Salt-Induced Formation of the Molten Globule State of Cytochrome C

Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10325-10329, October 1994 Biochemistry Salt-induced formation of the molten globule state of cytochrome c studied by isothermal titration calorimetry (compact denatured state/anion binding/protein folding/thermal unfolding) DAIZo HAMADA*, SHUN-ICHI KIDOKOROt, HARUMI FUKADA*, KATSUTADA TAKAHASHI*§, AND Yuji GOTo*§1 *Department of Biology, and IMicrocalorimetry Research Center, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan; tSagami Chemical Research Center, Nishiohuma 44-1, Sagamihara, Kanagawa 229, Japan; and tLaboratory of Biophysical Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan Communicated by Julian M. Sturtevant, June 30, 1994 (receivedfor review March 2, 1994)

ABSTRACT Although the molten globule state has been bic interaction during protein unfolding is probably divisible proposed as a major intermediate of protein folding, it has into two processes; one is the hydration of nonpolar groups, proven difficult to obtain thermodynamic data characterizing and the other is the disruption of van der Waals interactions this state. To explore another approach for characterizing the between nonpolar groups. In this paper, we use the term molten globule state, salt-induced formation of the molten hydrophobic interaction to mean the total interactions in- globule state of horse cytochrome c at pH 1.8 was studied by volved in the transfer of nonpolar substances from water to isothermal titration calorimetry. By titrating the acid-unfolded a nonpolar environment. Recently, it has been proposed that cytochrome c with sodium perchlorate, an exothermic reaction a significant proportion of the stabilization of protein native was observed. The titration curve obtained from the heat was structure arises from the van der Waals interactions between cooperative and agreed well with the conformational transition nonpolar groups and the hydrogen bonds between polar curve measured by CD at 222 nm. This result indicated that the groups and that the contribution ofnonpolar group hydration salt-induced conformation change is well approximated by a is much less significant than once imagined (9). two-state transition between the acid-unfolded and molten To understand the mechanism of protein folding, it is globule states. The heat for formation of the molten globule important to know how hydrophobic interactions contribute state estimated by isothermal titration calorimetry was consis- to the stability ofthe molten globule state. Thermal unfolding tent with the enthalpy change for unfolding of the sodium of the molten globule state of horse cytochrome c is a perchlorate-stabilzed molten globule state at pH 1.8, which cooperative process with a small but distinct ACpu and the was measured by differential snning calorimetry and CD. enthalpy change of unfolding, AHu (10, 11). Recently, Nishii These results indicate that the heat of titration largely reflects et al. (12) indicated that the molten globule states of apo- the enthalpy change of the conformational transition. From myoglobin exhibit cold-denaturation, demonstrating that these results, we consider that isothermal titration calorimetry ACp,u upon unfolding is positive. These results suggest that will become a useful approach for investigating the molten a small, but distinct, contribution ofhydrophobic interaction globule state. to the stability of the molten globule structure is common to many proteins. In addition, the theoretical calculation pre- The molten globule state, first identified for cytochrome c (1, dicts a relatively large value of ACp,u and AHu for the 2) and a-lactalbumin (3, 4), is a compact denatured state with unfolding of the molten globule states (13). a significant amount ofnative-like secondary structure, but a On the other hand, differential scanning calorimetry (DSC) largely disordered tertiary structure, and has since been showed that the thermal unfolding ofthe molten globule state recognized for various other proteins (3, 4). The location of of a-lactalbumin is accompanied by no distinct enthalpy and secondary structure in the equilibrium molten globule states heat capacity changes, suggesting that no significant hydro- of several proteins has been characterized using the two- phobic interaction is retained in the molten globule structure dimensional 1H-NMR and amide proton/deuterium exchange (refs. 3 and 4; see also ref. 14 for contradictory results). Griko techniques (4, 5). Kinetic studies of several proteins have and Privalov (15) reported that, although the thermal unfold- indicated that the equilibrium molten globule state is analo- ing ofthe molten globule ofapomyoglobin is accompanied by gous to the rapidly formed kinetic intermediate of protein a positive ACp,u, it proceeds without AHu, suggesting that refolding (3-6). Now, the molten globule state is assumed to the unfolding process represents a second-order phase tran- be a major intermediate state ofprotein folding. At the same sition. This situation indicates the necessity for an additional time, it is becoming apparent that there is a range of molten and sensitive approach, which should be complementary to globule states from the relatively disordered to the highly DSC, examining the calorimetric properties of the molten ordered. However, much remains unknown-particularly, globule state. the thermodynamic mechanism responsible for the confor- Recently, isothermal titration calorimetry (ITC) has mational stability. yielded a large amount of excellent data on the interactions The positive heat capacity change (ACpu) observed for the between proteins and their ligands (16, 17). The sensitivity thermal unfolding of native proteins indicates that hydropho- and reliability of ITC are so high that, under suitable condi- bic interaction is the dominant driving force stabilizing the tions, it is capable of detecting a reaction with a small native structure (7, 8). It should be mentioned that, whereas enthalpy change undetectable by DSC. the term hydrophobic interaction is generally used to explain Various basic proteins, including cytochrome c, are sig- various aspects of nonpolar substances, the definition of nificantly unfolded at pH 2 in the absence ofsalt, but addition hydrophobic interaction is sometimes controversial, and its of anions, from either salt or acid, causes stabilization of the exact mechanism is still unknown. The change in hydropho- compact molten globule state (18, 19). The mechanism can be

The publication costs of this article were defrayed in part by page charge Abbreviations: DSC, differential scanning calorimetry; ITC, isother- payment. This article must therefore be hereby marked "advertisement" mal titration calorimetry; Tm, midpoint temperature of unfolding. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 10325 Downloaded by guest on October 1, 2021 10326 Biochemistry: Hamada et al. Proc. Natl. Acad Sci. USA 91 (1994) interpreted in terms of preferential binding of the anions to 250 the compactly folded molten globule state compared with the expanded unfolded state (18). We expected that, by ITC 200 analysis of the anion-induced conformational change of proteins, we might be able to elucidate the calorimetric features ' of the molten globule state. In this paper, we report ITC 150 measurements of the salt-induced stabilization of the molten globule state of horse cytochrome c. o 100

MATERIALS AND METHODS 50 Materials. Horse cytochrome c (type VI) was from Sigma. Acetylated cytochrome c, of which 10 lysyl amino groups 0 were acetylated with acetic anhydride, was prepared as described (19). Methods. Most experiments in this study were done in the 250 presence of20 mM HCl, pH 1.8. The pH was measured using a Radiometer pH meter, model PHM83, at 20TC. Stock protein solution (10-20 mg-mli') was prepared by dialysis 200 against 20 mM HCl. V- Stepwise titration calorimetry was done by using an iso- 150 thermal titration calorimeter, OMEGA, from Microcal, Northampton, MA (16). Cell volume was 1.3 ml, the protein 0 100 concentration was 2 mg-ml-. Titration was done with injections of1-3 y1 each ofa 2-3 M salt solution in 20 mM HCl with a 100-iA syringe at temperatures from 200C to 40TC. The 50 recorded time course of the change in heat from the baseline corresponds to the heat effect due to the change in salt 0 concentration before and after each injection. The apparent 0 2000 4000 6000 heat change observed includes the heat effects due to several Time, sec possible reactions in addition to the heat of conformational change. The heat of dilution of added salt was corrected by UI II subtracting the heat of titration in the absence of protein. o 0000000 Consideration ofother heats will be described later. Although - the heat effect caused by the initial injection had a tendency to be slightly smaller than that expected from the later injections, presumably because of minute diffusion of the -2 0 0~~0 titrant from the syringe tip during the period of thermal E -8 0 0O~~~ equilibration, we did not correct it in the present experi- 0 ments. x Calorimetry ofthe thermal unfolding ofthe molten globule state at pH 1.8 in the presence of various concentrations of -10- 0 0 sodium perchlorate (NaClO4) was measured with a differen- 00 tial scanning calorimeter, DASM-4, from Mashpriborintorg, -12 I IA I_ H ! Moscow. Protein concentration was 5 mg-mli', which was 0 5 10 15 20 25 higher than that used for the ITC measurement. The temper- Injection number the ature was scanned at 1 K-min-1. The reversibility of FIG. 1. (A) Typical calorimetric titration of the acid-unfolded unfolding was checked by reheating; unfolding was found to cytochrome c (4 mg-ml-) with NaC04 at 200C in 20 mM HCl, pH be quantitatively reversible. Data analysis was based on the 1.8. One microliter of2 M NaCl04 was added for each injection. (B) method of Sturtevant (20), including the curve-resolution Control titration in the absence of protein. (C) Heat effect involved procedure. It was found that the transition between the with each injection, which was obtained after subtracting the effect molten globule and unfolded states could be adequately of salt dilution. represented by a two-state process. Data were analyzed by a non-linear least-squares curve-fitting program to obtain the RESULTS AND DISCUSSION midpoint temperature of unfolding (Tm), calorimetric AHu, Titration Calorimetry ofthe Molten Globule State. Whereas and van't Hoff AHu values. various salts can induce the refolding transition of acid- CD spectra were measured with a Jasco spectropolarime- unfolded cytochrome c to the molten globule state, the ter, model J-500A, as described (19). The results were potential of salt varies substantially depending on the anion expressed as mean residue ellipticities, [0]. For measure- species and follows the electroselectivity series, indicating ments ofthermal unfolding, the change in ellipticity at 222 nm the direct interaction of anions and positive charges on the was monitored. Protein concentration was 0.1 mgml-', and protein (18). Because a large volume of protein solution (1.3 a cell with a 1-mm light path was used. The temperature was ml) would be titrated with a small volume ofsalt solution (100 scanned at 1 K-min-1 with a thermostatically controlled cell A4 at most), the choice of salt species is of practical impor- holder and was monitored with a microprobe thermometer. tance. In the present study, we first used NaCl04; the The unfolding was >95% reversible. Unfolding transition midpoint salt concentration (Cm) ofthe refolding transition at curves were analyzed from the van't Hoff plot, assuming a pH 2 and 20TC measured by CD was 10 mM, being much less two-state transition and linear baselines. than that (100 mM) of NaCl (18). Downloaded by guest on October 1, 2021 Biochemistry: Hamada et al. Proc. Natl. Acad. Sci. USA 91 (1994) 10327 A control titration done in the absence ofprotein. Each peak in Fig. 1 A and B corresponds to the shift of heat, arising from the change in NaClO4 concentration before and after each injection. Fig. 1B shows that in the absence ofprotein, alarge positive heat of dilution was observed, indicating that the dilution ofNaClO4 solution is an endothermic reaction. In the E. presence of cytochrome c, the titration pattern indicated negative heat effect, in addition to the heat of dilution of NaClO4. Fig. 1C shows the net heat effect observed at each injection of NaCl04, which was obtained by subtracting the heat of salt dilution. As can be seen, the negative heat effect had a maximum and leveled off with the progress oftitration. E This result suggested that the net exothermic heat change E arose from the formation of the molten globule state and that [NaCIO4] (mM) it represented the enthalpy change for the formation of the molten globule state (&HF). B The net heat effect observed at each injection of NaClO4 was integrated to obtain the total heat ofreaction (QtotJ). Fig. -l' 2A shows the plots of Qtow against NaClO4 concentration at B) 20 and 300C, which represent a saturation curve with a ~0 sigmoidal characteristic. a.) Mechanism of the Salt-Induced Conformational Transition. Cr 0 Goto et al. (18) proposed that, because anions can interact with positive charges ofboth the unfolded and molten globule c'a states, the salt-induced formation of the molten globule state should be explained in terms ofpreferential binding ofanions to the compactly packed molten globule state compared with the expanded unfolded state. At pH 2, most titratable groups are protonated and, hence, the net charge of the molten [NaCIO4] (mM) globule state is essentially the same as that of the fully unfolded state at the same pH. However, because the anion C binding arises from an electrostatic interaction, the compact molten globule state with higher charge density binds anions more tightly than the unfolded state. This results in stabilization of the molten globule state with an increase in salt concentration. Assuming a two-state transition between the acid-unfolded state (U) and the molten globule state (MG),

U = MG, [1]

and the preferential anion binding to the molten globule state, the equilibrium constant (Kpp = [MG]/[U]) for formation of the molten globule state can be described as 0 20 40 60 80 100 120 [NaCIO4] (mM) nMG nU Kapp = >, [MG-Xi]III>. [UXi]J , [21 FIG. 2. NaC104-induced conformational transition of cytochrome c in 20 mM HCl, pH 1.8, at various temperatures. (A) where [MG-Xi] and [U-Xil indicate the concentrations of the Transition monitored by the calorimetric titration. (B) Transition in i monitored by the ellipticity at 222 nm. (C) Normalized transition molten globule and the unfolded states, which moles of curves measured by the calorimetric titration (clear symbols) and anion, X, are bound to 1 mol ofprotein, respectively (22). The ellipticity (solid symbols). Temperatures are at 200C (circles), 300C fraction ofthe molten globule state ('MG) can be calculated by (triangles), and 40"C (squares). Diamonds in A show the titration of cytochrome c with 10 acetylated amino groups at 200C. Dotted lines fMG = Kapp/(l + Kapp). [31 in A show the baselines for the normalization; solid lines show the theoretical curves calculated on the basis of an approximate equation On the basis of the preferential binding model, Qtot for K.pp:Kapp = Ko{(1 + kfdA])/(l + ku[A])}", where Ko is the observed by titration calorimetry consists of the heat of equilibrium constant in the absence of salt, kf and k. are the conformational change (QJzO including the heat of interac- anion-binding constants per binding site for the molten globule and with to unfolded states, respectively, [A] is the concentration ofperchlorate, tion water, and those of anion binding the molten and n is the number of binding sites. The Ko values at different globule (QdG) and the unfolded states (Qbsxd): temperatures were obtained from Hagihara et al. (21), and n was assumed to be the number of positive groups (+24). The kf and ku Qtotal = Qconf + bind+ QMind [41 values, which are =60 and 40 (M-1), respectively, were adjusted so that the calculated curve fits the observed curves. Although the With Eqs. 2 and 3 and an assumption that the enthalpy change fitness of the calculated curves for the experimental data points are (Ah) for the anion binding to each binding site is the same, fairly good, we do not discuss here the validity of the parameters. Qtot is expressed as / [MG raw i Fig. 1A shows the data for the titration of cytochrome Qtotal = AHF'fMG + AhifMG' ffMG-Ml / [MG-M] c with NaClO4 at pH 1.8 and 20TC, and Fig. 1B shows the Downloaded by guest on October 1, 2021 10328 Biochemistry: Hamada et al. Proc. Natl. Acad. Sci. USA 91 (1994) Table 1. AHF for the NaCl04 or Na2SO4-induced refolding + Ah-(l -fMG).() i[U.Xi])j(l U.Xi]). [5] transition of cytochrome c at pH 1.8 determined by ITC AHFo ACpF*, To obtain Qolf we need to know the contribution of the Temperature, 0C k.Fmol- kJ-moh'-K-1 heat of anion bindings (i.e., Qbli.d and Qblid). Although direct NaClO4 measurement of the heat of anion binding is difficult, its 20 -122.5 amplitude might be inferred from a control titration in which 25 -134.7 a protein binds anions but does not exhibit the conforma- 30 -177.7 -2.4 ± 1.0 tional change. For such a control experiment, we carried out 35 -160.6 calorimetric titration ofcytochrome c ofwhich 10 lysyl amino 40 -169.0 groups were acetylated. As described by Goto and Nishikiori Na2SO4 (19), an increase in the number of acetylations stabilizes the 20 -119.6 molten globule state at pH 2. The cytochrome c with 10 25 -140.9 I acetylated amino groups assumes a molten globule state, 30 -172.4 -1.9 ± 0.9 even in the absence of salt at pH 2, where the intact protein 35 -168.1 is unfolded (19). 40 -153.9 As shown in Fig. 2A, the excess heat upon titration of the *Heat capacity change upon formation of the molten globule state acetylated cytochrome c was very small, being within the error obtained from the slope for the plot of AHF against temperature. of the measurements. Because the net charge of intact cytochrome c at pH 2 is +24, that of the species with 10 Fig. 3 shows the plots ofAHF against temperature, which can acetylated amino groups is predicted to be +14. Therefore, the be approximated by a linear correlation. The slope of the acetylated species should bind perchlorate, even though the plots gives the heat capacity change upon formation of the affinity is weaker than that of the intact protein. This result molten globule state (ACP,F). The estimated ACP,F value was suggests that the heat ofanion binding (Q and QbU,.d) is small -2.4 ± 1.0 kUmol-l K-l. We also carried out ITC measure- compared with the heat of conformational change (Qca) and ments of the Na2SO4-induced stabilization of the molten that the heat of titration (Qtota) is approximated by AHFfMG. globule state. As shown in Table 1 and Fig. 3, the results were To confirm this idea, we measured the NaCl04-induced very similar to those obtained with NaCl04. These values transition by the ellipticity at 222 nm (Fig. 2B) and compared were comparable in magnitude to ACp,u of the chloride- it with that monitored by ITC. In the normalization ofthe ITC induced molten globule state measured by DSC (10) or CD results, because a gradual exothermic heat change followed (11, 21) and were much smaller than (5.3 K-1) the major transition, we assumed an asymptote for the ACp,u Ukmol-l for the native state see titration curve, as shown in the broken lines in Fig. 2A. The (ref. 21; also refs. 10 and 11). significance of the slight slope for the baseline is unclear at To compare the thermodynamic parameters obtained by present. It is possible that the slope arises from the anion ITC with those for thermally induced unfolding ofthe molten binding to the molten globule state (QbmjGd), although such a globule state, the scanning calorimetric and CD spectro- slope was not evident for the acetylated cytochrome c. scopic measurements were made on the NaCl04-stabilized Fig. 2C compares the normalized transition curves deter- molten globule state at pH 1.8. Fig. 4 A and B shows the mined by the heat and CD at 20, 30, and 40°C. With increase thermal unfolding of the molten globule state in the presence in temperature, the salt concentration necessary for stabiliz- of various concentrations of NaCl04 at pH 1.8 measured by ing the molten globule state increases. As can be seen, the DSC and CD at 222 nm, respectively. Although no excess transition monitored by heat agreed very well with that DSC signal was observed in the absence of salt, because the detected by CD, further supporting the idea that Qconf largely protein was unfolded at any temperature, a broad but distinct contributes to Qtota. The mechanism of the salt-induced stabilization of the molten globule state ofcytochrome c is controversial (23-26). Goto and coworkers (23, 24) proposed that the equilibrium transition at pH 2 is consistent with a two-state mechanism E on the basis ofthe high cooperativity and the agreement ofthe .200 transition curves measured by various methods, including small-angle x-ray scattering, which directly measures the size of a protein particle. On the other hand, Jeng and Englander AT (25) reported an accumulation of the extended helical state 0100 (premolten globule state) in 2H20 at p2Happ 2.2 (pH meter reading) under low salt conditions. The premolten globule state is located between the molten globule and fully unfolded ,, state, and its involvement is inconsistent with a two-state 10 20 30 40 50 60 mechanism. Karplus and Shakhnovich (26) argued that the IC apparent high cooperativity of the salt-induced stabilization Temperature, ofthe molten globule state does not necessarily mean that the FIG. 3. Dependence on temperature ofthe - HF (open symbols) transition is a first-order phase transition or two-state tran- and AHu (solid symbols) values of the molten globule state of horse sition but might be explainable in terms of a gradual confor- cytochrome c. AHF values are obtained from the calorimetric titra- mational change. As shown here, the agreement of the tion with NaCl04 (o) and Na2SO4 (0). AHu values are the van't Hoff transition curves determined by titration calorimetry and CD enthalpy change obtained from the thermal unfolding ofthe NaC104- spectroscopy (Fig. 2C) provides definitive evidence support- stabilized molten globule state, detected by DSC (*) or CD (A). The solid line is the one drawn by the least-squares analysis of the AHF ing the first-order phase transition at pH 2. values with NaC104, from which the heat capacity change for the Comparison of Salt-Induced Refolding with Heat-Induced formation of molten globule state was obtained to be -2.4 ± 1.0 Unfolding. Assuming that the heat of titration arises largely kJ-K1-mol1. The temperature dependence of the unfolding en- from the conformational change, the heat extrapolated to thalpy of native cytochrome c (21) is shown by the broken line for zero concentration of NaCl04 gives AHF (Fig. 2A, Table 1). comparison. Downloaded by guest on October 1, 2021 Biochemistry: Hamada et al. Proc. Natl. Acad. Sci. USA 91 (1994) 10329 A Table 2. Thermodynamic parameters for the thermal unfolding of 0 the molten globule state stabilized by NaCl04 determined by DSC and CD at 222 nm -5- NaCI04, Tm, Calorimetric van't Hoff ACp,u*, E mM OC A-u, kJ-mol-1 AHu, kJmoi-1 kJ.mol-LK1 -10 DSC 20 34.4 135.3 133.1 ut-15 30 40.0 131.5 131.5 1.3 ± 0.5 40 42.6 145.8 145.8 (1.1 ± 0.6) 50 44.1 141.1 141.1 -20 60 46.9 148.1 148.1 CD B 10 25.8 117.9 20 37.0 135.9 1.8 + 0.1 E -4 50 47.0 159.1 E 100 52.7 164.8

cs -6 *Heat capacity change obtained from the temperature dependence of E van't Hoff AHu. The value in parenthesis shows the heat capacity o change obtained with calorimetric AHu. a) ITC, as we demonstrated here with cytochrome c. To under- o -10 stand the mechanism ofprotein folding, it is critically important - 1 to elucidate the thermodynamic mechanism of the stability of yC 21 the molten globule state in comparison with that ofthe native 40 60 state. We consider that ITC measurement will become a prom- Temperature, 'C ising approach for this purpose. FIG. 4. Thermal unfolding of the molten globule state of horse We thank Prof. Julian M. Sturtevant ofYale University for critical cytochrome c at pH 1.8 in the presence of various concentrations of reading ofthe manuscript. This work was supported by the Ministry NaCl04, measured by DSC (A) and ellipticity at 222 nm (B). Numbers of Education, Science and Culture of Japan and by the Hyogo refer to the concentration of NaCl04 in millimolar units. Dots repre- Science and Technology Association. Contribution No. 83 from the sent observed data points; solid fines indicate the theoretical curves Microcalorimetry Research Center. calculated on the basis of the thermodynamic parameters given in 1. Ohgushi, M. & Wada, A. (1983) FEBS Lett. 164, 21-24. Table 2 and the equations in ref. 20 for A and the Gibbs-Helmholtz 2. Ohgushi, M. & Wada, A. (1984) Adv. Biophys. 18, 75-90. equation (21) with a constant ACpu value of Table 2 for B. Broken 3. Kuwajima, K. (1989) Proteins Struct. Funct. Genet. 6, 87-103. lines show the baselines used for the analyses described in text. 4. Ptitsyn, 0. B. (1992) in Protein Folding, ed. Creighton, T. E. (Freeman, New York), pp. 243-300. excess heat capacity curve was observed in the presence of 5. Baldwin, R. L. (1993) Curr. Opin. Struct. Biol. 3, 84-91. NaClO4 with the peak temperature being increased with the 6. Jennings, P. A. & Wright, P. E. (1993) Science 262, 892-896. increase in salt concentration (Fig. 4A). The apparent Tm 7. Privalov, P. L. & Makhatadze, G. I. (1992) J. Mol. Biol. 224, of 715-723. measured by CD was consistent with the peak temperature 8. Oobatake, M. & Ooi, T. (1993) Prog. Biophys. Mol. Biol. 59, the excess heat capacity curve (Fig. 4 A and B). 237-284. The DSC curves were analyzed on the basis of the method 9. Privalov, P. L. & Makhatadze, G. I. (1993) J. Mol. Biol. 232, described by Sturtevant (20), and it was found that the 660-679. unfolding could be adequately represented by a two-state 10. Potekhin, S. & Pfeil, W. (1989) Biophys. Chem. 34, 55-62. 11. Kuroda, Y., Kidokoro, S. & Wada, A. (1992) J. Mol. Biol. 223, process at all salt concentrations. The transition curves 1139-1153. observed by CD spectroscopic measurement were analyzed 12. Nishii, I., Kataoka, M., Tokunaga, F. & Goto, Y. (1994) on the basis of the van't Hoff relation. The Tm, calorimetric Biochemistry 33, 4903-4909. AHu, and van't Hoff AHu values obtained by these proce- 13. Haynie, D. & Freire, E. (1993) Proteins Struct. Funct. Genet. dures are summarized in Table 2. The ACp,u values were 16, 115-140. obtained from the temperature dependence of AHu and are 14. Xie, D., Bhakuni, V. & Freire, E. (1993).J. Mol. Biol. 232, 5-8. 15. Griko, Y. V. & Privalov, P. L. (1994) J. Mol. Biol. 235, also included in Table 2. 1318-1325. The AHu values obtained by DSC and CD measurements 16. Wiseman, T., Williston, S., Brandts, J. F. & Lin, L. N. (1989) are superimposed in Fig. 3. These AHu values were consis- Anal. Biochem. 179, 131-137. tent with the AHF values obtained with ITC. If we consider 17. Connelly, P. R., Varadarajan, R., Sturtevant, J. M. & Rich- the fact that the salt-induced refolding and the thermal ards, F. M. (1990) Biochemistry 29, 6108-6114. unfolding reactions follow a reaction in opposite directions, 18. Goto, Y., Takahashi, N. & Fink, A. L. (1990) Biochemistry 29, 3480-3488. the consistency of AHu and AHF or A-Cp,u and ACP,F is not 19. Goto, Y. & Nishikiori, S. (1991) J. Mol. Biol. 222, 679-686. surprising. However, if we consider that the parameters were 20. Sturtevant, J. M. (1987) Annu. Rev. Phys. Chem. 38, 463-488. obtained from different reactions using different techniques, 21. Hagihara, Y., Tan, Y. & Goto, Y. (1994) J. Mol. Biol. 237, this consistency would be remarkable. 336-348. Cnlusi. We have shown using horse cytochrome c that 22. Tanford, C. (1970) Adv. Protein Chem. 24, 1-95. ITC provides a reliable calorimetric enthalpy change for the 23. Kataoka, M., Hagihara, Y., Mihara, K. & Goto, Y. (1993) J. formation of the salt-induced molten globule state from the Mol. Biol. 229, 591-596. 24. Goto, Y., Hagihara, Y., Hamada, D., Hoshino, M. & Nishii, I. acid-unfolded state. A number ofproteins show salt-dependent (1993) Biochemistry 32, 11878-11885. conformational changes similar to that of cytochrome c (18). 25. Jeng, M. F. & Englander, S. W. (1991) J. Mol. Biol. 221, However, the cooperative thermal transition with distinct AHu 1045-1061. and ACpu has been established only for cytochrome c. It is 26. Karplus, M. & Shakhnovich, E. I. (1992) in Protein Folding, probable that these thermodynamic features can be explored by ed. Creighton, T. E. (Freeman, New York), pp. 127-195. Downloaded by guest on October 1, 2021