Complexation of Lysozyme with Sodium Poly(styrenesulfonate) via the

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Complexation of Lysozyme with Sodium Poly(styrenesulfonate) via the Two-State and Non-Two-State Unfoldings of Lysozyme Fu-Gen Wu, Yao-Wen Jiang, Hai-Yuan Sun, Jun-Jie Luo, and Zhi-Wu Yu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b07277 • Publication Date (Web): 21 Oct 2015 Downloaded from http://pubs.acs.org on October 23, 2015

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The Journal of Physical Chemistry

Complexation of Lysozyme with Sodium Poly(styrenesulfonate) via the Two-State and Non-Two-State Unfoldings of Lysozyme

Fu-Gen Wu,†,‡ Yao-Wen Jiang,‡ Hai-Yuan Sun,† Jun-Jie Luo,† 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, P. R. China

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ABSTRACT To provide an in-depth understanding on the complexation mechanism of protein and polyelectrolyte, a heating–cooling–reheating protocol was employed to study the unfolding and refolding behaviors of a model protein, lysozyme, in the presence of the negatively charged polyelectrolyte, sodium poly(styrenesulfonate) (PSS). It was found that with elevated PSS concentration, a new state (state I) was first formed via a “two-state” conversion process, and this state could further convert to a completely unfolded state (state II) via the “non-two-state” conversion. This “non-two-state” conversion process occurs without the coexistence of the two states I and II, but involves the formation of various intermediate unfolded protein structures. Different from the pure lysozyme that exhibited refolding upon cooling from its heat-denatured state, lysozyme in state I could undergo unfolding upon heating but no refolding upon cooling, while lysozyme in state II did not undergo unfolding or refolding upon thermal treatments. Besides, the effects of ionic strength and the molecular weight of the polyelectrolyte on the unfolding and refolding behaviors of lysozyme were also investigated. The present work provides a better understanding on the principles governing the protein–polyelectrolyte interactions and may have implications for the fabrication of biocolloids and biofilms.

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1. INTRODUCTION Water-soluble proteins have complex charged surfaces including ionizable side-chains, migrating protons, and bound water.1 These charged sites allow the adsorption of charged and non-charged polar molecules. Polyelectrolytes, representing important charged building blocks of self-assembled structures, can be absorbed onto the surfaces of oppositely charged proteins for various fundamental studies and engineering applications.2–4 The adsorption/immobilization/inclusion of proteins to polyelectrolytes can occur both in bulk systems and at surfaces or interfaces.2–4 It was reported that the complexation of polyelectrolytes with proteins can be used to prevent protein aggregation,5,6 modulate protein stability,6–8 and assist protein separation and purification.9,10 Besides, the protein–polyelectrolyte systems can be used in the layer-by-layer assembly for the construction of multi-component nanostructured films/coatings or nanoparticles/capsules aiming at biomedical or analytical applications such as enzyme immobilization, drug delivery, wound repair, and biosensors.5,6,11–21 Numerous efforts have been devoted to studying the complexation processes between proteins and polyelectrolytes,16,22–32 and the structures and properties of the protein–polyelectrolyte complexes/coacervates.14,33–45 There lacks, however, an in-depth understanding on the formation mechanism of different protein folding states induced by polyelectrolytes and the unfolding and refolding behaviors of proteins in the presence of polyelectrolytes. To this end, we selected a commonly used model protein, hen egg white lysozyme, and a negatively charged polyelectrolyte, sodium

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poly(styrenesulfonate) (PSS), to study their complexation behaviors by using nano differential scanning calorimetry (nanoDSC), dynamic light scattering (DLS), transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy. Hen egg white lysozyme is an extensively studied, small (14.3 kDa, 129 amino acids) globular enzyme with a well-documented conformational stability.46 It is a basic protein with a pI value of 10.7. Lysozyme was found to be one of the most thermostable proteins (enzymes). Besides, it is an effective antibiotic by attacking the peptidoglycans found in the cell walls of bacteria, especially Gram-positive bacteria. It has been reported that lysozyme can form amyloid nanofibers, nanocubes, or ribbons, which enables lysozyme to be used as a model system to investigate the protein fibrillation process, and for the fabrication of amyloid nanofiber materials.47,48 Thus the investigation of the interaction and assembly of lysozyme and other molecules is important for the understanding towards its folding mechanism, thermostability, and functions. Besides, it can also shed new light on the development of novel lysozyme-based self-assembled functional nanomaterials with desired properties and applications. Up to now, there have been reports regarding the interactions of lysozyme with polyelectrolytes,7,22,23,30,32 including the influence of PSS on the thermostability of lysozyme,8 and the structures and structural transitions of the lysozyme–PSS complexes.37,38,44,45 However, the dynamic complexation mechanism between lysozyme and PSS, and the unfolding and refolding mechanisms of lysozyme in the presence of PSS still remain uncovered. Related to this topic, we have examined in

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detail

the

thermal

denaturation

process

of

lysozyme

and

proposed

an

interruption–incubation protocol to differentiate the two-state and non-two-state unfolding processes.49 We have also found that the unfolded lysozyme can induce the lipid lateral redistribution of a mixed lipid bilayer.50 Besides, we have studied the unfolding and refolding behaviors of lysozyme in the presence of micelles composed of the unstructured amphiphilic β-casein proteins.51 In this work, by careful designing experiments, we found that low concentrations of PSS induced a two-state unfolding of lysozyme, whilst high contents of PSS caused a non-two-state denaturation of the protein. Two PSS-induced unfolded states of lysozyme (state I and state II) were identified and their unfolding and refolding abilities were studied by employing a heating–cooling–reheating protocol. 2. EXPERIMENTAL SECTION 2.1. Materials and Sample Preparation. High-purity hen egg white lysozyme was from Amresco. Sodium poly(styrenesulfonate) (PSS) with different average molecular weights of ~70,000, ~200,000, and ~1000,000 Da (abbreviated as PSS-70k, PSS-200k, and PSS-1000k) were purchased from Aldrich. Double deionized water with a resistivity of 18.2 MΩ·cm was used for the preparation of protein samples. To prepare the mixtures of lysozyme and PSS for DSC measurements, a PSS solution of twice the desired final concentration was added into an equal volume of a 10 mg/mL lysozyme solution. The final lysozyme concentration was 5 mg/mL in all the lysozyme–PSS mixtures. These mixtures were then immediately vortexed for 3 min, after which they were left for equilibration for at least 3 days at ~3 oC before

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examinations. 2.2. Nano Differential Scanning Calorimetry (nanoDSC). The microcalorimetric measurements were performed on a CSC Model 6300 Nano III differential scanning calorimeter (Calorimetry Sciences Corp, Lindon, UT, USA). The scan rate was 1 o

C/min.

2.3. Circular Dichroism (CD). CD spectra were measured with a Chirascan CD spectrometer (Applied Photophysics Ltd., Leatherhead, UK). For measurements in the far-UV region (185–260 nm) and near-UV region (240–400 nm), quartz cells with the path length of 0.1 cm and 0.5 cm were used, respectively. To avoid the saturation of the detector, the lysozyme–PSS mixtures were diluted with water in both the far- and near-UV CD experiments. For far-UV CD experiments, the final lysozyme concentration was 0.2 mg/mL; while for near-UV CD experiments, it was 1 mg/mL or 0.25 mg/mL. 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 PSS. 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.52,53 2.4. Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). To characterize the structures of the aggregates, DLS and TEM measurements were carried out for two representative samples (“5 mg/mL lysozyme and 1 mg/mL PSS-70k” and “5 mg/mL lysozyme and 5 mg/mL PSS-70k”). The

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samples were diluted to obtain the final protein concentration of 0.5 mg/mL using water. DLS experiments were carried out on a Malven Nano ZS Zetasizer at 25 oC. TEM was commonly used to study the self-assembly structures of the proteins in aqueous solutions.54 The detailed procedures of the TEM sample preparation is similar to that described in literature.54 Typically, 10 µL of lysozyme-containing solution was withdrawn and put on a 300-mesh copper grid, which had been covered by carbon-coated film. After 2 min, excess fluid was drawn out using a paper filter, 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. Unfolding and Refolding of Lysozyme. DSC is a frequently used technology that can derive information on the thermostability, phase transition behavior, structural change, and conformational rearrangement of various molecules through the record of heat flow.55–58 Figure 1 shows the heating–cooling–reheating nanoDSC results of lysozyme in water. During the first heating, a single endothermic peak centered at 77 o

C is seen, indicating the denaturation of native lysozyme. Upon cooling, an

exothermic peak resides in the temperature range of 82–56 oC, and upon reheating, an endothermic peak occurs in the same temperature range. By comparison of the peak positions and peak shapes of the cooling and reheating curves, we can see that the denatured lysozyme at high temperatures after the first heating process can undergo reversible refolding and re-unfolding processes during the cooling and reheating scans,

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respectively. The unfolding, refolding, and re-unfolding processes of pure lysozyme upon heating, cooling, and reheating treatments, respectively, have been previously observed.51,59 Such a refolding capability may account for the high thermostability of lysozyme. To acquire the information on the protein structural changes during thermal treatments, we carried out far- and near-UV CD experiments for the pure lysozyme solutions, and the results are shown in Figure 2. The far-UV CD spectra in Figure 2A reveal the changes of protein secondary structures. The difference in the two spectra labeled “(1) 20 oC” and “(2) 90 oC” indicates that after first heating, the secondary structure of the heat-denatured lysozyme is different from its native state. Specifically, data fitting revealed that when the native state converted to the denatured state, the content of

-helix decreased from 36.0 ± 2.1 % to 18.3 ± 1.4 % and that of β-sheet

increased from 14.2 ± 0.4 % to 26.1 ± 2.0 %. The nearly identical CD spectra of the native-state lysozyme (“(1) 20 oC”) and the refolded-state lysozyme (“(3) 20 oC”) indicate that the thermal denatured lysozyme can refold to regain the same secondary structure as the native state upon cooling, suggesting that lysozyme is highly reversible upon the heating/cooling treatments. The profiles of the near-UV CD spectra can show microenvironmental changes around the aromatic amino acids within the protein molecule,60 reflecting the protein tertiary structure changes. The presence of the CD peak in this spectral region can tell the existence of protein tertiary structures. On the other hand, the fully-denatured proteins do not have any characteristic CD band in the near-UV region, suggesting

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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.60 From the near-UV CD spectra of lysozyme shown in Figure 2B, we can see that lysozyme possesses a well-defined three-dimensional structure (tertiary structure) at 20 oC. After heating to 90 oC, almost no CD band is seen, showing that the heat-denatured state of lysozyme has no tertiary structure. However, by cooling to 20 oC, the obtained CD spectrum resembles the initial spectrum at 20 oC. That is, lysozyme refolded to regain the tertiary structure by cooling from the heat-denatured state. Combined with the far-UV CD results in Figure 2A, we conclude that the refolded state of lysozyme at 20 oC has nearly identical secondary and tertiary structures as compared with its native state. To sum up, both the DSC and CD studies show that the pure lysozyme in dilute solutions underwent unfolding and refolding upon thermal treatments. 3.2. Unfolding and Refolding of Lysozyme in the Presence of PSS. Figure 3 shows the first heating scans of lysozyme in the presence of various amounts of PSS-70k. When the concentration of PSS-70k (CPSS-70k) increases to 0.2 mg/mL PSS-70k, a small endothermic peak centered at 59 oC is seen, indicating the formation of a new state of lysozyme (state I) which is less thermostable than the native state. When CPSS-70k further increases, the peak at 59 oC increases in intensity at the expense of the peak intensity at ~77 oC, showing that in the samples before heating, the population of the native state decreases while the population of the state I increases. At CPSS-70k = 1 mg/mL, the DSC trace is exclusively composed of the new peak. Upon further

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increasing CPSS-70k, the endothermic peak shifts from 59 oC at CPSS-70k = 1 mg/mL to 42 oC at CPSS-70k = 3 mg/mL, which eventually vanishes at CPSS-70k = 5 mg/mL. The above DSC results reveal that two populations of lysozyme existed in the samples before heating at CPSS-70k = 0.2–1 mg/mL: the native state and the PSS-complexed state (state I). These two states of lysozyme have almost fixed denaturation temperatures at ~59 oC and ~77 oC, respectively, showing that these two populations of lysozyme have explicitly different structures and thermostabilities. An increase in CPSS-70k only changes the ratio of the two populations. Thus there exists a “two-state” unfolding transition upon addition of PSS (CPSS-70k = 0.2–1 mg/mL) to a lysozyme solution. However, the DSC curves from CPSS-70k = 1 mg/mL to CPSS-70k = 5 mg/mL show a gradual decrease in the denaturation temperature of lysozyme as CPSS-70k increases. If we pay our attention to the sample states at 20 oC before heating, the gradual decrease in the denaturation temperature upon further addition of PSS-70k means that lysozyme in state I has further converted, perhaps via various intermediate structures, to a final unfolded state (state II) that does not undergo any significant change upon heating. This is a “non-two-state” unfolding/denaturation process. Thus, two different mechanisms are observed in the PSS-induced unfolding of lysozyme in the two different CPSS-70k regions (0–1 mg/mL and 1–5 mg/mL). To more clearly investigate the unfolding and refolding behaviors of lysozyme in the presence of PSS-70k, we also give the nanoDSC results of the two selected samples at CPSS-70k = 1 and 5 mg/mL in Figure 4. From the heating–cooling–reheating scans of the lysozyme solution in the presence of 1 mg/mL PSS-70k (Figure 4A), we

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can see that after thermal denaturation, no DSC peaks during the subsequent cooling and reheating scans can be detected. This means that the new state (state I) of lysozyme exhibits unfolding upon heating but no refolding upon cooling. For the 5 mg/mL PSS-70k sample (Figure 4B), only baselines are seen during the heating–cooling–reheating scans. The results show that lysozyme in state II exhibits no unfolding and refolding under thermal treatments. As will be confirmed by our following CD results, lysozyme has already been completely unfolded after the addition of 5 mg/mL PSS-70k at room temperature, and thus upon heating/cooling treatment, no DSC signal can be observed. Since the thermograms of the reversible folding transitions of proteins are not affected by the different scan rates, while those of irreversible folding transitions of proteins will be dependent on heating scan rate, we have thus carried out additional DSC measurements for lysozyme (5 mg/mL) in the presence of different concentrations (0, 1, and 2 mg/mL) of PSS at different heating rates (2, 0.5, and 0.1 o

C/min), and the results are shown in Figure S1 and Table S1 (see Supporting

Information). The almost unchanged transition temperatures of pure lysozyme indicate that the unfolding transitions of lysozyme are reversible, while the heating rate-dependent transition temperatures of “5 mg/mL lysozyme + 1 mg/mL PSS” and “5 mg/mL lysozyme + 2 mg/mL PSS” samples reflect that the PSS-complexed lysozyme molecules undergo irreversible unfolding transitions during heating. These results agree well with the heating–cooling–reheating results shown in Figure 1 and Figure 4A.

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To see the secondary structural changes of lysozyme upon the gradual addition of PSS-70k at 20 oC, a series of far-UV CD experiments were carried out for the samples diluted from “5 mg/mL lysozyme and 0–5 mg/mL PSS-70k”. Figure 5A shows that for a 5 mg/mL lysozyme solution, when CPSS-70k is above 0.3 mg/mL, the CD curve changes significantly, indicative for the formation of a new state of lysozyme (state I). As revealed by nanoDSC, upon the addition of 1 mg/mL PSS-70k, the native state of lysozyme was completely replaced by the new state, and the CD curve at 1 mg/mL PSS-70k shows exclusively the secondary structures of the new state of lysozyme. Most interestingly, we observed a cross point (an isodichroic point) of the curves at 199 nm. This means that these CD spectra at various intermediate concentrations (0.5, 0.65, and 0.8 mg/mL) of PSS-70k can all be fitted with the linear combinations of the two spectra at CPSS-70k = 0 and 1 mg/mL. This suggests that the PSS-70k-induced change of lysozyme is two-state when CPSS-70k changed from 0 to 1 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.61,62 As shown in Figure 5B, at 1 mg/mL ≤ CPSS-70k ≤ 5 mg/mL, no cross point is seen. This means that the CD curves at various intermediate concentrations (1.5, 2, and 2.5 mg/mL) of PSS-70k can not be fitted with the linear combinations of the two spectra at CPSS-70k = 1 and 5 mg/mL. The curves of the samples at CPSS-70k = 1.5, 2, and 2.5 mg/mL in the wavelength range of 210–225 nm are not even between those of the samples at CPSS-70k = 1 and 5 mg/mL. This suggests that the PSS-70k-induced change

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of lysozyme is non-two-state when CPSS-70k changes from 1 mg/mL to 5 mg/mL, which also agrees with the conclusion drawn from the nanoDSC results. Next, to see the polyelectrolyte-induced changes of the protein secondary and tertiary structures upon thermal treatments, we carried out far- and near-UV CD experiments for the samples diluted from “5 mg/mL lysozyme and 1 mg/mL PSS-70k” and “5 mg/mL lysozyme and 5 mg/mL PSS-70k”. Figure 6A (left panel) shows that upon the addition of 1 mg/mL PSS-70k, the secondary structure of lysozyme at 20 oC is already different from that in its native state without PSS-70k. The contents of

-helix and β-sheet changes from 36% and 14% in the native state to

18% and 30% in state I, respectively. After heating to 90 oC, the far-UV CD spectrum exhibits a flat baseline, indicating that lysozyme completely lost its secondary structure after heat-denaturation. Subsequent cooling and reheating results also show only baselines, indicating that lysozyme cannot regain its original secondary structure after complexation with PSS-70k. The near-UV CD results (Figure 6A, right panel) of the sample at 20 oC shows a broad peak at ~290 nm. By comparison of this curve with that of the pure lysozyme at 20 oC in Figure 2B, we can see that with the presence of 1 mg/mL PSS-70k, lysozyme has a somewhat different tertiary structure as compared with that in its native state. Upon heating to 90 oC, an almost flat baseline was recorded, indicating that the tertiary structure of lysozyme was completely lost. The baselines observed in cooling and reheating show that lysozyme cannot regain its tertiary structure upon further thermal treatments.

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The above far- and near-UV CD results for the sample diluted from “5 mg/mL lysozyme and 1 mg/mL PSS-70k” suggest that PSS-70k can induce the formation of a new protein state (state I) with different secondary and tertiary structures as compared with those of the native-state lysozyme. After heating, lysozyme completely loses its secondary and tertiary structures, which cannot refold upon the subsequent cooling process. The CD results (Figure 6A) correlate well with the DSC results in Figure 4A. For the sample diluted from “5 mg/mL lysozyme and 5 mg/mL PSS-70k”, Figure 6B (left panel) shows that upon the addition of 5 mg/mL PSS-70k, the structure of lysozyme in state II at 20 oC is somewhat different from that in state I (shown in Figure 6A, left panel). By calculation, we can see that lysozyme in the sample diluted from “5 mg/mL lysozyme and 5 mg/mL PSS-70k” contains 19% α-helix, 29% β-sheet, 23% β-turn, and 29% random coil, while lysozyme in the sample diluted from “5 mg/mL lysozyme and 1 mg/mL PSS-70k” contains 19% α-helix, 26% β-sheet, 23% β-turn, and 32% random coil. Besides, by comparison of the near-UV CD results of the two curves labeled “(1) 20 oC” in the two right panels in Figure 6, we can see that the two states of lysozyme have different tertiary structures. The absence of a CD peak at ~290 nm for “(1) 20 oC” indicates that the state II of lysozyme does not have tertiary structure, which is consistent with the DSC result in Figure 3 where no DSC signal was recorded upon heating. After heating to 90 oC, cooling to 20 oC, and reheating to 90 oC, the obtained far- and near-UV CD spectra do not show significant differences as compared with that of the “(1) 20 oC” sample. This means that upon the addition of 5 mg/mL PSS-70k, the sample can withstand thermal treatments and

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maintain constant secondary and tertiary structures, which agrees with the flat baselines observed in the DSC curves in Figure 4B. To see if the molecular weight of PSS can affect the unfolding and refolding behaviors of lysozyme, we also carried out nanoDSC experiments for the PSS samples with the molecular weights of 200k and 1000k, and the results of the initial heating scans are shown in Figure 7. By careful comparisons of the DSC traces of 5 mg/mL lysozyme in the presence of 0.5, 1, 3, and 5 mg/mL PSS (including PSS-70k (see Figure 3), PSS-200k, and PSS-1000k), we can see that the difference in the molecular weight of PSS does not evidently change the unfolding behaviors of lysozyme in water. Besides, as revealed by the respective heating–cooling–reheating DSC scans of these mixtures (data not shown), the difference in the molecular weight of PSS also does not affect the refolding behavior of the heat-denatured lysozyme upon cooling. 3.3. Interaction Mechanisms between Lysozyme and PSS. The appearances of the lysozyme–PSS-70k samples and the measured pH values of these solutions are shown in Figure S2. Upon increasing CPSS-70k, the initial transparent and clear solution first became turbid suspensions at CPSS-70k > 0.2 mg/mL and then turned into clear solution again at CPSS-70k > 3 mg/mL. The occurrence of very large aggregates (or precipitates) that was visible to naked-eye at CPSS-70k > 0.2 mg/mL accompanied with the formation of the state I of lysozyme. The formation of large aggregates is also verified by the TEM result shown in Figure 8A, where we can see the presence of particles with sizes ranging from hundreds of nanometers to several micrometers in the sample diluted

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from “5 mg/mL lysozyme and 1 mg/mL PSS-70k”. The clear solution observed at CPSS-70k > 3 mg/mL also agrees well with the formation of another unfolded state (state II) that was inert to the change of temperature. As revealed by the CD data (Figure 2 and Figure 6), the two clear solutions diluted from “5 mg/mL lysozyme, 20 o

C” and “5 mg/mL lysozyme and 5 mg/mL PSS-70k, 20 oC” have different protein

secondary and tertiary structures. The lysozyme molecules in “5 mg/mL lysozyme and 5 mg/mL PSS-70k” existed in a completely unfolded state (Figure 6). The complete unfolding of lysozyme by excess PSS was also observed by Cousin et al., who have also derived the structure of the lysozyme/PSS mixture using SANS.45 Besides, our TEM result in Figure 8B shows that the clear solution of “5 mg/mL lysozyme and 5 mg/mL PSS-70k” is composed of very small particles with sizes of less than 5 nm. The TEM result agrees with the DLS result, which reveals that the hydrodynamic diameter of these particles is 4.0 ± 1.2 nm. According to previous work,45 the clear solution may be composed of co-assemblies of PSS chains and fully unfolded proteins, one decorating the other. Since the concentration of the polyelectrolyte in the unit of mg/mL as we used above is not very helpful in the interpretation of the experimental results, it could be valuable to discuss the interaction between lysozyme and polyelectrolyte based on the absolute value of the net charge ratio of PSS-70k/lysozyme (rPSS-70k/lysozyme). The net charge values for lysozyme at different pH values can be found in the literature.63 Although these values were obtained in a salt-containing dispersion, the values were almost independent on salt concentrations.63 Considering its pI value (10.7), lysozyme

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possesses positive net charges at all these pH values (4.0–6.5) of the samples. By assuming that PSS has a 100% dissociation in the pH range, we can work out the net charge ratios for the lysozyme–PSS-70k system at various pH values. The dependence of rPSS-70k/lysozyme on CPSS-70k (where the concentration of lysozyme was fixed at 5 mg/mL) calculated by using the pH values listed in Figure S2 was shown in Figure S3. We can see that upon increasing PSS-70k, rPSS-70k/lysozyme increases almost linearly from 0.067 at 0.05 mg/mL PSS-70k to 8.4 at 5 mg/mL PSS-70k. In particular, the beginning point of the coassembly (or coagulation) between lysozyme (5 mg/mL) and PSS-70k (0.2 mg/mL) corresponds to rPSS-70k/lysozyme = 0.27, which is the necessary amount of the negatively charged polyelectrolytes required for the induction of the new unfolded state of lysozyme. Besides, a close to unity (0.96) of the charge ratio value is at CPSS-70k = 0.65 mg/mL, which correlates well with the fact that large amounts of precipitates form for lysozyme/PSS mixtures around charge neutrality. The formation of large aggregates of the lysozyme/PSS mixture at low charge excess has also been reported by a previous work carried out by Cousin et al..45 Besides, the phenomenon that the coassembly between two oppositely charged molecules occurred at “unbalanced” charge ratios was also observed in the coassembly processes of the DNA–surfactant,64 lysozyme–DNA,65 and lysozyme–β-casein51 systems. To exclude the effect of pH on the unfolding of lysozyme, we have performed control experiments using buffered solutions (20 mM phosphate buffered saline) to prepare pure lysozyme solutions and the results (data not shown) show that the denaturation temperature (and unfolding-refolding behavior upon heating and cooling)

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of lysozyme did not change at pH range of 4–6.5. Thus, pH is not the main factor that affects the unfolding and refolding behavior of lysozyme in the presence of polyanions. For the lysozyme–PSS-70k mixtures, we selected the two samples (“5 mg/mL lysozyme and 1 mg/mL PSS-70k” and “5 mg/mL lysozyme and 5 mg/mL PSS-70k”), and carried out the nanoDSC experiments to study the addition of NaCl on the unfolding behaviors of lysozyme. We have first confirmed that the presence of NaCl to a concentration of 300 mM did not significantly change the denaturation temperature of 5 mg/mL lysoyzme (data not shown). For “5 mg/mL lysozyme and 1 mg/mL PSS-70k” in Figure 9A, we can see that with the addition of NaCl at concentrations above 50 mM, an endothermic peak at ~74 oC appears and it becomes larger as the concentration of NaCl increased. The occurrence of this peak represents the presence of free, native lysozyme in the sample before heating. It means that the addition of NaCl can cause the disassembly between lysozyme and PSS-70k through salt screening, which in turn proves the important role of electrostatic interactions between lysozyme and PSS-70k. Besides, we can see that, after the addition of 300 mM NaCl, the original endothermic peak at ~58 oC still predominates, indicating the strong complexation between lysozyme and PSS-70k. For “5 mg/mL lysozyme and 5 mg/mL PSS-70k” (Figure 9B), we can see that the addition of 50 mM NaCl produces a small endothermic peak at ~38 oC, which shifts to higher temperatures and becomes larger at elevated NaCl concentrations (e.g., ~42 o

C at 150 mM NaCl and ~44 oC at 300 mM NaCl). The results again indicate that the

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salt screening effect can change the structure and thermostability of lysozyme. However, in the presence of 300 mM NaCl, lysozyme in “5 mg/mL lysozyme and 5 mg/mL PSS-70k” sample is still much less stable than pure lysozyme, which has a denaturation temperature of ~75 oC in 300 mM NaCl. The results suggest that the complexation between lysozyme and PSS still exists in “5 mg/mL lysozyme and 5 mg/mL PSS-70k” system at a high salt concentration. The schematic model in Figure 10 summarizes the main findings of this work. The native-state globular lysozyme (~4 nm66) in water is shown as circles. The concentration of lysozyme in all the samples is fixed at 5 mg/mL and the temperature is at 20 oC. When PSS-70k is added, soluble complex forms at CPSS-70k < 0.2 mg/mL. The electrostatic forces have been found to play a crucial role in the primary formation of the soluble protein–polyelectrolyte complex.24 At CPSS-70k > 0.2 mg/mL, a new protein state (state I) forms with different secondary and tertiary structures as compared with the original native state (shown as ellipses). Actually, the state I can be regarded as a partially unfolded state of lysozyme if we compare its denaturation temperature with that of the native state. At 0.2 mg/mL < CPSS-70k < 1 mg/mL, two states of lysozyme (the native state and the state I) coexists in the mixture, and the increase in CPSS-70k facilitates the interactions between the soluble complex particles, thus inducing the formation of very large insoluble aggregates (precipitates). The formation of the insoluble aggregate can be considered as a process driven by the disorder of the ordered water molecules around the hydrophobic chain of the polymer.24 At CPSS-70k = 1 mg/mL, the state I forms exclusively in the dispersion,

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which is complexed tightly with the oppositely charged PSS-70k. As more PSS-70k is added, lysozyme in state I can be further destabilized and various intermediate structures forms at 1 mg/mL < CPSS-70k < 5 mg/mL. Finally, at CPSS-70k = 5 mg/mL, the sample becomes clear solution again and lysozyme in this dispersion (state II) has different secondary and tertiary structures as compared with that in the native state. The state II is a completely unfolded state since it does not show any DSC endothermic signal upon heating (Figure 3) and it does not have tertiary structure as revealed by the near-UV CD result (Figure 6B, right panel). The existence of excess water-soluble PSS-70k ensures the redissolution of the lysozyme–PSS complexes. As PSS is in excess, the lysozyme–PSS complexes are highly negatively charged. The electrostatic repulsive forces lead to the solubilization of the complexes and also force lysozyme to unfold. The repulsive force between the complexes is very strong and is nearly irrelevant with temperature. Therefore, no temperature-dependent unfolding or tertiary structure change of lysozyme can be observed. Taken together, we believe that state II is a state with soluble lysozyme/PSS complexes as depicted in Figure 10, and such an unfolded state may represent a common state when proteins unfold in the presence of polyelectrolytes or surfactants. It was reported that PSS is more effective to destabilize lysozyme than other negatively charged polyelectrolytes such as sodium poly(acrylate) (PAAS).8 We have also tested the influence of PAAS on the thermostability of lysozyme, and found that for 5 mg/mL lysozyme, PAAS at the concentration range of 0–50 mg/mL can at most decrease the unfolding temperature of lysozyme by ~27 oC, while PSS can completely

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unfold lysozyme at CPSS > 3 mg/mL. This may be attributed to the presence of the benzene group in the side chain of PSS, which makes it be more hydrophobic than PAAS. Thus the hydrophobic interaction between lysozyme and PSS also plays an important role in the complexation and unfolding/refolding behaviors. A previous study also revealed the role of the polyanion hydrophobicity on the protein stability in the protein–polyanion complex.7 It can thus be concluded that the electrostatic and hydrophobic interactions are the two dominating factors that control the interaction between lysozyme and PSS. Lysozyme is one of the most thermostable proteins. This is possibly related to its capability to refold and regain its tertiary structure after cooling from its denatured state, as revealed by our DSC and CD results in Figure 1 and Figure 2, respectively. The present investigation of the interaction between lysozyme and PSS is important for the understanding towards its folding mechanism, thermostability, and related functions. This work may also shed new light on the development of novel lysozyme-based self-assembled functional materials with desired properties and applications. For example, as a non-toxic protein used frequently as food additive, nanocomposites made of lysozyme fibrils have both fundamental and practical relevance. The lysozyme fibril is emerging as a unique building block for the design of functional materials.47,48 Since the amyloid nanofiber materials highly rely on the protein folding/unfolding behavior and the interaction between polyelectrolyte and protein, unraveling the self-assembly mechanism, structure and physical properties of amyloid fibrils made of lysozyme is thus of particular importance. Furthermore, the

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present work demonstrates that upon complexation with polyelectrolyte, some intermediate states of protein with altered secondary and tertiary structures are formed, which will be possibly accompanied by changes in the aggregation state and/or self-assembled structure. These intermediate states of naturally-occurring proteins can be used as conformationally flexible building blocks to construct novel protein-based nanomaterials or nanofilms, if we can finely tune the structure of these polyelectrolytes. An ongoing work in our laboratory has revealed that by linking with a polyethylene glycol (PEG) segment, a PEGylated polyelectrolyte (PEGylated polyamino acid) can form nanosized polyelectrolyte complex micelle via the electrostatic complexation between the polymer and lysozyme for efficient drug delivery applications. 4. CONCLUSION By using nanoDSC and CD spectroscopy, we studied the unfolding and refolding behaviors of the positively charged lysozyme in the presence of the negatively charged polyelectrolyte PSS. The most exciting result is that we observed two states of lysozyme (state I and state II) with different formation mechanisms upon increasing the concentration of PSS at room temperature. The formation of the state I upon the addition of PSS is a two-state process. While further increasing PSS induces the non-two-state conversion from the state I to the state II via the formation of various intermediate structures. To the best of our knowledge, the present work is the first discovery that a negatively charged polyelectrolyte can induce both the two-state and non-two-state unfolding processes of the positively charged protein depending on

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the relative amount of the added polyelectrolytes. Besides, with the help of the heating–cooling–reheating procedure, we can obtain the unfolding and refolding abilities of the three different protein states: native state, state I, and state II. Different from the native-state lysozyme that exhibits refolding upon cooling from its heat-denatured state, lysozyme in the state I can undergo unfolding upon heating but no refolding upon cooling, while lysozyme in the state II does not undergo unfolding or refolding upon thermal treatments. We would like to point out that the state II here is actually an “unfolded state”, which does not display any detectable unfolding transition. However, there is a very large difference between the normal heat-induced unfolded state of pure lysozyme that exhibits refolding and re-unfolding upon subsequent cooling and reheating processes, while the PSS-complexed unfolded state II here does not have unfolding and refolding behaviors. The results indicate that such a heating–cooling–reheating procedure can give us clues on the structures and properties of the polyelectrolyte-complexed proteins. Besides, this procedure can also be used to study the unfolding and refolding details of the target protein in other protein-containing mixtures. Furthermore, the effects of the ionic strength and the molecular weight of the polyelectrolyte on the unfolding and refolding behaviors of lysozyme were also investigated. The addition of salt weakened the complexation between lysozyme and PSS. However, the addition of NaCl up to a concentration of 300 mM could not completely disrupt the complexation between lysozyme and PSS. The difference in the molecular weight of the polyelectrolyte did not significantly change the unfolding

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and refolding behaviors of lysozyme upon thermal cycles. The present work shows that the complexation between lysozyme and PSS could significantly alter the unfolding and refolding behaviors of lysozyme. The thermostability of lysozyme was also largely affected by the addition of PSS, which accordingly changed the properties and functions of the protein and the protein–polyelectrolyte complex. The present work provides a better understanding of the principles governing the protein–polyelectrolyte interactions and may have implications for the fabrication of novel nanomaterials.

ASSOCIATED CONTENT Supporting Information Additional DSC data, photographs of lysozyme–PSS-70k mixtures at 20 oC, and the absolute value of the net charge ratio of PSS-70k/lysozyme versus the concentration of PSS-70k. This information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *(Z.-W.Y.) E-mail: [email protected]. Notes The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (21133009, 21273130, and 21303017), the Natural Science Foundation of Jiangsu Province (KB20130601), the Fundamental Research Funds for the Central Universities (2242015R30016), and the project sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

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REFERENCES (1) Careri, G. Cooperative Charge Fluctuations by Migrating Protons in Globular Proteins. Prog. Biophys. Mol. Biol. 1998, 70, 223–249. (2) Bittrich, E.; Rodenhausen, K. B.; Eichhorn, K. J.; Hofmann, T.; Schubert, M.; Stamm, M.; Uhlmann, P. Protein Adsorption on and Swelling of Polyelectrolyte Brushes: A Simultaneous Ellipsometry-Quartz Crystal Microbalance Study. Biointerphases 2010, 5, 159–167. (3) Silva, R. A.; Urzua, M. D.; Petri, D. F. S.; Dubin, P. L. Protein Adsorption onto Polyelectrolyte Layers: Effects of Protein Hydrophobicity and Charge Anisotropy. Langmuir 2010, 26, 14032–14038. (4) Lindhoud, S.; de Vries, R.; Schweins, R.; Cohen Stuart, M. A.; Norde, W. Salt-Induced Release of Lipase from Polyelectrolyte Complex Micelles. Soft Matter 2009, 5, 242–250. (5) Schwinté, P.; Ball, V.; Szalontai, B.; Haikel, Y.; Voegel, J. C.; Schaaf, P. Secondary Structure of Proteins Adsorbed onto or Embedded in Polyelectrolyte Multilayers. Biomacromolecules 2002, 3, 1135–1143. (6) Schwinté, P.; Voegel, J. C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B. Stabilizing Effects of Various Polyelectrolyte Multilayer Films on the Structure of Adsorbed/Embedded Fibrinogen Molecules: An ATR−FTIR Study. J. Phys. Chem. B 2001, 105, 11906–11916. (7) Sedlák, E.; Fedunová, D.; Veselá, V.; Sedláková, D.; Antalík, M. Polyanion Hydrophobicity

and

Protein

Basicity

Affect

Protein

Stability

in

Protein−Polyanion Complexes. Biomacromolecules 2009, 10, 2533–2538. (8) Ivinova, O. N.; Izumrudov, V. A.; Muronetz, V. I.; Galaev, I. Y.; Mattiasson, B. Influence of Complexing Polyanions on the Thermostability of Basic Proteins. Macromol. Biosci. 2003, 3, 210–215. (9) Xu, Y. S.; Mazzawi, M.; Chen, K.; Sun, L. H.; Dubin, P. L. Protein Purification by Polyelectrolyte Coacervation: Influence of Protein Charge Anisotropy on Selectivity. Biomacromolecules 2011, 12, 1512–1522. 26


Page 27 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(10) Jiang, J. W.; Prausnitz, J. M. Molecular Thermodynamics for Protein Precipitation with a Polyelectrolyte. J. Phys. Chem. B 1999, 103, 5560–5569. (11) Saurer, E. M.; Flessner, R. M.; Sullivan, S. P.; Prausnitz, M. R.; Lynn, D. M. Layer-by-Layer Assembly of DNA- and Protein-Containing Films on Microneedles for Drug Delivery to the Skin. Biomacromolecules 2010, 11, 3136–3143. (12) Karlsson, A. J.; Flessner, R. M.; Gellman, S. H.; Lynn, D. M.; Palecek, S. P. Polyelectrolyte Multilayers Fabricated from Antifungal β-Peptides: Design of Surfaces

that

Exhibit

Antifungal

Activity

Against

Candida

albicans.

Biomacromolecules 2010, 11, 2321–2328. (13) Jewell, C. M.; Fuchs, S. M.; Flessner, R. M.; Raines, R. T.; Lynn, D. M. Multilayered

Films

Fabricated

from

an

Oligoarginine-Conjugated

Protein Promote Efficient Surface-Mediated Protein Transduction. Biomacromolecules 2007, 8, 857–863. (14)

Cooper,

C.

L.;

Dubin,

P.

L.;

Kayitmazer,

A.

B.;

Turksen,

S.

Polyelectrolyte–Protein Complexes. Curr. Opin. Colloid Interface Sci. 2005, 10, 52–78. (15) Houska, M.; Brynda, E.; Bohatá, K. The Effect of Polyelectrolyte Chain Length on Layer-by-Layer Protein/Polyelectrolyte Assembly—an Experimental Study. J. Colloid Interface Sci. 2004, 273, 140–147. (16) Ladam, G.; Gergely, C.; Senger, B.; Decher, G.; Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. G. Protein Interactions with Polyelectrolyte Multilayers: Interactions

between

Human

Serum

Albumin

and

Polystyrene

Sulfonate/Polyallylamine Multilayers. Biomacromolecules 2000, 1, 674–687. (17) Caruso, F.; Furlong, D. N.; Ariga, K.; Ichinose, I.; Kunitake, T. Characterization of Polyelectrolyte−Protein Multilayer Films by Atomic Force Microscopy, Scanning

Electron

Microscopy,

and

Fourier

Transform

Infrared

Reflection−Absorption Spectroscopy. Langmuir 1998, 14, 4559–4565. (18) Kayitmazer, A. B.; Seeman, D.; Minsky, B. B.; Dubin,P. L.; Xu, Y. S. Protein–Polyelectrolyte Interactions. Soft Matter 2013, 9, 2553–2583. 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 41

(19) Jones, O. G.; McClements, D. J. Recent Progress in Biopolymer Nanoparticle and Microparticle Formation by Heat-Treating Electrostatic Protein–Polysaccharide Complexes. Adv. Colloid Interface Sci. 2011, 167, 49–62. (20) Baik, H. J.; Oh, N. M.; Oh, Y. T.; Yoo, N. Y.; Park, S. Y.; Oh, K. T.; Youn, Y. S.; Lee, E. S. 3-Diethylaminopropyl-Bearing Glycol Chitosan as a Protein Drug Carrier. Colloid Surf. B-Biointerfaces 2011, 84, 585–590. (21) Lee, E. S.; Park, K. H.; Park, I. S.; Na, K. Glycol Chitosan as a Stabilizer for Protein Encapsulated into Poly(lactide-co-glycolide) Microparticle. Int. J. Pharm. 2007, 338, 310–316. (22) Karayianni, M.; Pispas, S.; Chryssikos, G. D.; Gionis, V.; Giatrellis, S.; Nounesis, G.

Complexation

of

Lysozyme

with

Poly(sodium(sulfamate-carboxylate)isoprene). Biomacromolecules 2011, 12, 1697–1706. (23) da Silva, F. L. B.; Jönsson, B. Polyelectrolyte–Protein Complexation Driven by Charge Regulation. Soft Matter 2009, 5, 2862–2868. (24) Boeris, V.; Spelzini, D.; Salgado, J. P.; Picó, G.; Romanini, D.; Farruggia, B. Chymotrypsin–Poly Vinyl Sulfonate Interaction Studied by Dynamic Light Scattering and Turbidimetric Approaches. Biochim. Biophys. Acta-Gen. Subj. 2008, 1780, 1032–1037. (25) Matsunami, H.; Kikuchi, R.; Ogawa, K.; Kokufuta, E. Light Scattering Study of Complex Formation between Protein and Polyelectrolyte at Various Ionic Strengths. Colloid Surf. B 2007, 56, 142–148. (26) da Silva, F. L. B.; Lund, M.; Jönsson, B.; Åkesson, T. On the Complexation of Proteins and Polyelectrolytes. J. Phys. Chem. B 2006, 110, 4459–4464. (27) Carlsson, F.; Malmsten, M.; Linse, P. Protein-Polyelectrolyte Cluster Formation and Redissolution: A Monte Carlo Study. J. Am. Chem. Soc. 2003, 125, 3140–3149. (28) Seyrek, E.; Dubin, P. L.; Tribet, C.; Gamble, E. A. Ionic Strength Dependence of Protein-Polyelectrolyte Interactions. Biomacromolecules 2003, 4, 273–282. (29) Ball, V.; Winterhalter, M.; Schwinté, P.; Lavalle, Ph.; Voegel, J. C.; Schaaf, P. 28


Page 29 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Complexation Mechanism of Bovine Serum Albumin and Poly(allylamine hydrochloride). J. Phys. Chem. B 2002, 106, 2357–2364. (30) Takahashi, D.; Kubota, Y.; Kokai, K.; Izumi, T.; Hirata, M.; Kokufuta, E. Effects of Surface Charge Distribution of Proteins in Their Complexation with Polyelectrolytes in an Aqueous Salt-Free System. Langmuir 2000, 16, 3133–3140. (31) Mattison, K. W.; Dubin, P. L.; Brittain, I. J. Complex Formation between Bovine Serum Albumin and Strong Polyelectrolytes: Effect of Polymer Charge Density. J. Phys. Chem. B 1998, 102, 3830–3836. (32) Park, J. M.; Muhoberac, B. B.; Dubin, P. L.; Xia, J. L. Effects of Protein Charge Heterogeneity in Protein-Polyelectrolyte Complexation. Macromolecules 1992, 25, 290–295. (33) 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. (34) 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. (35) 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. (36) 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. (37) 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. (38) 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. 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 41

(39) 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. (40) 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. (41) 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. (42) 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. (43) 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. (44) 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. (45) Cousin, F.; Gummel, J.; Ung, D.; Boué, F. Polyelectrolyte−Protein Complexes: Structure and Conformation of Each Specie Revealed by SANS. Langmuir 2005, 21, 9675–9688. (46) Lundberg, D.; Carnerup, A. M.; Schillén, K.; Miguel, M. D.; Lindman, B. Phase Behavior and Coassembly of DNA and Lysozyme in Dilute Aqueous Mixtures: A Model Investigation of DNA−Protein Interactions. Langmuir 2010, 26, 2986–2988. (47) Meier, C.; Lifincev, I.; Welland, M. E. Conducting Core–Shell Nanowires by 30


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Amyloid Nanofiber Templated Polymerization. Biomacromolecules 2015, 16, 558–563. (48) Lara, C.; Reynolds, N. P.; Berryman, J. T.; Xu, A. Q.; Zhang, A. F.; Mezzenga, R. ILQINS Hexapeptide, Identified in Lysozyme Left-Handed Helical Ribbons and Nanotubes, Forms Right-Handed Helical Ribbons and Crystals. J. Am. Chem. Soc. 2014, 136, 4732–4739. (49) Luo, J. J.; Wu, F. G.; Yu, J. S.; Wang, R.; Yu, Z. W. Denaturation Behaviors of Two-State and Non-Two-State Proteins Examined by an Interruption–Incubation Protocol. J. Phys. Chem. B 2011, 115, 8901–8909. (50) Luo, J. J.; Wu, F. G.; Qin, S. S.; Yu, Z. W. In Situ Unfolded Lysozyme Induces the Lipid Lateral Redistribution of a Mixed Lipid Model Membrane. J. Phys. Chem. B 2012, 116, 12381–12388. (51) Wu, F. G.; Luo, J. J.; Yu, Z. W. Unfolding and Refolding Details of Lysozyme in the Presence of β-Casein Micelles. Phys. Chem. Chem. Phys. 2011, 13, 3429–3436. (52) Sreerama, N.; Venyaminov, S. Y.; Woody, R. W. Estimation of Protein Secondary Structure from Circular Dichroism Spectra: Inclusion of Denatured Proteins with Native Proteins in the Analysis. Anal. Biochem. 2000, 287, 243–251. (53) Sreerama, N.; Woody, R. W. Estimation of Protein Secondary Structure from Circular Dichroism Spectra: Comparison of CONTIN, SELCON, and CDSSTR Methods with an Expanded Reference Set. Anal. Biochem. 2000, 287, 252–260. (54) Morshedi, D.; Ebrahim-Habibi, A.; Moosavi-Movahedi, A. A.; Nemat-Gorgani, M. Chemical Modification of Lysine Residues in Lysozyme May Dramatically Influence Its Amyloid Fibrillation. Biochim. Biophys. Acta 2010, 1804, 714–722. (55) Wu, F. G.; Yu, J. S.; Sun, S. F.; Yu, Z. W. Comparative Studies on the Crystalline to Fluid Phase Transitions of Two Equimolar Cationic/Anionic Surfactant Mixtures Containing Dodecylsulfonate and Dodecylsulfate. Langmuir 2011, 27, 14740–14747. (56) Wu, F. G.; Wang, N. N.; Yu, J. S.; Luo, J. J.; Yu, Z. W. Nonsynchronicity 31



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Phenomenon Observed during the Lamellar−Micellar Phase Transitions of 1-Stearoyllysophosphatidylcholine Dispersed in Water. J. Phys. Chem. B 2010, 114, 2158–2164. (57) Wu, F. G.; Jia, Q.; Wu, R. G.; Yu, Z. W. Regional Cooperativity in the Phase Transitions of Dipalmitoylphosphatidylcholine Bilayers: The Lipid Tail Triggers the Isothermal Crystallization Process. J. Phys. Chem. B 2011, 115, 8559–8568. (58) Wu, F. G.; Wang, N. N.; Yu, Z. W. Nonsynchronous Change in the Head and Tail of Dioctadecyldimethylammonium Bromide Molecules during the Liquid Crystalline to Coagel Phase Transformation Process. Langmuir 2009, 25, 13394–13401. (59) Blumlein, A.; McManus, J. J. Reversible and Non-Reversible Thermal Denaturation of Lysozyme with Varying pH at Low Ionic Strength. Biochim. Biophys. Acta 2013, 1834, 2064–2070. (60) Tani, F.; Murata, M.; Higasa, T.; Goto, M.; Kitabatake, N.; Doi, E. Molten Globule State of Protein Molecules in Heat-Induced Transparent Food Gels. J. Agric. Food Chem. 1995, 43, 2325–2331. (61) Holtzer, M. E.; Holtzer, A. α-Helix to Random Coil Transitions: Determination of Peptide Concentration from the CD at the Isodichroic Point. Biopolymers 1992, 32, 1675–1677. (62) Johansson, J.; Gudmundsson, G. H.; Rottenberg, M. E.; Berndt, K. D.; Agerberth, B. Conformation-dependent Antibacterial Activity of the Naturally Occurring Human Peptide LL-37. J. Biol. Chem. 1998, 273, 3718–3724. (63) Kuehner, D. E.; Engmann, J.; Fergg, F.; Wernick, M.; Blanch, H. W.; Prausnitz, J. M. Lysozyme Net Charge and Ion Binding in Concentrated Aqueous Electrolyte Solutions. J. Phys. Chem. B 1999, 103, 1368–1374. (64) Dias, R.; Mel'nikov, S.; Lindman, B.; Miguel, M. G. DNA Phase Behavior in the Presence of Oppositely Charged Surfactants. Langmuir 2000, 16, 9577–9583. (65) Lundberg, D.; Carnerup, A. M.; Janiak, J.; Schillén, K.; Miguel, M. G.; Lindman, B. Size and Morphology of Assemblies Formed by DNA and Lysozyme in Dilute Aqueous Mixtures. Phys. Chem. Chem. Phys. 2011, 13, 3082–3091. 32


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(66) Vertegel, A. A.; Siegel, R. W.; Dordick, J. S. Silica Nanoparticle Size Influences the Structure and Enzymatic Activity of Adsorbed Lysozyme. Langmuir 2004, 20, 6800–6807.

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TOC Graphic

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Figure

1.

NanoDSC

results

of

the

lysozyme

(5

mg/mL)

during

the

heating–cooling–reheating processes. The curves are vertically shifted for better presentation.

Figure 2. Far-UV CD (A) and near-UV CD (B) results of lysozyme solutions at selected temperatures. The numbers (1), (2), (3), and (4) represent the sequential order of the thermal treatments.

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Figure 3. NanoDSC results of the lysozyme–PSS-70k mixtures during the initial heating processes. The curves are vertically shifted for better presentation.

Figure 4. Heating–cooling–reheating nanoDSC results of two lysozyme–PSS-70k mixtures. (A) 5 mg/mL lysozyme and 1 mg/mL PSS-70k, and (B) 5 mg/mL lysozyme and 5 mg/mL PSS-70k. The curves are vertically shifted for better presentation.

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Figure 5. Far-UV CD results of the samples diluted (25-fold) from (A) “5 mg/mL lysozyme and 0–1 mg/mL PSS-70k” and (B) “5 mg/mL lysozyme and 1–5 mg/mL PSS-70k”. These spectra were all obtained from the samples at 20 oC before thermal treatments.

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Figure 6. Far-UV and near-UV CD results of lysozyme–PSS-70k at selected temperatures. Samples in (A) are diluted from “5 mg/mL lysozyme and 1 mg/mL PSS-70k”, while samples in (B) are diluted from “5 mg/mL lysozyme and 5 mg/mL PSS-70k”. The numbers (1), (2), (3), and (4) represent the sequential order of the thermal treatments. The curves are vertically shifted for better presentation.

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Figure 7. NanoDSC results of lysozyme in the presence of various amounts of PSS-200k (A) and PSS-1000k (B). The concentration of lysozyme is 5 mg/mL in all samples. The curves are vertically shifted for better presentation.

Figure 8. TEM results of “0.5 mg/mL lysozyme and 0.1 mg/mL PSS-70k” (diluted from “5 mg/mL lysozyme and 1 mg/mL PSS-70k”) (A) and “0.5 mg/mL lysozyme and 0.5 mg/mL PSS-70k” (diluted from “5 mg/mL lysozyme and 5 mg/mL PSS-70k”) (B).

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Figure 9. Salt effect on the denaturation of lysozyme in the presence of PSS-70k. The curves are vertically shifted for better presentation.

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Figure 10. Schematic illustrations showing the complexations between lysozyme and PSS at different PSS concentrations at 20 oC in water. The concentration of lysozyme in all the mixtures is fixed at 5 mg/mL.

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Complexation of Lysozyme with Sodium Poly(styrenesulfonate) via the

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