Flow-assisted assembly of nanostructured protein microfibers

Abstract

Some of the most remarkable materials in nature are made from proteins. The properties of these materials are closely connected to the hierarchical assembly of the protein building blocks. In this perspective, amyloid-like protein nanofibrils (PNFs) have emerged as a promising foundation for the synthesis of novel bio-based materials for a variety of applications. Whereas recent advances have revealed the molecular structure of PNFs, the mechanisms associated with fibril-fibril interactions and their assembly into macroscale structures remain largely unexplored. Here, we show that whey PNFs can be assembled into microfibers using a flow-focusing approach and without the addition of plasticizers or cross-linkers. Microfocus small-angle X-ray scattering allows us to monitor the fibril orientation in the microchannel and compare the assembly processes of PNFs of distinct morphologies. We find that the strongest fiber is obtained with a sufficient balance between ordered nanostructure and fibril entanglement. The results provide insights in the behavior of protein nanostructures under laminar flow conditions and their assembly mechanism into hierarchical macroscopic structures.protein nanofibrils | amyloid | hierarchical assembly | flow focusing | small-angle X-ray scattering P roteins are widely used in nature to create high-performance materials that can have both extraordinary mechanical properties (similar to muscles, silks) and sophisticated functionalities (e.g., adhesion, biological signaling) (1). The characteristics of these materials are intimately connected to the hierarchical assembly of the protein building blocks with well-defined organization at all structural levels (2). Improved knowledge about how to control the assembly of protein molecules into higher-order structures would open the possibilities to create novel bio-based materials for a variety of applications. In this perspective, the ability of protein molecules to undergo nonnative self-assembly into protein nanofibrils (PNFs) with highly organized supramolecular structures is of significant interest (3). The formation of PNFs was initially observed in association with diseases, such as Alzheimer's and Parkinson's diseases, and type II diabetes, where human organs are impaired by fibrous protein inclusions referred to as amyloid (4). However, several non-disease-related proteins have also been shown to form amyloid-like fibrils, e.g., the bovine whey protein β-lactoglobulin (5), hen-egg lysozyme (6), and soybean proteins (7). The unique fibrous structures of PNFs have the potential for being the building block of protein-based nanomaterials and to be used as scaffolds for applications such as tissue engineering, drug delivery systems, and biosensors (3). PNFs are characterized by intermolecular β-sheet structures, where the peptide backbones are oriented perpendicularly to the fibril axis and connected through a network of hydrogen bonds (4,8). This provides them with stiffness equivalent to silk and strength comparable to steel (2, 8). Moreo...

On the role of peptide hydrolysis for fibrillation kinetics and amyloid fibril morphology

“…As previously reported by others for the brillation of pure b-lactoglobulin 24 and recently also by us for WPI brillation, 25 the morphologies of the brils formed at low and high concentrations differ. At lower concentrations (10-40 g l À1 ), straight brils that are up to a few micrometers long were observed (Fig.…”

“…We recently demonstrated that the same morphological switch is also observed for PNFs from WPI. 25 Interestingly, we also found that the two classes of PNFs display different behavior in the assembly of micrometer-sized laments. The fact that amyloidogenic polypeptides can form brils with different morphologies (strains) and also propagate the structural features through seeding is recognized.…”

“…In our previous work we analyzed a large set of AFM images in detail and found a signicant difference in the persistence lengths for the two classes of PNFs. 25 The persistence length of the straight brils was found to be ca. 1.9 mm while it is around 40 nm for the curved brils.…”

Robust Biological Fibers Based on Widely Available Proteins: Facile Fabrication and Suturing Application

“…In addition, the BSA fiber is superior to the pseudo‐protein and phage virus fibers in breaking strength . Additionally, the robust mechanical performance in the present BSA system is significantly stronger than that of any other reported globular protein‐based fibers …”

Modulating the Mechanical Performance of Macroscale Fibers through Shear‐Induced Alignment and Assembly of Protein Nanofibrils

“…In order to assemble nanofibrils into microsized fibers, wet‐spinning, dry‐spinning, and microfluidic techniques have previously been explored. In particular, microfluidic spinning has been demonstrated as a powerful technique for the assembly of nanoscale building blocks, such as cellulose fibrils, carbon nanotubes, and graphene fibers in a controlled manner, where the flow of solutions is confined within micrometer scale channels .…”