- Email: [email protected]
Molten globule-like state of cytochrome c induced by polyanion poly(vinylsulfate) in slightly acidic pH
Biochimica et Biophysica Acta 1434 (1999) 347^355 www.elsevier.com/locate/bba
Molten globule-like state of cytochrome c induced by polyanion poly(vinylsulfate) in slightly acidic pH Erik Sedla¨k a , Maria¨n Antal|¨k
b;
*
Department of Biochemistry, Faculty of Science, P.J. Síafa¨rik University, Moyzesova 11, 041 54 Kosice, Slovak Republic Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 043 53 Kosice, Slovak Republic a
b
Received 21 April 1999; received in revised form 6 July 1999; accepted 12 August 1999
Abstract The effect of polyanion, poly(vinylsulfate), used as a model of negatively charged surface, on ferric cytochrome c (ferricyt c) structure in acidic pH has been studied by absorbance spectroscopy, circular dichroism (CD), tryptophan (Trp) fluorescence and microcalorimetry. The polyanion induced only small changes in the native structure of the protein at neutral pH, but it profoundly shifted the acid induced high spin state of the heme in the active center of cyt c to a more neutral pH region. Cooperativity of the acidic transition of ferricyt c in the presence of the polyanion was disturbed, in comparison with uncomplexed protein, as followed from different apparent pKa values observed in a distinct regions of the ferricyt c electronic absorbance spectrum (4.55 þ 0.08 in the 620 nm band region and 5.47 þ 0.15 in the Soret region). The ferricyt c structure in the complex with the polyanion at acidic pH (below pH 5.0) has properties of a molten globule-like state. Its tertiary structure is strongly disturbed according to CD and microcalorimetry measurements ; however, its secondary structure, from CD, is still native-like and ferricyt c is in a compact state as evidenced by quenched Trp fluorescence. These findings are discussed in the context of the molten globule state of proteins induced on a negatively charged membrane surface under physiological conditions. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome c; Polyanion; Protein folding; Molten globule; Microcalorimetry ; Circular dichroism spectroscopy
1. Introduction The molten globule-like state, as well as other nonnative states of proteins, can exist in a living cell and can be involved in di¡erent physiological processes [1]. Proteins can be transformed in vitro into the molten globule state at low pH, moderate concentrations of denaturants, high temperature, i.e. at conditions which are far from usual physiological conditions [2]. On the other hand, it was shown that
* Corresponding author. Fax: +421 (95) 763754; E-mail: [email protected]
binding to a membrane surface can lead to a `partial denaturation' of proteins, transforming them into a non-native state [3]. A possible mechanism of how a negatively charged membrane surface can promote protein destabilization requires the consideration of local pH. The high density of negative charges on a membrane surface creates a strong electrostatic potential which leads to a decrease of local pH at the membrane surface. However, even in salt-free solutions, this local decrease of pH does not exceed apî from the membrane surprox. 2 pH units at 5^15 A face [4], which is usually insu¤cient for acid denaturation. In recent work [5] an additional denaturation ac-
0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 1 8 6 - 7
BBAPRO 35999 30-9-99
348
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
tion of the membrane surface was proposed ^ a local decrease of the dielectric constant near the membrane surface modeled by water-alcohol mixtures. Cytochrome (cyt) c, as a typical extrinsic membrane protein, was chosen for this modeling. Due to its location on the surface of the inner mitochondrial membrane and its ability to associate with negatively charged membrane phospholipids as well as negatively charged groups of the redox partners, a lot of e¡ort was devoted to the understanding of the e¡ect of negatively charged membrane surfaces on cyt c structure [6^11]. Because of the complexity of the interactions between biopolymers and the molecular assemblies, rigid heteropolytungstates were chosen as a suitable model of negatively charged surfaces [12^15]. Our group focused our attention on a di¡erent model of negatively charged surface, i.e., on linear polyanions such as heparin, polyglutamate, poly(vinylsulfate) (PVS), poly(4-styrene-sulfonate). The advantage of these polyanions is their stability and solubility in a wide range of pH values. Hybridization of cyt c with the £exible arti¢cial polyanions seemed to be a very fruitful model system for understanding the conformational changes of cyt c induced by the electrostatic ¢eld of negatively charged surfaces. It was found that they shift the pH value of the socalled alkaline isomerization of cyt c towards higher pH values, similar to what has been shown with cyt c oxidase and cardiolipin [16,17]. In other studies [18^ 21] polyanions were found to decrease the temperature transition for cyt c, which was similar to the e¡ect of its natural redox partners as well as anionic phospholipid bilayers [7,8,22^25]. Moreover, it is known that one likely structural change for the lipid, due to peripheral interaction of proteins with negatively charged lipids, is an increase in local surface curvature, which in turn could favor the conformational change in proteins [26]. The £exibility of the linear polyanions is therefore the other reason for its utilization as a model for the protein-membrane surface system. We believe that complex cyt c-£exible polyanion in an slightly acidic pH region may be a suitable model for the study of conformational changes of positively charged proteins induced by unspeci¢c interaction with negatively charged membrane surface. In the present paper, therefore, the properties of a complex
ferricyt c-polyanion PVS in an acidic pH region are discussed in this context. Subsequently the e¡ect of the linear polyanion with that of single anions on protein structure in an acidic pH is compared. The pH induced structural modi¢cations in a complexed ferricyt c have been investigated by means of electronic absorption, £uorescence and circular dichroism (CD) spectroscopic techniques and by di¡erential scanning calorimetry (DSC). 2. Materials and methods 2.1. Materials Horse heart cytochrome c, type III, was obtained from Sigma and used without further puri¢cation. Poly(vinylsulfate) was from Aldrich. Other chemicals were obtained from Lachema. 2.2. Bu¡ers For CD and DSC measurements in acidic pH: 10 mM glycine in pH 2.2^3.6, 10 mM acetate in pH 3.7^5.6, 10 mM cacodylate in pH 5.0^7.4, 10 mM phosphate in pH 5.8^8.0. 2.3. Absorption spectra Spectrophotometric observations were performed on a Shimadzu UV-3000 spectrophotometer. Absorption measurements were performed with a solution of 8.5 WM cyt c [27]. Acidic transitions were determined from di¡erences at 354^395 nm in the Soret region and from di¡erences at 556^622 nm in the region 500^800 nm with a 8.5 WM and 34.0 WM solution of cyt c, respectively. The spectrum of ferricyt c at pH 7.6 in 10 mM phosphate was taken as reference. 2.4. CD spectra CD measurements were obtained using a Jasco J-600 spectropolarimeter. CD measurements were performed with a solution of 22.9 WM cyt c containing 10 mM phosphate, pH 7.0, 5 WM potassium ferricyanide in 0.5 cm or 1 cm path cuvettes. PVS has neither absorbance nor CD contribution
BBAPRO 35999 30-9-99
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
into related spectra in the investigated regions under studied conditions. 2.5. Fluorescence Fluorescence measurements were performed on a Shimadzu RF-5000 spectro£uorometer. The excitation wavelength was 290 nm. Trp-59 £uorescence emission was followed at 350 nm. PVS does not signi¢cantly a¡ect the £uorescence of the free tryptophan in the investigated conditions. The temperature of the cell compartments was kept constant at 25³C by water circulation, pH was changed by addition of concentrated HCl. The pH values were measured with a Sensorex glass electrode. 2.6. DSC measurements Di¡erential scanning calorimetric measurements were made on a high-sensitivity DASM-4 microcalorimeter at a heating rate of 1 K/min. The protein concentration was 110 WM. Prior to use cyt c was converted to the fully oxidized form by addition of potassium ferricyanide. Ferricyt c-PVS complex was always prepared at pH 7.0 and then pH was adjusted to the required value. The reversibility of the ferricyt c-PVS complex at a low ionic strength (2 mM phosphate bu¡er) formed at a high concentration of polyanion (3.3 mg/ml) was more than 90% (not shown). PVS has no thermal transition under the investigated conditions. The pH value of the solution was measured always before and after cooling the sample. Only the measurements at which the pH change was less than 0.2 pH units were taken for further consideration. 3. Results 3.1. Acidic transition of ferricyt c-PVS complex In our very recent work it was shown that the acidic transition from a low to a high spin state of ferricyt c in the complex with PVS was profoundly shifted to more neutral pH in comparison with uncomplexed ferricyt c in a low ionic strength bu¡er [21]. In order to distinguish between the e¡ect of
349
polyanion PVS and free `small' anions on the acidic transition of ferricyt c we have compared the in£uence of high ionic strength with that of the polyanion on the midpoint pH value of the ferricyt c conformational transition. The apparent pKa of the free ferricyt c acidic transition in a low ionic strength bu¡er obtained from spectral changes observed in the Soret (354^394 nm) region and in the 620 nm (556^622) band regions (Fig. 1) was 2.46 þ 0.06 which was in good agreement with the published results [28]. High ionic strength (0.5 M NaCl) caused a shift of the pKa value of the acidic transition to a more neutral pH, apparent pKa was 3.36 þ 0.08, in both spectral regions (Fig. 1). Normalizing the dependencies of the ferricyt c spectral changes in the low and high ionic strength bu¡ers obtained from the di¡erent spectral regions shows the apparent pKa values did not signi¢cantly depend on which spectral region was investigated. From the e¡ect of high ionic strength on the midpoint pH of the ferricyt c acidic transition it might be expected that the polyanion PVS would induce a shift to more neutral pH, but in comparison with `small' anions this shift was much greater. The distinguishing di¡erence between the e¡ect of high ionic strength and the e¡ect of PVS on the ferricyt c acidic transition was a separation of the conformational changes occurring in the protein structure in the complex with polyanion. In the 620 nm band region the apparent pKa of the transition was 4.55 þ 0.08, while in the Soret region the apparent pKa was shifted to a more neutral pH 5.47 þ 0.15. Since di¡erent bands monitor di¡erent aspects of protein perturbation, a cooperativity of the acidic transition of ferricyt c was disturbed in the presence of polyanion PVS. In the e¡ort to assess structural perturbations of the heme region induced by acidi¢cation of bu¡er in the above investigated conditions the spectral changes in the Soret region were compared. Such a comparison was possible because a shape and a position of maxima and minima in the di¡erence spectra in all cases were very similar (not shown). Fig. 2 demonstrates this comparison: observed di¡erences in the Soret region at 354^394 nm were normalized to the maximum of the spectral change of ferricyt c in the low ionic strength bu¡er. As shown, conformational changes of ferricyt c re£ected by the
BBAPRO 35999 30-9-99
350
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
changes in the Soret region in the electronic absorbance spectrum were comparable and signi¢cantly smaller in the high ionic strength and in the presence of the polyanion PVS. The structural changes occurring in the deeply buried Trp-59 region may be detected by £uorescence intensity investigation. A single tryptophan in the polypeptide chain of cytochrome c is localized in the vicinity of the heme, which is responsible for a strong quenching through Fo«rster energy transfer in the native state of the protein (inset Fig. 2). The increase in the Trp distance from the heme results in an increase in the £uorescence intensity and this can be used to detect subtle conformational changes occurring around the heme [29]. From the midpoint pH of the acidic transition determined from £uorescence changes follows that ferricyt c underwent a highly cooperative transition to an expanded unfolded state due to the acidi¢cation of the low ionic strength bu¡er. On the other hand, Trp £uorescence of ferricyt c in high ionic strength, as in the presence of polyanion PVS, was quenched even at very acidic pH (inset Fig. 2) which indicates a compact state of
Fig. 2. Comparison of observed di¡erences of ferricyt c at acidic transition in low (E), high (a) ionic strength and in the presence of PVS (b) in the Soret region. Di¡erences were normalized on maximum di¡erence of ferricyt c di¡erence spectrum in low ionic strength. (Inset) Acidic transition of ferricyt c in low (E), high (a) ionic strength and in the presence of PVS (b) followed by Trp-59 £uorescence. Ionic strength conditions are the same as in Fig. 1.
ferricyt c in both cases. These results are in agreement with the decreased spectral changes observed in the Soret region of the absorbance spectrum of ferricyt c in acidic pH in these conditions (Fig. 2). It is not surprising as it is known that cytochrome c at pH approx. 2 and in high ionic strength is in the molten globule state that is characterized as a compact denatured state with a signi¢cant amount of native-like secondary structure, but a largely £exible and disordered tertiary structure [30^32]. 3.2. CD measurements
Fig. 1. Acidic transition of ferricyt c in low ionic strength solvent (10 mM phosphate) followed at 556^622 nm (E), 354^394 nm (F) of electronic absorption spectra and by Trp-59 £uorescence (P); ferricyt c in high ionic strength solvent (10 mM phosphate +0.5 M NaCl) followed at 556^622 nm (a), 354^394 nm (b) of electronic absorption spectra; ferricyt c-PVS complex in low ionic strength solvent (10 mM phosphate) followed at 556^622 nm (R), 354^394 nm (*) of electronic absorption spectra.
Circular dichroism was used to monitor the e¡ect of the acidi¢cation on ferricyt c secondary and tertiary structures in the complex with PVS. The far-UV CD spectrum of ferricyt c in solution shows the typical features of K-helical protein structure (Fig. 3A), in which the 222 nm dichroic band is predominantly associated with K-helical nCZ* amide transitions [33]. Neither interaction of PVS with ferricyt c nor following acidi¢cation of the bu¡er induced signi¢cant changes in the K-helix content. The spectral changes around the minimum at 208 nm, dichroic band corresponding to the ZCZ*
BBAPRO 35999 30-9-99
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
351
Fig. 3. Peptide (A), aromatic (B) and visible (C) circular dichroism spectra of ferricyt c at pH 7.0 (ööö) and ferricyt c in the presence of PVS at: pH 7.0 (999), pH 5.2 (W W W), pH 3.0 (^ ^ ^), pH 2.0 (- - -) in low ionic strength (10 mM bu¡er). (A) Peptide, deg.cm2 /mol of amide bonds; (B) aromatic and visible, deg.cm2 /dmol of heme.
amide transitions [34], may arise from changes in other secondary structure elements in the protein, or may be due to the presence of optically active heme transitions other than those associated with the amide transitions of the polypeptide chain. Therefore, an unambiguous interpretation of the spectral changes on this region of the spectrum was not possible [35]. Near-UV circular dichroism is a probe for protein tertiary structure changes that a¡ect the environment of aromatic side chains. In the aromatic region of a CD spectrum of ferricyt c two sharp minima are remarkable at 282 and 288 nm which are assigned to the Trp-59 side chain [34,36]. Upon binding to PVS at neutral pH, no signi¢cant changes are observed in the aromatic region of the CD spectrum of ferricyt c. However, opposite to the far-UV spectra of ferricyt c-PVS, the acidi¢cation of the bu¡er induced profound changes in this region of the CD spectrum: these near-UV CD spectral markers disappeared in an slightly acidic pH, with the apparent pKa = 4.6 þ 0.1 (not shown), which indicates a disruption of the tight packing of core residues in the cyt cPVS complex. Cytochrome c exhibits a characteristic CD doublet in the Soret region: the intensity of the negative band at 417 nm depends on heme-protein interactions [34] or, more exactly, on the distance and orientation of the phenylalanine residue positioned on the methionine side of the heme plane [37]. The addition of PVS
to the solution of ferricyt c at neutral pH resulted in insigni¢cant changes in the intensity of the CD signal of ferricyt c in the Soret region. Similar to the aromatic region, acidi¢cation of the solvent induced profound changes in the Soret CD ^ the negative Cotton e¡ect at 417 nm disappeared and the spectrum changed to a single positive band with maximum near 408 nm. Noteworthy, the changes observed upon acidi¢cation in the Soret and aromatic regions of the ferricyt c-PVS complex are also observed with urea-denatured cyt c, and are induced by elevated temperature or pH-induced denaturation [34], alcohol denaturation [38] and, as we have shown, also by complexation of ferricyt c with `hydrophobic' polyanions [20,21]. The observed changes in the Soret region clearly indicate a disruption of the coupling between ZCZ* transitions of the heme group and those of the aromatic amino acid residues in its proximity. This e¡ect is consistent with loosening of the tertiary structure, which removes the anisotropic character of the Soret CD band. 3.3. DSC measurements From the CD results it might be concluded that a perturbation of the interior of ferricyt c structure in the complex with PVS by the acidi¢cation of the solvent medium took place. The disturbance of the hydrophobic core of the protein paralleled by changes in its tertiary structure was therefore further
BBAPRO 35999 30-9-99
352
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
Fig. 4. DSC scans of ferricyt c-PVS in 10 mM phosphate at pH 6.1 and 7.0, in 10 mM acetate at pH 5.1, in 10 mM glycine bu¡er at pH 2.5. The PVS concentration in all samples was 1.0 mg/ml.
explored by DSC experiments. The endotherm of ferricyt c is characterized by a single peak at 84.9³C, calorimetric enthalpy (vHcal ) of 390 kJ/mol (not shown). The binding of ferricyt c to the polyanion PVS in a low ionic strength bu¡er induced a large decrease in the denaturation temperature from 84.9 to 52.8³C (Fig. 4). This shift of temperature was interpreted as an overall protein destabilization. However, ferricyt c remained in a compact form as indicated by only a small decrease of the calorimetric enthalpy transition (vHcal approx. 350 kJ/mol) even at high (3.3 mg/ml) concentration of PVS [21]. A decrease in the pH of the bu¡er slightly diminished the temperature of denaturation, as followed by DSC, of ferricyt c in the complex with PVS. This change was paralleled by an extensive reduction in the calorimetric enthalpy of the transition, with a loss of the thermal transition ferricyt c-PVS complex below pH approx. 4.9 (Fig. 4). No other thermal transition was observed in the temperature range from 2 to 120³C at the investigated conditions. 4. Discussion The interaction of extrinsic proteins with membrane surfaces is a common process during the course of various cellular functions. Although it is becoming apparent that the transition from a
water-soluble to a membrane-associated state involves major changes in the protein structure, the mechanism of the association is at present poorly understood [3,35,39,40]. Such systems are complex and have a tendency to precipitate, particularly in an acidic pH where the negative charges of the membrane are partially neutralized. As a possible mechanism of how a negatively charged membrane surface can promote protein conformational changes requires the consideration of a local pH this seems to be a major disadvantage in the investigation of pH e¡ect on protein structure in the complex with membrane surface. On the other hand linear polyanions, whose interaction with proteins is at the center of our attention, are charged and also in complex with most proteins soluble over wide pH range. In our recent work we were therefore capable of determining the apparent pKa of the acidic transition of ferricyt c in the presence of polyanions and thus showed that both heparin and PVS strongly shift the acidic transition of ferricyt c to a more neutral pH region [21,41]. This is interesting in light of the published observation that at pH 4.0 (the region of the pKa of ferricyt c-polyanion complex) the apparent a¤nity of cyt c for cardiolipin-containing liposomes was signi¢cantly increased [8]. Moreover, it was shown that heparin and PVS induced a decrease of the calorimetrically observed temperature of denaturation of ferricyt c to 59.7 and 52.8³C, respectively [18,21]. These results are analogous with those obtained in the system with ferricyt c and a membrane surface containing acidic phospholipids [7,10]. Based on these preliminary results we have chosen PVS as a suitable model of a negatively charged membrane, which might elucidate conformational changes of proteins connected with association to the membrane surface in the locally decreased pH. It has been shown previously that two di¡erent conformational states, I and II, are induced on binding ferricyt c to negatively charged surfaces [13,15] including anionic lipid dispersions [6,26]. While the conformation of state I is very similar to that of the protein in solution and the native six-coordinated low-spin con¢guration of the heme is preserved, in state II the heme crevice opens and the iron exists in a thermal equilibrium between a ¢ve-coordinated high-spin and a new six-coordinated low-spin con¢g-
BBAPRO 35999 30-9-99
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
uration. A native-like state of ferricyt c was also observed in the complex with £exible polyanions like heparin, polyglutamate and PVS evidenced by optical spectroscopic techniques [20,21,41^43]. Ferricyt c binding to polyanions is connected with a discontinuous decrease in the melting temperature of ferricyt c observed by microcalorimetry [18,20,21]. Interestingly, ferricyt c undergoes an analogous change in its melting temperature under complexation with a negatively charged membrane [7,10,22]. The calorimetric observations were interpreted as an overall destabilization of ferricyt c structure due to electrostatic interaction with negatively charged surfaces. On the other hand, the protein remained in a compact state as con¢rmed by only a small change in enthalpy of its thermal transition with even stabilized structure of the heme crevice evidenced by a shift of alkaline isomerization of ferricyt c in the complex to a more alkaline pH [16,17,19]. Our ¢ndings con¢rmed that the compact state of ferricyt c with native-like spectral features in the complex with PVS at neutral pH is a relatively labile state. A decrease in the pH of the solvent brought about large conformational changes in ferricyt c structure already at slightly acidic pH. From comparison of the observed changes of ferricyt c-PVS complex and ferricyt c in high ionic strength in the acidic pH in the Soret region with those in low ionic strength one could conclude that the heme region of ferricyt c is less perturbed in both former conditions. Moreover, quenching of the intrinsic probe of ferricyt c structure-Trp-59 by the heme suggests a compactness of the heme region and a capability of anions to partially preserve the structure of ferricyt c in the acidic pH in both cases. Although ferricyt c in the complex with PVS in neutral pH has preserved tertiary structure, it was very labile as expressed by the loss of cooperativity of its acidic transition (Fig. 1), and by the abrupt decrease in the calorimetric enthalpy of the thermal transition of ferricyt c in slightly acidic pH with its loss at pH approx. 4.9 (Fig. 4). All these ¢ndings together show that ferricyt c in the complex with polyanion PVS at the acidic pH is in `a compact structure compared to that of the unfolded state (Fig. 2), buried tryptophan residue (inset Fig. 2), with a high content of secondary structure (Fig. 3) and largely disordered tertiary structure (Fig. 4)', i.e. in the conformational state of proteins observed at
353
low pH in high ionic strength solution, de¢ned as a molten globule state [44]. At this point we need to stress the di¡erences between the e¡ects of `small' anions and polyanions, i.e. between free and covalently bound anions, on ferricyt c structure. While ferricyt c could attain a `classic' molten-like conformation only in the presence of high concentrations of anions, the polyanion a¡ected the protein structure at a concentration comparable to that of the protein. Moreover, the e¡ect of the polyanion on ferricyt c is stronger as seen in the pronounced shift of the acidic transition to more neutral pH, as well as the disturbance of the cooperativity of this transition and the loss of the calorimetrically observed thermal transition of ferricyt c in this `new' molten globule state [44^47]. Interestingly, our preliminary results from denaturant and temperature-unfolding experiments of the ferricyt c-PVS complex indicate that polyanion strongly stabilizes secondary structure and compact state of ferricytochrome c even at high concentration of the denaturant or high temperature at slightly acidic pH. The double e¡ect of the electrostatic ¢eld of the polyanion (destabilization of tertiary and stabilization of secondary structure and compactness of the protein) on the ferricytochrome c protein structure might be important for an understanding of a mechanism for conformational changes of proteins induced by negatively charged membrane surfaces (assemblies of negatively charged lipids `connected' with hydrophobic interaction) at physiological conditions, especially in the view of the proposed decrease in pH at the membrane surface and a `partial' unfolding as an inevitable condition for protein translocation [48^ 50]. Importantly, conformational changes of proteins might occur even at more neutral pH and/or be more profound in connection with the proposed local decrease of the dielectric constant near the membrane surface [5] that we have recently shown in the systems ferricyt c-`hydrophobic' polyanions [20,21,41]. In summary, in the present paper it is shown that ferricyt c in a complex with a negatively charged surface, the polyanion PVS, is relatively labile and can readily undergo a transition from a native-like to a molten globule-like state due to acidi¢cation of the bu¡er. These ¢ndings point to the critical importance of cooperativity among the negative charges represented by covalent (polyanion) or by hydrophobic
BBAPRO 35999 30-9-99
354
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
interaction (phospholipids in the membrane) in induction of a destabilizing e¡ect on protein structure. Based upon these results we believe that the proteinpolyanion system can provide useful information about conformational transitions of proteins connected with their association with negatively charged surface. This system can also provide an assessment of thermodynamic parameters of protein in such a complex from denaturant and temperature-induced unfolding experiments (work in preparation) which can lead to more general conclusions about interactions between macromolecular systems. Acknowledgements The authors gratefully acknowledge the ¢nancial support provided by the Slovak Grant Agency VEGA to this research through Grants No. 4173, No. 5053. We are indebted to Prof. F.X. Schmid for his generous permission to measure CD spectra in his laboratory and to Linda H. Sowdal for help with the manuscript.
References [1] O.B. Ptitsyn, Adv. Protein Chem. 47 (1995) 83^229. [2] O.B. Ptitsyn, T.E. Creighton (Ed.), Protein Folding, W.H. Freeman and Co., New York, 1992, pp. 243^300. [3] H.H.J. de Jongh, J.A. Killiam, B. de Kruij¡, Biochemistry 31 (1992) 1636^1643. [4] M. Prats, J. Teissie¨, J.-F. Tocanne, Nature 322 (1986) 756^ 758. [5] V.E. Bychkova, A.E. Dujsekina, S.I. Klenin, E.I. Tiktopulo, V.N. Uversky, O.B. Ptitsyn, Biochemistry 35 (1996) 6058^ 6063. [6] P. Hildebrandt, T. Heimburg, D. Marsh, Eur. Biophys. J. 18 (1990) 193^201. [7] A. Muga, H.H. Mantsch, W.K. Surewicz, Biochemistry 30 (1991) 7219^7224. [8] M. Ryto«maa, P. Mustonen, P.K.J. Kinnunen, J. Biol. Chem. 267 (1992) 22243^22248. [9] M. Ryto«maa, P.K.J. Kinnunen, J. Biol. Chem. 270 (1992) 3197^3202. [10] F. Zhang, E.S. Rowe, Biochim. Biophys. Acta 1193 (1994) 219^225. [11] I. Hamachi, A. Fujita, T.J. Kunitake, Am. Chem. Soc. 119 (1997) 9096^9102. [12] G. Chottard, M. Michelon, M. Herve¨, G. Herve¨, Biochim. Biophys. Acta 916 (1987) 402^410.
[13] P. Hildebrandt, M. Stockburger, Biochemistry 28 (1989) 6710^6721. [14] P. Hildebrandt, M. Stockburger, Biochemistry 28 (1989) 6722^6728. [15] P. Hildebrandt, Biochim. Biophys. Acta 1040 (1990) 175^ 186. [16] M. Antal|¨k, M. Bona, J. Ba¨gelova¨, Biochem. Int. 28 (1992) 675^682. [17] B. Soussi, A.-Ch. Bylund-Fellenius, T. Scherste¨n, J. Angstro«m, Biochem. J. 265 (1990) 227^232. [18] J. Ba¨gelova¨, M. Antal|¨k, M. Bona, Biochem. J. 297 (1994) 99^101. [19] J. Ba¨gelova¨, M. Antal|¨k, Z. Tomori, Biochem. Mol. Biol. Int. 43 (1997) 891^900. [20] E. Sedla¨k, M. Antal|¨k, J. Ba¨gelova¨, M. Fedurco, Biochim. Biophys. Acta 1319 (1997) 258^266. [21] E. Sedla¨k, M. Antal|¨k, Biopolymers 46 (1998) 145^154. [22] C.A. Yu, J. Steidl, L. Yu, Biochim. Biophys. Acta 736 (1983) 226^234. [23] C.A. Yu, S.H. Gwak, L. Yu, Biochim. Biophys. Acta 812 (1985) 656^664. [24] G.C. Kresheck, J.E. Erman, Biochemistry 27 (1988) 2490^ 2496. [25] T.J.T. Pinheiro, A. Watts, Biochemistry 33 (1994) 2451^ 2458. [26] T. Heimburg, P. Hildebrandt, D. Marsh, Biochemistry 30 (1991) 9084^9089. [27] E. Margoliash, N. Frohwirt, J. Biochem. 71 (1959) 570^ 572. [28] Y.P. Myer, A.F. Saturno, J. Protein Chem. 9 (1990) 379^ 387. [29] T.Y. Tsong, Biochemistry 15 (1967) 5747^5773. [30] Y. Goto, L.J. Calciano, A.L. Fink, Proc. Natl. Acad. Sci. USA 87 (1990) 573^577. [31] Y. Goto, S. Nishikiori, J. Mol. Biol. 222 (1991) 679^686. [32] M.-F. Jeng, S.W. Englander, J. Mol. Biol. 221 (1991) 1045^ 1061. [33] Y.P. Myer, J. Biol. Chem. 243 (1968) 2115^2122. [34] Y.P. Myer, Biochemistry 7 (1968) 765^776. [35] T.J.T. Pinheiro, G.A. Elo«ve, A. Watts, H. Roder, Biochemistry 36 (1997) 13122^13132. [36] A.M. Davies, J.G. Guillemette, M. Smith, C. Greenwood, A.G.P. Thurgood, A.G. Mauk, G.R. Moore, Biochemistry 32 (1993) 5431^5435. [37] G.J. Pielak, K. Oikawa, A.G. Mauk, M. Smith, M.K. Cyril, J. Am. Chem. Soc. 108 (1986) 2724^2727. [38] L.S. Kaminsky, F.C. Yong, T.E. King, J. Biol. Chem. 247 (1972) 1354^1359. [39] S. Ban¬uelos, A. Muga, J. Biol. Chem. 270 (1995) 29910^ 29915. [40] S. Ban¬uelos, A. Muga, FEBS Lett. 386 (1996) 21^25. [41] E. Sedla¨k, Biochem. Mol. Biol. Int. 41 (1997) 25^32. [42] M. Antal|¨k, M. Bona, Z. Gazova¨, A. Kucha¨r, Biochim. Biophys. Acta 1100 (1992) 155^159. [43] E.A.E. Garber, E. Margoliash, Biochim. Biophys. Acta 1187 (1994) 289^295.
BBAPRO 35999 30-9-99
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355 [44] Y. Goto, N. Takashi, A.L. Fink, Biochemistry 29 (1990) 3480^3488. [45] S. Potekhin, W. Pfeil, Biophys. Chem. 34 (1989) 55^62. [46] Y. Hagihara, Y. Tan, Y. Goto, J. Mol. Biol. 237 (1994) 336^ 348. [47] D. Hamada, S.-I. Kidokoro, H. Fukada, K. Takahashi, Y. Goto, Proc. Natl. Acad. Sci. USA 91 (1994) 10325^10329.
355
[48] M. Eilers, G. Schatz, Nature 322 (1986) 228^232. [49] M. Eilers, S. Hwang, G. Schatz, EMBO J. 7 (1988) 1139^ 1145. [50] T. Endo, G. Schatz, EMBO J. 7 (1988) 1153^1158.
BBAPRO 35999 30-9-99
Molten globule-like state of cytochrome c induced by polyanion poly(vinylsulfate) in slightly acidic pH Erik Sedla¨k a , Maria¨n Antal|¨k
b;
*
Department of Biochemistry, Faculty of Science, P.J. Síafa¨rik University, Moyzesova 11, 041 54 Kosice, Slovak Republic Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 043 53 Kosice, Slovak Republic a
b
Received 21 April 1999; received in revised form 6 July 1999; accepted 12 August 1999
Abstract The effect of polyanion, poly(vinylsulfate), used as a model of negatively charged surface, on ferric cytochrome c (ferricyt c) structure in acidic pH has been studied by absorbance spectroscopy, circular dichroism (CD), tryptophan (Trp) fluorescence and microcalorimetry. The polyanion induced only small changes in the native structure of the protein at neutral pH, but it profoundly shifted the acid induced high spin state of the heme in the active center of cyt c to a more neutral pH region. Cooperativity of the acidic transition of ferricyt c in the presence of the polyanion was disturbed, in comparison with uncomplexed protein, as followed from different apparent pKa values observed in a distinct regions of the ferricyt c electronic absorbance spectrum (4.55 þ 0.08 in the 620 nm band region and 5.47 þ 0.15 in the Soret region). The ferricyt c structure in the complex with the polyanion at acidic pH (below pH 5.0) has properties of a molten globule-like state. Its tertiary structure is strongly disturbed according to CD and microcalorimetry measurements ; however, its secondary structure, from CD, is still native-like and ferricyt c is in a compact state as evidenced by quenched Trp fluorescence. These findings are discussed in the context of the molten globule state of proteins induced on a negatively charged membrane surface under physiological conditions. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome c; Polyanion; Protein folding; Molten globule; Microcalorimetry ; Circular dichroism spectroscopy
1. Introduction The molten globule-like state, as well as other nonnative states of proteins, can exist in a living cell and can be involved in di¡erent physiological processes [1]. Proteins can be transformed in vitro into the molten globule state at low pH, moderate concentrations of denaturants, high temperature, i.e. at conditions which are far from usual physiological conditions [2]. On the other hand, it was shown that
* Corresponding author. Fax: +421 (95) 763754; E-mail: [email protected]
binding to a membrane surface can lead to a `partial denaturation' of proteins, transforming them into a non-native state [3]. A possible mechanism of how a negatively charged membrane surface can promote protein destabilization requires the consideration of local pH. The high density of negative charges on a membrane surface creates a strong electrostatic potential which leads to a decrease of local pH at the membrane surface. However, even in salt-free solutions, this local decrease of pH does not exceed apî from the membrane surprox. 2 pH units at 5^15 A face [4], which is usually insu¤cient for acid denaturation. In recent work [5] an additional denaturation ac-
0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 1 8 6 - 7
BBAPRO 35999 30-9-99
348
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
tion of the membrane surface was proposed ^ a local decrease of the dielectric constant near the membrane surface modeled by water-alcohol mixtures. Cytochrome (cyt) c, as a typical extrinsic membrane protein, was chosen for this modeling. Due to its location on the surface of the inner mitochondrial membrane and its ability to associate with negatively charged membrane phospholipids as well as negatively charged groups of the redox partners, a lot of e¡ort was devoted to the understanding of the e¡ect of negatively charged membrane surfaces on cyt c structure [6^11]. Because of the complexity of the interactions between biopolymers and the molecular assemblies, rigid heteropolytungstates were chosen as a suitable model of negatively charged surfaces [12^15]. Our group focused our attention on a di¡erent model of negatively charged surface, i.e., on linear polyanions such as heparin, polyglutamate, poly(vinylsulfate) (PVS), poly(4-styrene-sulfonate). The advantage of these polyanions is their stability and solubility in a wide range of pH values. Hybridization of cyt c with the £exible arti¢cial polyanions seemed to be a very fruitful model system for understanding the conformational changes of cyt c induced by the electrostatic ¢eld of negatively charged surfaces. It was found that they shift the pH value of the socalled alkaline isomerization of cyt c towards higher pH values, similar to what has been shown with cyt c oxidase and cardiolipin [16,17]. In other studies [18^ 21] polyanions were found to decrease the temperature transition for cyt c, which was similar to the e¡ect of its natural redox partners as well as anionic phospholipid bilayers [7,8,22^25]. Moreover, it is known that one likely structural change for the lipid, due to peripheral interaction of proteins with negatively charged lipids, is an increase in local surface curvature, which in turn could favor the conformational change in proteins [26]. The £exibility of the linear polyanions is therefore the other reason for its utilization as a model for the protein-membrane surface system. We believe that complex cyt c-£exible polyanion in an slightly acidic pH region may be a suitable model for the study of conformational changes of positively charged proteins induced by unspeci¢c interaction with negatively charged membrane surface. In the present paper, therefore, the properties of a complex
ferricyt c-polyanion PVS in an acidic pH region are discussed in this context. Subsequently the e¡ect of the linear polyanion with that of single anions on protein structure in an acidic pH is compared. The pH induced structural modi¢cations in a complexed ferricyt c have been investigated by means of electronic absorption, £uorescence and circular dichroism (CD) spectroscopic techniques and by di¡erential scanning calorimetry (DSC). 2. Materials and methods 2.1. Materials Horse heart cytochrome c, type III, was obtained from Sigma and used without further puri¢cation. Poly(vinylsulfate) was from Aldrich. Other chemicals were obtained from Lachema. 2.2. Bu¡ers For CD and DSC measurements in acidic pH: 10 mM glycine in pH 2.2^3.6, 10 mM acetate in pH 3.7^5.6, 10 mM cacodylate in pH 5.0^7.4, 10 mM phosphate in pH 5.8^8.0. 2.3. Absorption spectra Spectrophotometric observations were performed on a Shimadzu UV-3000 spectrophotometer. Absorption measurements were performed with a solution of 8.5 WM cyt c [27]. Acidic transitions were determined from di¡erences at 354^395 nm in the Soret region and from di¡erences at 556^622 nm in the region 500^800 nm with a 8.5 WM and 34.0 WM solution of cyt c, respectively. The spectrum of ferricyt c at pH 7.6 in 10 mM phosphate was taken as reference. 2.4. CD spectra CD measurements were obtained using a Jasco J-600 spectropolarimeter. CD measurements were performed with a solution of 22.9 WM cyt c containing 10 mM phosphate, pH 7.0, 5 WM potassium ferricyanide in 0.5 cm or 1 cm path cuvettes. PVS has neither absorbance nor CD contribution
BBAPRO 35999 30-9-99
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
into related spectra in the investigated regions under studied conditions. 2.5. Fluorescence Fluorescence measurements were performed on a Shimadzu RF-5000 spectro£uorometer. The excitation wavelength was 290 nm. Trp-59 £uorescence emission was followed at 350 nm. PVS does not signi¢cantly a¡ect the £uorescence of the free tryptophan in the investigated conditions. The temperature of the cell compartments was kept constant at 25³C by water circulation, pH was changed by addition of concentrated HCl. The pH values were measured with a Sensorex glass electrode. 2.6. DSC measurements Di¡erential scanning calorimetric measurements were made on a high-sensitivity DASM-4 microcalorimeter at a heating rate of 1 K/min. The protein concentration was 110 WM. Prior to use cyt c was converted to the fully oxidized form by addition of potassium ferricyanide. Ferricyt c-PVS complex was always prepared at pH 7.0 and then pH was adjusted to the required value. The reversibility of the ferricyt c-PVS complex at a low ionic strength (2 mM phosphate bu¡er) formed at a high concentration of polyanion (3.3 mg/ml) was more than 90% (not shown). PVS has no thermal transition under the investigated conditions. The pH value of the solution was measured always before and after cooling the sample. Only the measurements at which the pH change was less than 0.2 pH units were taken for further consideration. 3. Results 3.1. Acidic transition of ferricyt c-PVS complex In our very recent work it was shown that the acidic transition from a low to a high spin state of ferricyt c in the complex with PVS was profoundly shifted to more neutral pH in comparison with uncomplexed ferricyt c in a low ionic strength bu¡er [21]. In order to distinguish between the e¡ect of
349
polyanion PVS and free `small' anions on the acidic transition of ferricyt c we have compared the in£uence of high ionic strength with that of the polyanion on the midpoint pH value of the ferricyt c conformational transition. The apparent pKa of the free ferricyt c acidic transition in a low ionic strength bu¡er obtained from spectral changes observed in the Soret (354^394 nm) region and in the 620 nm (556^622) band regions (Fig. 1) was 2.46 þ 0.06 which was in good agreement with the published results [28]. High ionic strength (0.5 M NaCl) caused a shift of the pKa value of the acidic transition to a more neutral pH, apparent pKa was 3.36 þ 0.08, in both spectral regions (Fig. 1). Normalizing the dependencies of the ferricyt c spectral changes in the low and high ionic strength bu¡ers obtained from the di¡erent spectral regions shows the apparent pKa values did not signi¢cantly depend on which spectral region was investigated. From the e¡ect of high ionic strength on the midpoint pH of the ferricyt c acidic transition it might be expected that the polyanion PVS would induce a shift to more neutral pH, but in comparison with `small' anions this shift was much greater. The distinguishing di¡erence between the e¡ect of high ionic strength and the e¡ect of PVS on the ferricyt c acidic transition was a separation of the conformational changes occurring in the protein structure in the complex with polyanion. In the 620 nm band region the apparent pKa of the transition was 4.55 þ 0.08, while in the Soret region the apparent pKa was shifted to a more neutral pH 5.47 þ 0.15. Since di¡erent bands monitor di¡erent aspects of protein perturbation, a cooperativity of the acidic transition of ferricyt c was disturbed in the presence of polyanion PVS. In the e¡ort to assess structural perturbations of the heme region induced by acidi¢cation of bu¡er in the above investigated conditions the spectral changes in the Soret region were compared. Such a comparison was possible because a shape and a position of maxima and minima in the di¡erence spectra in all cases were very similar (not shown). Fig. 2 demonstrates this comparison: observed di¡erences in the Soret region at 354^394 nm were normalized to the maximum of the spectral change of ferricyt c in the low ionic strength bu¡er. As shown, conformational changes of ferricyt c re£ected by the
BBAPRO 35999 30-9-99
350
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
changes in the Soret region in the electronic absorbance spectrum were comparable and signi¢cantly smaller in the high ionic strength and in the presence of the polyanion PVS. The structural changes occurring in the deeply buried Trp-59 region may be detected by £uorescence intensity investigation. A single tryptophan in the polypeptide chain of cytochrome c is localized in the vicinity of the heme, which is responsible for a strong quenching through Fo«rster energy transfer in the native state of the protein (inset Fig. 2). The increase in the Trp distance from the heme results in an increase in the £uorescence intensity and this can be used to detect subtle conformational changes occurring around the heme [29]. From the midpoint pH of the acidic transition determined from £uorescence changes follows that ferricyt c underwent a highly cooperative transition to an expanded unfolded state due to the acidi¢cation of the low ionic strength bu¡er. On the other hand, Trp £uorescence of ferricyt c in high ionic strength, as in the presence of polyanion PVS, was quenched even at very acidic pH (inset Fig. 2) which indicates a compact state of
Fig. 2. Comparison of observed di¡erences of ferricyt c at acidic transition in low (E), high (a) ionic strength and in the presence of PVS (b) in the Soret region. Di¡erences were normalized on maximum di¡erence of ferricyt c di¡erence spectrum in low ionic strength. (Inset) Acidic transition of ferricyt c in low (E), high (a) ionic strength and in the presence of PVS (b) followed by Trp-59 £uorescence. Ionic strength conditions are the same as in Fig. 1.
ferricyt c in both cases. These results are in agreement with the decreased spectral changes observed in the Soret region of the absorbance spectrum of ferricyt c in acidic pH in these conditions (Fig. 2). It is not surprising as it is known that cytochrome c at pH approx. 2 and in high ionic strength is in the molten globule state that is characterized as a compact denatured state with a signi¢cant amount of native-like secondary structure, but a largely £exible and disordered tertiary structure [30^32]. 3.2. CD measurements
Fig. 1. Acidic transition of ferricyt c in low ionic strength solvent (10 mM phosphate) followed at 556^622 nm (E), 354^394 nm (F) of electronic absorption spectra and by Trp-59 £uorescence (P); ferricyt c in high ionic strength solvent (10 mM phosphate +0.5 M NaCl) followed at 556^622 nm (a), 354^394 nm (b) of electronic absorption spectra; ferricyt c-PVS complex in low ionic strength solvent (10 mM phosphate) followed at 556^622 nm (R), 354^394 nm (*) of electronic absorption spectra.
Circular dichroism was used to monitor the e¡ect of the acidi¢cation on ferricyt c secondary and tertiary structures in the complex with PVS. The far-UV CD spectrum of ferricyt c in solution shows the typical features of K-helical protein structure (Fig. 3A), in which the 222 nm dichroic band is predominantly associated with K-helical nCZ* amide transitions [33]. Neither interaction of PVS with ferricyt c nor following acidi¢cation of the bu¡er induced signi¢cant changes in the K-helix content. The spectral changes around the minimum at 208 nm, dichroic band corresponding to the ZCZ*
BBAPRO 35999 30-9-99
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
351
Fig. 3. Peptide (A), aromatic (B) and visible (C) circular dichroism spectra of ferricyt c at pH 7.0 (ööö) and ferricyt c in the presence of PVS at: pH 7.0 (999), pH 5.2 (W W W), pH 3.0 (^ ^ ^), pH 2.0 (- - -) in low ionic strength (10 mM bu¡er). (A) Peptide, deg.cm2 /mol of amide bonds; (B) aromatic and visible, deg.cm2 /dmol of heme.
amide transitions [34], may arise from changes in other secondary structure elements in the protein, or may be due to the presence of optically active heme transitions other than those associated with the amide transitions of the polypeptide chain. Therefore, an unambiguous interpretation of the spectral changes on this region of the spectrum was not possible [35]. Near-UV circular dichroism is a probe for protein tertiary structure changes that a¡ect the environment of aromatic side chains. In the aromatic region of a CD spectrum of ferricyt c two sharp minima are remarkable at 282 and 288 nm which are assigned to the Trp-59 side chain [34,36]. Upon binding to PVS at neutral pH, no signi¢cant changes are observed in the aromatic region of the CD spectrum of ferricyt c. However, opposite to the far-UV spectra of ferricyt c-PVS, the acidi¢cation of the bu¡er induced profound changes in this region of the CD spectrum: these near-UV CD spectral markers disappeared in an slightly acidic pH, with the apparent pKa = 4.6 þ 0.1 (not shown), which indicates a disruption of the tight packing of core residues in the cyt cPVS complex. Cytochrome c exhibits a characteristic CD doublet in the Soret region: the intensity of the negative band at 417 nm depends on heme-protein interactions [34] or, more exactly, on the distance and orientation of the phenylalanine residue positioned on the methionine side of the heme plane [37]. The addition of PVS
to the solution of ferricyt c at neutral pH resulted in insigni¢cant changes in the intensity of the CD signal of ferricyt c in the Soret region. Similar to the aromatic region, acidi¢cation of the solvent induced profound changes in the Soret CD ^ the negative Cotton e¡ect at 417 nm disappeared and the spectrum changed to a single positive band with maximum near 408 nm. Noteworthy, the changes observed upon acidi¢cation in the Soret and aromatic regions of the ferricyt c-PVS complex are also observed with urea-denatured cyt c, and are induced by elevated temperature or pH-induced denaturation [34], alcohol denaturation [38] and, as we have shown, also by complexation of ferricyt c with `hydrophobic' polyanions [20,21]. The observed changes in the Soret region clearly indicate a disruption of the coupling between ZCZ* transitions of the heme group and those of the aromatic amino acid residues in its proximity. This e¡ect is consistent with loosening of the tertiary structure, which removes the anisotropic character of the Soret CD band. 3.3. DSC measurements From the CD results it might be concluded that a perturbation of the interior of ferricyt c structure in the complex with PVS by the acidi¢cation of the solvent medium took place. The disturbance of the hydrophobic core of the protein paralleled by changes in its tertiary structure was therefore further
BBAPRO 35999 30-9-99
352
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
Fig. 4. DSC scans of ferricyt c-PVS in 10 mM phosphate at pH 6.1 and 7.0, in 10 mM acetate at pH 5.1, in 10 mM glycine bu¡er at pH 2.5. The PVS concentration in all samples was 1.0 mg/ml.
explored by DSC experiments. The endotherm of ferricyt c is characterized by a single peak at 84.9³C, calorimetric enthalpy (vHcal ) of 390 kJ/mol (not shown). The binding of ferricyt c to the polyanion PVS in a low ionic strength bu¡er induced a large decrease in the denaturation temperature from 84.9 to 52.8³C (Fig. 4). This shift of temperature was interpreted as an overall protein destabilization. However, ferricyt c remained in a compact form as indicated by only a small decrease of the calorimetric enthalpy transition (vHcal approx. 350 kJ/mol) even at high (3.3 mg/ml) concentration of PVS [21]. A decrease in the pH of the bu¡er slightly diminished the temperature of denaturation, as followed by DSC, of ferricyt c in the complex with PVS. This change was paralleled by an extensive reduction in the calorimetric enthalpy of the transition, with a loss of the thermal transition ferricyt c-PVS complex below pH approx. 4.9 (Fig. 4). No other thermal transition was observed in the temperature range from 2 to 120³C at the investigated conditions. 4. Discussion The interaction of extrinsic proteins with membrane surfaces is a common process during the course of various cellular functions. Although it is becoming apparent that the transition from a
water-soluble to a membrane-associated state involves major changes in the protein structure, the mechanism of the association is at present poorly understood [3,35,39,40]. Such systems are complex and have a tendency to precipitate, particularly in an acidic pH where the negative charges of the membrane are partially neutralized. As a possible mechanism of how a negatively charged membrane surface can promote protein conformational changes requires the consideration of a local pH this seems to be a major disadvantage in the investigation of pH e¡ect on protein structure in the complex with membrane surface. On the other hand linear polyanions, whose interaction with proteins is at the center of our attention, are charged and also in complex with most proteins soluble over wide pH range. In our recent work we were therefore capable of determining the apparent pKa of the acidic transition of ferricyt c in the presence of polyanions and thus showed that both heparin and PVS strongly shift the acidic transition of ferricyt c to a more neutral pH region [21,41]. This is interesting in light of the published observation that at pH 4.0 (the region of the pKa of ferricyt c-polyanion complex) the apparent a¤nity of cyt c for cardiolipin-containing liposomes was signi¢cantly increased [8]. Moreover, it was shown that heparin and PVS induced a decrease of the calorimetrically observed temperature of denaturation of ferricyt c to 59.7 and 52.8³C, respectively [18,21]. These results are analogous with those obtained in the system with ferricyt c and a membrane surface containing acidic phospholipids [7,10]. Based on these preliminary results we have chosen PVS as a suitable model of a negatively charged membrane, which might elucidate conformational changes of proteins connected with association to the membrane surface in the locally decreased pH. It has been shown previously that two di¡erent conformational states, I and II, are induced on binding ferricyt c to negatively charged surfaces [13,15] including anionic lipid dispersions [6,26]. While the conformation of state I is very similar to that of the protein in solution and the native six-coordinated low-spin con¢guration of the heme is preserved, in state II the heme crevice opens and the iron exists in a thermal equilibrium between a ¢ve-coordinated high-spin and a new six-coordinated low-spin con¢g-
BBAPRO 35999 30-9-99
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
uration. A native-like state of ferricyt c was also observed in the complex with £exible polyanions like heparin, polyglutamate and PVS evidenced by optical spectroscopic techniques [20,21,41^43]. Ferricyt c binding to polyanions is connected with a discontinuous decrease in the melting temperature of ferricyt c observed by microcalorimetry [18,20,21]. Interestingly, ferricyt c undergoes an analogous change in its melting temperature under complexation with a negatively charged membrane [7,10,22]. The calorimetric observations were interpreted as an overall destabilization of ferricyt c structure due to electrostatic interaction with negatively charged surfaces. On the other hand, the protein remained in a compact state as con¢rmed by only a small change in enthalpy of its thermal transition with even stabilized structure of the heme crevice evidenced by a shift of alkaline isomerization of ferricyt c in the complex to a more alkaline pH [16,17,19]. Our ¢ndings con¢rmed that the compact state of ferricyt c with native-like spectral features in the complex with PVS at neutral pH is a relatively labile state. A decrease in the pH of the solvent brought about large conformational changes in ferricyt c structure already at slightly acidic pH. From comparison of the observed changes of ferricyt c-PVS complex and ferricyt c in high ionic strength in the acidic pH in the Soret region with those in low ionic strength one could conclude that the heme region of ferricyt c is less perturbed in both former conditions. Moreover, quenching of the intrinsic probe of ferricyt c structure-Trp-59 by the heme suggests a compactness of the heme region and a capability of anions to partially preserve the structure of ferricyt c in the acidic pH in both cases. Although ferricyt c in the complex with PVS in neutral pH has preserved tertiary structure, it was very labile as expressed by the loss of cooperativity of its acidic transition (Fig. 1), and by the abrupt decrease in the calorimetric enthalpy of the thermal transition of ferricyt c in slightly acidic pH with its loss at pH approx. 4.9 (Fig. 4). All these ¢ndings together show that ferricyt c in the complex with polyanion PVS at the acidic pH is in `a compact structure compared to that of the unfolded state (Fig. 2), buried tryptophan residue (inset Fig. 2), with a high content of secondary structure (Fig. 3) and largely disordered tertiary structure (Fig. 4)', i.e. in the conformational state of proteins observed at
353
low pH in high ionic strength solution, de¢ned as a molten globule state [44]. At this point we need to stress the di¡erences between the e¡ects of `small' anions and polyanions, i.e. between free and covalently bound anions, on ferricyt c structure. While ferricyt c could attain a `classic' molten-like conformation only in the presence of high concentrations of anions, the polyanion a¡ected the protein structure at a concentration comparable to that of the protein. Moreover, the e¡ect of the polyanion on ferricyt c is stronger as seen in the pronounced shift of the acidic transition to more neutral pH, as well as the disturbance of the cooperativity of this transition and the loss of the calorimetrically observed thermal transition of ferricyt c in this `new' molten globule state [44^47]. Interestingly, our preliminary results from denaturant and temperature-unfolding experiments of the ferricyt c-PVS complex indicate that polyanion strongly stabilizes secondary structure and compact state of ferricytochrome c even at high concentration of the denaturant or high temperature at slightly acidic pH. The double e¡ect of the electrostatic ¢eld of the polyanion (destabilization of tertiary and stabilization of secondary structure and compactness of the protein) on the ferricytochrome c protein structure might be important for an understanding of a mechanism for conformational changes of proteins induced by negatively charged membrane surfaces (assemblies of negatively charged lipids `connected' with hydrophobic interaction) at physiological conditions, especially in the view of the proposed decrease in pH at the membrane surface and a `partial' unfolding as an inevitable condition for protein translocation [48^ 50]. Importantly, conformational changes of proteins might occur even at more neutral pH and/or be more profound in connection with the proposed local decrease of the dielectric constant near the membrane surface [5] that we have recently shown in the systems ferricyt c-`hydrophobic' polyanions [20,21,41]. In summary, in the present paper it is shown that ferricyt c in a complex with a negatively charged surface, the polyanion PVS, is relatively labile and can readily undergo a transition from a native-like to a molten globule-like state due to acidi¢cation of the bu¡er. These ¢ndings point to the critical importance of cooperativity among the negative charges represented by covalent (polyanion) or by hydrophobic
BBAPRO 35999 30-9-99
354
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355
interaction (phospholipids in the membrane) in induction of a destabilizing e¡ect on protein structure. Based upon these results we believe that the proteinpolyanion system can provide useful information about conformational transitions of proteins connected with their association with negatively charged surface. This system can also provide an assessment of thermodynamic parameters of protein in such a complex from denaturant and temperature-induced unfolding experiments (work in preparation) which can lead to more general conclusions about interactions between macromolecular systems. Acknowledgements The authors gratefully acknowledge the ¢nancial support provided by the Slovak Grant Agency VEGA to this research through Grants No. 4173, No. 5053. We are indebted to Prof. F.X. Schmid for his generous permission to measure CD spectra in his laboratory and to Linda H. Sowdal for help with the manuscript.
References [1] O.B. Ptitsyn, Adv. Protein Chem. 47 (1995) 83^229. [2] O.B. Ptitsyn, T.E. Creighton (Ed.), Protein Folding, W.H. Freeman and Co., New York, 1992, pp. 243^300. [3] H.H.J. de Jongh, J.A. Killiam, B. de Kruij¡, Biochemistry 31 (1992) 1636^1643. [4] M. Prats, J. Teissie¨, J.-F. Tocanne, Nature 322 (1986) 756^ 758. [5] V.E. Bychkova, A.E. Dujsekina, S.I. Klenin, E.I. Tiktopulo, V.N. Uversky, O.B. Ptitsyn, Biochemistry 35 (1996) 6058^ 6063. [6] P. Hildebrandt, T. Heimburg, D. Marsh, Eur. Biophys. J. 18 (1990) 193^201. [7] A. Muga, H.H. Mantsch, W.K. Surewicz, Biochemistry 30 (1991) 7219^7224. [8] M. Ryto«maa, P. Mustonen, P.K.J. Kinnunen, J. Biol. Chem. 267 (1992) 22243^22248. [9] M. Ryto«maa, P.K.J. Kinnunen, J. Biol. Chem. 270 (1992) 3197^3202. [10] F. Zhang, E.S. Rowe, Biochim. Biophys. Acta 1193 (1994) 219^225. [11] I. Hamachi, A. Fujita, T.J. Kunitake, Am. Chem. Soc. 119 (1997) 9096^9102. [12] G. Chottard, M. Michelon, M. Herve¨, G. Herve¨, Biochim. Biophys. Acta 916 (1987) 402^410.
[13] P. Hildebrandt, M. Stockburger, Biochemistry 28 (1989) 6710^6721. [14] P. Hildebrandt, M. Stockburger, Biochemistry 28 (1989) 6722^6728. [15] P. Hildebrandt, Biochim. Biophys. Acta 1040 (1990) 175^ 186. [16] M. Antal|¨k, M. Bona, J. Ba¨gelova¨, Biochem. Int. 28 (1992) 675^682. [17] B. Soussi, A.-Ch. Bylund-Fellenius, T. Scherste¨n, J. Angstro«m, Biochem. J. 265 (1990) 227^232. [18] J. Ba¨gelova¨, M. Antal|¨k, M. Bona, Biochem. J. 297 (1994) 99^101. [19] J. Ba¨gelova¨, M. Antal|¨k, Z. Tomori, Biochem. Mol. Biol. Int. 43 (1997) 891^900. [20] E. Sedla¨k, M. Antal|¨k, J. Ba¨gelova¨, M. Fedurco, Biochim. Biophys. Acta 1319 (1997) 258^266. [21] E. Sedla¨k, M. Antal|¨k, Biopolymers 46 (1998) 145^154. [22] C.A. Yu, J. Steidl, L. Yu, Biochim. Biophys. Acta 736 (1983) 226^234. [23] C.A. Yu, S.H. Gwak, L. Yu, Biochim. Biophys. Acta 812 (1985) 656^664. [24] G.C. Kresheck, J.E. Erman, Biochemistry 27 (1988) 2490^ 2496. [25] T.J.T. Pinheiro, A. Watts, Biochemistry 33 (1994) 2451^ 2458. [26] T. Heimburg, P. Hildebrandt, D. Marsh, Biochemistry 30 (1991) 9084^9089. [27] E. Margoliash, N. Frohwirt, J. Biochem. 71 (1959) 570^ 572. [28] Y.P. Myer, A.F. Saturno, J. Protein Chem. 9 (1990) 379^ 387. [29] T.Y. Tsong, Biochemistry 15 (1967) 5747^5773. [30] Y. Goto, L.J. Calciano, A.L. Fink, Proc. Natl. Acad. Sci. USA 87 (1990) 573^577. [31] Y. Goto, S. Nishikiori, J. Mol. Biol. 222 (1991) 679^686. [32] M.-F. Jeng, S.W. Englander, J. Mol. Biol. 221 (1991) 1045^ 1061. [33] Y.P. Myer, J. Biol. Chem. 243 (1968) 2115^2122. [34] Y.P. Myer, Biochemistry 7 (1968) 765^776. [35] T.J.T. Pinheiro, G.A. Elo«ve, A. Watts, H. Roder, Biochemistry 36 (1997) 13122^13132. [36] A.M. Davies, J.G. Guillemette, M. Smith, C. Greenwood, A.G.P. Thurgood, A.G. Mauk, G.R. Moore, Biochemistry 32 (1993) 5431^5435. [37] G.J. Pielak, K. Oikawa, A.G. Mauk, M. Smith, M.K. Cyril, J. Am. Chem. Soc. 108 (1986) 2724^2727. [38] L.S. Kaminsky, F.C. Yong, T.E. King, J. Biol. Chem. 247 (1972) 1354^1359. [39] S. Ban¬uelos, A. Muga, J. Biol. Chem. 270 (1995) 29910^ 29915. [40] S. Ban¬uelos, A. Muga, FEBS Lett. 386 (1996) 21^25. [41] E. Sedla¨k, Biochem. Mol. Biol. Int. 41 (1997) 25^32. [42] M. Antal|¨k, M. Bona, Z. Gazova¨, A. Kucha¨r, Biochim. Biophys. Acta 1100 (1992) 155^159. [43] E.A.E. Garber, E. Margoliash, Biochim. Biophys. Acta 1187 (1994) 289^295.
BBAPRO 35999 30-9-99
E. Sedla¨k, M. Antal|¨k / Biochimica et Biophysica Acta 1434 (1999) 347^355 [44] Y. Goto, N. Takashi, A.L. Fink, Biochemistry 29 (1990) 3480^3488. [45] S. Potekhin, W. Pfeil, Biophys. Chem. 34 (1989) 55^62. [46] Y. Hagihara, Y. Tan, Y. Goto, J. Mol. Biol. 237 (1994) 336^ 348. [47] D. Hamada, S.-I. Kidokoro, H. Fukada, K. Takahashi, Y. Goto, Proc. Natl. Acad. Sci. USA 91 (1994) 10325^10329.
355
[48] M. Eilers, G. Schatz, Nature 322 (1986) 228^232. [49] M. Eilers, S. Hwang, G. Schatz, EMBO J. 7 (1988) 1139^ 1145. [50] T. Endo, G. Schatz, EMBO J. 7 (1988) 1153^1158.
BBAPRO 35999 30-9-99