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Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme
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Biochimica et Biophysica Acta 1784 (2008) 472 – 480 www.elsevier.com/locate/bbapap
Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme Bing-Rui Zhou, Zheng Zhou, Qing-Lian Hu, Jie Chen, Yi Liang ⁎ State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China Received 30 August 2007; received in revised form 20 December 2007; accepted 8 January 2008 Available online 18 January 2008
Abstract The effects of two single macromolecular crowding agents, Ficoll 70 and bovine serum albumin (BSA), and one mixed macromolecular crowding agent containing both BSA and Ficoll 70, on amyloid formation of hen egg white lysozyme have been examined by thioflavin T binding, Congo red binding, transmission electron microscopy, and activity assay, as a function of crowder concentration and composition. Both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Further kinetic and isothermal titration calorimetry analyses indicate that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibits amyloid formation of lysozyme and maintains lysozyme activity via mixed macromolecular crowding as well as weak, nonspecific interactions between BSA and nonnative lysozyme. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism. © 2008 Elsevier B.V. All rights reserved. Keywords: Macromolecular crowding; Amyloid fibril; Lysozyme; Inhibition; Kinetics
1. Introduction The formation of amyloid fibrils is of intense medical interest because it is associated with neurodegenerative diseases such as Alzheimer's disease and transmissible spongiform encephalopathy, as well as nonneuropathic systemic amyloidosis [1–5]. Up to now, about 20 different proteins with unrelated sequences and tertiary structures, including prions [2] and α-synuclein [5], are involved in these amyloidoses. It is now evident that the ability to form amyloid structures is a general property of all proteins rather than a characteristic feature of the proteins associated with diseases [6–9]. Hen egg white lysozyme is amongst the proteins that form amyloid fibrils under appropriate conditions [10–15] but is not Abbreviations: BSA, bovine serum albumin; CR, Congo red; HSA, human serum albumin; ITC, isothermal titration calorimetry; TEM, transmission electron microscopy; ThT, thioflavin T ⁎ Corresponding author. Tel./fax: +86 27 6875 4902. E-mail address: [email protected] (Y. Liang). 1570-9639/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2008.01.004
linked to any disease, which makes it an ideal model to study the mechanism of amyloid formation of a non-disease-associated protein. Hen lysozyme is one of the proteins that have been studied most extensively and its physiochemical properties have been examined in detail [16–18]. The kinetics of amyloid formation and loss of activity of hen lysozyme upon incubation in dilute acid have been described [15]. Recently, it has been shown that hen lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways [19]. Furthermore, hen lysozyme is closely related to human lysozyme in structure and function, several naturally occurring variants of which form amyloid fibrils that are related to hereditary nonneuropathic systemic amyloidosis [1,4,20]. Amyloid formation has been traditionally studied in dilute solutions [7,21]. However, the inside of a cell is poorly modeled by such dilute solutions and biochemical reactions within cells differ much from those in dilute solutions [22–24]. Most biological fluids contain a high total concentration of macromolecules, which collectively occupy a lower limit of about 10% and an upper limit of about 40% of total fluid volume [25–28].
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A special term, macromolecular crowding, has been introduced to describe the effect of high concentrations of macromolecules on biochemical reactions [28–30]. The effect of macromolecular crowding upon protein stability and conformation has been described by a simple excluded volume theory [31,32]. Crowding can be mimicked experimentally by adding a high concentration of nonspecific crowding agents to the system in vitro [28,33]. Furthermore, mixed macromolecular crowding agents containing proteins, nucleic acids, and polysaccharides can be used to reflect the physiological environment more accurately than single crowding agents [34,35]. Several groups have discussed the effect of macromolecular crowding on the formation of amyloid fibrils. Hatters et al. [36] have reported that the rate of amyloid formation of human apolipoprotein C-II is 8-fold increased by the addition of a high concentration of an inert polymer, dextran T-10. Shtilerman et al. [8] and Uversky et al. [22,23] have demonstrated that macromolecular crowding significantly reduces the lag time for protofibril formation of α-synuclein and the conversion of protofibril to the fibril. Yamin et al. [37] have demonstrated that the metal-induced fibrillization of β-synuclein is further accelerated by the addition of high concentrations of crowding agents. The major factor responsible for the acceleration is excluded volume, and simple excluded volume theory predicts that an increase in the fractional volume occupancy of macromolecules in a physiological fluid can nonspecifically accelerate the formation of amyloid fibrils of any amyloidogenic protein [30,38,39]. However, McNulty et al. [40] have recently demonstrated that macromolecular crowding in the Escherichia coli periplasm or 300 g/l of bovine serum albumin (BSA) in vitro is sufficient to stabilize the disordered, monomeric structure of α-synuclein. Amyloid fibrils associated with neurodegenerative diseases such as Alzheimer's disease and transmissible spongiform encephalopathy, as well as nonneuropathic systemic amyloidosis, can be considered biologically relevant failures of posttranslational quality control maintained by molecular chaperones and proteases [41]. Cellular protection mechanisms, such as molecular chaperones and the protein degradation machinery, are crucial in the prevention of diseases in normally functioning living organisms [21]. In this study, we demonstrated that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixture of BSA and Ficoll 70, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism. 2. Materials and methods 2.1. Materials Hen egg white lysozyme was obtained from Sigma (Sigma-Aldrich Co, St. Louis, MO) and was used without further purification. The A1% 1 cm value of 26.5 at 280 nm [14] was used for protein concentration measurements. The two crowding agents, Ficoll 70 and BSA, were purchased from Sigma. Thioflavin T (ThT) and Congo red (CR) were also products of Sigma. All other chemicals used were made in China and of analytical grade. Aqueous solution of HCl at pH 2.0 containing 100 mM NaCl and 0.2% NaN3 was used for amyloid formation of lysozyme.
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20 mM sodium phosphate buffer at pH 7.0 was used for the activity assay of lysozyme. All buffers were filtered through a 0.22-μm pore size filter before use to remove insoluble particles.
2.2. Preparation for stock solutions of crowding agents Stock solutions of Ficoll (200 g/l) and BSA (200 g/l) were prepared in aqueous solution of HCl at pH 2.0 containing 100 mM NaCl and 0.2% NaN3. The pH values of these solutions were adjusted to 2.0 with HCl.
2.3. Amyloid formation Hen egg white lysozyme was first dissolved in aqueous solution of HCl at pH 2.0 containing 100 mM NaCl and 0.2% NaN3. After extensive dialysis and dilution to a final concentration of 25 g/l with the same solvent, hen lysozyme was unfolded by incubation at 37 °C for 3–5 days. Then the lysozyme solution was mixed with stock solutions of crowding agents, to yield a solution of 5 g/l lysozyme in HCl (pH 2.0) containing 100 mM NaCl, 0.2% NaN3, and a chosen concentration of a mixed or single crowding agent, followed by incubation at 37 °C for at least 20 days with a constant stirring rate at 220 rpm. At different time intervals, aliquots of sample were taken out for a series of biophysical or morphological measurements.
2.4. Thioflavin T binding assays The thioflavin T binding assays were performed according to Naiki et al. [42]. The fluorescence of ThT was excited at 440 nm with a slit-width of 10 nm and the emission was measured at 482 nm with slit-width of 5 nm on a LS-55 luminescence spectrometer (PerkinElmer Life Sciences, Shelton, CT). A 3 mM ThT stock solution was freshly prepared in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl, and passed through a 0.22-μm filter pore size filter before use to remove insoluble particles. Samples (10 μl) incubated under different conditions were diluted into 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 65 μM ThT, giving a final volume of 3 ml. The fluorescence intensity at 482 nm was averaged for 60 s to increase the signal-tonoise ratio of the measurements. Control experiments were performed to ensure that the crowding agents had no influence on the ThT binding assays.
2.5. Lysozyme activity assays Aliquots of the hen egg white lysozyme sample were diluted to 5 μM in HCl (pH 2.0) containing 100 mM NaCl and 0.2% NaN3, and the enzymatic activity was assayed by mixing 10 μl of diluted sample with 490 μl of 0.5 mg/ml Micrococcus lysodeikticus cell wall suspension in 20 mM sodium phosphate buffer at pH 7.0, and then by monitoring the decrease in absorbance at 450 nm during 60 s and at 25 °C. All enzymatic activities were normalized against and expressed as a percentage of 5 μM solution of native lysozyme in HCl at pH 2.0 containing 100 mM NaCl and 0.2% NaN3, and the specific activity of native lysozyme under such conditions was (5.10± 0.05) × 104 U mg− 1 (n = 3). Control experiments were performed to ensure that the crowding agents had no influence on the activity assay.
2.6. Transmission electron microscopy Aliquots of protein samples (100 μg/ml) under different conditions were produced by 50-fold dilution of the original samples and a drop was deposited on formvar-coated copper grids (200 mesh). The grids were then negatively stained with 2% (w/v) uranyl acetate for 45 s before examination using an H-8100 transmission electron microscope (Hitachi, Tokyo, Japan) operating at accelerating voltages of 100 kV.
2.7. Isothermal titration calorimetry ITC measurements were performed at 37.0 °C by using a VP-ITC titration calorimeter (MicroCal, Northampton, MA). All solutions were thoroughly degassed before use by stirring under vacuum. Before each experiment, the ITC sample cell was washed several times with HCl at pH 2.0. The sample cell was loaded with 1.43 ml of
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hen egg white lysozyme solution (17.4 μM) incubated at pH 2.0 and the reference cell contained doubly distilled water. Titration was carried out using a 280-μl syringe filled with BSA (or Ficoll 70) solution, with stirring at 300 rpm. The concentration of BSA (or Ficoll 70) was 139 μM. Injections were started after baseline stability had been achieved. Titrations were performed by adding an aliquot of 4 μl for the first injection and aliquots of 10 μl for the following injections with a 5-min interval between injections. Heats of dilution were determined by injecting BSA (or Ficoll 70) into the buffer alone and the total observed heats of binding were corrected for the heat of dilution. MicroCal ORIGIN software supplied with the instrument was used to determine the site binding model that gave a good fit (low χ 2 value) to the resulting data. From the various binding models tested (a single set of identical sites model, two sets of independent sites model, and sequential binding sites model), only the three sequential binding sites model fitted adequately to the binding isotherms, and the standard molar enthalpy change for the binding between BSA and nonnative lysozyme, Δb H 0m, and the binding constant, Kb, were thus obtained. The standard molar free energy change, Δb G 0m, and the standard molar entropy change, Δb S 0m, for the binding reaction were calculated by the fundamental equations of thermodynamics [43,44]: Db G0m ¼ RT ln Kb
ð1Þ
Db Sm0 ¼ Db Hm0 Db G0m =T :
ð2Þ
retained its native conformation under such mixed crowding conditions. In contrast, the activity of hen lysozyme in the absence of crowding agents was completely lost after incubation for 35 days (Fig. 2A). At the same time, amyloid formation of hen lysozyme was blocked by such mixed crowding agents and reached a maximum in the absence of crowding agents (Fig. 1A), suggesting that there is a negative correlation between amyloid content and residual activity change of lysozyme under different compositions and concentrations of crowding agents. Control experiments showed that the activity of lysozyme decreased nearly to 70% and 0 after incubation for 35 days in the presence of 5 g/l BSA (Fig. 2B) and in the presence of 25–100 g/l Ficoll 70 (Fig. 2C) respectively, indicating that neither 5 g/l of BSA alone nor 25–100 g/l Ficoll 70 alone stabilized lysozyme activity on the time scale of 35 days,
3. Results 3.1. Effect of mixed crowding agents on amyloid formation of lysozyme We have demonstrated that mixed macromolecular crowding reflects the physiological environments more accurately than individual crowding agents [34,35]. In this study, we employed such mixed crowding agents to test their effects on amyloid formation of hen lysozyme. Scale bar N in Fig. 1A represented amyloid formation of hen egg white lysozyme in the absence of crowding agents, and scale bar 5, 10, 25, 50, and 75 represented that in the presence of 100 g/l mixture of BSA and Ficoll 70, in which the weight ratio of BSA to Ficoll 70 is 5:95, 10:90, 25:75, 50:50, and 75:25, respectively. As can be seen from Fig. 1, ThT fluorescence intensity of hen egg white lysozyme in the absence of crowding agents increased to a maximum of about 130 U after incubation for 35 days. In contrast, ThT fluorescence for hen lysozyme in the presence of these mixed crowding agents had no obvious change and was below 10 U after incubation for 35 days (Fig. 1A), indicating that amyloid formation of lysozyme was blocked by such mixed crowding agents. As shown in Fig. 1B, ThT fluorescence for lysozyme incubated for 35 days in the presence of 5 or 10 g/l BSA increased to 57 or 21 U, indicating that 5 or 10 g/l of BSA alone did not block amyloid formation of lysozyme, although the protein crowding agent BSA at 100 g/l almost completely inhibited amyloid formation of lysozyme on the investigated time scale. Furthermore, 25–100 g/l Ficoll 70 alone did not impede amyloid formation of lysozyme effectively (Fig. 1C). We thus conclude that both BSA and Ficoll 70 cooperatively contribute to the inhibitory effect of mixed crowding agents. Activity measurements further supported our conclusion. As shown in Fig. 2A, the remaining activity of lysozyme in the presence of such mixed crowding agents decreased slightly after incubation for 35 days, indicating that the majority of lysozyme
Fig. 1. Effects of mixed or single crowding agents on amyloid formation of lysozyme monitored by ThT fluorescence. Lysozyme was incubated in HCl (pH 2.0) containing 100 mM NaCl and 0.2% NaN3 in the absence of crowding agents (N) and in the presence of 100 g/l mixture of BSA and Ficoll 70 (A), or single component crowding agents (B, BSA, and C, Ficoll 70). ThT Fluorescence of lysozyme was measured after incubation for 35 days as a function of the ratio (%) of the weight of BSA to the total weight of mixed crowding agents (A), or a function of the concentrations of BSA (B) or Ficoll 70 (C). The data with error bars were expressed as mean ± S.D. (n = 2–3).
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although the protein crowding agent BSA at 100 g/l stabilized lysozyme activity on the investigated time scale (Fig. 2B). We thus conclude that both BSA and Ficoll 70 cooperatively contribute to the stabilization effect of mixed crowding agents. 3.2. Inhibition of amyloid formation of lysozyme by delayed addition of mixed crowding agents We further investigated the effect of mixed macromolecular crowding agents on amyloid formation of lysozyme by delayed addition of such mixed crowding agents. The weight ratio of BSA to Ficoll 70 as 5:95 was chosen for the mixed crowding agent, and the addition time of such a mixed crowding agent was designed to cover all of the three different stages of amyloid
Fig. 3. Kinetics of fibrillization and remaining enzymatic activity of lysozyme after adding mixed macromolecular crowding agents at various time points. (A) Amyloid formation of lysozyme was monitored by ThT fluorescence in the absence of crowding agents (open square) and in the presence of 5 g/l BSA + 95 g/ l Ficoll 70 added at 1 (open triangle), 6 (inverted filled triangle), 10 (open circle), 12 (filled circle), and 14 (filled triangle) days, respectively. (B) Remaining enzymatic activity of lysozyme in the absence of crowding agents (a) and in the presence of 5 g/l BSA + 95 g/l Ficoll 70 added at 1 (b), 6 (c), 10 (d), 12 (e), and 14 (f) days was assayed after incubation for 22 days. The data with error bars were expressed as mean ± S.D. (n = 2–3).
Fig. 2. Effects of mixed or single crowding agents on remaining enzymatic activity of lysozyme in these crowded solutions during amyloid formation. Lysozyme was incubated in HCl (pH 2.0) containing 100 mM NaCl and 0.2% NaN3 in the absence of crowding agents (N) and in the presence of 100 g/l mixture of BSA and Ficoll 70 (A), or single component crowding agents (B, BSA, and C, Ficoll 70). Remaining enzymatic activity of lysozyme was assayed after incubation for 35 days as a function of the ratio (%) of the weight of BSA to the total weight of mixed crowding agents (A), or a function of the concentrations of BSA (B) or Ficoll 70 (C). The data with error bars were expressed as mean ± S.D. (n = 2–3).
formation. As shown in Fig. 3A, the mixture of 5 g/l BSA and 95 g/l Ficoll 70 added at 1, 6, and 10 days in the lag phase inhibited the increase of ThT fluorescence intensity of hen lysozyme. Interestingly, such a mixture added at 12 days in the growth phase showed a similar inhibitory effect. When added at 14 days in the final equilibrium phase, no obvious inhibitory effect of such a mixed crowding agent on ThT fluorescence intensity of lysozyme was observed, indicating that mixed macromolecular crowding has no effect on the mature fibrils of lysozyme. The remaining activity of hen lysozyme in the presence of the mixture of 5 g/l BSA and 95 g/l Ficoll 70 after 22day of incubation is shown in Fig. 3B, compared to that in the absence of crowding agents. Lysozyme incubated in such mixed crowded solution added at 1, 6, and 10 days in the lag phase remained about 99%, 89% and 87% relative enzymatic activity, respectively. The relative activity of lysozyme was 44% and 3% in the presence of this mixed crowding agent added at 12 and 14 days, suggesting that mixed macromolecular crowding stabilized the native structure of lysozyme and thus inhibited its structure transformation as well as amyloid formation, even in the growth phase. 3.3. Morphology of lysozyme samples monitored by TEM Transmission electron microscopy (TEM) was employed to study the morphology of hen egg white lysozyme samples
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Fig. 4. Transmission electron micrographs of lysozyme samples at pH 2.0 after incubation for 14 days in the presence of mixed or single crowding agents. Lysozyme was incubated in the presence of 100 g/l BSA (A), 100 g/l Ficoll 70 (B), 5 g/l BSA + 95 g/l Ficoll 70 (C), or 5 g/l BSA (D). A 2% (w/v) uranyl acetate solution was used to negatively stain the fibrils. The scale bars represent 200 nm.
incubated under different conditions. As shown in Fig. 4A and C, no fibril structure was observed in the presence of 100 g/l BSA or 100 g/l mixed crowding agent (5 g/l BSA + 95 g/l Ficoll 70). A mixture of protofibrils and separate fibrils [45,46] of hen lysozyme was formed in the presence of 5 g/l BSA (Fig. 4B), indicating that 5 g/l BSA only was not enough to inhibit amyloid formation of lysozyme. As a control study, the majority of separate fibrils were observed in the presence of 100 g/l Ficoll 70 (Fig. 4B). The results from TEM study are in accordance with those from ThT binding and activity assays. 3.4. Weak, nonspecific interactions between BSA and nonnative lysozyme detected by ITC Because weak, nonspecific interactions between macromolecular reactants and constituents of the local environment can greatly influence the equilibria and rates of reactions in which they participate [24,35], we investigated the effect of possible complex formation between BSA and nonnative lysozyme on amyloid formation of hen lysozyme. By using isothermal titration calorimetry (ITC), which is thought to be one of the most reliable and high-precision methods to quantitate protein interactions [43,47], we observed a weak, nonspecific interaction between BSA and nonnative lysozyme at pH 2.0. The ITC results are shown in Fig. 5. The best fit for the integrated heat data was obtained using a three sequential binding sites model
described by Hammann et al. [48] and Roufik et al. [44] with the lowest χ2 (26.50 cal mol− 1), yielding the thermodynamic parameters for the interaction between BSA and nonnative 0 lysozyme: Kb,1 = (1.03 ± 0.04) × 105 M− 1 , ΔbHm,1 = − 46.6 ± −1 0 −1 0.7 kcal mol , ΔbG m,1 = − 7.12 ± 0.10 kcal mol , ΔbS0m,1 = − 127 ± 2 cal mol − 1 K − 1 , Kb,2 = (1.01 ± 0.04) × 10 5 M − 1 , ΔbH0m,2 = − 6.1 ± 1.5 kcal mol− 1, ΔbG0m,2 = − 7.11 ± 0.11 kcal mol − 1 , Δb S 0 m,2 = 3.2 ± 4.6 cal mol − 1 K − 1 , Kb,3 = (1.02 ± 0.07) × 10 5 M − 1 , Δ b H 0 m , 3 = − 73.0 ± 2.2 kcal mol − 1 , ΔbG 0 m,3 = − 7.11 ± 0.12 kcal mol − 1 , and ΔbS0 m,3 = − 213 ± 7 cal mol− 1 K− 1. These results showed that the binding of BSA to nonnative lysozyme was driven entirely by large favorable enthalpy decreases but with unfavorable entropy decreases for the first and the third sequential binding sites of nonnative lysozyme, implying that BSA may bind to lysozyme oligomers to prevent the formation of prefibrillar lysozyme, and bind with the protofibrils to retard fibril elongation of lysozyme. Furthermore, we performed ITC experiments on the binding of Ficoll 70 to nonnative lysozyme at pH 2.0 and found that the calorimetric data were too small to be fitted to any binding model (Fig. 6). No optimal fit was found for Ficoll 70, indicating that Ficoll 70 had no specific binding affinity for nonnative lysozyme under such experimental conditions. The above kinetic and ITC analyses suggested that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibited amyloid formation of lysozyme via mixed macromolecular crowding as well as weak,
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stabilized lysozyme activity on the investigated time scale, suggesting that, in addition to nonspecific excluded volume effects, weak and nonspecific protein–protein interactions could greatly affect protein misfolding in the highly complex intracellular environment. By using the absorbance maximum of the protein in the presence of Congo red as another indicator of amyloid fibrils [50], we obtained qualitatively similar results (data not shown), further supporting the conclusion reached by ThT binding assays and TEM that BSA and Ficoll 70 cooperatively contribute to the inhibitory effect of mixed crowding agents. Milojevic et al. have found that human serum albumin (HSA) caps the exposed hydrophobic patches located at the growing and/or transiently exposed sites of the Aβ oligomers, thereby blocking the addition of further monomers and the growth of the prefibrillar assemblies [51]. Both ThT binding assays and CR binding assays have been widely used for the characterization of in vitro amyloid fibrils [9,12,13,42,46,50,52,53]. There are suggestions that the dyes ThT and CR bind to the crossed-β structure in the amyloid fibrils [50,52,53]. One of the differences between both assays is that Congo red is not specific for amyloid fibrils. CR can also bind to native or partially folded states of several different proteins, regardless of their secondary structures and induce oligomerization of native proteins [54]. It has been suggested that amyloid fibril formation can occur when the native structure of a protein is destabilized, favoring formation of partially folded conformations [7,55–57]. Simple Fig. 5. ITC profiles for the binding of BSA to lysozyme samples at pH 2.0 after incubation for 10 days in the absence of crowding agents at 37.0 °C. The top panels (A) represent the raw data for sequential 10-μl injections of BSA (139 μM) into lysozyme (17.4 μM) solution incubated for 10 days. The middle panel (B) represents the control experiment in which BSA (139 μM) was injected into the buffer alone. The bottom panels (C) show the plot of the heat evolved (kcal) per mole of BSA added, corrected for the heat of BSA dilution, against the molar ratio of BSA to lysozyme incubated for 10 days. The volume of the first injection of BSA was 4 μl (A and B). The data (filled square) were fitted to a three sequential binding sites model (C), and the solid lines represented the best fit.
nonspecific interactions between BSA and nonnative lysozyme observed in dilute solutions. 4. Discussion The cellular environment comprises a heterogeneous mixture of proteins, nucleic acids, ribosomes, and carbohydrates (polysaccharides), each of which is likely to affect the folding mechanism of different proteins in a distinctive fashion [49]. There may be hidden facts that proteins have evolved to function in such a mixed crowded milieu. We extended our idea of mixed macromolecular crowding [34,35] to study the effects of mixed crowding agents on amyloid formation of hen egg white lysozyme, compared with single crowding agents. Although macromolecular crowding accelerates amyloid formation of native disordered protein α-synuclein [8,22,23] and human apolipoprotein C-II [36], our data demonstrated that the mixed crowding agent containing both BSA and polysaccharide almost completely inhibited amyloid formation of lysozyme and
Fig. 6. ITC profiles for the binding of Ficoll 70 to lysozyme samples at pH 2.0 after incubation for 10 days in the absence of crowding agents at 37.0 °C. The top panels (A) represent the raw data for sequential 10-μl injections of Ficoll 70 (139 μM) into lysozyme (17.4 μM) solution incubated for 10 days. The bottom panels (B) show the plot of the heat evolved (kcal) per mole of Ficoll 70 added, corrected for the heat of Ficoll 70 dilution, against the molar ratio of Ficoll 70 to lysozyme incubated for 10 days. The volume of the first injection of Ficoll 70 was 4 μl (A). The data (filled square) were too small to be fitted, indicating that no binding was observed in the conditions used.
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excluded volume theory predicts that high concentration of ‘inert’ macromolecules should stabilize the compact native state relative to any less compact unfolded or partially folded state of the polypeptide [29–31,58]. In addition, it has been reported that at pH 2.0 the molten globule states of hen egg white lysozyme and horse heart myoglobin are stabilized relative to the fully unfolded forms by dextran at high concentrations [31,59]. In this study, the major reason for the inhibitory effect is that hen egg white lysozyme was stabilized in its compact, functional structure by the mixed macromolecular crowding agent used, via nonspecific excluded volume effects as well as weak, nonspecific interactions between BSA and nonnative lysozyme. However, the possibility that the inhibitory and stabilizing effects reflect the enhancement of nonspecific interactions between BSA and nonnative lysozyme by macromolecular crowding effected by Ficoll 70, BSA serving as a ‘chaperone’ [60] in lysozyme protection, cannot be excluded. In addition, BSA oligomerization due to macromolecular crowding may influence lysozyme activity. It has been reported that the lower stability of the native state of the amyloidogenic proteins, such as α-synuclein [22,23] and human lysozyme variants [20], is the primary factor resulting in their tendency to form amyloid deposits in crowded intracellular environments and hence to be associated with diseases [4,5,61,62]. Therefore it is interesting to assume that non-disease-associated proteins with stable structures, such as hen egg white lysozyme, could efficiently avoid the formation of fibrillar deposits in crowded physiological fluid. In vitro experiments have suggested that all peptides and proteins have the potential to form fibrils under appropriate conditions [6–9,63]. Amyloid formation of some stable proteins such as hen egg white lysozyme does occur in vitro under appropriate conditions [10–15], but they never occur in vivo. They have relative stable native conformation compared with mutant proteins and native disordered proteins related to serious diseases, and a posttranslational quality control mechanism maintains their structures throughout their functional life-time [41]. In this study, we demonstrated that the crowded intracellular environment has a remarkable inhibitory effect on amyloid formation of a stable protein in a stressed environment. It has been noted that an increased level of macromolecular crowding may not always promote diseases [8] and macromolecular crowding does enhance native state stability of globular proteins [64]. Combined with the results that chaperones are more effective in assisting protein refolding under crowding conditions than those in the absence of crowding agents [65], we conclude that in addition to the present knowledge of posttranslational quality control mechanism, mixed macromolecular crowding may play a role in the mechanism that regulates normally functioning proteins. In summary, both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and
the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism. Acknowledgments We sincerely thank Prof. Allen P. Minton (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health) for his helpful suggestions and Dr. Peter McPhie (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health) for his critical reading of the manuscript. This work was supported by National Key Basic Research Foundation of China Grant 2006CB910301, National Natural Science Foundation of China Grants 30770421 and 90408012, and Program for New Century Excellent Talents in University Grant NCET-04-0670 from the Ministry of Education of China. References [1] M.B. Pepys, P.N. Hawkins, D.R. Booth, D.M. Vigushin, G.A. Tennent, A.K. Soutar, N. Totty, O. Nguyen, C.C. Blake, C.J. Terry, T.G. Feest, A.M. Zalin, J.J. Hsuan, Human lysozyme gene mutations cause hereditary systemic amyloidosis, Nature 362 (1993) 553–557. [2] S.B. Prusiner, Prions, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 13363–13383. [3] C.M. Dobson, Protein folding and misfolding, Nature 426 (2003) 884–890. [4] M. Dumoulin, J.R. Kumita, C.M. Dobson, Normal and aberrant biological self-assembly: insights from studies of human lysozyme and its amyloidogenic variants, Acc. Chem. Res. 39 (2006) 603–610. [5] A.L. Fink, The aggregation and fibrillation of α-synuclein, Acc. Chem. Res. 39 (2006) 628–634. [6] J.I. Guijarro, M. Sunde, J.A. Jones, I.D. Campbell, C.M. Dobson, Amyloid fibril formation by an SH3 domain, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 4224–4228. [7] C.M. Dobson, Protein misfolding, evolution and disease, Trends Biochem. Sci. 24 (1999) 329–332. [8] M.D. Shtilerman, T.T. Ding, P.T. Lansbury, Molecular crowding accelerates fibrillization of α-synuclein: could an increase in the cytoplasmic protein concentration induce Parkinson's disease? Biochemistry 41 (2002) 3855–3860. [9] F. Yang Jr., M. Zhang, B.R. Zhou, J. Chen, Y. Liang, Oleic acid inhibits amyloid formation of the intermediate of α-lactabumin at moderately acidic pH, J. Mol. Biol. 362 (2006) 821–834. [10] S. Goda, K. Takano, Y. Yamagata, R. Nagata, H. Akutsu, S. Maki, K. Namba, K. Yutani, Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution, Protein Sci. 9 (2000) 369–375. [11] M.R. Krebs, D.K. Wilkins, E.W. Chung, M.C. Pitkeathly, A.K. Chamberlain, J. Zurdo, C.V. Robinson, C.M. Dobson, Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the β-domain, J. Mol. Biol. 300 (2000) 541–549. [12] A. Cao, D. Hu, L. Lai, Formation of amyloid fibrils from fully reduced hen egg white lysozyme, Protein Sci. 13 (2004) 319–324. [13] B.A. Vernaglia, J. Huang, E.D. Clark, Guanidine hydrochloride can induce amyloid fibril formation from hen egg-white lysozyme, Biomacromolecules 5 (2004) 1362–1370. [14] L.N. Arnaudov, R. de Vries, Thermally induced fibrillar aggregation of hen egg white lysozyme, Biophys. J. 88 (2005) 515–526. [15] T. Ohkuri, S. Shioi, T. Imoto, T. Ueda, Effect of the structure of the denatured state of lysozyme on the aggregation reaction at the early stages of folding from the reduced form, J. Mol. Biol. 347 (2005) 159–168. [16] S.E. Radford, C.M. Dobson, P.A. Evans, The folding of hen lysozyme involves partially structured intermediates and multiple pathways, Nature 358 (1992) 302–307.
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Biochimica et Biophysica Acta 1784 (2008) 472 – 480 www.elsevier.com/locate/bbapap
Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme Bing-Rui Zhou, Zheng Zhou, Qing-Lian Hu, Jie Chen, Yi Liang ⁎ State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China Received 30 August 2007; received in revised form 20 December 2007; accepted 8 January 2008 Available online 18 January 2008
Abstract The effects of two single macromolecular crowding agents, Ficoll 70 and bovine serum albumin (BSA), and one mixed macromolecular crowding agent containing both BSA and Ficoll 70, on amyloid formation of hen egg white lysozyme have been examined by thioflavin T binding, Congo red binding, transmission electron microscopy, and activity assay, as a function of crowder concentration and composition. Both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Further kinetic and isothermal titration calorimetry analyses indicate that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibits amyloid formation of lysozyme and maintains lysozyme activity via mixed macromolecular crowding as well as weak, nonspecific interactions between BSA and nonnative lysozyme. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism. © 2008 Elsevier B.V. All rights reserved. Keywords: Macromolecular crowding; Amyloid fibril; Lysozyme; Inhibition; Kinetics
1. Introduction The formation of amyloid fibrils is of intense medical interest because it is associated with neurodegenerative diseases such as Alzheimer's disease and transmissible spongiform encephalopathy, as well as nonneuropathic systemic amyloidosis [1–5]. Up to now, about 20 different proteins with unrelated sequences and tertiary structures, including prions [2] and α-synuclein [5], are involved in these amyloidoses. It is now evident that the ability to form amyloid structures is a general property of all proteins rather than a characteristic feature of the proteins associated with diseases [6–9]. Hen egg white lysozyme is amongst the proteins that form amyloid fibrils under appropriate conditions [10–15] but is not Abbreviations: BSA, bovine serum albumin; CR, Congo red; HSA, human serum albumin; ITC, isothermal titration calorimetry; TEM, transmission electron microscopy; ThT, thioflavin T ⁎ Corresponding author. Tel./fax: +86 27 6875 4902. E-mail address: [email protected] (Y. Liang). 1570-9639/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2008.01.004
linked to any disease, which makes it an ideal model to study the mechanism of amyloid formation of a non-disease-associated protein. Hen lysozyme is one of the proteins that have been studied most extensively and its physiochemical properties have been examined in detail [16–18]. The kinetics of amyloid formation and loss of activity of hen lysozyme upon incubation in dilute acid have been described [15]. Recently, it has been shown that hen lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways [19]. Furthermore, hen lysozyme is closely related to human lysozyme in structure and function, several naturally occurring variants of which form amyloid fibrils that are related to hereditary nonneuropathic systemic amyloidosis [1,4,20]. Amyloid formation has been traditionally studied in dilute solutions [7,21]. However, the inside of a cell is poorly modeled by such dilute solutions and biochemical reactions within cells differ much from those in dilute solutions [22–24]. Most biological fluids contain a high total concentration of macromolecules, which collectively occupy a lower limit of about 10% and an upper limit of about 40% of total fluid volume [25–28].
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A special term, macromolecular crowding, has been introduced to describe the effect of high concentrations of macromolecules on biochemical reactions [28–30]. The effect of macromolecular crowding upon protein stability and conformation has been described by a simple excluded volume theory [31,32]. Crowding can be mimicked experimentally by adding a high concentration of nonspecific crowding agents to the system in vitro [28,33]. Furthermore, mixed macromolecular crowding agents containing proteins, nucleic acids, and polysaccharides can be used to reflect the physiological environment more accurately than single crowding agents [34,35]. Several groups have discussed the effect of macromolecular crowding on the formation of amyloid fibrils. Hatters et al. [36] have reported that the rate of amyloid formation of human apolipoprotein C-II is 8-fold increased by the addition of a high concentration of an inert polymer, dextran T-10. Shtilerman et al. [8] and Uversky et al. [22,23] have demonstrated that macromolecular crowding significantly reduces the lag time for protofibril formation of α-synuclein and the conversion of protofibril to the fibril. Yamin et al. [37] have demonstrated that the metal-induced fibrillization of β-synuclein is further accelerated by the addition of high concentrations of crowding agents. The major factor responsible for the acceleration is excluded volume, and simple excluded volume theory predicts that an increase in the fractional volume occupancy of macromolecules in a physiological fluid can nonspecifically accelerate the formation of amyloid fibrils of any amyloidogenic protein [30,38,39]. However, McNulty et al. [40] have recently demonstrated that macromolecular crowding in the Escherichia coli periplasm or 300 g/l of bovine serum albumin (BSA) in vitro is sufficient to stabilize the disordered, monomeric structure of α-synuclein. Amyloid fibrils associated with neurodegenerative diseases such as Alzheimer's disease and transmissible spongiform encephalopathy, as well as nonneuropathic systemic amyloidosis, can be considered biologically relevant failures of posttranslational quality control maintained by molecular chaperones and proteases [41]. Cellular protection mechanisms, such as molecular chaperones and the protein degradation machinery, are crucial in the prevention of diseases in normally functioning living organisms [21]. In this study, we demonstrated that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixture of BSA and Ficoll 70, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism. 2. Materials and methods 2.1. Materials Hen egg white lysozyme was obtained from Sigma (Sigma-Aldrich Co, St. Louis, MO) and was used without further purification. The A1% 1 cm value of 26.5 at 280 nm [14] was used for protein concentration measurements. The two crowding agents, Ficoll 70 and BSA, were purchased from Sigma. Thioflavin T (ThT) and Congo red (CR) were also products of Sigma. All other chemicals used were made in China and of analytical grade. Aqueous solution of HCl at pH 2.0 containing 100 mM NaCl and 0.2% NaN3 was used for amyloid formation of lysozyme.
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20 mM sodium phosphate buffer at pH 7.0 was used for the activity assay of lysozyme. All buffers were filtered through a 0.22-μm pore size filter before use to remove insoluble particles.
2.2. Preparation for stock solutions of crowding agents Stock solutions of Ficoll (200 g/l) and BSA (200 g/l) were prepared in aqueous solution of HCl at pH 2.0 containing 100 mM NaCl and 0.2% NaN3. The pH values of these solutions were adjusted to 2.0 with HCl.
2.3. Amyloid formation Hen egg white lysozyme was first dissolved in aqueous solution of HCl at pH 2.0 containing 100 mM NaCl and 0.2% NaN3. After extensive dialysis and dilution to a final concentration of 25 g/l with the same solvent, hen lysozyme was unfolded by incubation at 37 °C for 3–5 days. Then the lysozyme solution was mixed with stock solutions of crowding agents, to yield a solution of 5 g/l lysozyme in HCl (pH 2.0) containing 100 mM NaCl, 0.2% NaN3, and a chosen concentration of a mixed or single crowding agent, followed by incubation at 37 °C for at least 20 days with a constant stirring rate at 220 rpm. At different time intervals, aliquots of sample were taken out for a series of biophysical or morphological measurements.
2.4. Thioflavin T binding assays The thioflavin T binding assays were performed according to Naiki et al. [42]. The fluorescence of ThT was excited at 440 nm with a slit-width of 10 nm and the emission was measured at 482 nm with slit-width of 5 nm on a LS-55 luminescence spectrometer (PerkinElmer Life Sciences, Shelton, CT). A 3 mM ThT stock solution was freshly prepared in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl, and passed through a 0.22-μm filter pore size filter before use to remove insoluble particles. Samples (10 μl) incubated under different conditions were diluted into 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 65 μM ThT, giving a final volume of 3 ml. The fluorescence intensity at 482 nm was averaged for 60 s to increase the signal-tonoise ratio of the measurements. Control experiments were performed to ensure that the crowding agents had no influence on the ThT binding assays.
2.5. Lysozyme activity assays Aliquots of the hen egg white lysozyme sample were diluted to 5 μM in HCl (pH 2.0) containing 100 mM NaCl and 0.2% NaN3, and the enzymatic activity was assayed by mixing 10 μl of diluted sample with 490 μl of 0.5 mg/ml Micrococcus lysodeikticus cell wall suspension in 20 mM sodium phosphate buffer at pH 7.0, and then by monitoring the decrease in absorbance at 450 nm during 60 s and at 25 °C. All enzymatic activities were normalized against and expressed as a percentage of 5 μM solution of native lysozyme in HCl at pH 2.0 containing 100 mM NaCl and 0.2% NaN3, and the specific activity of native lysozyme under such conditions was (5.10± 0.05) × 104 U mg− 1 (n = 3). Control experiments were performed to ensure that the crowding agents had no influence on the activity assay.
2.6. Transmission electron microscopy Aliquots of protein samples (100 μg/ml) under different conditions were produced by 50-fold dilution of the original samples and a drop was deposited on formvar-coated copper grids (200 mesh). The grids were then negatively stained with 2% (w/v) uranyl acetate for 45 s before examination using an H-8100 transmission electron microscope (Hitachi, Tokyo, Japan) operating at accelerating voltages of 100 kV.
2.7. Isothermal titration calorimetry ITC measurements were performed at 37.0 °C by using a VP-ITC titration calorimeter (MicroCal, Northampton, MA). All solutions were thoroughly degassed before use by stirring under vacuum. Before each experiment, the ITC sample cell was washed several times with HCl at pH 2.0. The sample cell was loaded with 1.43 ml of
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hen egg white lysozyme solution (17.4 μM) incubated at pH 2.0 and the reference cell contained doubly distilled water. Titration was carried out using a 280-μl syringe filled with BSA (or Ficoll 70) solution, with stirring at 300 rpm. The concentration of BSA (or Ficoll 70) was 139 μM. Injections were started after baseline stability had been achieved. Titrations were performed by adding an aliquot of 4 μl for the first injection and aliquots of 10 μl for the following injections with a 5-min interval between injections. Heats of dilution were determined by injecting BSA (or Ficoll 70) into the buffer alone and the total observed heats of binding were corrected for the heat of dilution. MicroCal ORIGIN software supplied with the instrument was used to determine the site binding model that gave a good fit (low χ 2 value) to the resulting data. From the various binding models tested (a single set of identical sites model, two sets of independent sites model, and sequential binding sites model), only the three sequential binding sites model fitted adequately to the binding isotherms, and the standard molar enthalpy change for the binding between BSA and nonnative lysozyme, Δb H 0m, and the binding constant, Kb, were thus obtained. The standard molar free energy change, Δb G 0m, and the standard molar entropy change, Δb S 0m, for the binding reaction were calculated by the fundamental equations of thermodynamics [43,44]: Db G0m ¼ RT ln Kb
ð1Þ
Db Sm0 ¼ Db Hm0 Db G0m =T :
ð2Þ
retained its native conformation under such mixed crowding conditions. In contrast, the activity of hen lysozyme in the absence of crowding agents was completely lost after incubation for 35 days (Fig. 2A). At the same time, amyloid formation of hen lysozyme was blocked by such mixed crowding agents and reached a maximum in the absence of crowding agents (Fig. 1A), suggesting that there is a negative correlation between amyloid content and residual activity change of lysozyme under different compositions and concentrations of crowding agents. Control experiments showed that the activity of lysozyme decreased nearly to 70% and 0 after incubation for 35 days in the presence of 5 g/l BSA (Fig. 2B) and in the presence of 25–100 g/l Ficoll 70 (Fig. 2C) respectively, indicating that neither 5 g/l of BSA alone nor 25–100 g/l Ficoll 70 alone stabilized lysozyme activity on the time scale of 35 days,
3. Results 3.1. Effect of mixed crowding agents on amyloid formation of lysozyme We have demonstrated that mixed macromolecular crowding reflects the physiological environments more accurately than individual crowding agents [34,35]. In this study, we employed such mixed crowding agents to test their effects on amyloid formation of hen lysozyme. Scale bar N in Fig. 1A represented amyloid formation of hen egg white lysozyme in the absence of crowding agents, and scale bar 5, 10, 25, 50, and 75 represented that in the presence of 100 g/l mixture of BSA and Ficoll 70, in which the weight ratio of BSA to Ficoll 70 is 5:95, 10:90, 25:75, 50:50, and 75:25, respectively. As can be seen from Fig. 1, ThT fluorescence intensity of hen egg white lysozyme in the absence of crowding agents increased to a maximum of about 130 U after incubation for 35 days. In contrast, ThT fluorescence for hen lysozyme in the presence of these mixed crowding agents had no obvious change and was below 10 U after incubation for 35 days (Fig. 1A), indicating that amyloid formation of lysozyme was blocked by such mixed crowding agents. As shown in Fig. 1B, ThT fluorescence for lysozyme incubated for 35 days in the presence of 5 or 10 g/l BSA increased to 57 or 21 U, indicating that 5 or 10 g/l of BSA alone did not block amyloid formation of lysozyme, although the protein crowding agent BSA at 100 g/l almost completely inhibited amyloid formation of lysozyme on the investigated time scale. Furthermore, 25–100 g/l Ficoll 70 alone did not impede amyloid formation of lysozyme effectively (Fig. 1C). We thus conclude that both BSA and Ficoll 70 cooperatively contribute to the inhibitory effect of mixed crowding agents. Activity measurements further supported our conclusion. As shown in Fig. 2A, the remaining activity of lysozyme in the presence of such mixed crowding agents decreased slightly after incubation for 35 days, indicating that the majority of lysozyme
Fig. 1. Effects of mixed or single crowding agents on amyloid formation of lysozyme monitored by ThT fluorescence. Lysozyme was incubated in HCl (pH 2.0) containing 100 mM NaCl and 0.2% NaN3 in the absence of crowding agents (N) and in the presence of 100 g/l mixture of BSA and Ficoll 70 (A), or single component crowding agents (B, BSA, and C, Ficoll 70). ThT Fluorescence of lysozyme was measured after incubation for 35 days as a function of the ratio (%) of the weight of BSA to the total weight of mixed crowding agents (A), or a function of the concentrations of BSA (B) or Ficoll 70 (C). The data with error bars were expressed as mean ± S.D. (n = 2–3).
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although the protein crowding agent BSA at 100 g/l stabilized lysozyme activity on the investigated time scale (Fig. 2B). We thus conclude that both BSA and Ficoll 70 cooperatively contribute to the stabilization effect of mixed crowding agents. 3.2. Inhibition of amyloid formation of lysozyme by delayed addition of mixed crowding agents We further investigated the effect of mixed macromolecular crowding agents on amyloid formation of lysozyme by delayed addition of such mixed crowding agents. The weight ratio of BSA to Ficoll 70 as 5:95 was chosen for the mixed crowding agent, and the addition time of such a mixed crowding agent was designed to cover all of the three different stages of amyloid
Fig. 3. Kinetics of fibrillization and remaining enzymatic activity of lysozyme after adding mixed macromolecular crowding agents at various time points. (A) Amyloid formation of lysozyme was monitored by ThT fluorescence in the absence of crowding agents (open square) and in the presence of 5 g/l BSA + 95 g/ l Ficoll 70 added at 1 (open triangle), 6 (inverted filled triangle), 10 (open circle), 12 (filled circle), and 14 (filled triangle) days, respectively. (B) Remaining enzymatic activity of lysozyme in the absence of crowding agents (a) and in the presence of 5 g/l BSA + 95 g/l Ficoll 70 added at 1 (b), 6 (c), 10 (d), 12 (e), and 14 (f) days was assayed after incubation for 22 days. The data with error bars were expressed as mean ± S.D. (n = 2–3).
Fig. 2. Effects of mixed or single crowding agents on remaining enzymatic activity of lysozyme in these crowded solutions during amyloid formation. Lysozyme was incubated in HCl (pH 2.0) containing 100 mM NaCl and 0.2% NaN3 in the absence of crowding agents (N) and in the presence of 100 g/l mixture of BSA and Ficoll 70 (A), or single component crowding agents (B, BSA, and C, Ficoll 70). Remaining enzymatic activity of lysozyme was assayed after incubation for 35 days as a function of the ratio (%) of the weight of BSA to the total weight of mixed crowding agents (A), or a function of the concentrations of BSA (B) or Ficoll 70 (C). The data with error bars were expressed as mean ± S.D. (n = 2–3).
formation. As shown in Fig. 3A, the mixture of 5 g/l BSA and 95 g/l Ficoll 70 added at 1, 6, and 10 days in the lag phase inhibited the increase of ThT fluorescence intensity of hen lysozyme. Interestingly, such a mixture added at 12 days in the growth phase showed a similar inhibitory effect. When added at 14 days in the final equilibrium phase, no obvious inhibitory effect of such a mixed crowding agent on ThT fluorescence intensity of lysozyme was observed, indicating that mixed macromolecular crowding has no effect on the mature fibrils of lysozyme. The remaining activity of hen lysozyme in the presence of the mixture of 5 g/l BSA and 95 g/l Ficoll 70 after 22day of incubation is shown in Fig. 3B, compared to that in the absence of crowding agents. Lysozyme incubated in such mixed crowded solution added at 1, 6, and 10 days in the lag phase remained about 99%, 89% and 87% relative enzymatic activity, respectively. The relative activity of lysozyme was 44% and 3% in the presence of this mixed crowding agent added at 12 and 14 days, suggesting that mixed macromolecular crowding stabilized the native structure of lysozyme and thus inhibited its structure transformation as well as amyloid formation, even in the growth phase. 3.3. Morphology of lysozyme samples monitored by TEM Transmission electron microscopy (TEM) was employed to study the morphology of hen egg white lysozyme samples
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Fig. 4. Transmission electron micrographs of lysozyme samples at pH 2.0 after incubation for 14 days in the presence of mixed or single crowding agents. Lysozyme was incubated in the presence of 100 g/l BSA (A), 100 g/l Ficoll 70 (B), 5 g/l BSA + 95 g/l Ficoll 70 (C), or 5 g/l BSA (D). A 2% (w/v) uranyl acetate solution was used to negatively stain the fibrils. The scale bars represent 200 nm.
incubated under different conditions. As shown in Fig. 4A and C, no fibril structure was observed in the presence of 100 g/l BSA or 100 g/l mixed crowding agent (5 g/l BSA + 95 g/l Ficoll 70). A mixture of protofibrils and separate fibrils [45,46] of hen lysozyme was formed in the presence of 5 g/l BSA (Fig. 4B), indicating that 5 g/l BSA only was not enough to inhibit amyloid formation of lysozyme. As a control study, the majority of separate fibrils were observed in the presence of 100 g/l Ficoll 70 (Fig. 4B). The results from TEM study are in accordance with those from ThT binding and activity assays. 3.4. Weak, nonspecific interactions between BSA and nonnative lysozyme detected by ITC Because weak, nonspecific interactions between macromolecular reactants and constituents of the local environment can greatly influence the equilibria and rates of reactions in which they participate [24,35], we investigated the effect of possible complex formation between BSA and nonnative lysozyme on amyloid formation of hen lysozyme. By using isothermal titration calorimetry (ITC), which is thought to be one of the most reliable and high-precision methods to quantitate protein interactions [43,47], we observed a weak, nonspecific interaction between BSA and nonnative lysozyme at pH 2.0. The ITC results are shown in Fig. 5. The best fit for the integrated heat data was obtained using a three sequential binding sites model
described by Hammann et al. [48] and Roufik et al. [44] with the lowest χ2 (26.50 cal mol− 1), yielding the thermodynamic parameters for the interaction between BSA and nonnative 0 lysozyme: Kb,1 = (1.03 ± 0.04) × 105 M− 1 , ΔbHm,1 = − 46.6 ± −1 0 −1 0.7 kcal mol , ΔbG m,1 = − 7.12 ± 0.10 kcal mol , ΔbS0m,1 = − 127 ± 2 cal mol − 1 K − 1 , Kb,2 = (1.01 ± 0.04) × 10 5 M − 1 , ΔbH0m,2 = − 6.1 ± 1.5 kcal mol− 1, ΔbG0m,2 = − 7.11 ± 0.11 kcal mol − 1 , Δb S 0 m,2 = 3.2 ± 4.6 cal mol − 1 K − 1 , Kb,3 = (1.02 ± 0.07) × 10 5 M − 1 , Δ b H 0 m , 3 = − 73.0 ± 2.2 kcal mol − 1 , ΔbG 0 m,3 = − 7.11 ± 0.12 kcal mol − 1 , and ΔbS0 m,3 = − 213 ± 7 cal mol− 1 K− 1. These results showed that the binding of BSA to nonnative lysozyme was driven entirely by large favorable enthalpy decreases but with unfavorable entropy decreases for the first and the third sequential binding sites of nonnative lysozyme, implying that BSA may bind to lysozyme oligomers to prevent the formation of prefibrillar lysozyme, and bind with the protofibrils to retard fibril elongation of lysozyme. Furthermore, we performed ITC experiments on the binding of Ficoll 70 to nonnative lysozyme at pH 2.0 and found that the calorimetric data were too small to be fitted to any binding model (Fig. 6). No optimal fit was found for Ficoll 70, indicating that Ficoll 70 had no specific binding affinity for nonnative lysozyme under such experimental conditions. The above kinetic and ITC analyses suggested that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibited amyloid formation of lysozyme via mixed macromolecular crowding as well as weak,
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stabilized lysozyme activity on the investigated time scale, suggesting that, in addition to nonspecific excluded volume effects, weak and nonspecific protein–protein interactions could greatly affect protein misfolding in the highly complex intracellular environment. By using the absorbance maximum of the protein in the presence of Congo red as another indicator of amyloid fibrils [50], we obtained qualitatively similar results (data not shown), further supporting the conclusion reached by ThT binding assays and TEM that BSA and Ficoll 70 cooperatively contribute to the inhibitory effect of mixed crowding agents. Milojevic et al. have found that human serum albumin (HSA) caps the exposed hydrophobic patches located at the growing and/or transiently exposed sites of the Aβ oligomers, thereby blocking the addition of further monomers and the growth of the prefibrillar assemblies [51]. Both ThT binding assays and CR binding assays have been widely used for the characterization of in vitro amyloid fibrils [9,12,13,42,46,50,52,53]. There are suggestions that the dyes ThT and CR bind to the crossed-β structure in the amyloid fibrils [50,52,53]. One of the differences between both assays is that Congo red is not specific for amyloid fibrils. CR can also bind to native or partially folded states of several different proteins, regardless of their secondary structures and induce oligomerization of native proteins [54]. It has been suggested that amyloid fibril formation can occur when the native structure of a protein is destabilized, favoring formation of partially folded conformations [7,55–57]. Simple Fig. 5. ITC profiles for the binding of BSA to lysozyme samples at pH 2.0 after incubation for 10 days in the absence of crowding agents at 37.0 °C. The top panels (A) represent the raw data for sequential 10-μl injections of BSA (139 μM) into lysozyme (17.4 μM) solution incubated for 10 days. The middle panel (B) represents the control experiment in which BSA (139 μM) was injected into the buffer alone. The bottom panels (C) show the plot of the heat evolved (kcal) per mole of BSA added, corrected for the heat of BSA dilution, against the molar ratio of BSA to lysozyme incubated for 10 days. The volume of the first injection of BSA was 4 μl (A and B). The data (filled square) were fitted to a three sequential binding sites model (C), and the solid lines represented the best fit.
nonspecific interactions between BSA and nonnative lysozyme observed in dilute solutions. 4. Discussion The cellular environment comprises a heterogeneous mixture of proteins, nucleic acids, ribosomes, and carbohydrates (polysaccharides), each of which is likely to affect the folding mechanism of different proteins in a distinctive fashion [49]. There may be hidden facts that proteins have evolved to function in such a mixed crowded milieu. We extended our idea of mixed macromolecular crowding [34,35] to study the effects of mixed crowding agents on amyloid formation of hen egg white lysozyme, compared with single crowding agents. Although macromolecular crowding accelerates amyloid formation of native disordered protein α-synuclein [8,22,23] and human apolipoprotein C-II [36], our data demonstrated that the mixed crowding agent containing both BSA and polysaccharide almost completely inhibited amyloid formation of lysozyme and
Fig. 6. ITC profiles for the binding of Ficoll 70 to lysozyme samples at pH 2.0 after incubation for 10 days in the absence of crowding agents at 37.0 °C. The top panels (A) represent the raw data for sequential 10-μl injections of Ficoll 70 (139 μM) into lysozyme (17.4 μM) solution incubated for 10 days. The bottom panels (B) show the plot of the heat evolved (kcal) per mole of Ficoll 70 added, corrected for the heat of Ficoll 70 dilution, against the molar ratio of Ficoll 70 to lysozyme incubated for 10 days. The volume of the first injection of Ficoll 70 was 4 μl (A). The data (filled square) were too small to be fitted, indicating that no binding was observed in the conditions used.
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excluded volume theory predicts that high concentration of ‘inert’ macromolecules should stabilize the compact native state relative to any less compact unfolded or partially folded state of the polypeptide [29–31,58]. In addition, it has been reported that at pH 2.0 the molten globule states of hen egg white lysozyme and horse heart myoglobin are stabilized relative to the fully unfolded forms by dextran at high concentrations [31,59]. In this study, the major reason for the inhibitory effect is that hen egg white lysozyme was stabilized in its compact, functional structure by the mixed macromolecular crowding agent used, via nonspecific excluded volume effects as well as weak, nonspecific interactions between BSA and nonnative lysozyme. However, the possibility that the inhibitory and stabilizing effects reflect the enhancement of nonspecific interactions between BSA and nonnative lysozyme by macromolecular crowding effected by Ficoll 70, BSA serving as a ‘chaperone’ [60] in lysozyme protection, cannot be excluded. In addition, BSA oligomerization due to macromolecular crowding may influence lysozyme activity. It has been reported that the lower stability of the native state of the amyloidogenic proteins, such as α-synuclein [22,23] and human lysozyme variants [20], is the primary factor resulting in their tendency to form amyloid deposits in crowded intracellular environments and hence to be associated with diseases [4,5,61,62]. Therefore it is interesting to assume that non-disease-associated proteins with stable structures, such as hen egg white lysozyme, could efficiently avoid the formation of fibrillar deposits in crowded physiological fluid. In vitro experiments have suggested that all peptides and proteins have the potential to form fibrils under appropriate conditions [6–9,63]. Amyloid formation of some stable proteins such as hen egg white lysozyme does occur in vitro under appropriate conditions [10–15], but they never occur in vivo. They have relative stable native conformation compared with mutant proteins and native disordered proteins related to serious diseases, and a posttranslational quality control mechanism maintains their structures throughout their functional life-time [41]. In this study, we demonstrated that the crowded intracellular environment has a remarkable inhibitory effect on amyloid formation of a stable protein in a stressed environment. It has been noted that an increased level of macromolecular crowding may not always promote diseases [8] and macromolecular crowding does enhance native state stability of globular proteins [64]. Combined with the results that chaperones are more effective in assisting protein refolding under crowding conditions than those in the absence of crowding agents [65], we conclude that in addition to the present knowledge of posttranslational quality control mechanism, mixed macromolecular crowding may play a role in the mechanism that regulates normally functioning proteins. In summary, both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and
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