Biomacromolecules 2008, 9, 3165–3172
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Net Charge Affects Morphology and Visual Properties of Ovalbumin Aggregates Mireille Weijers,†,§,#,3 Kerensa Broersen,*,†,‡,#,O Peter A. Barneveld,§ Martien A. Cohen Stuart,§ Rob J. Hamer,†,‡,| Harmen H. J. De Jongh,†,| and Ronald W. Visschers†,⊥ Wageningen Centre for Food Sciences/Top Institute Food and Nutrition, Nieuwe Kanaal 9a, 6709 PA Wageningen, The Netherlands, Laboratory for Food Chemistry, Wageningen University and Research Centre, Bomenweg 2, 6700 EV Wageningen, The Netherlands, Laboratory of Physical Chemistry and Colloid Science, Wageningen University and Research Centre, Dreijenplein 6, 6703 HB Wageningen, The Netherlands, TNO Quality for Life, Utrechtseweg 48, 3704 HE Zeist, The Netherlands, and NIZO Food Research, Kernhemseweg 2, P.O. Box 20, 6710BA Ede, The Netherlands Received July 7, 2008; Revised Manuscript Received September 9, 2008
The effect of ovalbumin net charge on aggregate morphology and visual properties was investigated using chromatography, electrophoresis, electron microscopy, and turbidity measurements. A range of differently charged ovalbumin variants (net charge ranging from -1 to -26 at pH 7) was produced using chemical engineering. With increasing net charge, the degree of branching and flexibility of the aggregates decreased. The turbidity of the solutions reflected the aggregate morphology that was observed with transmission electron microscopy. Increasing the stiffness of the aggregates transformed the solutions from turbid to transparent. Artificially shielding the introduced net charge by introducing salt in the solution resulted in an aggregate morphology that was similar to that for low-net-charge variants. The morphology of heat-induced aggregates and the visual appearance of the solutions were significantly affected by net charge. We also found that the morphology of ovalbumin aggregates can be rapidly probed by high-throughput turbidity experiments.
Introduction Aggregate formation by proteins has been a major topic for a number of decades in biology, medicine, and the food industry.1-5 The discovery that many neurodegenerative diseases are related to protein misfolding and aggregation has triggered an interest in understanding which factors regulate the aggregation properties of proteins. Many disease-related mutations in proteins are correlated with a neutralization of net charge.6,7 A number of computational methods that aim to predict the aggregation propensity of a wide range of polypeptide chains have appeared in literature.8-10 This reflects the thought that the capability to aggregate is strongly preserved among all polypeptide chains and may be assigned to specific characteristics of the sequence. An important factor found to affect the aggregation propensity of polypeptide sequences is the net charge of the protein.8-10 Even though every amino acid sequence can show aggregation in theory, the morphology of the formed aggregates can widely vary from ordered to disordered aggregates, or mixtures of these.10,11 The factors that underlie these differences have been assigned to differences in * Corresponding author. E-mail:
[email protected]. Tel: +32 (0) 26291996. † Wageningen Centre for Food Sciences/Top Institute Food and Nutrition. ‡ Laboratory for Food Chemistry, Wageningen University and Research Centre. § Laboratory of Physical Chemistry and Colloid Science, Wageningen University and Research Centre. | TNO Quality for Life. ⊥ NIZO Food Research. # Both authors contributed equally to the work. 3 Present address: Danone Research, P.O. Box 7005, 6700 CA Wageningen, The Netherlands. O Present address: Switch Laboratory, Flanders Institute for Biotechnology (VIB) and Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Brussels, Belgium.
amino acid sequence and environmental factors.12 Aso et al.12 investigated a number of non-disease-related proteins and reported major differences in aggregate morphology by using Atomic Force Microscopy. A number of these formed amyloid fibril structures under specific conditions. This so-called mature amyloid fiber morphology (unbranched fibers with a diameter of 6-12 nm and indeterminate length13) has been related to the end-stage of a number of neurodegenerative diseases.14 Also, from an industrial perspective, different types of network structures have been shown to provide variety in the texture of food products.15 Therefore, it is useful to investigate how the surface charge of a protein molecule may be able to affect aggregate morphology. It has been reported that aggregate morphology and the visual aspects of the formed aggregates may be related.16 For example, Mine16 showed that increasing the net charge changed the transparency and elasticity of ovalbumin gels. Because the visual appearance of a solution would provide for a very rapid assay for the evaluation of fibril morphology, as compared with the more labor-intensive screening of aggregate morphologies using high-resolution microscopy, we also report on the value of turbidity measurements as a rapid screening technique for ovalbumin aggregate morphology. Here we report on the consequences of the net charge modification of ovalbumin, a hen egg-white protein, on aggregate morphology and visual properties. The electrostatic repulsion of polypeptide chains can be altered by chemical modification of readily expressed proteins or by the introduction of charged residues into a polypeptide chain by point mutations. In a previous article, we showed that we can chemically modify ovalbumin within a wide range of charges by using methylation or succinylation.17 Successful charge modification in this way was later confirmed for green fluorescent protein and glutathione S-transferase by Lawrence et al.18
10.1021/bm800751e CCC: $40.75 2008 American Chemical Society Published on Web 10/10/2008
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Because protein aggregation is usually the result of irreversible unfolding, the (ir)reversibility of this process of the charge variants of ovalbumin has been described in detail in a previous article.17 It was found to be possible, under specific conditions, to use random charge engineering of proteins in a nondestructive manner with respect to the folded structure of the wild-type protein. By the use of succinylation and methylation, four ovalbumin variants were generated with charges of -1 (OVA1), -5 (OVA-5), -12 (OVA-12), and -26 (OVA-26). The numbers denote the net charges calculated at pH 7. It was previously demonstrated that these variants showed no changes in the overall topology (i.e., in protein conformation, surfaceexposed hydrophobicity, or disulphide interaction potential).17 Therefore, we consider this series to be an excellent model system for the study of the effect of electrostatic interactions on aggregation. This series of charge variants produced by succinylation or methylation can serve as a useful approach to investigating the effect of charge variation on aggregate morphology.
Results In an earlier article, we reported on the characterization of a series of ovalbumin variants with ranging net charge by chemical modification (either succinylation of lysine groups or methylation of carboxyl groups).17 The obtained variants were characterized in terms of size, hydrophobicity, sulphydryldisulphide exchange index (SEI), and structural integrity. On the basis of the results of this characterization, four variants with retained globular structures that have comparable molecular characteristics, as mentioned above, but differ in their net charge (OVA-1, OVA-5, OVA-12, and OVA-26, in which the numbers -1 to -26 represent the calculated net charges of the proteins at pH 7) were selected. Using these engineered ovalbumin variants, we expect to elucidate the way in which net charge affects aggregate morphology. Polydispersity of Ovalbumin Aggregates. Size exclusion chromatography (SEC) was used to study the effect of net charge on the polydispersity of the formed aggregates. All variants were heated to 72 °C, and aliquots were withdrawn at various incubation times ranging from 0 to 5000 min and analyzed by SEC. Figure 1 shows elution profiles of heated solutions of the ovalbumin variants (OVA-26, OVA-12, and OVA-5). Aggregates of ovalbumin showed a wide size distribution and eluted at volumes between 16.0 and 22.5 mL depending on the charge variant studied. With increasing incubation time, the fraction of monomers decreased, as was shown by the decreasing monomer peak height at 22 to 22.5 mL elution volume and by peaks appearing at lower elution volumes representing aggregated material. It was observed in the elution profile of OVA12 heated at 10 mg/mL that the aggregates formed are baselineseparated from the monomeric peak. This suggests that under these conditions, large, more discrete aggregates were formed. Analysis of SEC results did not suggest the formation of detectable fractions of dimers and trimers. These findings were also valid for OVA-5. However, the aggregates formed for OVA-5 show a larger polydispersity (Mw/Mn ≈ 3) in size compared with those formed for OVA-12. This was shown by the wide range of elution volumes at which the aggregates are eluted (18.0 to 22.4 and 20.0 to 22.0 mL for OVA-5 and OVA12, respectively). In the left upper panel, chromatograms of OVA-26 solutions heated at 40 mg/mL are shown. The inset demonstrates that no clear separation can be observed between the monomeric and aggregate elution positions; rather, the peaks
Figure 1. Kinetics of ovalbumin aggregation. Size exclusion chromatograms of differently charged ovalbumins at a concentration of 10 mg/mL (pH 7) (OVA-26 at 40 mg/mL) incubated at 72 °C. The inset is an enlargement of the SEC-MALLS data from the chromatogram of OVA-26. The peak at elution volumes between 22.5 and 24.5 mL corresponds to monomers.
merge. This suggests that the aggregation process involves the formation of small oligomers that could not be baselineseparated from the monomers. Microstructural Organization of Aggregates. Figure 2 shows cryo-TEM images of the microstructural organization of the aggregates obtained from the incubation of the ovalbumin variants at elevated temperature. It was found that net charge has an effect on fibril morphology and contour length (lc). The characteristics derived from analyzing the cryo-TEM images of the formed aggregates and the effects of net charge are summarized in Table 1. For OVA-1 and OVA-5, the aggregates appear to be very curved, and in some cases, particularly for OVA-1, they appear to be organized in clusters (lower panel of Figure 2). The aggregates formed by these variants also show a significant degree of branching. Upon increasing the net charge to -12 (OVA-12), the aggregates retain some of their curvature, although it is clearly decreased compared with the OVA-1 and OVA-5 aggregates, but the degree of branching is nearly negligible. The aggregates prepared from OVA-26 were again less curved and appeared to be short stiff rods without any observed branching. These results show that the morphology of aggregates is clearly affected by the net charge of the protein. Apparently, decreasing the net charge results in an increased degree of branching and a decreasing persistence length, which characterizes the curvature. The contour length of the aggregates shows an apparent maximum between a net charge of -12 and -5. At a higher net charge, the aggregates were significantly
Effect of Ovalbumin Net Charge
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Figure 2. Microstructural organization of aggregates formed by ovalbumin variants with varying net charge investigated by cryo-TEM after incubating for 24 h at 72 °C and pH 7. Ovalbumin variants were initially heated at 25 (OVA-1), 52 (OVA-5), 55 (OVA-12), and 42 mg/mL (OVA-26) and were diluted to 1 mg/mL before cryo-TEM images were recorded. The length of the bar represents 100 nm. Table 1. Aggregate Characteristics: Contour Length (lc), Morphology of Aggregates, and Critical Gelation Concentration (C*) of Ovalbumin Variants ovalbumin variant
lc (nm)a
morphologya
C* (mg/mL)
OVA-26 OVA-12 OVA-5
20-200 400-1100 400-1000
42 55 52
OVA-1
n.d.b
linear rods curved fibrils curved, branched fibrils clusters of branched aggregates
a
Determined by analysis of cryo-TEM images.
b
25
Not determined.
shorter. At a lower net charge, the aggregates formed clusters, which hampered a reliable determination of the contour length. Visual Properties of Heat-Induced Ovalbumin Aggregates. It was previously shown that the visual appearance (i.e., turbidity) of aggregated solutions is strongly associated with their morphological organization. Transparent samples primarily consist of fibrilar structures, whereas turbid samples are built up from branched structures.19 Therefore, we hypothesized that measuring the turbidity is a convenient technique to screen for the microstructure of aggregates rapidly. Solutions of OVA-12 were heated to 78 °C (pH 7) for 1 h, and the turbidity (τ) at 500 nm was measured spectrophotometrically as a function of protein concentration. Because literature suggested that a high net charge can be significantly shielded by the addition of a salt, we also studied the effect of NaCl concentration. If the effects of the charge variation are directly responsible for the observed effects on aggregate formation and morphology, then shielding these differences should lead to the possibility of forming similar aggregate morphologies for all ovalbumin charge variants (Figure 3). Figure 3a shows the diagram of states of OVA-12 for which the protein concentration varied between 10 and 100 mg/mL
and the NaCl concentration varied between 0 and 100 mM NaCl. We classified τ in three regimes: transparent, translucent, and turbid. (See Figure 3 and theExperimental Methods section for details). The individual measurements are presented by the various symbols, as explained in the legend. With increasing ovalbumin concentration, a lower concentration of NaCl is required to obtain a high turbidity of the aggregated solution. Also, the critical gelation concentration was affected by the charge variation of ovalbumin (Table 1). Figure 3a shows that at all protein concentrations transparent solutions or gels are formed when ovalbumin is incubated in the presence of
