conclusions of Desmet et al. (1989) and Kuroki et al. (1992a) are correct, EL would appear to be a rather unique calcium binding protein. Their conclusion, however, is based on the results of isothermal calorimetric titration of EL by CaCl2 at 25 and 30°C. As is clear from Figure 4, at 25°C and more so at 30°C, apo-EL is already partly denatured even in neutral solutions. Therefore the heat effect which is measured upon titration of apo-EL by Ca2+ at these temperatures is in fact not the heat effect of calcium binding by the native form of this protein but mainly the heat effect of renaturation of the protein molecules already denatured. This explains why the titration of EL by Ca2+ at 25°C was found to be accompanied by a change in the UV CD spectra (Desmet et al., 1989). A similar situation appears to hold for the experimental determination of the thermodynamic characteristics of Ca2+ binding by LA. The apo form of this protein is even less stable than that of EL and is in a largely unfolded (molten globule) state at room temperature (Griko et al., 1994a). Therefore, the large negative heat effect which has been measured calorimetrically upon titration of LA by calcium at 25°C (Van Ceunebroeck et al., 1985; Kuroki et al., 1992a) may in fact represent the heat effect associated with the refolding of this protein. The enthalpy and entropy of Ca2+ binding by EL obtained in this study is very similar to that found by Kuroki et al. (1992a,b) for the mutant human lysozyme D86/92. Measuring the heat effect by titration microcalorimetry at 30°C, they found that the enthalpy of Ca2+ binding by this protein is 6.7 kJ mol−1 and the entropy of binding is 124 JK−1 mol−1. The advantage of the mutant human lysozyme D86/92 in studying Ca2+ binding is that it is more stable than EL and AL and the titration calorimetric results are not contaminated with a refolding enthalpy. The binding of Ca2+ by the mutant human lysozyme is an entropically driven process, and the same is true for AL (see Griko et al., 1994a) and EL. Thus, thermodynamically EL is not an exceptional calcium binding protein. In calcium binding, the favorable entropic term certainly overbalances the unfavorable enthalpic contribution to the Gibbs energy of binding as it does in simple calcium chelation reactions. The enthalpy of Ca2+ binding by EL is very close to that found for the mutant human lysozyme, but the entropy, Gibbs energy, and correspondingly, the binding constant, are slightly lower. The most probable reason appears to be that our experiments were performed at low pH, in order to avoid aggregation of protein upon heating. Protonation of a binding site in acidic solutions should certainly decrease the entropy of binding and correspondingly the binding constant. Structural assignment of EL transitions As shown in Figure 1, the amino acid residues in EL are packed in two domains which are separated

Equine Lysozyme Unfolding

by a cleft. It is tempting to assume that the observed cooperative transitions correspond to the unfolding of these two domains. Since the first transition is affected by the presence of CaCl2 , it is clear that this transition is associated with the unfolding of the b-domain which is responsible for binding Ca2+. If this is the case, the second transition must be associated with the unfolding of the a-domain which is rich in a-helical conformation. This conclusion is in agreement with the experimental observation that the far UV CD spectra of EL changes significantly in the temperature region of the second transition (Van Dael et al., 1993). A similar conclusion was recently reached by Dobson and co-workers in the study of the refolding kinetics of HEWL by pulse NMR techniques (Dobson et al., 1994; Hooke et al., 1994). According to these authors the a-domain in HEWL folds first and appears to be more stable than the b-domain. In contrast, NMR experiments on EL indicate an extensive although not complete loss of highly ordered tertiary structure during the first transition suggesting that the partly folded intermediate lacks significant tertiary packing (Van Dael et al., 1993). On that basis, it was concluded that the EL unfolding intermediate represents a molten globule state (see also Nitta et al., 1993) such as the one existing in LA. In LA, the region of the molecule that undergoes cooperative unfolding is also the calcium binding b-domain. Under some experimental conditions (e.g. low pH), once this transition has occurred and the b-domain is unfolded the molecule exists in the so-called molten globule state. The molten globule state of a-lactalbumin is characterized by having the b-domain unfolded and by preserving most of the secondary structure of the a-domain but without having the tightly packed tertiary structure that this domain assumes in the native state (Baum et al., 1989; Alexandrescu et al., 1993; Peng & Kim, 1994; Xie & Freire, 1994). The NMR spectrum of the partly folded intermediate of EL, however, is more native like than that of apo-AL (Alexandrescu et al., 1992), which is usually regarded as a typical molten globule. Also, the main changes in the near UV CD, which is sensitive to changes in tertiary structure, occur in the temperature range of unfolding of the partly folded intermediate state (second transition; Morozova et al., 1991; Van Dael et al., 1993). Therefore, the intermediate state of EL certainly preserves a significant degree of tertiary structure. To reconcile this conclusion with the NMR observation one should take into account that the NMR studies of EL were performed at 50°C and pH 4.5. At this pH, the second transition in EL starts at 35°C (Figure 4). At 50°C the Gibbs energy difference between folded and unfolded molecules does not much exceed the energy of thermal motion (Figure 8). Therefore, it is not surprising that the structure observed by NMR at 50°C is strongly fluctuating and is unfolded a considerable fraction of the time.

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Equine Lysozyme Unfolding

Figure 10. Temperature dependence of the total unfolding enthalpy of (1) EL; (2) AL (Griko et al., 1994a), and (3) HEWL (Privalov & Khechinashvili, 1974). The enthalpy function is corrected for ionization effects and does not depend on pH. Figure 9. The temperature dependence of the partial heat capacity of (1) EL at pH 4.5; (2) AL at pH 3.5 (Griko et al., 1994a), and (3) HEWL at pH 2.0 (Privalov & Khechinashvili, 1974).

Cooperative transitions in EL The native structure of EL is very similar to that of HEWL and LA. However, the unfolding behavior of these three proteins is quite different (Figure 9). EL unfolds in two distinct cooperative stages with two distinct heat absorption peaks. LA also unfolds in two stages, however in this case only the first transition is highly cooperative; the second transition is poorly cooperative and occurs with only diffuse heat absorption (see Griko et al., 1994a). In the case of HEWL the unfolding transition always occurs in a single cooperative peak. In this case experimental conditions have never been found in which the two domains behave independently in equilibrium unfolding. Is the difference in unfolding behavior between HEWL, EL and LA due to the existence of a calcium binding site in the latter two proteins? The calcium binding site in the b-domain of EL is formed by three closely located Asp residues and two main-chain carbonyl groups (Tsuge et al., 1992). One can imagine that upon heating (or upon increasing the concentration of a denaturant) the domain with the cluster of negative charges might unfold first because of the destabilizing effect of repulsive electrostatic interactions. HEWL and human lysozyme do not have the calcium binding site and unfold as a single cooperative unit; also, the enthalpy of their unfolding is somewhat higher than that of EL and AL (Privalov & Khechinashvili, 1974; Kuroki et al., 1992b). However, the introduction of a calcium binding site into human lysozyme (HL) by replacement of Gly86Asp and Ala92Asp does not result in a loss of cooperativity and separate unfolding of its domains (Kuroki et al., 1992b). Also, the introduction of the cluster of charges in the structure of this protein leads to a significant decrease in stability but, surprisingly, it does not change the enthalpy function significantly, i.e. the

destabilization induced by the cluster of charges is of an entropic nature. A similar situation is found with pigeon egg white lysozyme. This protein contains an Asp at positions 86, 91 and 92 like EL, and binds calcium, but unfolds in one very cooperative two-state transition (Haezebrouck, 1992; Nitta et al., 1993). The above results indicate that it is not the calcium binding site, i.e. the cluster of negative charges, which are responsible for the loss of interdomain cooperativity in EL and AL. The structural origin of the cooperative behavior of EL According to our current understanding of protein folding, the extreme cooperativity which is specific to globular proteins, results from the tight and unique packing of amino acid residues in the native structure (Privalov, 1979, 1989; Shakhnovich & Finkelstein 1989; Karplus & Shakhnovich, 1992; Griko et al., 1994b). The molten globule state, on the other hand, does not possess a unique tertiary structure (Kuwajima, 1989; Ptitsyn, 1992). In the absence of tertiary interactions one cannot expect global cooperation between all the elements in a protein (Karplus & Shakhnovich, 1992; Shakhnovich & Finkelstein, 1989; Griko et al., 1994b; Freire, 1995). Therefore, unfolding of the molten globule state is expected to be a gradual process which has been regarded by some as a second order phase transition (Shakhnovich & Finkelstein, 1989; Karplus & Shakhnovich, 1992). This reduced cooperativity has been experimentally observed for the temperatureinduced unfolding of apo-myoglobin in acidic solutions (Griko & Privalov, 1994) and denatured apo-LA (Griko et al., 1994a) which are commonly regarded as examples of the molten globule state. The fact that after the second transition the partial heat capacity of EL reaches the value expected for the fully unfolded polypeptide chain shows that this transition results in the complete unfolding of the protein. The value of the heat capacity increment for

456

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(a)

(b) Figure 11. Densely packed regions of HEWL (a) and EL (b). The viewing position is similar to that of Figure 1. Wireframe convex polygons (shown in red) correspond to the regions with packing density higher than 0.7. The a and b domains of HEWL merge into a single dense cluster, while in EL the two domains are separated by a loosely packed cleft area.

457

Equine Lysozyme Unfolding

the second stage, which amounts to about 35% of the total heat capacity increment, makes it clear, that this stage results in the exposure to water of approximately 40% of the surface area of non-polar groups which are screened from water in the native state, i.e. it is connected with the unfolding of a compact nonpolar core. It is also clear that this structure is maintained by enthalpic forces since its disruption results in a significant increase of enthalpy. The positive enthalpy of protein unfolding at elevated temperatures is provided mainly by the disruption of van der Waals interactions between tightly packed protein groups. At 80 to 85°C the enthalpy effect of hydration of non-polar groups upon protein unfolding is very small and the enthalpy contribution of hydrogen bonds is negative (Makhatadze & Privalov, 1993, 1995). Therefore, analysis of the enthalpy functions may provide information about the packing of groups in the structure of EL which unfolds on the second stage. Figure 10 shows the temperature dependence of the unfolding enthalpy of EL, LA and HEWL. It is clear from this Figure that HEWL has the highest enthalpy, followed by EL whereas LA exhibits the lowest unfolding enthalpy of the three proteins. At 80°C the enthalpy of overall unfolding of LA equals 450 kJ mol−1 (Griko et al., 1994a), that of EL equals 513 kJ mol−1 and that of HEWL equals 575 kJ mol−1 (Privalov & Khechinashvili, 1974). It was already observed before that the heat capacity of the native state of LA is slightly higher than that of HEWL (Griko et al., 1994a) suggesting that its structure is somewhat looser. This conclusion can be verified by examining the crystallographic structure of the three proteins. The results of this analysis are presented in Figure 11 and reveal significant differences in the packing at the interface between the a and b domains. In HEWL the interface is tightly packed forming a single, extended densely packed cluster. In contrast, the groups at the interface in EL are loosely packed, defining two well defined densely packed clusters. The overall volume of these two clusters is lower than that of the single cluster in HEWL. These observations explain the differences in total unfolding enthalpy for these two proteins. It is also noteworthy, that in EL the densely packed cluster corresponding to the a-domain is smaller in volume than that of the b-domain. Accordingly, the unfolding enthalpy and corresponding heat capacity increment of the a-domain in EL are lower than those of the b-domain. Unfortunately, the atomic coordinates for bovine LA are not known and we can only assume that its structure is similar to that of HEWL and EL, since the structures of these two proteins are very similar to that of baboon LA which has been studied crystallographically at high resolution (Acharya et al., 1991). However, if we consider the calorimetric results obtained for bovine LA (Griko et al., 1994a) we can expect that this protein should also exhibit a low packing density at the domain interface and,

furthermore, a lower packing density in the adomain than that observed for EL. These structural characteristics would explain the low enthalpy and unfolding cooperativity observed for the a-domain of bovine LA. The last remaining question is why the a domains in EL and AL are more thermostable than in HEWL if they are more loosely packed and have fewer stabilizing contacts. The answer is obvious: they are more stable precisely because they are more loosely packed and therefore they gain less entropy upon unfolding.

Materials and Methods Equine lysozyme (EL) was obtained from Clydesdale horse milk as described before (Haezebrouck et al., 1992). The residual content of Ca2+ was less than 0.03 mol/mol of protein. The protein concentration was determined spectrophotometrically using the extinction coefficient 1% E280 = 23.5 (Desmet et al., 1989). Apo-EL denaturation was studied in acidic solutions at various pH values (10 mM Na-acetate and Glycine/HCl buffers) which did not contain calcium. In order to exclude calcium contamination all glassware was treated with 0.1 M HCl and EDTA. Calcium was introduced in solution when needed by long dialysis against calcium-containing buffer solution. The free calcium concentration in the protein sample was considered to be equal to the concentration in this solution. Calorimetric studies were performed using a DASM-4 scanning microcalorimeter and a DS92 scanning microcalorimeter built at the Biocalorimetry Center, at heating rates ranging from 0.5 to 2.0 K/min, using solutions with protein concentrations between 0.8 and 3.0 mg/ml. The partial specific heat capacity was determined as described by Privalov & Potekhin (1986). To expand the temperature range, all calorimetric experiments were performed under an excess pressure of 1.5 atm. The expected partial heat capacity for unfolded EL was calculated assuming that amino acids contribute additively to the heat capacity of the unfolded polypeptide chain, using the heat capacity values of individual amino acid residues given by Makhatadze & Privalov (1990). Deconvolution analysis of the excess heat absorption was carried out using the algorithm of Freire & Biltonen (1978) extended for explicit consideration of the heat capacity difference between states and the temperature dependence of the heat capacity (Montgomery et al., 1993; Griko et al., 1994a; Freire, 1994; Xie et al., 1995). Packing density was analyzed using a continuous packing density function algorithm (G.P. & E.F., unpublished). The images of high density clusters were generated by convex isosurfaces that localize the areas with packing density greater than any given number in the range of 0-1.

Acknowledgements The authors thank Professor Shintaro Sugai for providing the coordinates for the three-dimensional structure of equine lysozyme and Professor Christopher M. Dobson for helpful discussion of the results. This work

458 is supported by NIH grants GM48036-01, RR04328 and NSF grant MCB9118687. The research of H. Van D. is supported by grants from the Research Council of the K.U. Leuven and the Belgian F.G.W.O.

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Edited by P. E. Wright (Received 21 February 1995; accepted 5 July 1995)