Amyloid-like Fibrils Formed by ε-Poly-l-lysine - Biomacromolecules

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Amyloid-like Fibrils Formed by ε‑Poly‑L‑lysine Jingjing Lai,† Cui Zheng,‡ Dehai Liang,*,‡ and Yanbin Huang*,† †

Key Laboratory of Advanced Materials (MOE), Department of Chemical Engineering, Tsinghua University, Beijing 100084, China College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

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S Supporting Information *

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BI-200SM goniometer. A solid-state laser (CNI Changchun GXL-III, 532 nm, 100 mW) operating at 532 nm was used as the light source. Both incident light and scattered light were polarized in the vertical direction. Intensity autocorrelation functions G(2)(τ) in the self-beating mode were measured using a BI-TurboCorr digital correlator. The electric field time correlation function g(1)(τ) was obtained by the equation G(2)(τ) = A[1 + β|g(1)(τ)|2], where A is the measured baseline, β is a coherence factor, and τ is the delay time. The Γ distribution is calculated by a Laplace inversion program, CONTIN, and transformed into the diffusion coefficient distribution and further into the hydrodynamic radius distribution by the equations Γ = Dq2 and D = kBT/6πηRh, respectively, where Γ, D, q, kB, T, η, and Rh are the line width, the diffusion coefficient, the scattering vector, the Boltzmann constant, the absolute temperature, the viscosity of the solvent, and the hydrodynamic radius, respectively. The solutions were filtered through a hydrophilic filter (Millipore, 0.45 μm, polyvinyl difluoride) to remove dust. The DLS measurement was started immediately after the filtration. Atomic Force Microscopy (AFM). AFM was performed with a Shimadzu SPM-9500 instrument in tapping mode. The sample solution was dropped on a freshly prepared mica surface and airdried for more than 24 h before being observed. Scanning Electron Microscopy (SEM). SEM was performed by a Tescan VEGA3 instrument operated at an accelerating voltage of 10 kV. The sample solution was dropped on the silicon surface and airdried. Transmission Electron Microscopy (TEM) and Selected Area Electron Diffraction (SAED). TEM and SAED were performed with a JEOL JEM-2010 instrument operated at an accelerating voltage of 120 kV. The sample solution was diluted to ∼0.1 mg/mL, dropped on carbon-coated copper grids, and air-dried. Circular Dichroism (CD). CD spectra were recorded on a Pistar π180 instrument at 20 and 60 °C in a cuvette with a path length of 0.1 cm. The spectra were recorded at a 0.5 nm interval from 260 to 190 nm. X-ray Diffraction (XRD). The ε-PL solution was dried on PTFE films and then lifted off carefully to obtain films with thickness of ∼100 μm for two-dimensional XRD experiments. The X-ray pattern was then collected using a Rigaku R-Axis Spider instrument with a Mo target at wavelength of 0.708 Å. The X-ray beam is set perpendicular to the films.

INTRODUCTION Proteins and peptides tend to aggregate into amyloid fibrils where the chains form highly ordered β-sheets and the chain axis is perpendicular to the direction of fibril growth. In a typical amyloid fibril, multiple hydrogen bonds are formed between the amide groups of two adjacent peptide chains, while the side groups stack up above or below the β-sheet.1−4 Though the amyloid aggregate state was initially observed with several disease-related proteins such as Aβ peptides in Alzheimer’s disease, amylin in Type II diabetes, and α-synuclein in Parkinson’s disease, now it is regarded as a universal and intrinsic property of all proteins, irrespective of their amino acid sequences and native folded structures.5 Interestingly, a variety of proteins and peptides that do not share sequence homology were shown to self-assemble into amyloid fibrils with similar morphologies and β-sheet structures.6 Recently, even homopolymers of α-amino acids such as poly-L-lysine, poly-Lglutamic acid, and poly-L-threonine were found to form amyloid fibrils.7 Besides being important in biology, protein/ peptide aggregates are also interesting candidates as biomaterials with potential applications in tissue engineering and drug release.8−11 Therefore, it is interesting to see if this generality of amyloid structures can be pushed even further to be a universal property of all polyamides, i.e., not limited to α-amino acid polymers. In this study, ε-poly-L-lysine (ε-PL) was chosen as a model polyamide (Scheme 1). As a non-α-poly amino acid, ε-PL has a much lower amide group density along its backbone chain and has small amine groups in the side chain. Therefore, it can be regarded as an intermediate structure between proteins and nylon polymers (Scheme 1). With ε-PL, we should be able to study (1) whether a non-α-polypeptide can form amyloid fibrils and, if so, how similar or different the structures would be from the conventional amyloid and (2) how the amine side groups, which are very close to the backbone amide groups, participate in or interfere with the hydrogen bond networks of backbone chains.

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RESULTS AND DISCUSSION First, an ε-PL solution in deionized water (2.5 mg/mL, pH 7) was monitored by DLS at different temperatures. As shown in Figure 1, the scattering light intensity at 60 °C rapidly increases with time and reaches a plateau after 80 min, indicating a fast aggregation of ε-PL molecules. In contrast, the scattered

EXPERIMENTAL SECTION

Sample Preparation. ε-PL was purchased from Nanjing Zytex Biotechnology Co., dialyzed, and lyophilized before being used. The 1 H nuclear magnetic resonance (NMR) and mass spectrometry (MS) spectra of ε-PL are shown in the Supporting Information. The purified ε-PL was dissolved in deionized water; concentrated sodium hydroxide and hydrochloric acid solutions were used to adjust pH to the set value, and then the solution was filtered through a 220 nm syringe filter (PES) before incubation. Dynamic Light Scattering (DLS). DLS experiments were performed on a Brookhaven Instruments apparatus equipped with a © XXXX American Chemical Society

Received: September 5, 2013 Revised: November 7, 2013

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dx.doi.org/10.1021/bm4013259 | Biomacromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Chemical Structures of (A) α-PL, (B) ε-PL, and (C) Nylon 6

solution. The fraction of aggregates increases with time at the cost of the single chains (Figure 2B), and at 63 min, a new diffusive mode with an average size of >1 μm (Figure 2C) is observed, indicating the occurrence of secondary aggregation. The fraction of these larger aggregates also increases with time (Figure 2D). Next, the morphology of the aggregates formed by ε-PL was characterized by AFM, SEM, and TEM. Multiple microscopy techniques were used to demonstrate that the observed morphology was independent of the specific technique or substrate used in sample preparation. As shown in Figure 3A, the samples taken from ε-PL solutions before incubation show ∼100 nm particles on the mica surface. This is in agreement with the results from DLS (Figure 2A). After being incubated at 60 °C for 5 min (Figure 3B), filaments (∼20 nm high and ∼5 μm long) are observed, which correspond to the secondary aggregation observed in DLS. These ε-PL filaments are quite similar in size to conventional amyloid fibrils.12 With longer incubation times, filaments assemble to form larger fibrils (Figure 3C,E,F and Figure S6 of the Supporting Information), whose size surpasses the resolution of DLS. Finally, the thin fibrils grow into large fibrils with heights of several hundred nanometers and lengths of several hundred micrometers (Figure 3D). More details of fibril size are shown in Figures S4 and S5 of the Supporting Information. DLS and microscopy techniques also demonstrate that the filaments are derived from spherical aggregates, which has been observed in other systems.13 It should be noted that the fibrillar morphology is also observed at pH >7 but the formed fibrils will dissociate when the solution is made acidic (Figures S7 and S8 of the Supporting Information). Circular dichroism (CD) was used to analyze the chain conformations of ε-PL in aggregates. As shown in Figure 4, the ε-PL solution samples at different temperatures all exhibit similar CD spectra with a negative peak at ∼225 nm and positive bands at lower wavelengths, suggesting a β-sheet-rich conformation. Similar results showing that ε-PL adopts a βsheet conformation at pH ≥7 have been reported in the literature.14,15 Different from conventional peptides, the ε-PL chains in aqueous solutions are rich in β-type secondary structure even at room temperature, and little α-helix component is detected. This may explain the phenomenon that no lag time was observed in its further aggregation into amyloid-like fibrils. Stronger peaks are observed with an increase in the temperature and incubation time at 60 °C for 24 h, suggesting a better organized and/or dominating conformation of β-structure in the fibril aggregates. We also tested the binding of Congo red and thioflavin T to our samples, following the experimental protocol of Nilsson.16

Figure 1. Time dependence of the excess scattered intensity of the 2.5 mg/mL ε-PL solution at 25 (▲) and 60 °C (□) and pH 7.

intensity at 25 °C exhibits no prominent increase in the studied time period, suggesting that the aggregation of ε-PL is very slow at 25 °C. Therefore, an incubation temperature of 60 °C was adopted for all the studies. Figure 2 shows the hydrodynamic radius (Rh) distribution measured by DLS at different time points during the aggregation process at 60 °C. The Rh of the initial system shows a bimodal distribution (Figure 2A). The fast mode with a size between 1 and 10 nm is attributed to the diffusion of single ε-PL chains, and the peak around 100 nm is attributed to the diffusion of the aggregates formed by ε-PL in the initial

Figure 2. Size distribution of ε-PL at 2.5 mg/mL in an aqueous solution at 60 °C: (A) 1, (B) 34, (C) 63, and (D) 130 min. B

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Figure 3. Images of ε-PL aggregates from a 2.5 mg/mL ε-PL solution (pH 7), observed using AFM. (A) The ∼100 nm height particles were observed without incubation (scale bar of 5 μm). (B) Filaments appeared upon incubation at 60 °C for 5 min (scale bar of 5 μm). (C) Filaments assembled into fibrils at 30 min (scale bar of 5 μm). (D) After 48 h, larger fibrils were formed (scale bar of 50 μm). The morphology was also confirmed using (E) SEM at 30 min (scale bar of 20 μm) and (F) TEM at 60 min (scale bar of 1 μm).

As for the thioflavin T binding, no fluorescence enhancement was observed in our samples. In comparison, α-polylysine in a β-sheet conformation showed a typical Congo red binding profile but no thioflavin T fluorescence enhancement.17,18 We are interested in the effect of amine side groups on the fibril structure, as they may participate in hydrogen bonding. For example, Fulara and Dzwolak19 found that, in the β2 fibrils of poly-L-glutamic acid, the side carboxyl groups interact with amide groups in the backbone via three-center hydrogen bonds, forming side chain-involved amyloid-like fibrils. For ε-PL, we suspect that, with a smaller size and a shorter distance to the backbone chain, the side amine group may be more likely to form intersheet hydrogen bonds with a backbone amide from another chain. These additional hydrogen bonds may affect the chain arrangement in two ways. (1) In typical protein amyloid fibrils, the spacing between β-sheets is usually ∼10 Å, which is the average length of peptide side groups,20 but for ε-PL, it should be shorter because of the smaller side amine groups; with the intersheet hydrogen bonds, an even more compact structure should be possible. (2) As hydrogen bond formation requires the participating groups to be at certain distances and in certain orientations, the neighboring β-sheets would align with each other at an optimal relative position so that the strongest intersheet hydrogen bonds could be formed. We used simple computer modeling (ChemDraw 3D) to identify the most likely arrangement (Figure 5) and found that, to achieve the strongest hydrogen bonding interactions, the neighboring β-sheets should stagger along sheets so that the adjacent chains could be arranged in orthorhombic unit cells with an a

Figure 4. Far-UV CD spectra of a 1 mg/mL ε-PL solution (pH 7), recorded at room temperature (●) and 60 °C (○) and incubated for 24 h at 60 °C (▲). Samples at different temperatures all exhibit βconformation.

In the Congo red binding experiment, precipitation and a red shift of the UV absorption spectrum of Congo red were observed (Figure S9 of the Supporting Information), which is qualitatively similar to those of amyloid samples described in the literature. However, the characteristic apple-green birefringence was not observed in Congo red-stained samples. C

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Figure 5. Proposed structural model of ε-PL fibrils. To be clear, only the -NH-CO-CH(-NH2)- part of the chain is shown (nitrogen atoms colored blue and oxygen atoms red), and the green lines show the direction of β-sheets. The polymer chains (c axis) are perpendicular to the paper. For ε-PL, hydrogen bonds may be formed between side chain amine groups and backbone carbonyl groups from another chain, and the dotted lines represent hydrogen bonds involving the backbone carbonyl and side amine in two neighboring β-sheets. The distances between sheets and the relative position of adjacent sheets are adjusted to maximize hydrogen bond formation, thus forming an orthorhombic unit cell with an a dimension of ∼7.0 Å and a b dimension of ∼4.8 Å constituting the fibrils.

supporting the idea that the ε-PL chain orientation is perpendicular to the long axis of the fibril. In our model, an angle of ∼34° between β-sheets and the fibril axis exists in ε-PL fibrils. We believe the intersheet hydrogen bonds affect the direction of growth of fibrils. Usually, in amyloid fibrils of proteins/peptides, the side functional groups are too far from the backbone chains to interact with each other; thus, the hydrogen bonding direction is solely determined by the backbone chains, resulting in the backbone chains being perpendicular to the direction of growth of the βsheet, which is also the direction of growth of fibrils. However, for ε-PL, the summation of all hydrogen bonds has a direction different from that of the β-sheets (Figure 5), giving the fibrils a different dominant growth direction.

dimension of ∼7.0 Å and a b dimension of ∼4.8 Å. This structural model is similar to the β-form of poly-L-alanine.21,22 With the small methyl side groups, the hydrogen-bonded sheets in poly-L-alanine were able to pack in an orthorhombic or pseudo-orthorhombic manner to form a more closely packed crystal structure. This proposed structural model is supported by twodimensional XRD characterization of fibril samples (Figure 6A). According to this model, the two orthogonal diffractions at distances of 3.52 and 2.39 Å correspond to the (200) and (020) lattice planes, respectively, and the very strong diffraction at 4.0 Å should be indexed as (110) and (−110), corresponding to the intersheet distances. With these assumptions, an orthorhombic unit cell that exactly agrees with our structural model can be obtained. The resulting interplanar spacing fit very well with all the experimental diffraction peaks (Figure S10 and Table S1 of the Supporting Information, except for the strong diffraction ring at 2.82 Å, which we believe belongs to the sodium chloride crystal22). The model was also confirmed by SAED. As shown in Figure 6B and Figure S11 and Table S2 of the Supporting Information, the SAED pattern also agrees well with the proposed model. The fibril axis is shown in Figure 6B,

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CONCLUSION Similar to α-polypeptides, ε-PL also aggregates into a wellorganized fibrillar structure in aqueous solution. In conventional protein/peptide amyloids, hydrogen bonds among the backbone peptide chains determine the direction of growth of fibrils, explaining why almost all peptides share a common fibrillar structure. In ε-PL, it is possible for the amine side D

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Figure 6. (A) Two-dimensional XRD patterns of ε-PL aggregates. (B) SAED patterns of ε-PL fibrils (corresponding fibril shown in the inset). All patterns can well fit with the proposed model, except for the strong diffraction ring at 2.82 Å, which we believe belongs to the sodium chloride crystal. (6) Chiti, F.; Dobson, C. M. Annu. Rev. Biochem. 2006, 75, 333−366. (7) Fändrich, M.; Dobson, C. M. EMBO J. 2002, 21, 5682−5690. (8) Adamcik, J.; Mezzenga, R. Macromolecules 2012, 45, 1137−1150. (9) Yan, C.; Pochan, D. J. Chem. Soc. Rev. 2010, 39, 3528−3540. (10) Chen, C.; Wu, D.; Fu, W.; Li, Z. Biomacromolecules 2013, 14, 2494−2498. (11) Top, A.; Roberts, C. J.; Kiick, K. L. Biomacromolecules 2011, 12, 2184−2192. (12) Sipe, J. D.; Cohen, A. S. J. Struct. Biol. 2000, 130, 88−98. (13) Lin, W. R.; Zheng, C.; Wan, X. H.; Liang, D. H.; Zhou, Q. F. Macromolecules 2010, 43, 5405−5410. (14) Kushwaha, D. R. S.; Mathur, K. B. Biopolymers 1980, 19, 219− 229. (15) Lee, H.; Yamaguchi, H.; Fujimori, D. Spectrosc. Lett. 1995, 28, 177−190. (16) Nilsson, M. R. Methods 2004, 34, 151−160. (17) Benditt, E. P.; Eriksen, N.; Berglund, C. Proc. Natl. Acad. Sci. U.S.A. 1970, 66, 1044−1051. (18) LeVine, H. Amyloid 1995, 2, 1−6. (19) Fulara, A.; Dzwolak, W. J. Phys. Chem. B 2010, 114, 8278−8283. (20) Eanes, E. D.; Glenner, G. G. J. Histochem. Cytochem. 1968, 16, 673−677. (21) Brown, L.; Trotter, I. F. Trans. Faraday Soc. 1956, 52, 537−548. (22) Nguyen, J. T.; Inouye, H.; Baldwin, M. A.; Fletterick, R. J.; Cohen, F. E.; Prusiner, S. B.; Kirschner, D. A. J. Mol. Biol. 1995, 252, 412−422.

groups to interact with backbone amide groups and hence affect the intersheet distance and alignment and the direction of growth of fibrils. In terms of the chemical structure, ε-PL is closer to synthetic polymers (especially nylons) than to proteins. With a smaller number of amide linkages than proteins, its ability to form amyloid-like fibril structures suggests that amyloids may be more tolerant of the chemical structures of polyamides. It is also interesting to determine whether amyloid-like aggregates of synthetic polymers can be used as templates to induce protein amyloidosis, which will be very important to their biomedical applications.

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ASSOCIATED CONTENT

S Supporting Information *

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H NMR data, 13C NMR data, MALDI-MS data, AFM height measurements, SEM images, dye binding studies, two-dimensional XRD data, SAED data, and the comparison with α-PL. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-10-62756170. *E-mail: [email protected]. Phone: +86-10-62797572. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project is sponsored by the National Natural Science Foundation of China (Project 21074064 to Y.H.). REFERENCES

(1) Fraser, R. D. B.; MacRae, T. P. Conformations in Fibrous Proteins; Academic Press: New York, 1973. (2) Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. J. Mol. Biol. 1997, 273, 729−739. (3) Eisenberg, D.; Jucker, M. Cell 2012, 148, 1188−1203. (4) Shewmaker, F.; McGlinchey, R. P.; Wickner, R. B. J. Biol. Chem. 2011, 286, 16533−16540. (5) Dobson, C. M. Trends Biochem. Sci. 1999, 24, 329−332. E

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