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Specific Ion Effects on Protein Thermal Aggregation from Dilute Solutions to Crowded Environments Shuling Li, Shuji Ye, and Guangming Liu* Hefei National Laboratory for Physical Sciences at the Microscale, Department of Chemical Physics, University of Science and Technology of China, Hefei, P. R. China 230026 S Supporting Information *

ABSTRACT: We have investigated specific ion effects on protein thermal aggregation from dilute solutions to crowded environments. Ovalbumin and poly(ethylene glycol) have been employed as the model protein and crowding agent, respectively. Our studies demonstrate that the rate-limiting step of ovalbumin thermal aggregation is changed from the aggregation of unfolded protein molecules to the unfolding of the protein molecules, when the solution conditions are varied from a dilute solution to a crowded environment. The specific ion effects acting on the thermal aggregation of ovalbumin generated by kosmotropic and chaotropic ions are different. The thermal aggregation of ovalbumin molecules is promoted by kosmotropic anions in dilute solutions via an increase in protein hydrophobic interactions. In contrast, ovalbumin thermal aggregation is facilitated by chaotropic ions in crowded environments through accelerated unfolding of protein molecules. Therefore, there are distinct mechanisms causing the ion specificities of protein thermal aggregation between dilute solutions and crowded environments. The ion specificities are dominated by ion-specific hydrophobic interactions between protein molecules and ion-specific unfolding of protein molecules in dilute solutions and crowded environments, respectively.

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INTRODUCTION Protein aggregation has attracted great interest during past decades due to observations of a close correlation between protein aggregation and several fatal human diseases.1,2 Extensive investigations have been performed in dilute solutions, in vitro, to unravel the aggregation mechanism for different types of protein molecules.3−6 However, the aggregation of protein molecules often occurs within the intracellular environment, in which the volume of the cell is occupied by various biomacromolecules (e.g., proteins and polysaccharides) with a concentration up to ∼400 mg mL−1 and with a volume up to ∼40% of the cell.7−9 It is generally thought that the prerequisite for protein aggregation is misfolding or unfolding of the native protein conformation.8,10−13 In crowded environments, the native protein conformation may be stabilized by the presence of crowding agents via excluded volume effects that reduce the available space.14−16 This inhibits protein aggregation. Alternatively, the presence of macromolecular crowding agents may promote the association of protein molecules, through enhanced attractive depletion forces between the protein molecules.17−20 Consequently, the overall effect of crowded environments on protein aggregation depends on the competition between these stabilizing and destabilizing effects.8 The intracellular environment also contains various small ions with an ionic strength of ∼0.15 M in the intracellular fluid.21−23 It is well-known that different types of ions have © XXXX American Chemical Society

distinct effects on the aggregation and precipitation of protein molecules in aqueous solutions.24,25 These so-called “Hofmeister effects” were first discovered by Franz Hofmeister when performing experiments on the salt-induced precipitation of egg white protein.26,27 In the classical Hofmeister series, the effectiveness of anions to precipitate the protein decreases following the order SO42− > HPO42− > F− > CH3COO− > Cl− > Br− > NO3− > I− > ClO4− > SCN−.22,25 Generally, the strongly hydrated kosmotropes give rise to a higher stability of the native protein conformation via the salting-out effect, whereas the weakly hydrated chaotropes result in denaturation/ unfolding of protein molecules through the salting-in effect.23,28,29 From this perspective, kosmotropic and chaotropic ions should respectively suppress and facilitate protein aggregation. However, hydrophobic interactions between protein molecules may be strengthened in the presence of kosmotropic ions via the salting-out effect and weakened in the presence of chaotropic ions through the salting-in effect,30,31 leading to the expectation that protein aggregation should be promoted by kosmotrpic ions and suppressed by chaotropic ions. Consequently, the overall specific ion effects on protein aggregation are determined by whether the rate-limiting step of protein aggregation is the unfolding of the protein Received: January 28, 2018 Revised: February 27, 2018 Published: March 22, 2018 A

DOI: 10.1021/acs.langmuir.8b00294 Langmuir XXXX, XXX, XXX−XXX

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Figure 1. Cloud point temperature (Tcp) of ovalbumin as a function of PEG concentration (CPEG) in the presence of different anions, with Na+ as the common cation at different salt concentrations. Here, the ovalbumin concentration is fixed at 1.0 mg mL−1, and the pH of the protein solutions is fixed at ∼7.1. The salt concentrations for (a), (b), (c), and (d) are 0.2, 0.5, 0.8, and 1.0 M, respectively. The inset shows a close-up view of the Tcp of ovalbumin in dilute solutions as a function of the anion identity.

Figure 2. ATR-FTIR spectra of ovalbumin in the frequency range of 1300−1800 cm−1 in the presence of different anions with Na+ as the common cation, in dilute solutions and crowded environments. The salt concentration is fixed at 0.5 M for (a), (b), and (c) and at 1.0 M for (d), (e), and (f). (a) and (d): PEG-free solutions; (b) and (e): 50 mg mL−1 PEG; and (c) and (f): 100 mg mL−1 PEG. The intensities of the amide II′ band are compared by normalizing with the intensities of the amide I band.

model system to study specific ion effects on protein thermal aggregation in both dilute solutions and crowded environments. Thermal aggregation of protein molecules usually involves protein unfolding followed by aggregation of the unfolded protein molecules.3 Considering that both of these processes should be influenced by macromolecular crowding and specific ion effects, we are interested in understanding how protein thermal aggregation is affected by the interplay between the combined influences of macromolecular crowding and specific ion effects.

conformation or the aggregation by intermolecular hydrophobic interactions. Previously, protein aggregation studies have mostly been performed in either crowded environments, without considering specific ion effects, or in dilute solutions in the presence of different types of ions. 28,32−38 Very few studies have investigated specific ion effects on protein aggregation in crowded environments, despite the fact that macromolecular crowding agents and various small ions coexist in the intracellular environment, with strong influences on protein aggregation.39 The exact mechanism of the interplay between the macromolecular crowding and the specific ion effects on protein aggregation remains elusive. In the present work, we have employed the thermal aggregation of ovalbumin as a

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EXPERIMENTAL SECTION

Materials. Ovalbumin (isoelectric point ∼4.7) was purchased from Beijing HWRK Chemical Co., Ltd. and used as received. All the salts B

DOI: 10.1021/acs.langmuir.8b00294 Langmuir XXXX, XXX, XXX−XXX

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Figure 3. Relative fluorescence intensity (F/F0) of ovalbumin as a function of PEG concentration (CPEG) in the presence of different anions with Na+ as the common cation, at different salt concentrations. The salt concentrations for (a), (b), (c), and (d) are 0.2, 0.5, 0.8, and 1.0 M, respectively. Here, F represents the fluorescence intensity of ovalbumin in the relevant solutions, and F0 represents the fluorescence intensity of ovalbumin in the absence of both the salts and the crowding agents. phosphate buffer solutions (pH ∼ 7.1) to avoid the self-quenching of the ovalbumin fluorescence. The intrinsic tryptophan fluorescence was monitored by exciting the protein at 295 nm to avoid the excitation of tyrosine, and the emission was collected between 300 and 550 nm. Prior to each test, the protein solution was equilibrated for approximately 10 min at 25 °C, using a temperature-controlled, circulating water bath.

(AR grade) were purchased from Sinopharm or Aladdin and used as received. PEG (20 kDa) was purchased from Sinopharm and used as received. Deuterium oxide (D2O) was purchased from Sigma-Aldrich and used as received. The water used was purified by filtration through a Millipore Gradient system after predistillation, giving a resistivity of 18.2 MΩ cm. Cloud Point Measurements. Cloud points of the ovalbumin were determined by monitoring the turbidity of protein solutions when heated at a constant rate of 0.1 °C min−1 across the phase transition of ovalbumin, using a spectrophotometer (UNICO 2802 UV/vis) with the wavelength set at 500 nm. The temperature of the cell was controlled using a temperature-controlled, circulating bath, with an accuracy of ±0.1 °C and monitored by an electronic thermometer. For the turbidity measurements, the concentration of ovalbumin was fixed at 1.0 mg mL−1, and the pH of the ovalbumin solutions was fixed at ∼7.1 using phosphate buffer. The phosphate buffer was composed of disodium hydrogen phosphate (∼2.74 × 10−2 M) and sodium dihydrogen phosphate (∼1.77 × 10−2 M) with a total ionic strength of ∼0.1 M. The influences of ion specificities generated by the phosphate buffer could be neglected in the investigations of specific ion effects on the protein thermal aggregation in both dilute solutions and crowded environments because the phosphate buffer was employed as the common background solution to dissolve all the different types of salts. Herein, the cloud point was determined from the intersection of two straight lines drawn through the transmittance versus temperature curves during the initial stage of the phase transition of ovalbumin, and the error for the cloud point determinations is typically within ∼±1 °C (Figure S1, Supporting Information). Attenuated Total Reflectance−Fourier Transform Infrared (ATR-FTIR) Spectra Measurements. The ATR-FTIR spectra of the ovalbumin solutions were measured using a Shimadzu IRPrestige-21 spectrometer at 25 ± 1 °C. To increase the signal-to-noise ratio, the ovalbumin concentration was increased to 10 mg mL−1 in the phosphate buffer solutions (pH ∼ 7.1). The maximum concentration of crowding agents used here was 100 mg mL−1 due to the limited solubility of ovalbumin in the highly crowded environments (Figure S2). The spectra were collected over the range of 4000−700 cm−1 with a spectral resolution of 4 cm−1, and each spectrum was recorded as an average of 256 scans. Prior to each test, the protein solution was equilibrated for about 10 min. Deuterated water (D2O) was used as the solvent to remove the influence of the H2O bending mode on the amide I band. Fluorescence Spectra Measurements. Fluorescence measurements were conducted on a Hitachi F-4600 fluorescence spectrophotometer. The ovalbumin concentration was fixed at 0.1 mg mL−1 in the

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RESULTS AND DISCUSSION Figure 1 shows the cloud point temperature (Tcp), obtained from the turbidity measurements, of the ovalbumin as a function of PEG concentration (CPEG), in the presence of different anions. Here, three anions that are known to have different specific ion effects are employed to investigate the specific anion effects on the thermal aggregation of ovalbumin from dilute solutions to crowded environments. Perchlorate (ClO4−) is a strongly chaotropic anion, and nitrate (NO3−) is a mildly chaotropic anion, whereas acetate (Ac − ) is a kosmotropic anion.22,24,40 It is evident that the Tcp of ovalbumin decreases with increasing CPEG, irrespective of the salt concentration and the anion identity. This is attributed to the following two effects. (i) The strongly hydrated PEG competes for the water molecules in the hydration shells of the protein, thereby reducing the solubility of the protein.41 (ii) The increasing macromolecular crowding causes an increase in the attractive depletion forces between the protein molecules, facilitating aggregation.17−20 Although the macromolecular crowding could stabilize the native protein conformation, this effect is clearly dominated by the above two effects, as evidenced by the fact that the crowding of PEG promotes the thermal aggregation of ovalbumin. Specific anion effects can also be observed in the thermal aggregation of ovalbumin molecules. In dilution solutions, the Tcp of ovalbumin increases along the anion series Ac− < NO3− < ClO4−. However, the influence of the anions on Tcp is reversed in crowded environments, where Tcp increases along the anion series ClO4− < NO3− < Ac−. The observation is that the more kosmotropic anions are more effective in promoting the thermal aggregation of ovalbumin in dilute solutions, whereas the more chaotropic anions are more effective in promoting the thermal aggregation of ovalbumin in crowded C

DOI: 10.1021/acs.langmuir.8b00294 Langmuir XXXX, XXX, XXX−XXX

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versa. In terms of specific ion effects on protein aggregation, chaotropic ions should weaken inter-protein hydrophobic interactions via salting-in effects;23,28−31 they should also weaken intra-protein hydrophobic interactions and promote the unfolding of proteins. In contrast, kosmotropic ions should strengthen inter-protein hydrophobic interactions via saltingout effects;23,28−31 they should also strengthen the intra-protein hydrophobic interactions and stabilize the native protein conformation. In dilute solutions, kosmotropic anions are more effective than chaotropic anions at inducing thermal aggregation of ovalbumin molecules, suggesting that the ratelimiting step of ovalbumin thermal aggregation is the aggregation of unfolded ovalbumin molecules. That is, the anion-specific thermal aggregation of ovalbumin is dominated by hydrophobic interactions between the protein molecules in dilute solutions. The ordering of the anions in terms of the anion-specific Tcp observed in dilute solutions is reversed in crowded environments. In crowded environments, chaotropic anions are more effective than kosmotropic anions at inducing the thermal aggregation of ovalbumin, as indicated by the increasing Tcp along the anion series ClO4− < NO3− < Ac−. This suggests that the rate-limiting step of the thermal aggregation of ovalbumin changes from the aggregation of unfolded ovalbumin molecules to the unfolding of ovalbumin molecules as the solution conditions switch from a dilute to a crowded environment. The introduction of macromolecular crowding agents to dilute solutions accelerate the aggregation of the unfolded protein molecules by strengthening the attractive depletion interactions between the protein molecules. Meanwhile, the macromolecular crowding retards the unfolding of the protein molecules through excluded volume effects. As the more chaotropic anions are more effective in facilitating the unfolding of protein molecules, the fact that the Tcp increases following the anion series ClO4− < NO3− < Ac− suggests that specific anion effects on the thermal aggregation of ovalbumin in crowded environments are dominated by the anion-specific unfolding of the protein molecules.

Figure 4. Salt concentration dependence of cloud point temperature (Tcp) of ovalbumin as a function of PEG concentration (CPEG) for different anions. Here, the ovalbumin concentration is fixed at 1.0 mg mL−1, and the pH of the protein solutions is fixed at ∼7.1. (a) NaAc, (b) NaNO3, and (c) NaClO4.

environments.39 It is known that the thermal aggregation of proteins usually involves an initial unfolding stage followed by aggregation of the unfolded protein molecules.3 If the rate of protein unfolding is slower than the rate of aggregation of the unfolded protein molecules, the rate-limiting step of the overall aggregation will be the unfolding of protein molecules, and vice

Figure 5. Cloud point temperature (Tcp) of ovalbumin as a function of PEG concentration (CPEG) in the presence of different cations with Cl− as the common anion at different salt concentrations. Here, the ovalbumin concentration is fixed at 1.0 mg mL−1, and the pH of the protein solutions is fixed at ∼7.1. The salt concentrations for (a), (b), (c), and (d) are 0.2, 0.5, 0.8, and 1.0 M, respectively. D

DOI: 10.1021/acs.langmuir.8b00294 Langmuir XXXX, XXX, XXX−XXX

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Figure 6. Relative fluorescence intensity (F/F0) of ovalbumin as a function of PEG concentration (CPEG) in the presence of different cations with Cl− as the common anion, at different salt concentrations. The salt concentrations for (a), (b), (c), and (d) are 0.2, 0.5, 0.8, and 1.0 M, respectively. Here, F represents the fluorescence intensity of ovalbumin in the relevant solutions, and F0 represents the fluorescence intensity of ovalbumin in the absence of both the salts and the crowding agents.

tion.28,42−44 At the same time, the specific anion effects can also be amplified with increasing extent of crowding at the same salt concentration. In crowded environments, the effective concentration, or activity coefficient, of the protein molecules is enhanced due to excluded volume effects.21,23,45,46 As a consequence, amplification of specific anion effects with increased crowding can be easily understood because the different types of anions can be more effectively differentiated by increasing effective protein concentration through the ionspecific interactions between the anions and the protein molecules during the anion-assisted unfolding of the ovalbumin molecules. The ATR-FTIR technique has proven to be a powerful tool for the identification of conformational states of proteins.47 This technique has been widely applied to determine the structure and orientation of peptides and proteins in different chemical environments, by probing the spectral signals from the amide I and amide II vibrations.47,48 The amide I band appears at 1600−1700 cm−1 and is mainly due to CO stretching vibrations of the protein backbone, while the amide II band arises from the out-of-phase combination of C−N stretching and N−H bending.47 The amide II band is generally located between 1500 and 1600 cm−1 and shifts to 1400−1500 cm−1 (amide II′ band) upon hydrogen−deuterium exchange of the amide proton.47 Figure 2 shows the ATR-FTIR spectra of ovalbumin in the range of 1300−1800 cm−1 as a function of the anion identity in dilute solution and in the crowded environments. Here, D2O was used as the solvent to remove the influence of the H2O bending mode on the amide I band. No obvious changes are observed in the amide I spectral features, such as the frequency and peak profiles, in the presence of different anions in both dilute solution and crowded environments, indicating that the addition of anions does not cause a change in the secondary structure of the ovalbumin molecules. In contrast, the relative intensity ratio of amide II′ band to amide I band (Iamide II′/ Iamide I) increases along the anion series ClO4− ≈ NO3− < Ac− in the dilute solutions and in the crowded environments. It has been reported that the intensity ratio of Iamide II′/Iamide I is an indicator of the compactness of the conformation of the

Figure 7. Salt concentration dependence of cloud point temperature (Tcp) of ovalbumin as a function of PEG concentration (CPEG) in the presence of different cations with Cl− as the common anion. Here, the ovalbumin concentration is fixed at 1.0 mg mL−1, and the pH of the protein solutions is fixed at ∼7.1. (a) NaCl, (b) CsCl, and (c) N(CH3)4Cl.

The specific anion effects in crowded environments become more obvious as the salt concentration increases from 0.2 to 1.0 M, at the same extent of crowding. This may be because the ion-specific interactions between the ions and the protein molecules are strengthened with increasing salt concentraE

DOI: 10.1021/acs.langmuir.8b00294 Langmuir XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Illustration of the Specific Ion Effects on the Thermal Aggregation of Ovalbumin in Dilute Solutions and Crowded Environments

the fluorescence intensities for the Ac− are slightly lower than those for the ClO4− at the high extent of crowding. This interesting phenomenon becomes more obvious as the salt concentration increases from 0.5 to 1.0 M. The above results imply that the salting-out effect generated by the Ac− anion, in the low extent of crowding, renders the conformation of ovalbumin more compact in comparison with the ClO4− anion. On the other hand, the more extended conformation of the ovalbumin molecules in the presence of ClO4− would undergo a stronger volume exclusion effect than that in the presence of Ac− in the highly crowded environments.55 Therefore, the local protein conformation around the tryptophan may become more compact, though the global protein conformation becomes more extended in the presence of the ClO4− compared with the Ac− in the highly crowded environments. This may be why the fluorescence intensities for the ClO4− are higher than those for the Ac− in highly crowded environments, particularly at high salt concentrations. The combination of ATR-FTIR and fluorescence results suggests that the specific anion effects on the thermal aggregation of ovalbumin in crowded environments are correlated with the global conformational changes of the protein molecules rather than variations in the local conformation within the protein molecules. Figure 4 shows the salt concentration dependence of Tcp of ovalbumin as a function of CPEG for the different anions. For the case of Ac−, the Tcp decreases with increasing NaAc concentration (CNaAc) in dilute solutions, indicating that the addition of the kosmotropic Ac− anion facilitates the thermal aggregation of ovalbumin molecules in the absence of PEG (Figure 4a). As the hydrophobic interactions between the protein molecules could be strengthened with the addition of kosmotropic anions, this experimental fact further suggests that the rate-limiting step of the thermal aggregation of ovalbumin is the aggregation of the unfolded ovalbumin molecules through hydrophobic interactions in dilute solutions.30,31 As CPEG increases from 0 to 200 mg mL−1, the salt concentration dependence of Tcp is gradually varied from a downtrend to an uptrend with increasing CNaAc. That is the addition of the Ac− anion suppresses the thermal aggregation of ovalbumin in highly crowded environments. Because the addition of the Ac− anion stabilizes the native ovalbumin conformation, the result that the Tcp increases with increasing CNaAc suggests that the rate-limiting step of the thermal aggregation of ovalbumin in

protein, when the protein does not undergo secondary structure changes.48−50 The more compact the conformation of the protein the higher the ratio of Iamide II′/Iamide I. This indicates that the ovalbumin molecules adopt a more compact conformation in the presence of the kosmotropic Ac− anion compared with the chaotropic NO3− and ClO4− anions, regardless of the solution conditions. Consequently, the inversion of the specific anion trend on the thermal aggregation of ovalbumin between dilute solutions and crowded environments is not related to the anion-specific conformation of the ovalbumin molecules. Instead, the switching of the rate-limiting reaction step of the ovalbumin thermal aggregation from the aggregation of unfolded protein molecules to the unfolding of protein molecules triggered by the PEG crowding determines the inversion of the specific anion trend. To further explore the relationship between the thermal aggregation of ovalbumin and the conformation of the ovalbumin molecules, we have measured the fluorescence spectra of ovalbumin molecules in the presence of different types of anions in dilute solutions and crowded environments (Figure S3). The fluorescence of the ovalbumin intrinsic tryptophan is dynamically quenched (Figure S4). The quenching is closely related to the hydrophobicity of the microenvironment around the tryptophan.51,52 An increase in fluorescence intensity is an indication that the local environment near the tryptophan in the interior of the protein molecules is more hydrophobic. 51,53 In Figure 3, the fluorescence intensity increases with increased crowding irrespective of the anion identity and salt concentration, indicating that the microenvironment around the tryptophan becomes more hydrophobic as the extent of crowding increases. This is understandable because the ovalbumin molecules adopt a more compact global conformation associated with an increase in the compactness of the hydrophobic core induced by crowding. At the same extent of crowding, the fluorescence intensity of the ovalbumin molecules in the presence of NO3− is much lower than those for the ClO4− and Ac− at all the salt concentrations studied due to the strong quenching effect of the NO3− anion.54 At the salt concentration of 0.2 M, the fluorescence intensities of the ovalbumin molecules in the different crowded environments for Ac− are similar to those for the ClO4−. As the salt concentration increases from 0.2 to 0.5 M, the fluorescence intensities for the Ac− are slightly higher than those for the ClO4− at the low extent of crowding, while F

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crowding. This can be understood on the basis of the same mechanism that controls the anion specificities. Irrespective of the cation identity and salt concentration, the fluorescence intensity increases with increasing CPEG, indicating that the microenvironment around the tryptophan becomes more hydrophobic with increasing crowding (see Figure 6). The fluorescence intensities of the ovalbumin in the presence of Cs+ are lower than those for Na+ and N(CH3)4+ owing to the quenching effect of the Cs+ ion.57 The fluorescence intensities of the ovalbumin for Na+ are similar to those for N(CH3)4+ at the salt concentration of 0.2 M. As the salt concentration increases from 0.2 to 0.5 M, the fluorescence intensities of ovalbumin in the presence of N(CH3)4+ are slightly lower than those in the presence of Na+ at the same extent of crowding, and the fluorescence intensities for the former are obviously lower than those for the latter as the salt concentration increases to 0.8 and 1.0 M. This suggests that in crowded environments the addition of N(CH3)4+ ion makes the conformation of the ovalbumin molecules more extended through the salting-in effect in comparison to Na+. A similar conclusion can be obtained from the ATR-FTIR spectra (Figure S6). Thus, the specific cation effects on the thermal aggregation of ovalbumin in crowded environments are dominated by the cation-specific unfolding of the protein conformation. Note that the ClO4− may more effectively induce the unfolding of the ovalbumin conformation than the N(CH3)4+ (Figure S7). Therefore, the local protein conformation around the tryptophan may become more compact in the presence of ClO4− because the more extended global conformation of the ovalbumin would undergo a stronger volume exclusion effect in the highly crowded environments.55 This may be why the fluorescence intensities for the ClO4− are higher than those for the Ac− in the highly crowded environments, but the fluorescence intensities for the N(CH3)4+ are lower than those for the Na+ in the highly crowded environments. Figure 7 shows the salt concentration dependence of Tcp of ovalbumin as a function of CPEG for the different cations. In dilute solutions, the Tcp for Na+ remains almost unchanged with increasing NaCl concentration (CNaCl), suggesting that the addition of Na+ does not affect the aggregation of ovalbumin molecules (Figure 7a). In highly crowded environments, the measured increase in Tcp with increasing CNaCl is an indication that the thermal aggregation of ovalbumin is suppressed by the addition of Na+. In Figure 7b, the Tcp remains almost constant with increasing CsCl concentration (CCsCl) in all the solution conditions, indicating that the addition of Cs+ does not strongly affect the thermal aggregation of ovalbumin in either dilute solutions or crowded environments. In Figure 7c, the constancy of Tcp with increasing concentration of N(CH3)4Cl (CN(CH3)4Cl) in dilute solutions shows that the addition of N(CH3)4+ has no obvious effect on the aggregation of ovalbumin in dilute solutions. However, the Tcp exhibits a gradual decrease with increasing CN(CH3)4Cl in crowded environments. As increasing the N(CH3)4+ concentration would facilitate the unfolding of the protein through the salting-in effect, the decrease in Tcp with the addition of N(CH3)4+ indicates that the thermal aggregation of ovalbumin is dominated by protein unfolding in crowded environments. Therefore, both the anion and cation specificities demonstrated in this work suggest that the specific ion effects on the thermal aggregation of ovalbumin are dominated by the ion-specific hydrophobic interactions

crowded environments is the unfolding of the ovalbumin molecules. In Figure 4b, the Tcp is almost independent of the concentration of NaNO3 (CNaNO3) in both dilute solutions and crowded environments, suggesting that the addition of the NO3− anion does not strongly affect the thermal aggregation of ovalbumin through the modulation of either protein conformation or hydrophobic interactions between the protein molecules. In Figure 4c, the Tcp remains almost constant with changes in concentration of NaClO4 (CNaClO4) in the PEG-free solution, implying that the addition of ClO4− anion has no obvious influence on the aggregation of ovalbumin molecules in dilute solutions. Nevertheless, Tcp gradually decreases with increasing CNaClO4 under crowding by PEG. As the unfolding of the protein molecules could be facilitated by the addition of the ClO4− anion through the salting-in effect, the lower Tcp at the higher ClO4− concentration is an indication that the unfolding of ovalbumin promotes the thermal aggregation of ovalbumin. This result further suggests that the rate-limiting step is switched from the aggregation of unfolded ovalbumin molecules to the unfolding of the ovalbumin molecules with the change of solution conditions from a dilute solution to a crowded environment. Figure 5 shows the Tcp of ovalbumin, obtained from the turbidity measurements, as a function of CPEG in the presence of different types of cations. Three types of cations, i.e., Na+, Cs+, and N(CH3)4+, are employed to investigate the specific cation effects on the thermal aggregation of ovalbumin. The kosmotropic cations (e.g., Li+) have not been employed here because the strong cation−protein interactions lead to precipitation of the negatively charged ovalbumin molecules (Figure S5). The Tcp of ovalbumin decreases with increasing C PEG , regardless of the cation identity and the salt concentration, which is due to the same mechanisms operating in the presence of anions. Overall, the strength of the specific cation effects on the thermal aggregation of ovalbumin is weaker than that of the specific anion effects. No obvious specific cation effects on the thermal aggregation of ovalbumin can be observed in dilute solutions at all the salt concentrations studied. However, specific cation effects can be observed in crowded environments. Here, the Tcp decreases following the cation series Na+ > Cs+ > N(CH3)4+ for the same extent of crowding at all the salt concentrations studied. Thus, the more chaotropic cation can more effectively promote the aggregation of ovalbumin molecules in crowded environments. The unfolding of proteins by cations through the salting-in effect should increase following the cation series Na+ < Cs+ < N(CH3)4+.24,25 Meanwhile, ovalbumin is negatively charged; thus, the strength of interactions between the carboxylate groups in the protein and the cations should increase following the cation series N(CH3)4+ < Cs+ < Na+ according to the law of matching water affinities.56 As a consequence, the stability of the ovalbumin conformation should increase along the series N(CH3)4+ < Cs+ < Na+. Therefore, the results shown in Figure 5 confirm that the rate-limiting reaction step of the thermal aggregation of ovalbumin in crowded environments is the unfolding of the protein conformation, and the specific cation effects on the thermal aggregation of ovalbumin in crowded environments are dominated by the cation-specific unfolding of the protein molecules. Moreover, the cation specificities of the thermal aggregation of ovalbumin can be amplified by an increase in either the salt concentration or the extent of G

DOI: 10.1021/acs.langmuir.8b00294 Langmuir XXXX, XXX, XXX−XXX

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between protein molecules and the ion-specific unfolding of proteins in dilute solutions and crowded environments, respectively (Scheme 1).

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CONCLUSION In this work, we have investigated specific ion effects on the thermal aggregation of ovalbumin in both dilute solutions and crowded environments. The rate-limiting step of the thermal aggregation of ovalbumin changes from the aggregation of unfolded ovalbumin to the unfolding of ovalbumin as the solution conditions switch from a dilute solution to a crowded environment. The kosmotropic anions promote the thermal aggregation of ovalbumin in dilute solutions via strengthening of the hydrophobic interactions between the protein molecules, whereas both the chaotropic anions and the chaotropic cations facilitate the thermal aggregation of ovalbumin in crowded environments, by promoting protein unfolding. Therefore, the specific ion effects on the thermal aggregation of ovalbumin are dominated by the ion-specific hydrophobic interactions between the protein molecules and the ion-specific unfolding of the protein conformation in dilute solutions and crowded environments, respectively.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b00294. Measured temperature dependence transmittance, the fluorescence spectra, and the FTIR spectra of ovalbumin (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (G.L.). ORCID

Shuji Ye: 0000-0002-3286-5258 Guangming Liu: 0000-0003-2455-1395 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of the National Natural Science Foundation of China (21574121, 21622405, 21374110, and 21473177), the Youth Innovation Promotion Association of CAS (2013290), and the Fundamental Research Funds for the Central Universities (WK2340000066 and WK2340000064) is acknowledged. This work was partially carried out at the USTC Center for Micro and Nanoscale Research and Fabrication.

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REFERENCES

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DOI: 10.1021/acs.langmuir.8b00294 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.8b00294 Langmuir XXXX, XXX, XXX−XXX