Structural Transformation of Apocytochrome c Induced by Alternating

Apocytochrome c interacts with two copolymers: poly(isobutylene-alt-maleic acid) (PIMA) and poly(1-tetradecene-alt-maleic acid) (PTMA). The interactio...
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Biomacromolecules 2005, 6, 2748-2755

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Structural Transformation of Apocytochrome c Induced by Alternating Copolymers of Maleic Acid and Alkene Li Liang, Ping Yao,* and Ming Jiang Department of Macromolecular Science and the Key Laboratory of Molecular Engineering of Polymer, Fudan University, Shanghai 200433, China Received April 7, 2005; Revised Manuscript Received June 9, 2005

Apocytochrome c interacts with two copolymers: poly(isobutylene-alt-maleic acid) (PIMA) and poly(1tetradecene-alt-maleic acid) (PTMA). The interaction leads to apocytochrome c, a conformational change from random coil to R-helical structure. The R-helix content is influenced by the copolymer concentration, the length of alkyl chain of the copolymers, and pH of the medium. The electrostatic attraction between the copolymer and protein is an indispensable factor for the folding of the protein at acid pH. The hydrophobic interaction is an important factor over the entire pH range, especially when both the copolymer and protein carry negative charges at alkaline pH. The electrostatic and hydrophobic attractions between the copolymer and protein exclude water molecules, promoting the formation of hydrogen bonds within the helical structure. On the other hand, the hydrogen bonds formed between the ionized carboxyl of the copolymer and the amide of the protein partly restrain the formation of hydrogen bonds within the helical structure when the copolymer concentration is higher at pH 6.5 and 10.5. Introduction The correct folding of a protein is fundamental for its biological functions. It is widely recognized that the structure of a protein is a result of a delicate balance between the interactions inside and outside the protein. In vivo, protein chaperones can help protein folding by binding with unstructured protein and releasing folded protein to avoid its hydrophobic aggregation.1 In vitro, the addition of various molecules has been one of the few effective approaches to improve protein folding. The studies of the interactions of proteins and polymers by several research groups have shown that protein folding can be assisted by polymers: poly(ethylene glycol) (PEG),2 poly(propylene oxide)-phenylpoly(ethylene glycol) (PPOn-Ph-PEG) nanoassemblies,3 and temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm)4 assisted the refolding of bovine carbonic anhydrase; thiol-modified poly(styrene-co-glycidyl methacrylate) microspheres assisted the refolding of RNase A;5 PEG and PNIPAAm enhanced the renaturation of β-lactamase from inclusion bodies;6 nanogels of cholesterol-bearing pullulan assisted the refolding of carbonic anhydrase and citrate synthase;7 and nanoparticles of sulfonated polystyrene assisted the folding of apocytochrome c.8 Noncovalent bond interactions, that is, hydrogen-bonding, electrostatic, hydrophobic, and van der Waals interactions that exist inside and outside protein, are very important for the protein folding.9 These interactions are feasible to modulate by changing the composition and structure of polymers, as well as by changing the ionic strength, pH, and temperature of the solutions. Hence, synthetic polymers * Corresponding author: fax 86-21-65640293; e-mail yaoping@ fudan.edu.cn.

provide good models to investigate the influence of various interactions on the protein folding. Copolymers of maleic acid and alkene and their derivatives are a series of wellcharacterized polymers. The conformation of these copolymers is jointly determined by the degree of ionization and the length of alkyl chain. Copolymers with long alkyl side chains at low pH tend to have a compact conformation and form micelles, while those with short alkyl side chains at high pH trend to exhibit an extended conformation.10-12 The interactions of ethylene-maleic acid copolymers and derivatives with poly-L-lysine and poly-L-ornithine were investigated by studying the conditions of the coprecipitation, which showed that the complex formation was influenced by the ionization degree of the carboxylic groups and the hydrophobic interactions of alkyl side chain of the polyanion.13 Binding isotherms of β-lactoglobulin or bovine serum albumin interacting with a series of alternating copolymers of maleic acid and alkyl vinyl ethers with varying hydrophobicity by capillary electrophoresis were reported by Gao and Dubin.14 Their studies showed that a minimum alkyl chain length of 3-4 methylenes was required for significant hydrophobic interactions between the proteins and copolymers and that a competition existed between intrapolymer micelle formation and protein-polymer hydrophobic interactions. Cytochrome c (cyt c) plays an electron-transfer role in biological systems and also plays a role in programmed cell death. Because of its small size and stability, as well as the increasing evidence for its physiological roles in both soluble and membrane-bound forms, cyt c has been a subject of extensive studies in protein chemistry and protein folding. Apo cyt c is the precursor of the mitochondrial protein cyt c, which is encoded by nuclear DNA and synthesized on

10.1021/bm050250d CCC: $30.25 © 2005 American Chemical Society Published on Web 07/15/2005

Structural Transformation of Apocytochrome c Scheme 1. Structures of (A) PIMA and (B) PTMA

free cytoplasmic ribosomes. Apo cyt c inserts spontaneously and partially crosses the outer mitochondrial membrane. After or simultaneously with translocation across the outer membrane, apo cyt c binds with heme and then folds around the heme into the native structure.15-17 In contrast with cyt c, which has a compact well-defined structure in aqueous solution, the heme-free apo cyt c has a disordered structure in solution. The influence of electrostatic and hydrophobic interactions on the refolding of apo cyt c and denaturated cyt c has been well studied by interacting with various amphiphilic lipids and detergents at certain pH to mimic the interactions of the protein with mitochondrial membrane.15-21 Previous work in our laboratory found that the R-helical structure of apo cyt c was induced by highly sulfonated polystyrene nanoparticles.8 These studies showed that both electrostatic and hydrophobic interactions were involved in the conformational transition of the protein. The studies of the interactions of apo cyt c with lipid micelles showed that apo cyt c inserted into the lipid micelles after the formation of helical structure on the surface of the micelles.15-17 In addition, the interactions between ethanol and apo cyt c can decrease the hydrogen bonds between apo cyt c and water, increasing the intramolecular hydrogen bonds and then the helical structure of apo cyt c.22 In this study, we investigate the structural transformation of apo cyt c induced by two alternating copolymers, poly(isobutylene-alt-maleic acid) (PIMA) and poly(1-tetradecenealt-maleic acid) (PTMA), at different pHs to understand the influence of the various interactions on the conformational transition of the protein. The structures of PIMA and PTMA are shown in Scheme 1. The substantial difference in the length of the alkyl chains between the two copolymers makes it possible to estimate the effect of hydrophobic interaction between the copolymer and the protein on protein folding. Circular dichroism and steady-state fluorescence, including intrinsic tryptophan fluorescence and additional pyrene fluorescence, are measured to study the secondary structure of apo cyt c, the environment of tryptophan, and the aggregation of the copolymer. Experimental Section Samples. Horse heart cyt c was purchased from Sigma. Apo cyt c was prepared by chemically removing heme group of cyt c as described by Fisher et al.23 The concentration of the stock solution of apo cyt c was 100 µM, which was measured spectrophotometrically with a molar extinction coefficient of 10 580 M-1 cm-1 at 277 nm.24 Poly(isobutylene-alt-maleic anhydride), with MW 6000 and 39 repeat units, and poly(1-tetradecene-alt-maleic anhydride), with MW 9000 and 31 repeat units, were purchased from Aldrich. PIMA and PTMA were obtained by hydrolysis

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of poly(isobutylene-alt-maleic anhydride) in 0.8 mol/L NaOH and poly(1-tetradecene-alt-maleic anhydride) in 0.1 mol/L NaOH solution at 60 °C for 4 h, respectively, followed by dialysis against water repeatedly to remove small molecules. PIMA and PTMA stock solutions were prepared at the desired pH with a concentration of 4.5 × 10-4 g/mL. The pH of the solution was adjusted with HCl or NaOH. Potentiometric Titration. Potentiometric titration of PIMA and PTMA was monitored by a digital pH meter, model PHs-2ST with electrode E201. The concentration of PIMA and PTMA is 4.5 × 10-3 g/mL. The titration with 0.1000 mol/L NaOH and HCl was carried out under nitrogen at room temperature (26 ( 1 °C). The ionization degree was determined from the end point of the titration obtained by subtracting the blank titration according to the method of Kitano et al.25,26 Samples for Spectrophotometry. The samples were prepared by titrating PIMA or PTMA stock solution into desired pH solution, and then followed by titrating apo cyt c stock solution. The samples were prepared at least 12 h before measurement. Circular Dichroic Spectra Measurement. Circular dichroic (CD) spectra were recorded on a Jasco J-715 spectropolarimeter equipped with Naslab temperature controller. The cell length is 0.1 cm for the 190-250 nm region. The ellipticity was recorded at a speed of 100 nm/min, 0.2 nm resolution, 8 accumulations, 1.0 nm bandwidth, and 25 °C. Buffer background was subtracted from the original spectra. The secondary structure was calculated by Model SSE-338 Protein Secondary Structure Estimation Program equipped for the spectropolarimeter as described by Yang et al.27 Steady-State Fluorescence Measurement. The fluorescence was measured on a fluorescence spectrophotometer FLS-920 (Edinburgh Instruments). Pyrene was used as an additional fluorescence probe and its final concentration was 2 × 10-7 g/mL. Before the fluorescence measurement, the solutions were stirred for at least 12 h after the addition of pyrene. The protein intrinsic fluorescence, tryptophan fluorescence, was also measured. The spectral resolution of excitation and emission was 0.5 and 1 nm for pyrene and tryptophan, respectively. The emission spectra were recorded with excitation wavelengths of 335 nm for pyrene and 295 nm for tryptophan, respectively. Results and Discussion Behavior of the Alternating Copolymers in Aqueous Solution. PIMA and PTMA were obtained by hydrolyzing the corresponding alternating anhydride copolymers at alkali solution. The conversion of anhydride into carboxylate was confirmed by IR (Magna-550 infrared spectrophotometer, Nicolet) spectra of the polymers before and after hydrolysis. The strong absorbance at 3300 cm-1 of the stretching vibration of the carboxylate appears and the absorbance of the carbonyl shifts from 1760 to 1560 cm-l, suggesting the hydrolysis is accomplished.28 A number of studies found that poly(maleic acid) and its copolymers ionized in two steps, which is similar to poly(dicarboxylic acids).13,26,28 The ionization degree of the

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Table 1. Ionization Degree and Charges of the Copolymers at Different pH Values pH PIMA PIMA PTMA PTMA

ionization degree (%) negative charges/chain ionization degree (%) negative charges/chain

2.1

6.5

10.5

11.8

11 8 8 5

53 41 55 34

87 68 97 60

100 78 100 62

copolymers at different pH was calculated from the potentiometric titration curves according to the method of Kitano et al.25,26 The charges of each copolymer chain at different pH values were calculated from the ionization degree and the repeat units of the copolymers. These calculated results are shown in Table 1. In the following study of the conformational transition of apo cyt c induced by the copolymers, pH values of 2.1, 6.5, and 11.8 are used, where the copolymers are slightly, about 50%, and fully ionized, respectively. In addition, the pH of 10.5 is selected because it is close to the isoelectric point of cyt c 10.6.29 Pyrene has much lower solubility in water (about 10-7 mol/L) than in hydrocarbons (0.075 mol/L); it significantly transfers into hydrophobic regions once the hydrophobic association occurs in aqueous solution. Pyrene has been widely used to monitor the association and micellization of polymers in solutions because its photophysical character changes when it transfers from a polar environment to a nonpolar one.12,30-33 The hydrophobic association of PIMA and PTMA in aqueous solution is investigated by examining the intensity ratio of the first to third band (I1/I3) in pyrene emission spectra (Figure 1). For PIMA, the I1/I3 ratios of pyrene decrease a little with increasing copolymer concentration and the ratios at pH 2.1 are slightly lower than those at pH 6.5 and 10.5 with the same concentrations. These results suggest that PIMA chains are slightly coiled at pH 2.1. All the I1/I3 ratios in PIMA solutions are larger than 1.60, indicating that PIMA is very hydrophilic and generally in expanded structure in aqueous solution, which is consistent with the study of Kitano et al.28 that the apparent hydrophobicity of PIMA was too weak to cause a conformational transition from an extended coil to a compact globule. However, the I1/I3 ratios of pyrene in PTMA solution decrease evidently with an increase in PTMA concentration. When the concentration of PTMA reaches 2.8 × 10-4 g/mL, the I1/I3 ratios are in the range of 1.1-1.2 over the entire pH range, suggesting that the hydrophobic aggregation occurred for PTMA in aqueous solution. This is similar to the result for the copolymer of maleic acid and 1-octadecene (PA-18K2) which formed micelles over the entire pH range.12 But the ratios for PTMA are still larger than those for PA18K2, being less than 1.0, suggesting that PTMA particle cores are somewhat hydrophilic because the side alkyl chain of PTMA is shorter than that of PA-18K2 and some carboxylic groups and water molecules are trapped in the hydrophobic core. As the pH increases, the electrostatic repulsion among the ionized carboxylic groups trapped in the cores causes the I1/I3 ratio to increase as shown in Figure 1; that is to say, the hydrophobicity of PTMA particles decreases. Our dynamic light scattering study (to be published elsewhere) shows that PTMA particles do not dis-

Figure 1. I1/I3 ratio of pyrene fluorescence in (A) PIMA or (B) PTMA solutions as a function of the copolymer concentration at pH 2.1, 6.5, 10.5, or 11.8.

Figure 2. Far-UV CD spectra of apo cyt c at different pH. The concentration of apo cyt c is 10 µM.

sociate at the concentration of 4.5 × 10-6 g/mL at pH 2.1 but begin to dissociate at this concentration at pH 6.5; the dissociating concentration of the particles is 3.6 × 10-5 g/mL at pH 11.8, nearly 1 order of magnitude higher than that at pH 6.5 because the ionization degree of PTMA is 100% at pH 11.8 and the electrostatic repulsion is stronger than at pH 2.1 and 6.5. Structure of Apo Cyt c in Aqueous Solution. Figure 2 shows the influence of pH on the secondary structure of apo cyt c. The negative peak around 200 nm in CD spectra indicates that apo cyt c maintains its random coil structure over the pH range from 2.1 to 11.8. The negative peak is much smaller at pH 10.5, which is near the pI of the protein, so the protein tends to aggregate. The I1/I3 ratios of pyrene in apo cyt c solution are 1.83, 1.76, 1.46, and 1.46 at pH

Structural Transformation of Apocytochrome c

Figure 3. Far-UV CD spectra of apo cyt c in the presence of various concentrations of PIMA: (1) 4.5 × 10-6, (2) 9.0 × 10-6, (3) 1.4 × 10-5, (4) 1.8 × 10-5, (5) 2.7 × 10-5, (6) 3.6 × 10-5, (7) 5.4 × 10-5, and (8) 7.2 × 10-5 g/mL. The concentration of apo cyt c is 10 µM, and the pH is 2.1. (Inset) Comparison of experimental spectrum 8 with the spectrum reproduced with the Secondary Structure Estimation Program.

2.1, 6.5, 10.5, and 11.8, respectively. This result illuminates that the states of apo cyt c are different when the pH increases although its secondary structure does not change. The I1/I3 ratio of 1.83 suggests that the chain of apo cyt c is expanded at pH 2.1. Apo cyt c carries about 24 positive charges (19 Lys + 2 Arg + 3 His) at pH 2.118 and its net positive charges decrease when pH increases, causing the protein chains to shrink and the I1/I3 ratio to decrease. That is to say, the apo cyt c conformation of UA state, expanded random coil at pH 2.1, transforms to C state compact conformation with no specific secondary structure at higher pH. This conclusion is similar to the result of acid-induced conformational transition of N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine- (IAEDANS-) labeled apo cyt c derivative,34 which became compact as the pH increased with a transition midpoint of pH 4.5. The transition midpoint of apo cyt c is between pH 6.5 and 10.5, higher than that of IAEDANSlabeled apo cyt c. Obviously, the hydrophobic group IAEDANS can make apo cyt c chain shrink at a lower pH. Interaction of Apo Cyt c with PIMA. We studied the interaction of apo cyt c with PIMA and PTMA, respectively, at different pH values. Figure 3 shows far-UV CD spectra of the mixtures of 10 µM apo cyt c with different concentrations of PIMA at pH 2.1. Upon interacting with PIMA, apo cyt c shows a lessened intensity for the negative peak around 200 nm and an appearance of the bands at 208 and 222 nm. This indicates that PIMA induces a conformational transition of apo cyt c from random coil to R-helical structure. The R-helix content of apo cyt c induced by the copolymer is calculated with Model SSE-338 Protein Secondary Structure Estimation Program according to the experimental CD spectra and the results are summarized in Figure 4. Cyt c and apo cyt c were analyzed to confirm the validity of the calculation. The R-helix content calculated for cyt c is 39%, consistent with the result of Yang et al.,27 and the R-helical content calculated for apo cyt c is 0, the same as the report of Bryson et al.16 The inset in Figure 3 shows a comparison of an experimental spectrum with the spectrum reproduced with the Secondary Structure Estimation Program.

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Figure 4. R-Helix content of apo cyt c in the presence of various concentrations of PIMA at pH 2.1, 6.5, 10.5, and 11.8. The concentration of apo cyt c is 10 µM. Most of the error bars for R-helix content are smaller than (3, and the largest error bar is (6.3.

Figure 4 shows that at pH 2.1 there is no R-helical structure when the concentration of PIMA is lower than 1.4 × 10-5 g/mL. When the concentration of PIMA is increased to 7.2 × 10-5 g/mL, the R-helix content attains 42%. At this pH, each PIMA carries 8 negative charges and apo cyt c carries 24 positive charges, so the electrostatic interaction occurs between PIMA and apo cyt c. At pH 1.2, the negative charges of PIMA are very low, and the R-helix content induced decreases to about 10% (data not shown). This suggests that the electrostatic attraction between PIMA and apo cyt c is important to promote the folding of apo cyt c at low pH because the electrostatic attraction between PIMA and apo cyt c can screen the electrostatic repulsion within the polypeptide and then help the folding of the protein. The increase of pH from 2.1 to 6.5 causes two major structural changes, that is, an increase of PIMA negative charges and a decrease of the net positive charges of apo cyt c. The former favors the electrostatic interaction between PIMA and apo cyt c but the latter has complicated effects on the interaction, which depresses the electrostatic repulsion within apo cyt c and then favors its chain folding, but at the same time, the fewer positive charges weaken the polymerprotein electrostatic interaction. The various factors together result in a complicated dependence of R-helix content on PIMA concentrations at pH 6.5. The R-helix content is much larger than that at pH 2.1 and increases with PIMA concentration when the concentration is lower than 3.6 × 10-5 g/mL. When the concentration is above 3.6 × 10-5 g/mL, the R-helix content begins to decrease. This polymer concentration dependence of the R-helix content is different from that observed at pH 2.1. Besides, for the folding of apo cyt c induced by partly sulfonated polystyrene (SPS), where both electrostatic and hydrophobic interactions exist, or by 100% sulfonated polystyrene (PSS), where only electrostatic interaction exists, no such dependence was observed.8 Moreover, the concentration of the ionized carboxylic groups is only 0.5 mM when PIMA is 7.2 × 10-5 g/mL at pH 6.5, and we find that the addition of 10 mM NaCl does not decrease the helix content under these conditions; therefore the influence of the ionic strength can be neglected. A possible explanation is as follows. As we know, the helical structure results from the hydrogen bonds between CdO and NH groups of the polypeptide backbone.

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For PIMA, CdO in the ionized carboxylic groups is a stronger proton acceptor than either the carbonyl in the unionized carboxylic groups or the carbonyl in the protein backbone.35 Therefore, at a higher concentration of PIMA, the hydrogen bonds between the ionized carboxyl of PIMA and amide of apo cyt c may restrain those within the polypeptide backbone, causing the decrease in the R-helical structure. It is interesting to see that at pH 10.5, which is closest to the pI of cyt c, the R-helical structure can still be induced by PIMA (Figure 4). The net charge of apo cyt c is about 0, whereas the negative charges of PIMA are 68 at this pH. The electrostatic repulsion within apo cyt c almost disappears so it favors the folding within the protein, but apo cyt c cannot form R-helical structure by itself. The induction of PIMA at pH 10.5 is similar to that at pH 6.5. Obviously, the electrostatic attraction between local positive charges of apo cyt c and the negative charges of PIMA and the hydrophobic interaction between the isobutyl of PIMA and the hydrophobic residues of apo cyt c help the folding of the protein. At PIMA concentration as low as 4.5 × 10-6 g/mL, the R-helix content is 12% at pH 10.5, much higher than those at other pHs. When PIMA concentration increases to 1.8 × 10-5 g/mL, R-helix content reaches its maximum, 37%, which is smaller than the maximum of 50% at the concentration of 3.6 × 10-5 g/mL at pH 6.5. As the total negative charge of PIMA at pH 10.5 is about two-thirds more than that at pH 6.5, it is reasonable that the hydrogen bonds between the ionized carboxyl of PIMA and the amide of apo cyt c occur at lower PIMA concentration, the maximum of the R-helix curve appears at lower PIMA concentration, and the maximum helix content is smaller at pH 10.5. At pH 11.8, PIMA cannot induce any R-helical structure (Figure 4) because both the copolymer and protein carry negative charges and the electrostatic repulsion is strong enough to restrain the interaction of PIMA with apo cyt c. The studies of protein conformational transition induced by aliphatic alcohols found that the polar amide groups of the proteins were shielded from water and the intramolecularly hydrogen-bonded helical structure became more stable owing to an entropic reason.36-38 Compared with the interactions of aliphatic alcohols with proteins, the interactions of PIMA, a macromolecule, with apo cyt c are more entropyfavorable. Therefore, PIMA may also shield amide groups of the protein from water molecules that originally bond with apo cyt c through hydrogen bonds, promoting the formation of hydrogen bonds within polypeptide backbone and then the helical structure through the hydrophobic and electrostatic interactions between PIMA and apo cyt c. For the same reason, such a result may also exist in the interaction of PTMA with apo cyt c. Interaction of Apo Cyt c with PTMA. To understand the influence of hydrophobic interaction on the folding of apo cyt c, the structural transition of apo cyt c induced by PTMA is investigated (Figure 5). At pH 2.1, the total negative charges of PTMA are about a third those of PIMA at the same copolymer concentration. Compared with PIMA, 5-7 times higher PTMA concentration is needed to induce the same content of helical structure. Increasing the concen-

Liang et al.

Figure 5. R-Helix content of apo cyt c in the presence of various concentrations of PTMA at pH 2.1, 6.5, 10.5, and 11.8. The concentration of apo cyt c is 10 µM. Most of the error bars for R-helix content are smaller than (3, and the largest error bar is (6.3.

tration of PTMA to 4.0 × 10-4 g/mL, the R-helix content attains 36%, smaller than the 42% attained at a PIMA concentration of 7.2 × 10-5. At pH 1.2, the negative charges of PTMA are very low, and the R-helix content induced decreases to 18% (data not shown) at PTMA concentration of 4.0 × 10-4 g/mL. Therefore, the decrease of the electrostatic repulsion within the protein is a main factor for the refolding of apo cyt c at low pH. At pH 6.5, there is no significant induction when PTMA concentration is less than 9.0 × 10-6 g/mL (Figure 5). The long hydrophobic tetradecyl side chains may prohibit the electrostatic attraction between PTMA and apo cyt c at low PTMA concentration. As PTMA concentration increases, the R-helix content induced by PTMA becomes similar to that induced by PIMA. The R-helix content reaches its maximum, 52% at the concentration of 3.6 × 10-5 g/mL. Rankin et al.17 reported the folding of apo cyt c induced by negatively charged lipid micelles at pH 7.0. Their study found that the extent of lipid-induced secondary structure is dependent on the monomer lipid concentration because the free lipid molecules, which are not involved in the formation of the micelles, can interact with the protein through the ionic headgroups as well as through the hydrophobic tails of the lipid. For lipid concentrations above the critical micelle concentration, the helix content remains approximately constant. The recent study of SDS with cyt c denatured by urea at physiological pH also showed that the monomeric surfactant molecules interact with the protein and this leads to the formation of partially folded intermediates of cyt c.18 In this study, the data in Figure 1 show that the hydrophobic side chains of PTMA aggregate at such concentration. Dynamic light scattering study shows that the dissociating concentration of PTMA particles is 4.5 × 10-6 g/mL at pH 6.5, but Figure 5 shows that the helix content begins to increase after this concentration. Our studies of atomic force microscopy and dynamic light scattering show that the interaction between PTMA and apo cyt c can destroy the aggregates of PTMA (to be published elsewhere), suggesting that apo cyt c interacts not only with the hydrophilic carboxyl but also with the hydrophobic tetradecyl. Perhaps there is a competition between the hydrophobic aggregation of PTMA and PTMA-apo cyt c interaction, as reported for the interactions of β-lactoglobulin or bovine serum albumin with

Structural Transformation of Apocytochrome c

a series of alternating copolymers of maleic acid and alkyl vinyl ethers with varying hydrophobicities.14 Compared with PIMA, the interaction of the hydrophobic residues of apo cyt c with long tetradecyl side chains of PTMA may make the hydrogen bonds between ionized carboxyl of PTMA and amide of apo cyt c less favorable and/or the hydrophobic aggregation makes the effective concentration of PTMA decrease; therefore the helix content does not decrease much when the PTMA concentration is higher than 3.6 × 10-5 g/mL. At pH 10.5, the induction of PTMA at a very low concentration of 4.5 × 10-6 g/mL is lower than that of PIMA at the same condition. Over the concentration range from 4.5 × 10-6 to 5.4 × 10-5 g/mL, PTMA and PIMA show similar induction. At concentrations greater than 5.4 × 10-5 g/mL, the induction of PTMA increases with the concentration again, which is observed neither at lower pH nor for PIMA induction. As shown in Figure 1, hydrophobic aggregation of PTMA occurred around this concentration. Perhaps the hydrophobic aggregation of PTMA decreases the effective concentration of PTMA and makes the hydrogen bonds between ionized carboxyl of PTMA and amide of apo cyt c less favorable, so the induction increases again. At pH 11.8, there is 16% and 20% helical structure induced by PTMA at concentrations of 7.2 × 10-5 and 2.8 × 10-4 g/mL, respectively, whereas PIMA cannot do so. At this pH, PIMA, PTMA, and apo cyt c carry negative charges and there is electrostatic repulsion between the copolymer and protein. McGlade et al.39 observed that the hydrophobic modified polyanion, poly(1-octadecene-co-maleic acid) (POMA) or poly(1-decene-co-maleic acid) (PDMA) could interact with anionic surfactant, SDS while poly(1-ethyleneco-maleic acid) (PEMA) could not. Gao and Dubin14 studied the interaction of β-lactoglobulin or bovine serum albumin with a series of alternating copolymers of maleic acid and alkyl vinyl ethers at pH values greater than the pI of the proteins, showing that the hydrophobic interaction between the copolymer and protein can compensate for the electrostatic repulsion and intrapolymer micellization. Obviously, the tetradecyl side chains of PTMA play an important role in the induction of helical structure. Like DNA double chains, the negative charges of PTMA and apo cyt c may be separated from each other by the hydrophobic interaction between PTMA and the protein. This hydrophobic interaction excludes the water molecules that bond with apo cyt c originally, promoting the formation of hydrogen bonds within the helical structure as the aliphatic alcohols did.36 However, the electrostatic repulsion between PTMA and apo cyt c at this pH results in only 20% helical structure induced. Alcohols could induce polypeptides and unstructured proteins to form R-helix36,38 or disrupt the tertiary structure of native protein to form and enhance R-helical structure40-42 because alcohols could shield amide groups of the proteins from the solvent at high alcohol concentrations. The highly R-helical structure induced by alcohols was the extended helical rods.37,40 Like alcohols, PIMA and PTMA are able to induce more than native R-helical structure but not able to induce natural tertiary structure for apo cyt c (data not

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Figure 6. Fluorescence emission spectra of Trp59 of apo cyt c in the presence of various concentrations of PIMA: (0) 0, (1) 4.5 × 10-6, (2) 9.0 × 10-6, (3) 1.8 × 10-5, (4) 3.6 × 10-5, and (5) 7.2 × 10-5 g/mL. The concentration of apo cyt c is 10 µM, and the pH is 6.5.

shown). Similarly, the helical structure of apo cyt c induced by PIMA or PTMA is only an intermediate state of the folding. Tryptophan Fluorescence Study. Structural transformation of the protein can be monitored by intrinsic fluorescence. Apo cyt c has a single tryptophan at position 59 that has been widely used to examine the folding of apo cyt c.15-17 The interaction of apo cyt c with PIMA or PTMA is investigated by Trp59 fluorescence. Figure 6 shows the fluorescence emission spectra of mixtures of 10 µM apo cyt c with different concentrations of PIMA at pH 6.5. After interaction with the copolymer, Trp59 fluorescence produces a blue shift of 15 nm, from 343 to 328 nm, for its wavelength of maximum fluorescence (λmax), which is similar to the interaction of apo cyt c with lipids.15-17 This blue shift and the increase of fluorescence intensity indicate that Trp59 transforms from a hydrophilic environment in its unfolded state in aqueous solution to a hydrophobic environment upon folding caused by the interaction of the protein with the copolymer. Hence, the intensity ratio of 328 to 343 nm for Trp59 fluorescence is monitored to investigate the change of the hydrophobic/hydrophilic environment of Trp59 of apo cyt c (Figure 7). Generally, the copolymer concentration dependence of the ratios of I328/I343 for Trp59 correlates well with the changes of R-helix content monitored by far-UV CD (Figures 4 and 5). The ratio and λmax for apo cyt c alone are about 0.9 and 343 nm, respectively, independent of pH, which indicates that Trp59 is in a very hydrophilic environment although apo cyt c aggregates at higher pH. After interaction with PIMA or PTMA, the maximal ratio increases to about 1.2, except for the case of PIMA at pH 10.5, where the maximal ratio is 1.0 and the blue shift of λmax is only 8 nm. These results prove that Trp59 of apo cyt c is in a more hydrophobic environment after interacting with PIMA or PTMA. Both the helix contents shown in Figures 4 and 5 and the fluorescence ratios in Figure 7 have maxima in the curves of pH 6.5 and 10.5, although the corresponding copolymer concentrations are somewhat different. This suggests that by further increasing copolymer concentration at pH 6.5 and 10.5, the environment of Trp59 gradually turns back to hydrophilic. This phenomenon does not happen at pH 2.1, where the ionization degree is only 11% and 8% for PIMA and PTMA, respectively. Obviously, the electro-

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lated, and this is a very useful means to study the mechanism of protein folding. Conclusion

Figure 7. I328/I343 ratio of Trp59 fluorescence of apo cyt c in the presence of various concentrations of (A) PIMA and (B) PTMA at pH 2.1, 6.5, and 10.5 or 11.8. The concentration of apo cyt c is 10 µM.

static attraction and hydrophobic interactions of apo cyt c with PIMA or PTMA produce a more hydrophobic environment, leading to an increase of the I328/I343 ratios at pH 6.5 and 10.5; on the other hand, when the copolymer concentration is higher, the excess negative charges make the environment less hydrophobic, leading to a decrease of the I328/I343 ratios again. At pH 11.8, both PTMA and apo cyt c carry negative charges, but hydrophobic interaction between PTMA and the protein happens and then Trp59 is in a more hydrophobic environment when PTMA concentration increases. A number of studies found that small molecules could promote helical structural transition of apo cyt c under acid conditions, and the anions were responsible for bringing about this transition because anions reduced the repulsion within the protein.34,43 At neutral pH, sodium perchlorate at a high concentration could also induce helical transition of apo cyt c.34 So the structural transformations of apo cyt c induced by maleic acid, citric acid monohydrate, and R-methylacrylic acid, which have the same functional groups as PIMA and PTMA, were investigated. These small molecules do not show any effect on the conformational transition of apo cyt c when the concentration is 3.6 × 10-5 or 7.2 × 10-5 g/mL, although they may interact with apo cyt c. This clearly shows the importance of polymeric structure on the induction of structural transition. Polymers can offer a microenvironment that benefits protein folding at a very low concentration. Furthermore, the polymeric construction and hydrophobicity/hydrophilicity can be modu-

In aqueous solution, PIMA is in an expanded state over the entire pH range; PTMA forms hydrophobic aggregates, and the hydrophilicity of the aggregates increases with increasing pH. The negative charges of the copolymers depend on the pH of the solution. Apo cyt c transforms from the UA state, expanded random coil at pH 2.1 to the C state compact conformation with no secondary structure at higher pH. After interacting with PIMA or PTMA, apo cyt c undergoes a conformational transition from random coil to R-helical structure. The R-helical content induced by copolymers is influenced by the pH of the solution, the concentration of the copolymers, and the hydrophobic alkyl chain length of the copolymers. At pH 2.1, apo cyt c carries 24 positive charges and the decrease of the electrostatic repulsion within the polypeptide is a dominant factor for apo cyt c folding, so the electrostatic attraction between the copolymer and apo cyt c promotes the folding of apo cyt c. Over the entire pH range, the electrostatic and hydrophobic attractions between the copolymer and apo cyt c exclude water molecules that originally bond with apo cyt c through hydrogen bonds, promoting the formation of hydrogen bonds within the helical structure. On the other hand, the hydrogen bonds formed between the ionized carboxyl of the copolymer and the amide of apo cyt c partly restrain the hydrogen bonds formed between the carbonyl and amide within the polypeptide backbone, making helical structural decrease partly. The latter effect becomes substantial when the concentration of the copolymer is higher at pH 6.5 and 10.5. At pH 11.8, the hydrophobic attraction of PTMA and apo cyt c induces partial helical structure. The alkyl in PIMA is too short to compensate the electrostatic repulsion of PIMA and apo cyt c, so no helical structure is induced by PIMA at pH 11.8. Compared with the protein conformational transition induced by small molecules, use of polymers has several advantages: very low concentration can promote the conformational transition of the protein; electrostatic and hydrophobic interactions and hydrogen bonds formed between polymer and protein can be manipulated by the pH of the solution and the component of the polymer. Acknowledgment. The financial support of the National Natural Science Foundation of China (NSFC Projects 20074006 and 20474009) is gratefully acknowledged. References and Notes (1) Hartl, F. U.; Hayer-Hartl, M. Science 2002, 295, 1852-1858. (2) Cleland, J. L.; Hedgepeth, C.; Wang, D. I. C. J. Biol. Chem. 1992, 267, 13327-13334. (3) Yoshimoto, N.; Hashimoto, T.; Felix, M. M.; Umakoshi, H.; Kuboi, R. Biomacromolecules 2003, 4, 1530-1538. (4) Chen, Y.-J.; Huang, L.-W.; Chiu, H.-C.; Lin, S.-C. Enzyme Microb. Technol. 2003, 32, 120-130. (5) Shimizu, H.; Fujimoto, K.; Kawaguchi, H. Colloids Surf., A 1999, 153, 421-427. (6) Lin, S.-C.; Lin, K.-L.; Chiu, H.-C.; Lin, S. Biotechnol. Bioeng. 2000, 67, 505-512. (7) Nomura, Y.; Ikeda, M.; Yamaguchi, N.; Aoyama, Y.; Akiyoshi, K. FEBS Lett. 2003, 553, 271-276.

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