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Protonation favors aggregation of lysozyme with SDS Javed M. Khan,a Sumit K. Chaturvedi,a Shah K. Rahman,b Mohd. Ishtikhar,a Atiyatul Qadeer,a Ejaz Ahmada and Rizwan H. Khan*a Different proteins have different amino acid sequences as well as conformations, and therefore different propensities to aggregate. Electrostatic interactions have an important role in the aggregation of proteins as revealed by our previous report (J. M. Khan et al., PLoS One, 2012, 7, e29694). In this study, we designed and executed experiments to gain knowledge of the role of charge variations on proteins during the events of protein aggregation with lysozyme as a model protein. To impart positive and negative charges to proteins, we incubated lysozyme at different pH values of below and above the pI (11). Negatively charged SDS was used to ‘antagonize’ positive charges on lysozyme. We examined the effects of pH variations on SDS-induced amyloid fibril formation by lysozyme using methods such as farUV circular dichroism, Rayleigh scattering, turbidity measurements, dye binding assays and dynamic light scattering. We found that sub-micellar concentrations of SDS (0.1 to 0.6 mM) induced amyloid fibril formation by lysozyme in the pH range of 10.0–1.0 and maximum aggregation was observed at pH 1.0.
Received 16th September 2013 Accepted 18th December 2013
The morphology of aggregates was fibrillar in structure, as visualized by transmission electron microscopy. Isothermal titration calorimetry studies demonstrated that fibril formation is exothermic. To the best of our current understanding of the mechanism of aggregation, this study demonstrates the
DOI: 10.1039/c3sm52435c
crucial role of electrostatic interactions during amyloid fibril formation. The model proposed here will
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help in designing molecules that can prevent or reverse the amyloid fibril formation or the aggregation.
Introduction Proteins require correctly folded conformations for their biological function to be normal. Major diseases such as Alzheimer's, Parkinson's, Huntington's, Senile systematic amyloidoses, type II diabetes and many others are caused by the abnormal function of proteins through adoption of an incorrect conformation.1–3 In addition to in vivo studies, much in vitro work has been performed to gain insight into the mechanism of amyloid formation. However, the detailed mechanism of amyloid formation is still under debate.4 Under in vitro conditions amyloid brils are generated by employing harsh conditions such as high temperature, high pressure, acidic or alkaline pH, by the use of cosolvents, metal ions, lipid assemblies and surfactants.5–9 These conditions cause protein molecules to adopt partially folded conformations that facilitate brillation in a relatively short span of time. The characteristic features of amyloid structures are high b-sheet content, brillar morphology, enhanced ThT uorescence intensity and red shi in absorbance by Congo Red binding.10 In this context protein– a
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India. E-mail: [email protected]; [email protected]; Fax: +91 571 2721776; Tel: +91 571 2720388
b
Randall Division of Cell and Molecular Biophysics, King's College London, UK
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surfactant interactions have been widely studied due to their applications in pharmaceutical, chemical, and cosmetic industries.11–13 Anionic surfactants and, in particular SDS, are now widely used for amyloid bril formation under different conditions.14–16 SDS contains a negatively charged head group and a 12 carbon hydrophobic tail for which it is also said to mimic some characteristics of biological membranes. SDS can induce amyloid formation in proteins below its critical micellar concentration (CMC) and has the ability to induce helical conformation in many proteins above its CMC.17,18 The CMC of SDS in distilled water is around 8 mM. In our previous report, we have shown how SDS induces amyloid formation in a number of proteins when subjected to a pH two units below its isoelectric point (pI) and demonstrated that bril formation is determined mainly by electrostatic interactions.19 It is very interesting to investigate the interaction of proteins with detergent micelles to understand the role of such interactions in the bril formation of the proteins. However, the role of SDS (which provides some environments similar to lipid membranes) in the formation of brils is still very complex and questionable. Two types of arguments exist: one contends that membranes favor aggregation and bril formation while the other contends that membranes inhibit the bril formation. So it is very important to resolve the question. In this study we have tried to propose how SDS can possibly perform both the
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activities. The present study is aimed at exploring the response of protein conformation in the presence of SDS, upon varying the pH from alkaline to acidic. We also investigated the role of charge in amyloid induction using several spectroscopic techniques complemented with calorimetry and microscopy. In this study, we took hen egg white lysozyme as a model protein. Researchers have studied lysozyme before, using different methods to understand the mechanism of protein aggregation.20–23 While some groups have shown amyloid induction in lysozyme by SDS under alkaline conditions, others have reported that SDS can also induce bril formation in lysozyme in an acidic environment.24,25 However, thus far possible explanations for such behavior have not been discussed in detail. Hen egg white lysozyme shares 60% homology with its human counterpart, which is involved in hereditary non-neuropathic systemic amyloidosis.26 Hen egg white lysozyme belongs to the a + b class of proteins and contains 129 amino acid residues out of which nine are negatively charged while 17 are positively charged. It consists of two domains. Residues 1–36 and 87–129 belong to an alpha helical domain, and residues 37–86 belong to a b-sheet domain.27 In this study we demonstrate how lysozyme aggregates in the presence of SDS and elucidate the mechanism of amyloid bril formation. The pI of lysozyme is 11.0 which makes it a good choice for studying the effect of pH variation on SDS induced aggregation.
Material and methods Materials Hen egg white lysozyme, thioavin T (ThT), sodium dodecyl sulfate (SDS) and Congo Red (CR) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents used were of analytical grade. Double distilled water was used throughout the study. Methods Protein concentration determination. A stock solution of lysozyme (500 mM) was made in 20 mM Tris–HCl buffer pH 7.4 and its concentration was determined using a UV-Visible spectrophotometer (Perkin Elmer Lambda25) using a molar extinction coefficient (3M) of 37 970 M1 cm1 at 280 nm. The stock of lysozyme was further diluted 33.33 times in the respective buffer for the measurements and the pH of the sample did not change. pH measurements. pH was determined using a Mettler Toledo Seven Easy pH meter (model S20) with an Expert “Pro3 in 1” type electrode having a least count of 0.01 pH unit, with routine calibration with standard buffers. Experiments were performed in the pH range of 13.0–1.0 with the following 20 mM buffers: KCl–NaOH (pH 13.0–11.0); glycine–NaOH (pH 10.0– 9.0); Tris–HCl (pH 8.0–7.0); sodium phosphate (pH 6.0); sodium acetate (pH 5.0–3.0); glycine–HCl (pH 2.0) and KCl–HCl (pH 1.0). All the buffers used in the experiments were ltered through a 0.45 mm Millipore Millex-HV PVDF lter. For turbidity, Rayleigh light scattering (RLS), ThT, CR, circular dichroism (CD) and transmission electron microscopy
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(TEM) experiments, the protein concentration was xed at 15 mM and the SDS concentration at 300.0 mM, but for dynamic light scattering (DLS) experiments the protein concentration was xed at 70 mM and SDS at 1400.0 mM. Before being allowed to interact, lysozyme and SDS were incubated with a desired range of buffers for 12 h. Turbidity measurements. Turbidity measurements were performed on a Perkin Elmer double beam UV-Visible spectrophotometer model Lambda 25 in a cuvette of 1 cm path length. The turbidity of the lysozyme (15 mM) sample incubated at different values of pH in the absence as well as the presence of 300 mM SDS was determined by monitoring the change in absorbance at 350 nm. All the samples were incubated for 12 hours before measurements. RLS measurements. RLS measurements were carried out using a Hitachi F-4500 uorescence spectrouorometer at 25 C in a cuvette of 1 cm path length. Samples were excited at 350 nm and spectra were recorded from 300 to 400 nm. Both the excitation and emission slit widths were set at 5 nm. The lysozyme sample without SDS served as the control. In each case, the lysozyme concentration was xed at 15 mM. All the samples were incubated for 12 hours prior to measurements. The kinetics of the aggregation process was studied in the presence of SDS at different pH using a SHIMADZU RF 5301 PC uorescence spectrophotometer by exciting the samples at 350 nm and recording the light scattering at 350 nm for 15 minutes. The excitation and emission slit widths were set at 1.5 nm. The lysozyme concentration was 1.0 mM and that of SDS was 20 mM for kinetic studies in order to maintain the ratio of protein to SDS at 1 : 20. ThT binding assay. A stock solution of ThT was prepared in double distilled water and ltered with a 0.2 micron Millipore lter. The concentration of ThT was measured using 3M ¼ 36 000 M1 cm1 at 412 nm. The protein samples (15 mM), in the absence as well as the presence of 300 mM SDS, were incubated overnight at different pH values. Post incubation, samples were supplemented with 15 mM of ThT solution, and further incubated for 30 minutes in the dark. The ThT was excited at 440 nm and spectra were recorded from 450 to 600 nm. The excitation and emission slit widths were set at 10 nm. The spectra were subtracted from an appropriate blank. CR binding assay. A stock solution of CR was prepared in double distilled water and ltered for further use. The concentration was determined using 3M ¼ 45 000 M1 cm1 at 498 nm. The protein concentration was xed at 15.0 mM and incubated overnight at different values of pH (12 h). Aliquots of CR (15.0 mM) were mixed with protein (15.0 mM) in the absence and presence of 300 mM SDS at a molar ratio of 1 : 1 and kept for 15 minutes at 25 C. The absorbance spectra (300–900 nm) of the resulting samples were recorded with a UV-Visible spectrophotometer (Perkin Elmer Lambda 25) in a 1 cm path length cuvette. DLS measurements. The change in aggregation behavior of lysozyme at different pH values was determined using DLS techniques. The Rh measurements were taken using a protein concentration of 70 mM at 830 nm on a DynaPro–TC–04
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dynamic light scattering instrument (Protein Solutions, Wyatt Technology, Santa Barbara, CA) equipped with a temperaturecontrolled microsampler. All the solutions were ltered through a 0.22 mm pore sized microlter (Whatman International, Maidstone, UK). The measured hydrodynamic radius (Rh) was the average of 50 measurements taken at 25 C. The mean Rh and polydispersity (Pd) were estimated, on the basis of an autocorrelation analysis of scattered light intensity based on the translational diffusion coefficient, from the Stokes–Einstein equation: Rh ¼
kT C 6phD25 W
where Rh is the hydrodynamic radius, k is the Boltzmann's constant, T is the absolute temperature, h is the viscosity of C water and D25 is the translational diffusion coefficient. All the W samples were incubated overnight prior to measurements. Far-UV CD measurements. The CD measurements were performed on a JASCO spectropolarimeter (J-815) with a thermostatically controlled cell holder attached to a Peltier with a multitech water circulator. The experiments were carried out aer 12 hours of incubation at 25 C and spectra were scanned in the range of 200–250 nm in a cuvette of 0.1 cm path length. Each spectrum was an average of three scans. The spectra were smoothed by the Savitzky–Golay method with a convolution width of 25. The results were expressed as mean residual ellipticity (MRE) in deg cm2 dmol1 which is dened as: MRE ¼ qobs (mdeg)/10 n Cp l where qobs is the CD in millidegree, n is the number of amino acid residues l is the path length of the cell in centimeters and Cp is the molar fraction of protein. The percent secondary structure was calculated by online K2d soware. For all of the CD measurements the lysozyme concentration was invariably 15 mM. TEM. TEM images were taken using a Philips CM-10 transmission electron microscope operating at an accelerating voltage of 200 kV. The amyloid bril formation was assessed by applying 6 mL of lysozyme (15 mM) containing 300 mM SDS on a 200-mesh copper grid (CF 200-Cu, lot no110323) covered by carbon-stabilized formvar lm. Excess uid was removed aer 2 minutes and the grids were then negatively stained with 2% (w/v) uranyl acetate. Images were viewed at 10 000. Before taking the image all the samples were incubated overnight. Isothermal titration calorimetry (ITC). Calorimetric measurements were taken in the range of pH 11.0–1.0. Before running the experiment, all the solutions were ltered and degassed using a Thermovac. The sample cell was lled with 70 mM lysozyme at the desired pH values while the reference cell contained respective buffers. SDS (7 mM) was introduced into a syringe while ensuring the removal of any trapped air bubbles. The titration experiments consisted of 28 injections of 10 mL each of duration 20 s with a 2 s lter period and 180 s spacing between each injection. The stirring speed was 307 rpm. The
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analog input range was 21.25 V and the reference power was set at 20 mcal s1. Control experiments were performed by titrating SDS into the same buffer to obtain the heats of ligand dilution.
Results Turbidity measurements The increase in absorbance of a protein sample at 350 nm is a measure of the turbidity and gives an idea of the extent of aggregation in the solution. In many reports, the change in absorbance at 350 nm was found to be due to the change in the number and size of the protein–surfactant complexes.28,29 Fig. 1A shows the change in the turbidity of lysozyme at different pH values following incubation with or without 300 mM SDS. As can be seen the turbidity of the lysozyme increased in the presence of 300 mM SDS with decreasing pH values and attained a maximum turbidity at pH 1.0. On the other hand, the turbidity of the control samples (1) lysozyme + different pH buffer, (2) 300 mM SDS + different pH buffer was insignicant and more or less similar at all pH values, suggesting that SDS itself was not responsible for any turbidity at any of the pH values used in this study. In Fig. 1B lysozyme (15 mM) was incubated with varying concentrations of SDS as monitored by absorbance at 350 nm, at different pH values. The maximum change in absorbance was observed in the range of 0.1 to 0.6 mM of SDS at pH in the range of 10.0–5.0. But for pH in the range of 4.0–1.0 the maximum turbidity was found in the range of 0.1 to 2.5 mM SDS. This is because of the high level of protonation of lysozyme. Beyond 2.5 mM SDS concentration, the turbidity of the sample markedly reduces, and it can be concluded that SDS has the ability to induce aggregation in lysozyme upto 2.5 mM SDS concentration and that beyond these concentrations it suppresses aggregation. At lower concentrations, the interaction between SDS and lysozyme might be electrostatic but at higher concentrations the hydrophobic interaction was highly favorable.
Fig. 1 (A) Turbidity of the samples was measured by taking the optical density at 350 nm. Lysozyme (15 mM) was incubated in the absence ( ) and the presence of ( ) 300 mM SDS at different pH for 12 h. The turbidity was also checked only in the presence of 300 mM SDS at different pH ( ). The turbidity of lysozyme (15 mM) versus varying concentration of SDS (mM) at pH 11 ( ), pH 10 ( ), pH 7 ( ), pH 5 (+), pH 4 ( ), pH 3 ( ), pH 2 ( ), pH 1 ( ) were also measured (B).
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RLS Light scattering at 350 nm is another important parameter used to determine the extent of aggregation. The increase in scattering at this wavelength is due to a rise in aggregation. The changes in scattering of lysozyme at 350 nm in the absence as well as the presence of 300 mM SDS, at different pH values, are shown in Fig. 2. The results further support the fact that lowering of pH promotes aggregation of lysozyme by SDS. Besides, negative controls (samples at different pH values in the absence of SDS) showed negligible scattering, suggesting that aggregation was not pH dependent, but induced by SDS. Sometimes surfactants make micelles or large particles in a solution on their own and these scatter light during experiments. This can lead to erroneous readouts. This is why before performing the experiments, we ensured that there was no scattering of light by SDS solution up to 300 mM SDS at different pH. Kinetics of aggregate formation The SDS-induced aggregation of lysozyme was also followed as a function of time at different pH values under dened conditions as discussed earlier. The kinetics of lysozyme aggregation in the presence of 20 mM SDS in the pH range of 11.0–1.0 was studied using RLS at 350 nm. To make clear only a few points are shown in Fig. 3. At pH 11.0 in the presence of SDS no scattering was observed with respect to time while below pH 11.0 the scattering of light increased correspondingly with a maximum at pH 1.0 (Fig. 3). The aggregation induced by SDS is without any lag phase. ThT binding assay
Fig. 3 Study of kinetics of lysozyme by RLS at 350 nm at different pH conditions in the presence of 20 mM SDS.
brils.30,31 The ThT (15 mM) uorescence spectra of lysozyme (15 mM) at different pH values, in the absence and presence of 300 mM SDS, are shown in Fig. 4A and B. The binding of ThT to lysozyme in the absence of SDS was quite insignicant throughout the pH range, whereas in the presence of 300 mM SDS, a strong ThT binding was found at all pH values as shown in Fig. 4B. The ThT uorescence intensity at 485 nm was markedly increased in the presence of 300 mM SDS when pH values were lowered from 11.0 to 3.0. A slight decrease in uorescence intensity below pH 3.0 could be attributed to the charge–charge repulsion between positively charged protein as well as ThT. Similar ThT binding pattern was also reported below pH 3.32 We further conrmed whether ThT binding occurs to SDS micelles or SDS induced lysozyme aggregates. For this, we took appropriate controls (300 mM SDS + buffer + 15 mM
We further checked whether the nature of aggregation induced by SDS is brillar or amorphous. For this, we performed ThT binding assays in which SDS-induced aggregated lysozyme was incubated with ThT and the nature of binding was analysed by measuring changes in ThT uorescence intensity. ThT is commonly used as an amyloid detector as it is reported to bind specically to the characteristic b-sheet structure of amyloid
Fig. 2 RLS of lysozyme at 350 nm measured in the absence ( ) and presence ( ) of 300 mM of SDS at different pH. The RLS was also checked only in the presence of 300 mM SDS at different pH ( ). Prior to measurements lysozyme (15 mM) was incubated at different pH in the presence of 300 mM SDS for 12 h.
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Fig. 4 ThT fluorescence spectra of lysozyme (15 mM) in the absence (A) and presence (B) of 300 mM SDS at different pH. ThT fluorescence intensity of lysozyme at 485 nm as a function of pH in the absence ( ), presence ( ) of 300 mM SDS and also measured only in the presence of 300 mM SDS at different pH ( ). Before the ThT experiments the ThT was incubated with aggregated and non-aggregated samples for 30 minutes in the dark.
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ThT). ThT did not show signicant binding to SDS at different pH values, for which data is shown in Fig. 4C. The above results suggest that SDS induces aggregates of brillar structure in lysozyme and aggregation increases with decreases in pH.
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CR binding assay Since ThT also binds to the oligomers of proteins, we performed a CR binding assay to corroborate our hypothesis.33,34 CR is widely used to detect amyloid brils.35 The results from the CR binding assay are shown in Fig. 5A and B. The dye binding to protein in the absence of SDS showed a maximum absorbance at 495 nm. However in the presence of 300 mM SDS, the concentration at which brils are formed, the absorbance maxima were red shied to 505 nm at pH 7.0 and to 509 nm at pH 5.0. The data showing shi at other pH values are shown in Table 1. These results conrmed formation of characteristic brillar species and ruled out possibilities of simple oligomerization. The reason for this shi is that lysozyme becomes more compact, and this allows only hydrophobic interactions to take place with SDS under these pH conditions. Unfortunately, the assay could not be performed below pH 5.0 because CR changes colour under such conditions. DLS measurements DLS is a well exploited technique for monitoring changes in the sizes of aggregates.36 It was used to determine the pH dependent changes in the size of SDS-induced aggregates of lysozyme (Fig. 6, Table 1). It was found that the intensity of light scattered at 90 increased in the presence of 1400 mM SDS at all pH values studied below the pI of lysozyme. This observation further supports the conclusion that protein aggregates are formed. The increase in light scattering was less pronounced when the pH of the sample was close to pI, indicating the inability of SDS to induce aggregation under such conditions (Table 2). In the absence of SDS at pH values from 11.0 to 1.0 the light scattering was more or less the same. However there was an increase in the Rh value of protein under acidic pH which suggests partial unfolding of the molecule. The results from DLS experiments obtained at pH 7.0 and pH 3.0 are shown in Fig. 6 and the rest of the results are in Table 2. The Rh value of protein at pH 7.0 was 2.1 nm while at pH 3.0 it
Fig. 5 The absorbance spectra of lysozyme (15 mM) obtained from CR binding assay at pH 7.0 (A) and pH 5.0 (B) in the absence ( ) and presence ( ) of 300 mM SDS. CR (15 mM) was mixed with aggregated and non-aggregated sample and incubated for 30 minutes in the dark prior to measurements.
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Fig. 6 DLS measurements performed to determine the hydrodynamic radii (Rh) of lysozyme (70 mM) in the absence and presence of 1400 mM SDS at pH 7.0 and pH 3.0 at 25 C after 12 h incubation.
increased to 3.0 nm. Upon incubation with 1400.0 mM SDS, two types of species were generated with a size of 2.8 nm and 54.5 nm at pH 7.0, and 5.3 nm and 154.9 nm at pH 3.0. This observation suggests that the larger aggregates were formed at lower pH. CD measurements The change in the secondary structure of lysozyme (15 mM) was monitored at different pH values, in the absence and presence of 300 mM SDS, using far-UV CD. Lysozyme belongs to the a + b class of proteins and its far-UV CD spectra are characterized by two negative peaks centered around 208 nm and 222 nm.37 As shown in Fig. 7A, in the absence of SDS the spectral features were retained throughout the pH range (11.0–1.0) studied, with some increase in ellipticity at low pH, owing to the formation of some secondary structure elements. When lysozyme was incubated with 300.0 mM SDS, the far-UV CD spectra showed two types of changes. From pH 11.0 to pH 10.0, the protein showed a considerable decrease in ellipticity probably due to loss of structure but the overall spectra remained qualitatively unchanged (data not shown). In contrast, the spectra of proteins below pH 9.0 exhibited a transition from an a-helix to a b-sheet structure characterized by a single negative peak around 219 nm, which is a hallmark of amyloid formation (Fig. 7B). For the sake of clarity we have shown representative spectra at pH 1.0, 3.0, 5.0 and 7.0. The secondary structure content of lysozyme at different pH values was calculated by the K2d method and the results are listed in Table 3. As can be seen, a prominent increase in the b-sheet content of protein, occurring below pH 10.0 in the presence of SDS, clearly suggests that aggregates have a dened bril structure. TEM The morphology of lysozyme aggregates was analyzed by TEM. Prior to taking the TEM images lysozyme (15.0 mM) was Soft Matter, 2014, 10, 2591–2599 | 2595
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Table 1
Paper
Wavelength shift of lysozyme upon binding with CR at different pH
pH
Shi in lmax (nm) of lysozyme (15 mM)
Shi in lmax (nm) of (15 mM) lysozyme + 300 mM SDS
Difference in lmax (nm) shi
11 10 9 8 7 6 5
496 496 498 496 495 495 497
489 493 503 505 505 505 507
7 (blue) 3 (blue) 5 (red) 9 (red) 10 (red) 10 (red) 10 (red)
Table 2
Hydrodynamic radii of lysozyme under different conditions at
25 C Sample no.
Conditions
Hydrodynamic radii (nm)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
pH 11.0 pH 11.0 + 1400 mM SDS pH 10.0 pH 10.0 + SDS pH 9.0 pH 9.0 + 1400 mM SDS pH 8.0 pH 8.0 + 1400 mM SDS pH 7.0 pH 7.0 + 1400 mM SDS pH 6.0 pH 6.0 + 1400 mM SDS pH 5.0 pH 5.0 + 1400 mM SDS pH 4.0 pH 4.0 + 1400 mM SDS pH 3.0 pH 3.0 + 1400 mM SDS pH 2.0 pH 2.0 + 1400 mM SDS pH 1.0 pH 1.0 + 1400 mM SDS
3.0 0.1 2.0 0.09 2.0 0.1 2.0 0.1, 9.0 0.1 2.0 0.1 3.0 0.1, 41.0 0.2 2.0 0.1 3.0 0.1, 49.0 0.1 2.0 0.1 2.0 0.1, 54.0 0.2 2.0 0.1 3.0 0.1, 71.0 0.2 2.0 0.1 3.0 0.1, 79.0 0.3 2.0 0.1 3.0 0.1, 112.0 0.1 3.0 0.1 5.0 0.2, 122.0 0.2 3.0 0.1 5.0 0.2, 144.0 0.3 3.0 0.2 5.0 0.2, 150.0 0.5
values. In the absence of SDS, at a pH below the pI, no bril or aggregates were found (data not shown). Similar types of bril structures were also found in the other proteins as well.38–40 ITC When the ligand binds to a protein either heat is released or absorbed but it is very difficult to understand the binding process, in vivo, i.e., whether it is exothermic or endothermic. Therefore we studied the binding process in vitro using ITC. From the above spectroscopic results we found that lysozyme aggregates in the presence of 300.0 mM SDS, at different pH values. Here we used ITC to examine what type of interactions are taking place between lysozyme and SDS. In ITC experiments we injected 10 mL of 7.0 mM SDS 28 times into the reaction cell lled with 70 mM lysozyme at desired pH values. The ITC isotherms showed an exothermic peak when lysozyme was titrated with SDS pH below 10.0 (Fig. 9). These exothermic peaks reveal binding between SDS and lysozyme to be electrostatic. Similar types of exothermic peaks were also reported in some proteins at pH 7.4.41 In control experiments, the heat released was very low. We further checked whether the interactions between lysozyme and SDS were strong or weak. A less exothermic heat was released at pH 11.0, while below this pH a continuous increase in exothermic heat was observed and maximum heat was released at pH 1.0. The possible explanation for low heat released at pH 11.0 is that lysozyme becomes uncharged therefore SDS is not able to bind to it properly. However at lower pH values, lysozyme is highly protonated favoring binding of the negative head group of SDS and release of more heat.
Discussion Fig. 7 Far-UV CD spectra of lysozyme (15 mM) in the absence (A) and presence of 300 mM SDS (B) at pH 1.0, 3.0, 5.0 and 7.0 at 25 C. Only selected spectra are shown in panel A and B because some spectra are overlapping. Prior to CD measurements the entire sample was incubated for 12 h.
incubated with 300.0 mM of SDS at a pH below the pI for 12 hours incubation. Fibril formation was observed at all the pH values studied, below the pI. However, only four representative images are shown (Fig. 8). Mature brils were observed at all the pH
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Polypeptides and proteins have the generic property of forming amyloid brils, with the tendency originating in different parts of each protein.42 Here, lysozyme was incubated at different pH values ranging from 13.0–1.0. The positive charge starts to increase at a lysozyme pH below 11.0 and a maximum positive charge exists at pH 1.0. We observed that as the pH of lysozyme was lowered below the pI in the presence of SDS, the protein began to aggregate as conrmed by turbidity, light scattering, DLS and far-UV CD measurements. These results may be viewed together with the results of Wang et al. (2011) in which, lysozyme incubated with two phospholipids, D6PC and D7PC, showed enhanced light scattering due to the formation of larger
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Paper Table 3
Soft Matter % Secondary structure calculated by K2d method at different conditions
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Lysozyme (15 mM)
(15 mM) lysozyme + 300 mM SDS
Sample no.
pH
% a Helix
% b Sheet
% Random coil
% a Helix
% b Sheet
% Random coil
1 2 3 4 5 6 7 8 9 10 11
11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0
40.0 44.0 43.0 44.0 44.0 45.0 45.0 45.0 44.0 44.0 42.0
18.0 23.0 22.0 23.0 23.0 23.0 23.0 23.0 23.0 23.0 21.0
42.0 33.0 35.0 33.0 37.0 32.0 31.0 33.0 33.0 33.0 36.0
45.0 45.0 20.0 18.0 13.0 15.0 5.0 5.0 4.0 4.0 4.0
23.0 23.0 45.0 44.0 42.0 35.0 48.0 47.0 47.0 48.0 48.0
31.0 31.0 35.0 38.0 55.0 50.0 48.0 48 48.0 48.0 48.0
Fig. 8 TEM images of lysozyme (15 mM) in the presence of 300 mM SDS at pH 9.0 (A), pH 6.0 (B), pH 3.0 (C) and pH 1.0 (D). TEM images were taken after 12 h incubation.
Fig. 9 Plot of enthalpy change against molar ratio for titration of SDS with lysozyme at pH 11.0 ( ), 9.0 ( ), 7.0 ( ), pH 4.0 ( ), pH 2.0 ( ) and pH 1.0 ( ).
aggregates.12 The kinetics of the aggregation process was measured by RLS experiments; also the aggregation tendency of different conformations of lysozyme at varying pH values in the
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presence of SDS was determined. The rate of aggregation was so fast that no lag phase was observed at any of the pH conditions taken in this study. Similar results were obtained for amyloid formation in ApoMb where exponential growth was observed without any lag phase.43 In this context, it is also reported that amyloid bril formation of N47A Spc-SH3 is a rapid process without any lag phase. In our case, saturation took place within 1200 s, so we conclude that the process was fast and that amyloid nuclei are established early.44 We used the ThT binding assay to investigate whether aggregation was amorphous or brillar. We observed a continuous increase in ThT uorescence intensity in the presence of 300 mM SDS at pH below the pI of lysozyme suggesting that aggregates formed were amyloid brils. A slight decrease in ThT uorescence intensity at pH 1.0 and 2.0 could be attributed to the fact that at acidic pH the positively charge N-atom of a benzothiazole group of ThT repels the N-atom of arginine and lysine of lysozyme that may not allow strong binding to the amyloid bril due to charge–charge repulsion.32 This notion is also supported by the observation that ThT binding to insulin laments is greater in distilled water than at pH 1.6.45 We further reconrmed bril formation of our protein by the CR dye binding assay. CR binds strongly to the parallel and antiparallel b-sheet region of the amyloid crossb structures.46 The CR binding assay was performed to rule out the doubts of binding occurring to some non-amyloid structures. A noticeable red shi was observed at acidic pH indicating strong CR binding to protein corresponding to amyloid formation. These two dye binding experiments conrmed that aggregates of lysozyme were formed at all pH values below its pI and have well-ordered structures. Further, we followed changes in the secondary structure of lysozyme in the presence of SDS at different pH. Generally amyloid brils have ordered b-sheet structures.47,48 Lysozyme contains both a-helix and b-sheet structures at neutral pH. The a-helical content of lysozyme is converted into a b-sheet aer incubation with SDS at a pH below the pI. Due to this secondary structure transition we conclude that the aggregate induced by SDS has an ordered b-sheet structure which could be the case in amyloid brils. To conrm this further, we also employed TEM to observe the morphology of the aggregates. The TEM images
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revealed that the aggregates formed by SDS were typically brillar. It can be concluded, therefore, that lysozyme exhibits a strong propensity to form amyloids in the presence of SDS at any pH below its pI and the size of the aggregates increases with the increase in positive charge of the protein (as the pH moves down from the pI). In Fig. 10, we have proposed a hypothetical model that explains how SDS induces aggregation in lysozyme. In our previous study, we discussed how SDS induces aggregation in proteins at a pH below the pI. Since the pI of lysozyme is 11.0, below this pH the polypeptide chain will acquire a net positive charge. The net charge of lysozyme from pH 11.0–1.0 was calculated using PROTEIN CALCULATOR v3.3 soware (Table 4). As the pH of the protein samples decreases below 10.0, the net positive charge of the protein keeps increasing due to protonation of carboxylate (–COOH) and amino (–NH3+) groups of amino acid side chains. The negatively charged head group of SDS would be expected to neutralize more and more positively charged centers on the protein. Therefore, the aggregating propensity as well as the size of aggregate increases accordingly. On the other hand, at a pH above 10.0, the lysozyme develops a net negative charge on its surface, and this is expected not to bind to negatively charged SDS due to charge–charge repulsions. Ultimately, above pH 10.0, therefore, no aggregation is observed.
Conclusions From this study it can be concluded that SDS induces aggregation in lysozyme at values of pH below 11.0 and that the aggregation propensity is at a maximum at pH 1.0 because the net positive charge increase of lysozyme upon lowering of the pH is highest at this pH, allowing SDS to interact electrostatically with the oppositely charged surface of lysozyme, leading to the assembly of protein into characteristic amyloid brils.
Paper Table 4 Estimated charge over pH range by “PROTEIN CALCULATOR
v3.3” software Sample no.
pH
Total charge
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
19.0 18.9 18.2 15.5 10.8 8.9 7.9 5.5 4.9 0.2
Notes The authors declare no competing interest.
Author contributions Conceived and designed the experiments: JMK and RHK. Performed the experiments: JMK, SKC and MI. Performed TEM experiments: JMK and SKR. Analyzed the data: JMK and RHK. Contributed reagents/materials/analysis tools: JMK, EA, AQ and RHK. Paper was checked by JMK, SKC, SKR and RHK. Wrote the paper: JMK and RHK.
Acknowledgements The facilities provided by I.B.U, Aligarh Muslim University are gratefully acknowledged. We are also grateful to AIRF, AIIMS for TEM images. J.M.K and S.K.C are highly thankful to the Council of Scientic and Industrial Research, New Delhi, for nancial
Fig. 10 Schematic representation of the effect of SDS on the conformation of lysozyme below and above the pI of lysozyme.
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assistance in the form of senior research fellowships (SRF). We are thankful to DBT (BT/PR 13194/10/742/2009) and CSIR (37(1456)/10/EMR-II) for nancial support. M.I is highly thankful to the Indian Council of Medical Research for nancial assistance in the form of a senior research fellowship (SRF) New Delhi. We would like to thank Prof. P. Guptasarma from IISER Mohali and Paul Brown at the Randall Division of Cell and Molecular Biophysics, King's College London for Englishlanguage and technical editing of the manuscript.
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Protonation favors aggregation of lysozyme with SDS Javed M. Khan,a Sumit K. Chaturvedi,a Shah K. Rahman,b Mohd. Ishtikhar,a Atiyatul Qadeer,a Ejaz Ahmada and Rizwan H. Khan*a Different proteins have different amino acid sequences as well as conformations, and therefore different propensities to aggregate. Electrostatic interactions have an important role in the aggregation of proteins as revealed by our previous report (J. M. Khan et al., PLoS One, 2012, 7, e29694). In this study, we designed and executed experiments to gain knowledge of the role of charge variations on proteins during the events of protein aggregation with lysozyme as a model protein. To impart positive and negative charges to proteins, we incubated lysozyme at different pH values of below and above the pI (11). Negatively charged SDS was used to ‘antagonize’ positive charges on lysozyme. We examined the effects of pH variations on SDS-induced amyloid fibril formation by lysozyme using methods such as farUV circular dichroism, Rayleigh scattering, turbidity measurements, dye binding assays and dynamic light scattering. We found that sub-micellar concentrations of SDS (0.1 to 0.6 mM) induced amyloid fibril formation by lysozyme in the pH range of 10.0–1.0 and maximum aggregation was observed at pH 1.0.
Received 16th September 2013 Accepted 18th December 2013
The morphology of aggregates was fibrillar in structure, as visualized by transmission electron microscopy. Isothermal titration calorimetry studies demonstrated that fibril formation is exothermic. To the best of our current understanding of the mechanism of aggregation, this study demonstrates the
DOI: 10.1039/c3sm52435c
crucial role of electrostatic interactions during amyloid fibril formation. The model proposed here will
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help in designing molecules that can prevent or reverse the amyloid fibril formation or the aggregation.
Introduction Proteins require correctly folded conformations for their biological function to be normal. Major diseases such as Alzheimer's, Parkinson's, Huntington's, Senile systematic amyloidoses, type II diabetes and many others are caused by the abnormal function of proteins through adoption of an incorrect conformation.1–3 In addition to in vivo studies, much in vitro work has been performed to gain insight into the mechanism of amyloid formation. However, the detailed mechanism of amyloid formation is still under debate.4 Under in vitro conditions amyloid brils are generated by employing harsh conditions such as high temperature, high pressure, acidic or alkaline pH, by the use of cosolvents, metal ions, lipid assemblies and surfactants.5–9 These conditions cause protein molecules to adopt partially folded conformations that facilitate brillation in a relatively short span of time. The characteristic features of amyloid structures are high b-sheet content, brillar morphology, enhanced ThT uorescence intensity and red shi in absorbance by Congo Red binding.10 In this context protein– a
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India. E-mail: [email protected]; [email protected]; Fax: +91 571 2721776; Tel: +91 571 2720388
b
Randall Division of Cell and Molecular Biophysics, King's College London, UK
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surfactant interactions have been widely studied due to their applications in pharmaceutical, chemical, and cosmetic industries.11–13 Anionic surfactants and, in particular SDS, are now widely used for amyloid bril formation under different conditions.14–16 SDS contains a negatively charged head group and a 12 carbon hydrophobic tail for which it is also said to mimic some characteristics of biological membranes. SDS can induce amyloid formation in proteins below its critical micellar concentration (CMC) and has the ability to induce helical conformation in many proteins above its CMC.17,18 The CMC of SDS in distilled water is around 8 mM. In our previous report, we have shown how SDS induces amyloid formation in a number of proteins when subjected to a pH two units below its isoelectric point (pI) and demonstrated that bril formation is determined mainly by electrostatic interactions.19 It is very interesting to investigate the interaction of proteins with detergent micelles to understand the role of such interactions in the bril formation of the proteins. However, the role of SDS (which provides some environments similar to lipid membranes) in the formation of brils is still very complex and questionable. Two types of arguments exist: one contends that membranes favor aggregation and bril formation while the other contends that membranes inhibit the bril formation. So it is very important to resolve the question. In this study we have tried to propose how SDS can possibly perform both the
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activities. The present study is aimed at exploring the response of protein conformation in the presence of SDS, upon varying the pH from alkaline to acidic. We also investigated the role of charge in amyloid induction using several spectroscopic techniques complemented with calorimetry and microscopy. In this study, we took hen egg white lysozyme as a model protein. Researchers have studied lysozyme before, using different methods to understand the mechanism of protein aggregation.20–23 While some groups have shown amyloid induction in lysozyme by SDS under alkaline conditions, others have reported that SDS can also induce bril formation in lysozyme in an acidic environment.24,25 However, thus far possible explanations for such behavior have not been discussed in detail. Hen egg white lysozyme shares 60% homology with its human counterpart, which is involved in hereditary non-neuropathic systemic amyloidosis.26 Hen egg white lysozyme belongs to the a + b class of proteins and contains 129 amino acid residues out of which nine are negatively charged while 17 are positively charged. It consists of two domains. Residues 1–36 and 87–129 belong to an alpha helical domain, and residues 37–86 belong to a b-sheet domain.27 In this study we demonstrate how lysozyme aggregates in the presence of SDS and elucidate the mechanism of amyloid bril formation. The pI of lysozyme is 11.0 which makes it a good choice for studying the effect of pH variation on SDS induced aggregation.
Material and methods Materials Hen egg white lysozyme, thioavin T (ThT), sodium dodecyl sulfate (SDS) and Congo Red (CR) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents used were of analytical grade. Double distilled water was used throughout the study. Methods Protein concentration determination. A stock solution of lysozyme (500 mM) was made in 20 mM Tris–HCl buffer pH 7.4 and its concentration was determined using a UV-Visible spectrophotometer (Perkin Elmer Lambda25) using a molar extinction coefficient (3M) of 37 970 M1 cm1 at 280 nm. The stock of lysozyme was further diluted 33.33 times in the respective buffer for the measurements and the pH of the sample did not change. pH measurements. pH was determined using a Mettler Toledo Seven Easy pH meter (model S20) with an Expert “Pro3 in 1” type electrode having a least count of 0.01 pH unit, with routine calibration with standard buffers. Experiments were performed in the pH range of 13.0–1.0 with the following 20 mM buffers: KCl–NaOH (pH 13.0–11.0); glycine–NaOH (pH 10.0– 9.0); Tris–HCl (pH 8.0–7.0); sodium phosphate (pH 6.0); sodium acetate (pH 5.0–3.0); glycine–HCl (pH 2.0) and KCl–HCl (pH 1.0). All the buffers used in the experiments were ltered through a 0.45 mm Millipore Millex-HV PVDF lter. For turbidity, Rayleigh light scattering (RLS), ThT, CR, circular dichroism (CD) and transmission electron microscopy
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(TEM) experiments, the protein concentration was xed at 15 mM and the SDS concentration at 300.0 mM, but for dynamic light scattering (DLS) experiments the protein concentration was xed at 70 mM and SDS at 1400.0 mM. Before being allowed to interact, lysozyme and SDS were incubated with a desired range of buffers for 12 h. Turbidity measurements. Turbidity measurements were performed on a Perkin Elmer double beam UV-Visible spectrophotometer model Lambda 25 in a cuvette of 1 cm path length. The turbidity of the lysozyme (15 mM) sample incubated at different values of pH in the absence as well as the presence of 300 mM SDS was determined by monitoring the change in absorbance at 350 nm. All the samples were incubated for 12 hours before measurements. RLS measurements. RLS measurements were carried out using a Hitachi F-4500 uorescence spectrouorometer at 25 C in a cuvette of 1 cm path length. Samples were excited at 350 nm and spectra were recorded from 300 to 400 nm. Both the excitation and emission slit widths were set at 5 nm. The lysozyme sample without SDS served as the control. In each case, the lysozyme concentration was xed at 15 mM. All the samples were incubated for 12 hours prior to measurements. The kinetics of the aggregation process was studied in the presence of SDS at different pH using a SHIMADZU RF 5301 PC uorescence spectrophotometer by exciting the samples at 350 nm and recording the light scattering at 350 nm for 15 minutes. The excitation and emission slit widths were set at 1.5 nm. The lysozyme concentration was 1.0 mM and that of SDS was 20 mM for kinetic studies in order to maintain the ratio of protein to SDS at 1 : 20. ThT binding assay. A stock solution of ThT was prepared in double distilled water and ltered with a 0.2 micron Millipore lter. The concentration of ThT was measured using 3M ¼ 36 000 M1 cm1 at 412 nm. The protein samples (15 mM), in the absence as well as the presence of 300 mM SDS, were incubated overnight at different pH values. Post incubation, samples were supplemented with 15 mM of ThT solution, and further incubated for 30 minutes in the dark. The ThT was excited at 440 nm and spectra were recorded from 450 to 600 nm. The excitation and emission slit widths were set at 10 nm. The spectra were subtracted from an appropriate blank. CR binding assay. A stock solution of CR was prepared in double distilled water and ltered for further use. The concentration was determined using 3M ¼ 45 000 M1 cm1 at 498 nm. The protein concentration was xed at 15.0 mM and incubated overnight at different values of pH (12 h). Aliquots of CR (15.0 mM) were mixed with protein (15.0 mM) in the absence and presence of 300 mM SDS at a molar ratio of 1 : 1 and kept for 15 minutes at 25 C. The absorbance spectra (300–900 nm) of the resulting samples were recorded with a UV-Visible spectrophotometer (Perkin Elmer Lambda 25) in a 1 cm path length cuvette. DLS measurements. The change in aggregation behavior of lysozyme at different pH values was determined using DLS techniques. The Rh measurements were taken using a protein concentration of 70 mM at 830 nm on a DynaPro–TC–04
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dynamic light scattering instrument (Protein Solutions, Wyatt Technology, Santa Barbara, CA) equipped with a temperaturecontrolled microsampler. All the solutions were ltered through a 0.22 mm pore sized microlter (Whatman International, Maidstone, UK). The measured hydrodynamic radius (Rh) was the average of 50 measurements taken at 25 C. The mean Rh and polydispersity (Pd) were estimated, on the basis of an autocorrelation analysis of scattered light intensity based on the translational diffusion coefficient, from the Stokes–Einstein equation: Rh ¼
kT C 6phD25 W
where Rh is the hydrodynamic radius, k is the Boltzmann's constant, T is the absolute temperature, h is the viscosity of C water and D25 is the translational diffusion coefficient. All the W samples were incubated overnight prior to measurements. Far-UV CD measurements. The CD measurements were performed on a JASCO spectropolarimeter (J-815) with a thermostatically controlled cell holder attached to a Peltier with a multitech water circulator. The experiments were carried out aer 12 hours of incubation at 25 C and spectra were scanned in the range of 200–250 nm in a cuvette of 0.1 cm path length. Each spectrum was an average of three scans. The spectra were smoothed by the Savitzky–Golay method with a convolution width of 25. The results were expressed as mean residual ellipticity (MRE) in deg cm2 dmol1 which is dened as: MRE ¼ qobs (mdeg)/10 n Cp l where qobs is the CD in millidegree, n is the number of amino acid residues l is the path length of the cell in centimeters and Cp is the molar fraction of protein. The percent secondary structure was calculated by online K2d soware. For all of the CD measurements the lysozyme concentration was invariably 15 mM. TEM. TEM images were taken using a Philips CM-10 transmission electron microscope operating at an accelerating voltage of 200 kV. The amyloid bril formation was assessed by applying 6 mL of lysozyme (15 mM) containing 300 mM SDS on a 200-mesh copper grid (CF 200-Cu, lot no110323) covered by carbon-stabilized formvar lm. Excess uid was removed aer 2 minutes and the grids were then negatively stained with 2% (w/v) uranyl acetate. Images were viewed at 10 000. Before taking the image all the samples were incubated overnight. Isothermal titration calorimetry (ITC). Calorimetric measurements were taken in the range of pH 11.0–1.0. Before running the experiment, all the solutions were ltered and degassed using a Thermovac. The sample cell was lled with 70 mM lysozyme at the desired pH values while the reference cell contained respective buffers. SDS (7 mM) was introduced into a syringe while ensuring the removal of any trapped air bubbles. The titration experiments consisted of 28 injections of 10 mL each of duration 20 s with a 2 s lter period and 180 s spacing between each injection. The stirring speed was 307 rpm. The
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analog input range was 21.25 V and the reference power was set at 20 mcal s1. Control experiments were performed by titrating SDS into the same buffer to obtain the heats of ligand dilution.
Results Turbidity measurements The increase in absorbance of a protein sample at 350 nm is a measure of the turbidity and gives an idea of the extent of aggregation in the solution. In many reports, the change in absorbance at 350 nm was found to be due to the change in the number and size of the protein–surfactant complexes.28,29 Fig. 1A shows the change in the turbidity of lysozyme at different pH values following incubation with or without 300 mM SDS. As can be seen the turbidity of the lysozyme increased in the presence of 300 mM SDS with decreasing pH values and attained a maximum turbidity at pH 1.0. On the other hand, the turbidity of the control samples (1) lysozyme + different pH buffer, (2) 300 mM SDS + different pH buffer was insignicant and more or less similar at all pH values, suggesting that SDS itself was not responsible for any turbidity at any of the pH values used in this study. In Fig. 1B lysozyme (15 mM) was incubated with varying concentrations of SDS as monitored by absorbance at 350 nm, at different pH values. The maximum change in absorbance was observed in the range of 0.1 to 0.6 mM of SDS at pH in the range of 10.0–5.0. But for pH in the range of 4.0–1.0 the maximum turbidity was found in the range of 0.1 to 2.5 mM SDS. This is because of the high level of protonation of lysozyme. Beyond 2.5 mM SDS concentration, the turbidity of the sample markedly reduces, and it can be concluded that SDS has the ability to induce aggregation in lysozyme upto 2.5 mM SDS concentration and that beyond these concentrations it suppresses aggregation. At lower concentrations, the interaction between SDS and lysozyme might be electrostatic but at higher concentrations the hydrophobic interaction was highly favorable.
Fig. 1 (A) Turbidity of the samples was measured by taking the optical density at 350 nm. Lysozyme (15 mM) was incubated in the absence ( ) and the presence of ( ) 300 mM SDS at different pH for 12 h. The turbidity was also checked only in the presence of 300 mM SDS at different pH ( ). The turbidity of lysozyme (15 mM) versus varying concentration of SDS (mM) at pH 11 ( ), pH 10 ( ), pH 7 ( ), pH 5 (+), pH 4 ( ), pH 3 ( ), pH 2 ( ), pH 1 ( ) were also measured (B).
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RLS Light scattering at 350 nm is another important parameter used to determine the extent of aggregation. The increase in scattering at this wavelength is due to a rise in aggregation. The changes in scattering of lysozyme at 350 nm in the absence as well as the presence of 300 mM SDS, at different pH values, are shown in Fig. 2. The results further support the fact that lowering of pH promotes aggregation of lysozyme by SDS. Besides, negative controls (samples at different pH values in the absence of SDS) showed negligible scattering, suggesting that aggregation was not pH dependent, but induced by SDS. Sometimes surfactants make micelles or large particles in a solution on their own and these scatter light during experiments. This can lead to erroneous readouts. This is why before performing the experiments, we ensured that there was no scattering of light by SDS solution up to 300 mM SDS at different pH. Kinetics of aggregate formation The SDS-induced aggregation of lysozyme was also followed as a function of time at different pH values under dened conditions as discussed earlier. The kinetics of lysozyme aggregation in the presence of 20 mM SDS in the pH range of 11.0–1.0 was studied using RLS at 350 nm. To make clear only a few points are shown in Fig. 3. At pH 11.0 in the presence of SDS no scattering was observed with respect to time while below pH 11.0 the scattering of light increased correspondingly with a maximum at pH 1.0 (Fig. 3). The aggregation induced by SDS is without any lag phase. ThT binding assay
Fig. 3 Study of kinetics of lysozyme by RLS at 350 nm at different pH conditions in the presence of 20 mM SDS.
brils.30,31 The ThT (15 mM) uorescence spectra of lysozyme (15 mM) at different pH values, in the absence and presence of 300 mM SDS, are shown in Fig. 4A and B. The binding of ThT to lysozyme in the absence of SDS was quite insignicant throughout the pH range, whereas in the presence of 300 mM SDS, a strong ThT binding was found at all pH values as shown in Fig. 4B. The ThT uorescence intensity at 485 nm was markedly increased in the presence of 300 mM SDS when pH values were lowered from 11.0 to 3.0. A slight decrease in uorescence intensity below pH 3.0 could be attributed to the charge–charge repulsion between positively charged protein as well as ThT. Similar ThT binding pattern was also reported below pH 3.32 We further conrmed whether ThT binding occurs to SDS micelles or SDS induced lysozyme aggregates. For this, we took appropriate controls (300 mM SDS + buffer + 15 mM
We further checked whether the nature of aggregation induced by SDS is brillar or amorphous. For this, we performed ThT binding assays in which SDS-induced aggregated lysozyme was incubated with ThT and the nature of binding was analysed by measuring changes in ThT uorescence intensity. ThT is commonly used as an amyloid detector as it is reported to bind specically to the characteristic b-sheet structure of amyloid
Fig. 2 RLS of lysozyme at 350 nm measured in the absence ( ) and presence ( ) of 300 mM of SDS at different pH. The RLS was also checked only in the presence of 300 mM SDS at different pH ( ). Prior to measurements lysozyme (15 mM) was incubated at different pH in the presence of 300 mM SDS for 12 h.
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Fig. 4 ThT fluorescence spectra of lysozyme (15 mM) in the absence (A) and presence (B) of 300 mM SDS at different pH. ThT fluorescence intensity of lysozyme at 485 nm as a function of pH in the absence ( ), presence ( ) of 300 mM SDS and also measured only in the presence of 300 mM SDS at different pH ( ). Before the ThT experiments the ThT was incubated with aggregated and non-aggregated samples for 30 minutes in the dark.
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ThT). ThT did not show signicant binding to SDS at different pH values, for which data is shown in Fig. 4C. The above results suggest that SDS induces aggregates of brillar structure in lysozyme and aggregation increases with decreases in pH.
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CR binding assay Since ThT also binds to the oligomers of proteins, we performed a CR binding assay to corroborate our hypothesis.33,34 CR is widely used to detect amyloid brils.35 The results from the CR binding assay are shown in Fig. 5A and B. The dye binding to protein in the absence of SDS showed a maximum absorbance at 495 nm. However in the presence of 300 mM SDS, the concentration at which brils are formed, the absorbance maxima were red shied to 505 nm at pH 7.0 and to 509 nm at pH 5.0. The data showing shi at other pH values are shown in Table 1. These results conrmed formation of characteristic brillar species and ruled out possibilities of simple oligomerization. The reason for this shi is that lysozyme becomes more compact, and this allows only hydrophobic interactions to take place with SDS under these pH conditions. Unfortunately, the assay could not be performed below pH 5.0 because CR changes colour under such conditions. DLS measurements DLS is a well exploited technique for monitoring changes in the sizes of aggregates.36 It was used to determine the pH dependent changes in the size of SDS-induced aggregates of lysozyme (Fig. 6, Table 1). It was found that the intensity of light scattered at 90 increased in the presence of 1400 mM SDS at all pH values studied below the pI of lysozyme. This observation further supports the conclusion that protein aggregates are formed. The increase in light scattering was less pronounced when the pH of the sample was close to pI, indicating the inability of SDS to induce aggregation under such conditions (Table 2). In the absence of SDS at pH values from 11.0 to 1.0 the light scattering was more or less the same. However there was an increase in the Rh value of protein under acidic pH which suggests partial unfolding of the molecule. The results from DLS experiments obtained at pH 7.0 and pH 3.0 are shown in Fig. 6 and the rest of the results are in Table 2. The Rh value of protein at pH 7.0 was 2.1 nm while at pH 3.0 it
Fig. 5 The absorbance spectra of lysozyme (15 mM) obtained from CR binding assay at pH 7.0 (A) and pH 5.0 (B) in the absence ( ) and presence ( ) of 300 mM SDS. CR (15 mM) was mixed with aggregated and non-aggregated sample and incubated for 30 minutes in the dark prior to measurements.
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Fig. 6 DLS measurements performed to determine the hydrodynamic radii (Rh) of lysozyme (70 mM) in the absence and presence of 1400 mM SDS at pH 7.0 and pH 3.0 at 25 C after 12 h incubation.
increased to 3.0 nm. Upon incubation with 1400.0 mM SDS, two types of species were generated with a size of 2.8 nm and 54.5 nm at pH 7.0, and 5.3 nm and 154.9 nm at pH 3.0. This observation suggests that the larger aggregates were formed at lower pH. CD measurements The change in the secondary structure of lysozyme (15 mM) was monitored at different pH values, in the absence and presence of 300 mM SDS, using far-UV CD. Lysozyme belongs to the a + b class of proteins and its far-UV CD spectra are characterized by two negative peaks centered around 208 nm and 222 nm.37 As shown in Fig. 7A, in the absence of SDS the spectral features were retained throughout the pH range (11.0–1.0) studied, with some increase in ellipticity at low pH, owing to the formation of some secondary structure elements. When lysozyme was incubated with 300.0 mM SDS, the far-UV CD spectra showed two types of changes. From pH 11.0 to pH 10.0, the protein showed a considerable decrease in ellipticity probably due to loss of structure but the overall spectra remained qualitatively unchanged (data not shown). In contrast, the spectra of proteins below pH 9.0 exhibited a transition from an a-helix to a b-sheet structure characterized by a single negative peak around 219 nm, which is a hallmark of amyloid formation (Fig. 7B). For the sake of clarity we have shown representative spectra at pH 1.0, 3.0, 5.0 and 7.0. The secondary structure content of lysozyme at different pH values was calculated by the K2d method and the results are listed in Table 3. As can be seen, a prominent increase in the b-sheet content of protein, occurring below pH 10.0 in the presence of SDS, clearly suggests that aggregates have a dened bril structure. TEM The morphology of lysozyme aggregates was analyzed by TEM. Prior to taking the TEM images lysozyme (15.0 mM) was Soft Matter, 2014, 10, 2591–2599 | 2595
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Table 1
Paper
Wavelength shift of lysozyme upon binding with CR at different pH
pH
Shi in lmax (nm) of lysozyme (15 mM)
Shi in lmax (nm) of (15 mM) lysozyme + 300 mM SDS
Difference in lmax (nm) shi
11 10 9 8 7 6 5
496 496 498 496 495 495 497
489 493 503 505 505 505 507
7 (blue) 3 (blue) 5 (red) 9 (red) 10 (red) 10 (red) 10 (red)
Table 2
Hydrodynamic radii of lysozyme under different conditions at
25 C Sample no.
Conditions
Hydrodynamic radii (nm)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
pH 11.0 pH 11.0 + 1400 mM SDS pH 10.0 pH 10.0 + SDS pH 9.0 pH 9.0 + 1400 mM SDS pH 8.0 pH 8.0 + 1400 mM SDS pH 7.0 pH 7.0 + 1400 mM SDS pH 6.0 pH 6.0 + 1400 mM SDS pH 5.0 pH 5.0 + 1400 mM SDS pH 4.0 pH 4.0 + 1400 mM SDS pH 3.0 pH 3.0 + 1400 mM SDS pH 2.0 pH 2.0 + 1400 mM SDS pH 1.0 pH 1.0 + 1400 mM SDS
3.0 0.1 2.0 0.09 2.0 0.1 2.0 0.1, 9.0 0.1 2.0 0.1 3.0 0.1, 41.0 0.2 2.0 0.1 3.0 0.1, 49.0 0.1 2.0 0.1 2.0 0.1, 54.0 0.2 2.0 0.1 3.0 0.1, 71.0 0.2 2.0 0.1 3.0 0.1, 79.0 0.3 2.0 0.1 3.0 0.1, 112.0 0.1 3.0 0.1 5.0 0.2, 122.0 0.2 3.0 0.1 5.0 0.2, 144.0 0.3 3.0 0.2 5.0 0.2, 150.0 0.5
values. In the absence of SDS, at a pH below the pI, no bril or aggregates were found (data not shown). Similar types of bril structures were also found in the other proteins as well.38–40 ITC When the ligand binds to a protein either heat is released or absorbed but it is very difficult to understand the binding process, in vivo, i.e., whether it is exothermic or endothermic. Therefore we studied the binding process in vitro using ITC. From the above spectroscopic results we found that lysozyme aggregates in the presence of 300.0 mM SDS, at different pH values. Here we used ITC to examine what type of interactions are taking place between lysozyme and SDS. In ITC experiments we injected 10 mL of 7.0 mM SDS 28 times into the reaction cell lled with 70 mM lysozyme at desired pH values. The ITC isotherms showed an exothermic peak when lysozyme was titrated with SDS pH below 10.0 (Fig. 9). These exothermic peaks reveal binding between SDS and lysozyme to be electrostatic. Similar types of exothermic peaks were also reported in some proteins at pH 7.4.41 In control experiments, the heat released was very low. We further checked whether the interactions between lysozyme and SDS were strong or weak. A less exothermic heat was released at pH 11.0, while below this pH a continuous increase in exothermic heat was observed and maximum heat was released at pH 1.0. The possible explanation for low heat released at pH 11.0 is that lysozyme becomes uncharged therefore SDS is not able to bind to it properly. However at lower pH values, lysozyme is highly protonated favoring binding of the negative head group of SDS and release of more heat.
Discussion Fig. 7 Far-UV CD spectra of lysozyme (15 mM) in the absence (A) and presence of 300 mM SDS (B) at pH 1.0, 3.0, 5.0 and 7.0 at 25 C. Only selected spectra are shown in panel A and B because some spectra are overlapping. Prior to CD measurements the entire sample was incubated for 12 h.
incubated with 300.0 mM of SDS at a pH below the pI for 12 hours incubation. Fibril formation was observed at all the pH values studied, below the pI. However, only four representative images are shown (Fig. 8). Mature brils were observed at all the pH
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Polypeptides and proteins have the generic property of forming amyloid brils, with the tendency originating in different parts of each protein.42 Here, lysozyme was incubated at different pH values ranging from 13.0–1.0. The positive charge starts to increase at a lysozyme pH below 11.0 and a maximum positive charge exists at pH 1.0. We observed that as the pH of lysozyme was lowered below the pI in the presence of SDS, the protein began to aggregate as conrmed by turbidity, light scattering, DLS and far-UV CD measurements. These results may be viewed together with the results of Wang et al. (2011) in which, lysozyme incubated with two phospholipids, D6PC and D7PC, showed enhanced light scattering due to the formation of larger
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Paper Table 3
Soft Matter % Secondary structure calculated by K2d method at different conditions
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Lysozyme (15 mM)
(15 mM) lysozyme + 300 mM SDS
Sample no.
pH
% a Helix
% b Sheet
% Random coil
% a Helix
% b Sheet
% Random coil
1 2 3 4 5 6 7 8 9 10 11
11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0
40.0 44.0 43.0 44.0 44.0 45.0 45.0 45.0 44.0 44.0 42.0
18.0 23.0 22.0 23.0 23.0 23.0 23.0 23.0 23.0 23.0 21.0
42.0 33.0 35.0 33.0 37.0 32.0 31.0 33.0 33.0 33.0 36.0
45.0 45.0 20.0 18.0 13.0 15.0 5.0 5.0 4.0 4.0 4.0
23.0 23.0 45.0 44.0 42.0 35.0 48.0 47.0 47.0 48.0 48.0
31.0 31.0 35.0 38.0 55.0 50.0 48.0 48 48.0 48.0 48.0
Fig. 8 TEM images of lysozyme (15 mM) in the presence of 300 mM SDS at pH 9.0 (A), pH 6.0 (B), pH 3.0 (C) and pH 1.0 (D). TEM images were taken after 12 h incubation.
Fig. 9 Plot of enthalpy change against molar ratio for titration of SDS with lysozyme at pH 11.0 ( ), 9.0 ( ), 7.0 ( ), pH 4.0 ( ), pH 2.0 ( ) and pH 1.0 ( ).
aggregates.12 The kinetics of the aggregation process was measured by RLS experiments; also the aggregation tendency of different conformations of lysozyme at varying pH values in the
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presence of SDS was determined. The rate of aggregation was so fast that no lag phase was observed at any of the pH conditions taken in this study. Similar results were obtained for amyloid formation in ApoMb where exponential growth was observed without any lag phase.43 In this context, it is also reported that amyloid bril formation of N47A Spc-SH3 is a rapid process without any lag phase. In our case, saturation took place within 1200 s, so we conclude that the process was fast and that amyloid nuclei are established early.44 We used the ThT binding assay to investigate whether aggregation was amorphous or brillar. We observed a continuous increase in ThT uorescence intensity in the presence of 300 mM SDS at pH below the pI of lysozyme suggesting that aggregates formed were amyloid brils. A slight decrease in ThT uorescence intensity at pH 1.0 and 2.0 could be attributed to the fact that at acidic pH the positively charge N-atom of a benzothiazole group of ThT repels the N-atom of arginine and lysine of lysozyme that may not allow strong binding to the amyloid bril due to charge–charge repulsion.32 This notion is also supported by the observation that ThT binding to insulin laments is greater in distilled water than at pH 1.6.45 We further reconrmed bril formation of our protein by the CR dye binding assay. CR binds strongly to the parallel and antiparallel b-sheet region of the amyloid crossb structures.46 The CR binding assay was performed to rule out the doubts of binding occurring to some non-amyloid structures. A noticeable red shi was observed at acidic pH indicating strong CR binding to protein corresponding to amyloid formation. These two dye binding experiments conrmed that aggregates of lysozyme were formed at all pH values below its pI and have well-ordered structures. Further, we followed changes in the secondary structure of lysozyme in the presence of SDS at different pH. Generally amyloid brils have ordered b-sheet structures.47,48 Lysozyme contains both a-helix and b-sheet structures at neutral pH. The a-helical content of lysozyme is converted into a b-sheet aer incubation with SDS at a pH below the pI. Due to this secondary structure transition we conclude that the aggregate induced by SDS has an ordered b-sheet structure which could be the case in amyloid brils. To conrm this further, we also employed TEM to observe the morphology of the aggregates. The TEM images
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revealed that the aggregates formed by SDS were typically brillar. It can be concluded, therefore, that lysozyme exhibits a strong propensity to form amyloids in the presence of SDS at any pH below its pI and the size of the aggregates increases with the increase in positive charge of the protein (as the pH moves down from the pI). In Fig. 10, we have proposed a hypothetical model that explains how SDS induces aggregation in lysozyme. In our previous study, we discussed how SDS induces aggregation in proteins at a pH below the pI. Since the pI of lysozyme is 11.0, below this pH the polypeptide chain will acquire a net positive charge. The net charge of lysozyme from pH 11.0–1.0 was calculated using PROTEIN CALCULATOR v3.3 soware (Table 4). As the pH of the protein samples decreases below 10.0, the net positive charge of the protein keeps increasing due to protonation of carboxylate (–COOH) and amino (–NH3+) groups of amino acid side chains. The negatively charged head group of SDS would be expected to neutralize more and more positively charged centers on the protein. Therefore, the aggregating propensity as well as the size of aggregate increases accordingly. On the other hand, at a pH above 10.0, the lysozyme develops a net negative charge on its surface, and this is expected not to bind to negatively charged SDS due to charge–charge repulsions. Ultimately, above pH 10.0, therefore, no aggregation is observed.
Conclusions From this study it can be concluded that SDS induces aggregation in lysozyme at values of pH below 11.0 and that the aggregation propensity is at a maximum at pH 1.0 because the net positive charge increase of lysozyme upon lowering of the pH is highest at this pH, allowing SDS to interact electrostatically with the oppositely charged surface of lysozyme, leading to the assembly of protein into characteristic amyloid brils.
Paper Table 4 Estimated charge over pH range by “PROTEIN CALCULATOR
v3.3” software Sample no.
pH
Total charge
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
19.0 18.9 18.2 15.5 10.8 8.9 7.9 5.5 4.9 0.2
Notes The authors declare no competing interest.
Author contributions Conceived and designed the experiments: JMK and RHK. Performed the experiments: JMK, SKC and MI. Performed TEM experiments: JMK and SKR. Analyzed the data: JMK and RHK. Contributed reagents/materials/analysis tools: JMK, EA, AQ and RHK. Paper was checked by JMK, SKC, SKR and RHK. Wrote the paper: JMK and RHK.
Acknowledgements The facilities provided by I.B.U, Aligarh Muslim University are gratefully acknowledged. We are also grateful to AIRF, AIIMS for TEM images. J.M.K and S.K.C are highly thankful to the Council of Scientic and Industrial Research, New Delhi, for nancial
Fig. 10 Schematic representation of the effect of SDS on the conformation of lysozyme below and above the pI of lysozyme.
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assistance in the form of senior research fellowships (SRF). We are thankful to DBT (BT/PR 13194/10/742/2009) and CSIR (37(1456)/10/EMR-II) for nancial support. M.I is highly thankful to the Indian Council of Medical Research for nancial assistance in the form of a senior research fellowship (SRF) New Delhi. We would like to thank Prof. P. Guptasarma from IISER Mohali and Paul Brown at the Randall Division of Cell and Molecular Biophysics, King's College London for Englishlanguage and technical editing of the manuscript.
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