Mechanical Anisotropy in GNNQQNY Amyloid Crystals

Subscriber access provided by AUSTRALIAN NATIONAL UNIV

Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Mechanical Anisotropy in GNNQQNY Amyloid Crystals Roy Nassar, Eric Wong, Jennifer M. Bui, Calvin Yip, Hongbin Li, Joerg A. Gsponer, and Guillaume Lamour J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b02027 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 15, 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 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 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.

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 22 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

The Journal of Physical Chemistry Letters

Mechanical Anisotropy in GNNQQNY Amyloid Crystals Roy Nassar,†,‡ Eric Wong,†,‡ Jennifer M. Bui,†,‡ Calvin K. Yip,‡ Hongbin Li§, Jörg Gsponer,*,†,‡ and Guillaume Lamour*,†,‡,§ †

The University of British Colombia, Michael Smith Laboratories, Vancouver, BC Canada V6T 1Z4

‡

The University of British Colombia, Department of Biochemistry & Molecular Biology, Vancouver, BC,

Canada V6T 1Z3 §

The University of British Columbia, Department of Chemistry, Vancouver, BC, Canada V6T 1Z1

Present address of G.L.: Laboratoire d’Analyse et Modélisation pour la Biologie et l’Environnement LAMBE-CNRS, UMR 8587, Université d’Evry, 91025 Evry, France *Correspondence to: [email protected] and [email protected]

ABSTRACT Mapping the nanomechanical properties of amyloids can provide valuable insights into structure and assembly mechanisms of protein aggregates that underlie the development of various human diseases. Although it is well known that amyloids exhibit an intrinsic stiffness comparable to that of silk (1–10 GPa), a detailed understanding of the directional dependence (anisotropy) of the stiffness of amyloids and how it relates to structural features in these protein aggregates is missing.

Here we used steered

molecular dynamics (SMD) simulations and amplitude modulation-frequency modulation (AM-FM) atomic force microscopy to measure the directional variation in stiffness of GNNQQNY amyloid crystals. We reveal that individual crystals display significant mechanical anisotropy and relate this anisotropy to subtle but mechanically important differences in interactions between interfaces that define the crystal architecture. Our results provide detailed insights into the structure-mechanics relationship of amyloid that may help in designing amyloid-based nanomaterials with tailored mechanical properties. 1 ACS Paragon Plus Environment

The Journal of Physical Chemistry Letters 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

TOC FIGURE

Keywords: amyloid, GNNQQNY, nanomechanics, anisotropy, intermolecular forces, Young’s modulus, atomic force microscopy, molecular dynamics simulation

Amyloids are protein aggregates that can be found in plaques and other deposits of more than 40 debilitating diseases.1 Amyloids are defined as protein aggregates with a cross-beta structure in which beta-strands run perpendicular to the fibril axis. An interesting feature of amyloids is that they are very polymorphic, with morphologies ranging from single filaments to twisted/helical or tape-like fibrils to amyloid microcrystals. The latter have played an important role in the determination of the first models of amyloid-like structures via X-ray crystallography. Specifically, it was the structure of the amyloid crystal formed by the GNNQQNY peptide, corresponding to the amino-acids sequence Gly-Asn-Asn-Gln-GlnAsn-Tyr, that was first determined in 2005.2 The GNNQQNY sequence was studied because it forms the core of the Sup35 yeast fibril and can form both fibrillar and crystal structures.3,4 Recent experiments and theoretical calculations have shown that amyloid crystals arise as a result of untwisting of twisted fibrils once a critical number of filaments have been incorporated into the mature fibril.5,6 Nanoscale and mechanical properties of different amyloid morphologies have drawn considerable interest. As amyloids can self-assemble spontaneously to form very stiff fibrils, a detailed understanding of their nanostructures will help in exploiting amyloidogenic sequences as building blocks in novel biomaterials.7-10 Amyloid nanomechanics are also believed to play a key role in the conversion of soluble protein species to fibrillar ones, because fibril scission generates new fibril ends that can act as seeds for

2 ACS Paragon Plus Environment

Page 2 of 22

Page 3 of 22 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


structural conversion.1,11 Quantifying stiffnesses and strengths of different amyloid morphologies may thus help reveal whether amyloid nanomechanical properties affect the spreading of amyloid-based diseases. In addition, it has been difficult to determine the structural details of amyloids, at least for amyloids formed by long protein chains. Mechanical studies of fibrils provide indirect insights into their structural features, specifically into the hydrogen bonding network and van der Waals (vdW) interactions that define amyloid material properties.12-15 An important measure that has been used to characterize amyloid nanomechanics is the Young’s modulus, which reflects the stiffness of a material. Amyloids have axial Young’s moduli (in the direction corresponding to the fibril’s axis) in the range of 0.1–10 GPa.13 Young’s modulus measurements performed in the radial direction, that is, orthogonal to the fibril’s axis, have given values in the range of 0.05–5 GPa.14,16,17 For fibrils made by the GNNQQNY peptide, an axial modulus of ~10 GPa has been measured.12 Consistent with experimental measurements, steered molecular dynamics simulations (SMD) indicate that amyloid fibrils have a Young’s modulus on the order of gigapascals.13,18 However, the nanomechanics of proteins and protein assemblies are often anisotropic. Mechanical anisotropy has been observed at the single-protein level,19 and in higher-order supramolecular assemblies, anisotropy in the forces acting to stabilize amyloid at the atomic scale leads to length-dependent mechanical properties.20 In particular, an intrasheet H-bond network confers 20 times more rigidity to amyloid in the axial direction than intersheet side-chain interactions in the radial direction.20,21 However, one major question has yet to be addressed: is there mechanical anisotropy in the radial direction of amyloids, and if so, does it correlate with differences in filament interfaces and their molecular interactions? Here, we use all-atom SMD simulations and atomic force microscopy (AFM) to study mechanical anisotropy in amyloid crystals of the GNNQQNY peptide. We first measure deformability in a resolved 3D structure of the crystal, both along the fibril axis and in two distinct radial directions, using the same SMD compression protocol. Then we assess the mechanical anisotropy experimentally by performing direct Young’s modulus measurements on GNNQQNY crystals using AFM. By combining simulations



with experiments, we reveal how structural characteristics of the amyloid crystals of GNNQQNY give rise to differences in radial Young’s moduli. Finally, we show that these differences lead to mechanical anisotropy within individual amyloid crystals.

RESULTS AND DISCUSSION Compression simulations reveal mechanical anisotropy in GNNQQNY crystals. In order to investigate the mechanical anisotropy of GNNQQNY crystals, we first carried out all-atom steered molecular dynamics (SMD) simulations. We assembled a “3 x 3” model of the GNNQQNY crystal that consists of nine filaments with three filaments along each side-wall (total 180 peptides) (Fig. 1) and equilibrated it at 300 K in a TIP3P water box (see Material and Methods for details). After the backbone atoms RMSD had plateaued at 3 ns (Fig. 2A), we used SMD simulations to compress the model in 3 directions of space: along the fibril axis and along the 2 radial directions perpendicular to the fibril axis (Fig. 1A-C). We used stress-strain curves to derive and compare the radial and axial Young’s moduli of different crystal axes. The Young’s elastic modulus indicates the level of resistance to deformation of a structure whose integrity is maintained over the reversible deformation process. The stress-strain curves show linear responses of stress as a function of strain (Fig. 2B); hence we consider that the crystal models behave as homogenous elastic materials along each compression direction. Fits to the stress-strain curves within the elastic regime (where deformation responds linearly to stress) reveal that the crystal model has different Young’s elastic moduli according to the direction along which stress is applied. Compressing the crystal model along the fibril axis gives a Young’s modulus of 27 ± 2.3 GPa. This value is 3 to 4 times higher than the 6.3 ± 1.2 and 9.6 ± 1.8 GPa obtained by compressing the crystal model along the radial directions Radial-1 and Radial-2, respectively (Fig. 2C). Note that Radial-1 and Radial-2 moduli also differ significantly from each other. Next, using the same stress-strain curves, we assessed the in silico mechanical strength of the crystal model by measuring the amount of stress that is required to break the fibrils. We find that a stress of ~0.8 4 ACS Paragon Plus Environment

Page 4 of 22

Page 5 of 22 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


± 0.2 GPa is required to break the crystal model along the fibril axis, whereas 0.4 ± 0.2 GPa is sufficient to break it along any of the radial directions (Fig. 2C). These in silico experiments reveal that the crystal model has not only different elastic moduli but also different strengths across distinct orthogonal directions. The amyloid crystal model displays pronounced mechanical anisotropy.

Molecular interactions across different interfaces drive the structural response to compression. The observed results suggest that radial and axial interactions in the crystal structure differ significantly. In order to understand which interactions underlie the mechanical anisotropy, we first analyzed where the crystal models break at high stress. According to the simulations, stress along the axis direction ruptures primarily the “interlayer” interface that connects beta-strands in the fibril (i.e. all in planes oriented perpendicularly to the fibril axis) (Fig. 1A). Ruptures as a result of radial compression occur primarily between beta-sheets, at the different interfaces that hold the sheets together (i.e. all in planes oriented parallel to the fibril axis) (Fig. 1B and C). Notably, for the case of radial compression, three relevant interfaces can be distinguished: the “dry”, “wet” and “YG” interfaces. Within a filament, the dry interface connects beta-sheets through a “steric zipper”, in which asparagines and glutamines interact so closely that solvent molecules cannot have access to it, hence the “dry” qualification. The wet interface connects filaments with each other via tyrosines as well as asparagines and glutamines that are more loosely interacting than in the dry interface (Fig. 1B). The YG interface connects filaments via the terminal ends of peptides; therefore, it is composed essentially of tyrosines and glycines facing each other (Fig. 1C). Radial-1 compression leads to the rupture of the wet interface (Fig. 1B) while compression along the Radial-2 direction breaks the wet and YG interfaces, and sometimes the dry interface (Fig. 1C). Importantly, in a total of 8 compression simulations in the Radial-1 direction, we never observed the dry interface undergoing rupture; it is always the wet interface between the “rows” that is seen to deform and break (Fig. 1B). On the other hand, the Radial-2 compression direction shows mixed results. In 4 out of 8 compressions along Radial-2, the wet interface deformed first; this is evidenced by the initial increase in 5 ACS Paragon Plus Environment


the spaces between the filaments. Multiple YG interfaces in the crystal then break simultaneously (up to three in some cases). All the dry interfaces remained intact even after the crystal had fragmented in this scenario (Fig. 1C). In the other 4 compressions, the sequence of events seems more intricate; the simulations ended up with a broken dry interface in the middle filament of the first (top) row (Fig. 1D). The rupture that begins at the wet interface propagates to the YG and dry interfaces, where the bending and peeling back of the top beta sheet causes the dry interface to unzip. That is, the leading (corner) filament in the first row seems to have taken most of the load in this case and broke off from the crystal along with one of the beta sheets of the nearby (middle) filament. All radial simulations taken collectively hint that the wet interface seems much weaker than the YG and dry interfaces and rearranges or breaks first in most radial compressions. Previously, we and others have reported that axial Young’s modulus and strength of amyloid fibrils depend on the hydrogen bond density (HBD) in the interface between interacting beta-strands.12,13,18 Consistent with the differences of Young’s moduli and strengths that we measured here for axial and radial compression, we find the highest HBD across the interlayer interface, and significantly lower values across other interfaces (Fig. 2D). For Radial-1, it is clear that the loading force is mostly exerted on the wet and dry interfaces. For Radial-2, the loading geometry suggests that it is the YG interface that supports most of the load. The HBD of the YG (~5 nm-2) is in between that of the interlayer (~9 nm-2) and the wet interface, the latter having no HBs. This difference in HBD is consistent with the difference in Young’s moduli that we measured for compressions along Radial-2 (~10 GPa), the axial (~27 GPa) and Radial-1 (~6 GPa) directions, respectively. In contrast to the Young’s modulus, we measured similar strength for compressions in the Radial-1 and Radial-2 directions (~400 MPa). This result suggests that interactions other than hydrogen bonds modulate mechanical properties. Indeed, we have shown previously that vdW interactions can fine tune interlayer interactions and thus modulate Young’s modulus and strength of amyloid fibrils.13 Therefore, we performed additional energy calculations, which revealed that both the wet and YG interfaces have


Page 6 of 22

Page 7 of 22 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


low vdW energy densities, but that the dry interface has a high favorable vdW energy density, even higher than the one measured between layers in the axis direction (Fig. 2E). This high vdW energy density of the dry interface is the result of tight side- chain interactions in the “steric zipper”. Together with our finding that the dry interface rarely ruptures and that the wet one is, in most cases, the first to rupture during compression in both radial directions, these energy calculations suggest that the wet interface is the “weakest link” and plays a dominant role in defining the radial strength of the crystal. Overall, SMD simulations suggest that mechanical anisotropy in GNNQQNY crystals acts at two levels: (i) stronger interactions in the direction of the fibril axis lead to higher axial modulus compared to the radial one, and (ii) the radial structure is itself anisotropic such that compressions in two orthogonal directions result in distinct moduli but, interestingly, the same strength—a consequence of an interplay between HBD and vdW interactions. The simulations further reveal that compression leads to complex deformation processes, due to distinct responses of the interfaces to mechanical stress.



Figure 1. Compression simulations on GNNQQNY crystal structure. Different compression geometries lead to breakage of distinct interfaces in the crystal. Peptide backbone is displayed in cartoon representation (blue) and side-chains are shown as lines with colors corresponding to the various residues (e.g. TYR in green) to show the steric-zipper architecture of the inner interface of each fibril. (A) Side-view of the crystal showing compression along the fibril axis: the top layer of the crystal was held fixed while the center of mass of the three bottom layers was compressed upwards (as shown by the red arrow symbol); side chains are not displayed for clarity purposes. Dashed lines indicate interlayer spaces where hydrogen bonds connect the beta strands along the fibril axis. (B) Top-down view: compression along one of the first radial directions (Radial-1) in which the three neighboring sheets at the top of the panel are held fixed and the three bottom sheets are steered up to compress the crystal. (C) Top-down view: the two left-most residues of the left-most peptides were constrained and the two end residues of all the peptides on the far right side were pushed leftwards (Radial-2). Orange circle shows an example of a filament in the center of the crystal structure. (D) Top-down view: the two right-most residues of the right-most peptides were constrained and the two end residues of all the peptides on the far left side were pushed rightwards (Radial-2). No difference in measured mechanical properties could be noticed between pushing leftwards or rightwards.


Page 8 of 22

Page 9 of 22 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


Figure 2. Mechanical properties of GNNQQNY crystal structure derived from compression simulations. (A) Root mean square displacement (RMSD) calculated from the trajectory of the production run (backbone atoms). (B) Representative stressstrain curves for each of the compressing directions. (C) Ashby plot showing Young’s modulus and (yield) strength of the crystal structure along the distinct compressing directions. Young’s modulus p-values (unpaired t-test): p < 10-9 for both Axial-Radial1 and Axial-Radial2, p~9x10-4 for Radial1-Radial2. Strength p-values (unpaired t-test): p~1x10-3 for Axial-Radial1, p~2x10-3 for Axial-Radial2 and p~0.3 for Radial1-Radial2. (D) Linear relationship between hydrogen bond energy density and hydrogen bond density (correlation coefficient for the red line = 0.99). HBD and vdW p-values (unpaired t-test): p 1 indicates a fluid behavior, and loss tangent < 1 means that the


Page 10 of 22

Page 11 of 22 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


material is rather solid.23 Here, all the crystals have loss tangents of ~0.1, which indicates that energy dissipation is low during indentation. Hence, amyloid crystals can be considered very solid materials whose shape and position on the substrate are not significantly affected by the interaction with the AFM tip. We found several instances where all these conditions are satisfied. One of these instances, for which we carried out a section analysis on the AFM image (black line), is shown in Fig. 3B. The results of this section analysis for each imaging parameter are displayed in Fig. 3C. Both crystals that we analyzed in this instance have nearly identical heights of ~34 nm but different moduli of 0.4 and 1.6 GPa, respectively. Importantly, all other relevant scanning parameters have identical or similar values. In addition to AFM height, the tip scanning amplitude (of the first resonance) is similar at the top of the height peaks (green markers) and constant around that area according to the image (Amplitude panel). Only the indentation depth differs between the two crystals, which is consistent with different elasticity (i.e. lower modulus corresponding to higher indentation). Indentation depth is 1–3 nm, which is small compared to the crystal heights (> 30 nm), and ensures that the underlying substrate plays little or no role in the measured moduli.24 In total, we collected N = 105 measurements on as many different crystals whose heights were always above 30 nm and at locations where first mode amplitude was always 87 nm, as in Fig. 3. We found that, under these constraints, the measured values of the Young’s modulus are distributed rather homogenously in the range of 0.4–1.8 GPa (Fig. 4). These experimental results are fully consistent with the SMD simulation findings. They reveal that intrinsic stiffness in the radial direction of GNNQQNY crystals can vary, with the highest recorded modulus being more than two times higher than the lowest one. We note that the Young’s modulus measured experimentally is about an order of magnitude lower than that measured via SMD simulations. Most likely, this difference derives from limit-size effects as demonstrated in previous studies.25,26 It has been shown that the computed modulus decreases exponentially when increasing the number of monomers in a model of Aβ fibrils, and finally tends to match the experimental modulus quite closely.25



Figure 3. Morphology and mechanical properties of GNNQQNY crystals. (A) GNNQQNY crystals observed by transmission electron microscopy (TEM). The nanocrystals were obtained by sonication of the fibrils for 20 min (see Materials and Methods for details). (B) Crystals adsorbed on PS-LDPE surface and imaged by AFM using the AM-FM mode in ambient conditions. The AFM tip scanned the surface from right to left. Black line: section analysis, with green symbols reported in (C). (C) Section profiles with values corresponding to those measured along the black line in (A). Young’s modulus variation ∆Y = 1.2 GPa between two distinct crystals both having a height of around 34 nm (measured at the highest point for each crystal, green symbols). Note that the AFM image is an enlarged area of a larger image comprising several LDPE islands (one of them visible at the top-right of the image) therefore the modulus is properly calibrated because the crystals’ modulus is enclosed within the moduli of LDPE and PS (0.1 and 2.2 GPa, respectively).

Figure 4. Distribution of Young’s modulus of GNNQQNY crystals. A total of 105 measurements were made on crystals adsorbed on PS (see Fig. 3). For each measurement AFM first mode amplitude was equal to 87 nm and loss tangent was 0.10 ± 0.03.


Page 12 of 22

Page 13 of 22 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


Mechanical anisotropy within single crystals. Next, we sought to determine whether variations in the modulus could be found within the same crystal. Although we measured some level of variations in the modulus within the crystals analyzed in Fig. 3, we cannot rule out that these variations resulted from imaging artifacts due to first resonance amplitude changes in the peripheral regions of the crystals. To overcome some of the problems associated with imaging on PS-LDPE, we adsorbed crystals on mica for additional rounds of AM-FM AFM imaging (Fig. 5). Using mica, instead of PS-LDPE, provides two advantages: mica is much stiffer (Young’s modulus > 60 GPa) and it has higher surface energy, both of which should improve image resolution by enhancing crystals immobilization. We checked the immobilization of crystals by loss tangent measurements. We found a loss tangent of ~0.02 for mica alone and for the crystals on mica, which is half an order of magnitude lower than what we found for both PS and crystals on PS. The Young’s modulus of crystals on mica is in the same range as when measured on PS. However, AFM images of crystals on mica (Fig. 5) reveal higher contrasts for Young’s moduli than for heights within single crystals, highlighting differences in material properties. The spatial orientation of crystal filaments in relation to the crystal orientation is apparent from the new AFM images on mica (Fig. 5, bottom), which is consistent with the TEM images and our assumption that we are probing radial but not axial elastic modulus during AM-FM AFM imaging. Importantly, the new images on mica demonstrate that modulus variations between 1.3 and 2.0 GPa within a single crystal are not correlated with variations in height (Fig. 5C). The variations in modulus measured within a crystal on mica are comparable to the variations that we measured on many crystals on PS, although slightly lower in maximum amplitude. We note that the modulus is quite constant along what resembles filament core structures oriented along the axis of needle-shaped crystal. We hypothesize that varying structural features of the different filaments within the crystals cause the variation in measured moduli; specifically, the possibility that filaments displaying the most extreme moduli differences are not oriented the same way. In other words, it is possible that some of the “softest” filaments are oriented such that the AFM tip



indents in the Radial-1 direction, whereas the “stiffest” filaments are indented in the Radial-2 direction (see Fig. 1B and C). Our AFM results reveal that the GNNQQNY amyloid crystals have significant mechanical anisotropy. Whether the range of 1.3–2.0 GPa represents a significant variation is debatable, given the large variations in moduli (> 1 GPa) that have been reported in the literature for amyloid fibrils. In fibrils, single filaments can arrange and pack in different conformations, thus giving rise to different moduli for different fibrils made by the same peptide or protein. However, here we measure variations consistently within crystals in the range of 1.3–2.0 GPa, which indicates that there is variability in the modulus within the same assembly (Fig. 5). Although the range of variation is not very large, it is consistent with the SMD results. Combined with the SMD simulations, these findings suggest that this anisotropy emerges from differences in the structure of the different interfaces between filaments in the crystals. A very dense H-bond network connects layers of beta-strands in the direction of the fibril axis, whereas lower H-bond densities combine with rather weak van der Waals interactions to hold interfaces connecting filaments in the radial direction. The fact that mechanical anisotropy is present in a system as simple as the GNNQQNY amyloid crystals (only 7 residues per strand) suggests that larger and more diverse systems (e.g. fibrils associated with diseases) are likely to display mechanical anisotropy as well. Of particular interest in the disease context is whether features associated with anisotropy, especially the low radial strength, impact fibril fission and propagation. In fact, breakage of fibrils along their axis produces new seeds to recruit monomers and enhance polymerization, but it is not fully clear how radial breakage and separation of filaments affects the propagation of different fibril species.11,27


Page 14 of 22

Page 15 of 22 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


Figure 5. Mechanical heterogeneities within single GNNQQNY crystals. Crystals were adsorbed on mica surface and imaged using AM-FM AFM in air. (A) From top to bottom, height and Young’s modulus maps, respectively. Black square: zoomed-in area represented in (B). (B) Enlargement of images in (A). Black line: pixels selected for section analysis in (C). (C) Section analyzes of images in (B), with ∆Ymax = 0.8 GPa along the black line.

CONCLUSIONS In this paper, we have combined AM-FM atomic force microscopy with full-atom SMD simulations to study mechanical anisotropy in GNNQQNY amyloid crystals. We demonstrated that their axial and radial stiffness and strength differ significantly. Furthermore, SMD simulations predicted that radial elastic moduli within GNNQQNY amyloid crystals vary by a factor of 2. Direct measurement of radial moduli by AM-FM AFM confirmed this prediction. In addition, AFM experiments showed that mechanical anisotropy is present at the level of a single crystal, in which filaments sitting next to each 15 ACS Paragon Plus Environment


other have different radial Young’s moduli. Detailed analysis of the in silico deformation process revealed that the source of different radial stiffness is the presence of distinct interfaces. Calculations of interaction energies indicated that the wet interface, which holds filaments together via loose van der Waals interactions, is the “weakest link” in the crystal, while the other radial interfaces (YG and dry) display better resistance to deformation due to a combination of higher H-bond density and stronger van der Waals interactions. Given the cross-beta characteristic common to all amyloids, it is likely that many amyloids display mechanical anisotropy. However, further experiments are needed to prove this conjecture and investigate the impact of mechanical anisotropy on fibril formation and growth in the biological context.

MATERIALS AND METHODS Preparation of amyloid models. The crystal structure formed by the peptide GNNQQNY was obtained from the online fibril structure repository of Sawaya’s group.28 The structure files for the simulation were prepared using VMD as follows. In order to have the same number of filaments along the sides of the crystal, the ones on the far ends in the initial structure were removed, leaving a nine-filament structure comprised of three filaments along each side-wall; i.e. a 3 x 3 filament crystal model (Fig. 1). The crystal was elongated to ten layers along the fibril-axis direction by duplication and 4.8 Å translations of existing layers. Equilibration of amyloid models. Molecular dynamics simulations were carried out in NAMD 29 version 2.10 using the CHARMM22 all-hydrogen force field with CMAP correction.30 The simulations were run on a 2 GHz Genuine Intel(R) Xeon(R) CPU using CUDA extension running on a NVIDIA GeForce GTX 780 graphics card. Non-bonded interaction cutoff distance of 12 Å was used and the Particle Mesh Ewald method for long-range electrostatics calculation. The fibril structure was solvated in TIP3P water box with 25 Å buffer distance on each side. The system was minimized for 40,000 steps and heated to 300 K in 30 ps. The SHAKE algorithm allowed for 2 fs integration time step by fixing the covalent bonds 16 ACS Paragon Plus Environment

Page 16 of 22

Page 17 of 22 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


involving hydrogen atoms. Langevin dynamics and a Nosé-Hoover barostat were used to maintain the temperature at 300 K and the pressure at 1 bar, respectively. The system first underwent NVT equilibration for 100 ps with backbone positional restraints followed by a NPT equilibration for 120 ps where positional restraints were gradually released. The fibril was then simulated with no restraints for 10 ns (production run) under NPT conditions. To elucidate the stability of the structure used for SMD, we computed the backbone RMSD of the frames in the production MD to our model (Fig. 2A), and also to the starting model obtained from the literature, i.e. that before any minimization or equilibration in the force field. In this case, the RMSD shows a plateau around 1.2 Å, confirming the similarity between the starting model and that of the MD (plateau around 0.95 Å). Steered molecular dynamics. Frames from the production run were extracted at 6, 7, 8, 9 and 10 ns for the SMD simulations. The SMD simulations consisted of compressing the crystal along each of the three directions: axial (along the fibril axis) and two radial directions (perpendicular to the fibril axis). Deformation and breakage in the crystal along each direction was performed using constant velocity (0.025 Å.ps-1) compression where one side wall was held constrained while the other at the opposite end was pushed in towards the crystal along the direction of interest. We chose compression over pulling mode because compression is more consistent with the AFM measurements of amyloid crystals elasticity that were done through indentation. A spring constant of 5000 KJ.mol-1.nm-2 was used, and the force in the compression simulations was recorded and used for computing the mechanical properties. The stress was obtained from the force values after dividing by the cross-sectional area of the interface along the specific direction, and the engineering strain was calculated at each step as s = (l – l0)/l0, where l0 was the initial crystal length and l the instantaneous length during the SMD run. The ultimate strength was obtained from the peak of the stress-strain curve, and the slope provided an estimate of the Young’s (elastic) modulus. The values reported are averages from multiple SMD runs with the errors corresponding to the standard deviation.



Analysis of molecular interactions across the interfaces. Frames from the last 5 ns of the production run were analyzed in VMD to compute VdW energies and the number of hydrogen bonds between the layers using the cutoff values of 30° for donor-hydrogen-acceptor angle and 3.5 Å for H-bond donoracceptor distance. To obtain densities (nm-2), the number of H-bonds and the vdW energy values were normalized by the area of each interface that experienced compression, which took into account the different cross-sections of these interfaces. Each loading direction (Axial, Radial-1, and Radial-2) corresponded to a specific interface (interlayer, dry/wet, and YG, respectively; see Fig. 1). Due to the existence of dry and wet interfaces in the Radial-1 direction of compression, it was necessary to distinguish between the two when calculating interaction energies. Rosetta’s residue-energy-breakdown module was employed to compute energies of H-bond interactions between segment pairs across interfaces, where one segment referred to a single GNNQQNY peptide.31 VdW energies were also calculated using the Lennard-Jones term in Rosetta’s energy function. H-bond and/or vdW interaction energies between segment pairs belonging to each specific interface were summed to provide the total interaction energy per interface, which was then normalized by the cross-sectional area of that interface. Preparation of amyloid crystals. GNNQQNY peptide was purchased from Sigma. Fibrils were obtained by immersion of the peptide in deionized water adjusted to pH 2.0 with HCl and at a concentration of 10 mg/mL for 4 days at room temperature. Reaction was quenched by cooling the solution to 4°C. Crystals were made by sonicating the preformed fibrils for 20 min using a Qsonica Q500 sonicator operated with a 2 mm-diameter microtip whose vibrational amplitude and frequency were 25% and 20 kHz, respectively. 5 s on-3 s off pulse cycle and a cooling system were used to make sure the fibril/crystal solution remained at a temperature lower than 15°C during the whole sonication process. Transmission electron microscopy. Amyloid crystals were stained with uranyl formate and adsorbed to glow discharged carbon-coated copper grids as previously described.14,32 Specimens were examined using a Tecnai Spirit transmission electron microscope (FEI) equipped with a LaB6 filament and operated at an


Page 18 of 22

Page 19 of 22 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


accelerating voltage of 120 kV. Images were acquired at a nominal magnification of 49,000× on a 4K × 4K Eagle charge-coupled device (CCD) camera (FEI). Atomic-force microscopy. Amyloid crystals were adsorbed on mica and imaged by a Cypher AFM (Asylum Research) operated in AM-FM-mode using silicon nitride tips (AC160TS from Olympus; nominal spring constant: 40 N/m). Amplitude modulation-frequency modulation (AM-FM) mode is a recent bimodal technique in which the first vibrational mode (AM) provides topography measurement (analogous to standard tapping mode) and the second vibrational mode simultaneously provides a direct measurement of the Young’s modulus of elasticity via frequency shifts.14,22 Note that the AM-FM nanomechanical tool also allows for the acquisition of loss tangent and indentation depth for each and every pixel of the image simultaneously with the measurement of topography and elasticity.22,23 In AMFM AFM experiments, we used not only mica, but also a polymeric blend of polystyrene and low-density polyethylene (PS-LDPE) as a substrate, because the stiffnesses of PS (~2.2 GPa) and LDPE (~0.2 GPa) are comparable to that of amyloid (i.e. 0.1−5 GPa). Hence PS-LDPE is used not only as a substrate but also to calibrate the frequency shifts of the second resonance to proper moduli values (set to 2.2 and 0.2 GPa for PS and LDPE, respectively).

AUTHOR CONTRIBUTIONS R.N., J.G. and G.L. designed the study. R.N., E.W., J.M.B., C.K.Y. and G.L. performed the experiments. R.N., H.L., J.G. and G.L. analyzed data. R.N., J.G. and G.L. wrote the paper.

ACKNOWLEDGMENTS The authors acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR).



REFERENCES (1)

Knowles, T. P. J.; Vendruscolo, M.; Dobson, C. M. The Amyloid State and Its Association with Protein Misfolding Diseases. Nat. Rev. Mol. Cell Biol. 2014, 15, 384-396.

(2)

Nelson, R.; Sawaya, M. R.; Balbirnie, M.; Madsen, A. O.; Riekel, C.; Grothe, R.; Eisenberg, D. Structure of the Cross-Β Spine of Amyloid-Like Fibrils. Nature 2005, 435, 773-778.

(3)

Lewandowski, J. R.; van der Wel, P. C. A.; Rigney, M.; Grigorieff, N.; Griffin, R. G. Structural Complexity of a Composite Amyloid Fibril. J. Am. Chem. Soc. 2011, 133, 14686-14698.

(4)

Marshall, K. E.; Hicks, M. R.; Williams, T. L.; Hoffmann, S. V.; Rodger, A.; Dafforn, T. R.; Serpell, L. C. Characterizing the Assembly of the Sup35 Yeast Prion Fragment, Gnnqqny: Structural Changes Accompany a Fiber-to-Crystal Switch. Biophys. J. 2010, 98, 330-338.

(5)

Knowles, T. P. J.; De Simone, A.; Fitzpatrick, A. W.; Baldwin, A.; Meehan, S.; Rajah, L.; Vendruscolo, M.; Welland, M. E.; Dobson, C. M.; Terentjev, E. M. Twisting Transition between Crystalline and Fibrillar Phases of Aggregated Peptides. Phys. Rev. Lett. 2012, 109.

(6)

Reynolds, N. P.; Adamcik, J.; Berryman, J. T.; Handschin, S.; Zanjani, A. A. H.; Li, W.; Liu, K.; Zhang, A.; Mezzenga, R. Competition between Crystal and Fibril Formation in Molecular Mutations of Amyloidogenic Peptides. Nat. Commun. 2017, 8, 1338.

(7)

Das, S.; Zhou, K.; Ghosh, D.; Jha, N. N.; Singh, P. K.; Jacob, R. S.; Bernard, C. C.; Finkelstein, D. I.; Forsythe, J. S.; Maji, S. K. Implantable Amyloid Hydrogels for Promoting Stem Cell Differentiation to Neurons. Npg Asia Materials 2016, 8.

(8)

Knowles, T. P. J.; Mezzenga, R. Amyloid Fibrils as Building Blocks for Natural and Artificial Functional Materials. Adv. Mater. 2016, 28, 6546-6561.

(9)

Shen, Y.; Posavec, L.; Bolisetty, S.; Hilty, F. M.; Nystrom, G.; Kohlbrecher, J.; Hilbe, M.; Rossi, A.; Baumgartner, J.; Zimmermann, M. B.; Mezzenga, R. Amyloid Fibril Systems Reduce, Stabilize and Deliver Bioavailable Nanosized Iron. Nature Nanotechnol. 2017, 12, 642-+.

(10)

Knowles, T. P. J.; Buehler, M. J. Nanomechanics of Functional and Pathological Amyloid Materials. Nature Nanotechnol. 2011, 6, 469-479.

(11)

Knowles, T. P. J.; Waudby, C. A.; Devlin, G. L.; Cohen, S. I. A.; Aguzzi, A.; Vendruscolo, M.; Terentjev, E. M.; Welland, M. E.; Dobson, C. M. An Analytical Solution to the Kinetics of Breakable Filament Assembly. Science 2009, 326, 1533-1537.

(12)

Knowles, T. P.; Fitzpatrick, A. W.; Meehan, S.; Mott, H. R.; Vendruscolo, M.; Dobson, C. M.; Welland, M. E. Role of Intermolecular Forces in Defining Material Properties of Protein Nanofibrils. Science 2007, 318, 1900-1903.

(13)

Lamour, G.; Nassar, R.; Chan, P. H. W.; Bozkurt, G.; Li, J.; Bui, J. M.; Yip, C. K.; Mayor, T.; Li, H.; Wu, H.; Gsponer, J. Mapping the Broad Structural and Mechanical Properties of Amyloid Fibrils. Biophys. J. 2017, 112, 584-594.


Page 20 of 22

Page 21 of 22 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


(14)

Lamour, G.; Yip, C. K.; Li, H.; Gsponer, J. High Intrinsic Mechanical Flexibility of Mouse Prion Nanofibrils Revealed by Measurements of Axial and Radial Young's Moduli. ACS Nano 2014, 8, 3851-3861.

(15)

Ruggeri, F. S.; Adamcik, J.; Jeong, J. S.; Lashuel, H. A.; Mezzenga, R.; Dietler, G. Influence of the Beta-Sheet Content on the Mechanical Properties of Aggregates During Amyloid Fibrillization. Angew. Chem. Int. Ed. Engl. 2015, 54, 2462-2466.

(16)

Guo, S.; Akhremitchev, B. B. Packing Density and Structural Heterogeneity of Insulin Amyloid Fibrils Measured by Afm Nanoindentation. Biomacromolecules 2006, 7, 1630-1636.

(17)

Adamcik, J.; Lara, C.; Usov, I.; Jeong, J. S.; Ruggeri, F. S.; Dietler, G.; Lashuel, H. A.; Hamley, I. W.; Mezzenga, R. Measurement of Intrinsic Properties of Amyloid Fibrils by the Peak Force Qnm Method. Nanoscale 2012, 4, 4426-4429.

(18)

Solar, M.; Buehler, M. J. Tensile Deformation and Failure of Amyloid and Amyloid-Like Protein Fibrils. Nanotechnology 2014, 25, 105703.

(19)

Li, Y. D.; Lamour, G.; Gsponer, J.; Zheng, P.; Li, H. The Molecular Mechanism Underlying Mechanical Anisotropy of the Protein Gb1. Biophys. J. 2012, 103, 2361-2368.

(20)

Fitzpatrick, A. W. P.; Vanacore, G. M.; Zewail, A. H. Nanomechanics and Intermolecular Forces of Amyloid Revealed by Four-Dimensional Electron Microscopy. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 3380-3385.

(21)

Fitzpatrick, A. W. P.; Park, S. T.; Zewail, A. H. Exceptional Rigidity and Biomechanics of Amyloid Revealed by 4d Electron Microscopy. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 1097610981.

(22)

Labuda, A.; Kocun, M.; Meinhold, W.; Walters, D.; Proksch, R. Generalized Hertz Model for Bimodal Nanomechanical Mapping. Beilstein J. Nanotechnol. 2016, 7, 970-982.

(23)

Proksch, R.; Kocun, M.; Hurley, D.; Viani, M.; Labuda, A.; Meinhold, W.; Bemis, J. Practical Loss Tangent Imaging with Amplitude-Modulated Atomic Force Microscopy. J. Appl. Phys. 2016, 119.

(24)

Clifford, C. A.; Seah, M. P. Modelling of Nanomechanical Nanoindentation Measurements Using an Afm or Nanoindenter for Compliant Layers on Stiffer Substrates. Nanotechnology 2006, 17, 5283-5292.

(25)

Paul, T. J.; Hoffmann, Z.; Wang, C.; Shanmugasundaram, M.; DeJoannis, J.; Shekhtman, A.; Lednev, I. K.; Yadavalli, V. K.; Prabhakar, R. Structural and Mechanical Properties of Amyloid Beta Fibrils: A Combined Experimental and Theoretical Approach. J. Phys. Chem. Lett. 2016, 7, 2758-2764.

(26)

Xu, Z.; Paparcone, R.; Buehler, M. J. Alzheimer's Abeta(1-40) Amyloid Fibrils Feature SizeDependent Mechanical Properties. Biophys. J. 2010, 98, 2053-2062.

(27)

Mizuno, N.; Baxa, U.; Steven, A. C. Structural Dependence of Het-S Amyloid Fibril Infectivity Assessed by Cryoelectron Microscopy. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 3252-3257.



(28)

Sawaya, M. R. Cross Beta Crystal Structures. http://people.mbi.ucla.edu/sawaya/jmol/xtalpept/index.html.

(29)

Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kale, L.; Schulten, K. Scalable Molecular Dynamics with Namd. J. Comput. Chem. 2005, 26, 1781-1802.

(30)

Mackerell, A. D., Jr.; Feig, M.; Brooks, C. L., 3rd. Extending the Treatment of Backbone Energetics in Protein Force Fields: Limitations of Gas-Phase Quantum Mechanics in Reproducing Protein Conformational Distributions in Molecular Dynamics Simulations. J. Comput. Chem. 2004, 25, 1400-1415.

(31)

O'Meara, M. J.; Leaver-Fay, A.; Tyka, M. D.; Stein, A.; Houlihan, K.; DiMaio, F.; Bradley, P.; Kortemme, T.; Baker, D.; Snoeyink, J.; Kuhlman, B. Combined Covalent-Electrostatic Model of Hydrogen Bonding Improves Structure Prediction with Rosetta. J. Chem. Theory Comput. 2015, 11, 609-622.

(32)

Ohi, M.; Li, Y.; Cheng, Y.; Walz, T. Negative Staining and Image Classification - Powerful Tools in Modern Electron Microscopy. Biol. Proced. Online 2004, 6, 23-34.


Page 22 of 22

Mechanical Anisotropy in GNNQQNY Amyloid Crystals

Recommend Documents