Self-assembled Fibrils Research Articles

Formation of self-assembled fibril aggregates of trypsin-controllably hydrolyzed soy protein and its regulation on stability of high internal phase Pickering emulsions

The mechanisms of trypsin hydrolysis time on the structure of soy protein hydrolysate fibril aggregates (SPHFAs) and the stability of SPHFAs-high internal phase Pickering emulsions (HIPPEs) were investigated. SPHFAs were prepared using soy protein hydrolysate (SPH) with different trypsin hydrolysis time (0 min–120 min) to stabilize SPHFAs-HIPPEs. The results showed that moderate trypsin hydrolysis (30 min, hydrolysis degree of 2.31 %) induced SPH unfolding and increased the surface hydrophobicity of SPH, thereby promoting the formation of flexible SPHFAs with maximal thioflavin T intensity and ζ-potential. Moreover, moderate trypsin hydrolysis improved the viscoelasticity of SPHFAs-HIPPEs, and SPHFAs-HIPPEs remained stable after storage at 25 °C for 80 d and heating at 100 °C for 1 h. Excessive trypsin hydrolysis (> 30 min) decreased the stability of SPHFAs-HIPPEs. In conclusion, moderate trypsin hydrolysis promoted the formation of flexible SPHFAs with high surface charge by inducing SPH unfolding, thereby promoting the stability of SPHFAs-HIPPEs.

Antiviral fibrils of self-assembled peptides with tunable compositions

The lasting threat of viral pandemics necessitates the development of tailorable first-response antivirals with specific but adaptive architectures for treatment of novel viral infections. Here, such an antiviral platform has been developed based on a mixture of hetero-peptides self-assembled into functionalized β-sheets capable of specific multivalent binding to viral protein complexes. One domain of each hetero-peptide is designed to specifically bind to certain viral proteins, while another domain self-assembles into fibrils with epitope binding characteristics determined by the types of peptides and their molar fractions. The self-assembled fibrils maintain enhanced binding to viral protein complexes and retain high resilience to viral mutations. This method is experimentally and computationally tested using short peptides that specifically bind to Spike proteins of SARS-CoV-2. This platform is efficacious, inexpensive, and stable with excellent tolerability.

How Chromophore Labels Shape the Structure and Dynamics of a Peptide Hydrogel.

Biocompatible and functionalizable hydrogels have a wide range of (potential) medicinal applications. The hydrogelation process, particularly for systems with very low polymer weight percentages (<1 wt %), remains poorly understood, making it challenging to predict the self-assembly of a given molecular building block into a hydrogel. This severely hinders the rational design of self-assembled hydrogels. In this study, we demonstrate the impact of an N-terminal group on the self-assembly and rheology of the peptide hydrogel hFF03 (hydrogelating, fibril forming peptide 03) using molecular dynamics simulations, oscillatory shear rheology, and circular dichroism spectroscopy. We find that the chromophore and even its specific regioisomers have a significant influence on the microscopic structure and dynamics of the self-assembled fibril, and on the macroscopic mechanical properties. This is because the chromophore influences the possible salt bridges, which form and stabilize the fibril formation. Furthermore, we find that the solvation shell fibrils by itself cannot explain the viscoelasticity of hFF03 hydrogels. Our atomistic model of the hFF03 fibril formation enables a more rational design of these hydrogels. In particular, altering the N-terminal chromophore emerges as a design strategy to tune the mechanic properties of these self-assembled peptide hydrogels.

Modeling collagen fibril self-assembly from extracellular medium in embryonic tendon

Collagen is a key structural component of multicellular organisms and is arranged in a highly organized manner. In structural tissues such as tendons, collagen forms bundles of parallel fibers between cells, which appear within a 24-h window between embryonic day 13.5 (E13.5) and E14.5 during mouse embryonic development. Current models assume that the organized structure of collagen requires direct cellular control, whereby cells actively lay down collagen fibrils from cell surfaces. However, such models appear incompatible with the time and length scales of fibril formation. We propose a phase-transition model to account for the rapid development of ordered fibrils in embryonic tendon, reducing reliance on active cellular processes. We develop phase-field crystal simulations of collagen fibrillogenesis in domains derived from electron micrographs of inter-cellular spaces in embryonic tendon and compare results qualitatively and quantitatively to observed patterns of fibril formation. To test the prediction of this phase-transition model that free protomeric collagen should exist in the inter-cellular spaces before the formation of observable fibrils, we use laser-capture microdissection, coupled with mass spectrometry, which demonstrates steadily increasing free collagen in inter-cellular spaces up to E13.5, followed by a rapid reduction of free collagen that coincides with the appearance of less-soluble collagen fibrils. The model and measurements together provide evidence for extracellular self-assembly of collagen fibrils in embryonic mouse tendon, supporting an additional mechanism for rapid collagen fibril formation during embryonic development.

Governing the aggregation of type I collagen mediated through β-cyclodextrin

The effect of carbohydrates on collagen self-assembly behavior has been widely investigated because of their regulation on collagen fibrogenesis in vivo. In this paper, β-cyclodextrin (β-CD) was selected as an external disturbance to explore its intrinsic regulating mechanism on collagen self-assembly. The results of fibrogenesis kinetics indicated that β-CD had a bilateral regulation on collagen self-aggregation process, which was closely related to the content of β-CD: collagen protofibrils with low β-CD content were less aggregated compared to collagen protofibrils with high β-CD content. However, typical periodic stripes of ~67 nm on collagen fibrils were observed from transmission electron microscope (TEM), indicating that β-CD did not disturb the lateral arrangement of collagen molecules to form a 1/4 staggered structure. Correspondingly, the degree of aggregation of collagen self-assembled fibrils was closely correlated with the addition of β-CD content, as confirmed by field emission scanning electron microscopy (FESEM) and atomic force microscope (AFM). In addition, collagen/β-CD fibrillar hydrogel had good thermal stability and cytocompatibility. These results provide a better understanding of how to construct a structurally reliable collagen/β-CD fibrillar hydrogel as a biomedical material in a β-CD-regulated environment.

Self-Assembly of Amyloid Fibrils into 3D Gel Clusters versus 2D Sheets.

The deposition of dense fibril plaques represents the pathological hallmark for a multitude of human disorders, including many neurodegenerative diseases. Fibril plaques are predominately composed of amyloid fibrils, characterized by their underlying cross beta-sheet architecture. Research into the mechanisms of amyloid formation has mostly focused on characterizing and modeling the growth of individual fibrils and associated oligomers from their monomeric precursors. Much less is known about the mechanisms causing individual fibrils to assemble into ordered fibrillar suprastructures. Elucidating the mechanisms regulating this "secondary" self-assembly into distinct suprastructures is important for understanding how individual protein fibrils form the prominent macroscopic plaques observed in disease. Whether and how amyloid fibrils assemble into either 2D or 3D supramolecular structures also relates to ongoing efforts on using amyloid fibrils as substrates or scaffolds for self-assembling functional biomaterials. Here, we investigated the conditions under which preformed amyloid fibrils of a lysozyme assemble into larger superstructures as a function of charge screening or pH. Fibrils either assembled into three-dimensional gel clusters or two-dimensional fibril sheets. The latter displayed optical birefringence, diagnostic of amyloid plaques. We presume that pH and salt modulate fibril charge repulsion, which allows anisotropic fibril-fibril attraction to emerge and drive the transition from 3D to 2D fibril self-assembly.

Direct observation of peptide hydrogel self-assembly.

The characterization of self-assembling molecules presents significant experimental challenges, especially when associated with phase separation or precipitation. Transparent window infrared (IR) spectroscopy leverages site-specific probes that absorb in the “transparent window” region of the biomolecular IR spectrum. Carbon–deuterium (C–D) bonds are especially compelling transparent window probes since they are non-perturbative, can be readily introduced site selectively into peptides and proteins, and their stretch frequencies are sensitive to changes in the local molecular environment. Importantly, IR spectroscopy can be applied to a wide range of molecular samples regardless of solubility or physical state, making it an ideal technique for addressing the solubility challenges presented by self-assembling molecules. Here, we present the first continuous observation of transparent window probes following stopped-flow initiation. To demonstrate utility in a self-assembling system, we selected the MAX1 peptide hydrogel, a biocompatible material that has significant promise for use in drug delivery and medical applications. C–D labeled valine was synthetically introduced into five distinct positions of the twenty-residue MAX1 β-hairpin peptide. Consistent with current structural models, steady-state IR absorption frequencies and linewidths of C–D bonds at all labeled positions indicate that these side chains occupy a hydrophobic region of the hydrogel and that the motion of side chains located in the middle of the hairpin is more restricted than those located on the hairpin ends. Following a rapid change in ionic strength to initiate self-assembly, the peptide absorption spectra were monitored as function of time, allowing determination of site-specific time constants. We find that within the experimental resolution, MAX1 self-assembly occurs as a cooperative process. These studies suggest that stopped-flow transparent window FTIR can be extended to other time-resolved applications, such as protein folding and enzyme kinetics.

Oriented Crystallization of Hydroxyapatite in Self-Assembled Peptide Fibrils as a Bonelike Material.

Controlling oriented crystallization is key to producing bonelike composite materials with a well-organized structure. However, producing this type of composite material using synthetic biopolymers as scaffolds is challenging. Inspired by the molecular structure of collagen-I, a collagenlike peptide─(Pro-Hyp-Gly)10 (POG10)─was designed to produce self-assembled fibrils that resemble the structure of collagen-I fibrils. In addition, the oriented mineralization of HAP crystals is formed in the fibrils that reproduces a bonelike material similar to collagen-I fibril mineralization. Unlike collagen-I fibrils, POG10 fibrils do not contain gap spaces. The molecular simulation results indicate that in addition to space confinement, the molecular field generated by POG10 can also confine the orientation of HAP, enriching our understanding of physical confinement and shedding light on the design of synthetic biopolymer scaffolds for bonelike material fabrication.

Cover Feature: Asymmetric Organocatalysis Accelerated via Self‐Assembled Minimal Structures (Eur. J. Org. Chem. 39/2021)

The Cover Feature shows the catalytic activity exerted by a minimalistic peptide behaving as an organocatalyst in a Michael addition. A supramolecular arrangement on the catalyst in fibrils showed to remarkably improve the TOF of the catalyst. The aldehyde donors and nitroalkenes acceptors are represented in the top left corner and are largely converted to the adduct as the reaction proceeds, towards the bottom right. Across the image the self-assembled fibrils of a minimalistic tripeptide, catalyzing the reaction, are shown; the tripeptide is shown in detail, colored green, for one of the fibrils. Image credit: Nicola De Mitri. More information can be found in the Communication by A. Carlone et al.

Crystallization of a Self-Assembling Nucleator in Poly(l-lactide) Melt.

In the present work, crystallization of a soluble nucleator N, N′, N″-tricyclohexyl-1,3,5-benzenetricarboxylamide (TMC-328) in a poly(l-lactic acid) (PLLA) matrix has been studied at different temperatures. Based on the change in solubility with temperature, different levels of supersaturation of TMC-328 in a PLLA matrix can be obtained. This nucleator presents a fibrous structure produced via self-assembling and develops into an interconnected network when the temperature is lowered. The TMC-328 crystal nuclei density is quantified via optical microscopy, using the average distance of the adjacent fibrillar structure, which shows a steady decrease with the decrease in temperature. The crystallization rates of TMC-328 were assessed through rheological measurements of network formation. Both fibrils’ density and crystallization kinetics display a power law dependence on supersaturation. For the first time, the solid–melt interfacial energy, the size of the critical nucleus, and the number of molecules making up the critical nucleus of the nucleator TMC-328 in the PLLA matrix have been determined by adopting the classical nucleation theory. The subsequent crystallization of PLLA induced by this nucleator was investigated as a function of the fibrils’ spatial density. The crystallization rate of PLLA is enhanced with the increase in the TMC-328 fibrils’ density because of the availability of a larger nucleating surface. The self-assembled fibril of TMC-328 can serve as shish to form a hybrid shish-kebab structure after the crystallization of PLLA, regardless of the number of nucleation sites.

Short Peptides as Tunable, Switchable, and Strong Gelators.

This Perspective outlines our current understanding of molecular gels composed of short and ultrashort peptides over the past 20 years. We discuss in detail the state of the art regarding self-assembly mechanisms, structure, thermal stability, and kinetics of fibril and/or network formation. Emphasis is put on the importance of the combined use of spectroscopy and rheology for characterizing and validating self-assembly models. While a range of peptide chemistries are reviewed, we focus our discussion on a unique new class of ultrashort peptide gelators, denoted GxG peptides (x: guest residue), which are capable of forming self-assembled fibril networks. The storage moduli of GxG gels are tunable up to 100 kPa depending on concentration, pH, and/or cosolvent. The sheet structures of the fibrils differ from canonical β-sheets. When appropriate, each section highlights opportunities for additional research and technologies that would further our understanding.

EGFR Aggregation in the Brain

Recent findings showed that preformed fibrils (PFFs) of misfolded proteins, including α-synuclein (α-syn) and amyloid-β (Aβ), activate EGFR in cell cultures and animal models of amyloid propagation. Comparing these supramolecular structures to normal EGFR ligands such as EGF and HB-EGF suggests that these PFFs might trigger the formation of high order clustering of EGFR that stimulates the aggregation of EGFR tyrosine kinase domain (EGFR-TKD) which is known to form fibrils. Subsequently, self-assembled fibril of EGFR-TKDs itself can serve as a seed to induce aggregation of monomeric forms of misfolded proteins in cytoplasm or endosomes. In this model, EGFR serves as an amyloidogenic receptor to facilitate (1) cellular uptake of exogenous PFFs and (2) seeding of endogenous misfolded proteins.

Directionality of growth and kinetics of branched fibril formation.

The self-assembly of fibrils is a subject of intense interest, primarily due to its relevance to the formation of pathological structures. Some fibrils develop branches via the so-called secondary nucleation. In this paper, we use the master equation approach to model the kinetics of formation of branched fibrils. In our model, a branched fibril consists of one mother branch and several daughter branches. We consider five basic processes of fibril formation, namely, nucleation, elongation, branching, fragmentation, and dissociation of the primary nucleus of fibrils into free monomers. Our main focus is on the effect of the directionality of growth on the kinetics of fibril formation. We consider several cases. At first, the mother branch may elongate from one or from both ends, while the daughter branch elongates only from one end. We also study the case of branched fibrils with bidirectionally growing daughter branches, tangentially to the main stem, which resembles the intertwining process. We derive a set of ordinary differential equations for the moments of the number concentration of fibrils, which can be solved numerically. Assuming that the primary nucleus of fibrils dissociates with the fragmentation rate, in the limit of the zero branching rate, our model reproduces the results of a previous model that considers only the three basic processes of nucleation, elongation, and fragmentation. We also use the experimental parameters for the fibril formation of Huntingtin fragments to investigate the effect of unidirectional vs bidirectional elongation of the filaments on the kinetics of fibrillogenesis.

Dairy-Inspired Coatings for Bone Implants from Whey Protein Isolate-Derived Self-Assembled Fibrils.

To improve the integration of a biomaterial with surrounding tissue, its surface properties may be modified by adsorption of biomacromolecules, e.g., fibrils. Whey protein isolate (WPI), a dairy industry by-product, supports osteoblastic cell growth. WPI’s main component, β-lactoglobulin, forms fibrils in acidic solutions. In this study, aiming to develop coatings for biomaterials for bone contact, substrates were coated with WPI fibrils obtained at pH 2 or 3.5. Importantly, WPI fibrils coatings withstood autoclave sterilization and appeared to promote spreading and differentiation of human bone marrow stromal cells (hBMSC). In the future, WPI fibrils coatings could facilitate immobilization of biomolecules with growth stimulating or antimicrobial properties.

Rational Design and Self-Assembly of Coiled-Coil Linked SasG Protein Fibrils.

Protein engineering is an attractive approach for the self-assembly of nanometer-scale architectures for a range of potential nanotechnologies. Using the versatile chemistry provided by protein folding and assembly, coupled with amino acid side-chain functionality, allows for the construction of precise molecular "protein origami" hierarchical patterned structures for a range of nanoapplications such as stand-alone enzymatic pathways and molecular machines. The Staphyloccocus aureus surface protein SasG is a rigid, rod-like structure shown to have high mechanical strength due to "clamp-like" intradomain features and a stabilizing interface between the G5 and E domains, making it an excellent building block for molecular self-assembly. Here we characterize a new two subunit system composed of the SasG rod protein genetically conjugated with de novo designed coiled-coils, resulting in the self-assembly of fibrils. Circular dichroism (CD) and quartz-crystal microbalance with dissipation (QCM-D) are used to show the specific, alternating binding between the two subunits. Furthermore, we use atomic force microscopy (AFM) to study the extent of subunit polymerization in a liquid environment, demonstrating self-assembly culminating in the formation of linear macromolecular fibrils.

6-acylamino nicotinic acid-based hydrogelators applicable in phase selective gelation, reproducible mat formation and toxic dye removal

Formation of nanofibers and nanovesicles in the self-assembled state of small amphiphilic molecules has applications in versatile fields such as tissue engineering, controlled delivery of drug molecules, etc. This paper demonstrated the self-aggregation behavior of three synthesized 6-acylamino nicotinic acid amphiphiles named 6-octanoylamino-nicotinic acid (OANA), 6-decanoylamino-nicotinic acid (DANA) and 6-dodecanoylamino-nicotinic acid (DDANA) in water and basic aqueous solution. The result showed that the amphiphiles successfully self-organize into vesicles and twisted ribbons in water. FT-IR study revealed existence of mixtures of handedness in the fibrous structures. CD spectroscopy and TEM study elucidated formation of chiral structures through aggregation. Results showed that DDANA forms thermoreversible hydrogel in aqueous solutions of NaOH and Na2CO3, whereas other two amphiphiles form hydrogel only in the presence of NaOH. Morphological investigation revealed that the hydrogel is formed due to self-assembly of fibrils of micron length. The elastic fibrillar networks are quite stable to external forces. Existence of bilayer columnar square packing arrangement in the self-assembled state was recognized by XRD measurement. Spectroscopy and theoretical studies established that hydrogen bonding interactions are responsible to self-assemble the amphiphilic molecules. The amphiphiles are efficient phase selective gelators of mineral oils in water–mineral oil mixtures and excellent remover of rhodamine 6G, eosin Y and rose bengal from water. The amphiphiles successfully create reproducible fiber mat applicable in tissue engineering field.

Mechanoresponsive Alignment of Molecular Self-Assembled Negatively Charged Nanofibrils.

Inspired by the mechanoresponsive orientation of actin filaments in cell, we introduce a design paradigm of synthetic molecular self-assembling fibrils that respond to external mechanical force by transforming from a macroscopically disorder state to a highly ordered uniaxial aligned state. The incorporation of aromatic-containing amino acids and negatively charged amino acids lead to self-assembly motifs that transform into uniform nanofibrils in acidic solution. Adjusting the pH level of aqueous solution introduces optimal negative charge to the surface of self-assembling nanofibrils inducing long-range electrostatic repulsion forming a nematic phase. Upon external mechanical force, nanofibrils align in the force direction. Via evaporation casting in capillary confinement, the solvated synthetic self-assembling nanofibrils transform into scalable lamellar domains. Adjusting capillary geometry and drying procedure offers further parameters for tuning the mesoscale alignment of nanofibrils generating a variety of interference colors. The design paradigm of mechanoresponsive alignment of self-assembled nanofibrils as an addition of nanofabrication techniques is potentially employable for realizing biomimetic optical structures.

Algorithms for Parameterizing Network Hamiltonians for Simulations of Amyloid Fibril Self-assembly

Experimentally relevant ensembles of protein monomers that self-assemble into amyloid fibrils involve both system sizes and timescales that lie many orders of magnitude beyond the capabilities of even the most robust atomistic simulations. Motivated by this, we have previously introduced coarse-grained simulation schemes for amyloid fibril formation using a graph theoretic topological representation, where the interaction energy between monomers is governed by a network Hamiltonian rooted in exponential-family random graph models (ERGMs).

A Novel Method to Measure the Effective Change of the Interfacial Energy due to Kinetic Self-Assembly of Amyloid Fibrils.

Adsorbates growing a self-assembled layer on a solid-liquid interface can significantly change the effective interfacial energy at the solid surface. However, measuring the changes in the effective surface energy while these adsorbates accumulate is challenging, as static contact angle measurements can be affected by the motion and accumulation of these adsorbates at the droplet's boundary (coffee stain effects). In this report, we utilize a novel method that takes advantage of spin-induced dewetting to measure the change in the effective surface energy as the self-assembly progresses. We use a previously well-studied model system of self-assembled fibrils of amyloid-β (Aβ) peptides on the mica substrate to demonstrate the feasibility of this method. Using variations of terminal spin speeds and acceleration rates, we measure the terminal spin speed at which a wetting-dewetting transition (WDT) occurs on a surface that hosts self-assembled Aβ12-28 fibrils. By comparing this speed with the WDT speed on the bare mica substrate, we can quantify the spreading coefficient and thus the effective change of the substrate's interfacial energy due to the adsorption of mobile peptides at various stages of the self-assembly. These measurements show that the surface becomes more hydrophilic as the self-assembly progresses and thus can explain previous observations that the self-assembly of this particular peptide system is self-limiting and stops before full surface coverage.

Fibril Self-Assembly of Amyloid-Spider Silk Block Polypeptides.

Because of their association with debilitating diseases and their potential applications in developing novel bionanomaterials, highly ordered amyloid fibrils have recently received considerable attention. While many studies have thus far focused on amyloid fibrils made with short peptides containing just one steric zipper-forming segment of native amyloid proteins, the self-assembly of proteins containing multiple steric zipper-forming segments has been rarely explored. Here we develop a strategy to create four block polypeptides, each containing 16 repeats of a zipper-forming segment from four different amyloid morphological classes. All four block polypeptides self-assemble into fibrils that display the cross-β structure characteristic of amyloids. These amyloid-spider silk block polypeptides displayed fast self-assembly kinetics, and their fibrils exhibited high thermal stability. These novel synthetic amyloids provide insights into the self-assembly of proteins containing multiple zipper-forming segments, and our approach of creating block polypeptide fibrils could be used to expand the capability of amyloid-based bionanomaterials.