Membrane Determinants Affect Fibrillation Processes of Î²-Sheet

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Membrane Determinants Affect Fibrillation Processes of #-sheet Charged Peptides Elad Arad, Ravit Malishev, Hanna Rapaport, and Raz Jelinek Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01318 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 17, 2017

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Membrane Determinants Affect Fibrillation Processes of β-sheet Charged Peptides Elad Arad, a# Ravit Malishev, a# Hanna Rapaport b*, and Raz Jelinek a* a Department of Chemistry, the Ben Gurion University of the Negev, Beer Sheva 84105, Israel b Avram and Stella Goldstein-Goren Department of Biotechnology Engineering and Ilse Katz Institute for Nano-Science and Technology (IKI), Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. KEYWORDS (Word Style “BG_Keywords”). Peptide aggregation, charged peptides, charged membranes, membrane interactions, beta-sheet peptides, amyloids,

ABSTRACT:

Assembly of fibrillar peptide structures is dependent both upon their intrinsic propensities

towards β-structure formation, as well as on structural modulation by external molecular factors. β-sheet structures may either be designed to form useful assemblies or be the undesired consequence of protein denaturation to toxic amyloid structures in several neurodegenerative diseases. Membrane bilayers have been implicated as primary initiators and modulators of amyloid fibrillation and the reasons for this effect are yet to be elucidated. Here, we employed a set of three charged peptides having the tendency to form β-sheet fibrils, to investigate the effect of zwitterionic and negatively-charged bilayer vesicles on their assembly structures. Microscopic and spectroscopic experiments revealed intimate relationship between peptide/membrane charges and fibrillation properties. Electrostatic attraction was apparent between oppositely-charged peptides and vesicles however such interactions did not appear to significantly modulate fibril morphologies of either the net anionic peptide or the cationic one. Yet, a 1 Environment ACS Paragon Plus

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dramatic structural effect was observed when the nominal zwitterionic peptide underwent fibrillation in presence of negatively-charged vesicles. Assemblies of this peptide display a net positive charge which facilitated the counter ionic interactions with the vesicles. Furthermore, these interactions templated a unique twisted fiber morphology demonstrating the dramatic effect membrane mediated interactions exert on fibril morphologies.

Introduction

In recent years, there has been growing interest in utilizing peptides as building blocks in certain secondary structures capable of assembling into nanostructures, for chemical, biological and medicinal applications.1–4 Tailoring the exact sequence of amino acids in designed peptide assemblies enables tuning the assemblies' kinetics and their mechanical and chemical properties. Peptides sharing the amphiphilic motif of alternating hydrophilic and hydrophobic amino acids tend to form β-sheet structures and assemble as either monolayers at interfaces and as sheets or elongated bilayer fibrils in bulk solutions5. Under appropriate conditions of peptide concentrations, salinity, and pH, fibril assemblies in aqueous solutions can be stabilized by hydrophobic interactions between the layers, πstacking between aromatic amino acids, electrostatic interactions and cross-strand hydrogen bonds along each layer3 (Scheme 1).

In these assemblies the hydrophobic side chains are shielded and the

hydrophilic side chains extend to the surrounding solution6.

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Scheme 1. Possible pathways to fibrillation of charged amphiphilic β-sheet peptides in aqueous solutions. At low concentrations peptides tend to be unfolded. The formation of soluble β-sheet oligomers can be facilitated by increase in peptide concentration, and upon external factors such as ionic interactions, change in pH, and membranes as templating surfaces. With enhanced effects of these factors, fibrils may phase-separate out of the solution and in some cases also stabilize a hydrogel phase. All assemblies are generally composed of bilayers that shield the hydrophobic amino acids from solution and expose the hydrophilic ones to the aqueous surrounding.

Designed amphiphilic β-sheet peptides comprising equal number of cationic and anionic amino acids, firstly introduced by Zhang, have been shown to form hydrogels that enhance cellular activities and regeneration of various tissues in vivo7,8. Amphiphilic peptides with a β-hairpin motif and with all positively charged hydrophilic amino acids, firstly designed by Schneider et al. could be triggered to form self-supporting hydrogels on exposure to cell culture media9,10. Anionic β-sheet peptides were shown to form stable hydrogels at near neutral pH, induce biomineralization11,12 and bone tissue regeneration4. All these amphiphilic and charged peptides exhibit similar basic motif with bilayer fibril assemblies. β-sheet amphiphilic peptides, among wide range of other primary sequences, may adopt amyloid-like fibril formation, defined by the structural property of β-sheet aggregation by misfolded proteins13. The 3 Environment ACS Paragon Plus


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different mechanistic avenues, outlined in Scheme 1, above, point to different interrelated factors affecting fibrillation pathways.

Furthermore, the presence of membrane interfaces was shown to

significantly affect fibrillation pathways, see below. Studies have shown that among many of these amyloids, phenylalanine plays a significant role in the fibrillation process, implying that π-stacking has major influence on stabilizing the assembly14–16. Membranes constitute in many instances targets for binding of amyloid proteins, and were shown to intimately affect misfolding and fibrillation pathways of such proteins17,18. The interactions with lipid bilayers which take place either on the lipid surface or inside the membrane may induce aggregation of oligomers19,20 and either accelerate or inhibit fibril formation21,22. Moreover, molecular modeling of amyloids have shown that electrostatics and hydrogen bonds between amyloid peptides and membrane headgroups play significant role in their interactions23. In this study, we examine the assembly of three model β-sheet peptides displaying charged amino acids in the presence of two types of membrane vesicles – comprising zwitterionic phospholipids and zwitterionic/negative lipids, respectively. A combination of zwitterionic and negatively-charged phospholipids was chosen to model membrane electrostatic interactions, designed to mimic neuronal membranes23. The peptides studied share the same sequence, Pro-X-(Phe-X)5-Pro, where X is either all Asp (denoted anionic FD, or an-FD), all Lys (cationic-FK, cat-FK), or alternating Asp / Lys (zwitterionic, zwi-FDFK). Multipronged experiments were carried out, designed to shed light on the effects of different lipid bilayers on the fibrillation properties of the peptides, morphologies, and membrane interactions. The experiments reveal distinct structural interplay between the bilayer interface and peptide assembly.


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Scheme 2. Structures of the fibril-forming model peptides.

Experimental Section

Materials Peptides. Pro-Asp-(Phe-Asp)5-Pro, (Mw of 1638.65), Pro-Lys-(Phe-Lys)5-Pro (Mw of 1717.16), and Pro-Asp-(Phe-Lys-Phe-Asp)2-Phe-Lys-Pro (Mw of 1677.90) denoted An-FD, Cat-FK and Zwi-FDFK, respectively, were all custom synthesized, purified by high performance liquid chromatography to 95 % and supplied as lyophilized powders (An-FD by American Peptide, Sunnyvale, CA, Cat-FK and ZwiFDFK by GenScript, Piscataway, NJ). 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline (POPC), 1palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG),1,2-dimyristoyl-snglycero-3-phosphoethanolamine-N-(7-

nitro-2-1,3-benzoxadiazol-4-yl)

(N-NBD-PE),

and

1,2-

dimyristoyl-sn-glycero-3-phosphoethanolamin-e-N-(lissamine rhodamine B sulfonyl) (N-Rh-PE) were purchased from Avanti Polar Lipids. 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), were purchased from Sigma-Aldrich (Rehovot, Israel). 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMADPH) was obtained from Molecular Probes, Inc. (Eugene, Oregon). Peptide and Vesicle preparation Peptide solutions were prepared by dissolving lyophilized peptide powders in deionized water (DI water, 18.2 MΩ cm, Thermo Scientific) and applying 30 seconds sonication using probe-sonicator at 5 Environment ACS Paragon Plus


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30% amplitude (Sonics Vibra-cell VCX-130, Newtown, CT, USA) to accelerate dissolution. Solutions of cat-FK were prepared by dissolving the lyophilized peptide powder in 2 M KCl water solution (that was subsequently diluted, 1:1 v/v, in either vesicle solution or DI water), and then sonicated. Vesicles consisting of POPC or POPC:POPG, 1:1 mol:mol, were prepared by dissolving the lipid components in chloroform/ethanol (1:1, v/v) and drying together in vacuum. Small unilamellar vesicles (SUVs; POPC and POPC:POPG 1:1, mol:mol) were prepared in DIW by sonication using Sonics Vibra-cell VCX-130 sonicator (Newtown, CT, USA) at room temperature for 10 min at 20% amplitude. Vesicle suspensions were allowed to anneal for 1 h at room temperature prior to usage. Samples' pH values were measured after dissolution and adjusted to pH=6. Circular Dichroism (CD) Spectroscopy CD spectra were recorded in a range of 190–260 nm on a Jasco J-715 spectropolarimeter (Tokyo, Japan), with 0.01 cm quartz cuvettes. Concentration-dependent measurements were performed with a 1 mM peptide stock solution diluted with DIW to a set of final concentrations in the range of 0.1 – 0.5 mM. To test the effect of lipid vesicles concentration on the peptides' secondary structures POPC or POPC:POPG vesicles were added to the peptide solutions at 0.1-0.6 mM final concentrations. Background CD signals recorded from vesicles were subtracted from the corresponding spectra. Phenylalanine fluorescence assay Phenylalanine fluorescence measurements were conducted at 25°C, λex = 250 and λem = 280 nm in 96-well path cell culture plates on a BioTek SynergyMX plate reader (Vermont, USA). The samples contained 0.05 mM peptide of either an-FD, cat-FK or zwi-FDFK and 0.25 mM Phe (the single amino acid) as a control, in absence or presence of POPC and POPC:POPG vesicles (final concentration 0.5 mM). A 100 µL sample of the reaction was measured in each well. Atomic force microscopy (AFM) Peptide samples were scanned by atomic force microscopy (Cypher ES, Asylum Research, an Oxford Instruments company, Goleta, CA), in tapping mode, following deposition of 5 µL peptide solution (0.3 mM peptide concentration/ 0.3 mM lipid concentration) on a freshly cleaved mica substrate. After five 6 Environment ACS Paragon Plus

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minutes, 100 µL DIW was added and the sample was imaged under aqueous solution. All images were acquired using a silicon probe (AC 240, Olympus) with a spring constant of 2 N/m, frequency 70 kHz and a tip with a radius of 9 nm. Transmission Electron Microscopy (TEM) Peptide solutions were prepared as mentioned above at 0.6 mM aqueous/2M KCl solutions and were diluted up to 0.3 mM concentration with DIW/lipid vesicle solution (0.6 mM lipid concentration). Following 30 min. incubation at room temperature, 5 µL of the sample's solution was placed on 400mesh copper grids covered with a carbon-stabilized Formvar film. Excess solutions were removed after 2 min. of incubation. Dry TEM samples were negatively stained for 30 sec. with a 1 % uranyl acetate solution. Samples were viewed in an FEI Tecnai 12 TWIN TEM operating at 120 kV.

ζ potential ζ potential values of peptide aqueous/KCl solutions 0.3 mM were measured by Zetasizer (Zetasizer Nano ZS, Malvern, Worcestershire, UK). Förster resonance energy transfer (FRET) Lipid vesicles were prepared by the procedure described above. Prior to drying, the lipid vesicles were supplemented with N-NBD-PE and N-Rh-PE (at a molar ratio of POPC:N-NBD-PE:N-Rh-PE or POPC:POPG:N-NBD-PE:N-Rh-PE, 500:500:1 and 500:500:1:1 respectively). 0.6 mM an-FD, cat-FK or zwi-FDFK were added to POPC or POPC:POPG lipid vesicles (final vesicle concentration 1 mM). Fluorescence emission spectra were acquired (λex = 469 nm) in the range of 500 – 650 nm on a FL920 spectrofluorimeter (Edinburgh Co., Edinburgh, UK). FRET efficiency was calculated as follows:

R in general is the ratio of fluorescence emission of NBD-PE (536 nm)/Rhodamine B-PE (593 nm) where Ri is that ratio in the peptide/vesicles mixtures, R100% is measured following the addition of 20% Triton X-100 to the vesicles (causing complete dissolution of the vesicles) and R0 corresponds to



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the ratio recorded for the vesicles only24. The data is presented as mean ± SEM of at least three independent experiments. Fluorescence anisotropy The fluorescence probe TMA-DPH dissolved in THF (1 mM) was incorporated into the SUVs (POPC or POPC:POPG) by adding the dye solution to the vesicles, at a final concentration of 1.25 µM. After 30 min. of incubation at room temperature TMA-DPH fluorescence anisotropy was measured (λex = 355 nm, λem = 430 nm) before and after addition of an-FD, cat-FK or zwi-FDFK peptides to vesicle solution (final lipid concentration 0.3mM, final peptide concentration 0.3mM). this was measured on a FL920 spectrofluorimeter (Edinburgh Co., Edinburgh, UK). Anisotropy values were calculated by the embedded spectrofluorimeter software.

RESULTS AND DISCUSSION

Peptide aggregation and fibril structures modulated by lipid vesicles. Scheme 2 depicts the structures of the three peptides, illustrating the segregation of hydrophobic and hydrophilic amino acids along the backbone of the peptides in the β-sheet structure. The two proline residues at both C and N terminals contribute to the stability of their β-sheet assembly (Fig. S1)25. The peptides an-FD and cat-FK exhibit all aspartate or lysine residues, respectively, whereas the zwitterionic peptide, zwi-FDFK contains three alternating lysine and aspartate residues upon the hydrophilic face of the peptide. Moreover, the three peptides form dense and stable fibrillar structures in aqueous solution with very similar morphologies (Figure S1). Circular dichroism (CD) spectroscopy was utilized to examine the secondary structures of 0.3 mM peptides in water and in the presence of vesicles comprising only the zwitterionic phospholipid 1palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline (POPC), or vesicles comprising equimolar ratio of POPC and the anionic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) (Fig. 1). All spectra feature absorbance minima at 215 nm characteristic of β-sheet structures 8 Environment ACS Paragon Plus

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(denoted in Scheme 1 as soluble folded oligomers), and at 203 nm attributed to unfolded or polyproline II conformation26, which is typical of highly charged peptides.

Figure 1. Peptide conformations by circular dichroism (CD). Spectra of 0.3 mM an-FD (A), cat-FK in 1M KCl (B) and zwi-FDFK (C) in DI water (black line); 0.3 mM POPC (blue line); 0.3 mM POPC:POPG (red line). (D) The fraction of β-sheet relative to unfolded structure represented by the ellipticities θ215/θ203 ratios, as a function of POPC:POPG lipid molar concentration (0-0.5 mM).

The anionic peptide, an-FD, adopts these β-sheet structures in deionized water, with no added counter ions (Fig. 1A). It has been shown previously that anionic β-sheet peptide assemblies may show a nominal pKa value higher than that of single Asp residues, due to their ability to form stabilizing hydrogen bonds within β-sheet assemblies. The positively-charged peptide, cat-FK, exhibits weak CD signals in distilled water reflecting unfolded structures (SI. 2). However, in 1M KCl, the peptide yielded a similar CD spectrum to an-FD (Fig. 1B). The zwitterionic peptide, zwi-FDFK, in DI water



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features a similar spectrum to that of the two other peptides yet, with overall much higher intensities indicative of its higher tendency to form β-sheet structures (Fig. 1C). In the presence of POPC vesicles, all three peptides gave rise to almost the same spectra as in aqueous solution (Fig. 1, blue lines). Nonetheless, the CD data of the peptides in the presence of POPC:POPG vesicles attest to significant interactions of cat-FK and zwi-FDFK with these vesicles. Specifically, the two peptides formed insoluble fibrillar structures, likely comprising β-sheet assemblies in solutions containing higher concentrations of the negatively-charged vesicles, reflected in the decrease in unfolded random intensity and higher ratio between the ellipticities at 215 nm compared to 203 nm (Fig. 1A-B, red spectra). The conformation of an-FD, on the other hand, was not affected by the presence of POPC:POPG vesicles (Fig. 1A). Figure 1D depicts the CD intensity ratio θ215/θ203, corresponding to the extent of β-sheet conformation versus unfolded structure, recorded for 0.3 mM peptides with different concentrations of POPC:POPG vesicles. The graph in Figure 1D indicates, somewhat surprisingly, that zwi-FDFK adopted more pronounced β-sheet conformation as compared to cat-FK, although the latter peptide is more positivelycharged as it contains three more lysine residues. The fact that POPC:POPG favored interactions with the weaker cationic charged zwi-FDFK reflect the existence of an optimal balance between the charge of the β-sheet assembly and that of the lipid vesicles. To assure that this effect is not specific to POPG containing vesicles, POPG was replaced with phosphatidylserine (PS) which also displays a negativelycharged headgroup (Figure S3). Indeed, a similar pattern of increased β/R ratio in the presence of the charged lipid bilayer was recorded (Figure S3). Furthermore, it should be noted that in all these experiments the stabilization of cat-FK in β-sheet structure necessitated screening by 1M KCl ions. To confirm the significance of the bilayer rather than ionic strength, cat-FK's CD spectra in presence of POPC:POPG vesicles with and without KCl addition were recorded (Figure S2). To investigate the fibrillation kinetics of the peptides in water and upon addition of the lipid vesicles, we recorded the peptides' phenylalanine fluorescence over time after dissolution in water and in the 10 Environment ACS Paragon Plus

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presence of vesicles (Fig. 2).

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Phenylalanine fluorescence is highly sensitive to the chemical

environment of the amino acid; in particular, the intensity of phenylalanine fluorescence was previously shown to be intimately enhanced by both hydrophobic environment and π-stacking interactions in assembly processes of phenylalanine-containing peptides27,28. While the fluorescence intensity of zwiFDFK remained constant in DI water and in the presence of POPC vesicles, a dramatic fluorescence increase was observed when POPC:POPG vesicles were added to the peptide solution (Fig. 2A). The increase of phenylalanine fluorescence likely corresponds to stacking interactions promoted by the POPC:POPG vesicles during fibril formation29. Importantly, both cat-FK (in 1M KCl) and an-FD exhibited constant phenylalanine fluorescence in the POPC:POPG vesicle solutions in parallel with their assembly into fibrillar structures; this result is consistent with the CD measurements in Figure 1A-B indicating no significant effect of the negatively-charged vesicles upon assemblies of these peptide. It should be emphasized that all three peptides contain the same Phe hydrophobic backbone promoting βsheet assembly due to hydrophobic interactions. Yet, in case of zwi-FDFK, the (electrostatically-based) interaction with POPC:POPG vesicles induced significant reorganization of the Phe residues, giving rise to the increase in their fluorescence, apparent in Figure 2.



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Figure 2. Phenylalanine Fluorescence kinetics in an-FD, cat-FK and zwi-FDFK peptides. Fluorescence (excitation=250 ݊݉, emission=280 ݊݉) was recorded in 50 µM peptide concentrations, in water and in the presence of POPC or POPC:POPG vesicles, respectively. A. zwi-FDFK peptide; B. cat-FK; C. an-FD.


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Atomic force microscopy (AFM) measurements were applied to examine the peptide fibrils assembled in presence of the vesicles to further probe the effects of the lipid bilayers on fibril morphology30 (Fig. 3). The AFM images complement the CD and phenylalanine fluorescence experiments (Fig. 1 and 2) and underscore the distinct structural transformation zwi-FDFK undergo following interactions with POPC:POPG vesicles. While fibril morphology of cat-FK appears similar in 1M KCl solution and in the presence of POPC or POPC:POPG vesicles (Fig. 3A-C), zwi-FDFK assembled into much thicker fibrils in the presence of POPC:POPG vesicle and apparently to less extent in presence of POPC (Fig. 3D-F).

Figure 3. Microscopic analysis of peptide fibrils. AFM images of cat-FK (in aqueous 1M KCl solution) (A-C) and zwi-FDFK (D-F). The images were recorded after 30 min incubation of 0.3 mM peptide and 0.3 mM lipids. Scale bars correspond to 50 nm. Cryo-transmission electron microscopy (cryo-TEM) images of (G) zwi-FDFK incubated with POPC:POPG vesicles. Arrows point to vesicles bound along peptide fibrils demonstrating interactions between the two components. (H) Zwi-FDFK incubated with POPC vesicles showing coexistence with no apparent mutual interactions. Scale bars correspond to 50 nm (10nm in the insets). 13 Environment ACS Paragon Plus


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Notably, the AFM images in Figure 3E reveal that incubation of zwi-FDFK with POPC:POPG vesicles, and to lesser extent with POPC vesicles, gave rise to screw-shape fibrils which were not observed in samples deposited from aqueous solutions (Fig. 3D-F). While the lipid vesicle environment may lead to specific peptide morphology, it should be noted that the mica substrate may also affect the fibril assembly and organization. Mica is slightly negative, and thus electrostatic interactions between its surface and positively-charged residues is possible. Yet in the current system the mica apparently had negligible structural effects compared to the vesicles. It should be further noted that the an-FD samples were not compatible with the AFM analysis as both the peptide and mica substrates are negatively charged thereby preventing peptide assembly on the surface. Transmission electron microscopy (TEM) image acquired for zwi-FDFK co-incubated with POPC:POPG vesicles (Fig. 3G) provides vivid evidence for the significant effect of the negatively-charged vesicles on the peptide fibrils, which appear twisted in shape. Figure 3H, on the other hand, shows POPC vesicles exhibiting no apparent interaction within a mesh of zwi-FDFK fibrils. To evaluate the peptides' effective charge at the relevant assembly states, ζ-potential was measured (Fig. 4A). As expected, the ζ-potentials of both an-FD and cat-FK (in presence of 1M KCl) exhibited high negative and positive effective fibril charges, respectively. The ζ-potential of zwi-FDFK was also found to be positive at pH ~ 6 in which all experiments were performed. To further elucidate the evolution of surface charge of zwi-FDFK assemblies, the peptides (at a concentration of 0.03 mM) were titrated while monitoring the pH of the system (Fig. 4B). The titration curves reflect the protonation states of the peptides in each pH. In particular, protonation (or deprotonation) can change the hydrogen bonds and salt bridges, affecting the assembly and fibrillation process. In case of cat-FK, the lysine's amine protonation induces same positive charge repulsion, and conformation change likely occurred at around pH 8. For an-FD, the protonation pH is reduced. Zwi-FDFK shows two pKa regions (red circled). The basic pH of 9.2 likely corresponds to the protonation of the Lys amines, while the pKa region at around pH 6.5 is responsible for protonation of the carboxylic acid group (as previously observed, this apparent pKa of Asp residues in the β-sheet structure is about 2 pH units higher than free 14 Environment ACS Paragon Plus

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Asp in solution31). Therefore, at pH ~6 most of the zwi-FDFK side chains are in their protonated state, the peptide is positively-charged and favors interactions with the negatively-charged POPC:POPG vesicles.

Figure 4. Effective surface charge of the peptide fibrils. (A) ζ-potential measurement of the three peptides in aqueous solution. 0.3 mM peptide solutions were measured in aqueous solution in fibril forming conditions (cat-FK in 1M KCl solution) after 30-minute incubation at room temperature. CatFK exhibits slightly positive charge due to salt shielding effect while zwi-FDFK displays positive fibril surface charge. (B) pH titrations of the three peptides (0.03mM), showing the protonation pH steps.



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Effects of peptide assemblies on lipid bilayer properties. While Figures 1-4 illuminate the effects of membrane vesicles having different lipid compositions and surface charge upon the structural properties of the peptide assemblies, the reciprocal effects of the peptides upon bilayer organization and dynamics were also investigated (Fig. 5 and 6). Figure 5 presents Förster resonance energy transfer (FRET) experiments, carried out on POPC vesicles and POPC:POPG vesicles, respectively, which further incorporated the fluorescent donor, 1,2-dimyristoylsn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadi-azol-4-yl)

(N-NBD-PE),

and

fluorescence acceptor, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamin-e-N-(lissamine rhodamine B sulfonyl) (N-Rh-PE)32. FRET experiments reveal the effect of peptide interactions upon membrane rigidity and organization. Figure 5 reveals significant differences in FRET response both between the two vesicle compositions as well as between the three peptides. Importantly, both in N-NBD-PE/ N-Rh-PE/POPC vesicles (Fig. 5A) and N-NBD-PE/N-Rh-PE/POPC:POPG vesicles (Fig. 5B), an-FD seemed not to induce lipid reorganization as the FRET efficiency was identical (at around 1.0) to the control vesicles (not incubated with the peptides). Cat-FK (in 1M KCl solution), however, induced significant increase in FRET efficiency to almost 1.15 (Fig. 5B), particularly in case of the N-NBD-PE/N-Rh-PE/POPC:POPG vesicles. Greater FRET can be ascribed to enhanced rigidity and/or clustering of the phospholipids due to binding of the positively-charged peptide assemblies to the bilayer interface22,33. In contrast to the FRET increase induced by cat-FK, Figure 5 indicates that zwi-FDFK had the opposite effect upon the bilayers, giving rise to slight reduction of FRET efficiency, to approximately 0.99 and 0.98 in NBD-PE/Rh-PE/POPC and NBD-PE/Rh-PE/POPC:POPG vesicles, respectively. Lower FRET in lipid bilayer systems induced by membrane-active compounds has been ascribed to bilayer insertion of the compounds and consequent disruption of lipid organization34. Notably, lower FRET was apparent in the N-NBD-PE/N-Rh-PE/POPC:POPG vesicles (Fig. 5B) – consistent with the spectroscopy and microscopy experiments in Figures 2-4 which point to distinct interactions between zwi-FDFK and the negatively-charged POPC:POPG vesicle bilayers. 16 Environment ACS Paragon Plus

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Figure 6 presents fluorescence anisotropy measurements designed to further probe the effects of the peptides upon bilayer fluidity35,36. The experiments outlined in Figure 6 utilized POPC vesicles and POPC:POPG vesicles, respectively, which also contained the fluorescent dye trimethylammoniumdiphenylhexatriene (TMA-DPH). TMA-DPH is embedded in the hydrophobic environment of the bilayer, anchored close to the vesicle interface by the TMA residue; accordingly, changes to the fluorescence anisotropy of the DPH probe can be correlated to modulation of bilayer fluidity induced by membrane-active compounds37,38.

Figure 5. Forster resonance energy transfer (FRET) between fluorescent donor and acceptor embedded within membrane bilayers. Förster resonance energy transfer (FRET) efficiency measured in (A) NBD-PE/Rh-PE/POPC vesicles (1/1:500 mole ratio), and (B) NBD-PE/Rh-PE/POPC:POPG vesicles (1/1/500:500), in presence of 0.3mM peptide solution. FRET efficiency value of 1.00 represents the control vesicles prior to addition of the peptides. Error bars represent SD (triplicate experiments).



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Figure 6. Effect of the peptides upon bilayer dynamics properties. Fluorescence anisotropy of TMADPH embedded in (A) POPC or (B) POPC:POPG vesicles after 5 min. incubation with peptide solution (at concentration of 0.3mM). Error bars were calculated from triplicate experiments.

The fluorescence anisotropy measurements in Figure 6 corroborate the FRET data and further illuminate the effect of cat-FK, an-FD, and zwi-FDFK interactions upon bilayer dynamics. Figure 6A indicates that, in case of POPC vesicles, none of the peptides significantly altered the bilayer fluidity. However, for POPC:POPG vesicles (Fig. 6B), zwi-FDFK gave rise to small, albeit experimentallysignificant increase in bilayer fluidity. Cat-FK (at 1M KCl) affected a significantly higher fluorescence anisotropy of 0.28 corresponding to lower fluidity of the bilayer, consistent with its pronounced interaction with the bilayer, as discerned in the FRET data (Figure 5).


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Scheme 3. Schematic illustration of zwi-FDFK peptides in the absence or presence of membrane. Effect of (left) negatively-charged and (right) zwitterionic bilayer vesicles upon the assembly and morphologies of fibrils formed by zwi-FDFK.

While pronounced electrostatic attraction between cat-FK and the negatively-charged vesicles is expected to result in tight binding and consequent bilayer rigidity apparent in Figure 6B, the interaction between zwi-FDFK and the zwitterionic vesicles is unique and echoes the distinctive interactions and structural reorganization emanating from interactions between the peptide and the vesicles. Scheme 3 illustrates a possible model accounting for the experimental data in Figures 2-6.

In

particular, the negatively-charged bilayers exert significant effect upon the zwitterion zwi-FDFK peptide assembly, enhancing β-structure formation and modifying fibril morphology. This outcome is probably due to the affinity between the mildly-charged zwi-FDFK at pH of 6 and the negativelycharged POPC:POPG vesicles. The vesicles likely serve as nucleating templates that locally increase the peptide concentrations due to the electrostatic attraction, thereby promoting β-sheet formation and screw-shaped morphology. In comparison, no morphology differences were observed in case of an-FD and cat-FK in the absence or presence of the vesicle bilayers. The major effect of cat-FK on POPC:POPG vesicles, reflected in the FRET and anisotropy measurements (Fig. 5 and 6, respectively) may be related to electrostatic attraction. This may indicate the importance of the fibril surface's charge for interactions with the bilayer, suggesting correlation between assembly state and effective charge. A recent study showed distinct differences between the effect of the three peptides studied here on blood 19 Environment ACS Paragon Plus


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clotting that were attributed to electrostatic interactions between the fibrils and blood clotting proteins39. This work points to possible influences these peptides may have on cell membranes such as red blood cells (RBCs)40. This study also underscores distinctive differences between negatively-charged and positivelycharged peptide assemblies. Cat-FK fibrillation was studied in 1M KCl - a solution which induced fibril formation and apparently even supported the interactions between this peptide fibrils and the negativelycharged vesicles. Hence, this peptide requires strong counterbalancing electrostatic interactions for stabilizing fibril assemblies that may interact and also stabilized by negatively-charged membranes. zwi-FDFK which could apparently be considered overall uncharged was, in fact, positively-charged at near neutral pH conditions. The mildly-charged characteristics of this peptide rendered it susceptible to the effect of the charged vesicles. The membranes appeared to have both an enhancement effect on the extent of β-sheet formation and a dramatic influence, relying on electrostatic interactions and possibly involving hydrophobic interactions with the lipid tails that led to the thick screw-shaped fibrils.

Conclusions Lipid bilayers can induce and intimately modulate cooperative peptide assemblies, particularly fibril morphologies.

This study demonstrates that a negatively-charged lipid bilayer induced distinct

morphological change in fibrils assembled by a zwitterion peptide. This structural modulation cannot be described only by electrostatics, and points to intimate relationship between peptide assembly and interfacial properties of the bilayer. In particular, the presented data suggest that charged bilayer interfaces may promote β-sheet folding in case of zwitterion peptides. This work would contribute to a better understanding of the impact of membranes upon peptide fibrillation, and would aid the design and identification of biomedical applications of fibrillar peptides, particularly in physiological membrane environments. Future studies should examine other important factors pertaining to peptide fibrillation, such as local peptide-peptide interactions at the membrane interface, and the effect of peptide length. It is also 20 Environment ACS Paragon Plus

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expected that additional experiments will focus upon hydrophilic but not charged peptides that are rich in residues such as histidine, threonine and serine. Applications of such peptide/vesicle architectures are expected to contribute to both providing a better understanding of amyloid peptide aggregation, as well employed in novel drug delivery routes.

SUPPORTING INFORMATION INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI:

SI1. Transmission Electron Microscopy images of the an-FD, cat-FK and zwi-FDFK peptides in aqueous solution without any vesicles. SI2. Circular Dichroism and AFM comparison of cat-FK in unsalted solution and in 1M KCl solution. SI3.POPC:PS lipid vesicles effect on β-sheet assembly of cat-FK and zwi-FDFK peptides. SI4. FRET measurement containing unordered cat-FK effect on membrane bilayer. SI5. Titration pattern of cat-FK in presence of 1M KCl.

ACKNOWLEDGMENT

We thank Dr. Alexander Upcher for help with TEM. We are grateful to Jurgen Jopp for help with AFM. ABBREVIATIONS An-FD, peptide sequence PDFDFDFDFDFDP; zwi-FDFK, peptide sequence PDFKFDFKFDFKP; cat-FK, peptide sequence PKFKFKFKFKFKP; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3phospho-(1'-rac-glycerol) (sodium salt).

ASSOCIATED CONTENT

This material is available free of charge via the Internet at http://pubs.acs.org. 21 Environment ACS Paragon Plus


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AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]; *E-mail: [email protected] Hanna Rapaport and Raz Jelinek contributed equally. NOTES

The authors declare no competing financial interest. AUTHOR CONTRIBUTIONS

All authors have given approval to the final version of the manuscript.

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Graphical Abstract

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Membrane Determinants Affect Fibrillation Processes of Î²-Sheet

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