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

Subscriber access provided by READING UNIV

Article

To be fibrils or to be nanofilms? Oligomers are building blocks for fibrils and nanofilms formation of fragments of A# peptide Olga Michailovna Selivanova, Alexey Konstantinovich Surin, Yuriy Ryzhykau, Anna Vladimirovna Glyakina, Mariya Yu. Suvorina, Alexander Kuklin, Vadim Rogachevsky, and Oxana Valerianovna Galzitskaya Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03393 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

To be fibrils or to be nanofilms? Oligomers are building blocks for fibrils and nanofilms formation of fragments of Aβ peptide Olga M. Selivanova1, Alexey K. Surin1,2, Yuriy L. Ryzhykau3, Anna V. Glyakina1,4, Mariya Yu. Suvorina1, Alexander I. Kuklin3,5, Vadim V. Rogachevsky6, Oxana V. Galzitskaya1*

1

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Russia

2

State Research Center for Applied Microbiology & Biotechnology, Obolensk, Russia

3

Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation

4

Institute of Mathematical Problems of Biology RAS, Keldysh Institute of Applied Mathematics of Russian

Academy of Sciences, Pushchino, Russia 5

Joint Institute for Nuclear Research, Dubna, Russian Federation

6

Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Russia

*Correspondence: Oxana V. Galzitskaya, Group of Bioinformatics, Institute of Protein Research, Institutskaya Street 4, 142290, Pushchino, Russia. E-mail: [email protected]

Keywords: Aβ peptide; Alzheimer’s disease; amyloid fibril; biofilm; oligomer, X-ray analysis, tandem mass spectrometry

1 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 33

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 6-7 nm, the internal diameter of 2-3 nm and the height of about 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 amyloid-related diseases.

INTRODUCTION Aβ(1-42) peptide is linked to a severe neurodegenerative anomaly in humans - the 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 clear yet. 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


Page 3 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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). 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, form bands, bundles, clusters. The average width (diameter) of filaments/fibrils is about 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, β-myosin) 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 has remained the basic representation for explaining the structure of amyloid structures3. With the beginning of the use of the electron microscope for the study of biological objects, 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 β-sheets, 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 samples19. Recently, however, there appeared works that offer other models of fibril formation. For example, it has been shown in 20, when studying the formation of Aβ1-42 fibrils by the peptide with the help of the 3 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 33

EM method, that fibrils are formed from oligomers. And in 21, in the study of fibrillation of α-synuclein, 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), the structure of Aβ(1-42) peptide fragments being also analyzed in recent years 23–32. Further understanding of the properties of the peptide fragments will allow the development of high resolution biological models of the conformational changes in amyloidogenic proteins. We have investigated the properties of 3 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. Based on 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 ringlike 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.

EXPERIMENTAL SECTION 4 ACS Paragon Plus Environment

Page 5 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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-Val-Phe-Phe-Ala-GluAsp-Val-Gly-CОО- (16-25); +H3N-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-CОО- (31-40); +H3N-Gly-Leu-MetVal-Gly-Gly-Val-Val-Ile-Ala-CОО- (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. 5-1.0 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 40,000X magnification on a Kodak film (SO163) 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), 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 (~1-1.5 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 X-ray emitter with HELIOX optics equipped with a Platinum 135 CCD detector (X8 Proteum System, Bruker AXS). Cu Kα radiation (λ=1.54 Ǻ) was used (the sample was oriented perpendicular to the X-ray beam). 5 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 33

Small angle X-ray scattering analyses. All small angle X-ray scattering (SAXS) measurements were performed in Grenoble (France), on the beam-line BM-29 (synchrotron ESRF). The Aβ(16-25), Aβ(31-40), 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 meters. 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 pre-dissolved 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 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, 10000xG) 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 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 6 ACS Paragon Plus Environment

Page 7 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

7.5 program (Bioinformatics Solutions Inc.). 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 to102, 106 to 111, 116 to 121, 127 to 131, 134 to 138, 142 to 147 were taken from the pdb-file 4UZR. All these fragments correspond to the β-structure. β-Strands inside each β-sheet 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.

RESULTS AND DISCUSSION Electron microscopy analysis. The EM analysis shows that all studied fragments of the Aβ(142) peptide quickly form fibrils with the length up to 100-300 nm and the diameter of the thinnest fibril about 6-7 nm. After 9-10 h incubation at 37°C, in 5% DMSO, 50mM Tris-HCl, pH7.5, mature fibrils form, up to 1µm 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 incubation forms aggregates of mature fibrils in the shape of bundles and ribbons (Fig. 1, top row). The thinnest fibrils in the aggregates (single fibrils) have the diameter of about 6-7 nm. Under larger magnification (Fig. 1 bottom row), it becomes noticeable that the fibrils are formed from some ring-shaped oligomeric structures, with the external diameter of 6-7 nm, the inner diameter of the ring of 2-3 nm, and the height of 3 nm (indirectly estimated from the bending point of the 7 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 33

thinnest fibrils). After 10 h 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) vs. 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 ringshaped oligomeric particles with the diameter of about 6-7 nm (Fig. 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 incubation, some fibrils are formed in the shape of a film. Their length reaches 1µm or more, while their width is about 80 nm. These films are very thin, and only just detectable against the background (Fig. 1, top row). After 24 h, the length of these films increases insignificantly, but their width increases up to 170 nm. At larger magnification it is possible to see that the films are also formed by association of some ring-like structures with the diameter of about 6-7 nm (Fig. 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. Further, the polymers grow not only by increasing their length, but also by broadening. Initially, the polymers increase their length through the association of the ring oligomers in a ring-on-ring manner, with some shift against each other. At 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 surfaces of each ring is associated with the support film, producing a honeycomb pattern (Fig. 1, bottom row). 8 ACS Paragon Plus Environment

Page 9 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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: ring-to-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), ring-to-ring, associating irregularly, resulting in the formation of fibrils with a rough surface, in the case of Aβ(33-42) (Fig. 2). To better reveal morphological features of fibrils, we used the modified method known as the Markham rotation techniques. It was proposed by Markham et al. in 1963

34

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 (Fig. 3, A), which enables averaging the image of a separate element with lowering the background “noise” (Fig. 3, B). 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 shows clearly 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 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 Fig. 1, the mean diameter of a ring oligomer of the Аβ(33-42) peptide is about 6-8 nm, whereas the diameter of the cavity inside the ring is about 2-3 nm. It is worth noting that the amyloidogenic regions are localized in the functionally distinct parts of the Аβ(1-42) peptide: the Аβ(31-40) and Аβ(33-42) fragments correspond to the transmembrane region (2942), while the fragment Аβ(16-25) belongs to the extracellular domain Аβ(1-28). We have found that 9 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

when the last two amino acid residues are included in the peptide fragment, the amyloid structures formed by the latter mimic remarkably those formed by the whole peptide Аβ(1-42), the only difference being that no branching occurs within the Аβ(33-42) fibrils. Of importance is the fact that the extracellular domain Аβ(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 Аβ(15-28) and Аβ(17-28) are capable of forming structures in the shape of wide ribbons under different pH conditions 44. Stimulated by the fast development of nano-biotechnology, non-conventional 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 nano-films 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 non-physiological conditions (pH2) 44. The oligomers were observed as a ubiquitous key participant of fibrillogenesis. The presence of some oligomers with the diameter of about 10 nm could be found in EM images in numerous publications 46–48. These oligomers often possess an inner cavity, i.e. they are ring-shaped 46–49. But even when the attention is drawn to such structures, only 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 mentioned50. 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 1.5-2 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(140) than for the wild type Aβ(1-40) peptide50. In our view, this indirectly indicates that exactly anular particles play the major role in the formation of fibrils. However, in 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 10 ACS Paragon Plus Environment

Page 10 of 33

Page 11 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

publication

52

the authors even proposed a new mechanism of formation of α-synuclein fibrils via the

association of some pre-formed granules, which are the structural units of the fibrils (a doubleconcerted fibrillation model). The assembly of fibrils from oligomers was demonstrated for lysozyme53. 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) peptide55. 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 about 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.

X-ray analysis.

As early as in 1968, the X-ray diffraction analysis allowed for the first time to

identify the cross-β structure of amyloids (pleated sheets) in amyloid deposits from liver and spleen tissues 57. Since then, the 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

. Based on this well-established 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 11 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 33

ways of packing of β-sheets within fibrils, 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 amyloids

61

(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 (4.5-4.8 and 8-12 Å), which indicated the presence of cross-β structures (Fig. 4). However, on top of the two characteristic reflections, a large number of additional reflections were observed, they being 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 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; 3) the purity, source and method of preparation of the

sample 35,66. There is 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, the meridional and equatorial reflections being assessed separately 60,64,65

. For a precise interpretation of the data, not only very high quality of the diffraction pictures is

required, but also more advanced knowledge of the organization of fibrils on the molecular level. An interesting result derived from the X-ray analysis for fragments of Aβ peptides, similar in length to our studied peptides, namely Аβ(18-28), Аβ(17-28), Аβ(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 diminished between 1/40 and 1/20 Å, suggesting that ~30Å thick plates are stacked with a 40Å 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, VIYKI 67. The authors interpreted this as a protofilament width. 12 ACS Paragon Plus Environment

Page 13 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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

60

). Perutz et al.

68

give interesting

interpretation of their X-ray data, including that for the fragment Аβ(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 fibrils diameter in this case was measured to be about 6 nm. According to the optical reconstruction, the fibrils are hollow cylinders, with the diameter of about 57 Å, the wall thickness of 37 Å, and the central cavity of 19.5 Å in diameter. If this interpretation is correct, 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 Аβ(18-28) and Аβ(17-28), produce totally dissimilar diffraction patterns, despite the peptides differ only by 1 amino acid residue 64. This suggests that though 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 section “Molecular structure of nanofilms and amyloid fibrils …”). The tubular cylinder includes more than 40 β-strands and has a hollow with the diameter of ~ 20 Å.

Small angle X-ray scattering analyses.

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), i.e. the distance 13 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

between two points furthest from one another. Using the method of mass assessment

Page 14 of 33

69

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 is represented in Table 1. 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.

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 h and 8 h. After 1 h of incubation of Aβ(16-25), the predominant peptides were determined: the whole peptide, the peptides missing 2 to 4 C-terminal amino acid residues (glutamate, aspartate, valine, glycine), and the ones missing 1 to 3 N-terminal amino acid residues (lysine, leucine, valine) (Fig. 7, A). After 4 h and 8 h of proteolysis, peptides missing four N-terminal amino acid residues (lysine, leucine, valine, and phenylalanine) were recorded (Fig. 5, A). In the case of Aβ(31-40) peptide amyloids, all the amino acid residues were inaccessible for proteases even after 8 h of treatment (Fig. 7, B). Proteolysis of the amyloids formed by Aβ(33-42) peptides produced a great variety of truncated peptides. After 1 h of 14 ACS Paragon Plus Environment

Page 15 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

treatment, there were fragments missing between 1 and 4 N-terminal and between 2 and 4 C-terminal amino acid residues (separately and in combinations, Fig. 7, C). After 4 h of incubation the set of peptides did not change. After 8 h the number of truncated peptides from both termini decreased (Fig. 7, C). 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 Аβ(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(33-22). 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 about 6 nm, an inner diameter of about 2 nm (the hole diameter in the ring), and a ring height (measurement at the point of inflection of a single fibril) of about 3 nm. Thus, the ring oligomer is a short tubular cylinder with a wall width of about 2 nm We have proposed the molecular structure for this type of arrangement of oligomer structures using the method of molecular modeling (Fig.8). Amyloid fibrils for Aβ(31-40) 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) (single fibrils). The fibril Aβ(31-40) consisting of ring-like structures is presented in Fig. 8 (А, В). The Aβ(16-25) peptide aggregates into honeycomb nanofilms, which are not characteristic of an amyloid formation (Fig.8С)70. The annular oligomer can consist of 48 peptide molecules that form 12 β-sheets stacked in a circle (Fig.8). The orientation of β-strands in β-sheets in oligomers is compatible with the positions of strands and sheets in the model described in 71. Previously we have demonstrated that such antiparallel organization of βstrands is more favorable than others using Molecular Dynamics simulations 72.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 33

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, 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, the β-strand will be approximately standing up on top of the X-Y plane (i.e., along the zaxis). 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 residue long. The theoretical length of a peptide is about 3 nm, which is consistent with the height estimated from the EM (between 2 and 3 nm) and AFM experiments (1.5-2.3 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 oligomer structure.

CONCLUSIONS At present, the available information allows using experimental data, as well as theoretical predictions, studying the amyloid-forming 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. 16 ACS Paragon Plus Environment

Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Herein we have investigated the following amyloidogenic fragments of the Аβ42 peptide: Аβ(31-40), Аβ(33-42), and Аβ(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 the external diameter of 6-7 nm, the internal diameter of 2-3 nm, and the height of about 3 nm. According to a simplified model, the formation of fibrils goes through the following stages: destabilized monomer → oligomer → mature fibril (Fig. 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, i.e. the oligomers are ring-shaped 46–49. Based on 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, 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 selfassociation 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 predict the enormous length of the fibrils (up to 15-20 µm), nor can it convincingly explain some of the key features of fibrils, such as their polymorphism, splitting up, and branching. Our model does not contradict the Xray 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 Xray analysis, solid-state NMR, becomes increasingly important for the studies of amyloidogenesis, particularly the techniques of sample preparation and adequate interpretation of the results. The use of


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 33

methods of bioinformatics (including molecular dynamics) to aid the final interpretation of the results is also highly advantageous. Recent data suggests 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; 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 amyloidrelated 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.

Competing financial interests The authors declare no competing financial interests.

ACKNOWLEDGMENTS We are grateful T.B. Kuvshinkina, N. Lissin, E.I. Grigorashvili, E.Yu. Gorbunova, L.G. Mustaeva, N.V. Dovidchenko, A.D. Nikulin, N.A. Ryabova N.A., and A.G. Gabdulkhakov for assistance in preparation of the manuscript. This study was supported by the Russian Science Foundation (14-14-00536).


Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

REFERENCES (1) (2) (3) (4) (5) (6) (7)

(8) (9)

(10) (11) (12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

Dobson, C. M. Principles of Protein Folding, Misfolding and Aggregation. Semin. Cell Dev. Biol. 2004, 15 (1), 3–16. Astbury, W. T.; Dickinson, S.; Bailey, K. The X-Ray Interpretation of Denaturation and the Structure of the Seed Globulins. Biochem. J. 1935, 29 (10), 2351–2360.1. Eanes, E. D.; Glenner, G. G. X-Ray Diffraction Studies on Amyloid Filaments. J. Histochem. Cytochem. Off. J. Histochem. Soc. 1968, 16 (11), 673–677. Cohen, A. S.; Calkins, E. Electron Microscopic Observations on a Fibrous Component in Amyloid of Diverse Origins. Nature 1959, 183 (4669), 1202–1203. Shirahama, T.; Cohen, A. S. High-Resolution Electron Microscopic Analysis of the Amyloid Fibril. J. Cell Biol. 1967, 33 (3), 679–708. Benditt, E. P.; Eriksen, N. Amyloid. 3. A Protein Related to the Subunit Structure of Human Amyloid Fibrils. Proc. Natl. Acad. Sci. U. S. A. 1966, 55 (2), 308–316. Lührs, T.; Ritter, C.; Adrian, M.; Riek-Loher, D.; Bohrmann, B.; Döbeli, H.; Schubert, D.; Riek, R. 3D Structure of Alzheimer’s Amyloid-beta(1-42) Fibrils. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (48), 17342–17347. Petkova, A. T.; Yau, W.-M.; Tycko, R. Experimental Constraints on Quaternary Structure in Alzheimer’s Beta-Amyloid Fibrils. Biochemistry (Mosc.) 2006, 45 (2), 498–512. Paravastu, A. K.; Leapman, R. D.; Yau, W.-M.; Tycko, R. Molecular Structural Basis for Polymorphism in Alzheimer’s Beta-Amyloid Fibrils. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (47), 18349–18354. Qiang, W.; Yau, W.-M.; Luo, Y.; Mattson, M. P.; Tycko, R. Antiparallel β-Sheet Architecture in Iowa-Mutant β-Amyloid Fibrils. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (12), 4443–4448. Lu, J.-X.; Qiang, W.; Yau, W.-M.; Schwieters, C. D.; Meredith, S. C.; Tycko, R. Molecular Structure of β-Amyloid Fibrils in Alzheimer’s Disease Brain Tissue. Cell 2013, 154 (6), 1257–1268. Schütz, A. K.; Vagt, T.; Huber, M.; Ovchinnikova, O. Y.; Cadalbert, R.; Wall, J.; Güntert, P.; Böckmann, A.; Glockshuber, R.; Meier, B. H. Atomic-Resolution Three-Dimensional Structure of Amyloid β Fibrils Bearing the Osaka Mutation. Angew. Chem. Int. Ed Engl. 2015, 54 (1), 331–335. Xiao, Y.; Ma, B.; McElheny, D.; Parthasarathy, S.; Long, F.; Hoshi, M.; Nussinov, R.; Ishii, Y. Aβ(1– 42) Fibril Structure Illuminates Self-Recognition and Replication of Amyloid in Alzheimer’s Disease. Nat. Struct. Mol. Biol. 2015, 22 (6), 499–505. Schmidt, M.; Rohou, A.; Lasker, K.; Yadav, J. K.; Schiene-Fischer, C.; Fändrich, M.; Grigorieff, N. Peptide Dimer Structure in an Aβ(1-42) Fibril Visualized with Cryo-EM. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (38), 11858–11863. Sgourakis, N. G.; Yau, W.-M.; Qiang, W. Modeling an in-Register, Parallel “iowa” aβ Fibril Structure Using Solid-State NMR Data from Labeled Samples with Rosetta. Struct. Lond. Engl. 1993 2015, 23 (1), 216–227. Wälti, M. A.; Ravotti, F.; Arai, H.; Glabe, C. G.; Wall, J. S.; Böckmann, A.; Güntert, P.; Meier, B. H.; Riek, R. Atomic-Resolution Structure of a Disease-Relevant Aβ(1-42) Amyloid Fibril. Proc. Natl. Acad. Sci. U. S. A. 2016. Colvin, M. T.; Silvers, R.; Ni, Q. Z.; Can, T. V.; Sergeyev, I.; Rosay, M.; Donovan, K. J.; Michael, B.; Wall, J.; Linse, S.; Griffin, R. G. Atomic Resolution Structure of Monomorphic Aβ 42 Amyloid Fibrils. J. Am. Chem. Soc. 2016, 138 (30), 9663–9674. Gremer, L.; Schölzel, D.; Schenk, C.; Reinartz, E.; Labahn, J.; Ravelli, R. B. G.; Tusche, M.; LopezIglesias, C.; Hoyer, W.; Heise, H.; Willbold, D.; Schröder, G. F. Fibril Structure of Amyloid-β(1–42) by Cryo–electron Microscopy. Science 2017, 358 (6359), 116–119. Finder, V. H.; Glockshuber, R. Amyloid-Beta Aggregation. Neurodegener. Dis. 2007, 4 (1), 13–27. 19 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(20) (21) (22)

(23)

(24)

(25)

(26) (27)

(28) (29)

(30) (31) (32)

(33) (34) (35) (36)

(37) (38)

(39)

Page 20 of 33

Nielsen, E. H.; Nybo, M.; Svehag, S. E. Electron Microscopy of Prefibrillar Structures and Amyloid Fibrils. Methods Enzymol. 1999, 309, 491–496. Bhak, G.; Lee, J.-H.; Hahn, J.-S.; Paik, S. R. Granular Assembly of Alpha-Synuclein Leading to the Accelerated Amyloid Fibril Formation with Shear Stress. PloS One 2009, 4 (1), e4177. Do, T. D.; LaPointe, N. E.; Nelson, R.; Krotee, P.; Hayden, E. Y.; Ulrich, B.; Quan, S.; Feinstein, S. C.; Teplow, D. B.; Eisenberg, D.; Shea, J.-E.; Bowers, M. T. Amyloid β-Protein C-Terminal Fragments: Formation of Cylindrins and β-Barrels. J. Am. Chem. Soc. 2016, 138 (2), 549–557. Shankar, G. M.; Li, S.; Mehta, T. H.; Garcia-Munoz, A.; Shepardson, N. E.; Smith, I.; Brett, F. M.; Farrell, M. A.; Rowan, M. J.; Lemere, C. A.; Regan, C. M.; Walsh, D. M.; Sabatini, B. L.; Selkoe, D. J. Amyloid-Beta Protein Dimers Isolated Directly from Alzheimer’s Brains Impair Synaptic Plasticity and Memory. Nat. Med. 2008, 14 (8), 837–842. Wu, J. W.; Breydo, L.; Isas, J. M.; Lee, J.; Kuznetsov, Y. G.; Langen, R.; Glabe, C. Fibrillar Oligomers Nucleate the Oligomerization of Monomeric Amyloid Beta but Do Not Seed Fibril Formation. J. Biol. Chem. 2010, 285 (9), 6071–6079. Jan, A.; Adolfsson, O.; Allaman, I.; Buccarello, A.-L.; Magistretti, P. J.; Pfeifer, A.; Muhs, A.; Lashuel, H. A. Abeta42 Neurotoxicity Is Mediated by Ongoing Nucleated Polymerization Process rather than by Discrete Abeta42 Species. J. Biol. Chem. 2011, 286 (10), 8585–8596. Stroud, J. C.; Liu, C.; Teng, P. K.; Eisenberg, D. Toxic Fibrillar Oligomers of Amyloid-β Have Cross-β Structure. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (20), 7717–7722. Hong, S.; Ostaszewski, B. L.; Yang, T.; O’Malley, T. T.; Jin, M.; Yanagisawa, K.; Li, S.; Bartels, T.; Selkoe, D. J. Soluble Aβ Oligomers Are Rapidly Sequestered from Brain ISF in Vivo and Bind GM1 Ganglioside on Cellular Membranes. Neuron 2014, 82 (2), 308–319. Jin, M.; Selkoe, D. J. Systematic Analysis of Time-Dependent Neural Effects of Soluble Amyloid β Oligomers in Culture and in Vivo: Prevention by Scyllo-Inositol. Neurobiol. Dis. 2015, 82, 152–163. Breydo, L.; Kurouski, D.; Rasool, S.; Milton, S.; Wu, J. W.; Uversky, V. N.; Lednev, I. K.; Glabe, C. G. Structural Differences between Amyloid Beta Oligomers. Biochem. Biophys. Res. Commun. 2016, 477 (4), 700–705. Truex, N. L.; Nowick, J. S. Coassembly of Peptides Derived from β-Sheet Regions of β-Amyloid. J. Am. Chem. Soc. 2016, 138 (42), 13891–13900. Truex, N. L.; Wang, Y.; Nowick, J. S. Assembly of Peptides Derived from β-Sheet Regions of βAmyloid. J. Am. Chem. Soc. 2016, 138 (42), 13882–13890. Kreutzer, A. G.; Hamza, I. L.; Spencer, R. K.; Nowick, J. S. X-Ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17-36 β-Hairpin. J. Am. Chem. Soc. 2016, 138 (13), 4634–4642. Beyermann, M.; Henklein, P.; Klose, A.; Sohr, R.; Bienert, M. Effect of Tertiary Amine on the Carbodiimide-Mediated Peptide Synthesis. Int. J. Pept. Protein Res. 1991, 37 (4), 252–256. Markham, R.; Frey, S.; Hills, G. J. Methods for the Enhancement of Image Detail and Accentuation of Structure in Electron Microscopy. Virology 1963, 20 (1), 88–102. Serpell, L. C.; Fraser, P. E.; Sunde, M. X-Ray Fiber Diffraction of Amyloid Fibrils. Methods Enzymol. 1999, 309, 526–536. Petoukhov, M. V.; Franke, D.; Shkumatov, A. V.; Tria, G.; Kikhney, A. G.; Gajda, M.; Gorba, C.; Mertens, H. D. T.; Konarev, P. V.; Svergun, D. I. New Developments in the ATSAS Program Package for Small-Angle Scattering Data Analysis. J. Appl. Crystallogr. 2012, 45 (Pt 2), 342–350. Franke, D.; Svergun, D. I. DAMMIF, a Program for Rapid Ab-Initio Shape Determination in SmallAngle Scattering. J. Appl. Crystallogr. 2009, 42 (Pt 2), 342–346. Selivanova, O. M.; Glyakina, A. V.; Gorbunova, E. Y.; Mustaeva, L. G.; Suvorina, M. Y.; Grigorashvili, E. I.; Nikulin, A. D.; Dovidchenko, N. V.; Rekstina, V. V.; Kalebina, T. S.; Surin, A. K.; Galzitskaya, O. V. Structural Model of Amyloid Fibrils for Amyloidogenic Peptide from Bgl2p– glucantransferase of S. Cerevisiae Cell Wall and Its Modifying Analog. New Morphology of Amyloid Fibrils. Biochim. Biophys. Acta BBA - Proteins Proteomics 2016, 1864 (11), 1489–1499. Lansbury, P. T.; Costa, P. R.; Griffiths, J. M.; Simon, E. J.; Auger, M.; Halverson, K. J.; Kocisko, D. A.; Hendsch, Z. S.; Ashburn, T. T.; Spencer, R. G. Structural Model for the Beta-Amyloid Fibril Based


Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(40)

(41) (42)

(43)

(44) (45)

(46) (47) (48)

(49)

(50)

(51) (52) (53) (54)

(55) (56) (57) (58)

on Interstrand Alignment of an Antiparallel-Sheet Comprising a C-Terminal Peptide. Nat. Struct. Biol. 1995, 2 (11), 990–998. Sawaya, M. R.; Sambashivan, S.; Nelson, R.; Ivanova, M. I.; Sievers, S. A.; Apostol, M. I.; Thompson, M. J.; Balbirnie, M.; Wiltzius, J. J. W.; McFarlane, H. T.; Madsen, A. Ø.; Riekel, C.; Eisenberg, D. Atomic Structures of Amyloid Cross-Beta Spines Reveal Varied Steric Zippers. Nature 2007, 447 (7143), 453–457. Krieger, E.; Koraimann, G.; Vriend, G. Increasing the Precision of Comparative Models with YASARA NOVA--a Self-Parameterizing Force Field. Proteins 2002, 47 (3), 393–402. Dovidchenko, N. V.; Glyakina, A. V.; Selivanova, O. M.; Grigorashvili, E. I.; Suvorina, M. Y.; Dzhus, U. F.; Mikhailina, A. O.; Shiliaev, N. G.; Marchenkov, V. V.; Surin, A. K.; Galzitskaya, O. V. One of the Possible Mechanisms of Amyloid Fibrils Formation Based on the Sizes of Primary and Secondary Folding Nuclei of Aβ40 and Aβ42. J. Struct. Biol. 2016, 194 (3), 404–414. Selivanova, O. M.; Surin, A. K.; Marchenkov, V. V.; Dzhus, U. F.; Grigorashvili, E. I.; Suvorina, M. Y.; Glyakina, A. V.; Dovidchenko, N. V.; Galzitskaya, O. V. The Mechanism Underlying Amyloid Polymorphism Is Opened for Alzheimer’s Disease Amyloid-β Peptide. J. Alzheimers Dis. JAD 2016, 54 (2), 821–830. Fraser, P. E.; Nguyen, J. T.; Surewicz, W. K.; Kirschner, D. A. pH-Dependent Structural Transitions of Alzheimer Amyloid Peptides. Biophys. J. 1991, 60 (5), 1190–1201. Dai, B.; Li, D.; Xi, W.; Luo, F.; Zhang, X.; Zou, M.; Cao, M.; Hu, J.; Wang, W.; Wei, G.; Zhang, Y.; Liu, C. Tunable Assembly of Amyloid-Forming Peptides into Nanosheets as a Retrovirus Carrier. Proc. Natl. Acad. Sci. 2015, 112 (10), 2996–3001. Benditt, E. P.; Eriksen, N. Amyloid. 3. A Protein Related to the Subunit Structure of Human Amyloid Fibrils. Proc. Natl. Acad. Sci. U. S. A. 1966, 55 (2), 308–316. Goldsbury, C.; Frey, P.; Olivieri, V.; Aebi, U.; Müller, S. A. Multiple Assembly Pathways Underlie Amyloid-Beta Fibril Polymorphisms. J. Mol. Biol. 2005, 352 (2), 282–298. Goldsbury, C. S.; Wirtz, S.; Müller, S. A.; Sunderji, S.; Wicki, P.; Aebi, U.; Frey, P. Studies on the in Vitro Assembly of a Beta 1-40: Implications for the Search for a Beta Fibril Formation Inhibitors. J. Struct. Biol. 2000, 130 (2–3), 217–231. Quist, A.; Doudevski, I.; Lin, H.; Azimova, R.; Ng, D.; Frangione, B.; Kagan, B.; Ghiso, J.; Lal, R. Amyloid Ion Channels: A Common Structural Link for Protein-Misfolding Disease. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (30), 10427–10432. Lashuel, H. A.; Hartley, D. M.; Petre, B. M.; Wall, J. S.; Simon, M. N.; Walz, T.; Lansbury, P. T. Mixtures of Wild-Type and a Pathogenic (E22G) Form of Abeta40 in Vitro Accumulate Protofibrils, Including Amyloid Pores. J. Mol. Biol. 2003, 332 (4), 795–808. Nielsen, E. H.; Nybo, M.; Svehag, S. E. Electron Microscopy of Prefibrillar Structures and Amyloid Fibrils. Methods Enzymol. 1999, 309, 491–496. Bhak, G.; Lee, J.-H.; Hahn, J.-S.; Paik, S. R. Granular Assembly of Alpha-Synuclein Leading to the Accelerated Amyloid Fibril Formation with Shear Stress. PloS One 2009, 4 (1), e4177. Hill, S. E.; Robinson, J.; Matthews, G.; Muschol, M. Amyloid Protofibrils of Lysozyme Nucleate and Grow via Oligomer Fusion. Biophys. J. 2009, 96 (9), 3781–3790. Jeong, J. S.; Ansaloni, A.; Mezzenga, R.; Lashuel, H. A.; Dietler, G. Novel Mechanistic Insight into the Molecular Basis of Amyloid Polymorphism and Secondary Nucleation during Amyloid Formation. J. Mol. Biol. 2013, 425 (10), 1765–1781. Dean, D. N.; Das, P. K.; Rana, P.; Burg, F.; Levites, Y.; Morgan, S. E.; Ghosh, P.; Rangachari, V. Strain-Specific Fibril Propagation by an A? Dodecamer. Sci. Rep. 2017, 7, 40787. Chiti, F.; Dobson, C. M. Protein Misfolding, Functional Amyloid, and Human Disease. Annu. Rev. Biochem. 2006, 75, 333–366. Eanes, E. D.; Glenner, G. G. X-Ray Diffraction Studies on Amyloid Filaments. J. Histochem. Cytochem. Off. J. Histochem. Soc. 1968, 16 (11), 673–677. Glenner, G. G.; Eanes, E. D.; Bladen, H. A.; Linke, R. P.; Termine, J. D. Beta-Pleated Sheet Fibrils. A Comparison of Native Amyloid with Synthetic Protein Fibrils. J. Histochem. Cytochem. Off. J. Histochem. Soc. 1974, 22 (12), 1141–1158.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(59) (60) (61)

(62)

(63)

(64)

(65) (66) (67)

(68) (69) (70) (71)

(72)

(73)

(74)

(75)

Page 22 of 33

Sunde, M.; Blake, C. The Structure of Amyloid Fibrils by Electron Microscopy and X-Ray Diffraction. Adv. Protein Chem. 1997, 50, 123–159. Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. Common Core Structure of Amyloid Fibrils by Synchrotron X-Ray Diffraction. J. Mol. Biol. 1997, 273 (3), 729–739. Smaoui, M. R.; Poitevin, F.; Delarue, M.; Koehl, P.; Orland, H.; Waldispühl, J. Computational Assembly of Polymorphic Amyloid Fibrils Reveals Stable Aggregates. Biophys. J. 2013, 104 (3), 683–693. Colvin, M. T.; Silvers, R.; Ni, Q. Z.; Can, T. V.; Sergeyev, I.; Rosay, M.; Donovan, K. J.; Michael, B.; Wall, J.; Linse, S.; Griffin, R. G. Atomic Resolution Structure of Monomorphic Aβ 42 Amyloid Fibrils. J. Am. Chem. Soc. 2016, 138 (30), 9663–9674. Wälti, M. A.; Ravotti, F.; Arai, H.; Glabe, C. G.; Wall, J. S.; Böckmann, A.; Güntert, P.; Meier, B. H.; Riek, R. Atomic-Resolution Structure of a Disease-Relevant Aβ(1-42) Amyloid Fibril. Proc. Natl. Acad. Sci. U. S. A. 2016. Inouye, H.; Fraser, P. E.; Kirschner, D. A. Structure of Beta-Crystallite Assemblies Formed by Alzheimer Beta-Amyloid Protein Analogues: Analysis by X-Ray Diffraction. Biophys. J. 1993, 64 (2), 502–519. Malinchik, S. B.; Inouye, H.; Szumowski, K. E.; Kirschner, D. A. Structural Analysis of Alzheimer’s beta(1-40) Amyloid: Protofilament Assembly of Tubular Fibrils. Biophys. J. 1998, 74 (1), 537–545. Makin, O. S.; Serpell, L. C. X-Ray Diffraction Studies of Amyloid Structure. Methods Mol. Biol. Clifton NJ 2005, 299, 67–80. Morris, K. L.; Rodger, A.; Hicks, M. R.; Debulpaep, M.; Schymkowitz, J.; Rousseau, F.; Serpell, L. C. Exploring the Sequence–structure Relationship for Amyloid Peptides. Biochem. J. 2013, 450 (2), 275–283. Perutz, M. F.; Finch, J. T.; Berriman, J.; Lesk, A. Amyloid Fibers Are Water-Filled Nanotubes. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (8), 5591–5595. Rambo, R. P.; Tainer, J. A. Accurate Assessment of Mass, Models and Resolution by Small-Angle Scattering. Nature 2013, 496 (7446), 477–481. Abb, S.; Harnau, L.; Gutzler, R.; Rauschenbach, S.; Kern, K. Two-Dimensional Honeycomb Network through Sequence-Controlled Self-Assembly of Oligopeptides. Nat. Commun. 2016, 7, 10335. Dai, B.; Li, D.; Xi, W.; Luo, F.; Zhang, X.; Zou, M.; Cao, M.; Hu, J.; Wang, W.; Wei, G.; Zhang, Y.; Liu, C. Tunable Assembly of Amyloid-Forming Peptides into Nanosheets as a Retrovirus Carrier. Proc. Natl. Acad. Sci. 2015, 112 (10), 2996–3001. Glyakina, A. V.; Balabaev, N. K.; Galzitskaya, O. V. Dataset of the Molecular Dynamics Simulations of Bilayers Consisting of Short Amyloidogenic Peptide VDSWNVLVAG from Bgl2pGlucantransferase of S. Cerevisiae Cell Wall. Data Brief 2016, 9, 597–601. Suvorina, M. Y.; Selivanova, O. M.; Grigorashvili, E. I.; Nikulin, A. D.; Marchenkov, V. V.; Surin, A. K.; Galzitskaya, O. V. Studies of Polymorphism of Amyloid-β 42 Peptide from Different Suppliers. J. Alzheimers Dis. 2015, 47 (3), 583–593. Dovidchenko, N. V.; Glyakina, A. V.; Selivanova, O. M.; Grigorashvili, E. I.; Suvorina, M. Y.; Dzhus, U. F.; Mikhailina, A. O.; Shiliaev, N. G.; Marchenkov, V. V.; Surin, A. K.; Galzitskaya, O. V. One of the Possible Mechanisms of Amyloid Fibrils Formation Based on the Sizes of Primary and Secondary Folding Nuclei of Aβ40 and Aβ42. J. Struct. Biol. 2016, 194 (3), 404–414. Galzitskaya, O. V.; Selivanova, O. M. Rosetta Stone for Amyloid Fibrils: The Key Role of Ring-Like Oligomers in Amyloidogenesis. J. Alzheimers Dis. JAD 2017, 59 (3), 785–795.


Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 1. EM images of Aβ β(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 0.25−0.5 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 Аβ(33-42) peptide fragments – the ring oligomer with the outer diameter of about 6−7 nm and the inner diameter (hole) of about 2−3 nm.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 33

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


Page 25 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 3. Illustration of the method used for extraction of periodic structures from indistinct images of negatively stained amyloid fibrils of the Аβ(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 A) equally contrasted (upper image in D) but with twofold descending opacity of each subsequent overlapping layer (reduced opacity of each subsequent superimposed layer) (scheme B). Panel C represents interference of semitransparent layers gradually shifted by fixed step (2 pixels, about 0.5 nm) alongside of the fiber in one direction. At the step of about 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 2-3 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 D).


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 33

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


Page 27 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 5. Small angle scattering curves. 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.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 33

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


Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

Figure 8. Molecular structures for amyloid fibrils formed by Aβ(31-40) and for nanofilm Aβ(1625). (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 6−7 nm. The diameter of the hole of a ring is about 2 nm and the height is about 3 nm.


Page 31 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 33

Graphical abstract


Page 33 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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 form ab-initio structure V S Lm M

Aβ(16-25)

Aβ(33-42)

Aβ(31-40)

280 Å -36 Å 900 Å 1800 Å 11 × 106 Å3 930 × 103 Å2 100Å 9.7 MDa

150 Å 185 Å -500 Å 1200 Å 7.2 × 106 Å3 280 × 103 Å2 140 Å 3.6 MDa

410 Å 176 Å -1200 Å 2700 Å 96 × 106 Å3 4500 × 103 Å2 230 Å 88 MDa


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

Recommend Documents