Mechanic Insight into Aggregation of Lysozyme by

Dec 3, 2015 - Differential Scanning Calorimetry and Sedimentation Velocity. Sha Wu,. † ... dilute solution, no aggregation but unfolding happens in ...
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Mechanic Insight into Aggregation of Lysozyme by Ultrasensitive Differential Scanning Calorimetry and Sedimentation Velocity Sha Wu,† Yanwei Ding,*,† and Guangzhao Zhang*,†,‡ †

Hefei National Laboratory for Physical Sciences at the Microscale, Department of Chemical Physics, University of Science and Technology of China, Hefei, 230026, China ‡ Faculty of Materials Science and Engineering, South China University of Technology, Guangzhou, P. R. China 510640 S Supporting Information *

ABSTRACT: Folding and aggregation of proteins profoundly influence their functions. We have investigated the effects of thermal history, concentration and pH on the denaturation and refolding of lysozyme by using ultrasensitive differential scanning calorimetry (US-DSC) and sedimentation velocity (SV) via analytical ultracentrifugation (AUC). The former is sensitive to small energy change whereas the latter can differentiate the oligomers such as dimer and trimer from individual protein molecules. Our studies reveal that the degree of denaturation irreversibility increases as heating times increases. The denaturation temperature (Td) and enthalpy change (ΔH) are influenced by heating rate since the denaturation is not in equilibrium during the heating. We can obtain Td and ΔH in equilibrium by extrapolation of heating rate to zero. In a dilute solution, no aggregation but unfolding happens in the denaturation. However, when the concentration is above a critical value (∼15.0 mg/mL), lysozyme molecules readily form trimers or other oligomers. Lysozyme molecules unfold into stretched chains at pH > 6.0, which would further forms large aggregates. The formation of aggregates makes the refolding of lysozyme impossible.



INTRODUCTION Protein folding is one of the most important fundamental issues in life science. Proteins fold into a defined tertiary structure via a specific pathway to execute their biological function. If they mis-fold, they would form aggregates leading to some diseases.1−3 So far, it is still a challenge to study the kinetics of protein folding in vivo. One has to do it in vitro to obtain useful information. As a typical globular protein, lysozyme is often used for such a purpose.4,5 Lysozyme would denature upon acid,6 heating,7−9 or denaturants,10−12 that is, its conformation changes from folded state to unfolded state. Conversely, it may refold from an unfolded state. Whether it can refold depends on the structure in the unfolded state, which is profoundly influenced by the denaturation way.13 Surprisingly, a recent report shows that even tangled aggregates and misfolded lysozyme can refold into native state under shear stress.14 Privalov and co-workers15−17 investigated the pH effect on thermal denaturation of lysozyme with high sensitivity differential scanning calorimetry and revealed a reversible transition between the thermodynamic states. Their studies also show that the denaturation by guanidine hydrochloride (denaturant) is thermodynamically indistinguishable from that induced by heating, but they are quite different in kinetics. Besides, ionic strength also significantly influences the denaturation and refolding.18 Regarding the denaturation and refolding mechanism, some studies suggest that the thermal denaturation of lysozyme © 2015 American Chemical Society

undergoes two processes with an intermediate state where the unfolded lysozymes with various conformations coexist.19,20 Small-angle X-ray scattering (SAXS) studies reveal that the tertiary structure undergoes a two-state transition whereas the intramolecular structure has a multistate transition. The former is accompanied by a large heat absorption and the latter is associated with a small change of free energy.21,22 On the other hand, some studies show that lysozyme unfolds into β-sheets upon heating, which may interact with each other to form aggregates.23,24 Such aggregates are generally inactive proteins with modified or toxic functionality.23 However, whether the proteins form dimers, trimers or large aggregates and how the aggregation influences their refolding remain unknown. In the present work we have investigated the effects of thermal history, heating rate, pH and lysozyme concentration on its denaturation and refolding by using ultrasensitive differential scanning calorimetric (US-DSC) that is sensitive to small energy change. Meanwhile, we examined the species in denaturation by using sedimentation velocity (SV) in analytical ultracentrifugation (AUC), which can differentiate protein unimers from their oligomers such as dimers, trimers and tetramers. We attempt to understand the mechanism of protein aggregation. Received: August 22, 2015 Revised: December 1, 2015 Published: December 3, 2015 15789

DOI: 10.1021/acs.jpcb.5b08190 J. Phys. Chem. B 2015, 119, 15789−15795

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The Journal of Physical Chemistry B



EXPERIMENTAL SECTION Materials. Hen egg white lysozyme (99%) from Fluka and Micrococcus lysodeikticus cells, bromophenol blue, Coomassie Brilliant Blue R-250, protein marker from Sangon Biological Engineering (Shanghai) were used as received. Sodium chloride (NaCl), phosphoric acid, sodium dihydrogen phosphate, sodium hydrogen phosphate, acetic acid, sodium acetate, methyl green, acrylamide and ammonium persulfate were from Sinopharm. Bis-acrylamide and tetramethylethylenediamine were from Aladdin. Milli-Q water with a resistivity of 18.2 MΩ·cm purified by filtration through a Millipore Gradient system after distillation was used in all experiments. Solution Preparation. Buffer solution with different pH values were prepared by using different salts. Solutions with pH 2.0 and 3.0 were prepared with phosphoric acid and sodium dihydrogen phosphate. Acetic acid and sodium acetate were used to prepare solutions with pH 4.0 and 5.0. Sodium dihydrogen phosphate and sodium hydrogen phosphate were for solutions with pH 6.2 and pH 7.2. Each buffer solution had a pH below the isoelectric point (pI) of lysozyme. The concentration of the buffer solution was 0.02 M. The ionic strength (I) of the buffer adjusted by addition of NaCl was kept at 0.15 M so that the net charges on lysozyme were completely screened. The protein solution was prepared by dissolving a certain amount of lysozyme in buffer solution. The lysozyme solutions were stored in a refrigerator at 277 K for 24 h before use. US-DSC Measurements. Lysozyme solution was measured on a VP-DSC microcalorimeter from Microcal Inc. with the corresponding buffer solution as the reference over the temperature range from 10 to 95 °C. Each solution was first degassed by stirring under vacuum at 10.0 °C for 30 min before scanning and then equilibrated at 10.0 °C for 120 min before heating. Our preliminary experiments demonstrate that such stirring does not lead lysozyme to denature. The measurements were performed in triplicate and the data were analyzed using Microcal Origin software package. The raw thermograms were corrected by subtraction of the baseline signals and normalization for concentration and heating rate. Cubic baselines were generated and subtracted from the data prior to determining the denaturation temperature (Td) and the enthalpy change (ΔH). Td was taken as that corresponding to the maximum apparent partial heat capacity (Cp) during the transition. ΔH was estimated from the area under each peak. The excess heat capacity DSC curves were obtained after the above baseline subtraction, concentration and heating rate normalization and the cubic baseline correction. SV Measurements. A Proteomelab XL-A analytical ultracentrifuge (Beckman Coulter Instruments) was used to perform sedimentation velocity (SV) experiments. The detector used was UV−vis absorption optics which could monitor the time-dependent radial concentration of lysozyme. Three cells equipped with two-sector, charcoal-filled Epon centerpiece having quartz windows with a volume of 410 μL of buffer solution as the reference and 400 μL of lysozyme in corresponding buffer solution as the sample, and a counterbalance were loaded into an An-60 Ti 4-hole rotor. Before the angular velocity was increased to the final rotational speed of 58,000 rpm, lysozyme solution was thermostated at 20 °C and 0 rpm for at least 2 h. For each solution, the wavelength was chosen to ensure the absorbance was always lower than unit, which obeys Lambert−Beer’s law. For each lysozyme sample,

about 200 scans of data were obtained at a time interval of 3 min. The SEDFIT software developed by Schuck25 was used for data analysis. The sedimentation coefficient (s), diffusion coefficient (D) and molar mass (Mw) were fit by using the continuous c(s) distribution model with maximum entropy regularization which followed the CONTIN method provided by Provencher.26,27 In the present study, the confidence level was set to p = 0.95. For linear macromolecules, a distorted diffusion coefficient distribution is obtained because the continuous c(s) distribution model is strictly only valid for rigid spheres.28 The sedimentation coefficient is defined as s = u/ω2r where u is the sedimentation velocity of the solute. Assuming that all species in solution have the same weightaverage frictional ratio, their sedimentation and diffusion can be described by Svedberg equation and Stokes−Einstein equation: M=

D=

skBNAT D(1 − νρ ̅ s)

(1)

kBT kT = B f 6πηR h

(2)

where M, kB, NA, T, ν̅, ρs, f, η, and and Rh are the molar mass, Boltzmann constant, Avogadro’s number, absolute temperature, partial specific volume of the solute, solvent density, frictional coefficient, solvent viscosity and hydrodynamic radius, respectively. The partial specific volume of lysozyme used here was 0.72 mL/g.29 ρs and η were calculated by using SEDNTERP program.30



RESULTS AND DISCUSSION We first examined the effect of thermal history or heating times on the denaturation and refolding of lysozyme. The first heating was stopped at the temperature (75 °C) corresponding to the maximum of the endothermic peak due to the denaturation, and then the solution was cooled from 75 to 10 °C. After it was equilibrated for 30 min, the second heating started from 10 to 90 °C and then the solution was cooled from 90 to 10 °C. After it was equilibrated for 30 min, the third heating started from 10 to 90 °C. Likewise, the fourth to eighth heating were performed. Figure 1 shows the temperature

Figure 1. Temperature dependence of apparent partial heat capacity (Cp) of lysozyme as a function of heating times, where the heating rate is 1.0 °C/min, C = 3.0 mg/mL, and pH 3.0.

dependence of apparent partial heat capacity (Cp) of lysozyme solution for each heating. US-DSC curves for the first and second heating make little difference, suggesting that the denaturation and refolding are reversible if the heating is 15790

DOI: 10.1021/acs.jpcb.5b08190 J. Phys. Chem. B 2015, 119, 15789−15795

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The Journal of Physical Chemistry B stopped at a temperature below the denaturation temperatures (Td). In other words, most of the unfolded lysozyme molecules can refold into native state. The degree of denaturation estimated from the peak area is ∼50% at T = Td. In other words, the native structure is only partially destroyed at T < Td. The unfolded lysozyme molecules can refold upon cooling if the balance between hydrogen bonds, electrostatic interactions and hydrophobic interactions still holds there, which sustains the protein spatial structure. However, after heating more than twice, the denaturation is no longer reversible at T > Td, where over 50% lysozyme molecules are denatured. This is because the disulfide bond is broken and the spatial structure of the protein is destroyed and more hydrophobic moieties are exposed.31 The balance between the interactions sustaining the spatial structure no longer holds, and the denatured lysozyme cannot recover. We also examined the activity and tertiary structure as a function of heating times by using turbidity decrease of lysozyme substrate and circular dichroism (CD), respectively (see Figure 1s and Figure 2s in the Supporting Information). As the heating times increases, the activity of lysozyme decreases and its tertiary structure is gradually destroyed. The effect of heating times on lysozyme was further investigated by using polyacrylamide gel electrophoresis (PAGE) measurements (Figure 3s). It is known that both the molecular weight and conformation of a protein can influence its migration. As revealed by SV measurements below, lysozyme molecules exist as unimers in the solutions. Thus, the change in the band in PAGE measurement arises from its conformational change. As heating times increases, the migration becomes slower, indicating that the lysozyme molecules become more stretched. All the facts indicate that more times of heating makes the denatuation irreversible. However, the activity and tertiary structure and the band in PAGE for lysozyme molecules almost do not change after the first heating-and-cooling cycle in comparison with the unheated one, further suggesting that the denaturation is reversible when the heating is stopped below Td. Actually, the denaturation is not completely reversible. We will come back to this point later. Figure 1 also shows that the endothermic peak becomes bimodal in the third heating and Td shifts to a lower temperature (∼75 °C). As the heating times increases, the endothermic peak located at about 60 °C becomes stronger, while the endothermic peak located at about 75 °C becomes weaker. After the eighth heating, the endothermic peak height at about 75 °C is only ∼10% of that in the first heating since the heating promotes the partially denatured lysozyme molecules to further unfold. The endothermic peak at higher temperature is an indicative of the refolded lysozyme molecules. The endothermic peak at lower temperature was attributed to the partially denatured lysozyme molecules.32 However, its nature is not clear. We will examine the denaturation of lysozyme by using SV to clarify it below. Figure 2 shows the sedimentation coefficient distribution of lysozyme as a function of heating times. Clearly, lysozyme has a unimodal distribution and the distribution profile slightly varies even after eighth heating, indicating that only lysozyme unimers exist regardless of thermal history. In other words, no aggregation but only unfolding of individual chains happens during the denaturation. On the other hand, as the heating times increases, the sedimentation coefficient decreases. The sedimentation is mainly determined by the hydrodynamic force here in that the charges on lysozyme molecule are screened in the buffer. The unfolded lysozyme is more extended than a

Figure 2. Sedimentation coefficient (s) distribution of lysozyme as a function of heating times at pH 3.0, where C = 3.0 mg/mL.

folded one. As the degree of unfolding increases, the hydrodynamic friction increases, leading the sedimentation coefficient to decrease.33 Note that even after the first heatingand-cooling cycle, the sedimentation coefficient has a small change in comparison with that of the unheated lysozyme. This indicates that the refolding is not completely reversible. In other words, the first heating already leads to a little destruction in the spatial structure of lysozyme, which would evolve as the heating times increases. Accordingly, sedimentation velocity measurement is more sensitive than US-DSC, CD, and PAGE. Because of the effect of heating times, we focus on the events in the first heating-and-cooling cycle when we study the effects of heating rate and lysozyme concentration (C). Figure 3 shows the heating rate dependence of denaturation temperature (Td) and enthalpy change (ΔH) of lysozyme in

Figure 3. Heating rate dependence of denaturation temperature (Td) and the enthalpy change (ΔH) of lysozyme, where C = 3.0 mg/mL and pH 3.0.

the first heating. As the heating rate increases, ΔH and Td linearly increase. This is because the unfolding or denaturation of lysozyme is slower than the heating. The denaturation cannot be completed in the period of scanning time, and less denaturation happens in a faster heating. In other words, slow heating is favorable to the denaturation or unfolding. Similar phenomenon was observed about plant proteins by Grinberg et al.34 15791

DOI: 10.1021/acs.jpcb.5b08190 J. Phys. Chem. B 2015, 119, 15789−15795

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The Journal of Physical Chemistry B The heating rate dependence indicates that the denaturation in the heating process is not in equilibrium but a kinetic process. Anyhow, by extrapolation of heating rate to zero, we can obtain the Td (69.6 °C) and ΔH (426.3 kJ/mol) in equilibrium. We examined the effect of lysozyme concentration. Figure 4 shows that Td increases with lysozyme concentration (C) at

Figure 5. Sedimentation coefficient (s) distribution of lysozyme as a function of concentration at pH 3.0.

mass (∼4.1 × 104 g/mol), we know the aggregates are trimers. The trimers are minority reflected from the peak area. Such small amount of trimers cannot be detected by PAGE measurement. Actually, lysozyme molecules only form low molar mass aggregates or oligomers which do not precipitate in the range ( 5.0. On the other hand, ΔH increases with pH in the range pH < 5.0 in that lysozymes contain more α-helixes at higher pH. ΔH gradually decreases in the range pH > 5.0, that is, the endothermic peak reflecting ΔH becomes weak when pH > 5.0. As discussed below, this is because lysozyme molecules form precipitate in the heating. 15792

DOI: 10.1021/acs.jpcb.5b08190 J. Phys. Chem. B 2015, 119, 15789−15795

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structure of lysozyme is seriously destroyed at higher pH so that the hydrophobic moieties in the interior are exposed. Actually, the lysozyme solution becomes turbid at pH ∼ 6.0 and even precipitates at pH ∼ 7.0 in the second heating, indicating that the unfolded or denatured lysozyme molecules form large aggregates due to hydrophobic interactions. The protein aggregation which is influenced by its conformational stability, colloid stability and solution conditions would lead to the irreversibility of denaturation.42 Klibanov et al. revealed that deamidation of Asn residues, hydrolysis of Asp-X peptide bond, destruction of cysteine residues and formation of incorrect structures are also responsible for the irreversible denaturation of lysozyme.43−45 The present studies clearly demonstrate either aggregation or destruction of the spatial structure of the protein would make the denaturation irreversible. Figure 8 schematically summarizes the effects of heating times, lysozyme concentration and pH on the lysozyme denaturation and aggregation.

Figure 7. pH dependence of sedimentation coefficient (s) distribution of lysozyme, where C = 3.0 mg/mL.

Figure 7 shows the pH dependence of sedimentation coefficient distribution. Before heating, only a unimodal distribution can be observed, indicating the sedimentation of individual lysozyme molecules. After the first heating, a new peak with lower sedimentation coefficient appears at pH ∼ 7.0. Such a peak can be observed at pH ∼ 6.0 in the second heating. However, the peak is absent at pH < 5.0 in either the first or second heating. On the other hand, the sedimentation coefficient increases with pH regardless of the heating. This is understandable because the α-helix content of lysozyme increases as pH increases and it becomes more compact at higher pH.40 Such a conformation allows the hydrodynamic friction to decrease so that the sedimentation coefficient increases. Similar phenomenon was also observed about LeIF-A before.41 To be clear about the nature of the new peak, we examined the lysozyme solution by using PAGE (Figure 4s). Only one band with a molar mass close to that of lysozyme is observed regardless of heating times at pH 7.0, indicating only individual lysozyme molecules in the solution. Thus, the species corresponding to the new peak is attributed to lysozyme with changed conformation. Namely, some lysozyme molecules unfold from globules into extended chains, leading the hydrodynamic friction to increase, so that the extended lysozyme molecules have a smaller s. As discussed above, the unimodal distribution at pH< 5.0 indicates that the spatial structure of lysozyme is slightly destroyed even after the second heating. The biomodal distributions at pH 6.0 and pH 7.0 indicate that only part of lysozyme molecules denature in the heating. Yet, the spatial

Figure 8. Illustration of denaturation and aggregation of lysozyme.



CONCLUSIONS The present study leads to the following conclusions. The heating times, heating rate and lysozyme concentration profoundly influence its denaturation and aggregation. More times of heating can destroy the spatial structure of the protein and make the refolding irreversible. The denaturation is generally not in equilibrium at a certain heating rate during the DSC measurements. We can obtain the denaturation temperature (Td) and enthalpy change (ΔH) in equilibrium by extrapolation of heating rate to zero. Lysozyme molecules would aggregate into oligomers at a high concentration. Lysozyme molecules unfold into extended conformation and form large aggregates at high pH. The aggregation makes the denaturation irreversible.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b08190. 15793

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Results of the lysozyme activity measurements and circular dichroism (CD) and polyacrylamide gel electrophoresis (PAGE) measurements (PDF)

AUTHOR INFORMATION

Corresponding Authors

*(G.Z.) E-mail: [email protected]. *(Y.D.) E-mail: [email protected]. Telephone: +86-55163606123. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The financial support of the National Natural Science Foundation of China (21234003) and the Ministry of Science and Technology of China (2012CB933802) is acknowledged.



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