DOI: 10.1002/chem.201402934
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& Nanotechnology
Amyloid Transition of Ubiquitin on Silver Nanoparticles Produced by Pulsed Laser Ablation in Liquid as a Function of Stabilizer and Single-Point Mutations Vincenzo Mangini,[a] Marcella Dell’Aglio,[b] Angelo De Stradis,[c] Alessandro De Giacomo,[a, b] Olga De Pascale,[b] Giovanni Natile,[a] and Fabio Arnesano*[a]
Abstract: The interaction of nanoparticles with proteins has emerged as a key issue in addressing the problem of nanotoxicity. We investigated the interaction of silver nanoparticles (AgNPs), produced by laser ablation with human ubiquitin (Ub), a protein essential for degradative processes in cells. The surface plasmon resonance peak of AgNPs indicates that Ub is rapidly adsorbed on the AgNP surface yielding a protein corona; the Ub-coated AgNPs then evolve into clusters held together by an amyloid form of the protein, as
Introduction The interaction of nanoparticles (NPs) with proteins has emerged as a key issue in addressing the problem of nanotoxicity[1–3] and a number of studies have proven that, once entered in the bloodstream, NPs immediately interact with the abundant plasma proteins forming the so-called protein corona.[1, 4–6] This process is governed by molecular interactions between the NP surface and the amino acid residues of the proteins.[7] When proteins interact with NPs, their native conformation can be altered and new epitopes can be exposed on the surface, giving rise to unexpected biological responses.[1] Silver nanoparticles (AgNPs) have gained increasing interest for their antibacterial, antifungal, and antiviral properties.[8–10] The most diffuse method for the production of AgNPs is the chemical reduction of silver salts that requires chemical additives (for controlling the growth) and the use of a stabilizer. The latter can deeply affect the chemical activity of the NPs surface. Pulsed Laser Ablation in Liquid (PLAL) can also be [a] Dr. V. Mangini, Dr. A. D. Giacomo, Prof. G. Natile, Prof. F. Arnesano Department of Chemistry University of Bari “Aldo Moro” via Orabona, 4, 70125, Bari (Italy) E-mail: [email protected] [b] Dr. M. Dell’Aglio, Dr. A. D. Giacomo, Dr. O. D. Pascale CNR-IMIP, Section of Bari, via Amendola 122/D, 70126, Bari (Italy) [c] Dr. A. D. Stradis CNR-IVV, Section of Bari, via Amendola 165A, 70126, Bari (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201402934. Chem. Eur. J. 2014, 20, 10745 – 10751
revealed by binding of thioflavin T fluorescent dye. Transthyretin, an inhibitor of amyloid-type aggregation, impedes aggregate formation and disrupts preformed AgNP clusters. In the presence of sodium citrate, a common stabilizer that confers an overall negative charge to the NPs, Ub is still adsorbed on the AgNP surface, but no clustering is observed. Ub mutants bearing a single mutation at one edge b strand (i.e. Glu16Val) or in loop (Glu18Val) behave in a radically different manner.
used for producing stable AgNPs, with spherical shape and narrow size distribution, without the use of chemical reactant or stabilizer.[11, 12] Other advantages of PLAL are the environmental sustainability, the simple and flexible set-up, and the NPs stability.[12–14] In this work, AgNPs produced by PLAL have been allowed to react with human ubiquitin (Ub), a highly conserved regulatory protein of 76 amino acids that is widely expressed in eukaryotes,[12] which, among other functions, flags proteins for degradation by the proteasome.[12, 15, 16] It is found that, unlike AgNPs stabilized with citrate that interact with Ub leading only to formation of a protein corona around individual nanoparticles,[17] naked (i.e. stabilizer-free) AgNPs, after the protein corona formation, cluster into large aggregates held together by the protein. The aggregation process has been investigated by surface plasmon resonance (SPR), TEM, staining with thioflavin T, and NMR spectroscopy. The investigation has also been extended to two Ub mutants, E16V and E18V. An amyloid transition of Ub is found to be responsible for clustering of the AgNPs.
Results Characterization of silver nanoparticles produced by laser ablation Naked AgNPs have been produced through PLAL consisting of nanosecond laser pulses focused on a silver solid target submerged in water.[11, 14] The experimental setup is shown in Figure S1 of the Supporting Information, while details of the PLAL experiments are given in the Experimental Section. 10745
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Full Paper As already demonstrated,[11, 12, 14, 18, 19] the NPs are obtained with good reproducibility of particle shape (usually spherical), size, concentration, and stability. The AgNPs were characterized by SPR, dynamic light scattering (DLS), and TEM. The wavelength of the SPR peak is directly proportional to the size of NPs of spherical shape, as those produced by PLAL,[11] while the absorbance obeys the Lambert Beer law (scattering contribution can be neglected for NPs smaller than 20 nm),[20, 21] and can be used to determine the NPs concentration. The SPR spectrum showed a band centred at 398 nm that indicates a particle size of approximately 10 nm (Figure 1). The wavelength of the absorption maximum was found to increase by 1 nm in the first day, and then to remain constant over a time of several weeks (Figure 1). All experiments performed in this work used AgNPs (of about 2 nm concentration, see the Experimental Section) with an average size of 10.5 1.7 nm and spherical shape, as determined by DLS (Figure S2, Supporting Information) and TEM (Figure 1).
Figure 1. SPR spectra (A) and TEM images (B,C) of AgNPs produced by PLAL with a time of ablation of 3 min. SPR spectra were recorded at the indicated time intervals from production, and TEM images, with B or without C negative staining, were taken after 24 h. Scale bars: 10 (B) and 50 nm (C).
Interaction of ubiquitin with silver nanoparticles coated with citrate: Formation of protein corona Initially, we investigated the interaction of Ub with commercially available citrate-coated AgNPs of 10 nm size synthesized by wet chemical methods. By incubation with 25 mm Ub, the intense SPR band initially centered at 398 nm, underwent a 5 nm redshift, which indicates that Ub binds to the NPs and forms the protein corona. A similar behavior was observed when our PLAL-generated AgNPs were coated with citrate immediately after production and treated with 25 mm Ub (Figure 2). The redshift of the SPR peak (5 nm) increases by 1 nm after standing at room temperature for 12 h and then remains constant over time. Negative-stain TEM images confirm adsorption of Ub onto the NP surface (compare Figure 1B and 2B). The protein corona effect is shown for a single NP representative of the ensemble. Large field views indicate comparable sample dispersion in the absence and in the presence of Ub (Figure 1 C and 2C). Interaction of ubiquitin with naked silver nanoparticles: Amyloid transition
Figure 2. SPR spectra (A) and TEM images (B,C) of AgNPs produced by PLAL with a time of ablation of 3 min, coated with citrate, and treated with Ub. SPR spectra were recorded at the indicated time intervals from production, whereas TEM images, with B or without C negative staining, were taken after 24 h. Scale bars: 10 (B) and 50 nm (C).
Figure 3. SPR spectra of naked AgNPs incubated with 25 mm wild-type Ub (A). TEM images (with B or without negative staining C) taken after 24 h incubation confirm the presence of large aggregates. Scale bars: 25 (B) and 50 nm (C).
Addition of Ub (25 mm) to naked AgNPs produced by PLAL (2 nm) causes a gradual color change from yellow to orange. Aggregation and formation of a precipitate was observed after 48 h incubation (Figure S3, Supporting Information). The SPR band shifts slightly towards higher wavelength and broadens while a shoulder, shifted several tens of nm at higher wavelengths, appears, the intensity of which increases with time (Figure 3). These observations Chem. Eur. J. 2014, 20, 10745 – 10751
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suggest that the NPs size distribution changes and larger aggregates form. An independent confirmation of aggregate formation is provided by DLS measurements, which indicate an average hydrodynamic diameter of 190 nm and a doubling of the derived
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Full Paper count rate (number of photons detected on a “per second” basis) already after 2 h incubation of AgNPs with Ub (Figure S2, Supporting Information). The SPR bands were deconvoluted into their Gaussian components (Figure S4, Supporting Information) and the wavelength of the two resulting peak maxima were plotted as a function of time (Figure S5, Supporting Information). The wavelength of the first peak decreases slightly with time, while the wavelength of the second peak undergoes a strong increase. This is probably a consequence of a preferential aggregation of NPs at the upper end of the size distribution, with consequent decrease of the average size of the remaining NPs. After 2 h incubation, the wavelength of peak 1 remains constant, while the wavelength of the peak 2 continues to increase until it reaches a critical value at which the aggregates begin to precipitate. The precipitate formed after 48 h incubation, analyzed by TEM, consists of large protein deposits surrounding the NPs (Figure S6, Supporting Information). The formation of aggregates, as a function of Ub concentration, was also investigated (Figure 4). For a AgNPs concentration of 2 nm, a 0.050 mm Ub concentration is required for a significant formation of the large aggregates (Figure 4, top panel).
The effect of transthyretin (TTR), a widely known Ab aggregation inhibitor,[22, 23] on the aggregation process was investigated. AgNPs (2 nm) were preincubated with TTR (3 mm) and then treated with Ub (25 mm). TTR completely prevents the formation of AgNPs clusters, eliciting no detectable precipitation. UV/Vis spectroscopy and TEM images confirmed the inhibition (Figure S7, Supporting Information). For a deeper insight, the wavelength of peak 2 (indicative of the aggregates size) was plotted as a function of time for two AgNPs + Ub solutions only one of which was treated with TTR (3 mm) five minutes after incubation had started (Figure 5). The solution treated
Figure 5. Wavelength of SPR peak 2 as a function of time for two AgNP (2 nm) + Ub (25 mm) solutions, one of which (gray circles) was treated with TTR (3 mm) 5 min after incubation had started.
Figure 4. Top) Time-dependent SPR spectra of AgNPs (2 nm concentration) treated with various concentrations of Ub (from 0.0025 to 100 mm). Bottom) time-dependent wavelength of SPR peak 2 for different Ub concentrations.
For Ub concentrations 0.050 mm, the wavelength of peak 2 increases with time reaching a plateau after 12 h. It is interesting to note that the wavelength (and hence the size of the aggregates) is at a maximum for a Ub concentration of 0.050 mm (the minimum Ub concentration required for significant formation of larger aggregates) and decreases when increasing the Ub concentration up to a value of 2.5 mm, and then remains nearly constant. This unexpected behavior indicates that the nucleation of the NP aggregates is favored by the Ub concentration in solution, until it reaches a critical value. Chem. Eur. J. 2014, 20, 10745 – 10751
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with TTR shows a further increase of the aggregates size up to 6 h incubation, and then the aggregates size starts to decrease slowly and, after one week, reaches the dimensions of individual particles with the protein corona (with only a residual second peak assignable to small-size aggregates; Figure S8, Supporting Information). These results confirm that TTR not only inhibits the formation of new aggregates (which might explain the initial further increase of the already existing aggregates) but also disrupts preformed Ub–AgNPs clusters. The presence of amyloid-like structures in the AgNP–Ub aggregates was also assessed by a ThT fluorescence assay. The sample in which the AgNPs were only treated with Ub gave an intense fluorescence, indicative of amyloid-type transition, whereas the sample in which the AgNPs were treated with Ub and TTR did not exhibit any positive response (Figure 6). The interaction between AgNPs and Ub was also checked by NMR spectroscopy. The NMR samples were prepared by
Figure 6. Representative fluorescence microscopy images of AgNPs + Ub (A) and AgNPs + TTR + Ub (B) samples stained with ThT. Scale bars: 15 mm.
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Full Paper adding 15N-labeled Ub (30 mL of a 500 mm solution, 15 nmol) to the AgNPs (500 mL of a 2 nm solution, 1 pmol). Assuming that each NP becomes decorated with a corona of about 100 molecules of Ub, there are in solution about 150 molecules of free Ub per Ub molecule adsorbed on the NPs. Under these conditions, we did not expect to observe any significant change in chemical shifts of the protein since any change caused by the NP on the adsorbed protein would be divided by 150 in a regime of fast exchange. In the first 3 h, we observed only very small chemical shift changes. The [15N-1H]-HSQC NMR spectra of Ub and Ub with
AgNPs are shown in Figure 7. Each cross-peak represents a NH group. The observed chemical shift changes concern few cross-peaks (residues 3, 6–7, 13-14, 17, 39, 42–44, 55, 61, 64, 66–67, 69–70), while the remaining residues are unchanged. The affected amino acids are all positioned on a b-sheet region of the protein (Figure 7C). It is not possible to discriminate if such a change is a consequence of the adhesion between Ub molecules and the AgNPs surface (formation of the protein corona, a fast process), or a report of the protein–protein interaction starting on the AgNPs surface and which will lead to amyloid transition.
Interaction of ubiquitin mutants with naked silver nanoparticles Since negative charges (like those of citrate) appear to play a role in the adhesion of substrates to AgNPs, we have expressed and purified two Ub mutants, E16V and E18V, lacking a negatively charged residue (in both cases a Glu was replaced by a Val) localized on the edge of a b-strand and in a loop, respectively. Interestingly, while the E16V mutant interacts with the AgNPs forming a corona but with no signs of AgNP clustering in bigger aggregates (Figure 8); in contrast, the E18V mutant induces the formation of AgNP clusters, similarly to wild-type Ub (Figure 9). The different behavior of E16V, which does not promote clustering of AgNPs into large aggregates, with respect to E18V was also confirmed by the ThT fluorescence assay. Thus it is possible to conclude that Glu16 is required for amyloid transition.
Figure 8. SPR spectra of naked AgNPs incubated with 25 mm Ub mutant E16V (A). TEM (B) and fluorescence microscopy (C) images confirm the absence of aggregates. Scale bars: 50 nm (B) and 15 mm (C).
Discussion
Figure 7. A) Overlay of 2D [15N-1H]-HSQC NMR spectra of 15N-labeled Ub before (blue) and after incubation (3 h) with AgNPs (red). B) Weighted average chemical shift differences, Ddavg(HN). C) Mapping of the effects of AgNPs on Ub NMR spectroscopic signals. Residues undergoing significant chemical shift perturbation are colored in red, the side chains of Glu16 and Glu18 are shown in blue (PDB ID 1UBQ). Chem. Eur. J. 2014, 20, 10745 – 10751
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In this study we prepared metal NPs by the PLAL technique. Such a method does not require reductants and stabilizers, allows the preparation of clean solutions of the NPs of desired size and concentration, and could be amenable to produce NPs for biomedical applications. When citrate-coated AgNPs are incubated with Ub, the SPR peak undergoes a stable, small, redshift indicative of formation
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Figure 9. SPR spectra of naked AgNPs incubated with 25 mm Ub mutant E18V (A). The TEM (B) and fluorescence microscopy (C) images confirm the presence of large aggregates similar to those formed by wild-type Ub. Scale bars: 50 nm (B) and 15 mm (C).
of a protein corona. TEM images confirm the adsorption of Ub onto the NP surface. When uncoated AgNPs are incubated with Ub, after initial formation of a protein corona, the system evolves into clusters of AgNP held together by an amyloid form of Ub (TEM and ThT fluorescence experiments). Specific structural changes, consequent to the interaction with the AgNP surface, and the close proximity between Ub molecules achieved upon corona formation, can foster amyloidogenic transition and aggregation. AgNPs clustering is abolished by TTR, a known inhibitor of amyloid-type aggregation. NMR spectroscopy revealed only very small changes in protein chemical shifts, as expected for the presence of a large excess of solution protein in fast exchange with the adsorbed one (about 150:1 molecular ratio). The amino acids undergoing chemical-shift changes feature a surface patch corresponding to the protein b-sheet. Owing to the role of negative charges in promoting the adhesion to the surface of AgNPs, two one-point Ub mutants (E16V and E18V) were synthesized and their interaction with AgNPs investigated. While the interaction of E16V mutant with AgNPs is limited to corona formation, the analogous E18V mutant behaves similarly to wild-type Ub, ultimately leading to colloidal instability and clustering of AgNPs in larger aggregates. The net surface charge modification is similar for the two mutants and cannot explain their different behavior. Instead, the specific mutation site (edge of the b-strand for Glu16 and loop for Glu18) appears to be critical, thus highlighting how localized surface modifications can have great impact on supramolecular interactions. Our results indicate that Glu16 is dispensable for adsorption of Ub on the AgNP surface, but is required for amyloid conversion, being either directly involved in the protein–protein aggregation interface or just needed to properly orient the adsorbed protein molecules so to prime their amyloidogenic interactions. In contrast, Glu18 is neither required for protein–AgNP interaction, nor for subsequent amyloid conversion. It is worth comparing our results with previously reported NMR and computational studies.[17, 24] When the NPs (Ag, Au) Chem. Eur. J. 2014, 20, 10745 – 10751
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were coated with citrate, no aggregation phenomena with NP clustering were observed, but only protein corona formation (with slight destabilization of the a-helix and small increase in the b-sheet content of adsorbed Ub molecules).[24] In the case of AuNPs reacted with Ub, three Ub residues (2, 15, and 18) were found to undergo quite large NMR spectroscopy chemical-shift perturbations (in a fast-exchange regime).[17] These residues are close to one another in the 3D structure and could identify the site of interaction with the AuNP. Unlike citrate-coated NPs, uncoated AgNPs produced by PLAL promote a remarkable amyloid transition of the protein and clustering of the NPs in large aggregates. So far the role of metal NPs in protein aggregation, either catalyzing or inhibiting the process,[25–27] has been investigated only on amyloidogenic sequences, such as the well-known Ab peptide involved in Alzheimers’ disease, for which AuNPs were reported to promote aggregation and precipitation.[28] In a small number of cases self-assembly of NPs mediated by protein–protein interactions was also observed. Two possible mechanisms of bridging were proposed: one mechanism is based on electrostatic interactions between protein molecules in the absence of any structural perturbation, the other mechanism involves partial unfolding of the adsorbed proteins. The two mechanisms have been found in the case of lysozyme interacting with silica NPs[29] and AuNPs,[30] respectively. Our evidence indicates that the latter mechanism can also apply to Ub interacting with AgNPs, with the important difference that Ub is a stable protein with a compact fold and no tendency to aggregate. Available unfolding studies indicate that all of the described forms of destabilization of Ub native state seem to lead to a similar partially unfolded-state ensemble, in which the C-terminal half of the protein has a high propensity for a non-native conformation.[31] In contrast, native secondary structural elements are largely conserved in the Nterminal half of the protein, comprising the first b-hairpin, our mutation sites, and the a-helix. Ub mutants illustrate that a protein scaffold differing by a single amino acid can exhibit macroscopically distinct behaviors upon interaction with NPs and the same mutation (valine for glutamate) shifted by only two positions, but in a structurally different context in the native as well as in the partially unfolded state, can make a great difference.
Experimental Section General Silver targets for PLAL experiments were purchased from Goodfellow Cambridge Limited (99.95 % purity, 6 mm thickness). Sodium citrate, water (American Chemical Society reagent grade), Thioflavin T, and Transthyretin were purchased from Sigma Aldrich. Human 15N-labeled Ub was purchased from Giotto Biotech and used without further purification. The standard solution of stabilized AgNPs were purchased from NanoComposix (10 nm Citrate NanoXactTM Silver, 0.022 g L 1).
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Full Paper Protein expression and purification
TEM
The full-length wild-type Ub and Ub mutants (E16 V and E18 V) genes were cloned into pET-3a vectors. pET-3a-Ub, pET-3a-UbE16 V and pET-3a-Ub-E18 V were expressed in BL21(DE3) cells. The proteins were purified through selective protein precipitation with perchloric acid, cationic exchange chromatography (HiPrep SP FF 16/10), and size-exclusion chromatography (Superdex 75 10/300 GL). Complete removal of the chromatographic buffer salts was obtained with the column HiTrap Desalting. Chromatographic purifications were carried out using an Amersham Akta Purifier chromatographic system. Purified proteins were lyophilized using a Lio 5P freeze-drier. The purity of the proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ESI-MS.
The same samples used for the SPR spectroscopy were used for the TEM analysis. A little drop (20 mL) of incubated sample solution (an aliquot of the re-suspended sample in the case of the precipitated solution) was applied to a carbon-coated copper/rhodium grid (400 mesh; TAAB Laboratories Equipment Ltd, Aldermaston, Berks, GB). The coated grid was floated for 2 min on the sample drop and rinsed with 200 mL of doubly distilled water. Staining for protein revelation was performed with 200 mL of 2 % w/v uranyl acetate water solution (TAAB Laboratories Equipment Ltd). After draining off the excess of staining solution by means of a filter paper, the specimen was transferred for examination in a Philips Morgagni 282D transmission electron microscope, operating at 60 kV. Electron micrographs of all samples were photographed on Kodak electron microscope film 4489 (Kodak Company, New York, USA).
Production of silver nanoparticles The experimental setup (Figure S1) of PLAL consists of a nanosecond laser operating with second harmonic (532 nm) of Nd-YAG (Quanta System PILS-GIANT) with a repetition rate of 10 Hz and a nominal pulse duration of 8 ns, a 4 cm focusing lens in air, a silver target and a cuvette filled with ultrapure water. The Ag colloidal solutions were prepared by focusing the laser directly onto the silver target placed in a cuvette filled with 3 mL of ultrapure Milli-Q water. For each sample preparation the laser ablation time was fixed to 3 min and laser irradiance was 131 GW/cm2. AgNPs solutions were diluted 1:2.5 with ultrapure water and left to rest in the quartz cuvette for 24 h in order to stabilize the NPs (see Figure S9 and S10 and Supporting Methods in the Supporting Information).
Incubation of silver nanoparticles with ubiquitin and its mutants The lyophilized proteins (wild-type Ub, E16 V, or E18 V mutants) were dissolved in ultrapure water, and an aliquot (30 mL of a 500 mm solution) was added to the 2 nm AgNP solution to a final protein concentration of 25 mm, and incubated in the quartz cuvette at room temperature. All solutions were prepared by using ultrapure Milli-Q water from Millipore purification system.
UV/Vis absorption analysis The colloidal AgNPs solutions were characterized by Surface Plasmon Resonance (SPR) absorption spectroscopy using an Ocean Optics (USB2000 + XR) spectrometer with a light source (Mini Deuterium Halogen Light Source DT-Mini-2-GS). Absorption spectra of free AgNPs were recorded at room temperature in the 250– 600 nm range by using 1 cm path length quartz cuvettes and 1.5 nm spectral resolution. The AgNPs concentration was calculated by UV absorbance spectra: the extinction coefficient of AgNPs in water was measured by means of a calibration curve drawn using standard solutions of colloidal AgNPs with known concentration and particle-size distribution.
Dynamic light scattering Size distribution by number (%) of the scattered light was obtained using a Zetasizer Nano ZS dynamic light scattering (DLS) device from Malvern Instruments. The DTS0112 low-volume disposable cuvettes (pathlength 1 cm) were used. The solutions were filtered immediately before use to eliminate any impurity using Whatman Anotop 10 syringe filters with 0.02 mm pore size. Chem. Eur. J. 2014, 20, 10745 – 10751
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Thioflavin T assay A little drop of the sample solution (after 24 h of incubation) was deposited on a SiO2 support followed by a gently air-drying. Subsequently a drop of Thioflavin T (ThT) (5 mm aqueous solution) was added and, after 10 min of incubation, washed with deionized water before imaging. For all analyses, the excitation wavelength was set at 488 nm and the ThT dye emission was collected at 530 nm through a 40X objective. Only the fraction of the ThT dye interacting with amyloid-like fibrils gives the fluorescence signal.
NMR spectroscopy NMR spectroscopic experiments were performed at 25 8C on two 25 mm solutions of 15N-enriched Ub in water (90 % H2O and 10 % D2O) only one of which contained AgNPs (1.4 nm). Resonance assignment of the apoprotein was carried out with the aid of 2D TOCSY and NOESY by using available 1H and 15N chemical shift data.[32] All spectra were collected on a Bruker Avance 600 with an Ultra Shield Plus magnet using a triple resonance probe equipped with z axis self-shielded gradient coils, and processed using the standard Bruker software (TOPSPIN). Cross-peaks affected by the presence of AgNPs were identified by comparing their chemical shifts in the presence and in the absence of AgNPs. Chemical shift changes were reported as weighted average chemical shift differences Ddavg(HN) to account for differences in spectral widths between 1H and 15N resonances (i.e., Ddavg(HN) = [(DdH2 + (DdN/5)2)/ 2]1/2, in which DdH and DdN are chemical shift differences for 1H and 15N, respectively) and plotted as a function of the protein sequence.
Acknowledgements This work was supported by the Italian “Ministero dell’Universit e della Ricerca” (FIRB 2011—Rete Integrata per la Nano Medicina, RBAP114AMK). We also thank the University of Bari and the Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRCMSB). We thank Dr. F. Pisani and M. De Bellis for assistance in fluorescence microscopy measurements. Keywords: amyloids · nanoparticles · ubiquitin
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Received: April 3, 2014 Published online on July 24, 2014
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& Nanotechnology
Amyloid Transition of Ubiquitin on Silver Nanoparticles Produced by Pulsed Laser Ablation in Liquid as a Function of Stabilizer and Single-Point Mutations Vincenzo Mangini,[a] Marcella Dell’Aglio,[b] Angelo De Stradis,[c] Alessandro De Giacomo,[a, b] Olga De Pascale,[b] Giovanni Natile,[a] and Fabio Arnesano*[a]
Abstract: The interaction of nanoparticles with proteins has emerged as a key issue in addressing the problem of nanotoxicity. We investigated the interaction of silver nanoparticles (AgNPs), produced by laser ablation with human ubiquitin (Ub), a protein essential for degradative processes in cells. The surface plasmon resonance peak of AgNPs indicates that Ub is rapidly adsorbed on the AgNP surface yielding a protein corona; the Ub-coated AgNPs then evolve into clusters held together by an amyloid form of the protein, as
Introduction The interaction of nanoparticles (NPs) with proteins has emerged as a key issue in addressing the problem of nanotoxicity[1–3] and a number of studies have proven that, once entered in the bloodstream, NPs immediately interact with the abundant plasma proteins forming the so-called protein corona.[1, 4–6] This process is governed by molecular interactions between the NP surface and the amino acid residues of the proteins.[7] When proteins interact with NPs, their native conformation can be altered and new epitopes can be exposed on the surface, giving rise to unexpected biological responses.[1] Silver nanoparticles (AgNPs) have gained increasing interest for their antibacterial, antifungal, and antiviral properties.[8–10] The most diffuse method for the production of AgNPs is the chemical reduction of silver salts that requires chemical additives (for controlling the growth) and the use of a stabilizer. The latter can deeply affect the chemical activity of the NPs surface. Pulsed Laser Ablation in Liquid (PLAL) can also be [a] Dr. V. Mangini, Dr. A. D. Giacomo, Prof. G. Natile, Prof. F. Arnesano Department of Chemistry University of Bari “Aldo Moro” via Orabona, 4, 70125, Bari (Italy) E-mail: [email protected] [b] Dr. M. Dell’Aglio, Dr. A. D. Giacomo, Dr. O. D. Pascale CNR-IMIP, Section of Bari, via Amendola 122/D, 70126, Bari (Italy) [c] Dr. A. D. Stradis CNR-IVV, Section of Bari, via Amendola 165A, 70126, Bari (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201402934. Chem. Eur. J. 2014, 20, 10745 – 10751
revealed by binding of thioflavin T fluorescent dye. Transthyretin, an inhibitor of amyloid-type aggregation, impedes aggregate formation and disrupts preformed AgNP clusters. In the presence of sodium citrate, a common stabilizer that confers an overall negative charge to the NPs, Ub is still adsorbed on the AgNP surface, but no clustering is observed. Ub mutants bearing a single mutation at one edge b strand (i.e. Glu16Val) or in loop (Glu18Val) behave in a radically different manner.
used for producing stable AgNPs, with spherical shape and narrow size distribution, without the use of chemical reactant or stabilizer.[11, 12] Other advantages of PLAL are the environmental sustainability, the simple and flexible set-up, and the NPs stability.[12–14] In this work, AgNPs produced by PLAL have been allowed to react with human ubiquitin (Ub), a highly conserved regulatory protein of 76 amino acids that is widely expressed in eukaryotes,[12] which, among other functions, flags proteins for degradation by the proteasome.[12, 15, 16] It is found that, unlike AgNPs stabilized with citrate that interact with Ub leading only to formation of a protein corona around individual nanoparticles,[17] naked (i.e. stabilizer-free) AgNPs, after the protein corona formation, cluster into large aggregates held together by the protein. The aggregation process has been investigated by surface plasmon resonance (SPR), TEM, staining with thioflavin T, and NMR spectroscopy. The investigation has also been extended to two Ub mutants, E16V and E18V. An amyloid transition of Ub is found to be responsible for clustering of the AgNPs.
Results Characterization of silver nanoparticles produced by laser ablation Naked AgNPs have been produced through PLAL consisting of nanosecond laser pulses focused on a silver solid target submerged in water.[11, 14] The experimental setup is shown in Figure S1 of the Supporting Information, while details of the PLAL experiments are given in the Experimental Section. 10745
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Full Paper As already demonstrated,[11, 12, 14, 18, 19] the NPs are obtained with good reproducibility of particle shape (usually spherical), size, concentration, and stability. The AgNPs were characterized by SPR, dynamic light scattering (DLS), and TEM. The wavelength of the SPR peak is directly proportional to the size of NPs of spherical shape, as those produced by PLAL,[11] while the absorbance obeys the Lambert Beer law (scattering contribution can be neglected for NPs smaller than 20 nm),[20, 21] and can be used to determine the NPs concentration. The SPR spectrum showed a band centred at 398 nm that indicates a particle size of approximately 10 nm (Figure 1). The wavelength of the absorption maximum was found to increase by 1 nm in the first day, and then to remain constant over a time of several weeks (Figure 1). All experiments performed in this work used AgNPs (of about 2 nm concentration, see the Experimental Section) with an average size of 10.5 1.7 nm and spherical shape, as determined by DLS (Figure S2, Supporting Information) and TEM (Figure 1).
Figure 1. SPR spectra (A) and TEM images (B,C) of AgNPs produced by PLAL with a time of ablation of 3 min. SPR spectra were recorded at the indicated time intervals from production, and TEM images, with B or without C negative staining, were taken after 24 h. Scale bars: 10 (B) and 50 nm (C).
Interaction of ubiquitin with silver nanoparticles coated with citrate: Formation of protein corona Initially, we investigated the interaction of Ub with commercially available citrate-coated AgNPs of 10 nm size synthesized by wet chemical methods. By incubation with 25 mm Ub, the intense SPR band initially centered at 398 nm, underwent a 5 nm redshift, which indicates that Ub binds to the NPs and forms the protein corona. A similar behavior was observed when our PLAL-generated AgNPs were coated with citrate immediately after production and treated with 25 mm Ub (Figure 2). The redshift of the SPR peak (5 nm) increases by 1 nm after standing at room temperature for 12 h and then remains constant over time. Negative-stain TEM images confirm adsorption of Ub onto the NP surface (compare Figure 1B and 2B). The protein corona effect is shown for a single NP representative of the ensemble. Large field views indicate comparable sample dispersion in the absence and in the presence of Ub (Figure 1 C and 2C). Interaction of ubiquitin with naked silver nanoparticles: Amyloid transition
Figure 2. SPR spectra (A) and TEM images (B,C) of AgNPs produced by PLAL with a time of ablation of 3 min, coated with citrate, and treated with Ub. SPR spectra were recorded at the indicated time intervals from production, whereas TEM images, with B or without C negative staining, were taken after 24 h. Scale bars: 10 (B) and 50 nm (C).
Figure 3. SPR spectra of naked AgNPs incubated with 25 mm wild-type Ub (A). TEM images (with B or without negative staining C) taken after 24 h incubation confirm the presence of large aggregates. Scale bars: 25 (B) and 50 nm (C).
Addition of Ub (25 mm) to naked AgNPs produced by PLAL (2 nm) causes a gradual color change from yellow to orange. Aggregation and formation of a precipitate was observed after 48 h incubation (Figure S3, Supporting Information). The SPR band shifts slightly towards higher wavelength and broadens while a shoulder, shifted several tens of nm at higher wavelengths, appears, the intensity of which increases with time (Figure 3). These observations Chem. Eur. J. 2014, 20, 10745 – 10751
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suggest that the NPs size distribution changes and larger aggregates form. An independent confirmation of aggregate formation is provided by DLS measurements, which indicate an average hydrodynamic diameter of 190 nm and a doubling of the derived
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Full Paper count rate (number of photons detected on a “per second” basis) already after 2 h incubation of AgNPs with Ub (Figure S2, Supporting Information). The SPR bands were deconvoluted into their Gaussian components (Figure S4, Supporting Information) and the wavelength of the two resulting peak maxima were plotted as a function of time (Figure S5, Supporting Information). The wavelength of the first peak decreases slightly with time, while the wavelength of the second peak undergoes a strong increase. This is probably a consequence of a preferential aggregation of NPs at the upper end of the size distribution, with consequent decrease of the average size of the remaining NPs. After 2 h incubation, the wavelength of peak 1 remains constant, while the wavelength of the peak 2 continues to increase until it reaches a critical value at which the aggregates begin to precipitate. The precipitate formed after 48 h incubation, analyzed by TEM, consists of large protein deposits surrounding the NPs (Figure S6, Supporting Information). The formation of aggregates, as a function of Ub concentration, was also investigated (Figure 4). For a AgNPs concentration of 2 nm, a 0.050 mm Ub concentration is required for a significant formation of the large aggregates (Figure 4, top panel).
The effect of transthyretin (TTR), a widely known Ab aggregation inhibitor,[22, 23] on the aggregation process was investigated. AgNPs (2 nm) were preincubated with TTR (3 mm) and then treated with Ub (25 mm). TTR completely prevents the formation of AgNPs clusters, eliciting no detectable precipitation. UV/Vis spectroscopy and TEM images confirmed the inhibition (Figure S7, Supporting Information). For a deeper insight, the wavelength of peak 2 (indicative of the aggregates size) was plotted as a function of time for two AgNPs + Ub solutions only one of which was treated with TTR (3 mm) five minutes after incubation had started (Figure 5). The solution treated
Figure 5. Wavelength of SPR peak 2 as a function of time for two AgNP (2 nm) + Ub (25 mm) solutions, one of which (gray circles) was treated with TTR (3 mm) 5 min after incubation had started.
Figure 4. Top) Time-dependent SPR spectra of AgNPs (2 nm concentration) treated with various concentrations of Ub (from 0.0025 to 100 mm). Bottom) time-dependent wavelength of SPR peak 2 for different Ub concentrations.
For Ub concentrations 0.050 mm, the wavelength of peak 2 increases with time reaching a plateau after 12 h. It is interesting to note that the wavelength (and hence the size of the aggregates) is at a maximum for a Ub concentration of 0.050 mm (the minimum Ub concentration required for significant formation of larger aggregates) and decreases when increasing the Ub concentration up to a value of 2.5 mm, and then remains nearly constant. This unexpected behavior indicates that the nucleation of the NP aggregates is favored by the Ub concentration in solution, until it reaches a critical value. Chem. Eur. J. 2014, 20, 10745 – 10751
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with TTR shows a further increase of the aggregates size up to 6 h incubation, and then the aggregates size starts to decrease slowly and, after one week, reaches the dimensions of individual particles with the protein corona (with only a residual second peak assignable to small-size aggregates; Figure S8, Supporting Information). These results confirm that TTR not only inhibits the formation of new aggregates (which might explain the initial further increase of the already existing aggregates) but also disrupts preformed Ub–AgNPs clusters. The presence of amyloid-like structures in the AgNP–Ub aggregates was also assessed by a ThT fluorescence assay. The sample in which the AgNPs were only treated with Ub gave an intense fluorescence, indicative of amyloid-type transition, whereas the sample in which the AgNPs were treated with Ub and TTR did not exhibit any positive response (Figure 6). The interaction between AgNPs and Ub was also checked by NMR spectroscopy. The NMR samples were prepared by
Figure 6. Representative fluorescence microscopy images of AgNPs + Ub (A) and AgNPs + TTR + Ub (B) samples stained with ThT. Scale bars: 15 mm.
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Full Paper adding 15N-labeled Ub (30 mL of a 500 mm solution, 15 nmol) to the AgNPs (500 mL of a 2 nm solution, 1 pmol). Assuming that each NP becomes decorated with a corona of about 100 molecules of Ub, there are in solution about 150 molecules of free Ub per Ub molecule adsorbed on the NPs. Under these conditions, we did not expect to observe any significant change in chemical shifts of the protein since any change caused by the NP on the adsorbed protein would be divided by 150 in a regime of fast exchange. In the first 3 h, we observed only very small chemical shift changes. The [15N-1H]-HSQC NMR spectra of Ub and Ub with
AgNPs are shown in Figure 7. Each cross-peak represents a NH group. The observed chemical shift changes concern few cross-peaks (residues 3, 6–7, 13-14, 17, 39, 42–44, 55, 61, 64, 66–67, 69–70), while the remaining residues are unchanged. The affected amino acids are all positioned on a b-sheet region of the protein (Figure 7C). It is not possible to discriminate if such a change is a consequence of the adhesion between Ub molecules and the AgNPs surface (formation of the protein corona, a fast process), or a report of the protein–protein interaction starting on the AgNPs surface and which will lead to amyloid transition.
Interaction of ubiquitin mutants with naked silver nanoparticles Since negative charges (like those of citrate) appear to play a role in the adhesion of substrates to AgNPs, we have expressed and purified two Ub mutants, E16V and E18V, lacking a negatively charged residue (in both cases a Glu was replaced by a Val) localized on the edge of a b-strand and in a loop, respectively. Interestingly, while the E16V mutant interacts with the AgNPs forming a corona but with no signs of AgNP clustering in bigger aggregates (Figure 8); in contrast, the E18V mutant induces the formation of AgNP clusters, similarly to wild-type Ub (Figure 9). The different behavior of E16V, which does not promote clustering of AgNPs into large aggregates, with respect to E18V was also confirmed by the ThT fluorescence assay. Thus it is possible to conclude that Glu16 is required for amyloid transition.
Figure 8. SPR spectra of naked AgNPs incubated with 25 mm Ub mutant E16V (A). TEM (B) and fluorescence microscopy (C) images confirm the absence of aggregates. Scale bars: 50 nm (B) and 15 mm (C).
Discussion
Figure 7. A) Overlay of 2D [15N-1H]-HSQC NMR spectra of 15N-labeled Ub before (blue) and after incubation (3 h) with AgNPs (red). B) Weighted average chemical shift differences, Ddavg(HN). C) Mapping of the effects of AgNPs on Ub NMR spectroscopic signals. Residues undergoing significant chemical shift perturbation are colored in red, the side chains of Glu16 and Glu18 are shown in blue (PDB ID 1UBQ). Chem. Eur. J. 2014, 20, 10745 – 10751
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In this study we prepared metal NPs by the PLAL technique. Such a method does not require reductants and stabilizers, allows the preparation of clean solutions of the NPs of desired size and concentration, and could be amenable to produce NPs for biomedical applications. When citrate-coated AgNPs are incubated with Ub, the SPR peak undergoes a stable, small, redshift indicative of formation
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Figure 9. SPR spectra of naked AgNPs incubated with 25 mm Ub mutant E18V (A). The TEM (B) and fluorescence microscopy (C) images confirm the presence of large aggregates similar to those formed by wild-type Ub. Scale bars: 50 nm (B) and 15 mm (C).
of a protein corona. TEM images confirm the adsorption of Ub onto the NP surface. When uncoated AgNPs are incubated with Ub, after initial formation of a protein corona, the system evolves into clusters of AgNP held together by an amyloid form of Ub (TEM and ThT fluorescence experiments). Specific structural changes, consequent to the interaction with the AgNP surface, and the close proximity between Ub molecules achieved upon corona formation, can foster amyloidogenic transition and aggregation. AgNPs clustering is abolished by TTR, a known inhibitor of amyloid-type aggregation. NMR spectroscopy revealed only very small changes in protein chemical shifts, as expected for the presence of a large excess of solution protein in fast exchange with the adsorbed one (about 150:1 molecular ratio). The amino acids undergoing chemical-shift changes feature a surface patch corresponding to the protein b-sheet. Owing to the role of negative charges in promoting the adhesion to the surface of AgNPs, two one-point Ub mutants (E16V and E18V) were synthesized and their interaction with AgNPs investigated. While the interaction of E16V mutant with AgNPs is limited to corona formation, the analogous E18V mutant behaves similarly to wild-type Ub, ultimately leading to colloidal instability and clustering of AgNPs in larger aggregates. The net surface charge modification is similar for the two mutants and cannot explain their different behavior. Instead, the specific mutation site (edge of the b-strand for Glu16 and loop for Glu18) appears to be critical, thus highlighting how localized surface modifications can have great impact on supramolecular interactions. Our results indicate that Glu16 is dispensable for adsorption of Ub on the AgNP surface, but is required for amyloid conversion, being either directly involved in the protein–protein aggregation interface or just needed to properly orient the adsorbed protein molecules so to prime their amyloidogenic interactions. In contrast, Glu18 is neither required for protein–AgNP interaction, nor for subsequent amyloid conversion. It is worth comparing our results with previously reported NMR and computational studies.[17, 24] When the NPs (Ag, Au) Chem. Eur. J. 2014, 20, 10745 – 10751
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were coated with citrate, no aggregation phenomena with NP clustering were observed, but only protein corona formation (with slight destabilization of the a-helix and small increase in the b-sheet content of adsorbed Ub molecules).[24] In the case of AuNPs reacted with Ub, three Ub residues (2, 15, and 18) were found to undergo quite large NMR spectroscopy chemical-shift perturbations (in a fast-exchange regime).[17] These residues are close to one another in the 3D structure and could identify the site of interaction with the AuNP. Unlike citrate-coated NPs, uncoated AgNPs produced by PLAL promote a remarkable amyloid transition of the protein and clustering of the NPs in large aggregates. So far the role of metal NPs in protein aggregation, either catalyzing or inhibiting the process,[25–27] has been investigated only on amyloidogenic sequences, such as the well-known Ab peptide involved in Alzheimers’ disease, for which AuNPs were reported to promote aggregation and precipitation.[28] In a small number of cases self-assembly of NPs mediated by protein–protein interactions was also observed. Two possible mechanisms of bridging were proposed: one mechanism is based on electrostatic interactions between protein molecules in the absence of any structural perturbation, the other mechanism involves partial unfolding of the adsorbed proteins. The two mechanisms have been found in the case of lysozyme interacting with silica NPs[29] and AuNPs,[30] respectively. Our evidence indicates that the latter mechanism can also apply to Ub interacting with AgNPs, with the important difference that Ub is a stable protein with a compact fold and no tendency to aggregate. Available unfolding studies indicate that all of the described forms of destabilization of Ub native state seem to lead to a similar partially unfolded-state ensemble, in which the C-terminal half of the protein has a high propensity for a non-native conformation.[31] In contrast, native secondary structural elements are largely conserved in the Nterminal half of the protein, comprising the first b-hairpin, our mutation sites, and the a-helix. Ub mutants illustrate that a protein scaffold differing by a single amino acid can exhibit macroscopically distinct behaviors upon interaction with NPs and the same mutation (valine for glutamate) shifted by only two positions, but in a structurally different context in the native as well as in the partially unfolded state, can make a great difference.
Experimental Section General Silver targets for PLAL experiments were purchased from Goodfellow Cambridge Limited (99.95 % purity, 6 mm thickness). Sodium citrate, water (American Chemical Society reagent grade), Thioflavin T, and Transthyretin were purchased from Sigma Aldrich. Human 15N-labeled Ub was purchased from Giotto Biotech and used without further purification. The standard solution of stabilized AgNPs were purchased from NanoComposix (10 nm Citrate NanoXactTM Silver, 0.022 g L 1).
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Full Paper Protein expression and purification
TEM
The full-length wild-type Ub and Ub mutants (E16 V and E18 V) genes were cloned into pET-3a vectors. pET-3a-Ub, pET-3a-UbE16 V and pET-3a-Ub-E18 V were expressed in BL21(DE3) cells. The proteins were purified through selective protein precipitation with perchloric acid, cationic exchange chromatography (HiPrep SP FF 16/10), and size-exclusion chromatography (Superdex 75 10/300 GL). Complete removal of the chromatographic buffer salts was obtained with the column HiTrap Desalting. Chromatographic purifications were carried out using an Amersham Akta Purifier chromatographic system. Purified proteins were lyophilized using a Lio 5P freeze-drier. The purity of the proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ESI-MS.
The same samples used for the SPR spectroscopy were used for the TEM analysis. A little drop (20 mL) of incubated sample solution (an aliquot of the re-suspended sample in the case of the precipitated solution) was applied to a carbon-coated copper/rhodium grid (400 mesh; TAAB Laboratories Equipment Ltd, Aldermaston, Berks, GB). The coated grid was floated for 2 min on the sample drop and rinsed with 200 mL of doubly distilled water. Staining for protein revelation was performed with 200 mL of 2 % w/v uranyl acetate water solution (TAAB Laboratories Equipment Ltd). After draining off the excess of staining solution by means of a filter paper, the specimen was transferred for examination in a Philips Morgagni 282D transmission electron microscope, operating at 60 kV. Electron micrographs of all samples were photographed on Kodak electron microscope film 4489 (Kodak Company, New York, USA).
Production of silver nanoparticles The experimental setup (Figure S1) of PLAL consists of a nanosecond laser operating with second harmonic (532 nm) of Nd-YAG (Quanta System PILS-GIANT) with a repetition rate of 10 Hz and a nominal pulse duration of 8 ns, a 4 cm focusing lens in air, a silver target and a cuvette filled with ultrapure water. The Ag colloidal solutions were prepared by focusing the laser directly onto the silver target placed in a cuvette filled with 3 mL of ultrapure Milli-Q water. For each sample preparation the laser ablation time was fixed to 3 min and laser irradiance was 131 GW/cm2. AgNPs solutions were diluted 1:2.5 with ultrapure water and left to rest in the quartz cuvette for 24 h in order to stabilize the NPs (see Figure S9 and S10 and Supporting Methods in the Supporting Information).
Incubation of silver nanoparticles with ubiquitin and its mutants The lyophilized proteins (wild-type Ub, E16 V, or E18 V mutants) were dissolved in ultrapure water, and an aliquot (30 mL of a 500 mm solution) was added to the 2 nm AgNP solution to a final protein concentration of 25 mm, and incubated in the quartz cuvette at room temperature. All solutions were prepared by using ultrapure Milli-Q water from Millipore purification system.
UV/Vis absorption analysis The colloidal AgNPs solutions were characterized by Surface Plasmon Resonance (SPR) absorption spectroscopy using an Ocean Optics (USB2000 + XR) spectrometer with a light source (Mini Deuterium Halogen Light Source DT-Mini-2-GS). Absorption spectra of free AgNPs were recorded at room temperature in the 250– 600 nm range by using 1 cm path length quartz cuvettes and 1.5 nm spectral resolution. The AgNPs concentration was calculated by UV absorbance spectra: the extinction coefficient of AgNPs in water was measured by means of a calibration curve drawn using standard solutions of colloidal AgNPs with known concentration and particle-size distribution.
Dynamic light scattering Size distribution by number (%) of the scattered light was obtained using a Zetasizer Nano ZS dynamic light scattering (DLS) device from Malvern Instruments. The DTS0112 low-volume disposable cuvettes (pathlength 1 cm) were used. The solutions were filtered immediately before use to eliminate any impurity using Whatman Anotop 10 syringe filters with 0.02 mm pore size. Chem. Eur. J. 2014, 20, 10745 – 10751
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Thioflavin T assay A little drop of the sample solution (after 24 h of incubation) was deposited on a SiO2 support followed by a gently air-drying. Subsequently a drop of Thioflavin T (ThT) (5 mm aqueous solution) was added and, after 10 min of incubation, washed with deionized water before imaging. For all analyses, the excitation wavelength was set at 488 nm and the ThT dye emission was collected at 530 nm through a 40X objective. Only the fraction of the ThT dye interacting with amyloid-like fibrils gives the fluorescence signal.
NMR spectroscopy NMR spectroscopic experiments were performed at 25 8C on two 25 mm solutions of 15N-enriched Ub in water (90 % H2O and 10 % D2O) only one of which contained AgNPs (1.4 nm). Resonance assignment of the apoprotein was carried out with the aid of 2D TOCSY and NOESY by using available 1H and 15N chemical shift data.[32] All spectra were collected on a Bruker Avance 600 with an Ultra Shield Plus magnet using a triple resonance probe equipped with z axis self-shielded gradient coils, and processed using the standard Bruker software (TOPSPIN). Cross-peaks affected by the presence of AgNPs were identified by comparing their chemical shifts in the presence and in the absence of AgNPs. Chemical shift changes were reported as weighted average chemical shift differences Ddavg(HN) to account for differences in spectral widths between 1H and 15N resonances (i.e., Ddavg(HN) = [(DdH2 + (DdN/5)2)/ 2]1/2, in which DdH and DdN are chemical shift differences for 1H and 15N, respectively) and plotted as a function of the protein sequence.
Acknowledgements This work was supported by the Italian “Ministero dell’Universit e della Ricerca” (FIRB 2011—Rete Integrata per la Nano Medicina, RBAP114AMK). We also thank the University of Bari and the Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRCMSB). We thank Dr. F. Pisani and M. De Bellis for assistance in fluorescence microscopy measurements. Keywords: amyloids · nanoparticles · ubiquitin
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Received: April 3, 2014 Published online on July 24, 2014
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