Effect of the Surface Charge of Artificial Chaperones on the Refolding

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Effect of the Surface Charge of Artificial Chaperones on the Refolding of Thermally Denatured Lysozymes Fan Huang, Liangliang Shen, Jianzu Wang, Aoting Qu, Huiru Yang, Zhenkun Zhang, Yingli An, and Linqi Shi* State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) Nankai University, Tianjin 300071, China S Supporting Information *

ABSTRACT: Artificial chaperones are of great interest in fighting protein misfolding and aggregation for the protection of protein bioactivity. A comprehensive understanding of the interaction between artificial chaperones and proteins is critical for the effective utilization of these materials in biomedicine. In this work, we fabricated three kinds of artificial chaperones with different surface charges based on mixed-shell polymeric micelles (MSPMs), and investigated their protective effect for lysozymes under thermal stress. It was found that MSPMs with different surface charges showed distinct chaperone-like behavior, and the neutral MSPM with PEG shell and PMEO2MA hydrophobic domain at high temperature is superior to the negatively and positively charged one, because of the excessive electrostatic interactions between the protein and charged MSPMs. The results may benefit to optimize this kind of artificial chaperone with enhanced properties and expand their application in the future. KEYWORDS: artificial chaperones, mixed-shell polymeric micelle, protein folding, surface charge, self-assembly and protein engineering,23,24 have been reported to protect protein activity from misfolding and aggregation. Nevertheless, a majority of above methods displayed only a moderate protection efficiency and weak renaturation ability, because of some impediments such as the complicated separation process and the structural complexity of proteins. Molecular chaperones, as an important cellular component, play a critical role in assisting de novo protein folding and refolding of stress-denatured proteins, as well as preventing undesired protein aggregation in vivo.25−27 Since the concept “molecular chaperone” was initially proposed by Laskey et al.28 in 1978, numerous different types of structurally unrelated chaperones have been depicted and the GroEL-GroES chaperonin system was the most extensively studied.29−32 Typically, GroEL has cylinder-like structure with a large central cavity and functionally cooperate with GroES, which form the cap of the folding barrel.33 In the interior of the hollow cylinder, GroEL presents some hydrophobic binding sites for capturing the non-native substrate proteins to prevent the irreversible protein aggregation.4,34 Then the GroEL cylinder can undergo a

1. INTRODUCTION Protein folding has been considered as one of the most significant issues of the “21st century biophysics”, which is an urgent biological problem in the central dogma of molecular biology.1−3 As the material basis of life, proteins must properly fold into precise three-dimensional structures to fulfill their specific biological functions under physiological conditions.4 Universally, proteins are synthesized on ribosomes after transcription and translation in the cell. Then only through hydrophobic collapse, space twisting, and other folding processes step-by-step can nascent polypeptide chains form the correct spatial structure.5 However, because of the extremely crowded cellular milieu where total cytosolic protein could reach 300−400 g/L, the correct folding of proteins is considerably challenging in vivo.6 Moreover, proteins are very vulnerable to the external environmental stresses (e.g., high temperature) in vitro or in vivo, which could induce protein misfolding and irreversible aggregation, thereby resulting in the loss of biological activity and even occurrence of numerous human diseases, such as Alzheimer’s diseases, Parkinson’s disease, and Type II diabetes.7−9 Hence, exploring effective strategies for the inhibition of unfavorable protein aggregation is an increasingly pressing problem in biotechnology. To this end, considerable efforts, including the use of refolding additives (e.g., surfactants,10−12 cyclodextrin,13 amino acids,14,15 trehalose,16 and polyethylene glycol17), chemical modification,18,19 immobilization in matrixes,20−22 © XXXX American Chemical Society

Special Issue: Applied Materials and Interfaces in China Received: September 18, 2015 Accepted: November 9, 2015

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DOI: 10.1021/acsami.5b08843 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Schematic Illustration of Artificial Chaperones Based on MSPMs with Different Surface Charges

capturing the unfolded proteins into the cavity-like spaces and binding them onto the hydrophobic domains, it would result in the formation of thermostable MSPM/protein complexes and prevent protein aggregations just like GroEL-GroES system. Upon cooling, the PNIPAM turned into hydrophilic state and the refolded proteins were subsequently released, which led to an excellent chaperone-like activity as a one-pot system. To the best of knowledge, this kind of artificial chaperone was the first example to concurrently mimic the structure and function of natural molecular chaperone. Artificial chaperone systems based on the hydrophobic interaction as mentioned above is not the only way for protein renaturation. A plethora of studies indicate that electrostatic interactions also play a key role in stabilizing the protein structure and thereby maintaining protein bioactivity.40−44 Nagasaki et al.45 successfully prevented the heat induced irreversible aggregation of lysozyme and protected its enzymatic activity via the complex formation with a cationic copolymer, poly(N,Ndiethylaminoethyl methacrylate)-graf t-poly(ethylene glycol) (PEAMA-g-PEG). Rotello and co-workers46 reported 2-(10mercaptodecyl)malonic acid functionalized gold nanoparticles (AuDA) with high negative charge density could act as an efficient synthetic “chaperone” for the refolding of thermally denatured proteins. These results demonstrated that the surface charge of artificial chaperones might be of great importance for the interaction between artificial chaperones and proteins. Therefore, it would be critically significant to develop a new method to clarify the relationship between the surface charge of artificial chaperones and protein refolding. Herein, influenced and inspired by above works, we report the effect of surface charge of MSPM-based chaperones on the refolding of thermally denatured lysozymes. A thermoresponsive amphiphilic diblock copolymer poly(ε-caprolactone)-block-poly[2-(2-methoxyethoxy) ethyl methacrylate] (PCL-b-PMEO2MA)

marked conformational change triggered by binding ATP and GroES, and create a hydrophilic environment that is conducive to protein folding and refolding. Finally, the perfectly folded protein is released after GroES dissociation, which is also induced by ATP binding.35 This process is an excellent example of the chaperone machinery. Inspired by the natural molecular chaperone systems, a variety of biomimetic protein folding/refolding strategies, namely the “artificial chaperones”, have been developed. The earliest artificial chaperone system is put forward by Gellman and Rozema, which is a “two-step” method similar to the GroELGroES system.36 The main idea of this approach is that a capturer such as surfactant is first added to bind with denatured proteins through hydrophobic interaction and, by doing so, form stable protein-capturer complex and block the protein aggregation. Then the stripper is introduced to release the refolded proteins by strong competitive interaction which causes the dissociation of protein-capturer complex. This type of artificial chaperone system achieves high refolding yields via two separate processes. However, in natural molecular chaperone systems, the two steps proceed simultaneously with the help of ATP and cochaperone.37 Thus, Akiyoshi et al.38 presented dynamic polysaccharide nanogels, which are composed of cholesteryl-group-bearing pullulan (CHP) and β-cyclodextrin (CD), could control the dynamics of trapping and releasing proteins in a one-pot system. Recently, we fabricated an innovative artificial chaperone, mixedshell polymeric micelle(MSPM) with temperature-responsive poly(N-isopropylacrylamide) (PNIPAM) domains on the surface, which could closely simulate the marvelous GroEL-GroES complex.39 At high temperature, the PNIPAM chains in the mixed shell of MSPM would transform from hydrophilic to hydrophobic and collapse, thus forming hydrophobic domains on the micelle core and leaving behind some cavity-like spaces which were encircled by the hydrophilic PEG chains. Through B


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ACS Applied Materials & Interfaces

Figure 1. Synthesis routes of (A) PEG-b-PCL, (B) PCL-b-PAsp, (C) PCL-b-PLys, and (D) PCL-b-PMEO2MA.

and three kinds of biocompatible block copolymers with different charges: uncharged poly(ε-caprolactone)-block-poly(ethylene oxide) (PCL-b-PEG), negatively charged poly(ε-caprolactone)block-poly(aspartic acid) (PCL-b-PAsp), and positively charged poly(ε-caprolactone)-block-poly(L-lysine) (PCL-b-PLys) were synthesized. Three types of artificial chaperones with different surface charges based on MSPM were obtained via the selfassembly of above four block copolymers in aqueous solution (Scheme 1): PEG-MSPM, PAsp-MSPM, and PLys-MSPM. Then the chaperone-like behavior of these different charged micelles was investigated by examining the efficacy of MSPMs to

renature unfolded lysozymes. Interactions with lysozyme and the mechanism of these different charged artificial chaperones were also described, which would help us optimize our design of MSPM-based chaperones with enhanced properties and expand their application in the future.

2. MATERIALS AND METHODS 2.1. Materials. Poly(ethylene glycol) monomethyl ether (CH3OPEG45-OH, Mn = 2000, Mw/Mn = 1.05) was purchased from Fluka and used after dried under vacuum. ε-Caprolactone (ε-CL, 99%) from Alfa Asear was dried with calcium hydride (CaH2) and then purified by C


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ACS Applied Materials & Interfaces distillation under reduced pressure before use. 4-Hydroxybutyl αbromoisobutyrate (HBBiB, ≥ 97%) was purchased from Changzhou Yi Ping Tang Chemical Co., Ltd. β-Benzyl L-aspartate-N-carboxyanhydride (BLA-NCA) and ε-(benzyloxycarbonyl)-L-lysine N-carboxyanhydride (Lys(Z)-NCA) were synthesized by the Fuchs−Farthing method using bis(trichloromethyl) carbonate (triphosgene) according to ref 47. 2-(2Methoxyethoxy) ethyl methacrylate (MEO2MA, 97%) was purchased from J&K Chemical Company (Beijing, China) and was purified by passing through a column filled with basic aluminum oxide (Al2O3) to remove the inhibitor. CuCl was purchased from Sigma-Aldrich and purified prior to use. Tris[2-(dimethylamino)-ethyl]amine (Me6TREN) used as an atom transfer radical polymerization (ATRP) ligand was purchased from J&K Chemical Company. Stannous octoate (Sn(Oct)2, 96%), trifluoroacetic acid (TFA, 98%), hydrogen bromide (HBr, 45% in acetic acid), t-Boc-aminoethyl alcohol (99%), and trifluoromethanesulfonic acid (CF3SO3H, 98%) were purchased from Alfa Asear and used as-received. Lysozyme (from chicken egg white) and Micrococcus lysodeikticus was purchased from Beijing Dingguochangsheng Biotechnology (Beijing, China) and Shijiazhuang Huarui Biotechnology (Shijiazhuang, China), respectively. All the organic solvents were redistilled before use. All aqueous solutions were prepared with ultrapure Milli-Q water (resistance >18 MΩ cm−1). 2.2. Synthesis of the Block Copolymers. The synthesis routes of four block copolymers are shown in Figure 1. PEG-b-PCL was synthesized by ring-opening polymerization (ROP) of ε-CL monomer using CH3O-PEG−OH as the macroinitiator and Sn(Oct)2 as catalyst according to literature procedures.48,49 Briefly, CH3O-PEG45−OH (1.0 g, 0.5 mmol), ε-CL (5.0 g, 43.9 mmol) and one drop of Sn(Oct)2 were dissolved into 15 mL of toluene in a round-bottom flask. After three cycles of freeze−pump−thaw to remove moisture and oxygen, the polymerization was carried out at 110 °C for 12h. Then the reaction mixture was diluted with an appropriate amount of dichloromethane, and precipitated into excess diethyl ether. The precipitate was dried under vacuum to obtain a white powder, i.e., PEG-b-PCL. PCL-b-PAsp was synthesized through deprotection of benzyl groups of poly(ε-caprolactone)-block-poly(β-benzyl-L-aspartate) (PCL-bPBLA), which was prepared by ring-opening polymerization (ROP) of β-benzyl L-aspartate-N-carboxyanhydride (BLA-NCA) in the presence of a PCL macroinitiator (PCL80-NH2).50 First, prepared BLA-NCA (2.5 g, 10 mmol) was added to PCL-NH2 (2.0 g, 0.2 mmol) solution in CH2Cl2 under a dry argon atmosphere and stirred for 24h at 30 °C. The solution was then precipitated into excessive diethyl ether, filtered, and dried under vacuum to obtain PCL-b-PBLA. Subsequently, 1.0 g of PCL-b-PBLA was treated with a mixture of trifluoroacetic acid/ trifluoromethanesulfonic acid/anisole to remove the benzyl group. The mixed solution was gently stirred for 2h at 0 °C and precipitated into excessive diethyl ether to obtain PCL-b-PAsp. Similar to the synthesis of PCL-b-PAsp, PCL-b-PLys was synthesized via the ROP of lysine NCA (Lys(Z)-NCA) with PCL-NH2 as the macroinitiator and the deprotection of benzyloxycarbonyl groups by following the literature method.51 In brief, Lys(Z)-NCA (1.5 g, 4.9 mmol) was dispersed in dry DMF followed by addition of PCL-NH2 (1.0 g, 0.1 mmol). The reaction mixture was stirred for 72h at 40 °C under a dry argon atmosphere. Then, the solution was diluted with DMF and precipitated into excess cold diethyl ether to obtain PCL-b-PLys(Z). Subsequently, 0.5 g PCL-b-PLys(Z) was dissolved in 5 mL of trifluoroacetic acid at room temperature, and then CHCl3 was added dropwise with stirring until a slight cloudiness was observed. Then, a few drops (1 mL) of hydrogen bromide (HBr; 45% in acetic acid) was added to the solution and slowly stirred for further 24 h at room temperature. The reaction mixture was precipitated into excessive diethyl ether and isolated by filtration. After washed twice with diethyl ether, the product was dried under vacuum to obtain PCL-b-PLys. PCL-b-PMEO2MA was synthesized by the combination of ROP of εCL and atom transfer radical polymerization (ATRP) of MEO2MA using HBBiB as bifunctional initiator.52 First, HBBiB (0.12g, 0.5 mmol), ε-CL (5.0 g, 43.9 mmol) and one one drop of Sn(Oct)2 were dissolved into 12 mL of toluene in a round-bottom flask. After freeze-degas-thaw cycles for three times, the polymerization was conducted at 110 °C for 12h. Then the reaction mixture was diluted with dichloromethane and

precipitated into excess diethyl ether to obtain macroinitiator PCL-Br. Subsequently, 1.0 g of PCL-Br, 0.012g CuCl, 0.046g Me6TREN and 1.0 g MEO2MA were charged into a Schlenk flask. Then 3 mL DMF was added to dissolve the monomer and initiator. The flask was degassed by three freezing−thawing cycles. Then, the reaction mixture was stirred at 60 °C for 12h. After polymerization, the reaction mixture was diluted with THF and passed through a neutral Al2O3 column to remove the copper catalyst followed by precipitation in excess cold diethyl ether. Finally, the PCL-b-PMEO2MA was obtained by filtered and dried under vacuum. 2.3. Preparation of Mixed-Shell Polymeric Micelles (MSPMs). To afford the MSPMs with different surface charges, we mixed 1 mg PCL-b-PMEO2MA with equal mass of PEG-b-PCL, PCL-b-PAsp and PCL-b-PLys, respectively. And they were then dissolved in 1 mL of DMF to prepare the polymer solution with concentration of 2.0 mg/mL. Subsequently, these three kinds of different polymer solution were added dropwise into 15 mL of deionized water under vigorous stirring. The MSPMs were formed immediately, and the resulting solutions were stirred overnight at room temperature to make the micelles stable. Then, the solutions were dialyzed against deionized water for 3 days to remove the DMF and MSPMs were thus obtained. 2.4. Characterization. 1H NMR spectra of these block copolymers were recorded on a Varian UNITY-plus 400 M NMR spectrometer at room temperature using CDCl3 and DMSO-d6 as solvents. Dynamic light scattering (DLS) experiments were performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) at 636 nm at required temperature. All samples (about 1 mL) were obtained by filtering through 0.2 μm Millipore filter into a clean scintillation vial. Transmission electron microscopy (TEM) measurements were performed with a commercial Philips T20ST electron microscope at an acceleration voltage of 100 kV. To prepare the TEM samples, we dropped 10 μL of sample solution onto a carboncoated copper grid and dried it in the air. The zeta potential values were measured on a Brookhaven ZetaPALS (Brookhaven Instrument, USA). The instrument utilizes phase analysis light scattering at 25 °C to provide an average over multiple particles. 2.5. Determination of Protein Concentration. The protein concentration of lysozyme was determined from the absorbance at 280 nm with an extinction coefficient of 2.63(mg/mL protein)−1 cm−1 using UV-2550 UV−visible spectrophotometer (Shimadzu, Japan). 2.6. Thermal Denaturation of Lysozymes. Lysozymes were dissolved in 10 mM sodium phosphate buffer (pH 7.4) to a concentration of 1 mg/mL. Then, 50 μL of the above enzyme solution was mixed with 450 μL of three kinds of MSPMs with different surface charges and deionized water, respectively. Subsequently, the obtained mixture samples were incubated at 50 °C for 1h to make sure the transformation of PMEO2MA from hydrophilic to hydrophobic. After this step, the mixture was further heated to 80 °C for 10 min to denature lysozymes. Following thermally denaturation, the solutions were transferred to a refrigerator to quench the denaturation process and stored at 4 °C for 12h to make enzymes renaturation. The enzymatic activity assay and Circular dichroism study of the enzyme solutions was then performed. 2.7. Enzymatic Activity Assay. The measurement of lysozyme activity was performed on the basis of bacteriolysis reaction with Micrococcus lysodeikticus. Freeze-dried Micrococcus lysodeikticus cells were resuspended at 10 mM sodium phosphate buffer (pH 7.4) to act as the substrate solution. The 0.3 mg/mL Micrococcus lysodeikticus cell suspension (420 μL) was added to lysozyme solution (80 μL), and the activity was followed by measuring the decrease of absorbance at 450 nm using UV-2550 UV−visible spectrophotometer (Shimadzu, Japan) at room temperature. The obtained activity was normalized to that of native lysozyme. Each sample was measured in triplicate and the average was reported. 2.8. Circular Dichroism Measurement. Circular dichroism (CD) spectra were recorded by Bio-Logic MOS-500 circular dichroism spectrophotometer (France). For the CD experiments, 200 μL of the sample solution was added into a quartz cuvette of 1 mm path length and spectra was recorded from 190 to 260 nm with a scan speed of 100 nm/ min. Each spectra was an average of 3 scans. D


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Figure 2. (A) Hydrodynamic diameter distribution of three kinds of MSPMs with different surface charges (PEG-MSPM, PAsp-MSPM, and PLysMSPM) measured by DLS at 25 °C. The scattering angle was 90°. (B) ζ-potentials of three kinds of MSPMs with different surface charges: (blue) PEGMSPM, (orange) PLys-MSPM, and (green) PAsp-MSPM.

Figure 3. TEM images of (A) PEG-MSPM, (B) PAsp-MSPM, and (C) PLys-MSPM at 25 °C (scale bar: 200 nm).

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Mixed-Shell Polymeric Micelles with Different Surface Charges. In the present study, three kinds of mixed-shell polymeric micelles (MSPMs) with different surface charges were fabricated through the straightforward self-assembly of amphiphilic block copolymers PCL-b-PMEO2MA, PCL-b-PEG, PCL-b-PAsp, PCL-bPLys, which were synthesized as shown in Figure 1 and characterized in the Supporting Information. These MSPMs had same hydrophobic cores formed by PCL chains while different mixed shells composed of PEG/PMEO2MA, PAsp/PMEO2MA, and PLys/PMEO2MA, which were named as PEG-MSPM, PAsp-MSPM, and PLys-MSPM respectively. It is worth emphasizing that the mixed shells of three MSPMs possess thermal responsiveness because of the PMEO2MA segments. Dynamic light scattering (DLS) with the scattering angle of 90° was exploited to measure the hydrodynamic diameter distribution of three MSPMs. As demonstrated in Figure 2A, the average hydrodynamic diameter (Dh) of PEG-MSPM, PAsp-MSPM and PLys-MSPM were 75 ± 1 nm, 87 ± 1 nm, and 120 ± 2 nm, respectively. Additionally, they all had narrow particle size distributions. We also investigated the size and morphology of the MSPMs by TEM and their images were shown in Figure 3. Interestingly, it was found that these MSPMs all showed welldefined spherical structures, whereas their sizes were similar to each other around 80 nm, which was quite different with the DLS results. We attribute this divergence to the electrostatic repulsion of charged chains themselves which made them more outstretched in aqueous solution, thus led to the micelles swollen and larger size than that neutral one. The surface charge

properties of these three kinds of MSPMs can be confirmed by the zeta potential measurements. As shown in Figure 2B, the zeta potentials of the PEG-MSPM, PLys-MSPM and PAsp-MSPM were −4.45 ± 0.84, 16.67 ± 0.67, and −20.89 ± 0.62 mV, respectively. These above results fully proved that three kinds of MSPMs with different surface charges were successfully prepared. The most important attribute of these MSPMs to simulate the structure and function of molecular chaperone is that capturing unfolded proteins at high temperature and releasing refolded proteins at cooling temperature. To testify such thermal responsiveness of their mixed shells, we adopted DLS to monitor the size distribution change of these three MSPMs at different solution temperatures (Figure 4). Upon heating, the average Dh of PEG-MSPM, PAsp-MSPM, and PLys-MSPM were all decreased by about 10 nm. When cooled down to room temperature, the particle sizes of these three MSPMs returned to their original levels. This is because the thermoresponsive PMEO2MA chains in the mixed shell became hydrophobic and collapsed above its LCST (around 26 °C),53 leading to hydrophobic domains on the micelle core and a core−shell− corona (PCL−PMEO2 MA−PEG/PAsp/PLys) triple-layer structure, which made micelles more compact and thus smaller size. In the cooling process, the PMEO2MA chains would turn into the hydrophilic state and be restretched, so as to make the MSPMs recover back to their original core−mixed shell structure. Therefore, it can be concluded that these three kinds of MSPMs with different surface charges all possess thermoresponsive properties and their temperature-mediated structural E


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Figure 4. Size distribution of three kinds of MSPMs with different surface charges measured by DLS at 25 °C (black line), 80 °C (red line), and 25 °C* (blue line): (A) PEG-MSPM, (B) PAsp-MSPM, (C) PLys-MSPM. The scattering angle was 90°. The * indicates the measurements were performed on a sample that was kept at 80 °C and then cooled back to 25 °C. (D) Schematic illustration of the reversible structural transformation of the three MSPMs mediated by temperature.

Figure 5. (A) Enzymatic activity of thermally denatured lysozyme (D-LZM) in the presence of three kinds of MSPMs with different surface charges (PEG-MSPM, PAsp-MSPM, and PLys-MSPM) and 100 mM NaCl solution in 10 mM sodium phosphate buffer (pH 7.4). (B) Influence of the concentration of PEG-MSPM on the enzyme activity of thermally denatured lysozyme.

different sample solutions were incubated at 50 °C for 1h and subsequently heated to 80 °C for 10 min followed by a cooling process at room temperature, which is according to the procedure reported previously by our group.39 Then the enzymatic activity assay of lysozyme was carried out and results were summarized in Figure 5A. Apparently, without any MSPM, the residual enzymatic activity of lysozyme alone after the heating−cooling procedure was only 15%. Upon the introduc-

transformation is reversible, which play a pivotal role in realizing the chaperone-like functionalities for the MSPMs. 3.2. Thermally Denatured Lysozymes Protection of MSPMs with Different Surface Charges. To investigate the chaperone role of MSPMs with different surface charges, we selected lysozyme as a model protein and the recoverd enzymatic activity after heat treatment in the presence of differnt surface charged MSPMs were evaluated. During the measurements, the F


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Figure 6. CD spectra of native lysozyme, thermally denatured lysozyme, denatured lysozyme with different surface charged MSPMs and in the presence of 100 mM NaCl after 12h incubation. (A−C) CD spectra for PEG-MSPM, PAsp-MSPM, and PLys-MSPM, respectively.

Figure 7. Size distribution of three kinds of MSPMs with different surface charges (black line) and MSPMs/lysozyme mixtures (red line) at 25 °C: (A) PEG-MSPM, (B) PAsp-MSPM, (C) PLys-MSPM.

Table 1. ζ-Potentials of Three Kinds of MSPMs with Different Surface Charges and Lysozyme/MSPMs in 10 mM Sodium Phosphate Buffer (pH 7.4) at 25°C sample

ζ-potential (mV)

PEG-MSPM PEG-MSPM+lysozyme PLys-MSPM PLys-MSPM+lysozyme PAsp-MSPM PAsp-MSPM+lysozyme

−4.45 ± 0.84 −2.58 ± 0.37 16.67 ± 0.67 5.86 ± 0.92 −20.89 ± 0.62 −5.41 ± 0.11

that disrupting the electrostatic interactions between protein and charged MSPMs should be a crucial step for charged MSPMs releasing protein and assisting their refolding. According to the above results, it is found that PEG-MSPM with neutral surface exhibited the most effective protection ability for lysozyme under thermal stress. Therefore, we further considerd the effect of the concentration of PEG-MSPM on the refolding of thermally denatured lysozyme. Figure 5B showed the results when a fixed amount (0.1 mg/mL) of PEG-MSPM and different concentrations of the lysozyme were mixed and subjected to the heating−cooling treatment. Clearly, the higher concentration of PEG-MSPM in the mixture, the greater protective effect for the proteins under the thermal stress. It could be attributed that more PEG-MSPMs provided more available hydrophobic binding sites for catching the denatured enzymes and thus improved the refolding yield of lysozyme. 3.3. Secondary Structure of Protein in the Presence of Different Surface Charged MSPMs. To elucidate the conformational changes in the enzymes, CD spectra of lysozyme during heated-induced denaturation and renaturation were measured, both in the absence and presence of different charged MSPMs (Figure 6). It is worth mentioning that the CD spectra of samples with 100 mM NaCl could not be detected below the wavelengths of 200 nm because of their high absorbance from the increased salt concentration. The native lysozyme adopted αhelical dominated conformation with two negative bands around 208 and 222 nm, as widely observed before.46 After heating− cooling treatment, the signal intensity of α-helical conformation for lysozyme alone decreased sharply and the addition of 100 mM NaCl could hardly result in any changes in the CD spectra, suggesting the damage of α-helical structure and the irreversible inactivation of lysozyme. In the presence of neutral PEG-MSPM (Figure 6A), the CD spectra before heating was identical to that

tion of uncharged PEG-MSPM, a significant increase (∼82%) in the enzymatic activity was observed, which was consistent with our previous results on the similar system,39 indicating that the hydrophobic domains arised from phase transition of PMEO2MA could trap the thermally denatured lysozyme and then promote correct refolding of lysozyme in the cooling stage. However, in the presence of positively charged Plys-MSPM and negatively charged PAsp-MSPM, the residual enzymatic activity were only 23 and 10%, respectively. This result suggested that these two kinds of charged MSPMs barely displayed beneficial effects on the refolding of denatured lysozyme, which might be due to the excessive electrostatic interactions between charged MSPMs and lysozyme. It is widely known that increasing the ionic strength of the solution could attenuate electrostatic protein-particle interactions. So we attempted to investigate whether there would be changes in the residual enzymatic activity by adding 100 mM NaCl to the solutions. As expected, after adding the NaCl, the residual enzymatic activity of PLys-MSPM/ lysozyme and PAsp-MSPM/lysozyme mixture were increased to 33 and 38% respectively while little variation for lysozyme alone and PEG-MSPM/lysozyme. These observations demonstrated G


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Scheme 2. Schematic Illustration of the Possible Mechanism of Interactions between Thermally Denatured Lysozyme and Three Kinds of MSPMs with Different Surface Charges: (top) PAsp-MSPM, (middle) PEG-MSPM, (bottom) PLys-MSPM

interaction between lysozyme and MSPMs with different surface charges. Figure 7 illustrated the size distributions of three kinds of MSPM/lysozyme mixtures at room temperature. As seen in Figure 7A, negligible change in the average Dh was observed when lysozyme was added to the neutral PEG-MSPM, indicating that there was almost no interaction between lysozyme and PEGMSPM. Whereas in the case of charged MSPMs/lysozyme mixtures (Figure 7B, C), the Dh values were increased to 300− 400 nm from around 100 nm, suggesting that negatively charged PAsp-MSPM or positively charged PLys-MSPM could both adsorb lysozyme through strong electrostatic interaction and form MSPMs/lysozyme complexes. Moreover, the zeta potentials of different MSPM/lysozyme mixtures was measured and the results were shown in Table 1. It could be found that the zeta potential of PEG-MSPM/lysozyme mixture almost remained unchanged, whereas there was pronounced decline the absolute value of zeta potentials for the mixtures of PAspMSPM and PLys-MSPM with lysozyme, further verifying the fact that the lysozyme electrostatically interacted with PAsp-MSPM and PLys-MSPM. According to the all results discussed above, we speculated the possible mechanism of different surface charged MSPMs for refolding thermally denatured lysozyme, which is schematically illustrated in Scheme 2. With the isoelectric point (PI) of 11.35, lysozyme is positively charged at pH 7.4 which prevent their aggregation due to the electrostatic repulsion of them each other. When subjected to the heat treatment, the hydrophobic sites buried in the core of native lysozyme would be exposed and thereby caused their aggregation and inactivation through hydrophobic interaction between protein molecules. While in the presence of the neutral PEG-MSPM at high temperature, the unfolded lysozyme could bind to the PEMO2MA hydrophobic domains on micelle surface and resulted in the formation of PEGMSPM/lysozyme complexes. Meanwhile, the outstanding

of native lysozyme alone, signifying that PEG-MSPM did not disturb the secondary structure of lysozyme. Nevertheless, after the heating−cooling procedure, the secondary structure of lysozyme in the PEG-MSPM/lysozyme mixture seldom changed compared with that of natural lysozyme, which was quite different with that lysozyme alone and revealed that PEG-MSPM were capable of assisting in refolding of lysozyme effectively. In contrast, when native lysozyme was mixed with the negatively charged PAsp-MSPM at room temperature (Figure 6B), the fractions of their α-helix structure began to reduce. This finding illustrated that the second structure of lysozyme was partially destroyed by the strong electrostatic interaction between negatively charged PAsp-MSPM and positively charged lysozyme. Subsequent heat treatment caused the further destruction of the second structure while the addition of 100 mM NaCl led to the restoration of α-helix conformation in part. It could be explained that increasing the ionic strength could weaken the electrostatic enzyme-micelle interaction to a certain extent, thus resulting in the releasing of lysozyme and recovering partially secondary structure. It is noteworthy that the CD spectra of PLys-MSPM/lysozyme mixture was similar to that of native lysozyme alone at room temperature and did not change significantly after heating (Figure 6C). However, the above results of enzymatic activity assay showed that PLys-MSPM displayed low protection efficiency, even adding the NaCl to the solution. According to the report by Nagasaki and co-workers,45 it was probably because that the negatively charged residues in the active site of lysozyme bound to the positively charged surface of PLys-MSPM through electrostatic interactions, thereby hindering the attack of substrate into the active site and inhibiting the enzymatic activity of lysozyme. 3.4. Mechanism of Different Surface Charged MSPMs for Refolding Thermally Denatured Protein. On the basis of the above results, we finally conducted studies to explore the H


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hydrophilicity of PEG chains endowed the complexes remarkable dispersion stability and consequently prevented lysozyme from intermolecular aggregation. With cooling, the collapsed PEMO2MA became hydrophilic and stretched again, during which the unfolded lysozyme was automatically released and then refolded into their native state. As for the negatively charged PAsp-MSPM, although anionic PAsp chains and hydrophobic PEMO2MA domains could both interact with unfolded lysozyme via electrostatic and hydrophobic force respectively, the enzyme could not depart from the micelle surface in cooling step because of the excessive electrostatic interactions. Only increasing the salt concentration could induce the partially release and refold of lysozyme. In the case of positively charged PLys-MSPM, there were also strong electrostatic interactions between cationic PLys chains and negatively charged active center of lysozyme, inhibiting the substrate attack into the active center and leading to a decrease in enzyme activity. Similar with that of PAspMSPM, only a little mount of lysozyme could be released from the MSPM/lysozyme complexes by raising the ion strength at cooling temperature.

4. CONCLUSION In summary, we have demonstrated that MSPMs with different surface charges exhibited distinct chaperone-like behavior and neutral PEG-MSPM possessed the best capacity for the refolding of denatured lysozymes, benefiting from the synergistic effect of hydrophilically nonionic PEG chains and hydrophobic PMEO2MA domains. As for charged MSPMs, the unfolded lysozyme could not be autonomously released from the surfaces of these MSPMs because of the excessive electrostatic interactions between the lysozyme and charged shells. Only disrupted electrostatic interactions by increasing the ion strength of the system could induce the release of lysozyme, and their renaturation effects were markedly inferior to that of PEGMSPM. Therefore, this work could contribute to the further understanding of complex process of protein refolding and provide valuable guidance for the design of advanced artificial chaperones.

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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/acsami.5b08843. Characterizations of block copolymers (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 22 23503510. Tel: +86 22 23506103. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (91127045, 51390483, 21274067, 81171371, 51203189), the National Basic Research Program of China (973 Program, 2011CB932503), and PCSIRT (IRT1257).

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REFERENCES

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