- Email: [email protected]
Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c
BI()('HIMICA ET BIOPHYSICA AC'IA
ELSEVIER
Biochimica et BiophysicaActa 1298 (1996) 102-108
BB3
Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c T o m m a s o F e r r i a, A l e s s a n d r o P o s c i a a, F r a n c a A s c o l i b, R o b e r t o S a n t u c c i b,* a Dipartimento di Chimica, Universit~ di Roma 'La Sapienza ', 00185 Roma, Italy b Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universit2t di Roma 'Tor Vergata ', 00133 Roma, Italy
Received 21 May 1996; revised 22 July 1996; accepted 24 July 1996
Abstract
This paper reports a voltammetric and spectroscopic investigation of the guanidine-induced unfolding of cytochrome c at neutral pH and 25°C. Electrochemical data provide direct evidence for the presence of an equilibrium intermediate (form I) strictly dependent on the denaturant concentration. The midpoint potential of form I has been determined (Ej/2 = + 0.010 V vs. NHE) and its structural features defined from analysis of the circular dichroism and absorbance spectroscopy data obtained under the same experimental conditions. From the correlation of electrochemical and spectroscopic data, we propose that the features detected by the intermediate conform to the molten globule state. Keywords: Cytochrome c; Hemoprotein; Unfolding intermediate; Voltammetry; Circular dichroism
1. Introduction
In the last years great interest has been focused on the process of protein folding, in order to clarify the mechanism(s) controlling the formation of the biologically active three-dimensional structure from the unfolded chain during biosynthesis [1]. The hypothesis that folding does not occur as a single cooperative event (even in simple proteins), but rather in a stepwise manner with formation of well populated structural intermediates, is widely accepted today [2-5]; thus, a major interest in studies on protein folding concerns the characterization of these intermediate forms. A number of investigations on globular pro-
* Corresponding author. Fax: + 39 6 2042 7292.
teins demonstrated that folding intermediates possess common characteristics, as a compact globularity with native secondary structure and tertiary structure fluctuating; such a physical state was defined as m o l t e n g l o b u l e [2,6-9]. Denaturation of cytochrome c, induced by a number of chemicals (such as acids, guanidine hydrochloride (Gnd-HC1), urea, calcium chloride) a n d / o r heat, has been extensively investigated [10-18]; modified forms of the protein were also studied, with the aim to better clarify the characteristics of equilibrium folding intermediates [19-23]. However, despite the effort gone into understanding the structural features of the intermediates detected, some of their properties still remain poorly defined, such as their electron transfer properties (and the value of the midpoint potential).
0167-4838/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S0 167-4838(96)00 122-7
T. Ferri et al. / Biochimica et Biophysica Acta 1298 (1996) 102-108
In this paper we have investigated the guanidineinduced unfolding of cytochrome c at neutral pH and room temperature by DC cyclic voltammetry (c.v.) and differential pulse voltammetry (d.p.v.) techniques; the data obtained give a detailed picture of the process and direct evidence for the existence of an equilibrium intermediate of the protein with distinct redox properties, thus indicating that the twostate theory [2,24] cannot describe adequately the denaturation process of cytochrome c, clearly resulting an oversimplification. Further, the midpoint potential of the intermediate was determined and the structural features defined through additional circular dichroism (c.d.) and absorbance spectroscopy data obtained under the same experimental conditions. On the whole, analysis of the experimental data provides novel information on the equilibrium intermediate which structural features well conform to the molten globule state [2,6-9]. To our knowledge, this is the first case where the structural and electron transfer properties of an equilibrium unfolding intermediate of cytochrome c have been directly correlated.
2. Materials and methods
Horse heart cytochrome c (type VI), obtained from Sigma (St. Louis, MO), was further purified as described by Brautigan et al. [25]. High purity (99.99%) guanidinium hydrochloride was purchased from ICN (Costa Mesa, CA). All the reagents used were of analytical grade. Experiments were performed at neutral pH, in 50 mM phosphate buffer containing 100 mM sodium perchlorate as supporting electrolyte, and 25°C. An Amel 472 multipolarograph was used for voltammetric measurements. A gold electrode (2 mm diameter), chemically modified with 4,4'-bipyridine (Merck, Germany) [26], was the working electrode (the more stable 4S,4S'-bipyridine could not be utilized as promoter being electroactive in part of the potential range investigated [27]); an A g / A g C 1 / 3 M KC1 electrode was the reference ( E = + 0.220 V vs. NHE at 25°C) and a Pt wire the counter-electrode. Electrochemical measurements were carried out under anaerobic conditions; oxygen was removed under mild stirring by a gentle flow of pure, CO2-free
103
nitrogen maintained just above the solution surface and previously passed through a 0.2 M NaOH solution. A Jasco J-710 spectropolarimeter interfaced with a PC as data processor was used for c.d. measurements. Optical measurements were carried out on a Cary 219 spectrophotometer. Reversibility of the denaturation process of cytochrome c was tested by dialyzing extensively the solution vs. denaturant-free buffer and then running both the cyclic voltammogram and the c.d. spectrum of the dialyzed samples.
3. Results
3.1. Electrochemical measurements DC cyclic voltammograms of cytochrome c were run at several scan rates, from 5 m V / s to 100 m V / s , in the absence and in the presence of increasing amounts of Gnd-HC1; a well-defined electrochemistry was always observed. Fig. 1 shows a sequence of DC cyclic (left panel) and differential pulse (right panel) voltammograms run, at the same scan rate, in the presence of increasing concentrations of Gnd-HC1. As illustrated in Fig. 1A, the voltammograms evidence the presence of a single species in solution with behaviour typical of native cytochrome c (dotted line, both panels) up to a 1.0 M Gnd-HC1 concentration; on the other hand two distinct redox processes, indicating the presence of two forms in solution, are observed as the denaturant concentration is increased: at 2.0 M Gnd-HC1, for example, two quasi-reversible dc cyclic voltammograms (left panel of Fig. 1A, solid line) with cathodic waves centered at approx. + 220 mV vs. NHE (assigned to the native form, or form N [26]) and at approx. - 4 0 mV vs. NHE (that we assign to the intermediate form, or form I), respectively, are observed; similar results were obtained as differential pulse voltammograms were run under the same experimental conditions (right panel of Fig. IA, solid line). By stepwise addition of denaturant, progressive conversion of native cytochrome c to the intermediate form I occurs: as shown in Fig. 1B, right panel, at 2.6 M Gnd-HC1 two d.p.v, waves of comparable intensity were observed that we assign to the native and the intermediate form, respectively; in
T. Ferri et al. / Biochimica et Biophysica Acta 1298 (1996) 102-108
104
II I I
o.. A/
,-,_
1.e~A
I / /#
/I
S ,
A
/
--
I!
6
// J
+
rS oi.
~
0
2
3
4
5
6
[Gnd-HCI] (M) +
-o.'4
--0.4
0.4
E vs Ag/AgCI/3M KCI
Fig. I. DC cyclic (left panel) and differential pulse (right panel) voltammograms of cytochrome c (0.4 mM) at a chemically modified gold electrode in the presence of increasing Gnd-HCI concentration. Gnd-HCI concentration: absent (dotted line), 2.0 M (solid line) (A); 2.6 M (B); 4.0 M and 6.0 M (C). Other experimental conditions: 20 mM phosphate buffer (pH 7.0) + 0.1 M NaC104 as supporting electrolyte; temperature: 25°C. Electrochemical parameters: scan rate = 20 m V / s (left panel); scan r a t e = 2 0 mV/s; pulse width = - 4 0 mV; sampling time= I0 ms; pulse time = 50 ms (right panel).
addition, a (not well resolved) wave with peak centered at approx. - 2 3 0 mV vs. NHE was detected, assigned to the unfolded state D. Upon further addition of Gnd-HC1 (up to 6.0 M) the native form disappears, while form I progressively converts towards the unfolded state D of the protein; as shown in Fig. 1C, at denaturant concentrations > 3.5 M, form D is the only species detected by electrochemTable 1 Redox potentials of the forms detected by electrochemistry in the guanidine-induced unfolding process of cytochrome c E~/2 vs. NHEa(mV)
1
Form N
Form I
Form D
+250_+10
+10+10
-200_+25
From d.p.v, measurements carried out under the experimental conditions described in Fig. 1.
Fig. 2. Distribution profiles of the native ( • ) , the intermediate ( O ) and the denatured (zx) forms of cytochrome c as a function of Gnd-HCl concentration. Experimental data were obtained by calculating the current peak intensities of differential pulse voltammograms. Experimental conditions as for Fig. 1.
istry. The three forms observed all were able to re-convert to the native state as the denaturant was removed by dialysis. Further, analysis of the voltammetric data indicates that the electron-transfer process is diffusion-controlled [28]; the midpoint potentials determined (from d.p.v.) for each species are reported in Table 1. From the d.p.v, data, we graphed the probable distribution of the three forms as a function of the denaturant concentration; the results are shown in Fig. 2. The estimation made is to be considered not strictly quantitative, because of the distinct electrochemical behaviour of each species at the electrode (strictly depending on electroactivity, electron-transfer reversibility, degree of absorbance at the electrode surface, structural 'degradation'); however, it is indicative of a stepwise denaturant-dependent process. 3.2. Circular dichroism measurements
With the aim to better characterize the intermediate form detected by electrochemistry (and to find out a direct correlation between the protein structure and its functional properties), c.d. spectra of cytochrome c were carried out under the same experimental
T. Ferri et al. / Biochimica et Biophysica Acta 1298 (1996) 102-108
the secondary (a-helical) structure [13,16,17]. From the analysis of the unfolding curves, in the 0.5-2.0 M Gnd-HC1 concentration range we estimate that the species in solution (i.e. form N and form I, respectively, as determined by electrochemistry) both possess native secondary structures but distinct tertiary conformations; on the other hand, at Gnd-HC1 concentration > 2.2 M the presence of (at least) one species lacking ordered secondary and tertiary structures is to be expected.
100
80
£
60
o n Z
105
/
4o i I
3.3. Optical density measurements
20
0
1
2
3
4
5
[Gnd. HCL] (a)
Fig. 3. Unfolding profiles of cytochrome c in the presence of increasing Gnd-HC1 concentration. The dichroic measurements were carried out at 408 nm (zx, curve a in the text) and 222 nm (~7, curve b in the text). The filled circles (0) refer to the percentage of unfolding of the native form, as obtained from d.p.v, measurements (see Fig. 1, right panel) taking as endpoints the current intensity of the voltammetric peak centered at approx. +50 mV vs Ag/AgC1/3 M KC1 electrode in the absence of denaturant (0% unfolding) and the current intensity in the absence of the d.p.v, wave (100% unfolding). Experimental conditions: 20 mM phosphate buffer (pH 7.0)+0.1 M NaC104; the temperature was 25°C. conditions of cyclic voltammograms. Fig. 3 illustrates the unfolding titration curves of cytochrome c as obtained from measurements carried out at 222 nm (which is a probe for the secondary structure) [29], and at 408 nm (directly related to the structure at the heme pocket) [13,16], as a function of denaturant concentration. The ellipticity values are reported as percentage of unfolding, using as endpoints the values obtained in the absence (corresponding to 0% unfolding) and in the presence of 4.0 M Gnd-HC1 (corresponding to the fully reversible, 100% unfolding [13,16,30]). The denaturant-dependent curve referring to the far-UV region (curve b) indicates that the c.d. spectrum typical of the native protein remains unaltered at denaturant concentration lower than 2.4 M; on the other hand, the unfolding profile referring to the Soret region (curve a) indicates that guanidine, at concentration > 0.5 M, already induces alterations in the dichroic band, thus confirming that the heine pocket region is more sensitive to the denaturant than
Met(80), the axial ligand of the heme iron in cytochrome c under physiological-like conditions [31], is thought to play a relevant role for the relatively high reduction potential of cytochrome c [31,32]; the rupture of the heine Fe(III)-Met(80) bond is in fact coupled to a drastic lowering of the redox potential of the protein, indicative for dramatic changes occurring at the heine pocket domain. Spectrophotometric evidence for the Fe(III)-Met(80) bond is provided by the optical band with peak centered at 695 nm [33], which is very sensitive and practically disappears as the axial bond is broken, With the aim to determine the conditions required to induce rupture of the sixth coordination bond of the heme-iron in cytochrome c, absorbance measurements were carried out in the 650-730 nm wavelength range, in the presence of increasing Gnd-HC1 concentration. The titration curve obtained, illustrated in Fig. 4, clearly indicates that rupture of the coordination bond starts to occur at denaturant concentration > 1 M; the trend of the curve is extremely similar to that of curve a in Fig. 3 (i.e. the unfolding profile obtained from c.d. measurements in the Soret), thus suggesting that: (i) the rupture of the axial bond is the main cause inducing alteration of the heme pocket in cytochrome c; (ii) both the (optical and c.d.) curves give a direct correlation between the amount of denaturant in solution and the corresponding loss of native tertiary structure of the protein.
4. Discussion Folding of cytochrome c is a complex process which involves formation of intermediate forms when
T. Ferri et al. / Biochimica et Biophysica Acta 1298 (1996) 102-108
106
converting into the native state during biosynthesis. However, studies on the unfolding process of the protein performed to determine the mechanism(s) governing the event, are generally complicated from the difficulty to characterize folding intermediates: the transient species, very unstable, can be revealed only by fast kinetics techniques; on the other hand, equilibrium intermediates are even not easily detectable. Electrochemical techniques, such as c.v. and d.p,v., may thus represent a powerful tool to define some aspects of the unfolding process of redox proteins (as described here for cytochrome c), by detecting the presence of multiple forms on the basis of their differing electron transfer properties. We have shown here that electrochemical measurements give direct evidence for the presence of one equilibrium folding intermediate I of cytochrome c with a midpoint potential, E~/2, intermediate between those of the native and unfolded states (see Table 1). The picture emerging is that three equilibrium species are involved in the process at neutral pH: the native form, one intermediate state and the denaturated form, as illustrated by the following scheme: form N ~ form I ~ form D 0.6
0.5
0.4
0.3 O <1 0.2
-
0.1
0.0
-0.1
r 0
1
--
~
i
2
3
--
I 4
[Gnd-HCI] (M)
Fig. 4. Stability of the heme Fe(III)-Met(80) axial bond of cytochrome c as a function of Gnd-HC1 concentration. The absorbance measurements were carried out at A = 695 nm; protein concentration = 100 /~M. Other experimental conditions as for Fig. 3.
Table 2 Amount (as percentage) of the forms detected as a function of denaturant concentration, in the guanidine-induced unfolding process of cytochrome c Gnd-HCI (M)
%N ~
%D b
%1 c
0.5 1.0 1.5 2.0 2.2 2.4 2.5 2.6 2.7 2.8 3.0
I O0 95 91 (94) 85 (86) 82 65 (70) 50 40 (46) 34 24 (29) 10(13)
0 0 0 0 0 5 14 26 37 54 72
0 5 9 15 18 30 36 34 29 22 18
3.2
2 (5)
87
11
3.5 4.0 6.0
0 0 0
98 100 100
2 0 0
~' From Soret c.d. (Fig. 3, curve a) and optical (in brackets) measurements. b From far-UV c.d. measurements (Fig. 3, curve b). c Obtained as: 100 - (%N + %D), see text.
On the basis of the experimental results, we have determined the most probable distribution of the equilibrium forms of cytochrome c as a function of the denaturant concentration. The amount (as percentage) calculated for each species in solution, is reported in Table 2. The values were collected from c.d. data, taking into account the model above described; in fact, from the curve obtained from c.d. measurements in the Soret (curve a of Fig. 3), which gives indication for the loss of native tertiary structure of the protein, the amount of native protein still present in solution at any Gnd-HC1 concentration can be determined. Further, the amount of the unfolded conformer in solution can be determined from c.d. far-UV data (see curve b of Fig. 3, which is indicative for the loss of ordered secondary structure of the protein as a function of denaturant). Once calculated the amounts of form N and form D, respectively, the amount of form I has been determined by difference: %form I = 1 0 0 - (%form N + %form D). Our analysis is based on the following assumptions: (i) three equilibrium species are present in the guanidine-induced unfolding process of cytochrome c at neutral pH and room temperature (as indicated by electrochemistry); (ii) the amount of each species in solution is strictly
T. F erri et al. / Biochimica et Biophysica Acta 1298 (1996) 102-108
100
80
5_o
60
.Q
r~
40
20
0 o
1
2
3
4
5
[Gnd HCl] (M)
Fig. 5. Distribution profiles of the native ( • ) , the intermediate ( O ) and the denatured (zx) forms of cytochrome c in the presence of increasing Gnd-HC1 concentration. The data (reported in detail in Table 2) were obtained from analysis of the c.d. measurements carried out at 408 nm (curve a) and 222 nm (curve b), respectively (see text).
dependent from the denaturant concentration (as shown by voltammetric, c.d. and optical measurements); (iii) two of the three species detected (form N and form I) differ in the tertiary conformations but both possess native secondary structures (see the unfolding curves of Fig. 3); (iv) form D, the unfolded state of the protein, lacks ordered secondary and tertiary structures (as shown from c.d.). By utilizing the data of Table 2, we have plotted the probable distribution of the three equilibrium forms detected (by electrochemistry) as a function of Gnd-HC1, illustrated in Fig. 5. We like to point out: (i) the excellent agreement between c.d. and optical data (as reported in Table 2); (ii) the close likeness of the 'species distribution' graphs illustrated in Figs. 2 and 5, as obtained from voltammetric and c.d. data, respectively. Finally, we suggest that the structural features of the intermediate form I resemble those of molten globule state; this hypothesis is supported by the following considerations. As above described, the two species observed in the 0.5-2.0 M denaturant concentration range (i.e., form N and form I) show native secondary structures; however, their tertiary
107
conformations differ. This is clearly evidenced by the fact that the Sorer c.d. spectra recorded in the absence and in the presence of 2.0 M Gnd-HC1 (not shown) are significantly different, to indicate that changes occur in the heme pocket region upon formation of the intermediate form I. These changes involve the rupture of the Met(80)-heme iron bond (see Fig. 4) and likely favour the formation of a heine-His ligation ([34,35]; see also, below, our hypothesis basing on voltammetric data). This event provides significant rearrangement within the tertiary structure of the protein, given that either His(26) or His(33), being placed on the opposite side of the heme plane with respect to the Met(80)-heme ligation site, must rotate around the heme carrying along a not irrelevant portion of the main chain [34]. Further, the protein conformation should be expected less packed being the wllue of the midpoint potential, El~2, intermediate between those of form N and form D. Thus, form I seems to conform to the characteristics of molten globule, a globular state with native secondary structure but less packed tertiary conformation [2,6-9]. The transition to form I affects significantly the behavior of the active site: the midpoint potential, E~/2, drops down of approx. 230 mV with respect to that typical of the native state (E~/2 = + 10 mV vs. NHE). However, from the value of the midpoint potential, we expect that in form I the heme group still remains buried to the solvent [36]; microperoxidase, the heme-peptide obtained from peptic hydrolysis of cytochrome c which is characterized by high exposure of the active site to the solvent [37], shows a midpoint potential E~/2 = - 160 mV vs. NHE under the same experimental conditions [38]. In form I, Met(80) should be replaced by a nitrogenous base, possibly a histidine: a close value of the midpoint potential (E~/2 = + 41 mV vs. NHE) was determined for a modified (Met(80) --* His) form of cytochrome c (cyt c-His(80)) [39,40]. The hypothesis of an intermediate state characterized by a not-native His-iron bond at the Met(80) site, is in good agreement with fast-kinetics data of Sosnick et al. [35], who identify this incorrect ligation as a kinetic barrier for rapid folding to native cytochrome c. These authors hypothesize that the faster folding reaction rate of cytochrome c from the acidic equilibrium molten globule state is mainly due to the absence of incorrect heme-His ligation in the initial
108
T. Ferri et al. / Biochimica et Biophysica Acta 1298 (I 996) 102-108
state, being histidines protonated; conversely, under conditions close to neutrality the folding rate is slowed by the kinetic intermediate, which acts as 'trapped state' because of the incorrect heme-His (His(26) or His (33)) ligation that takes place at the Met(80) site. In conclusion, our results indicate that electrochemical techniques represent a powerful tool for investigating the unfolding process of redox proteins; as shown here for cytochrome c, these techniques give precious information on equilibrium unfolding intermediates by revealing their presence in solution and allowing determination of their redox properties. We believe that the detailed analysis of voltammetric and spectroscopic data obtained under the same experimental conditions may contribute significantly to shed deeper light on the mechanism(s) governing the folding process of redox proteins and to characterize equilibrium intermediates.
Acknowledgements
Work partially supported by grants from CNR (95.02934 CT14 to R.S.).
References [1] Dill, K.A. (1990) Biochemistry 29, 7135-7155. [2] Kuwajima, K., (1989) Proteins Struct. Funct. Genet. 6, 87-103. [3] Creighton, T.E. (1991) Current Biol. 1, 8-10. [4] Kim, P.S. and Baldwin, R.L. (1990) Annu. Rev. Biochem. 59, 631-660. [5] Oas, T.G. and Kim, P.S. (1988) Nature 336, 42-48. [6] Dolgikh, D.A., Gilmanshin, R.I., Brazhnikov, E.V., Bychkova, V.E., Semisotnov, G.V., Venyaminov, S., Yu, Y. and Ptitsyn, O.B. (1981) FEBS Lett. 136, 311-315. [7] Dolgikh, D.A., Kolomiets, A.P., Bolotina, I.A. and Ptitsyn, O.B. (1984) FEBS Lett. 165, 88-92. [8] Ptitsyn, O.B. (1987) J. Protein Chem. 6, 272-293. [9] Barrick, D. and Baldwin, R.L. (1993) Protein Sci. 2, 869876. [10] Privalov, P.L. and Khechinashvili, N.N. (1974) J. Mol. Biol. 86, 665-684. [11] Pace, C.N. (1975) CRC Crit. Rev. Biochem. 3, 1-43. [12] Bryant, C., Strottmann, J.M. and Stellwagen, E. (1985) Biochemistry 24, 3459-3464. [13] Myer, Y.P., Kumar, S., Kinnally, K. and Pande, J. (1987) J. Protein Chem. 6, 321-342.
[14] Jeng, M.F. and Englander, W. (1991) J. Mol. Biol. 221, 1045-1061. [15] Goto, Y. and Nishikiori, S. (1991) J. Mol. Biol. 222, 679-686. [16] Bixler, J., Bakker, G. and McLendon, G. (1992) J. Am. Chem. Soc. 114, 6938-6939. [17] Kataoka, M., Hagihara, Y., Mihara, K. and Goto, Y. (1993) J. Mol. Biol. 229, 591-596. [18] Ahamad, Z. and Ahamad, F. (1994) Biochim. Biophys. Acta 1207, 223-230. [19] Santucci, R., Brunori, M. and Ascoli, F. (1987) Biochim. Biophys. Acta 907, 223-230. [20] Santucci, R., Giartosio, A. and Ascoli, F. (1989) Arch. Biochem. Biophys. 275, 496-504. [21] Moreira, I., Sun, J., Cho, M.O.K., Wishart, J.F. and Isied, S.S. (1994)J. Am. Chem. Soc. 116, 8396-8397. [22] Marmorino, J.L. and Pielak, G.J. (1995) Biochemistry 34, 3140-3143. [23] Hildebrandt, P. and Stockburger, M. (1989) Biochemistry 28, 6710-6721. [24] Privalov, P.L. (1979) Adv. Protein Chem. 33, 167-241. [25] Brautigan, D.L., Ferguson-Miller, S. and Margoliash, E. (1978) Methods Enzymol. 53, 128-164. [26] Eddows, M.J. and Hill, H.A.O. (1979) J. Am. Chem. Soc. 101,4461-4464. [27] Hinnen, C. and Niki, K. (1989) J. Electroanal. Chem. 264, 157-165. [28] Bard, A.J. and Faulkner, L.R. (1980) Electrochemical Methods: Fundamentals and Applications, pp. 215-231, John Wiley, New York. [29] Greenfield, N. and Fasman, G.D. (1969) Biochemistry 8, 4108-4116. [30] Knapp, J.A. and Pace, C.N. (1974) Biochemistry 13, 12891294. [31] Dickerson, R.E. and Timkovich, R. (1975) in The Enzymes, Vol. XI (Boyer, P., ed.), pp. 397-492, Academic Press, New York. [32] Senn, H. and Wuthrich, K. (1985) Q. Rev. Biophys. 18, 111-134. [33] Schejter, A. and Aviram, I. (1970) J. Biol. Chem. 245, 1552-1557. [34] Elove, G.A. and Roder, H. (1991) in Protein Refolding (Georgiou, G. and De Bernardez-Clark, E., eds.), pp. 50-63, ACS Symposium Series, Washington, DC,. [35] Sosnick, T.R., Mayne, L., Hiller, R. and Englander, S.W. (1994) Nature Struct. Biol. 1, 149-156. [36] Stellwagen, E. (1978) Nature 275, 73-74. [37] Harbury, H.A. and Loach, P.A. (1959) Proc. Natl. Acad. Sci. USA 45, 1344-1359. [38] Santucci, R., Reinhard, H. and Brunori, M. (1988) J. Am. Chem. Soc. 110, 8536-8537. [39] Raphael, A.L. and Gray, H.B. (1989) Proteins Struct. Funct. Genet. 6, 338-340. [40] Raphael, A.L. and Gray, H.B. (1991) J. Am. Chem. Soc. 113, 1038-1040.
ELSEVIER
Biochimica et BiophysicaActa 1298 (1996) 102-108
BB3
Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c T o m m a s o F e r r i a, A l e s s a n d r o P o s c i a a, F r a n c a A s c o l i b, R o b e r t o S a n t u c c i b,* a Dipartimento di Chimica, Universit~ di Roma 'La Sapienza ', 00185 Roma, Italy b Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universit2t di Roma 'Tor Vergata ', 00133 Roma, Italy
Received 21 May 1996; revised 22 July 1996; accepted 24 July 1996
Abstract
This paper reports a voltammetric and spectroscopic investigation of the guanidine-induced unfolding of cytochrome c at neutral pH and 25°C. Electrochemical data provide direct evidence for the presence of an equilibrium intermediate (form I) strictly dependent on the denaturant concentration. The midpoint potential of form I has been determined (Ej/2 = + 0.010 V vs. NHE) and its structural features defined from analysis of the circular dichroism and absorbance spectroscopy data obtained under the same experimental conditions. From the correlation of electrochemical and spectroscopic data, we propose that the features detected by the intermediate conform to the molten globule state. Keywords: Cytochrome c; Hemoprotein; Unfolding intermediate; Voltammetry; Circular dichroism
1. Introduction
In the last years great interest has been focused on the process of protein folding, in order to clarify the mechanism(s) controlling the formation of the biologically active three-dimensional structure from the unfolded chain during biosynthesis [1]. The hypothesis that folding does not occur as a single cooperative event (even in simple proteins), but rather in a stepwise manner with formation of well populated structural intermediates, is widely accepted today [2-5]; thus, a major interest in studies on protein folding concerns the characterization of these intermediate forms. A number of investigations on globular pro-
* Corresponding author. Fax: + 39 6 2042 7292.
teins demonstrated that folding intermediates possess common characteristics, as a compact globularity with native secondary structure and tertiary structure fluctuating; such a physical state was defined as m o l t e n g l o b u l e [2,6-9]. Denaturation of cytochrome c, induced by a number of chemicals (such as acids, guanidine hydrochloride (Gnd-HC1), urea, calcium chloride) a n d / o r heat, has been extensively investigated [10-18]; modified forms of the protein were also studied, with the aim to better clarify the characteristics of equilibrium folding intermediates [19-23]. However, despite the effort gone into understanding the structural features of the intermediates detected, some of their properties still remain poorly defined, such as their electron transfer properties (and the value of the midpoint potential).
0167-4838/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S0 167-4838(96)00 122-7
T. Ferri et al. / Biochimica et Biophysica Acta 1298 (1996) 102-108
In this paper we have investigated the guanidineinduced unfolding of cytochrome c at neutral pH and room temperature by DC cyclic voltammetry (c.v.) and differential pulse voltammetry (d.p.v.) techniques; the data obtained give a detailed picture of the process and direct evidence for the existence of an equilibrium intermediate of the protein with distinct redox properties, thus indicating that the twostate theory [2,24] cannot describe adequately the denaturation process of cytochrome c, clearly resulting an oversimplification. Further, the midpoint potential of the intermediate was determined and the structural features defined through additional circular dichroism (c.d.) and absorbance spectroscopy data obtained under the same experimental conditions. On the whole, analysis of the experimental data provides novel information on the equilibrium intermediate which structural features well conform to the molten globule state [2,6-9]. To our knowledge, this is the first case where the structural and electron transfer properties of an equilibrium unfolding intermediate of cytochrome c have been directly correlated.
2. Materials and methods
Horse heart cytochrome c (type VI), obtained from Sigma (St. Louis, MO), was further purified as described by Brautigan et al. [25]. High purity (99.99%) guanidinium hydrochloride was purchased from ICN (Costa Mesa, CA). All the reagents used were of analytical grade. Experiments were performed at neutral pH, in 50 mM phosphate buffer containing 100 mM sodium perchlorate as supporting electrolyte, and 25°C. An Amel 472 multipolarograph was used for voltammetric measurements. A gold electrode (2 mm diameter), chemically modified with 4,4'-bipyridine (Merck, Germany) [26], was the working electrode (the more stable 4S,4S'-bipyridine could not be utilized as promoter being electroactive in part of the potential range investigated [27]); an A g / A g C 1 / 3 M KC1 electrode was the reference ( E = + 0.220 V vs. NHE at 25°C) and a Pt wire the counter-electrode. Electrochemical measurements were carried out under anaerobic conditions; oxygen was removed under mild stirring by a gentle flow of pure, CO2-free
103
nitrogen maintained just above the solution surface and previously passed through a 0.2 M NaOH solution. A Jasco J-710 spectropolarimeter interfaced with a PC as data processor was used for c.d. measurements. Optical measurements were carried out on a Cary 219 spectrophotometer. Reversibility of the denaturation process of cytochrome c was tested by dialyzing extensively the solution vs. denaturant-free buffer and then running both the cyclic voltammogram and the c.d. spectrum of the dialyzed samples.
3. Results
3.1. Electrochemical measurements DC cyclic voltammograms of cytochrome c were run at several scan rates, from 5 m V / s to 100 m V / s , in the absence and in the presence of increasing amounts of Gnd-HC1; a well-defined electrochemistry was always observed. Fig. 1 shows a sequence of DC cyclic (left panel) and differential pulse (right panel) voltammograms run, at the same scan rate, in the presence of increasing concentrations of Gnd-HC1. As illustrated in Fig. 1A, the voltammograms evidence the presence of a single species in solution with behaviour typical of native cytochrome c (dotted line, both panels) up to a 1.0 M Gnd-HC1 concentration; on the other hand two distinct redox processes, indicating the presence of two forms in solution, are observed as the denaturant concentration is increased: at 2.0 M Gnd-HC1, for example, two quasi-reversible dc cyclic voltammograms (left panel of Fig. 1A, solid line) with cathodic waves centered at approx. + 220 mV vs. NHE (assigned to the native form, or form N [26]) and at approx. - 4 0 mV vs. NHE (that we assign to the intermediate form, or form I), respectively, are observed; similar results were obtained as differential pulse voltammograms were run under the same experimental conditions (right panel of Fig. IA, solid line). By stepwise addition of denaturant, progressive conversion of native cytochrome c to the intermediate form I occurs: as shown in Fig. 1B, right panel, at 2.6 M Gnd-HC1 two d.p.v, waves of comparable intensity were observed that we assign to the native and the intermediate form, respectively; in
T. Ferri et al. / Biochimica et Biophysica Acta 1298 (1996) 102-108
104
II I I
o.. A/
,-,_
1.e~A
I / /#
/I
S ,
A
/
--
I!
6
// J
+
rS oi.
~
0
2
3
4
5
6
[Gnd-HCI] (M) +
-o.'4
--0.4
0.4
E vs Ag/AgCI/3M KCI
Fig. I. DC cyclic (left panel) and differential pulse (right panel) voltammograms of cytochrome c (0.4 mM) at a chemically modified gold electrode in the presence of increasing Gnd-HCI concentration. Gnd-HCI concentration: absent (dotted line), 2.0 M (solid line) (A); 2.6 M (B); 4.0 M and 6.0 M (C). Other experimental conditions: 20 mM phosphate buffer (pH 7.0) + 0.1 M NaC104 as supporting electrolyte; temperature: 25°C. Electrochemical parameters: scan rate = 20 m V / s (left panel); scan r a t e = 2 0 mV/s; pulse width = - 4 0 mV; sampling time= I0 ms; pulse time = 50 ms (right panel).
addition, a (not well resolved) wave with peak centered at approx. - 2 3 0 mV vs. NHE was detected, assigned to the unfolded state D. Upon further addition of Gnd-HC1 (up to 6.0 M) the native form disappears, while form I progressively converts towards the unfolded state D of the protein; as shown in Fig. 1C, at denaturant concentrations > 3.5 M, form D is the only species detected by electrochemTable 1 Redox potentials of the forms detected by electrochemistry in the guanidine-induced unfolding process of cytochrome c E~/2 vs. NHEa(mV)
1
Form N
Form I
Form D
+250_+10
+10+10
-200_+25
From d.p.v, measurements carried out under the experimental conditions described in Fig. 1.
Fig. 2. Distribution profiles of the native ( • ) , the intermediate ( O ) and the denatured (zx) forms of cytochrome c as a function of Gnd-HCl concentration. Experimental data were obtained by calculating the current peak intensities of differential pulse voltammograms. Experimental conditions as for Fig. 1.
istry. The three forms observed all were able to re-convert to the native state as the denaturant was removed by dialysis. Further, analysis of the voltammetric data indicates that the electron-transfer process is diffusion-controlled [28]; the midpoint potentials determined (from d.p.v.) for each species are reported in Table 1. From the d.p.v, data, we graphed the probable distribution of the three forms as a function of the denaturant concentration; the results are shown in Fig. 2. The estimation made is to be considered not strictly quantitative, because of the distinct electrochemical behaviour of each species at the electrode (strictly depending on electroactivity, electron-transfer reversibility, degree of absorbance at the electrode surface, structural 'degradation'); however, it is indicative of a stepwise denaturant-dependent process. 3.2. Circular dichroism measurements
With the aim to better characterize the intermediate form detected by electrochemistry (and to find out a direct correlation between the protein structure and its functional properties), c.d. spectra of cytochrome c were carried out under the same experimental
T. Ferri et al. / Biochimica et Biophysica Acta 1298 (1996) 102-108
the secondary (a-helical) structure [13,16,17]. From the analysis of the unfolding curves, in the 0.5-2.0 M Gnd-HC1 concentration range we estimate that the species in solution (i.e. form N and form I, respectively, as determined by electrochemistry) both possess native secondary structures but distinct tertiary conformations; on the other hand, at Gnd-HC1 concentration > 2.2 M the presence of (at least) one species lacking ordered secondary and tertiary structures is to be expected.
100
80
£
60
o n Z
105
/
4o i I
3.3. Optical density measurements
20
0
1
2
3
4
5
[Gnd. HCL] (a)
Fig. 3. Unfolding profiles of cytochrome c in the presence of increasing Gnd-HC1 concentration. The dichroic measurements were carried out at 408 nm (zx, curve a in the text) and 222 nm (~7, curve b in the text). The filled circles (0) refer to the percentage of unfolding of the native form, as obtained from d.p.v, measurements (see Fig. 1, right panel) taking as endpoints the current intensity of the voltammetric peak centered at approx. +50 mV vs Ag/AgC1/3 M KC1 electrode in the absence of denaturant (0% unfolding) and the current intensity in the absence of the d.p.v, wave (100% unfolding). Experimental conditions: 20 mM phosphate buffer (pH 7.0)+0.1 M NaC104; the temperature was 25°C. conditions of cyclic voltammograms. Fig. 3 illustrates the unfolding titration curves of cytochrome c as obtained from measurements carried out at 222 nm (which is a probe for the secondary structure) [29], and at 408 nm (directly related to the structure at the heme pocket) [13,16], as a function of denaturant concentration. The ellipticity values are reported as percentage of unfolding, using as endpoints the values obtained in the absence (corresponding to 0% unfolding) and in the presence of 4.0 M Gnd-HC1 (corresponding to the fully reversible, 100% unfolding [13,16,30]). The denaturant-dependent curve referring to the far-UV region (curve b) indicates that the c.d. spectrum typical of the native protein remains unaltered at denaturant concentration lower than 2.4 M; on the other hand, the unfolding profile referring to the Soret region (curve a) indicates that guanidine, at concentration > 0.5 M, already induces alterations in the dichroic band, thus confirming that the heine pocket region is more sensitive to the denaturant than
Met(80), the axial ligand of the heme iron in cytochrome c under physiological-like conditions [31], is thought to play a relevant role for the relatively high reduction potential of cytochrome c [31,32]; the rupture of the heine Fe(III)-Met(80) bond is in fact coupled to a drastic lowering of the redox potential of the protein, indicative for dramatic changes occurring at the heine pocket domain. Spectrophotometric evidence for the Fe(III)-Met(80) bond is provided by the optical band with peak centered at 695 nm [33], which is very sensitive and practically disappears as the axial bond is broken, With the aim to determine the conditions required to induce rupture of the sixth coordination bond of the heme-iron in cytochrome c, absorbance measurements were carried out in the 650-730 nm wavelength range, in the presence of increasing Gnd-HC1 concentration. The titration curve obtained, illustrated in Fig. 4, clearly indicates that rupture of the coordination bond starts to occur at denaturant concentration > 1 M; the trend of the curve is extremely similar to that of curve a in Fig. 3 (i.e. the unfolding profile obtained from c.d. measurements in the Soret), thus suggesting that: (i) the rupture of the axial bond is the main cause inducing alteration of the heme pocket in cytochrome c; (ii) both the (optical and c.d.) curves give a direct correlation between the amount of denaturant in solution and the corresponding loss of native tertiary structure of the protein.
4. Discussion Folding of cytochrome c is a complex process which involves formation of intermediate forms when
T. Ferri et al. / Biochimica et Biophysica Acta 1298 (1996) 102-108
106
converting into the native state during biosynthesis. However, studies on the unfolding process of the protein performed to determine the mechanism(s) governing the event, are generally complicated from the difficulty to characterize folding intermediates: the transient species, very unstable, can be revealed only by fast kinetics techniques; on the other hand, equilibrium intermediates are even not easily detectable. Electrochemical techniques, such as c.v. and d.p,v., may thus represent a powerful tool to define some aspects of the unfolding process of redox proteins (as described here for cytochrome c), by detecting the presence of multiple forms on the basis of their differing electron transfer properties. We have shown here that electrochemical measurements give direct evidence for the presence of one equilibrium folding intermediate I of cytochrome c with a midpoint potential, E~/2, intermediate between those of the native and unfolded states (see Table 1). The picture emerging is that three equilibrium species are involved in the process at neutral pH: the native form, one intermediate state and the denaturated form, as illustrated by the following scheme: form N ~ form I ~ form D 0.6
0.5
0.4
0.3 O <1 0.2
-
0.1
0.0
-0.1
r 0
1
--
~
i
2
3
--
I 4
[Gnd-HCI] (M)
Fig. 4. Stability of the heme Fe(III)-Met(80) axial bond of cytochrome c as a function of Gnd-HC1 concentration. The absorbance measurements were carried out at A = 695 nm; protein concentration = 100 /~M. Other experimental conditions as for Fig. 3.
Table 2 Amount (as percentage) of the forms detected as a function of denaturant concentration, in the guanidine-induced unfolding process of cytochrome c Gnd-HCI (M)
%N ~
%D b
%1 c
0.5 1.0 1.5 2.0 2.2 2.4 2.5 2.6 2.7 2.8 3.0
I O0 95 91 (94) 85 (86) 82 65 (70) 50 40 (46) 34 24 (29) 10(13)
0 0 0 0 0 5 14 26 37 54 72
0 5 9 15 18 30 36 34 29 22 18
3.2
2 (5)
87
11
3.5 4.0 6.0
0 0 0
98 100 100
2 0 0
~' From Soret c.d. (Fig. 3, curve a) and optical (in brackets) measurements. b From far-UV c.d. measurements (Fig. 3, curve b). c Obtained as: 100 - (%N + %D), see text.
On the basis of the experimental results, we have determined the most probable distribution of the equilibrium forms of cytochrome c as a function of the denaturant concentration. The amount (as percentage) calculated for each species in solution, is reported in Table 2. The values were collected from c.d. data, taking into account the model above described; in fact, from the curve obtained from c.d. measurements in the Soret (curve a of Fig. 3), which gives indication for the loss of native tertiary structure of the protein, the amount of native protein still present in solution at any Gnd-HC1 concentration can be determined. Further, the amount of the unfolded conformer in solution can be determined from c.d. far-UV data (see curve b of Fig. 3, which is indicative for the loss of ordered secondary structure of the protein as a function of denaturant). Once calculated the amounts of form N and form D, respectively, the amount of form I has been determined by difference: %form I = 1 0 0 - (%form N + %form D). Our analysis is based on the following assumptions: (i) three equilibrium species are present in the guanidine-induced unfolding process of cytochrome c at neutral pH and room temperature (as indicated by electrochemistry); (ii) the amount of each species in solution is strictly
T. F erri et al. / Biochimica et Biophysica Acta 1298 (1996) 102-108
100
80
5_o
60
.Q
r~
40
20
0 o
1
2
3
4
5
[Gnd HCl] (M)
Fig. 5. Distribution profiles of the native ( • ) , the intermediate ( O ) and the denatured (zx) forms of cytochrome c in the presence of increasing Gnd-HC1 concentration. The data (reported in detail in Table 2) were obtained from analysis of the c.d. measurements carried out at 408 nm (curve a) and 222 nm (curve b), respectively (see text).
dependent from the denaturant concentration (as shown by voltammetric, c.d. and optical measurements); (iii) two of the three species detected (form N and form I) differ in the tertiary conformations but both possess native secondary structures (see the unfolding curves of Fig. 3); (iv) form D, the unfolded state of the protein, lacks ordered secondary and tertiary structures (as shown from c.d.). By utilizing the data of Table 2, we have plotted the probable distribution of the three equilibrium forms detected (by electrochemistry) as a function of Gnd-HC1, illustrated in Fig. 5. We like to point out: (i) the excellent agreement between c.d. and optical data (as reported in Table 2); (ii) the close likeness of the 'species distribution' graphs illustrated in Figs. 2 and 5, as obtained from voltammetric and c.d. data, respectively. Finally, we suggest that the structural features of the intermediate form I resemble those of molten globule state; this hypothesis is supported by the following considerations. As above described, the two species observed in the 0.5-2.0 M denaturant concentration range (i.e., form N and form I) show native secondary structures; however, their tertiary
107
conformations differ. This is clearly evidenced by the fact that the Sorer c.d. spectra recorded in the absence and in the presence of 2.0 M Gnd-HC1 (not shown) are significantly different, to indicate that changes occur in the heme pocket region upon formation of the intermediate form I. These changes involve the rupture of the Met(80)-heme iron bond (see Fig. 4) and likely favour the formation of a heine-His ligation ([34,35]; see also, below, our hypothesis basing on voltammetric data). This event provides significant rearrangement within the tertiary structure of the protein, given that either His(26) or His(33), being placed on the opposite side of the heme plane with respect to the Met(80)-heme ligation site, must rotate around the heme carrying along a not irrelevant portion of the main chain [34]. Further, the protein conformation should be expected less packed being the wllue of the midpoint potential, El~2, intermediate between those of form N and form D. Thus, form I seems to conform to the characteristics of molten globule, a globular state with native secondary structure but less packed tertiary conformation [2,6-9]. The transition to form I affects significantly the behavior of the active site: the midpoint potential, E~/2, drops down of approx. 230 mV with respect to that typical of the native state (E~/2 = + 10 mV vs. NHE). However, from the value of the midpoint potential, we expect that in form I the heme group still remains buried to the solvent [36]; microperoxidase, the heme-peptide obtained from peptic hydrolysis of cytochrome c which is characterized by high exposure of the active site to the solvent [37], shows a midpoint potential E~/2 = - 160 mV vs. NHE under the same experimental conditions [38]. In form I, Met(80) should be replaced by a nitrogenous base, possibly a histidine: a close value of the midpoint potential (E~/2 = + 41 mV vs. NHE) was determined for a modified (Met(80) --* His) form of cytochrome c (cyt c-His(80)) [39,40]. The hypothesis of an intermediate state characterized by a not-native His-iron bond at the Met(80) site, is in good agreement with fast-kinetics data of Sosnick et al. [35], who identify this incorrect ligation as a kinetic barrier for rapid folding to native cytochrome c. These authors hypothesize that the faster folding reaction rate of cytochrome c from the acidic equilibrium molten globule state is mainly due to the absence of incorrect heme-His ligation in the initial
108
T. Ferri et al. / Biochimica et Biophysica Acta 1298 (I 996) 102-108
state, being histidines protonated; conversely, under conditions close to neutrality the folding rate is slowed by the kinetic intermediate, which acts as 'trapped state' because of the incorrect heme-His (His(26) or His (33)) ligation that takes place at the Met(80) site. In conclusion, our results indicate that electrochemical techniques represent a powerful tool for investigating the unfolding process of redox proteins; as shown here for cytochrome c, these techniques give precious information on equilibrium unfolding intermediates by revealing their presence in solution and allowing determination of their redox properties. We believe that the detailed analysis of voltammetric and spectroscopic data obtained under the same experimental conditions may contribute significantly to shed deeper light on the mechanism(s) governing the folding process of redox proteins and to characterize equilibrium intermediates.
Acknowledgements
Work partially supported by grants from CNR (95.02934 CT14 to R.S.).
References [1] Dill, K.A. (1990) Biochemistry 29, 7135-7155. [2] Kuwajima, K., (1989) Proteins Struct. Funct. Genet. 6, 87-103. [3] Creighton, T.E. (1991) Current Biol. 1, 8-10. [4] Kim, P.S. and Baldwin, R.L. (1990) Annu. Rev. Biochem. 59, 631-660. [5] Oas, T.G. and Kim, P.S. (1988) Nature 336, 42-48. [6] Dolgikh, D.A., Gilmanshin, R.I., Brazhnikov, E.V., Bychkova, V.E., Semisotnov, G.V., Venyaminov, S., Yu, Y. and Ptitsyn, O.B. (1981) FEBS Lett. 136, 311-315. [7] Dolgikh, D.A., Kolomiets, A.P., Bolotina, I.A. and Ptitsyn, O.B. (1984) FEBS Lett. 165, 88-92. [8] Ptitsyn, O.B. (1987) J. Protein Chem. 6, 272-293. [9] Barrick, D. and Baldwin, R.L. (1993) Protein Sci. 2, 869876. [10] Privalov, P.L. and Khechinashvili, N.N. (1974) J. Mol. Biol. 86, 665-684. [11] Pace, C.N. (1975) CRC Crit. Rev. Biochem. 3, 1-43. [12] Bryant, C., Strottmann, J.M. and Stellwagen, E. (1985) Biochemistry 24, 3459-3464. [13] Myer, Y.P., Kumar, S., Kinnally, K. and Pande, J. (1987) J. Protein Chem. 6, 321-342.
[14] Jeng, M.F. and Englander, W. (1991) J. Mol. Biol. 221, 1045-1061. [15] Goto, Y. and Nishikiori, S. (1991) J. Mol. Biol. 222, 679-686. [16] Bixler, J., Bakker, G. and McLendon, G. (1992) J. Am. Chem. Soc. 114, 6938-6939. [17] Kataoka, M., Hagihara, Y., Mihara, K. and Goto, Y. (1993) J. Mol. Biol. 229, 591-596. [18] Ahamad, Z. and Ahamad, F. (1994) Biochim. Biophys. Acta 1207, 223-230. [19] Santucci, R., Brunori, M. and Ascoli, F. (1987) Biochim. Biophys. Acta 907, 223-230. [20] Santucci, R., Giartosio, A. and Ascoli, F. (1989) Arch. Biochem. Biophys. 275, 496-504. [21] Moreira, I., Sun, J., Cho, M.O.K., Wishart, J.F. and Isied, S.S. (1994)J. Am. Chem. Soc. 116, 8396-8397. [22] Marmorino, J.L. and Pielak, G.J. (1995) Biochemistry 34, 3140-3143. [23] Hildebrandt, P. and Stockburger, M. (1989) Biochemistry 28, 6710-6721. [24] Privalov, P.L. (1979) Adv. Protein Chem. 33, 167-241. [25] Brautigan, D.L., Ferguson-Miller, S. and Margoliash, E. (1978) Methods Enzymol. 53, 128-164. [26] Eddows, M.J. and Hill, H.A.O. (1979) J. Am. Chem. Soc. 101,4461-4464. [27] Hinnen, C. and Niki, K. (1989) J. Electroanal. Chem. 264, 157-165. [28] Bard, A.J. and Faulkner, L.R. (1980) Electrochemical Methods: Fundamentals and Applications, pp. 215-231, John Wiley, New York. [29] Greenfield, N. and Fasman, G.D. (1969) Biochemistry 8, 4108-4116. [30] Knapp, J.A. and Pace, C.N. (1974) Biochemistry 13, 12891294. [31] Dickerson, R.E. and Timkovich, R. (1975) in The Enzymes, Vol. XI (Boyer, P., ed.), pp. 397-492, Academic Press, New York. [32] Senn, H. and Wuthrich, K. (1985) Q. Rev. Biophys. 18, 111-134. [33] Schejter, A. and Aviram, I. (1970) J. Biol. Chem. 245, 1552-1557. [34] Elove, G.A. and Roder, H. (1991) in Protein Refolding (Georgiou, G. and De Bernardez-Clark, E., eds.), pp. 50-63, ACS Symposium Series, Washington, DC,. [35] Sosnick, T.R., Mayne, L., Hiller, R. and Englander, S.W. (1994) Nature Struct. Biol. 1, 149-156. [36] Stellwagen, E. (1978) Nature 275, 73-74. [37] Harbury, H.A. and Loach, P.A. (1959) Proc. Natl. Acad. Sci. USA 45, 1344-1359. [38] Santucci, R., Reinhard, H. and Brunori, M. (1988) J. Am. Chem. Soc. 110, 8536-8537. [39] Raphael, A.L. and Gray, H.B. (1989) Proteins Struct. Funct. Genet. 6, 338-340. [40] Raphael, A.L. and Gray, H.B. (1991) J. Am. Chem. Soc. 113, 1038-1040.