Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte

Mar 29, 2016 - State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, Chi...
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Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte Complex Micelle Fu-Gen Wu,*,†,‡ Yao-Wen Jiang,‡ Zhan Chen,§ and Zhi-Wu Yu*,† †

Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China ‡ State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China § Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: The folding/unfolding behavior of proteins (enzymes) in confined space is important for their properties and functions, but such a behavior remains largely unexplored. In this article, we reported our finding that lysozyme and a double hydrophilic block copolymer, methoxypoly(ethylene glycol)5K-block-poly(L-aspartic acid sodium salt)10 (mPEG5K-b-PLD10), can form a polyelectrolyte complex micelle with a particle size of ∼30 nm, as verified by dynamic light scattering and transmission electron microscopy. The unfolding and refolding behaviors of lysozyme molecules in the presence of the copolymer were studied by microcalorimetry and circular dichroism spectroscopy. Upon complex formation with mPEG5K-b-PLD10, lysozyme changed from its initial native state to a new partially unfolded state. Compared with its native state, this copolymer-complexed new folding state of lysozyme has different secondary and tertiary structures, a decreased thermostability, and significantly altered unfolding/refolding behaviors. It was found that the native lysozyme exhibited reversible unfolding and refolding upon heating and subsequent cooling, while lysozyme in the new folding state (complexed with the oppositely charged PLD segments of the polymer) could unfold upon heating but could not refold upon subsequent cooling. By employing the heating−cooling−reheating procedure, the prevention of complex formation between lysozyme and polymer due to the salt screening effect was observed, and the resulting uncomplexed lysozyme regained its proper unfolding and refolding abilities upon heating and subsequent cooling. Besides, we also pointed out the important role the length of the PLD segment played during the formation of micelles and the monodispersity of the formed micelles. Furthermore, the lysozyme−mPEG5K-b-PLD10 mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates in solution as frequently observed in other protein−polyelectrolyte systems. Hence, the present protein−PEGylated poly(amino acid) mixture provides an ideal water-soluble model system to study the important role of electrostatic interaction in the complexation between proteins and polymers, leading to important new knowledge on the protein−polymer interactions. Moreover, the polyelectrolyte complex micelle formed between protein and PEGylated polymer may provide a good drug delivery vehicle for therapeutic proteins.

1. INTRODUCTION Proteins are natural polyelectrolytes and possess unique properties including the folding and unfolding behaviors, the abilities to form gel, emulsion, and nanofibers, and the encapsulation of bioactive compounds, such as small drugs, peptides, and short-chain DNA/RNA. The interactions of proteins and non-natural polyelectrolytes can create various new structures and have diverse applications in food science, biomaterials, enzyme immobilization, protein purification, drug delivery, and biosensors.1 Numerous efforts have been devoted to studying the complexation processes between proteins and polyelectrolytes2−10 and the structures and properties of the protein−polyelectrolyte complexes/coacervates.11−26 There lacks, however, an in-depth understanding on the formation mechanism of different protein folding states induced by © XXXX American Chemical Society

polyelectrolytes and the unfolding and refolding behaviors of proteins in the presence of polyelectrolytes. Electrostatic interaction is one of the most important intermolecular forces to complex the oppositely charged proteins with polyelectrolytes, and the driving force for this electrostatic interaction may be mainly entropy due to the release of bound counterions from the two charged species. The electrostatic interaction is also the primary reason for the large aggregates (precipitates) frequently to be observed in the electrostatically complexed protein−polyelectrolyte systems,5,12,14,27 causing difficulties to characterize such complexes using various solution-based Received: January 21, 2016 Revised: March 27, 2016

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Figure 1. (A) NanoDSC results of mPEG5K-b-PLD10−lysozyme mixtures obtained from the initial heating processes and (B) the corresponding relationship between the melting temperature (Tm) of lysozyme and the concentration of mPEG5K-b-PLD10, C(mPEG5K-b-PLD10). The concentration of lysozyme in all the mixtures is 2.0 mg/mL. The vertical dashed lines are visual guides for the two transition temperatures, and the arrow indicates the new peak appearing at 0.3 mg/mL mPEG5K-b-PLD10. The nanoDSC curves are vertically shifted for better presentation.

calorimetry (nanoDSC), circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). It was found that the complexation between mPEG5K-b-PLD10 and lysozyme led to a two-state conversion (from native state to polymer-complexed state) of the protein and resulted in the formation of polyelectrolyte complex micelle with a particle size of ∼30 nm. Besides, the polymer-complexed lysozyme was found to be in a partially unfolded state, undergoing unfolding upon heating but no refolding upon subsequent cooling, which was different from native lysozyme which could unfold upon heating and refold upon cooling.

techniques. Here we designed and investigated a water-soluble protein−polyelectrolyte system to elucidate the influences of polyelectrolytes on the protein structure, complexation mechanism, and unfolding and refolding behaviors. Specifically, a poly(ethylene glycol) (PEG)-containing negatively charged poly(amino acid), methoxypoly(ethylene glycol)5K-block-poly(L-aspartic acid sodium salt)10 (abbreviated as mPEG5K-b-PLD10), was chosen to study its complexation behavior with a positively charged protein, lysozyme. PEGylated poly(amino acid)s have recently been widely used for the construction of various supramolecular structures (such as micelles and vesicles) for various applications such as drug delivery, cell adhesion, and surface coating.28−37 The graft of PEG onto a polymer can endow it with protein-resistant ability, which in this case can reduce the interaction of lysozyme with this PEG segment of the copolymer during complexation. On the other hand, by introducing a PEG segment, the copolymer would possess much better water solubility, which is especially useful to ensure that the complexes formed by the oppositely charged proteins and the PEG-containing polyelectrolytes will not precipitate. Hen egg white lysozyme is a small (14.3 kDa, 129 amino acids) globular enzyme. It is a basic protein with a pI value of ∼10.7. Lysozyme was found to be one of the most thermostable proteins (enzymes), which may be related to its refolding ability upon cooling from a heat-denatured state.27,38 Lysozyme has been widely used as a model physicochemical system to study protein folding behavior.27,38−40 Besides, as a naturally occurring protein, lysozyme is being considered as a potential antimicrobial agent to replace the currently used synthetic food preservatives.41 It is an effective antibiotic because it can hydrolyze or dissolve the peptidoglycans found in bacterial cell walls, especially for Gram-positive bacteria.41 We believe that the investigation of the interactions between lysozyme and other molecules is important to understand its folding mechanism, thermostability, and functions. Besides, it can also shed new light on the development of novel lysozymebased self-assembled functional nanomaterials with desired properties and applications. In this work, the interaction between lysozyme and mPEG5Kb-PLD10 was studied using nano differential scanning

2. EXPERIMENTAL SECTION 2.1. Sample Preparation. Methoxypoly(ethylene glycol)5K-blockpoly(L-aspartic acid sodium salt)10 (mPEG5K-b-PLD10) and methoxypoly(ethylene glycol)5K-block-poly(L-aspartic acid sodium salt)50 (mPEG5K-b-PLD50) were purchased from Alamanda Polymers, with an average molecular weight of 6400 and 11 800 Da, respectively. The mPEG5K-b-PLD10 polymer has ∼113 average number of ethylene glycol repeating units (the average molecular weight of PEG is 5000 Da) and ∼10 average number of aspartic acid repeating units (the average molecular weight of PLD is 1400 Da), while the mPEG5K-bPLD50 polymer has ∼113 average number of ethylene glycol repeating units (the average molecular weight of PEG is 5000 Da) and ∼50 average number of aspartic acid repeating units (the average molecular weight of PLD is 6800 Da). High-purity hen egg white lysozyme was ordered from Amresco. Double deionized water with a resistivity of 18.2 MΩ·cm was used for the preparation of protein samples. To prepare the mixtures of mPEG5K-b-PLD10 and lysozyme, an mPEG5Kb-PLD10 solution (with twice of the desired final concentration) was added to an equal volume of lysozyme solution with a concentration of 4.0 mg/mL. Using this procedure, we prepared a series of mPEG5K-bPLD10−lysozyme mixture solutions with the concentrations of mPEG5K-b-PLD10 ranging from 0.0 to 1.3 mg/mL (with an increment of 0.1 mg/mL). All such mixture solutions have a final lysozyme concentration of 2.0 mg/mL. The solutions were vortex-mixed for 3 min and were then stored at ∼3 °C for more than 24 h before use. 2.2. NanoDSC. The microcalorimetric measurements were performed on a CSC Model 6300 Nano III differential scanning calorimeter (Calorimetry Sciences Corp, Lindon, UT) using the DSCRun software. The scan rate was 1 °C/min. 2.3. CD. CD spectra were measured with a Chirascan CD spectrometer (Applied Photophysics Ltd., Leatherhead, UK). To B

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Langmuir avoid the saturation of the detector, the lysozyme-containing solutions were diluted with water or buffer for both the far- and near-UV CD experiments. We have confirmed that the dilution did not affect the structure of the complexes as confirmed by dynamic light scattering (DLS) and CD results. For measurements conducted in the far-UV region (185−260 nm), a quartz cell with a path length of 0.1 cm and a lysozyme concentration of 0.2 mg/mL was used. For measurements in the near-UV region (240−400 nm), a quartz cell with a path length of 0.5 cm and a lysozyme concentration of 1.0 mg/mL was used. The sample temperature was controlled using a circulating water bath connected to the water-jacketed quartz cuvettes. The CD spectra were obtained by subtracting the buffer solution containing the same concentration of mPEG5K-b-PLD10. The percentages of the secondary structures in lysozyme were obtained by fitting the far-UV CD spectra using the CONTIN/LL method in the CDPro software package. 2.4. Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). DLS and TEM measurements were carried out to characterize the aggregate structures for the 2.0 mg/mL lysozyme + 1.3 mg/mL mPEG5K-b-PLD10 sample. DLS experiments were performed on a Malven Nano ZS Zetasizer at 25 °C. TEM was commonly used to study self-assembled structures in aqueous solutions. The detailed procedures of the TEM sample preparation used in this research are similar to that described in the literature.27 A lysozyme-containing solution of 10 μL was placed on a 300-mesh copper grid, which had been covered by a carbon-coated film. After 2 min, excess fluid was eliminated using a filter paper, and 1% uranyl acetate was added. After another 2 min, excess dye was removed. Finally, the grid was viewed by a JEM-2100 transmission electron microscope (JEOL).

3. RESULTS AND DISCUSSION 3.1. Complexation of Lysozyme with mPEG5K-b-PLD10 Destabilizes the Protein and Leads to the Formation of Micelles. DSC has been frequently used to study the thermal stability of proteins through monitoring the heat flow of the corresponding thermotropic transitions.38−40 Figure 1 shows the nanoDSC results of the mPEG5K-b-PLD10−lysozyme mixture solutions with a fixed lysozyme concentration of 2.0 mg/mL and varied mPEG5K-b-PLD10 concentrations. As mPEG5K-b-PLD10 alone exhibits a linear DSC curve in the temperature range of 25−100 °C (Figure S1), the endothermic peaks observed in Figure 1 are contributed by the denaturation process of lysozyme upon heating. It can be seen that in the absence of mPEG5K-b-PLD10 a single endothermic peak with the peak transition temperature at 77 °C occurs, which corresponds to the denaturation of pure lysozyme in solution.27,38 The addition of mPEG5K-b-PLD10 to reach a concentration of 0.2 mg/mL in the mixture does not result in any noticeable change in the DSC peak, indicating that a small amount of the polymer in the mixture solution does not affect the unfolding behavior of lysozyme upon heating. At C(mPEG5K-b-PLD10) = 0.3 mg/mL, a small peak at around 66 °C occurs (indicated by the arrow in Figure 1A). As C(mPEG5K-b-PLD10) increases further, this new peak gradually becomes larger at the expense of the original peak detected at 75−77 °C, and the position of this new peak also gradually shifts to lower temperatures: 64 °C at 0.5−0.6 mg/mL and 60− 62 °C at 0.7−1.3 mg/mL. The new transition peaks at 60−66 °C observed for the samples with C(mPEG5K-b-PLD10) = 0.3− 0.8 mg/mL suggest that addition of the copolymer led to the destabilization of the protein. The presence of a range of the denaturation temperature of for the copolymer-complexed lysozyme indicates that the protein may have a slight difference in its complexation degree with the polymer at different mPEG5K-b-PLD10 concentrations.

Figure 2. (A) pH value of the mPEG5K-b-PLD10−lysozyme mixtures as a function of mPEG5K-b-PLD10 concentration, (B) absolute value of the mPEG5K-b-PLD10/lysozyme charge ratio as a function of the concentration of mPEG5K-b-PLD10, and (C) absolute value of the mPEG5K-b-PLD10/lysozyme charge ratio as a function of the molar ratio of mPEG5K-b-PLD10/lysozyme.

The above DSC data show that the polymer concentration influences the interactions between polymers and lysozyme molecules. Such interactions should be dependent on the absolute value of the net charge ratio of mPEG5K-b-PLD10 to lysozyme defined as R. The net charge values for lysozyme at various pH values can be found in the literature.42 Although these values were obtained in a salt-containing dispersion, they were nearly independent of the salt concentration. Considering its pI value (10.7), lysozyme possesses positive net charges in the mPEG5K-b-PLD10−lysozyme mixture solutions with the pH range of 4.4−6.2 (as shown in Figure 2A, the y-axis). The deionization degree (α) of COONa in the PLD segment can be C

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PLD10/lysozyme is 1.2, which suggests that the binding of mPEG5K-b-PLD10 and lysozyme is approximately 1:1. The complexation between mPEG5K-b-PLD10 and lysozyme was confirmed by the DLS and TEM experiments for the “lysozyme (2.0 mg/mL) and mPEG5K-b-PLD10 (1.3 mg/mL)” sample (Figure 3). Such a complexation leads to the formation of polyelectrolyte complex micelles. Both the DLS and TEM data reveal that the micelles have an average diameter of ∼30 nm. The polydispersity index (PDI) of these micelles (obtained from DLS) is 0.053, reflecting that the micelles have a very good monodispersity. Besides, the strong Tyndall effect of the solution further supports the formation of nanocolloids (or nanomicelles) in the solution (see the inset of Figure 3). In contrast, the hydrodiameters of pure lysozyme and pure copolymer were measured to be below 3 nm (Figure S2), and no Tyndall effect was observed for both samples. Furthermore, we found that the micelles were stable for at least 6 months at ∼4 °C as confirmed by the almost unchanged DLS and TEM results. To study the role the length of PLD segment plays during the formation of the micelles, we have also investigated the formation of micelles between lysozyme and mPEG5K-b-PLD50. The mPEG5K-b-PLD50 polymer differs only in the length of the PLD segment as compared with mPEG5K-b-PLD10. The DLS results (Figure S3A) reveal that at a low mPEG5K-b-PLD50 concentration (the mPEG5K-b-PLD50/lysozyme molar ratio is 0.2; the corresponding charge ratio is ∼1.2) lysozyme can be well complexed with the polymer to form micelles with an average diameter of ∼93 nm. Increasing the concentration of mPEG5K-b-PLD50 to the mPEG5K-b-PLD50/lysozyme molar ratio of 1.0 (the corresponding charge ratio is ∼5.9) leads to the disassembly of the micelles (Figure S3B). This is because there is not enough lysozyme molecules to neutralize the negatively charged mPEG5K-b-PLD50 molecules, and the electrostatic repulsive forces between these still highly negatively charged polymers prevent the formation of micelles. These results clearly demonstrate the importance of the charge ratio in regulating the formation of micelles between the oppositely charged protein and polymer. Moreover, the size distribution (92.8 ± 89.0 nm; PDI = 0.492) of the lysozyme− mPEG5K-b-PLD50 micelles is much wider than that (30.6 ± 7.0 nm; PDI = 0.053) of the lysozyme−mPEG5K-b-PLD10 micelles, indicating that a proper PLD length is also important to control the monodispersity of the formed nanostructures. 3.2. Unfolding and Refolding of Lysozyme in the Presence of mPEG5K-b-PLD10. DSC has been applied to study the unfolding and refolding behaviors of lysozyme previously.27,38,40 Our results on such lysozyme behaviors are well correlated to the published results: The present nanoDSC results of lysozyme also show that heat-denatured lysozyme can refold during cooling and can reunfold during reheating (Figure S4A). The DSC cooling and reheating curves show that the refolding and reunfolding processes are almost reversible, and the difference between the cooling/reheating curve and the first heating curve may be due to the aggregation of the unfolded proteins at the elevated temperatures. To study the secondary and tertiary structural changes of pure lysozyme during the thermal treatments, we carried out far-UV CD (Figure S4B) and near-UV CD (Figure S4C) experiments, respectively. The far-UV CD curves (Figure S4B) show that lysozyme molecules in the native-state (25 °C) and the refolded state (25 °C, cooled from 95 °C) contain similar secondary structures. Lysozyme molecules in the heat-denatured state (95 °C) have a

Figure 3. DLS and TEM (the right inset) results demonstrating the formation of polyelectrolyte complex micelle between lysozyme (2.0 mg/mL) and mPEG5K-b-PLD10 (1.3 mg/mL) at 25 °C. Also shown as the left inset is the Tyndall effect of the micellar solution pictured under the irradiation of a red laser pointer.

Figure 4. Far-UV CD results of lysozyme (2 mg/mL) after the addition of various concentrations (0.0−1.3 mg/mL) of mPEG5K-bPLD10 in water at 25 °C.

calculated by pK′ = 3.86 and the pH value of the sample (plotted in Figure 2A) using the equation α = 1/[1 + 10 × exp(pK′ − pH)]. Then, by considering the molar ratio of mPEG5K-b-PLD10/lysozyme (r) and the number of −COONa per polymer (N = 10), we can get R = αrN/ρlyso, where ρlyso is the charge value of lysozyme. Figures 2B and 2C present the dependency of the charge ratio R on C(mPEG5K-b-PLD10) (Figure 2B) and r (Figure 2C). We can see from the figure that upon increasing mPEG5K-b-PLD10 concentration, R increases from 0.085 at C(mPEG5K-b-PLD10) = 0.1 mg/mL to 1.8 at C(mPEG5K-b-PLD10) = 1.3 mg/mL. In particular, the beginning point of the coassembly between lysozyme and mPEG5K-b-PLD10 at C(mPEG5K-b-PLD10) = 0.3 mg/mL corresponds to R = 0.30, and a close to unity (0.98) of the charge ratio value is obtained at C(mPEG5K-b-PLD10) = 0.8 mg/mL. These calculation results agree well with the nanoDSC results showing an almost fixed denaturation peak of lysozyme at 60−62 °C at C(mPEG5K-b-PLD10) > 0.7 mg/mL, indicating the complete complexation between the protein and the polymer. The above analyses point out that the ratio of the negative charge of mPEG5K-b-PLD10 to the positive charge of lysozyme is crucial for complex formation to mediate the unfolding and refolding behaviors of lysozyme. Besides, from Figure 2C, we can see that at the equimolar ratio of mPEG5K-bPLD10/lysozyme (r = 1.0) the charge ratio of mPEG5K-bD

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Figure 5. Effect of NaCl concentration on the initial heating results of (A) 2.0 mg/mL lysozyme and 0.6 mg/mL mPEG5K-b-PLD10 and (B) 2.0 mg/ mL lysozyme and 1.3 mg/mL mPEG5K-b-PLD10 samples measured by nanoDSC. (C) The melting temperature (Tm) of lysozyme vs the salt concentration derived from (B). The nanoDSC curves in both (A) and (B) are vertically shifted for better presentation.

different secondary structure compared to those in the native/ refolded state. Specifically, data fitting revealed that when the native state converted to the denatured state, the contents of αhelix, β-sheet, β-turn, and unordered (including random coil) structures changed from 34.9 ± 3.6%, 15.2 ± 1.6%, 22.2 ± 1.3%, and 27.7 ± 2.6% to 18.6 ± 1.3%, 26.5 ± 2.2%, 22.5 ± 1.5%, and 33.3 ± 2.2%, respectively. That is the contents of βsheet and unordered structures increased while that of α-helix decreased upon denaturation of lysozyme upon heating. The profiles of the near-UV CD spectra in Figure S4C can show microenvironmental changes around the aromatic amino acids within the protein molecule43 and can reflect the protein tertiary structure changes. The presence or absence of CD peaks in this spectral region can be used to analyze whether the protein tertiary structures exist or not. The fully denatured proteins do not have any characteristic CD bands in the nearUV region, suggesting that the configuration of aromatic amino acid residues within each native molecule is disrupted and randomized, and the integrity of the tertiary structure in native protein is completely lost.43 The near-UV CD results acquired here show that the native-state lysozyme possesses a welldefined three-dimensional structure (tertiary structure). After heating to 95 °C, almost no CD signal can be observed, indicating that the lysozyme at the heat-denatured state was completely unfolded without any tertiary structure. However, by subsequent cooling to 25 °C, the obtained CD spectrum resembles the initial spectrum collected at 25 °C, which shows that lysozyme can refold to regain the tertiary structure. The above CD results show that no significant differences in the secondary and tertiary structures of the native and refolded state of lysozyme can be observed.

To see the secondary structural changes of lysozyme upon the gradual addition of the copolymer at 25 °C, a series of farUV CD experiments were carried out. Figure 4 shows that the addition of various concentrations of mPEG5K-b-PLD10 to the 2 mg/mL lysozyme solution did not cause large deviations of the CD curves, indicating that the copolymer did not significantly change the protein secondary structures. At C(mPEG5K-bPLD10) = 0.6 mg/mL, the CD curve changes noticeably as compared with those of 0.0 and 0.3 mg/mL, indicative for the formation of a new state of lysozyme. When C(mPEG5K-bPLD10) is above 0.8 mg/mL, the native state of lysozyme was almost completely replaced by the new state, and the CD curves at 1.0 and 1.3 mg/mL mPEG5K-b-PLD10 show exclusively the secondary structures of the new state of lysozyme. These far-UV CD results correlated well with the above nanoDSC data shown in Figure 1A. Besides, we observed a cross point (an isodichroic point) of the curves at around 198 nm, suggesting that these CD spectra at various intermediate concentrations (0.3, 0.6, 0.8, and 1.0 mg/mL) of mPEG5K-bPLD10 can all be fitted with the linear combinations of the two spectra at C(mPEG5K-b-PLD10) = 0.0 and 1.3 mg/mL. The results indicate that the copolymer-induced change of lysozyme may be a two-state process when C(mPEG5K-b-PLD10) changes from 0.0 to 1.3 mg/mL, in consistence with the conclusion drawn from the nanoDSC results. Similar reasoning has been used before, where the presence of an isodichroic point of CD curves was explained as the existence of a two-state helix−coil equilibrium.27,44 Furthermore, we would like to study the properties of lysozyme complexed with mPEG5K-b-PLD10. First, we investigated how the addition of salt to the mixture affected the E

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Figure 6. Heating−cooling−reheating nanoDSC results of 2.0 mg/mL lysozyme and 1.3 mg/mL mPEG5K-b-PLD10 mixtures at various concentrations (0, 50, 100, 150, 300, and 500 mM) of NaCl. The nanoDSC cooling traces are vertically shifted for better presentation.

unfolding behavior of the sample with C(mPEG5K-b-PLD10) = 0.6 mg/mL. Without salt, two endothermic DSC peaks at 64 and 76 °C were observed at the first heating scan. Differently, with the presence of 150 mM NaCl in the mixture, only one endothermic peak at 75 °C was seen (Figure 5A). This indicates that lysozyme at the new folding state in complex with mPEG5K-b-PLD10 can revert back to native lysozyme because of the salt screening effect. Next, we carried out nanoDSC experiments for the sample at C(mPEG5K-b-PLD10) = 1.3 mg/mL. Figures 5B and 5C show that lysozyme in the mixture solution with the presence of 0, 50, 100, 150, 300, and 500 mM NaCl has a denaturation temperature of 59, 66, 70, 73, 75, and 75 °C, respectively. The gradual upshift in the denaturation temperature of lysozyme at increasing concentration of NaCl suggests the increase in the thermostability of lysozyme at elevated NaCl concentrations. This result implies that the highly concentrated salt solutions can screen the electrostatic interaction between the oppositely charged protein and polymer so that they could not form a complex. The absence of the complex formation was confirmed by (1) no observation of the Tyndall effect of the “2.0 mg/mL

lysozyme + 1.3 mg/mL mPEG5K-b-PLD10, 500 mM NaCl” solution (Figure S5A) and (2) the very small hydrodynamic diameter (∼3 nm) of the solution detected by DLS (Figure S5B). Besides, we have also carried out the corresponding farand near-UV CD experiments for the sample at C(mPEG5K-bPLD10) = 1.3 mg/mL at various NaCl concentrations (0, 50, 100, 150, 300, and 500 mM) (Figure S6). The results show that the increasing concentration of NaCl did not change the secondary structures significantly but could increase the content of tertiary structures of the protein especially at above 150 mM NaCl. Therefore, the CD data suggest that addition of salt could help lysozyme to regain its tertiary structure. To understand how the addition of salt to the mixture solution affects the unfolding and refolding behaviors of the complexed lysozyme, we carried out the heating−cooling− reheating DSC experiments for the mixture solutions with the mPEG5K-b-PLD10 concentration of 1.3 mg/mL at various NaCl concentrations (Figure 6). The samples with 0, 50, and 100 mM NaCl show similar DSC results: once lysozyme was heatdenatured, it could not refold upon cooling and could not reunfold upon reheating. However, at 150 mM NaCl, the heatF

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Figure 7. Far-UV CD (A, C) and near-UV CD (B, D) results of the 2.0 mg/mL lysozyme and 1.3 mg/mL mPEG5K-b-PLD10 sample (A, B) and the 2.0 mg/mL lysozyme and 1.3 mg/mL mPEG5K-b-PLD10, 500 mM NaCl sample (C, D).

absence of 500 mM NaCl. The calculated secondary structure contents (including α-helix, β-sheet, β-turn, and unordered structures) derived from the far-UV CD curves are listed in Table 1, which shows that the addition of salt can result in some changes of protein secondary structures. From the nearUV CD results, we can see that the CD peak of the mixture (Figure 7B) is lower than that of the native lysozyme (Figure S4C), indicating that partial tertiary structure of native lysozyme was lost upon the complex formation with the copolymer. This shows that the polymer-complexed lysozyme is in a partially unfolded state as compared to the native state of lysozyme, which can explain the decreased thermostability of the polymer-complexed lysozyme (Figure 1). Besides, from the comparison of the near-UV CD curves of the 2.0 mg/mL lysozyme + 1.3 mg/mL mPEG5K-b-PLD10 sample (Figure 7B) and the 2 mg/mL lysozyme + 1.3 mg/mL mPEG5K-b-PLD10, 500 mM NaCl sample (Figure 7D), we can see that the addition of salt led to the refolding of lysozyme upon cooling from the heat-denatured state, confirming that salt screening plays an important role in regulating the interaction between the protein and the polymer (or complex formation) and the refolding behavior of lysozyme upon cooling. 3.3. Formation of Polyelectrolyte Complex Micelles by Electrostatic Complexation between Lysozyme and mPEG5K-b-PLD10. The denaturation processes of globular proteins have been extensively investigated, and many factors such as covalent bonding, electrostatic interaction, hydrogen bonding, hydrophobic interaction, and van der Waals forces are

Table 1. Secondary Structural Contents of the Samples Diluted from “2.0 mg/mL Lysozyme”, “2.0 mg/mL Lysozyme and 1.3 mg/mL mPEG5K-b-PLD10”, and “2.0 mg/ mL Lysozyme and 1.3 mg/mL mPEG5K-b-PLD10, 500 mM NaCl” at 25 °Ca sample

α-helix (%)

β-sheet (%)

β-turn (%)

unordered (%)

2.0 mg/mL lysozyme 2.0 mg/mL lysozyme + 1.3 mg/mL mPEG5K-b-PLD10 2.0 mg/mL lysozyme + 1.3 mg/mL mPEG5K-b-PLD10, 500 mM NaCl

34.9 ± 3.6 29.9 ± 2.3

15.2 ± 1.6 21.4 ± 2.1

22.2 ± 1.3 21.1 ± 1.6

27.7 ± 2.6 27.6 ± 1.7

36.7 ± 2.3

14.8 ± 1.4

21.8 ± 1.0

26.7 ± 1.8

a

These data were derived from far-UV CD curves.

denatured lysozyme exhibited a small degree of refolding and reunfolding upon cooling and reheating, respectively. As the NaCl concentration further increased (300 and 500 mM), the degrees of the refolding and reunfolding also increased. The heating−cooling−reheating protocol employed here further provided us important clues for the refolding ability of lysozyme in the presence of mPEG5K-b-PLD10 and/or salt before heating, while these samples only exhibited difference in the denaturation temperature during the first heating scan in DSC curves. Figure 7 shows the CD results of the 2.0 mg/mL lysozyme and 1.3 mg/mL mPEG5K-b-PLD10 sample in the presence and G

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Figure 8. Schematic showing the formation of mPEG5K-b-PLD10−lysozyme complex and its disassembly upon salt addition.

drug delivery. Although the activity and stability of proteins (enzymes) encapsulated in the polyelectrolyte complex micelles have been investigated, the folding/unfolding details of the confined proteins remain unclear. To the best of our knowledge, the work reported here presents the first example to uncover the folding/unfolding mechanisms of proteins encapsulated in the polyelectrolyte complex micelles. Previous protein−polyelectrolyte systems investigated usually encountered the precipitation problem,5,12,14,27 which affects the accurate determination of the structures and properties of the complexes. In the present work, the long PEG segments contained in the mPEG5K-b-PLD10 copolymer prevent the formation of any precipitates even after several monthsall the solutions prepared were transparent. The system reported here provides an ideal water-soluble model system for various physicochemical and materials studies.

involved in the regulations of protein structures and properties. Our present work emphasized the importance of electrostatic interactions in mediating the unfolding and refolding processes of a typical globular protein, lysozyme, in the presence of the copolymer, mPEG5K-b-PLD10. By tuning the net charge ratio of mPEG5K-b-PLD10/lysozyme and the ionic strength of the dispersions, we are able to modulate the electrostatic interactions between the two compounds, which can in turn significantly change the structure, stability, and activity of the protein. Therefore, we believe that our work reported here should be able to provide new knowledge regarding the mechanisms of unfolding, refolding, complexation, and aggregation of proteins undergoing heating/cooling cycles and may be helpful to the design, preparation, and manipulation of nanoparticles involving proteins. We have constructed a novel protein-containing polyelectrolyte complex micelle system formed by the electrostatic complexation between lysozyme and mPEG5K-b-PLD10. The possible structure of the micelle is depicted in Figure 8 according to the electrostatic attractive interactions between lysozyme and mPEG5K-b-PLD10. The thus constructed micelle contains a lysozyme−PLD-complexed core and a PEG shell. Such a self-assembled structure may be used as an excellent drug delivery system in the future. Besides, the formed polyelectrolyte complex micelle has a PEG shell, which can effectively avoid the nonspecific adsorption of proteins and can escape the macrophage uptake in vivo, making the nanosized micelles be “stealthy” and bear a long in vivo circulation time. Researches on encapsulating proteins (enzymes) into the cores of polyelectrolyte complex micelles (or polyion complex micelles) have been reported.45−60 Such an encapsulation method has recently attracted much attention in pharmaceutics, since it may modulate the stability and activity of the encapsulated proteins and thereby affect the potential applications of these protein-based drug delivery systems. It has been shown that the activity of the enzymes (such as lysozyme, LysK, and lipase) confined in polyelectrolyte complex micelles is higher than that of the free enzymes in solution.54−56 However, there is also another report showing that lysozyme can be complexed with a block copolymer, sodium (sulfamate carboxylate) isoprene/ethylene oxide, and its structure was partially disturbed and its activity was significantly lost.57 The decreased stability or activity of the encapsulated protein may restore its activity when the complex disassembles triggered by a certain stimulus (such as salt). Nevertheless, these protein-encapsulated polymeric nanocapsules hold great promise in biocatalysis, biodetection, and

4. CONCLUSIONS In summary, we mixed lysozyme and a copolymer in solution to fabricate a novel type of nanosized micelle and studied the unfolding and refolding details of lysozyme in the presence of the copolymer using nanoDSC, CD, DLS, and TEM techniques. The nanoDSC and CD data indicated that lysozyme in the protein−polymer complex partially unfolded (compared to that in the native state), losing partial tertiary structure and changing its secondary structure. Different from the native-state lysozyme that can refold and regain its tertiary structure upon cooling from the denatured state at a high temperature (90 or 95 °C), this partially unfolded of lysozyme formed by complexation with polymer cannot refold upon cooling from its heat-denatured state. The salt screening effect can modulate the intermolecular electrostatic interaction between protein and polymer, and a high concentration of salt (500 mM) can prevent the complex formation. Besides, by comparison between mPEG5K-b-PLD10 and mPEG5K-b-PLD50, we have also pointed out the important role the length of the PLD segment plays during the formation of micelles and the monodispersity of the formed micelles. Besides, the lysozyme− mPEG5K-b-PLD10 mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates as frequently observed in other protein−polyelectrolyte systems. Hence, the present protein−PEGylated poly(amino acid) mixture provides an ideal water-soluble model system to study the electrostatic interaction between proteins and polyelectrolytes, leading to in-depth understanding of the principles governing protein−polymer interH

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Poly(allylamine hydrochloride). J. Phys. Chem. B 2002, 106, 2357− 2364. (9) Lindhoud, S.; Voorhaar, L.; de Vries, R.; Schweins, R.; Cohen Stuart, M. A.; Norde, W. Salt-Induced Disintegration of LysozymeContaining Polyelectrolyte Complex Micelles. Langmuir 2009, 25, 11425−11430. (10) Zhang, Y.; Han, K.; Lu, D.; Liu, Z. Reversible Encapsulation of Lysozyme Within mPEGB-PMAA: Experimental Observation and Molecular Dynamics Simulation. Soft Matter 2013, 9, 8723−8729. (11) Lindhoud, S.; de Vries, R.; Norde, W.; Cohen Stuart, M. A. Structure and Stability of Complex Coacervate Core Micelles with Lysozyme. Biomacromolecules 2007, 8, 2219−2227. (12) Cooper, C. L.; Dubin, P. L.; Kayitmazer, A. B.; Turksen, S. Polyelectrolyte−Protein Complexes. Curr. Opin. Colloid Interface Sci. 2005, 10, 52−78. (13) Morfin, I.; Buhler, E.; Cousin, F.; Grillo, I.; Boué, F. Rodlike Complexes of a Polyelectrolyte (Hyaluronan) and a Protein (Lysozyme) Observed by SANS. Biomacromolecules 2011, 12, 859− 870. (14) Cousin, F.; Gummel, J.; Clemens, D.; Grillo, I.; Boué, F. Multiple Scale Reorganization of Electrostatic Complexes of Poly(styrenesulfonate) and Lysozyme. Langmuir 2010, 26, 7078−7085. (15) Antonov, M.; Mazzawi, M.; Dubin, P. L. Entering and Exiting the Protein−Polyelectrolyte Coacervate Phase via Nonmonotonic Salt Dependence of Critical Conditions. Biomacromolecules 2010, 11, 51− 59. (16) Schmidt, I.; Cousin, F.; Huchon, C.; Boué, F.; Axelos, M. A. V. Spatial Structure and Composition of Polysaccharide−Protein Complexes from Small Angle Neutron Scattering. Biomacromolecules 2009, 10, 1346−1357. (17) Gummel, J.; Boué, F.; Clemens, D.; Cousin, F. Finite Size and Inner Structure Controlled by Electrostatic Screening in Globular Complexes of Proteins and Polyelectrolytes. Soft Matter 2008, 4, 1653−1664. (18) Gummel, J.; Cousin, F.; Boué, F. Structure Transition in PSS/ Lysozyme Complexes: A Chain-Conformation-Driven Process, as Directly Seen by Small Angle Neutron Scattering. Macromolecules 2008, 41, 2898−2907. (19) Kayitmazer, A. B.; Strand, S. P.; Tribet, C.; Jaeger, W.; Dubin, P. L. Effect of Polyelectrolyte Structure on Protein−Polyelectrolyte Coacervates: Coacervates of Bovine Serum Albumin with Poly(diallyldimethylammonium chloride) versus Chitosan. Biomacromolecules 2007, 8, 3568−3577. (20) Kayitmazer, A. B.; Bohidar, H. B.; Mattison, K. W.; Bose, A.; Sarkar, J.; Hashidzume, A.; Russo, P. S.; Jaeger, W.; Dubin, P. L. Mesophase Separation and Probe Dynamics in Protein−Polyelectrolyte Coacervates. Soft Matter 2007, 3, 1064−1076. (21) Sotiropoulou, M.; Bokias, G.; Staikos, G. Water-Soluble Complexes through Coulombic Interactions between Bovine Serum Albumin and Anionic Polyelectrolytes Grafted with Hydrophilic Nonionic Side Chains. Biomacromolecules 2005, 6, 1835−1838. (22) Bohidar, H.; Dubin, P. L.; Majhi, P. R.; Tribet, C.; Jaeger, W. Effects of Protein−Polyelectrolyte Affinity and Polyelectrolyte Molecular Weight on Dynamic Properties of Bovine Serum Albumin−Poly(diallyldimethylammonium chloride) Coacervates. Biomacromolecules 2005, 6, 1573−1585. (23) Xia, J. L.; Dubin, P. L.; Kokufuta, E.; Havel, H.; Muhoberac, B. B. Light Scattering, CD, and Ligand Binding Studies of Ferrihemoglobin−Polyelectrolyte Complexes. Biopolymers 1999, 50, 153−161. (24) Gao, J. Y.; Dubin, P. L.; Muhoberac, B. B. Capillary Electrophoresis and Dynamic Light Scattering Studies of Structure and Binding Characteristics of Protein−Polyelectrolyte Complexes. J. Phys. Chem. B 1998, 102, 5529−5535. (25) Cousin, F.; Gummel, J.; Combet, S.; Boué, F. The Model Lysozyme−PSSNa System for Electrostatic Complexation: Similarities and Differences with Complex Coacervation. Adv. Colloid Interface Sci. 2011, 167, 71−84.

actions. It is also expected that the polyelectrolyte complex micelle formed between protein and PEGylated polymer may be a good drug delivery vehicle for therapeutic proteins.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b00235. NanoDSC result of mPEG5K-b-PLD10; DLS results of pure lysozyme and pure mPEG5K-b-PLD10; DLS results of lysozyme−mPEG5K-b-PLD50 mixtures; nanoDSC, farUV CD, and near-UV CD (C) results of lysozyme; photograph of lysozyme−mPEG5K-b-PLD10 solution (with 500 mM NaCl) under laser irradiation and the corresponding DLS result; far- and near-UV CD results of 2.0 mg/mL lysozyme and 1.3 mg/mL mPEG5K-bPLD10 mixtures at various NaCl concentrations (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.-W.Y.). *E-mail: [email protected] (F.-G.W.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21133009, 21273130, and 21303017), Natural Science Foundation of Jiangsu Province (KB20130601), Fundamental Research Funds for the Central Universities (2242015R30016), Six Talents Peak Project in Jiangsu Province (2015-SWYY-003), and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

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REFERENCES

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