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Cysteine-capped gold nanoparticles suppress aggregation of proteins exposed to heat stress
Article first published online: 22 FEB 2013
Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.
Volume 65, Issue 5, pages 454–461, May 2013
How to Cite
Luthuli, S. D., Chili, M. M., Revaprasadu, N. and Shonhai, A. (2013), Cysteine-capped gold nanoparticles suppress aggregation of proteins exposed to heat stress. IUBMB Life, 65: 454–461. doi: 10.1002/iub.1146
- Issue published online: 22 APR 2013
- Article first published online: 22 FEB 2013
- Manuscript Accepted: 9 JAN 2013
- Manuscript Received: 27 NOV 2012
- gold nanoparticles;
- malate dehydrogenase;
- bovine serum albumin;
- heat shock protein 70;
- heat-induced aggregation
Gold nanoparticles show a lot of promise as potential agents for drug delivery and disease diagnosis. Because of this, it is important that the interaction between gold nanoparticles and biomolecules be well characterized to avoid undesirable consequences. In this study, gold nanoparticles were synthesized by the reduction of gold salt by sodium borohydride in the presence of cysteine as the capping agent. The physical features of the nanoparticles were analyzed using Ultraviolet–Visible spectrophotometry and transmission electron microscopy. The interaction between gold nanoparticles and the following proteins: bovine serum albumin, citrate synthase, malate dehydrogenase, and human heat shock protein 70 was investigated by UV–Vis spectrophotometry. The stability of the proteins against heat stress was assessed by monitoring their aggregation at 48 °C, either in the presence or absence of gold nanoparticles. The gold nanoparticles were capable of suppressing the heat-induced aggregation of the proteins. Furthermore, apart from possessing independent protein-aggregation suppression function, the AuNPs also augmented the chaperone function of human heat shock protein 70. Findings from this study demonstrate that cyteine-coated gold nanoparticles exhibit chaperone-like activity and have the capability to stabilize proteins to which they may be conjugated. © 2013 IUBMB Life, 65(5):454–461, 2013.
Nanoparticles possess significant adsorption capacities due to their relatively large surface area that allows them to bind and/or carry other substances such as chemicals, drugs, probes, and proteins attached to their surface by covalent bonds or through adsorption. The physiochemical properties of nanoparticles such as charge and hydrophobicity can be altered by attaching specific chemical compounds, peptides, or proteins to their surfaces to design them for specific biological tasks. Gold nanoparticles (AuNPs) have the prospects for application in diverse biomedical applications, such as in drug delivery and diagnostic kits (1). They possess a high extinction coefficient in the visible region, making them an ideal colour reporting group for signaling molecular recognition events (2). AuNPs typically have dimensions ranging from 1 to 100 nm and display distinctive, valuable electric, and optical properties that make them attractive to use in biomedical applications.
One of the major draw-backs in the field of nanotoxicology is evidence-based understanding of the interaction between nanoparticles and biomolecules. It is believed that most physical materials that travel through blood get wrapped up by serum proteins immediately upon coming into contact with the blood (3, 4). Thus, under physiological conditions, nanoparticles may be perceived as complexes of the original nanoparticles physically coated by proteins. The composition of the nanoparticle corona suspended in the blood is determined by the concentration of serum proteins to which it is exposed under the prevailing physiological conditions (3).
The binding of proteins to a particular planar surface is often associated with significant changes to the secondary structure of the former. The small sizes of nanoparticles present a relatively large surface curvature that facilitates attachment of proteins (5). Furthermore, the degree of the structural perturbation that proteins undergo in response to their interaction with particular nanoparticles varies across protein species. The complete plasma proteome is composed of approximately 3700 different proteins and about 50 of these have been shown to associate with nanoparticles (6, 7). Therefore, there are concerns regarding the safety of nanoparticles and whether modifications that biological materials undergo upon their interaction with nanoparticles may alter their intended function (8).
Heat shock proteins (Hsps) are ubiquitous and highly conserved molecules, whose main role is to facilitate folding of other proteins to their native states (9). Some Hsps are constitutively expressed in cells, while others are upregulated in response to physiological stress. Their relative abundance in cells and in the blood circulatory system suggests that they may associate with nanoparticles administered for biomedical interventions. Heat shock protein 70 (Hsp70) is one of the most abundant members of this family of proteins. It is involved in the folding of other proteins (chaperone function) and operates in an ATP-dependent fashion (10). Hsp70 is capable of suppressing the heat-induced aggregation of model proteins such as malate dehydrogenase (MDH) in vitro (11).
The interaction between nanoparticles and biological materials is poorly understood. In light of the prospects of their use in biomedical applications, it is important to understand how AuNPs interact and influence the structure and function of proteins. In this study, we synthesized cysteine-coated AuNPs and investigated how these nanoparticles influence the structural and functional features of select proteins (MDH, citrate synthase [CS], human Hsp70 [hHsp70], and bovine serum albumin [BSA]).
It has been previously suggested that AuNPs may promote protein folding and this study was based on using AuNPs functionalized with 2-(10-mercaptodecyl)malonic acid (AuDA) (12). Heat stress promotes protein misfolding, and consequently promotes the aggregation of proteins. Within the cell, misfolded proteins tend to expose their hydrophobic motifs on the surface, thus facilitating their interaction with molecular chaperones whose peptide binding domains possess hydrophobic charges. Cysteine is commonly used as a coating agent that wraps AuNPs intended for biomedical application. In this study, we hypothesized that because of the hydrophobic character of cysteine, this molecule must be able to facilitate interaction of cysteine-coated AuNPs with proteins, thereby modifying the structure and stability of the latter.
We established that AuNPs were capable of inhibiting the heat-induced aggregation of proteins in a manner that is to some extent reminiscent of molecular chaperones. Furthermore, the cysteine-coated AuNPs complemented the chaperone function of human Hsp70 (hHsp70) in vitro. These findings suggest that AuNPs may promote the stability of proteins with which they associate. We discuss the implications of these findings with respect to the application of AuNPs in the biomedical and nanobiotechnology fields.
Materials and Methods
Gold (III) chloride (AuCl3), sodium borohydride (NaBH4), Bradford reagent, cysteine, BSA, CS, pig heart MDH, and hHsp70 were purchased from Sigma–Aldrich (Germany). Tris, hydrochloric acid, potassium chloride (KCl), disodium hydrogen phosphate (Na2HPO4), potassium dihyrogen phosphate (KH2PO4), and sodium chloride (NaCl) were purchased from Merck (Germany). Phosphate-buffered saline (PBS; pH 7.4) was made up of the following constituents that were dissolved in triple distilled water: 137 mM NaCl, 27 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4. All the protein suspensions were prepared in PBS. The concentration of proteins was determined using the Bradford assay (13). An assay buffer (20 mM Tris, pH 7.4; 100 mM NaCl) was prepared for use during the determination of heat-induced protein aggregation.
Synthesis of AuNPs
AuNPs were synthesized by reducing 0.1 mM AuCl3 with 1 M NaBH4 in the presence of 0.033 M cysteine, the capping agent. The growth and morphology of the formed product was monitored by sampling at 5 min, 30 min, and 2 H. The final product was centrifuged and stored for use in subsequent studies.
Characterization of AuNPs by UV–Vis Absorption Spectrophotometry and TEM
The absorption spectra of the AuNPs were carried out using a Perkin Elmer Lambda 20 spectrophotometer. Imaging was conducted using Philips CM 120 Biotwin transmission electron microscope (TEM) and the images were viewed and captured on 80 K self-imaging system mega-view III digital copper grade. Using TEM analysis, the number of gold atoms per nanoparticle was determined as previously described (14). We assumed 100% reduction in the gold (III) ions to gold atoms. Using this information, we estimated the molar concentration of the nanoparticles by dividing the total number of gold atoms in the original amount of gold salt added to the reaction mixture over the average number of gold atoms per nanoparticle (14).
Analysis of the Interaction of AuNPs with BSA and hHsp70
BSA and hHsp70 suspensions were prepared using PBS (pH 7.4) to final concentrations of 0.01 mg mL−1. AuNPs were added to the protein solution at a final concentration of 1. 086 × 10−5 mol L−1. The solution was left to stand for 5 min before being placed in a quartz cuvette and subjected to UV–Vis spectroscopic analysis. A control experiment involved the determination of the absorption spectra of AuNPs in the absence of protein. The optical spectra of the nanoparticles were determined within 200–800 nm range. To validate whether the interaction between AuNPs and BSA was specific, the experiment was repeated at varying concentrations of BSA (0.02, 0.05, and 0.1 mg mL−1), followed by spectroscopic analysis. At least three independent experiments were conducted for each assay.
Analysis of the Interaction of AuNPs with BSA Under Different pH Conditions
BSA was prepared at a final concentration of 0.1 mg mL−1 at various pH points (1.6, 4.6, and 7.4) suspended in PBS. To this, AuNPs were added at make a final concentration of 1. 086 × 10−5 mol L−1. The mixture was allowed to equilibrate before samples were transferred to a 1-mL quartz cuvette and analyzed using a UV–Vis spectrophotometer. The optical spectra of the protein were determined within the wavelength range of 200–800 nm. The investigations were conducted three times in duplicates.
Investigation of the effect of AuNPs on the Thermal Stability of MDH, CS, BSA, and hHsp70
The stability of MDH, BSA, CS, and hHsp70 to heat stress (48 °C) in the presence and absence of AuNPs was investigated. First, the aggregation of the respective proteins in the absence of AuNPs was investigated as follows; 1 mL of assay buffer (20 mM Tris, pH 7.4; 100 mM NaCl) was prepared into which the respective proteins were added in separate tubes to a final concentration of 1.3 μM for BSA and hHsp70 and 0.65 μM for MDH and CS, following a method described previously (11). The solutions were placed in a quartz cuvette that was positioned on a port at 48 °C and protein aggregation was monitored based on the development of turbidity at 360 nm.
The effect of AuNPs (at an estimated concentration of 1.086 × 10−5 mol L−1) on the stability of proteins at 48 °C was investigated in the presence of the respective proteins at the following concentrations: 0.65 μM for MDH and CS and 1.3 μM for BSA and hHsp70. As a positive control, the heat-induced aggregation of MDH in response was investigated in the presence of hHsp70 as it is a known chaperone (10, 11). As a negative control, AuNPs alone (without protein added to them) were subjected to heat stress at 48 °C and turbidity was similarly determined at 360 nm. For the rest of the proteins, the reactions were allowed to proceed for 30 min, but for CS the reaction was left to proceed for 45 min. To further confirm the capability of AuNPs to suppress MDH and CS aggregation in response to heat stress, the experiment was repeated at the following variable concentrations of AuNPs in mol L−1: 0.13 × 10−5, 0.54 × 10−5, 0.64 × 10−5, and 0.86 × 10−5.
Analysis of the Effect of AuNPs on Solubility of MDH and CS Exposed to Thermal Stress
The capability of AuNPs to suppress MDH and CS aggregation was further assessed using a slightly modified protocol (11). In a typical reaction, 20 μL sample containing 1 μM of CS or MDH was preincubated in the presence of AuNPs at a final concentration of 1.086 × 10−5 mol L−1. Control experiments were set up in which AuNPs were excluded. The reaction mix was incubated at 48 °C for 20 min. The protein solubility was verified by centrifugation for 10 min at 14,000g. A total of 15 μL of the supernatant was removed (soluble protein) and mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. The remaining pellet was resuspended in SDS-PAGE loading buffer. The samples were boiled for 5 min before analysis by SDS-PAGE.
Synthesis and Characterization of Cysteine Coated AuNPs
The UV–Vis absorption spectra of cysteine-capped AuNPs synthesized at various time intervals is shown in Fig. 1. All the spectra obtained at various time intervals display an absorption wavelength peak at approximately 530 nm, which is attributed to the surface plasmon resonance (SPR) mode of spherical AuNPs (Fig. 1). The SPR peak depends on the dielectric properties, size, and shape of the AuNPs and is also influenced by the preparation medium and resonance energy (15). The AuNPs sampled after 2 H presented high intensity and absorption peaks at 535 nm compared with intermediate intensity of AuNPs synthesized for 30 min with absorption peak at 530 nm and reduced intensity with red shifted absorption peak at 540 nm for AuNPs synthesized for 5 min (Fig. 1). The AuNPs obtained in this study displayed features resembling those previously obtained by Chili and Revaprasadu (2).
The observed wavelength peaks of the AuNPs obtained suggest the presence of a mixture of AuNPs dominated by spherical shapes. This is thought to be due to isotropic oscillations that are the same in all directions, as explained by the principle of SPR (16). The UV–Vis absorption spectra peaks of AuNPs did not change significantly for a storage period of at least 1 month signifying that the AuNPs were stable. Although the yield of AuNPs was dominated by spherically shaped products, it was noted that the variation in time of synthesis resulted in slight modification in the particle size and morphology as well as their absorption wavelength peak. Spherically shaped AuNPs dominated in products sampled after 30 min and 5 min of synthesis (Fig. 2). AuNPs exhibiting hexagonal shape were dominant in product sampled after 2 H of the synthesis reaction (Fig. 2). TEM micrographs of batches prepared for 5 and 30 min presented particles that were predominantly spherical in shape with diameters ranging from 35 nm to 55 nm (Fig. 2). The batch prepared for 2 H was dominated by a mixture of hexagonal, pentagonal, and spherically shaped AuNPs with diameters ranging from 25 to 45 nm (Fig. 2). For subsequent biochemical studies, we chose to use the batch of product that was synthesized for 30 min as this was dominantly made up of largely spherical particles that were relatively uniformly distributed.
Interaction of AuNPs With BSA and hHsp70
Next, we investigated the capability of the cysteine-capped AuNPs to interact with BSA and hHsp70 at pH 7.4. Using UV–Vis spectrophotometry, we confirmed that the nanoparticles interacted with BSA and hHsp70 as confirmed by the absorption wavelength of the AuNPs (alone), compared with the AuNP–hHsp70 and AuNP–BSA complexes, which registered wavelength shifts from 530 nm to 538 and 539 nm, respectively (Table 1). The shift in wavelength must have been due to an increase in the size of nanoparticles as they absorbed protein. Furthermore, using TEM analysis, we observed the formation of a ‘coat’ made up of BSA protein that formed around the surfaces of the AuNPs (Fig. 2). The formation of the protein coat masked the otherwise distinct crystal-lines that are visible in AuNPs that were not exposed to the BSA (Fig. 2). The coating of the AuNPs by the BSA resulted in particles that were slightly larger in appearance compared with those that were not exposed to the BSA (Fig. 2).
It has been reported previously that AuNPs are capable of interacting with BSA in a pH sensitive fashion (17). To confirm that the interaction between BSA and AuNPs that we observed occurred through the formation of specific ionic contacts, we explored the effect of pH variation in the interaction of AuNPs with BSA. Variation in pH about the neutral level was associated with significant shifts in absorption peaks of the BSA–AuNP complexes (Table 2). This suggests that the interaction between AuNPs and BSA was influenced by the structural conformation of the protein as regulated by pH.
AuNPs Were Capable of Reversing Heat-Induced Aggregation of MDH
MDH is a protein that readily aggregates in response to heat stress. The protein aggregates at 48 °C, and this aggregation is suppressed in the presence of molecular chaperones, and due to this reason, the heat-induced aggregation of MDH was an appropriate model to investigate the effect of cysteine-coated AuNPs on protein stability (11). We therefore evaluated the effect of nanoparticles on the stability of MDH to thermal stress. MDH (alone) was exposed to heat stress at 48 °C. At this temperature, the protein did aggregate as was evidenced by the increase in turbidity (Fig. 3). On the other hand, exposing AuNPs to thermal stress did not result in change in turbidity for 30 min, suggesting that the nanoparticles are stable at this temperature. However, exposure of MDH to heat stress in the presence of increased AuNPs led to the improved suppression of the former's aggregation (Fig. 3).
Molecular chaperones are proteins that prevent and reverse the aggregation of other proteins in the cell (9). Thus by suppressing MDH aggregation, AuNPs appeared to act in a similar fashion to molecular chaperones. Molecular chaperones facilitate protein folding by binding to hydrophobic patches of non-native protein thereby giving the misfolded protein a chance to refold (9). The heat-induced aggregation of MDH was suppressed in the presence of hHsp70, a known molecular chaperone (Fig. 3). We investigated the effect of AuNPs on the function of hHsp70 with respect to its ability to suppress the heat-induced aggregation of MDH. We noticed that AuNPs augmented the function of hHsp70 (Fig. 3), as MDH aggregation was further suppressed in the presence of both AuNPs and hHsp70 compared with hHsp70 alone.
AuNPs Maintained MDH and CS in Soluble Form Under Heat Stress Conditions
We were curious to evaluate the effect of AuNPs on the solubility of MDH and another aggregation prone protein CS. MDH and CS were subjected to thermal stress at 48 °C both in the presence and absence of AuNPs. The proteins were then separated into soluble and pellet fractions before being subjected to SDS-PAGE analysis. MDH appeared as a single band with an apparent molecular weight of of 34.5 kDa. On the other hand, CS is a hexamer whose subunits are 46 kDa in size as confirmed by SDS-PAGE analysis. In the presence of AuNPs, approximately equal amounts of both MDH and CS were observed in the pellet and soluble fractions (Fig. 4; lanes P1; S1 and P3; S3). On the other hand, in the absence of AuNPs, both MDH and CS completely aggregated (Fig. 4, lanes P2; S2; and P4; S4). Based on these findings, AuNPs maintained a fraction of both MDH and CS in soluble form when the proteins were subjected to thermal stress.
In this study, cysteine-coated AuNPs were successfully synthesized and their interaction with various proteins was investigated. This study provides evidence that cysteine-coated AuNPs were capable of suppressing heat-induced aggregation of protein. Hsps are ubiquitous molecules that occur in the cells of living organisms (9). Some Hsps are known to possess protein aggregation suppression capabilities (9, 11). Thus, Hsps are generally referred to as ‘molecular chaperones’, because some of them have the capability to hold and maintain their peptide substrates in native form, and therefore the role of Hsps is particularly important in the wake of physiological stress (9, 11). Based on this study, it appears that cysteine-coated AuNPs may have a function that mirrors that of select Hsps as they were able to suppress the aggregation of proteins in vitro. The capability of cysteine-coated AuNPs to bind proteins and maintain them in native form suggests that these nanoparticles may have significant utility value in wide-ranging nanobiomedical applications. It has been reported previously that AuNPs whose surface coat had a net anionic charge were capable of facilitating refolding of heat-denatured cationic proteins (12). In the previous study, the AuNPs were functionalized with 2-(10-mercaptodecyl)malonic acid. In this study, we sought to establish the effect of cysteine-coated AuNPS on protein stability. We coated the AuNPs with cysteine because the cysteine side group confers a hydrophobic character to this amino acid. Typically, the interaction between molecular chaperones and their client protein involves contacts that are made between hydrophobic motifs of the molecular chaperones and hydrophobic patches exposed by the misfolded proteins (9). As the side chain of cysteine imparts a hydrophobic character to the amino acid, we proposed that cysteine-coated AuNPs would bind hydrophobic segments of misfolded protein, thereby inhibiting aggregation of the protein. To the best of our knowledge, this is the first study to demonstrate that cysteine-coated AuNPs are capable of inhibiting the heat-induced aggregation of proteins.
In this study, we explored the interaction of AuNPs with various proteins. BSA is known to be sensitive to conformational changes when it interacts with other molecules, and because of this it is useful in studies involving interaction of protein and nanoparticles (17). hHsp70 is a molecular chaperone that is ubiquitous in the cell and its expression is upregulated by physiological stress (10). We also investigated the capability of AuNPs to suppress the heat-induced aggregation of two aggregation prone proteins, MDH and CS.
It has been observed previously that BSA interacts with AuNPs (17). In this study, we observed the interaction of AuNPs with BSA and hHsp70 as confirmed by UV–Vis spectrophotometry (Tables 1 and 2). Therefore, this study confirms that cysteine-coated AuNPs are likely to interact with some of the most ubiquitous proteins that occur in cells and in the blood circulation system. Furthermore, this interaction is a function of pH and our observation supports a previously proposed view that extreme pH variation promotes the interaction of protein with nanoparticles (17). However, we also observed that AuNPs interacted with BSA under physiological pH conditions (Table 1). Given the predisposition of nanoparticles to interact with proteins that lurk in their vicinity, it has been proposed that in effect, the nanoparticle unit must be described in the context of the core nanoparticle material and its protein corona (3, 4). This is because the protein coat has a big effect on the function of the nanoparticle. Similarly, nanoparticles are expected to modify the structure and function of their protein interactors. In this study, we sought to understand how AuNPs are likely to influence the structural and functional integrity of proteins.
The side chain of cysteine is thought to impart a hydrophobic character to this amino acid (18). Therefore, the cysteine used to coat the AuNPs must be capable of binding hydrophobic moieties of misfolding proteins, thereby preventing their aggregation and thus creating an environment for their refolding. It was observed previously that AuNPs are capable of reversing protein aggregation when combined with light energy (19). Based on the previous study and our current study, it appears that AuNPs may possess the capability to suppress aggregation of proteins, and may thus possibly facilitate their refolding. Furthermore, conjugation of AuNPs with trypsin has been found to increase the stability and activity of the enzyme (20). Altogether, these findings suggest that AuNPs may possess chaperone-like activity provided they are coated with appropriate agents that promote their interaction with proteins. However, other factors such as size of the nanoparticle and surface curvature play a crucial role in determining the fate of the bound protein.
Various small molecules are known to modulate the function of Hsp70 molecular chaperones (21, 22). We were curious to understand the effect of AuNPs on the chaperone function of hHsp70. Our findings suggest that the chaperone effect conferred to MDH in the presence of both hHsp70 and AuNPs appeared to be additive. This suggests that hHsp70 and AuNPs may have simply inhibited the heat-induced aggregation of MDH independently. Could AuNPs have protected both hHsp70 and MDH from aggregating in response to thermal stress? Hsp70 proteins are generally heat stable, and in fact, one of their roles is to prevent aggregation of other proteins in the event of physiological stress (10, 11). For this reason, it is unlikely that AuNPs acted by stabilizing hHsp70 to enhance its chaperone activity as Hsp70 is inherently a stable protein (10). Therefore, the most likely explanation is that the AuNPs independently protected MDH from the effect of thermal stress, thus complementing the function of hHsp70. As the activity of Hsp70 protein is inhibited by several small molecules and some of these have been characterized (21, 22). Thus, it is important to note that AuNPs did not inhibit the activity of hHsp70, and this confirms that AuNPs may not adversely affect the action of the protein folding machinery of the cell, further affirming their safety.
The fact that AuNPs seem to confer protection to proteins against adverse physiological conditions is important as this suggests these particles may be compatible with biological systems. Furthermore, the prospects of AuNPs serving as some form of artificial chaperone systems have broad implications in the biomedical and protein biotechnology sectors. For example, AuNPs have been proposed as potential therapies as exposure of protein aggregates to these nanoparticles with accompanying application of photothermal treatment led to the disaggregation of the protein complexes suggesting that AuNPs could be used to treat conditions such as Alzheimer's disease (19). In a previous study, it was observed that AuNPs improved the efficiency of amplifying DNA by the polymerase chain reaction (23). It is possible that the AuNPs may have improved the efficiency of the polymerase chain reaction by conferring stability to the polymerase.
The cysteine-capped AuNPs analyzed in this study suppressed heat-induced aggregation of MDH and CS. Furthermore, the nanoparticles complemented the chaperone function of hHsp70. The findings from this study demonstrate the potential amenability of AuNPs to their possible application in biomedical applications. In future, it will be important to investigate the kinetics of AuNPs with proteins to predict their preferred protein binding candidates under given physiological conditions. In addition, the specific mechanisms by which AuNPs protect proteins from thermal aggregation remains to be fully understood.
The authors thank Dr. Rajasekhar Pullabhotla (Chemistry Department, University of Zululand) for providing technical guidance with the TEM work. We further wish to acknowledge the Department of Science and Technology and National Research Foundation (NRF) of South Africa for awarding AS an equipment grant that made this work possible. A.S. is a recipient of the Georg Forster Research Fellowship awarded by the Alexander von Humboldt Foundation, Germany. SDL received a scholarship from the National Research Foundation of South Africa. This work was partly supported by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation. The authors further acknowledge the University of Zululand Research Committee for funding this study.