To Be Fibrils or To Be Nanofilms? Oligomers Are Building Blocks for

Article Cite This: Langmuir 2018, 34, 2332−2343

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To Be Fibrils or To Be Nanofilms? Oligomers Are Building Blocks for Fibril and Nanofilm Formation of Fragments of Aβ Peptide Olga M. Selivanova,† Alexey K. Surin,†,‡ Yury L. Ryzhykau,§ Anna V. Glyakina,†,∥ Mariya Yu. Suvorina,† Alexander I. Kuklin,§,⊥ Vadim V. Rogachevsky,# and Oxana V. Galzitskaya*,† †

Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Russia State Research Center for Applied Microbiology & Biotechnology, Obolensk 142279, Russia § Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russian Federation ∥ Institute of Mathematical Problems of Biology RAS, Keldysh Institute of Applied Mathematics of Russian Academy of Sciences, Pushchino 142290, Russia ⊥ Joint Institute for Nuclear Research, Dubna 141980, Russian Federation # Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino 142290, Russia

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‡

ABSTRACT: To identify the key stages in the amyloid fibril formation we studied the aggregation of amyloidogenic fragments of Aβ peptide, Aβ(16− 25), Aβ(31−40), and Aβ(33−42), using the methods of electron microscopy, X-ray analysis, mass spectrometry, and structural modeling. We have found that fragments Aβ(31−40) and Aβ(33−42) form amyloid fibrils in the shape of bundles and ribbons, while fragment Aβ(16−25) forms only nanofilms. We are the first who performed 2D reconstruction of amyloid fibrils by the Markham rotation technique on electron micrographs of negatively stained fragments of Aβ peptide. Combined analysis of the data allows us to speculate that both the fibrils and the films are formed via association of ring-shaped oligomers with the external diameter of about 6 to 7 nm, the internal diameter of 2 to 3 nm, and the height of ∼3 nm. We conclude that such oligomers are the main building blocks in fibrils of any morphology. The interaction of ring oligomers with each other in different ways makes it possible to explain their polymorphism. The new mechanism of polymerization of amyloidogenic proteins and peptides, described here, could stimulate new approaches in the development of future therapeutics for the treatment of amyloidrelated diseases.

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By the 1980s, there was a general idea that fibrils were formed from filaments (from 2 to 6), which interact with each other laterally, can be twisted at different times, and form bands, bundles, clusters. The average width (diameter) of filaments/fibrils is ∼10 nm, and the length can exceed 15 μm.1 The filaments are β-sheets extending along the entire axis of the fibril. Such a view comes from 1935,2 when X-ray diffraction data from fibrils of fibrillar proteins (fibroin, β-keratin, βmyosin) and partially denatured globular protein albumin were studied. For these objects, the similar X-ray diffraction patterns with characteristic reflections (4.65 and 9.8 Å) were obtained. From that time the interpretation of the structural organization of fibrils as folded sheets (“pleated sheet”) stemmed. For the first time the term cross-β structure appeared in 1968, and since then it has remained the basic representation for explaining the structure of amyloid structures.3 With the beginning of the use of the electron microscope for the study of biological objects,

INTRODUCTION

Aβ(1−42) peptide is linked to a severe neurodegenerative anomaly in humansAlzheimer’s disease. Large aggregates (plaques) formed from fibrils consisting of the Aβ(1−42) peptide are found in the brain tissue of the sufferers. Considering its role in the disease, the Aβ peptide has become one of the most intensely studied amyloidogenic peptides. Despite the large number of publications devoted to studying various fragments of the Aβ peptide, the understanding of the molecular mechanism of the formation of Aβ fibrils is not yet clear. One of the approaches in studying of formation of Aβ peptide amyloids is the comparative analysis of properties of its amyloidogenic fragments and various mutant forms. Such studies showed that Aβ peptide fragments of different lengths exhibited differences in their key amyloid-forming characteristics (rate, lag period, fibril morphology, X-ray diffraction patterns) and made it possible to associate some particular regions of the peptide with the amyloidogenic properties (the propensity for aggregation, the morphology of the formed fibrils). © 2018 American Chemical Society

Received: September 27, 2017 Revised: January 12, 2018 Published: January 16, 2018 2332

DOI: 10.1021/acs.langmuir.7b03393 Langmuir 2018, 34, 2332−2343

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work began in the 1960s to study sections of tissues damaged by amyloidosis (liver, kidneys, spleen),4 and then extracts from the same tissues.5 Although some authors have already observed ring oligomeric structures,6 the basic interpretation of the EM images still suggests that the ring oligomer is a longitudinal section of the fibril. We imagine that it was easier to reconcile the existing and mysterious data of X-ray and EM analyses. To date, a large number of molecular organization models for amyloid fibrils have been proposed, including those for the whole Aβ(1−42) peptide and its fragments.7−18 The basis of all models is the idea of various forms of organized βsheets, running along the entire axis of the fibril. However, it should be noted that in the formation of fibrils, rounded oligomers (often annular) are always observed at early times, which disappear when forming mature fibrils. That is, oligomers are invariable participants in the amyloidogenesis of many proteins/peptides. At the same time, it is shown that it is the oligomers, starting with dimers, that are the most toxic samples.19 Recently, however, there appeared works that offer other models of fibril formation. For example, it has been shown in ref 20, when studying the formation of Aβ1−42 fibrils by the peptide with the help of the EM method, that fibrils are formed from oligomers. In ref 21, in the study of fibrillation of αsynuclein, a granular fibrillation mechanism is proposed (“a double-concerted fibrillation model”), in which monomers form granules and then the granules are associated with fibrils. In both cases, the diameter of the oligomers and granules coincides with the diameter of the thinnest fibrils. Recently, it has been demonstrated using Aβ40 and Aβ42 recombinant and synthetic peptides that their corresponding fibrils are formed from complete oligomeric ring-like structures.1 These oligomers associate in a fibril in such a way that they interact with each other, overlapping slightly. It has also been shown that the fragments of Aβ peptides Aβ(24− 34), Aβ(25−35), and Aβ(26−36) can form oligomeric structures resembling cylindrins and β-barrels.22 Numerous literature data demonstrate the toxicity of oligomer samples, from small oligomer formations (dimers) to large supramolecular complexes (annular pores), with the structure of Aβ(1−42) peptide fragments also being analyzed in recent years.23−32 Further understanding of the properties of the peptide fragments will allow the development of highresolution biological models of the conformational changes in amyloidogenic proteins. We have investigated the properties of three amyloidogenic fragments of Aβ peptide and studied the process of amyloid formation by these peptides. It has been demonstrated that one of them (16−25) forms nanofilms, while the others (31−40 and 33−42) form amyloid fibrils. All three fragments have the cross-β structure. On the basis of the EM images, X-ray diffraction patterns, tandem mass spectrometry, and structural modeling of the peptides, we have suggested the molecular structures for nanofilms formed by Aβ(16−25) and for amyloid fibrils formed by Aβ(31−40) and Aβ(33−42). We have shown that the ring-like oligomers act as a growth unit in the formation of both fibrils and nanofilms. Thus the process of formation of amyloid fibrils from ring oligomers that interact with each other in various ways and form polymorphic fibrils can be another possible way of forming amyloid fibrils.

Article

EXPERIMENTAL SECTION

Chemical Synthesis of Three Fragments Aβ(16−25), Aβ(31− 40), Aβ(33−42). Solid-phase chemical synthesis was used to obtain preparative amounts of three peptide fragments: +H3N-Lys-Leu-ValPhe-Phe-Ala-Glu-Asp-Val-Gly-COO− (16−25), +H3N-Ile-Ile-Gly-LeuMet-Val-Gly-Gly-Val-Val-COO− (31−40), and +H3N-Gly-Leu-MetVal-Gly-Gly-Val-Val-Ile-Ala-COO− (33−42). The chemical synthesis and purification of such peptides are a complex task due to low solubility of the peptides in water and organic solvents. The synthesis was performed using the Fmoc methodology.33 The purified peptide was tested using an Orbitrap Elite mass spectrometer (Thermo Scientific, Germany). The estimated peptide mass coincided with the calculated one. Electron Microscopy. The samples of the three fragments prepared in 5% DMSO, 50 mM Tris-HCl, pH 7.5 and concentration 0.5 to 1.0 mg/mL were incubated at 37 °C. In fixed time intervals, aliquots were taken for the EM analysis. The preparations for this analysis were negatively stained. A copper grid (400 Mesh) covered with a Formvar film (0.2% Formvar solution in chloroform) was placed on a preparation drop (∼10 μL). After adsorption, the grids with the preparation were stained by 1% (weight/volume) aqueous solution of uranyl acetate. The excess of the preparation and uranyl acetate was removed with filter paper. The preparations were analyzed on a JEM-1200EX microscope (JEOL, Japan) at an accelerating voltage of 80 kV. The recording was made at 40 000× magnification on a Kodak film (SO-163) for electron microscopy studies. To better reveal the morphological features of amyloid fibrils, we used the modified Markham rotation techniques.34 X-ray Analysis. The Aβ(16−25), Aβ(31−40), and Aβ(33−42) peptides were incubated in 5% DMSO, 50 mM Tris-HCl, pH 7.5 and concentration 1.0 mg/mL at 37 °C during 48 h. After that their concentrations were adjusted to 5 mg/mL using centrifugation at 12 000 rpm during 30 min. The supernatant was removed and 5−7 μL of the pellet of mature fibrils was placed in the clearance (∼1 to 1.5 mm) between the tips of two glass rods (of ∼1 mm in diameter) covered with wax.35 The preparation was dried for several hours. The X-ray analysis of the preparations was conducted using a Microstar Xray emitter with HELIOX optics equipped with a Platinum 135 CCD detector (X8 Proteum System, Bruker AXS). Cu Kα radiation (λ = 1.54 Å) was used (the sample was oriented perpendicular to the X-ray beam). Small-Angle X-ray Scattering Analyses. All small-angle X-ray scattering (SAXS) measurements were performed in Grenoble (France), on the beamline BM-29 (synchrotron ESRF). The Aβ(16−25), Aβ(31−40), and Aβ(33−42) peptides were incubated in 5% DMSO, 50 mM Tris-HCl, pH 7.5 and concentration 0.5 mg/mL at 37 °C during 20 days without stirring. All data from the run were collected at a wavelength of 0.9919 Å using a sample-to-detector distance (PILATUS 1M, DECTRIS) of 2.876 m. Integral parameters of peptide oligomers, such as radii of gyration, thickness of oblate particles, and diameter of prolate particles, were calculated using the ATSAS software package.36 The pair−distance distribution function p(r) is obtained with the program PRIMUS using GNOM. GNOM output was used for ab initio structure determination with the help of the DAMMIF program.37 Limited Proteolysis and Mass Spectrometry Analysis. Dry samples of fragments were predissolved in 100% DMSO (to achieve the final concentration of 5%) and adjusted to the concentration of 0.4 mg/mL with 50 mM Tris-HCl (pH 7.5). All of the procedures were conducted on ice. After dissolution, the samples were incubated at 37 °C for 24 h. After the incubation, the mature fibrils were isolated from oligomers and monomers by centrifugation (30 min, 10 000g) and washed twice with ammonium acetate (10 mM, pH 7.5). Then, isolated fibrils were treated with a mixture of proteases (trypsin, chymotrypsin, and proteinase K) for 8 h. The proteases were added so that the ratio of the enzyme to peptide was 1:25. To maintain the activity of proteinase K, CaCl2 was also added to the final concentration of 5 mM. Conditions of limited proteolysis were chosen so that the monomer molecules would be completely degraded 2333


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Figure 1. EM images of Aβ(1−42) peptide fragments. Bottom row: Fragments of fields at large magnification. Arrows indicate ring oligomers. All experiments were performed at concentrations of the preparations 0.25 to 0.5 mg/mL in 5% DMSO, 50 mM Tris-HCl (pH 7.5) and incubation at 37 °C for 24 h. The preparations were negatively stained with 1% aqueous solution of uranyl acetate. Arrows indicate the general structure element of the Aβ(1−42) peptide fragments: the ring oligomer with the outer diameter of about 6 to 7 nm and the inner diameter (hole) of about 2 to 3 nm. while the fibril moiety would retain long fragments. The fragments set apart of the proteases were centrifuged and washed, and the remaining fragments included in fibril structures were pelleted once again and isolated. Then, the isolated residual fibril structures were dried using a vacuum concentrator, after which they were dissolved in a small formic acid volume and thereafter in 10 mM ammonium acetate buffer. The obtained sets of peptides were separated by HPLC (EASY-nLC 1200 System, ThermoFisher Scientific, USA) and analyzed using a high-resolution mass spectrometer (Orbitrap Elite mass spectrometer, Thermo Scientific, Germany). Then, the peptides were identified using the Peaks Studio 7.5 program (Bioinformatics Solutions). This program makes it possible not only to identify peptides but also to estimate their relative concentration in the probe. Molecular Modeling. Structure 4UZR (chains A, B, C, D, E, F) from the Protein Data Bank was taken as a template to construct the arrangement of β-sheets. Initially, the fragments of amino acid residues from 79 to 84, 88 to 94, 97 to 102, 106 to 111, 116 to 121, 127 to 131, 134 to 138, and 142 to 147 were taken from the pdb file 4UZR. All of these fragments correspond to the β-structure. β-Strands inside each βsheet were arranged antiparallel relative to each other. Such organization is the most stable in comparison with the parallel arrangement of β-strands.38 Antiparallel β-sheets have been found in fibrils formed by short fragments of full-length amyloid-forming proteins.39,40 Using the YASARA program,41 two or three amino acid residues were added at the N- and C-termini of each β-strand. Then, the following amino acid sequences, 16−25 (KLVFFAEDVG), 31−40 (IIGLMVGGVV), and 33−42 (GLMVGGVVIA), corresponding to the three fragments of Aβ(1−42) peptide were fitted in each of these 48 β-strands. So, three structures with different amino acid sequences were obtained and minimized by the YASARA program. The principal geometric criterion of construction was the lack of steric overlaps between the van der Waals radii of the atoms.

(Figure 1 bottom row), it becomes noticeable that the fibrils are formed from some ring-shaped oligomeric structures, with the external diameter of 6 to 7 nm, the inner diameter of the ring of 2 to 3 nm, and the height of 3 nm (indirectly estimated from the bending point of the thinnest fibrils). After 10 h of incubation, the Aβ(33−42) peptide fragment forms fibrils and aggregates of fibrils dissimilar to those formed by the Aβ(31− 40) peptide, the main difference being pronounced polymorphism of the fibrils in their diameter (7−20 nm or wider). Moreover, no ribbons are present. The morphology of the fibrils formed by the Aβ(33−42) fragment resembles that of the fibrils formed by the complete Aβ(1−42) peptide.42,43 The characteristic morphological features in the case of the Aβ(1− 42) peptide are the rough surface and a pronounced variation in the diameter of the mature fibrils. The difference, however, in the morphology of the fibrils formed by Aβ(33−42) versus Aβ(1−42) is the absence of branching in the case of Aβ(33− 42). At larger magnification, it becomes noticeable that the fibrils produced from both Aβ(33−42) and Aβ(31−40) fragments are generated through the association of ring-shaped oligomeric particles with the diameter of about 6 to 7 nm (Figure 1, bottom row). However, in the case of the Aβ(33− 42) fragment, this association is more chaotic, leading to the formation of fibrils with a rough surface and a large variation in their diameter. The morphology of fibrils formed by the Aβ(16−25) fragment is quite dissimilar to that of the fibrils of both Aβ(31−40) and Aβ(33−42). After 10 h of incubation, some fibrils are formed in the shape of a film. Their length reaches 1 μm or more, while their width is ∼80 nm. These films are very thin and only just detectable against the background (Figure 1, top row). After 24 h, the length of these films increases insignificantly, but their width increases to 170 nm. At larger magnification it is possible to see that the films are also formed by the association of some ring-like structures with the diameter of about 6 to 7 nm (Figure 1, bottom row). These ring oligomers are arranged within the film in a honeycomb-like manner. The films are very thin; their thickness could be estimated from the points of bending or at the edges of the film. This thickness has been estimated to be 3 nm, and it suggests, indirectly, the height of the ring oligomer. It must be noted that only one edge of the film is clearly visible. Furthermore, the polymers grow not only by increasing their length but also by broadening. Initially, the polymers increase their length through

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RESULTS AND DISCUSSION Electron Microscopy Analysis. The EM analysis shows that all studied fragments of the Aβ(1−42) peptide quickly form fibrils with the length up to 100−300 nm and the diameter of the thinnest fibril about 6 to 7 nm. After 9 to 10 h of incubation at 37 °C, in 5% DMSO, 50 mM Tris-HCl, pH 7.5, mature fibrils form, up to 1 μm in length or even longer. After 24 h, the number of fibrils increases, and they form large aggregates. However, the morphology of the fibrils formed by different peptide fragments varies. The Aβ(31−40) peptide, after 10 h of incubation, forms aggregates of mature fibrils in the shape of bundles and ribbons (Figure 1, top row). The thinnest fibrils in the aggregates (single fibrils) have the diameter of about 6 to 7 nm. Under larger magnification 2334


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Figure 2. Schematic representation of the possible mechanism of fibril formation by fragments of Aβ peptide.

Figure 3. Illustration of the method used for extraction of periodic structures from indistinct images of negatively stained amyloid fibrils of the Aβ(33−42) peptide. The method of averaging along a single fiber is based on the Markham rotation technique and fixed shift of several copied layers of source image (selected area in panel A) equally contrasted (upper image in panel D) but with two-fold descending opacity of each subsequent overlapping layer (reduced opacity of each subsequent superimposed layer) (panel B). Panel C represents interference of semitransparent layers gradually shifted by fixed step (2 pixels, ∼0.5 nm) alongside the fiber in one direction. At the step of ∼13 pix (6.5 nm) interfered layers elucidate several visibly distinct annular structures (asterisk). Note that the neighboring fiber is twisted and the shift does not reveal any annular structures (arrowhead). (This is the place of inflection of the fibril, along which it is possible to determine the height of the ring oligomer, about 2 to 3 nm.) To extend the scope of distinct annular structures along the fiber, layers were shifted equally in both directions and united/merged (bottom image in panel D).

the association of the ring oligomers in a ring-on-ring manner, with some shift against each other. In a later stage, the growth

of the polymer continues mostly by broadening, when more ring oligomers associate by their sides (while one of the end 2335


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Langmuir surfaces of each ring is associated with the support film, producing a honeycomb pattern (Figure 1, bottom row)). In conclusion, the three studied fragments of the Aβ(1−42) peptide formed polymers of a dissimilar morphology. The ring oligomers could interact with each other in various ways: ringto-ring (sideways association) resulting in the formation of films, like in the case of Aβ(16−25); ring-on-ring with a slight shift, forming bundles and ribbons, like with Aβ(31−40); and ring-to-ring, associating irregularly, resulting in the formation of fibrils with a rough surface, in the case of Aβ(33−42) (Figure 2). To better reveal morphological features of fibrils, we used the modified method known as the Markham rotation technique. It was proposed by Markham et al. in 196334 when they studied structures of negatively stained viral particles using electron microscopy. The method allows revealing a more detailed structure of the object studied by upgrading the electron microscopy patterns. This method can be used for studying objects with axial and linear symmetry having recurring structure elements that are weakly identified against the background. The images of the recurring structure elements are upgraded as a result of multiple super positioning with a shift (rotation) of the semitransparent copy of the origin (Figure 3A), which enables averaging the image of a separate element with lowering the background “noise” (Figure 3B). The conception of averaging is widely used at present and underlies the up-to-date software to upgrade images for 3D reconstruction of macromolecules and their complexes using cryoEM and EM patters of negatively stained objects. Figure 3 clearly shows the recurrence of overlapping ring structures, which supports the correctness of our assumption on the role of ring oligomers as the main building blocks upon the formation of amyloid structures. Attention should be drawn to panel C, in which the fibril indicated by the white arrow is twisted and does not reveal the periodicity of the ring structures. This place of inflection of the fibril and its width can be used to estimate approximately the height of fibril. In compliance with the data given in Figure 1, the mean diameter of a ring oligomer of the Aβ(33−42) peptide is about 6−8 nm, whereas the diameter of the cavity inside the ring is about 2 to 3 nm. It is worth noting that the amyloidogenic regions are localized in the functionally distinct parts of the Aβ(1−42) peptide: The Aβ(31−40) and Aβ(33−42) fragments correspond to the transmembrane region (29−42), while the fragment Aβ(16−25) belongs to the extracellular domain Aβ(1−28). We have found that when the last two amino acid residues are included in the peptide fragment, the amyloid structures formed by the latter remarkably mimic those formed by the whole peptide Aβ(1−42), the only difference being that no branching occurs within the Aβ(33−42) fibrils. Of importance is the fact that the extracellular domain Aβ(1− 28) is charged, and so its behavior depends strongly on the pH environment, while the transmembrane domain is not charged. Consequently, the whole extracellular domain, as well as its truncations Aβ(15−28) and Aβ(17−28), is capable of forming structures in the shape of wide ribbons under different pH conditions.44 Stimulated by the fast development of nanobiotechnology, nonconventional structures, such as amyloid fibrils of various types, attract a lot of attention. In particular, peptide fragment Aβ(16−22) (KLVFFAK) has recently been studied; it forms nanofilms wider than 200 nm.45 The term “amyloid-like nano-

layers” was coined by the authors to describe such structures. However, these structures have been obtained under some nonphysiological conditions (pH 2).44 The oligomers were observed as a ubiquitous key participant of fibrillogenesis. The presence of some oligomers with the diameter of ∼10 nm could be found in EM images in numerous publications.46−48 These oligomers often possess an inner cavity; that is, they are ring-shaped.46−49 But even when the attention is drawn to such structures, only a few authors describe the way of the fibril formation through the association of ring-like oligomers into long polymers. In this connection, the work of Lashuel et al. should be mentioned.50 When studying the process of fibrillation of the pathogenic mutant Aβ(1−40) peptide (E22G is the Arctic mutation) it has been shown that both the wild-type Aβ40 and the mutant in the initial fibrillation period form annular pore-like particles with an external diameter of 6−9 nm and an internal diameter of 1.5 to 2 nm. When the particles mature, chain-like short protofibrils/ fibrils of a similar diameter are formed as pore-like particles. It is interesting to note that the formation of annular pores and, correspondingly, fibrils occurs much more rapidly for the mutant Aβ(1−40) than for the wild-type Aβ(1−40) peptide.50 In our view, this indirectly indicates that exactly annular particles play the major role in the formation of fibrils. However, in ref 51, the EM analysis was used to study fibrilogenesis using the Aβ(1−42) peptide. It was shown that the process of fibrilogenesis occurs through association of oligomeric particles with the diameter equal to that of the mature fibrils. In another publication52 the authors even proposed a new mechanism of formation of α-synuclein fibrils via the association of some preformed granules, which are the structural units of the fibrils (a double-concerted fibrillation model). The assembly of fibrils from oligomers was demonstrated for lysozyme.53 A new mechanism for the formation of fibrils through preformed oligomers was also discussed by Jeong et al. (2013).54 These articles on AFM images clearly show that fibrils have a beadlike morphology and the diameter of mature fibrils coincides with the diameter of oligomeric particles. Recently, Dean et al. have suggested a mechanism for the formation of fibrils through the interaction of preformed dodecamers (called large fatty-acid-derived oligomers (LFAOs)) for the modified Aβ(1−42) peptide.55 Thus there is a sufficient amount of data indicating that different mechanisms for the formation of amyloid fibrils can be realized. However, at present, the basic interpretation of the molecular organization of amyloid fibrils is that a fibril consists of a network of protofilaments (from 2 to 6), each of them being an in-sheet running parallel along the entire axis of the fibril. In this case, the β-sheet is formed from β-strands, which run perpendicular to the axis of the fibril. The average diameter of the fibril is ∼10 nm, and the length can reach up to 10−15 μm.56 In interpreting the results obtained, a number of models for the formation of fibrils from β-sheets were proposed.7−18 A β-sheet arranged in a different way but invariably passing along the entire axis of the fibril remains common for all models. We believe that the presence of different interpretations of the method of fibril formation may indicate the existence of different fibrillation mechanisms, and it is necessary to take this circumstance into account to develop approaches that can affect polymerization of amyloid proteins/peptides at its earliest stages. 2336


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Figure 4. Data of X-ray analysis for fragments of Aβ(1−42) peptide. (A) Aβ(16−25), (B) Aβ(31−40), and (C) Aβ(33−42). Prepaations were obtained in 50 mM Tris-HCl, pH 7.5, 48 h of incubation at 37 °C, 5 mg/mL.

diminished between 1/40 and 1/20 Å, suggesting that ∼30 Å thick plates are stacked with a 40 Å period. This size corresponds to β-strands that are 10 residues long in the chain direction. Equatorial reflections (∼40 and 20 Å) were obtained also for other short peptides: HYFNIF, RVFNIM, and VIYKI.67 The authors interpreted this as a protofilament width. Diffraction patterns between 2.90 and 2.39 Å correspond to 40−48 orders (to check), suggesting that the amyloid core contains 40−48 β-strands (see table 2 in ref 60). Perutz et al.68 give an interesting interpretation of their X-ray data, including that for the fragment Aβ(11−25), as well as its mutant form (Asp23Lys). According to the proposed reconstruction from the EM and X-ray data, the fibrils produce a complex diffraction pattern but with two characteristic reflections invariably present. The fibril diameter in this case was measured to be ∼6 nm. According to the optical reconstruction, the fibrils are hollow cylinders, with diameter of ∼57 Å, wall thickness of 37 Å, and central cavity of 19.5 Å in diameter. If this interpretation is correct, then amyloids consist of narrow tubes (nanotubes) with a central water-filled cavity. We would like to note that the X-ray diffraction pictures for different peptides and proteins, found in the literature, as well as our own ones, possess some particular “signature” characteristics. Also, even small changes in the sequence of the peptides, or their length, could drastically change the diffraction pattern. For example, the peptide fragments Aβ(18−28) and Aβ(17− 28) produce totally dissimilar diffraction patterns, despite the peptides differing by only one amino acid residue.64 This suggests that although many peptide fragments produce fibrils with roughly the same diameter of the thinnest fibrils, their inner organization should be different. Summarizing the above, we can make the following conclusion. In agreement with the literature data for peptides of a similar length, our own model structure for the fibrils is an assembly of tubular cylinders stacked with a 40 Å period, which corresponds to our model distance (see Molecular Structure of Nanofilms and Amyloid Fibrils Formed by Aβ(16−25), Aβ(31−40), and Aβ(33−42) section). The tubular cylinder includes more than 40 β-strands and has a hollow with a diameter of ∼20 Å. Small-Angle X-ray Scattering Analysis. Using a modified method of indirect Fourier transform (IFT), geometrical parameters were obtained: the correlation length (Lm), the scattering volume (V), the surface area (S), the gyration radius (Rg), and the size of particle (D), that is, the distance between two points furthest from one another. Using the method of

X-ray Analysis. As early as in 1968, X-ray diffraction analysis allowed for the first time identification of the cross-β structure of amyloids (pleated sheets) in amyloid deposits from liver and spleen tissues.57 Since then, X-ray diffraction has become a key method for studying all amyloid structures. It has been shown, by using this method, that the cross-β structure is a common feature of amyloids.58−60 On the basis of this wellestablished identification of the molecular organization of amyloids, a number of models were proposed to describe the formation of fibrils from β-sheets. These models assume various ways of packing of β-sheets within fibrils, with a common feature of all of them being the assumption that the β-sheet(s) runs all the way along the length of the fibril. No unified model exists at present to describe the tertiary structure of amyloids61 (this publication describes a possible packing of β-sheets within fibrils).62,63 In our X-ray diffraction analysis, the polymers formed from all three studied peptide fragments demonstrated the presence of two dominant reflections (4.5 to 4.8 and 8−12 Å), which indicated the presence of cross-β structures (Figure 4). However, on top of the two characteristic reflections, a large number of additional reflections were observed; they were dissimilar between the samples produced from different peptides, which suggests different structural organization of the polymers. Previously, such complex X-ray diffraction patterns had been observed by other authors.64,65 Currently, there is no precise interpretation of all of these reflections for amyloid preparations. However, it is noted that the diffraction pattern could be influenced by (1) the mode of alignment of the fibrils (stretch frame alignment, alignment in glass capillary, magnetic alignment, thin film alignment);60,64−66 (2) the concentration of the sample;44 and (3) the purity, source, and method of preparation of the sample.35,66 There are data showing that well-aligned samples produce a more complex pattern of sharp reflections, thus yielding more information.66 Varying explanations for the presence of extra reflections could be found in the literature, with the meridional and equatorial reflections being assessed separately.60,64,65 For a precise interpretation of the data, very high quality of the diffraction pictures in addition to more advanced knowledge of the organization of fibrils on the molecular level is required. An interesting result derived from the X-ray analysis for fragments of Aβ peptides, similar in length to our studied peptides, namely, Aβ(18−28), Aβ(17−28), and Aβ(15−28), should be mentioned.64 The equatorial reflections are indexed unidimensionally, with periods of 38, 37, and 40 Å for peptides 18−28, 17−28, and 15−28, respectively. The intensity 2337


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Figure 5. Small-angle scattering curves I(q) and Patterson functions P(r) for different Aβ aggregates. Cyan, orange, and blue curves correspond to Aβ(16−25), Aβ(33−42), and Aβ(31−40), respectively.

mass assessment69 and parameters Lm, V, and Rg, the masses of Aβ peptide oligomers were calculated. Small-angle X-ray scattering data measured for samples of Aβ(16−25), Aβ(33−42), and Aβ(31−40) and Patterson functions calculated based on these data are presented in Figure 5. Using experimental SAXS curves I(q) and Patterson functions we determined the set of structural parameters for aggregated particles in solution. The data are represented in Table 1.

Determination of Regions within Aβ Peptide Fragments Responsible for the Formation of Amyloid Structures: Limited Proteolysis and the Mass-Spectrometry Analysis. After incubation of peptides at 37 °C for 24 h the mature fibrils were treated with a mixture of proteases (trypsin, chymotrypsin, and proteinase K) for 8 h. The probes were analyzed after 1 and 8 h. After 1 h of incubation of Aβ(16−25), the predominant peptides were determined: the whole peptide, the peptides missing two to four C-terminal amino acid residues (glutamate, aspartate, valine, glycine), and the ones missing one to three N-terminal amino acid residues (lysine, leucine, valine) (Figure 7A). After 4 and 8 h of proteolysis, peptides missing four N-terminal amino acid residues (lysine, leucine, valine, and phenylalanine) were recorded (Figure 5A). In the case of Aβ(31−40) peptide amyloids, all of the amino acid residues were inaccessible for proteases, even after 8 h of treatment (Figure 7B). Proteolysis of the amyloids formed by Aβ(33−42) peptides produced a great variety of truncated peptides. After 1 h of treatment, there were fragments missing between one and four N-terminals and between two and four C-terminal amino acid residues (separately and in combination, Figure 7C). After 4 h of incubation the set of peptides did not change. After 8 h the number of truncated peptides from both termini decreased (Figure 7C). The limited proteolysis data demonstrate that the most protected from the proteolysis are the structures formed by the Aβ(31−40) peptide, which forms fibrils in the shape of bundles and ribbons. The most susceptible to proteolysis is Aβ(33−42), which forms uneven fibrils with a rough surface. The data indirectly support our model because the side surfaces of the oligomers could be critical for their association. Molecular Structure of Nanofilms and Amyloid Fibrils Formed by Aβ(16−25), Aβ(31−40), and Aβ(33−42). According to the EM data, all three fragments of Aβ(1−42) peptide are capable of self-organization into polymeric structures. At high magnification it is noticeable that all polymers are constructed from ring-like oligomers with an outer diameter of ∼6 nm, an inner diameter of ∼2 nm (the hole diameter in the ring), and a ring height (measurement at the point of inflection of a single fibril) of ∼3 nm. Thus the ring oligomer is a short tubular cylinder with a wall width of ∼2 nm We have proposed the molecular structure for this type of arrangement of oligomer structures using the method of molecular modeling (Figure 8). Amyloid fibrils for Aβ(31−40)

Table 1. Values of Radius of Gyration (Rg), Diameter (d) of Prolate Objects and Thickness (th) of Oblate Objects, Size of Particle (D), Scattering Volume (V), Surface Area (S), Correlation Length (Lm), and Molecular Mass (M) Obtained Using the Guinier Approximation and Methods of Indirect Fourier Transform for Conglomerates of the Three Aβ Fragments sample parameter Rg d th D from P(r) D from ab initio structure V S Lm M

Aβ(16−25) 280 Å

Aβ(33−42)

Aβ(31−40)

150 Å 185 Å

410 Å 176 Å

36 Å 900 Å 1800 Å

500 Å 1200 Å

1200 Å 2700 Å

11 × 106 Å3 930 × 103 Å2 100 Å 9.7 MDa

7.2 × 106 Å3 280 × 103 Å2 140 Å 3.6 MDa

96 × 106 Å3 4500 × 103 Å2 230 Å 88 MDa

The diameter (d) and thickness (th) were calculated on the assumption that the particles have a prolate or oblate shape, respectively. The method of calculating these quantities is analogous to the Guinier approximation method. Relative errors for values of the radius of gyration, diameter of prolate objects, and thickness of oblate objects are 5%. For values of the scattering volume, surface area, correlation length, and molecular mass, relative error is 10%. It is shown that part of the curves (for small values of the modulus of the scattering vector) indicates aggregation of these structures into larger agglomerates whose dimensions exceed 100 nm. Projections of ab initio models obtained in the framework of the ATSAS software package are presented in Figure 6. 2338


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Figure 6. Projections of 3D structure for fragments of Aβ peptide aggregates, obtained by the DAMMIF program (ATSAS).

(single fibrils). The fibril Aβ(31−40) consisting of ring-like structures is presented in Figure 8A,B. The Aβ(16−25) peptide aggregates into honeycomb nanofilms, which are not characteristic of an amyloid formation (Figure 8C).70 The annular oligomer can consist of 48 peptide molecules that form 12 βsheets stacked in a circle (Figure 8). The orientation of βstrands in β-sheets in oligomers is compatible with the positions of strands and sheets in the model described in ref 71. Previously we have demonstrated that such antiparallel organization of β-strands is more favorable than others using molecular dynamics simulations.72 It should be mentioned that the packing of oligomers depends on the amino acid sequence. In the case of Aβ(16− 25), the interactions of aromatic residues (Phe with Phe) probably will determine the honeycomb-like model. According to the conventional model, if we put an amyloid fibril on the X−Y plane, then the β-strand will be roughly lying on top of the X−Y plane. In contrast, according to our model, if we put an amyloid fibril on the X−Y plane, then the β-strand will be approximately standing up on top of the X−Y plane (i.e., along the z-axis). So the height of the unit fibril (oligomer structure) according to the model will be roughly the length of the β-strand. The Aβ(16−25), Aβ(31−40), and Aβ(33−42) peptides in our work are 10 amino acid residues long. The theoretical length of a peptide is ∼3 nm, which is consistent with the height estimated from the EM (between 2 and 3 nm) and AFM experiments (1.5 to 2.3 nm, data are not presented). The molecular model of amyloid fibrils consisting of ring-like structures agrees with the data of X-ray analysis because ring oligomer structures contain both β-strands and β-sheets. Moreover, the tandem mass spectrometry analysis also supported our molecular structure. Fibrils of peptide Aβ(1−42) are also formed of ring-like rounded oligomers,73,74 but the molecular organization of the ring-like oligomers is different. The β-strands are lying on top of the X−Y plane.43,75 Consequently, the internal organization of oligomers depends on the amino acid sequence, forming an oligomer structure.

Figure 7. Limited proteolysis and the mass-spectrometry analysis. Fragments (shown as blue bands) of Aβ(16−25) (A), Aβ(31−40) (B), and Aβ(33−42) (C) identified by Peaks Studio 7.5 after limited proteolysis and tandem mass spectrometry of fibrils from these peptides. Small letter “o” denominates “oxygen” and means that this fragment has oxygenated methionine. Small letter “f” denominates “formate” and means that this fragment has a formylated amino acid residue. These modifications were induced by the conditions of the experiment.

and Aβ(33−42) are formed from oligomer structures packed either directly ring-to-ring or ring-on-ring with a slight shift. Resulting from this packing are the thinnest fibrils (6 nm) 2339


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Figure 8. Molecular structures for amyloid fibrils formed by Aβ(31−40) and for nanofilm Aβ(16−25). (A) Top and (B) side views of organization of ring-like oligomer structures for amyloid fibrils formed by Aβ(31−40). (C) Arrangement of ring-like oligomer structures is similar to honeycomb for Aβ(16−25). The outer diameter of the ring is about 6 to 7 nm. The diameter of the hole of a ring is ∼2 nm and the height is ∼3 nm.

Figure 9. Schematic representation of the possible molecular mechanism of fibril formation by fragments of Aβ peptide.

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fibril (Figure 9). The least explained stage is the transition from the oligomer to fibrils. The oligomers could be seen in EM images represented in various publications. The diameter of such oligomers is similar to that of the thin fibrils, and it is often possible to observe an internal cavity; that is, the oligomers are ring-shaped.46−49 On the basis of our experimental data, we suggest that the main building block in the process of formation of various polymeric structures (e.g., fibrils of varying morphology, films) by amyloidogenic proteins and peptides is a ring oligomer. The characteristics of such oligomers and their internal organization depend on the properties of particular proteins and peptides, from which the oligomers were formed. The ring oligomers interact with each other in multiple ways, which leads to varying morphological properties of the resulting fibrils and films. Most of the existing models explain the formation of fibrils as self-association of β-sheets. The cross-β structure model implies that a continuous β-sheet, perpendicular to the axis of a fibril, runs through the whole length of the latter. From a purely mechanistic point of view, such molecular organization of amyloid fibrils seems to be improbable, as it can neither

CONCLUSIONS At present, the available information allows using experimental data, as well as theoretical predictions, studying the amyloidforming capacity of most proteins, and describing the mechanism of the formation of amyloid fibrils. It seems possible that despite a number of contradictions among some theoretical and experimental data, the process of amyloidogenesis should possess general key features common to all proteins and peptides. To negate the effect of varying contexts in proteins, it is convenient to make use of model peptides, whose sequence mostly comprises well-characterized amyloidogenic regions. Herein we have investigated the following amyloidogenic fragments of the Aβ42 peptide: Aβ(31−40), Aβ(33−42), and Aβ(16−25). The morphology of the amyloids is different between these three peptides. However, the EM data demonstrate one common feature, namely, the presence of ring oligomers, with external diameter of 6 to 7 nm, internal diameter of 2 to 3 nm, and height of ∼3 nm. According to a simplified model, the formation of fibrils goes through the following stages: destabilized monomer → oligomer → mature 2340


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Langmuir predict the enormous length of the fibrils (up to 15−20 μm) nor convincingly explain some of the key features of fibrils, such as their polymorphism, splitting up, and branching. Our model does not contradict the X-ray analysis data, as the structural organization of the ring oligomers allows for both β-layers and short β-sheets. Alongside the imaging methods of microscopy (EM, AFM), the use of structural methods, such as X-ray analysis and solidstate NMR, becomes increasingly important for the studies of amyloidogenesis, particularly the techniques of sample preparation and adequate interpretation of the results. The use of methods of bioinformatics (including molecular dynamics) to aid the final interpretation of the results is also highly advantageous. Recent data suggest that the process of fibril formation of amyloid fibrils is still not fully clarified. To date, there are at least two groups of studies with different interpretations of the mechanism of fibril formation: (a) The formation of filaments/ fibrils occurs due to the elongation of β-sheets passing along the entire axis of the fibril and (b) the formation of fibrils takes place due to the interaction of oligomers, which are building blocks in the construction of fibrils. Correct interpretation of such molecular processes is paramount for the understanding, prophylactics, and future treatment of the amyloid-related neurodegenerative conditions in humans. In that connection, we believe that a key effort must currently be concentrated on studying the mechanisms of the transition from the monomeric state to the formation of oligomers.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Oxana V. Galzitskaya: 0000-0002-3962-1520 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to T. B. Kuvshinkina, N. Lissin, E. I. Grigorashvili, E. Yu. Gorbunova, L. G. Mustaeva, N. V. Dovidchenko, A. D. Nikulin, N. A. Ryabova, and A. G. Gabdulkhakov for assistance in preparation of the manuscript. This study was supported by the Russian Science Foundation (14-14-00536). Research was supported by facilities of United Pushchino Center for Electron Microscopy (No. 507648).

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