In-situ Observation of Amyloid Nucleation and Fibrillation by Fast-Scan

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Surfaces, Interfaces, and Catalysis; Physical Properties of Nanomaterials and Materials

In-situ Observation of Amyloid Nucleation and Fibrillation by Fast-Scan Atomic Force Microscopy Qunxing Huang, Huayi Wang, Houqian Gao, Peng Cheng, Ling Zhu, Chen Wang, and Yanlian Yang J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b03143 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 16, 2018

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The Journal of Physical Chemistry Letters

In-situ Observation of Amyloid Nucleation and Fibrillation by FastScan Atomic Force Microscopy

Qunxing Huang, † Huayi Wang, † Houqian Gao, † Peng Cheng, ‡ Ling Zhu, † * Chen Wang, † * Yanlian Yang, † * †CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Key Laboratory of Biological Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China ‡State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing 100084, China

ABSTRACT:

Amyloidogenic proteins are key components in various Amyloid diseases. The aggregation process and

the local structural changes of the toxic species from toxic oligomers to protofibrils and subsequently to mature fibrils are crucial for understanding the molecular mechanism of amyloidgenic process and also for developing treatment strategy. Exploration on amyloid aggregation dynamics in situ in real liquid condition is feasible for reflection of the whole process with biological correlations. Herein we report the in-situ dynamic study and structure exploration of Amylin1-37 aggregation by FastScan atomic force microscopy (AFM). Amylin1-37 nucleation process was observed in which smaller oligomers or monomers were assimilated by the surrounding big oligomers. Amylin1-37 protofibril aggregation was positively correlated with monomer concentration, while no direct relationship was observed between fibril elongation and monomer concentration. Growing end and passivated end were found during Amylin1-37 fibrillation. In the assembly process, the growing end kept its structure, and its stiffness was lower than the aggregate body, while the passivated end might experience re-arrangements of β-structures, which eventually enabled fibril growth from this end. This work is beneficial to the insights of amyloid fibrillation, and may shed light on the development of drugs targeting the specific phase of amyloid aggregation.

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Molecular Dynamics (MD) simulations on Aβ1-4041 and Amylin1-3721 showed that amyloid monomers aggregated into crystallized oligomers, and monomers packed orderly onto the pre-formed oligomers to form protofibrils with β-sheet structures.9, 30 Seo et al. reported oligomers of amyloid peptide segments contained a significant amount of β-structures, by combining ion mobility spectrometry–mass spectrometry (IMS-MS) and gas-phase infrared spectroscopy (IR).42 Fink et al. revealed that structural changes began from oligomers to protofibrils by means of Nuclear Magnetic Resonance (NMR).28, 43 While Caflisch et al. regarded oligomers to be grossly amorphous, oligomers collide and cohere together by mainly Van De Valls force to form protofibrils..29 Furthermore, Teplow et al. studied Aβ1-40 and Aβ1-42 protofibril elongation patterns, and found that these periods were grossly amorphous, and concluded the process as bead-on-a-string morphology.5 Similarly, Massimo et al. observed that RADA 16-I fibrils elongate via an “end-to-end aggregation mechanism” at pH 2.0-4.5.44 To explain dynamic process of aggregation, Knowles et al. provided a unified framework which contained a set of coupled kinetic equations. Based on previous studies of amyloid aggregation, visual detection of the process will be of great help. 45

INTRODUCTION

Amyloid degenerative diseases are characterized of extracellular and/or intracellular aggregation of amyloid proteins. Representative amyloid degenerative diseases include Alzheimer's disease, type-Ⅱ diabetes, mad cow disease and Göttingen syndrome.1-3 These pathogenic proteins are closely related to misfolding of amyloid peptides which are derived from human body.1, 4 In vivo, amyloid aggregates assemble from the soluble monomers to the oligomers, then further to the formation of protofibrils and fibrils, finally mature fibrils deposit in the islet tissues to form amyloid plaques.4-7 Different cytotoxicity were found in all stages of assemblies, previous studies have shown that, soluble amyloid oligomers are the most toxic ones by disrupting the cell membrane, and penetrating cells.1, 4, 8-10 Research on structures of amyloid aggregates is key to decipher the pathogenesis of amyloid degenerative diseases. Amyloid oligomers were considered the first detectable stage, and more and more evidences showed the existence of β-structures. Infrared vibration spectrum showed that Aβ1-42 oligomers were composed of loosely aggregated strands.11 Eisenberg et al. obtained X-ray–derived atomic structure of Aβ segment and explained it as a cylindrical barrel, formed from six antiparallel protein strands.9 Amyloid protofibrils were viewed to be featured with secondary structures.12 Clore et al. used solution NMR techniques to identify that hydrophobic region of Aβ1-40 and Aβ1-42 were in direct contact with protofibril.13 Amyloid fibrils are believed to be polypeptide aggregates with a core structure consisting of β-sheets whose strands are perpendicular to the fibril axis, and the backbone hydrogen bonds are parallel to it.2, 4, 14-24 Cryo-electron microscopy (cryo-EM) revealed “LS”-shaped topography of subunit in Aβ1-42 fibril,25 and combined “cross beta/beta helix” structure of tau filaments.26 When amyloid aggregates grow from oligomers to fibrils, the beta contents increase simultaneously.11 While so far, the important questions about the formation kinetics of the early ordered aggregates and the local structural transition still remain unanswered.

The patterns of protofibril and fibril elongation were once believed to be in accordance with Symmetrical structural model46, which was supposed that monomers locked and docked in aggregates end to form β-sheets in elongation process, and the elongation pattern of the two ends should be identical. Based on Symmetrical Structural Model, Aebi and coworkers revealed similar growing velocities in two ends of Amylin1-37 protofibrils,47 and elongation velocity had a linear relationship with monomer concentrations.33 In recent years, this idea was partially updated. Dong et al. observed that glucagon protofibrils could form three different interwoven fibrils in amyloid fibrillation by AFM.48 Yamada et al. observed fast end and slow end in Aβ1-42 fibrillation using high-speed Atomic Force Microscopy (HSAFM).38 Cryo-EM studies of Aβ1-42 revealed different fibril ends.25, 49 After all, the dynamics of amyloid aggregation is still unclear. Even though static characterizations, such as solid state NMR and cryo-EM, have high resolution, it is better to explore aggregation dynamics in liquid phase, because water takes part in practical fibril formation.7, 18, 32, 50 Hence a method to combine morphology and structure characterization in real-time under liquid circumstance is

Many efforts have been made to understand the dynamics of the amyloid aggregation process from oligomers to protofibrils and fibrils in the recent 20 years. Previous work pointed out that amyloid aggregation began with structured oligomers.5-7, 14, 16, 18, 21, 24, 27-40 2


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The Journal of Physical Chemistry Letters seconds. The images consist of 512 ×512 pixels. Data collected by Dimension Fast-Scan AFM were handled by Nanoscope Analysis. Height distributions of peptide oligomers were analyzed by Depth. The individual fibril length was revised by tip radius deconvolution. The fibrils were computationally straightened. The growing positions of each aggregate end were marked, and the modulus data of every pixel in the region of growing ends were collected. Positions of the passivated ends were determined by the positions of their original oligomers, and the modulus data were collected in the same way. The lengths of individual fibril were recorded by time. Surface coverage was analyzed by using Nanoscope particle analysis. The AFM images were processed by Nanoscope lowpass filtering to reduce the noise from high frequency before the analysis of surface coverage. Data collected by Asylum Cypher were handled by Gwyddion in same way as Nanoscope Analysis.

essential to decipher both dynamics and structural changes in amyloid aggregating periods. In the present work, Amylin1-37, a peptide associated with type Ⅱ diabetes, is chosen as a model amyloid peptide. In-situ monitoring of the nucleation and growth kinetics from oligomer to proto-fibrillation and fibrillation of Amylin1-37 is performed by FastScan AFM combined with quantitative nanomechanics (QNM) measurement at water/mica interface. By real-time scanning, we not only obtained the growth rate, morphology and directionality, but also the modulus changes of each protofibrils and fibrils. The different growth rate and the structural differences among the growing end, the passivated end, and the fibril body lead to the understanding of the aggregation dynamics by in situ visualization. Moreover, statistics gave details of how Amylin1-37 nucleated and in which way protofibrils grew into fibrils. Statistical analysis of modulus provided evidence of when β-structure changes took place in amyloid aggregation and how it could influence the growth of Amylin1-37.

Force measurements and data analysis The force curves of polypeptide fibrillation in liquid phase were obtained by Dimension FastScan AFM (Bruker, MA, US) at Peakforce QNM mode with FASTSCAN-D cantilevers (Bruker, CA). Deflection sensitivity, spring constant and tip radius of FASTSCAN-D cantilevers were calculated on sapphire reference sample in MilliQ water in all the experiments. The deflection sensitivities were 29.25-39.67 nm/V, the spring constants were 0.07-0.12 N/m measured by thermal method and the calibrated tip radius were 5.7-9.5 nm. We chose peptides in different locations on the surface to guarantee the reliability. The temperature of experiments was 20℃. The noise threshold was set by 0.1 nm during scanning. All the recorded AFM images consist of 512 ×512 pixels and several images were obtained at separate locations across the mica surfaces to ensure a high degree of reproducibility of the recorded molecular nanostructures. The data were handled by Nanoscope Analysis. The individual fibril topography was calibrated by tip radius deconvolution, the modulus of different fibril regions were recorded by time.

MATERIALS AND METHODS Preparation of Amylin1-37 Amylin1-37(KCNTATCATQRLANFLVHSSNNFGAILSSTNV GSNTYN-H2) was bought from Shanghai Science Peptide Biological Technology Co., LTD. The purity of the polypeptide (95%) was verified by high-performance liquid chromatography (HPLC) and mass spectrometry analysis. Lyophilized powder of Amylin1-37 was dispersed in hexafluoroisopropanol (HFIP) for 8 hours and HFIP was then evaporated under the condition of nitrogen gas. After that, the peptide was dissolved in MilliQ water to a series of concentrations (2 μM, 5 μM, 10 μM, 20 μM, 30 μM, 50 μM) prior to AFM imaging. In-situ AFM Imaging and data analysis The freshly prepared Amylin1-37 peptide solution was deposited onto freshly cleaved mica. The nucleation process of Amylin1-37 was observed by Dimension FastScan AFM (Bruker, MA, US) in liquid using FASTSCAN-C cantilevers (Bruker, CA) with a rated 300 kHz resonate frequency with 0.8 N/m force constant. The temperature of experiments was 30℃. The noise threshold was set to 0.1 nm during scanning. The temperature of experiments was 30℃. We chose peptides in different locations on the surface to guarantee the reliability. The morphology changes of Amylin1-37 during the nucleation process were captured by collecting AFM images every 80 seconds. Images were obtained at separate locations across the mica surfaces to ensure a high degree of reproducibility of the recorded molecular nanostructures. All the recorded AFM images consist of 512 ×512 pixels. In-situ scanning of 50 μM solution was obtained by Asylum Cypher using a cantilever with the resonate frequency of 110 kHz and the spring constant 0.25 N/m. Images were acquired every 10

RESULTS AND DISCUSSION Amylin1-37 nucleation in water We first investigated the nucleation process of Amylin1-37 by in-situ AFM imaging. The adsorption and aggregation of amyloid peptides have been previously studied on both hydrophilic and hydrophobic surfaces.51-56 Here we took the hydrophilic mica surface as a model system to study the nucleation and fibrillation of Amylin1-37. Freshly prepared Amylin1-37 monomer solution was deposited onto freshly cleaved mica at the concentration of 5 μM and was imaged by AFM at 30℃ immediately. To check if the observations at 30℃ could represent the self-assembly process of Amylin1-37 at the physiological condition, we compared the aggregation of Amylin1-37 on the water/mica interface at different temperatures. Freshly prepared 3



Amylin1-37 solution was deposited onto freshly cleaved mica and was incubated for 10 minutes at different temperatures (10 ℃, 20 ℃ , 30 ℃ , 37 ℃ ) before being dried and characterized by AFM in air. We found that fibrillation could be observed above 20 ℃ . Though increased temperature could accelerate the aggregation process of Amylin1-37, the morphologies of amylin fibrils at 30 ℃ and 37 ℃ were similar (Figure S1), suggesting that our observation by real-time AFM imaging at 30 ℃ was similar to the one at the physiological temperature. The small amount of impurities in the synthesized peptides was believed to have little effect on the aggregation process of Amylin1-37 57 as modulating of the self-assembly of amyloid peptides normally required short peptides with specific sequences at certain concentrations and their binding to certain regions of the amyloid peptides.58-60 At 30 seconds after sample deposition, we found that the surface was covered by a layer of small particles well-distributed on the surface (Figure 1a), indicating the adsorption of Amylin1-37 on the surface. Considering the small size and the short incubation time after sample preparation, these particles are believed to be the individual monomers of Amylin1-37.17, 20-21, 61 At 270 seconds after sample deposition, we observed that the amount of the small particles decreased while larger particles began to appear on the surface (Figure 1b), indicating the aggregation of Amylin1-37. This nucleation process was faster than the reported aggregation process of amylin.62-64 This might be due to the acceleration of the self-assembling process by tip agitation,65-67 and the surface-induced catalyzing of amyloid aggregation.68, 69 We then analyzed the size distribution of the particles on the surface. The heights of Amylin1-37 aggregates which approximately equal to their diameters9 were measured. We found that at 30 seconds after sample deposition, the particles were relatively homogenous with an average diameter of 1.7 ± 0.2 nm, while at 270 seconds after sample deposition, the average diameter of the particles increased to size ranging from 1.7 nm to 2.9 nm and the size distribution of particles was significantly wider compared to the one at 30 seconds (Figure 1c). These data further demonstrated the accumulation of Amylin1-37 from monomers to oligomers. The wider distribution of aggregate diameters at 270 seconds could be attributed to the diversity of oligomer species as a result of the dynamic aggregation of amyloid peptides.29 We further calculated the density of the particles (oligomers and monomers) on the surface and found that the particle density reduced from 1.8 × 103 /μm2 at 30 seconds to 1.1 × 103 /μm2 at 270 seconds, indicating the vanish of monomers or small nuclei and the generation of larger oligomers. The newly generated aggregates might come from the addition of monomers or oligomers in the solution to the small nuclei, or the depletion of monomers or oligomers at the near vicinity of the nuclei.

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Figure 1. In-situ AFM imaging of Amylin1-37 nucleation. (a and b) AFM images of the nucleation of Amylin1-37 at 30 (a, z scale: 2 nm) and 270 (b, z scale: 3.5 nm) seconds after sample deposition. Inset: zoomed in AFM images of the red squares marked in the figures. Experimental temperature was 30℃. Freshly prepared Amylin1-37 solution was deposited onto freshly cleaved mica at the concentration of 5 μM. AFM imaging was performed immediately after sample deposition. Experimental temperature was 30℃. (c) Size distribution of different species of Amylin1-37 at different incubation time (black curve for 30 seconds and red curve for 270 seconds after sample deposition). The diameters of the oligomers were measured from AFM images (a and b). The percentage of the number of oligomer species with different diameters was calculated. (d) The density of different species of Amylin1-37 on the surface at different incubation time. The number of oligomers per μm2 was count from AFM images (a and b).

Amylin1-37 fibrillation in water We then investigated the fibrillation process of Amylin1-37 by in-situ AFM imaging. Freshly prepared Amylin1-37 at the concentration of 50 μM was deposited onto freshly cleaved mica and was imaged by AFM immediately. We found that after sample deposition, amyloid oligomers started to appear on the surface. The lengths of the aggregates increased with time until the oligomers grew into protofibrils and fibrils. Meanwhile, new aggregates continued to form and grow on the surface (Figure 2a-f, Video S1). We calculated the surface coverage of the aggregates, and found that the surface coverage increased dramatically (Figure S2, olive curve), indicating the self-assembly of Amylin1-37 on the surface. Then the surface coverage reached a plateau afterwards (Figure S2, olive curve). This might be due to the termination of Amylin1-37 fibrillation as a result of the exhaustion of monomers, or the overlap of aggregate species at different stages. This dynamic process was in accordance with the growth of amyloid fibrils as was previously described.7, 33 We also compared the changes of the surface coverage of 4


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The Journal of Physical Chemistry Letters the late stage of fibril growth, the passivated end began to grow (Figure 2f, inset, green arrow; Figure 2h, green frame). The phenomenon was contrary to the classical Symmetrical Structural Model45 or “fast/slow ends” patterns38 which suggest that the β-sheet structures make amyloid aggregates grow identically towards both ends, or the asymmetrical nucleus make the two ends grow in different rates. Our results implied that β-sheet structures were critical for the elongation process. The elongation of the passivated end at the late stage of fibril growth could be explained in the way that the aggregates in passivated ends eventually crossed the surface energy barrier and evolved into ordered β-sheet structures for fibril elongation.73

aggregates at different concentrations of Amylin1-37. We found that the surface coverage increased with the concentration of Amylin1-37 monomers deposited on the surface (Figure S2), indicating increased aggregation of Amylin1-37 with monomer concentration. We further measured the diameter of the aggregates, and found that in the first 40 seconds after detection, the diameter of aggregates increased rapidly by 2 to 3 folds (Figure 2g, red dotted frame), indicating that at the early stage of Amylin1-37 aggregation, diameter increase could not be restricted by the elongation of the protofibril. Monomers might absorb to protofibril bodies at any directions in this rapid growth period. At 40 seconds, the average diameter reached to around 6 nm, corresponding to the diameter of fibril with two-fold intertwining protofibrils.42, 70, 71 Then the average diameter reached to the first plateau period (Figure 2g, blue dotted frame), indicating that the aggregates had completed the formation of fibril. After this, the diameter increased again at the fibril stage, but the growth rate was much slower than that at the protofibril stage. This implied that the lateral association might take place. At 250 seconds, the growth of the average diameter reached the second plateau period (Figure 2g, green dotted frame). This might be because that the fibril contains three-fold intertwining protofibrils.58 Furthermore, we found that the two ends of Amylin1-37 protofibrils exhibited different growing modes. We took one position in the aggregate as the base point (indicated with pink dot in Figure 2a-f, inset), and measured the length of the fibril toward both ends. We observed that the aggregate elongated towards one direction (Figure 2a-f, indicated with red arrows in the inset, Figure 2h red curve), while kept static in the opposite direction (Figure 2a-f, indicated with blue arrows in the inset, Figure 2h blue curve). We explained this two-mode elongation in the way that one end of the nucleus surface was active for protofibril elongation while the opposite end might have imperfect lattice structure and low β-sheet content and was therefore inactivated. We define the active end as the “growing end” while the inactive end as the “passivated end”. In the growing end, the length of the aggregate increased rapidly to 150 nm at 40 seconds (indicated with purple frame Figure 2h) and then much slower afterwards (indicated with brown frame, Figure 2h). This was in accordance with the diameter growth of the aggregates showing that the diameter increased rapidly before 40 seconds, and then reached a plateau afterwards (Figure 2g). These evidences suggested that the fibril length of 150 nm at 40 seconds could be regarded as the dividing line between protofibrils and fibrils. The aggregation of Amylin1-37 was the at the protofibril stage before 40 seconds and was at the fibril stage after 40 seconds. This was in agreement with the previous MD studies suggesting that the length of 100-150 nm was the boundary to maintain the equilibrated structures of amyloid aggregates and to distinguish protofibrils from fibrils.2, 7, 16, 27-29, 41, 43, 47, 61, 72. What is interesting is that at

Figure 2. In-situ AFM imaging of Amylin1-37 fibrillation. (a-f) AFM images of the fibrillation of Amylin1-37. Freshly prepared Amylin1-37 solution (50 μM) was deposited onto freshly cleaved mica. AFM imaging was performed immediately after sample deposition. Images were captured at 0 (a), 20 (b), 60 (c), 100 (d), 190 (e) and 300 (f) seconds after AFM imaging, respectively. Z scale: 15 nm. Experimental temperature was 30℃. Inset: zoomed in AFM images of the blue squares marked in the figures. Scale bar, 50 nm. Red arrows represent the growing direction of the aggregates, blue arrows represent the passivated direction of the aggregates, green arrows represent the elongation of the passivated end in the late fibril growth stage. (g) The time-dependent growth of the diameter of Amylin1-37 fibril. Diameter growth of protofibril was shown in red dotted frame. Two distinguished plateau periods for growth curve of fibril diameter were shown by blue/green dotted frames, and highlighted by solid guidelines. (h) The time-dependent growth of the length of Amylin1-37 fibril at the growing and the passivated directions. Directions of the growing and passivated ends were shown by red and blue arrows respectively. In the growing end curve, stages of protofibril and fibril were marked with purple and brown frames respectively. The elongation stage in the passivated end is marked with green frame.

Amylin1-37 protofibril and fibril elongation analysis 5



After discriminating between the protofibril and fibril stages during the aggregation of Amylin1-37, we further explored the dynamics of Amylin1-37 fibrillation at the two stages. Amylin1-37 fibrillation at different concentrations was investigated by in-situ AFM imaging (Video S1-S6). We randomly chose 30 mature fibrils at each Amylin1-37 concentration, de-convoluted the image data, and measured the changes of the length and height of the aggregates from the protofibril stage to the fibril stage. We found that at the protofibril stage, the elongation speed increased significantly with Amylin1-37 monomer concentration (from 16 nm/min at 2 μM of Amylin1-37 to 220 nm/min at 50 μM of Amylin1-37, Figure 3a-f, red curve), and the elongation speed was linearly dependent on monomer density (Figure 3g, red curve). At the fibril stage, the elongation speed increased much slower than the one at the protofibril stage (from 40 nm/min at 2 μM of Amylin1-37 to 51 nm/min at 50 μM of Amylin1-37, Figure 3a-f, blue curve). The elongation speed was not directly dependent on peptide concentration, and it kept still when the peptide concentration was above 10 μM (Figure 3g, blue curve). Taken together, these results showed that Amylin1-37 aggregation exhibited different dynamic pathways in the protofibril and fibril stages. This is in agreement with the research by Knowles et al.45 showing a tentative model for the two different dynamic pathways in the protofibril and fibril stages was proposed for Amylin1-37 amyloidogenic aggregation. The elongation of the protofibrils could be a first-order kinetics in which the elongation speed was linearly correlated with monomer concentration. When there are excessive monomers in the system, the elongation speed of protofibrils increased with monomer concentration. On the contrary, it seems that the elongation speed of fibrils is independent on monomer concentration. Even when there are excessive monomers in the system, the elongation speed of fibrils maintains still. This could be attributed to a zero-order kinetics with a fixed elongation velocity of around 50 nm/min.

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Figure 3. Statistical analysis of the elongation velocities of Amylin1-37 protofibrils and fibrils. (a-f) Time-dependent length of Amylin1-37 protofibrils and fibrils at different concentrations of Amylin1-37:(a) 2 μM, (b) 5 μM ， (c) 10 μM ， (d) 20 μM, (e) 30 μM and (f) 50 μM. 0 independent fibrils at each Amylin1-37 monomer concentration were analyzed. Red lines stand for protofibril elongation and blue lines stand for fibril elongations. The slopes of the red and blue lines that stand for the elongation velocities of protofibrils and fibrils were labelled in the plots. (g) Concentration dependent elongation velocities of Amylin1-37 protofibrils and fibrils.

In-situ AFM QNM imaging of the structural changes of

Amylin1-37 during aggregation

To further explore the aggregation mechanism of Amylin1-37, we investigated the structure changes of Amylin1-37 during fibril elongation. High resolution 3D AFM image revealed that the morphology of the growing end was more curved than the fibril body (Figure 4a). Since the curved fibril segments have been reported to have less β-structure content,38 it could be inferred that the growing end lacked adequate β-sheet packing to maintain the rigid structure as was in the fibril body, and the aggregates experienced a transition from less ordered structure to ordered β-structure during fibril elongation. To explore this hypothesis, we tend to compare the β-sheet content between the growing end and the fibril body. Previous studies have proposed that the stiffness of the folded β-sheet is higher than that of the amorphous structure.32, 36, 74,75 Therefore, even though the two fibrils are similar in topography, their structure difference could be detected by AFM force measurements (Figure 4b, c)23, 32, 36, 42. Based on this hypothesis, we investigated the Derjaguin–Muller–Toporov (DMT) modulus 76 of the growing ends and the fibril bodies of Amylin1-37 using Bruker Peakforce QNM mode. 40 growing fibrils were tracked and the modulus of the growing ends and the fibril bodies were collected. Statistical analysis showed that the average modulus of the fibril bodies (26 ± 3 MPa) 6


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The Journal of Physical Chemistry Letters 11 (Figure 5d) minutes, respectively. We found that the overall modulous of the aggregates increased with time (Figure 5a-d). This was further confirmed by the zoom in time-lapse QNM images that showed increased modulous in each aggregate (Figure 5e, f). These results indicated increased ordered β-structure in the aggregates during the self-assembly process. Moreover, the modulous of the growing/passivated ends of the aggregates was also lower than the protofibril body (Figure 5e, f). We further tracked 30 fibrils in the continuously captured imagesduring the self-assembly process and recored the modulus of the growing/passivated ends and bodies of the aggregates. Statistical analysis showed that at the early stage (5 minutes) of the self-assembly process, the average modulus of the passivated ends was 18.5 MPa, lower than that of the growing ends (21.6 MPa) (Figure 5g, h). At the late stage of the self-assembly process, the average modulus of the passivated ends increased by 17% (21.2 MPa) (Figure 5g), while the growing ends maintained their stiffness (22.4 MPa) (Figure 5h). Structural changes also slightly took place in the fibril bodies, and the modulus increased from 26.6 MPa at 5 minutes to 28.6 MPa at 11 minutes. Those results indicated increased β-sheet content during the self-assembly process of Amylin1-37. During the fibrillation process, a number of passivated ends were found re-activated and growing (Figure 2f). It could be inferred that passivated ends were relatively amorphous in the early phase, and increased their β-structure content by local structural rearrangement. In the previous studies, cross-beta subunits were believed to be the foundation in proliferation of amyloid fibrils.23,24,34 Therefore, regularization of the fibril top ends might eventually cause a chance of turning into growing ends (Figure 2f). In the elongation period, growing ends were stable in structure, implying that they were continuous providers of β-sheet structures. Amylin1-37 monomers or oligomers assembled on the surface of growing end. The stiffness increase in the bodies indicated more β-structures in fibrils than protofibrils.

was slightly higher than that of the growing ends (22 ± 3 MPa) (Figure 4d), suggesting more ordered β-structure in the fibril bodies compared to the growing ends. We further performed paired comparison of the DMT modulus of the growing end and the fibril body in each fibril and found that the DMT modulus of fibril body was 16% higher than that of its growing end (Figure 4e), indicating higher stiffness of the fibril body than the growing end. These data suggested that the fibril body had more β-sheet content than the growing end, and a structure transition from less ordered structures in fibril growing end to more ordered β-structures in the fibril body took place during the elongation process. Amylin1-37 oligomers absorbed on the growing end and transformed from the amorphous aggregates to ordered β-structure. Closely packed Amylin1-37 molecules facilitated the decreased system energy to maintain the structural stability and mechanical strength.

Figure 4. Mechanical property of Amylin1-37 fibril. (a) High resolution 3D AFM image of Amylin1-37 fibril growing end (z scale: 10 nm). Inset: wide-field AFM image of Amylin1-37 fibrils. Freshly prepared Amylin1-37 solution (5 μM) was deposited onto freshly cleaved mica. (b and c) Schematic illustration of the relationship between the structure and the elastic modulus of fibril. Fibril with ordered β-sheet structure (b) reveals higher elastic modulus than that with amorphous structure (c). (d) Statistical data of DMT modulus on the growing end and the body of the fibril. (e) Paired comparison of the DMT modulus of fibril bodies and their growing ends. 40 growing fibrils were collected. The DMT modulus ratio of fibril body to growing end Amylin1-37 concentration was 5 μM. Experimental temperature was 20℃. AFM scan rate was 0.75 Hz. Z scale bar was 10 nm.

Previous studies showed that Young’ modulus was positively correlated with the content of β-sheet in the amyloid aggregates, and could be used to investigate the structural changes of the amyloid aggregates during the self-asembly process.77 We therefore used in-situ AFM QNM imaging to study the structural changes of Amylin1-37 during fibrillation.. Freshly prepared Amylin1-37 (10 μM) solution was quickly deposited onto freshly cleaved mica surface. QNM imaging was performed at 20 ℃ to slow down the aggregation process for a better capture of the force curves at every time point. DMT modulus mapping of the aggregation of Amylin1-37 was captured at 5 (Figure 5a), 7 (Figure 5b), 9 (Figure 5c) and

Figure 5. On-site modulus changes of passivated/ growing ends in

fibrillation. DMT modulus mapping of Amylin1-37 aggregates (10 μM) from protofibrils to fibrils were captured at 5 minutes (a), 7 minutes (b), 9 minutes (c) and 11 minutes (d) after sample deposition on mica. Experimental temperature was 20℃. (e) and (f) showed zoom in pictures of one growing aggregates, growing ends and passivated ends were represented by white arrows and blue arrows respectively. (g) The modulus increase of passivated end from protofibril to fibril.

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can be explained by molecular rearrangements and β-sheets optimization, which may be the bottleneck of growing velocity. This work could deepen our understanding of dynamic progress of amyloid aggregation, which may shed light on design of anti-Type II diabetes medicines. Investigation of Amylin1-37 aggregation on the biological surfaces or biomimetic surfaces, such as the charged lipid surfaces would provide insights into the interaction between the amyloid peptides and the cell membrane in the biological environment, and would therefore help to understand the mechanisms of amylin-induced toxicity. These studies would be included in our follow-up work.

(h) The slight modulus changes of growing end from protofibril to fibril.

Combined with deductions from previous experiments, a tentative model for amyloid fibrillation dynamics is proposed. Amylin1-37 grows from disperse monomers to oligomeric aggregates. After that, oligomers assemble together with the force of hydrogen bonds14-15, 43 and hydrophobic interactions14, 22. Then the most active regions of the aggregates may become the growing point (Figure 1). On one side, irregular structure and poor β-sheet crystallinity of oligomer nucleus (7.7 MPa on average) make the ends of early protofibrils passivated (Figure S3). In the meantime, re-arrangements of molecular chains have been in progress inside passivated ends and fiber backbones, which form more β-sheet and eventually lead to β-sheet crystalline fibers. The changes account for the activation of passivated ends in late period. On the other side, surface of growing ends undertake the responsibility for formation of β-sheet structures. Moreover, packing velocities in surface growing ends are positively correlated with monomer concentrations, while optimizations inside amyloid structures may be uncorrelated with monomer concentration (Figure 3).

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Authors * [email protected]; [email protected]; [email protected];

Funding Sources This work has been supported by the National Basic Research Program of China (2016YFF0203803) and the National Natural Science Foundation of China (No. 21773042, 21673055, 31600803).

CONCLUSIONS In this work, in-situ AFM characterization was performed Notes to explore the dynamic progress of amyloid fibrillation on The authors declare no competing financial interests. mica. A tentative model is developed based on the experimental results. In the initial phase of aggregation, ACKNOWLEDGMENT monomers develop into oligomeric nucleus. Afterwards, The authors thank Dr. Zhenwen Huang and Dr. Dengli Qiu one active region of oligomer begins to grow and forms from Surface Measurement Department in Bruker β-structures, while the opposite end becomes passivated Corporation for their support in FastScan AFM because of low β-sheet content. During fibrillation instrumentation. process, protofibrils experience local structural optimization inside of the aggregates; growing ends maintain their structure and keep increasing β-sheets; passivated ends have structure rearrangements, form more β-sheets, which eventually enable them to grow. The elongation of protofibrils and fibrils are different in dynamic pathways. Velocity of protofibril growing process depends on packing speed on growing end surface, hence elongation speed is positively correlated with monomer REFERENCE concentration. However, fibril elongation is not easily influenced by monomer concentration, that phenomenon 1. Uversky, V. N.; Oldfield, C. J.; Dunker, A. K., Dunker A K. Intrinsically disordered proteins in human diseases: introducing the D2 concept. Annu. Rev. Biophys. 2012, 37, 215-246. 2. Straub, J. E.; Thirumalai, D. Toward a molecular theory of early and late events in monomer to amyloid fibril formation. Annu. Rev. Phys. Chem. 2011, 62, 437-463. 3. Eisenberg, D.; Jucker, M. The amyloid state of proteins in human diseases. Cell 2012, 148, 1188-1203. 4. Per, W.; Christer, W.; Eric; David, W. H.; Timothy, D. O.; Kenneth, H. J. Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells. Proc. Nati. Acad. Sci. U.S.A. 1987, 84, 3881-3885. 5. B., G.; Marina, D. K.; Aleksey, L.; Sabrina, S. V.; George, B. B.; David, B. T. Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 330-335. 6. Caughey, B.; Lansbury, P. T. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu. Rev. Neurosci. 2003, 26, 267-298. 7. Cinar, G.; Ceylan, H.; Urel, M.; Erkal, T. S.; Deniz Tekin, E.; Tekinay, A. B.; Dana, A.; Guler, M. O. Amyloid inspired self-assembled peptide nanofibers. Biomacromolecules 2012, 13, 3377-87. 8


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In-situ AFM imaging of Amylin1-37 nucleation. (a and b) AFM images of the nucleation of Amylin1-37 at 30 (a, z scale: 2 nm) and 270 (b, z scale: 3.5 nm) seconds after sample deposition. Inset: zoomed in AFM images of the red squares marked in the figures. Experimental temperature was 30℃. Freshly prepared Amylin1-37 solution was deposited onto freshly cleaved mica at the concentration of 5 μM. AFM imaging was performed immediately after sample deposition. Experimental temperature was 30℃. (c) Size distribution of different species of Amylin1-37 at different incubation time (black curve for 30 seconds and red curve for 270 seconds after sample deposition). The diameters of the oligomers were measured from AFM images (a and b). The percentage of the number of oligomer species with different diameters was calculated. (d) The density of different species of Amylin1-37 on the surface at different incubation time. The number of oligomers per μm2 was count from AFM images (a and b).


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In-situ AFM imaging of Amylin1-37 fibrillation. (a-f) AFM images of the fibrillation of Amylin1-37. Freshly prepared Amylin1-37 solution (50 μM) was deposited onto freshly cleaved mica. AFM imaging was performed immediately after sample deposition. Images were captured at 0 (a), 20 (b), 60 (c), 100 s (d), 190 (e) and 300 (f) seconds after AFM imaging, respectively. Z scale: 15 nm. Experimental temperature was 30℃. Inset: zoomed in AFM images of the blue squares marked in the figures. Scale bar, 50 nm. Red arrows represent the growing direction of the aggregates, blue arrows represent the passivated direction of the aggregates, green arrows represent the elongation of the passivated end in the late fibril growth stage. (g) The time-dependent growth of the diameter of Amylin1-37 fibril. Diameter growth of protofibril was shown in red dotted frame. Two distinguished plateau periods for growth curve of fibril diameter were shown by blue/green dotted frames, and highlighted by solid guidelines. (h) The time-dependent growth of the length of Amylin1-37 fibril at the growing and the passivated directions. Directions of the growing and passivated ends were shown by red and blue arrows respectively. In the growing end curve, stages of protofibril and fibril were marked with purple and brown frames respectively. The elongation stage in the passivated end is marked with green frame.



Statistical analysis of the elongation velocities of Amylin1-37 protofibrils and fibrils. (a-f) Time-dependent length of Amylin1-37 protofibrils and fibrils at different concentrations of Amylin1-37:(a) 2 μM, (b) 5 μM，(c) 10 μM，(d) 20 μM, (e) 30 μM and (f) 50 μM. 0 independent fibrils at each Amylin1-37 monomer concentration were analyzed. Red lines stand for protofibril elongation and blue lines stand for fibril elongations. The slopes of the red and blue lines that stand for the elongation velocities of protofibrils and fibrils were labelled in the plots. (g) Concentration dependent elongation velocities of Amylin1-37 protofibrils and fibrils.


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Mechanical property of Amylin1-37 fibril. (a) High resolution 3D AFM image of Amylin1-37 fibril growing end (z scale: 10 nm). Inset: wide-field AFM image of Amylin1-37 fibrils. Freshly prepared Amylin1-37 solution (5 μM) was deposited onto freshly cleaved mica. (b and c) Schematic illustration of the relationship between the structure and the elastic modulus of fibril. Fibril with ordered β-sheet structure (b) reveals higher elastic modulus than that with amorphous structure (c). (d) Statistical data of DMT modulus on the growing end and the body of the fibril. (e) Paired comparison of the DMT modulus of fibril bodies and their growing ends. 40 growing fibrils were collected. The DMT modulus ratio of fibril body to growing end Amylin1-37 concentration was 5 μM. Experimental temperature was 20℃. AFM scan rate was 0.75 Hz. Z scale bar was 10 nm.



On-site modulus changes of passivated/ growing ends in fibrillation. DMT modulus mapping of Amylin1-37 aggregates (10 μM) from protofibrils to fibrils were captured at 5 minutes (a), 7 minutes (b), 9 minutes (c) and 11 minutes (d) after sample deposition on mica. Experimental temperature was 20℃. (e) and (f) showed zoom in pictures of one growing aggregates, growing ends and passivated ends were represented by white arrows and blue arrows respectively. (g) The modulus increase of passivated end from protofibril to fibril. (h) The slight modulus changes of growing end from protofibril to fibril.


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In-situ dynamic study and structure exploration of amyloid aggregation by FastScan atomic force microscopy (AFM) to reveal the amyloid nucleation and fibrillation pathway. 324x175mm (120 x 120 DPI)


In-situ Observation of Amyloid Nucleation and Fibrillation by Fast-Scan

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