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- Materials and methods
Despite the widespread use of sonication in medicine, industry, and research, the effects of sonication on proteins remain poorly characterized. We report that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been implicated in, causing of a wide range of protein conformational disorders including Alzheimer's disease, Huntington's disease, Parkinson's disease, and prion diseases. The aggregates cause large enhancements in fluorescence of the dye thioflavin T, exhibit green-gold birefringence upon binding the dye Congo red, and cause a red-shift in the absorbance spectrum of Congo red. In addition, circular dichroism reveals that sonication-induced aggregates have high β-content, and proteins with significant native α-helical structure show increased β-structure in the aggregates. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication-induced aggregates, including fibrils with diameters of 5–20 nm. The addition of preformed aggregates to unsonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. The dye-binding and structural characteristics, as well as the ability of the sonication-induced aggregates to seed the formation of new aggregates are all similar to the properties of amyloid. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders.
Loomis and Wood first reported the damaging effects of ultrasound radiation on biological systems in 1927 (Wood and Loomis 1927). In the intervening years, myriad applications of ultrasound in medicine and industry have been developed; however, the details of ultrasound-induced damage to biomolecules, especially proteins, remain poorly characterized. Such characterization is difficult, owing to the potentially complex mechanisms of sonication-induced damage. These may include the formation of liquid–gas interfaces, local heating effects, mechanical/sheer stresses, and free radical reactions (Hawkins and Davies 2001; Mason and Peters 2002). The functional native structure of proteins is determined by the subtle balance between many noncovalent interactions; this balance can be easily disrupted by the above mechanisms, leading to protein denaturation and aggregation.
Many applications of ultrasound in common use today may alter protein structures (Mason and Peters 2002). For example, sonication is used to prepare proteinaceous micro-spheres of human serum albumin (Grinstaff and Suslick 1991) (e.g., Albunex and Optison); these are widely used as ultrasound contrast agents, and are being investigated as possible gene transfer vehicles (Li et al. 2003). Sonication is also employed in procedures to encapsulate therapeutic proteins, such as asparaginase, insulin, and erythropoietin, in biodegradable poly(D,L-lactide-co-glycolide) microspheres for controlled release in vivo (Bittner et al. 1998; Jiang et al. 2003; Wolf et al. 2003). These microsphere protein-loading techniques are known to result in some protein inactivity and aggregation (van de Weert et al. 2000). Sonication is also used to sterilize surgical and dental instruments, for dental descaling; in water treatment for inactivation of chemical and biological pollutants; in the food industry for the preparation of emulsions; and in laboratories for cell disruption and, recently, for studies of protein conformational disorders. In these disorders, which include, for example, Alzheimer's disease, Huntington's disease, prion diseases, immunoglobulin light chain disorders and serpinopathies, naturally occurring proteins are altered or mutated, and the variant proteins misfold to form aggregates that may be causative agents in the diseases (Soto 2001; Lomas and Carrell 2002; Stefani and Dobson 2003). Soto and coworkers have reported a new method, termed protein misfolding cyclic amplification (PMCA), for possible diagnosis of prion disorders (Saborio et al. 2001; Soto et al. 2002). In PMCA, tiny quantities of prion aggregates can be detected with high sensitivity by amplifying the aggregates through cycles of sonication (to break existing aggregates into smaller pieces) followed by incubation periods (in which the small aggregates act as seeds for the formation of new aggregate from soluble prion protein). Sonication is also frequently used to break aggregates into smaller pieces for seeding new aggregate growth in laboratory studies of proteins and peptides associated with various conformational disorders, such as Alzheimer's (Jarrett et al. 1993; O'Nuallain et al. 2004), Huntington's (Chen et al. 2001), prion diseases (Saborio et al. 2001; Soto et al. 2002), and serpinopathies (Crowther et al. 2003), as well as non-disease-associated proteins (Jarrett and Lansbury 1992; Ramirez-Alvarado et al. 2000).
Amyloid is a common aggregate structure that has been observed for numerous disease- and nondisease-associated proteins (Stefani and Dobson 2003), and has been proposed to be an alternative structure that may be adopted by all proteins under conditions where the native state is destabilized (Fandrich et al. 2001). We report here that sonication of a range of structurally diverse proteins results in the formation of aggregates that have tinctorial, structural, and seeding properties similar to those of amyloid. These results have important and far-reaching implications for the use of sonication in medicine, biotechnology, and research.
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- Materials and methods
To obtain representative information on the effects of sonication on proteins, a range of unrelated proteins with diverse structures was chosen for study (Table 1). Protein structures included mainly α-helical (bovine serum albumin [BSA], myoglobin), mixed α-helix and β-sheet (lysozyme, Tm0979), and mainly β-sheet (hisactophilin, Cu/Zn super-oxide dismutase [SOD]). Cysteine residues are a particularly common site for free radical reactions in proteins (Hawkins and Davies 2001), and play a central role in the sonication-induced formation of human serum albumin proteinaceous microspheres (Grinstaff and Suslick 1991). Thus, proteins were chosen to have different cysteine contents (Table 1): no cysteine (myoglobin, Tm0979), 1 free thiol and 0 or 17 disulfides (hisactophilin and BSA), and no free thiols and 2 or 4 disulfides (SOD and lysozyme). The effects of sonication were characterized by light scattering, binding of the dyes thioflavin T (ThT) and Congo red (CR), measurements of protein secondary structure using circular dichroism (CD) spectropolarimetry, determination of ultra-structure using transmission electron microscopy (TEM), assessing aggregate stability by denaturing gel electrophoresis (SDS-PAGE), and examining the seeding ability of sonicated protein solutions.
Cycle-dependence of aggregation
The conditions used herein for sonication were similar to those used in the PMCA methodology (Saborio et al. 2001; Soto et al. 2002); proteins were typically subjected to 40 cycles of sonication (5 pulses of 1 sec) followed by incubation periods (1 min). After 40 rounds of sonication, small amounts of visible precipitate were observed in all the protein solutions. Using this protocol >90% of the protein remained in solution after sonication, based on measurements of supernatant protein concentration after centrifugation of sonicated solutions. The relationship between the extent of sonication and amount of protein aggregation was investigated by measuring 90° light-scattering intensity and ThT binding as a function of number of sonication cycles (Fig. 1). The formation of protein aggregates that are large relative to the wavelength of incident light results in scattering of light; aggregate formation can also be monitored by enhanced fluorescence upon binding of ThT (see following section). A linear dependence was observed between light-scattering intensity and the number of sonication cycles or ThT fluorescence (Fig. 1A,B), suggesting that the amount of sonication-induced aggregation is proportional to the extent of the sonication treatment. To investigate the contribution of aggregate size to the total light scattering intensity as a function of sonication cycle number, additional dynamic light scattering (DLS) and TEM experiments were performed. The distribution of particle sizes measured by DLS (Fig. 1C) and the particle sizes and morphologies observed by TEM (data not shown) do not change significantly above 5 sonication cycles, consistent with amount of aggregate formation being proportional to the number of sonication cycles.
Thioflavin T and Congo red binding to aggregates
ThT (LeVine 1999) and CR (Klunk et al. 1999) are standard dyes used to monitor the formation of amyloid structure. Amyloid binding to ThT causes a marked enhancement of ThT fluorescence, while binding to CR causes a red shift in the absorbance spectrum of the dye and green-gold birefringence of aggregates under polarized light. ThT fluorescence of amyloid is typically measured using an excitation wavelength of 440 nm, giving an emission maximum at ∼482 nm. The proteins studied here do not absorb significantly in this wavelength range, except for myoglobin, in which the heme cofactor has significant absorbance (maximum at 407–410 nm). As a consequence, ThT fluorescence enhancement for myoglobin may be underestimated; consistent with this, the fluorescence values for both unsonicated and sonicated myoglobin are relatively low (Fig. 2; Table 1). Sonication caused significant enhancement of ThT fluorescence for all the proteins studied, ranging from 2.2-fold enhancement for myoglobin to 38-fold for Tm0979 (Fig. 2; Table 1). These enhancements are comparable to the 2–60-fold enhancements reported for amyloid and other amyloid-like fibrils (Ismail et al. 1992; LeVine 1993; Schmittschmitt and Scholtz 2003).
To investigate whether the different levels of ThT enhancement are related to a different mode of ThT binding by different aggregates, a ThT titration was conducted on aggregates of an all-α protein (BSA) and an all-β protein (SOD) (Fig. 3). The titration data transformed as Scatchard plots are linear for aggregates of both proteins, consistent with a single type of binding site in both cases. The apparent dissociation constants (Kd) and magnitudes of the fluorescence enhancements for both proteins agree within an order of magnitude, suggestive of roughly similar binding modes. Interestingly, however, ThT appears to bind more tightly upon binding to aggregates of the all-β protein.
Bright-field microscopy of aggregates treated with CR showed that aggregates of all the proteins bind the dye (Fig. 4, left panels) and exhibit birefringence under cross-polarized light (Fig. 4, right panels). The birefringence varies somewhat in color, but is often green-gold, as is characteristic for CR binding to amyloid (Westermark et al. 1999). Upon incubation of protein solutions for longer periods of time after sonication, further aggregate assembly was observed, as evidenced by increased formation of large tangles of fibrillar aggregates with strong birefringence (Fig. 4G–I), with a concomitant decrease in sample turbidity. The more pronounced birefringence in these samples is likely due to the changes in sample thickness, which is a critical determinant of CR birefringence (Wolman and Bubis 1965). Spectral shifts for CR upon aggregate binding were found to be maximal at 526–543 nm (Fig. 5; Table 1). These shifts are similar to the characteristic maximum difference at ∼541 nm observed for amyloid (Klunk et al. 1999), and the differential absorbance values are comparable to levels observed for other amyloid and amyloid-like fibrils (Gross et al. 1999; Chiti et al. 2001; Sirangelo et al. 2002; Koscielska-Kasprzak and Otlewski 2003).
Secondary structure of aggregates
Amyloid has a characteristic “cross-β” structure, in which a fibrillar structure is formed by β-strands that run perpendicular to the axis of the fibril (Sunde and Blake 1997). A change in CD spectrum characteristic of increased β-structure has been observed when various native proteins, including myoglobin (Fandrich et al. 2001) and lysozyme (Goda et al. 2000), convert to an amyloid structure. Secondary structure analysis of CD spectra of the native proteins studied here and of the aggregates formed after sonication is given in Table 1. For the proteins that contain substantial helical structure in the native state (myoglobin, BSA, and lysozyme), sonication causes an increase in β-structure with a concomitant decrease in α-helical structure. For the proteins with predominantly β-features in native CD spectra (SOD, hisactophilin, Tm0979), spectral changes upon sonication are less pronounced, and there is little apparent change in secondary structure.
Ultrastructure of aggregates
The ultrastructure of the sonication-induced aggregates was investigated by TEM; typical structures are shown in Figure 6. For all the proteins studied, the aggregates exhibit a range of morphologies, including apparently amorphous structures (Fig. 6, left panels) and fibrillar species (Fig. 6, right panels). The structures of these aggregates have a strong resemblance to structures reported for other unsonicated and sonicated amyloid aggregates (O'Nuallain et al. 2004). Fibril diameters are typically between ∼5 and 20 nm (Fig. 6, right panels), and fibrils are often observed in bundles (Fig. 6D–F). Within the 20 nm fibrils, coiling together of thinner fibrils is often apparent. These structures and diameters are similar to those reported for amyloid fibrils (Sunde and Blake 1997).
Amyloid fibrils and associated structures are generally very stable and resistant to resolubilization through various physical and chemical treatments, such as heat, reducing agents, and detergents (Glover et al. 1997; Scherzinger et al. 1997; Tanaka et al. 2002; Stefani and Dobson 2003). The aggregates formed here by sonication also appear to be very stable. Aggregates pelleted by centrifugation and resuspended in buffer did not redissolve over a timescale of weeks. The stability of pelleted aggregates was also tested by SDS-PAGE of samples boiled in buffer containing 7 M urea, 2% (w/v) of the detergent SDS, and in the presence and absence of the reducing agent, 5% (v/v) β-mercapto-ethanol. Substantial quantities of aggregate did not redissolve, and could be observed visually in the boiled samples. There appeared to be some limited resolubilization of the aggregates, because weak bands were observed at molecular weights corresponding to the monomer, after silver staining (data not shown). However, the observed monomeric bands may have been the result of protein adhering to or trapped within pellets, despite extensive washing prior to experiments.
Seeding with sonicated protein
Amyloid aggregates have been shown to act as seeds for the formation of new aggregate from soluble protein (Rochet and Lansbury 2000). In general, seeding effects tend to increase with increasing temperature, although they have been reported to decrease again at temperatures well above the thermal melting points of proteins (Ramirez-Alvarado et al. 2000; Fandrich et al. 2003). Seeding of further aggregate formation by sonication-induced aggregates was investigated by incubating native proteins at various temperatures in the presence and absence of aggregates and monitoring aggregate formation by ThT fluorescence (Fig. 7). Small increases in ThT fluorescence were observed for proteins incubated in the absence of aggregates; this is consistent with previous studies showing formation of amyloid at elevated temperatures by various proteins including myoglobin (Fandrich et al. 2003) and lysozyme (Morozova-Roche et al. 2000). When proteins were incubated in the presence of aggregates, the increases in ThT fluorescence were larger in magnitude and occurred more rapidly than in the absence of aggregates (Fig. 7). The enhancement and acceleration of aggregation generally increased with increasing temperature at temperatures below the thermal melting point of the proteins, and then decreased as temperature was increased further. Thus, sonication-induced aggregates appear to seed the formation of further amyloid-like aggregates from soluble protein in a similar way to that observed for amyloid aggregates.