Research at the Laboratory of Molecular Self-Assembly

Summary

Two complementary strategies can be employed in the fabrication of molecular biological materials. In the ‘top-down’ approach, biomaterials are generated by stripping down a complex entity into its component parts. This contrasts sharply with the ‘bottom-up’ approach, in which materials are assembled molecule by molecule and in some cases even atom by atom to produce novel supramolecular architectures. The latter approach is likely to become an integral part of nanomaterials manufacture and requires a deep understanding of individual molecular building blocks, their structures, assembling properties and dynamic behaviors. Two key elements in molecular fabrication are chemical complementarity and structural compatibility, both of which confer the weak and noncovalent interactions that bind building blocks together during self-assembly. Significant advances have been achieved at the interface of biology and materials science, including the fabrication of nanofiber materials for 3-D cell cultures, tissue engineering and regenerative medicine, the peptide detergents for stabilizing, and crystallizing membrane proteins as well as nanocoating molecular for cell organizations. Molecular fabrications of nanobiomateirals have fostered diverse scientific discoveries and technological innovations.

Fabricate various peptide materials. (a) The ionic self-complementary peptide has 16 amino acids, ~5 nm in size, with an alternating polar and nonpolar pattern. They form stable b-strand and b-sheet structures, thus the side chains partition into two sides, one polar and the other nonpolar. They undergo self-assembly to form nanofibers with the nonpolar residues inside (green) and + (blue) and – (red) charged residues form complementary ionic interactions, like a checkerboard. These nanofibers form interwoven matrices that further form a scaffold hydrogel with very high water content, >99.5% water. This is similar to agarose gel and other hydrogels. (b) A type of surfactant-like peptide, ~2 nm in size, which has a distinct head charged group, either positively charged or negatively charged, and a nonpolar tail consisting of six hydrophobic amino acids. They can self-assemble into nanotube and nanovesicles with a diameter of ~30–50 nm. These nanotubes go on to form an inter-connected network, which has similar been observed in carbon nanotubes. (c) Surface nanocoating peptide. This type of peptide has three distinct segments: a functional segment where it interacts with other proteins and cells; a linker segment that can not only be flexible or stiff, but also sets the distance from the surface, and an anchor for covalent attachment to the surface. These peptides can be used as ink for an inkjet printer to directly print on a surface, instantly creating any arbitrary pattern, as shown here. Neural cells from rat hippocampal tissue form defined patterns. (d) Molecular switch peptide. A type of peptides with strong dipoles that can undergo drastic conformation changes, between a-helix and b-strand/sheet, under external stimuli. It is conceivable that metal nanocrystals could be attached onto these dipolar peptides in order to fabricate them into tiny switches.

Self-assembling peptides form a three-dimensional scaffold woven from nanofibers ~10 nm in diameter. The scaffolds have been applied in several three-dimensional cell culture studies and in tissue engineering applications. (a) Rat hippocampal neurons form active nerve connections, each green dot represents a single synapsis. (b) Neural cells from rat hippocampal tissue slide migrate on the three-dimensional peptide scaffold. Cells on the polymer membrane (left) and on the peptide scaffold (right) are shown. Both glia cells (green) and neural progenitors (red) migrate into the three-dimensional peptide scaffold. (c) Brain damage repair in hamster. The peptide scaffold was injected into the optical nerve area of brain that was first severed with a knife. The cut was sealed by the migrating cells after two days. A great number of neurons form synapses. (d) Chondrocytes from young and adult bovine encapsulated in the peptide scaffold. These cells not only produce a large amount of glycosaminoglycans (purple) and type II collagen (yellow), characteristic materials found in the cartilage, but also a cartilage-like tissue in vitro. (e) Adult rat liver progenitor cells encapsulated in the peptide scaffold. The cells on the two-dimensional dish did not produce cytochrome P450 type enzymes (left-panel). However, cells in three-dimensional scaffolds exhibited cytochrome P450 activity (right panel).

PHOSPHOSERINE PEPTIDES MIMICKING PHOSPHOLIPID SURFACTANTS

Steve Yang and Shuguang Zhang

Surfactants have not only had an enormous impact on biological, chemical and physical research, but have also spurred diverse industries. It is important to continue the discovery and development of new surfactants. We designed two phospholipid inspired peptide surfactants that contain a phosphoserine moiety as a hydrophilic head and hydrophobic amino acid tails, pSAAAAAA (pSA6) and pSVVVVVV (pSV6). These phosphopeptide surfactants are able to undergo self-assembly in aqueous solution above certain critical concentrations and exhibit circular dichroism signals characteristic of random-coil or eta-sheet peptides according to the identity of the hydrophobic tail. Transmission electron microscopy revealed various nanostructures. This novel peptide surfactant system is not only versatile, easy to design and scale-up for diverse uses, but also may be useful as an innovative detergent material to stabilize membrane proteins. Since the general chemistry and structural properties of the peptide surfactant system reported here are similar to other zwitterionic molecules, small molecular detergents and phospholipids, it will be interesting to adopt the designed peptide surfactant system as a stabilization agent for purifying, stabilizing and crystallizing a broad range of functionally diverse membrane proteins.

Quick-freeze / deep-etch TEM images of pSA6 and pSV6. An overview of a region on the replica, showing the presence of tubular structures and vesicles. The red arrow points to a vesicle budding at the end of a tube. A magnified image showing the budding vesicles. An area where many budding vesicles can be discerned. TEM images of pSV6 at various magnifications.

A schematic drawing of the formation, expansion and dissociation of an internal bud observed in both pSA6 and pSV6. The red exterior represent the hydrophilic surface formed by the self-assembly of the surfactant peptides. On the left is a cut-away view, revealing the possible formation of a bilayer consisting of parallel arrangement of the peptides.

DYNAMIC NANOSTRUCTURES FROM A CATIONIC SELF-ASSEMBLING PEPTIDE SURFACTANT

Hongjing Qu, Peng Jiao, Hidenori Yokoi, Shuguang Zhang

We studied the dynamic molecular self-assembly behavior of the peptide surfactant A6K (AAAAAAK) that formed nanotubes and flat sheets in aqueous solution at as a function of pH changes. The flat sheets underwent rearrangement into nanotubes during the process of self-assembly over time. Simultaneously, the pH of the solution slowly increases and approaches the peptide A6K pI value. We studied the correlation between the dynamic nanostructural behaviors and the pH changes. These findings may not only have implications for the study of pH-dependent programmed and controlled molecular assemblies, but also for the fabrication of a broad range of peptide nanomaterials. These studies provided new information for dynamic behaviors of pH-dependent self-assembly. Unlike previous studies focused on pH induced structural changes, our current study provided experimental observations suggesting that dynamic nanostructure formation of surfactant self-assembling peptides is more complex than we previously realized. These findings may not only have implications for the study of controlled and programmed self-assemblies but also for fabrications of a wide range of peptide nanomaterials.

The nanostructures at various magnifications of A6K peptides after 20 min (images A-C, pH=4.28) and 1 day (images D-F, pH=5.81) dissolved in water at original pH4.0. The insert in image (A) depicts the molecular model of A6K. The arrows in the images (B) and (D) show the structures of nanotubes and sheets. The arrow in the image F shows the two openings of a nanotube. The bars in the images are 100nm. The openings of the nanotubes are clearly visible in the AFM images. Some tubes are imaged in the vertical direction as seen in (E, F).

SELF-ASSEMBLING PEPTIDE SURFACTANTS STABILIZE SPINACH PHOTOSYSTEM I ON A DRY SURFACE FOR AN EXTENDED TIME

Patrick Kiley, Xiaojun Zhao, Marc Baldo, Barry Bruce and Shuguang Zhang

We have used a previously designed class of simple peptide surfactants to stabilize the integral membrane protein complex Photosystem 1 (PS-I) from spinach on a dry surface. The PS-I complex has a unique steady-state emission spectrum between 650 nm and 800 nm that can be readily followed. In the absence of peptide surfactants, large spectral changes that indicate complex disassembly are evident on a dry surface. In the presence of commonly used chemical surfactants, such as DM and n-Octyl-beta-D-Glucoside (OG), spectroscopic changes are still present. However, peptide surfactants, AAAAAAK (A₆K) stabilized the complex in a concentration dependent manner. At 0.5% (w/v) of A₆K, it stabilized the PS-I complex for up to three weeks. Another peptide surfactant VVVVVVD (V₆D) also stabilized the complex but to a lesser extent. These observations suggest that these simple peptide surfactants may be effective materials for the study and manipulation of some intractable membrane proteins and membrane protein complexes. Photosystem I belong to a class of membrane proteins that convert energy. It is estimated that approximately 30% of genomes code for membrane proteins. However, despite considerable effort, only ~80 membrane protein structures have been elucidated. PS-I is among them. In order to study more photosystems and other membranes, additional biological detergents, such as the designed peptide detergents, will be extremely important and beneficial because this class of peptide detergents can be designed and synthesized at the single molecular level.

Molecular structures of peptide surfactants A₆K and V₆D. This class of peptide surfactants has no sequence homology. Rather, they share a common feature, a hydrophilic head with any charged or polar residues and a hydrophobic tail with various nonpolar residues. Extended time of PS-I stability on dry surface. Fluorescence spectrum of a PS-I film containing 0.5% A₆K was monitored for 3 weeks. Negligible increase in fluorescence was observed at 683 nm. A slightly insignificant blue shift of the 730 nm fluorescence was observed. Thus, it suggests that the PS-I complex retained its structural integrity under such conditions examined.

A NEW CLASS OF SELF-ASSEMBLING PEPTIDE DETERGENTS SIGNIFICANTLY STABILIZED BOVINE RHODOPSIN

Xiaojun Zhao, Yusuke Nagai, Philip Reeves and Shuguang Zhang

One of the grand challenges in biology is to systematically determine the structures of many more membrane proteins. That daunting task requires new materials and tools. We have designed a new class of peptide detergents to conquer this frontier of membrane proteins. The integral membrane protein rhodopsin from the visual photoreceptor in the retina belongs to a typical G-protein coupled receptor (GPCR). A number of detergents including octyl-D-glucoside (OG) and n-dodecyl-D-maltoside (DM) are commonly used to solubilize rhodopsin from rod outer segments (ROS). These detergents stabilize rhodopsin by attaching their hydrophobic site with the transmembrane domain of rhodopsin. A great deal has been learned about rhodopsin using the simple detergents. However, in order to further gain detailed structural information of rhodopsin, it is necessary to use other detergents that can facilitate the high-resolution structural analyses. This new class of peptide detergents consists of a hydrophilic head of aspartic acid or lysine with a hydrophobic tail of consecutive Gly, Ala, Val, Ile and Leu. The individual peptide consists of ~7 residues and numerous peptides self-assemble to form nanotubes and nanovesicles in the absence of proteins. We here demonstrate that one of the peptide detergents A₆D (Ac-AAAAAAD) significantly stabilized bovine rhodopsin for an extended time at 50°C. In the control experiment of the same concentration, DM did not significantly stabilize rhodopsin under identical condition. Moreover, when OG was replaced with A₆D in the exchange reaction, bovine rhodopsin was further stabilized even at 55°C. These results demonstrate that peptide detergents are effective materials for stabilizing and studying membrane proteins.

A proposed plausible model of peptide surfactants interacting with membrane proteins. These small and simple peptide surfactants have two distinct parts. The hydrophobic tails are likely to interact with the belt-region of membrane proteins that are usually embedded in lipid bilayer. In the case of the PS-I complex that is directly coupled on surface, the peptide surfactants can surround the anchored proteins to stabilize them in a manner yet clearly understood at present. In the absence of membrane proteins, the peptide surfactants themselves can form a variety of nanostructures including micelles, vesicles and nanotubes to sequester their hydrophobic tails from water.

SCAFFOLD FORMATION OF THE RADA16 AND RADA 8 PEPTIDES COMPOSITE MATERIALS

Xiaojun Zhao, Kranthi Vistakula and Shuguang Zhang

Nanofiber structures of some peptides and proteins as biomaterials have been studied extensively, but the molecular mechanism of self-assembly and reassembly still remains unclear. We here report the reassembly of an ionic self-complementary peptide RADARADARADARADA (RADA16-I) that forms a well-defined nanofiber scaffold. The 16-residue peptide forms stable beta-sheet structure and undergoes molecular self-assembly into few nanofibers and eventually a scaffold hydrogel consisting of >99.5% water. In this study, the nanofiber scaffold was sonicated into smaller fragments. Atomic force microscopy (AFM), circular dichroism and rheology were used to follow the kinitics of re-assembly. These sonicated fragments quickly reassemble into nanofibers that are indistinguishable from the original material. These small fragments serve as nucleation units and are also able to self-assemble into long fibers. Using RADA 8 peptides as inhibitors, we inhibited fiber growth significantly. The nanofibers formed from the RADA 16 and RADA 8 composite peptides are much shorter than that of the RADA 16 pure nanofibers. The hydrogels formed by these shorter fibers displaced much stronger mechanical property with a 1,000 Pa. These shorter fibers maintain the short fibers stable for more than one month and can reassemble into the similar short fibers after sonication breakage. Our data clearly showed that the fibers length are reversely correlated with mechanical strength of hydrogel containing >99% water. The disassembly and re-assembly processes were repeated three times and, each time, the reassembly reached their original length. We proposed plausible model to interpret the re-assembly involving complementary nanofiber cohesive ends. This reassembly process is important for fabrication of new scaffolds for 3-D cell culture, tissue engineering and regenerative medicine.

AFM images of RADA16-I and RADA 8 mixture. A-D, RADA 16 and RADA 8 peptide powers were mixed at ratios of 1:4 (A1-A2), 1:8 (B1-B2), 1:16 (C1-C2), 1:32 (D1-D2).

Schematic molecular model of a segment of RADA16/RADA8 self-assembly peptides.

THE FABRICATION OF SELF-ASSEMBLING PEPTIDES INTO NANOFIBER SCAFFOLDS THROUGH MOLECULAR SELF-ASSEMBLY

Xiaojun Zhao, Jessica Dai and Shuguang Zhang

We designed and fabricated a class of self-assembling peptides into nanofiber scaffolds. KLDL-12 has been shown to be a permissible nanofiber scaffold for chondrocytes in cartilage 3-D cell cultures. However, the biochemical, structural, and biophysical properties of KLDL-12 remain unclear. We show that KLDL-12 peptides form stable b-sheet structures at different pH values and that KLDL-12 and RIDI-12 self-assemble into nanofibers. The nanofiber length, though, is sensitive to pH changes. These results not only suggest the importance of electrostatic attraction or repulsion affecting the fiber lengths but also provide us with useful information for rational design and fabrication of peptide scaffolds. We used AFM, CD and dynamic light scattering techniques to study the biochemical, structural, and biophysical properties of KLDL-12 peptides. Our data showed that KLDL-12 can form stable b-sheet structures between pH 3-10 and further self-assemble into nanofibers. Electrostatic interactions are important for peptide aggregation to form nanofibers as well as hydrophobic interactions. This explains why KLDL-12 peptide-derived hydrogels formed at neutral pH. This information provides future rationale to design peptides and develop applications of peptide-derived hydrogels for tissue engineering.

AFM images of self-assembling peptide KLD12 scaffold that was used to produce 3-D cell culture chondrocytes for regeneration medicine.

DYNAMIC REASSEMBLY OF PEPTIDE RADA16 NANOFIBER SCAFFOLD

Hidenori Yokoi and Shuguang Zhang

AFM images of RADA16-I nanofiber at various time points after sonication. The observations were made using AFM immediately after sample preparation. a, 1 min after sonication; b, 2 min; c, 4 min; d, 8 min; e, 16 min; f, 32 min; g, 64 min; h, 2 hr; i, 4 hr; j, 24 hr. Note the elongation and reassembly of the peptide nanofibers over time.

A proposed reassembly molecular model of self-assembling RADA16-I peptides. When the peptides form stable b-sheets in water, they form intermolecular hydrogen bonds along the peptide backbones. The b-sheets have two distinctive sides, one hydrophobic with an array of alanines and the other with negatively charged aspartic acids and positively charged arginines (see Fig. 1a). These peptides form anti-parallel b-sheet structures (gray arrows indicate the directions). The alanines form overlap packed hydrophobic interactions in water, a structure that is found in silk fibroin from silkworm and spiders. On the charged sides, both positive and negative charges are packed together through intermolecular ionic interactions in a checkerboard-like manner. These nanofiber fragments can form various assemblies similar to restriction digested DNA fragments: a, blunt ends; b, semi-protruding ends (see Fig. 1d). c, These fragments with protruding ends could reassemble readily through hydrophobic interactions. d, The fragments with semi-protruding and various protruding ends. e, These fragments can reassemble readily. Color code: green, alanines; red, negatively charged aspartic acids; blue, positively charged arginines. For clarity, these b-sheets are not presented as twisted strands.

HIERARCHICAL SELF-ASSEMBLY OF A COILED-COIL PEPTIDE INTO FRACTAL STRUCTURE

Andrea Lomander, Wonmuk Huang and Shuguang Zhang

We studied the hierarchical self-assembly of a coiled-coil peptide containing an internal cysteine. Atomic Force Microscopy (AFM) experiments revealed the fractal structure of the assemblies and molecular simulations showed that the peptides cross-linked to form clusters of coiled-coils, which further assembled to form globules of tens of nanometers in diameter. Such hierarchical organization was modulated by pH or thiol-reducing agent. Exploitation of the fractal structures through chemical methods may be valuable for the fabrication of materials spanning multiple length-scales. Using fractal systems to fabricate structures and continuum length-scale materials is a powerful tool because it can span a wide range of dimensions. It is possible to use many conventional biochemical methods to fine tune the self-assembly of the coiled-coil peptides and its derivatives as scaffolds not only for fabricating nanostructures, but also for other multiple length-scale materials.

Morphology of aggregates at different pH. (b-g are AFM images). (a) Large ramified patterns formed by the peptide at pH 1. Parts of the image were captured on the video monitor used in connection with the AFM. Such patterns were always visible when ramified patterns on the micrometer scale could be imaged (pH 1 and 2). (b) pH 1. (c) Sized up image from (b), pH 1. (d) pH 2. (e) pH 3. (f) pH7. (g) pH 1, with a reduction agent. (h) Quick-freeze deep-etch transmission electron microscopy image of the peptide at pH 5. Due to low contrast, numerous small globules of diameter 10-20 nm were only visible directly on the film.

A proposed model of the self-assembly process. Coiled-coils crosslink into concatemers, up to hexamers. In solution, they further aggregate to form globules and clusters consisting of more than hundreds of coiled-coils. When deposited on a substrate, the globules form DLA clusters. Addition of a reducing agent, denaturant, or a hydrophobic solvent modifies this process. (The diagrams are not to the scale).

COMPARATIVE AND QUANTITATIVE STUDIES OF A SPECTRUM OF BIOMATERIALS USING MOUSE ADULT NEURAL STEM CELLS

Neural Stem Cells are potential therapeutic source for cellular transplantations in nervous system injuries. They are currently studied in cell-based therapy and tissue engineering. They also provide a good in vitro model for developing and regenerating nervous system, helpful in testing for cytotoxicity, cellular adhesion, and differentiation properties of biological and synthetic biomaterials. We systematically studied ten biomaterials, including, including various commonly used biopolymers and newly discovered peptide scaffolds for four weeks. Additionally, the outcome of a surface treatment coating with laminin was also examined. For each condition, cell viability, differentiation and maturation of the differentiated stem cell progeny (i.e. progenitor cells, astrocytes, oligodendrocytes and neurons) were evaluated. It is anticipated that the optimal biomaterial should show high % of living and differentiated cells. We quantitatively showed that Collagen IV, Matrigel or Laminin were better than Collagen I, Fibronectin, PLLA, PCLA, PLGA and a self-assembling peptide RADA16. In all cases, the coating protocol dramatically improved the performance of the biomaterials, but without altering their ranking, hence showing the importance of a surface treatment in scaffold transplant procedures. Collagen IV, Laminin and Matrigel showed the best performances for NSCs adhesion, surviving and differentiation. However, a surface treatment is fully capable to improve these performances. The self-assembling peptide scaffold RADA16 was used as representative of a new class of biomaterials for its fully defined molecular structure with considerable potential for further functionalization and slow-drug release. Because of its comparable performance with the FDA approved biomaterials and because of its tailor-made potential, it is promising to be useful as a molecular designed material not only for improving cell adhesion and delivering drugs in vitro but also for future 3-D cell culture studies as well as clinical trials in regenerative medicine.

NSCs differentiation and maturation 14 days after plating. NSCs seeded on RADA16 coated (A column), PCLA coated (B column), collagen I coated (C column), collagen IV coated (D column) and matrigel (E column). Cell nuclei were stained in blue with the nucleic acid stain DAPI . In the first row, NSCs were stained for a progenitor marker (Nestin+ in green) and a neuronal one (bTubulin+ in red). The presence of GFAP+ and Galactocerebroside+ markers were also tested for to detect, respectively, astrocytes (second row in green) and oligodendrocytes (third row in green). Cells concentration is variable due to both cell surviving and irregularities in biomaterials surfaces. Neurons and astrocytes showed different morphologies ranging from, respectively, bipolar and multipolar immature shapes (A and C), to extensively branched and spread cells (B, D and E). Oligodendrocytes as well look importantly branched in B, D and E. Scale bars 100 mm.

MOLECULARLY ENGINEERED PEPTIDE-DNA COMPLEX TRANSLOCATES ACROSS CELL MEMBRANES

Amrita Ghosh, Jay Schwartz, Shuguang Zhang

A tryptophan-arginine (WR16) peptide was designed and synthesized following principles from natural translocating peptides. WR16 was shown to successfully translocate into COS7 mammalian tissue culture cells. Kinetics of translocation was studied to give clues to the mechanism of translocation. Variations on peptide motif and sequence were made in order to study efficacy of particular amino acid moieties. The results of translocation studies provide insights into the non-receptor-mediated translocation mechanism, hypothesized as a unique interaction between residues of tryptophan and arginine and the phospholipid bilayer.

Translocation of 10.0 mg pEGFP-C1 using different concentrations of WR16 (mM) into COS7 cells. A) Images are plain light view merged with UV light view, showing cells that have translocated pEGFP-C1, containing the GFP gene. Green glow of cells indicates the GFP gene has been transcribed and translated into green fluorescent protein. a) no peptide with 10.0 mg pEGFP-C1 showing DNA does not enter cells; b) 0.4 mM WR16; c) 0.6 mM WR16; d) 0.8 mM WR16; e) 1.5 mM WR16; f) 2.0 mM WR16.

SYNTHESIS OF MONOFUNCTIONALIZED GOLD NANOPARTICLES BY F-MOC SOLID-PHASE REACTIONS

Kie-Moon Sung, David Mosley, Beau Peelle, Shuguang Zhang and Joseph Jacobson

We have studied a versatile monofunctionalization method for gold NCs with L-lysine (Lys) as potential building blocks for one-dimensional NC-peptide chains (nanopearls). Peptides are ubiquitous in biology and provide well-defined directionality and 3D spatial structures specified by amino acid sequences. Precise sequential positioning NCs can be designed using peptide synthesis protocols. For this purpose an amino acid-NC building block should meet the requirements of (a) single amino acid functionality per particle, (b) sufficiently small and uniform core diameters of ~2 nm, (c) amphiphilic solubility in aqueous and organic media enabling manual or programmed peptide synthesis, and (d) ease of synthesis with minimized effort for purification. We have synthesized ~2 nm sized Lys-monofunctionalized gold NCs 5 in a highly selective manner by solid-phase reaction using PS Wang resin or HMPA-PEGA. Productivity and reaction efficiency are highly dependant on the swelling properties of the solid support, which is not common in solid phase organic synthesis. We are currently heading to search for the use of these NC building blocks in the linear arrangement of nanonecklace using the peptide synthesis strategy.

Kinetic control of dimer structures in amyloid fibrillogenesis

Wonmuk Hwang, Shuguang Zhang, Roger Kamm, and Martin Karplus

Amyloid fibril formation in neurodegenerative diseases involves distinct intermediates that act as possible neurotoxic species. We have used multiple molecular dynamics simulations of five b-sheet forming peptides to investigate the first step in aggregate formation. It was found that the dimer structures are kinetically determined by local hydrophobic contacts and become trapped in energetically unfavorable conformations. These results suggest that kinetic trapping may play an important role in the structural evolution of early aggregates in amyloidosis. Kinetic trapping mechanism playing a previosuly unrealized critical role in early steps of amyloid fibrillogenesis has been uncovered using molecular simulations on five diferent beta-sheet forming peptides. One of the most important of protein aggregation in amyloid disease is the initial nucleation. The first step for the event to occur is mostly through beta-sheet packing, dimers, trimers, tetramers and beyond. Currently, there is no instrumentation that can precisely measure such a dynamic event in such a short time scale. Our study through molecular dynamics suggests that the critical initial molecular assembly event can be simulated using well-developed tools and biochemical and biophysical knowledge. Thus, our study shows that it is possible to use an alternative route to study the seemingly intractable nucleation in amyloid aggregation and propagation. It will likely to have a general importance for additional studies and hopefully, our study can be confirmed through high precision instrumentations in the future.

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