We here explore confinement-induced assembly of whey protein nanofibrils (PNFs) into microscale fibers using micro-focused synchrotron X-ray scattering. Solvent evaporation aligns the PNFs into anisotropic fibers and the process is followed in situ by scattering experiments in a droplet of PNF dispersion. We find an optimal temperature at which the order of the protein fiber has a maximum, suggesting that the degree of order results from a balance between the time scales of the forced alignment and the rotational diffusion of the fibrils. Moreover, we observe that the assembly process depends on the nano-scale morphology of the PNFs. Stiff PNFs with a persistence length in the micrometer scale are aligned at the air-water interface and the anisotropy gradually decrease towards the center of the droplet. Marangoni flows often increase entanglements toward the center, leading to complex patterns in the droplet. Flexible fibrils with a short persistence length (< 100 nm) tends to align uniformly throughout the droplet, possibly due to stronger local entanglements. Straight PNFs form smaller clusters with shorter inter-cluster distances due to their tight packing and consistent linear structure. In contrast, curved PNFs form intricate networks with larger characteristic distances and more varied structures because of their flexibility and adaptability.

Protein-based materials, with their unique properties of combining high strength, while maintaining elasticity, and their inherent biocompatibility, hold immense potential for various applications. In order to harness these properties, we need to understand and control how protein building blocks come together to form hierarchically structured materials. Using critical thinking when combining different proteins may lead to advanced materials with synergistic effects that can tackle complex problems such as targeted drug delivery. This thesis presents an investigation into the behavior of some protein-based materials, specifically whey protein isolate nanofibrils, β-lactoglobulin peptide fragment assemblies, and recombinant spider silk microspheres.

In Paper I, the hierarchical assembly and packing behavior of whey protein nanofibrils were in situ investigated using a Liquid Bridge Induced Assembly setup and X-ray Scattering, along with Atomic Force Microscopy and Scanning Electron Microscopy. The results demonstrated that the alignment of straight and curved fibrils was affected by temperature and fibril size, providing insights into assembly dynamics for future material production.

In Paper II, the impact of nanoscale features of whey protein nanofibrils on the morphology of films was investigated. It was found that controlling fibril size and employing fast-drying protocols could manipulate macroscale features without sacrificing the functional properties of the nanofibrils.

In Paper III, the nanoscale morphology, molecular arrangement, and polymorphism of protein nanofibrils formed by a synthetic peptide fragment derived from β-lactoglobulin were examined. Β-lactoglobulin is the only nanofibril forming component in whey protein isolate. Results suggested that polymorphism stems from protofilament packing differences at the nanoscale, and a possible parallel steric zipper packing.

In Paper IV, the self-assembly behavior of a recombinant spider silk protein in physiological like buffer and with the addition of hyaluronic acid was explored. The self-assembled FN-silk microspheres demonstrated a fibrillar or porous mesostructure. 2D and 3D cell culture trials show that the microspheres could have potential applications in biomedicine.

Taken together, the acquired knowledge will contribute to our fundamental understanding of protein-based materials, especially those similar to PNF-based and recombinant spider silk-based materials and inform the design of improved and innovative materials in biomanufacturing, such as functional textiles and surface biofunctionalization.

Proteinbaserade material med sin unika kombination av styrka, elasticitet och biokompatibilitet har enorm potential för tillämpningar inom olika branscher. För att utnyttja dessa egenskaper måste vi förstå och kontrollera hur proteinbyggstenar sammanfogas för att bilda hierarkiskt strukturerade material. Samtidigt kan kombinationen av olika proteiner med kritiskt tänkande leda till avancerade material med synergistiska effekter som kan ta itu med komplexa problem som in vitro-vävnadsutveckling. Denna avhandling presenterar en undersökning av beteendet hos vissa proteinbaserade material, specifikt vassleproteinisolat (WPI) proteinnanofibriller (PNF), β-laktoglobulinprotein peptidfragmentförsamlingar och rekombinerade spindelsilkesmikrosfärer.

I Artikel I undersöktes den hierarkiska sammansättningen och packningsbeteendet hos WPI-nanofibrer med hjälp av en Liquid Bridge Induced Assembly (LBIA) uppställning. Resultaten visade att inriktningen av raka och böjda fibrer påverkades av temperatur och storlek vilket ger insikter i sammansättningsdynamiken för framtida materialproduktion. 

I Artikel II studerades effekten av WPI PNFs nanoskaliga funktioner på filmernas morfologi. Det visade sig att kontroll av fibrilstorlek och användning av snabbtorkningsprotokoll kunde manipulera makroskaliga funktioner utan att offra de funktionella egenskaperna hos PNF:erna.

I Artikel III undersöktes nanoskalig morfologi, molekylärt arrangemang och polymorfism hos PNF:er bildade av ett syntetiskt peptidfragment härstammande från β-laktoglobulinprotein (β-LG11–20). Resultaten tyder på att polymorfismen härrör från protofilament packningsdifferenser på nanoskalan. 

I Artikel IV utforskades självmonteringsbeteendet hos FN-silke i fysiologisk liknande buffert och i hyaluronsyralösning. FN-silkesmikrosfärerna visade en fibrillär eller porös mesostruktur, med potentiella tillämpningar inom optimering av cellkulturer och läkemedelsleverans.

Biomaterials made of self-assembling protein building blocks are widely explored for biomedical applications, for example, as drug carriers, tissue engineering scaffolds, and functionalized coatings. It has previously been shown that a recombinant spider silk protein functionalized with a cell binding motif from fibronectin, FN-4RepCT (FN-silk), self-assembles into fibrillar structures at interfaces, i.e., membranes, fibers, or foams at liquid/air interfaces, and fibrillar coatings at liquid/solid interfaces. Recently, we observed that FN-silk also assembles into microspheres in the bulk of a physiological buffer (PBS) solution. Herein, we investigate the self-assembly process of FN-silk into microspheres in the bulk and how its progression is affected by the presence of hyaluronic acid (HA), both in solution and in a cross-linked HA hydrogel. Moreover, we characterize the size, morphology, mesostructure, and protein secondary structure of the FN-silk microspheres prepared in PBS and HA. Finally, we examine how the FN-silk microspheres can be used to mediate cell adhesion and spreading of human mesenchymal stem cells (hMSCs) during cell culture. These investigations contribute to our fundamental understanding of the self-assembly of silk protein into materials and demonstrate the use of silk microspheres as additives for cell culture applications.

Natural high-performance materials have inspired the exploration of novel materials from protein building blocks. The ability of proteins to self-organize into amyloid-like nanofibrils has opened an avenue to new materials by hierarchical assembly processes. As the mechanisms by which proteins form nanofibrils are becoming clear, the challenge now is to understand how the nanofibrils can be designed to form larger structures with defined order. We here report the spontaneous and reproducible formation of ordered microstructure in solution cast films from whey protein nanofibrils. The structural features are directly connected to the nanostructure of the protein fibrils, which is itself determined by the molecular structure of the building blocks. Hence, a hierarchical assembly process ranging over more than six orders of magnitude in size is described. The fibril length distribution is found to be the main determinant of the microstructure and the assembly process originates in restricted capillary flow induced by the solvent evaporation. We demonstrate that the structural features can be switched on and off by controlling the length distribution or the evaporation rate without losing the functional properties of the protein nanofibrils.

Protein nanofibrils (PNFs) represent a promising class of biobased nanomaterials for biomedical and materials science applications. In the design of such materials, a fundamental understanding of the structure-function relationship at both molecular and nanoscale levels is essential. Here we report investigations of the nanoscale morphology and molecular arrangement of amyloid-like PNFs of a synthetic peptide fragment consisting of residues 11-20 of the protein beta-lactoglobulin (beta-LG(11-20)), an important model system for PNF materials. Nanoscale fibril morphology was analysed by atomic force microscopy (AFM) that indicates the presence of polymorphic self-assembly of protofilaments. However, observation of a single set of C-13 and N-15 resonances in the solid-state NMR spectra for the beta-LG(11-20) fibrils suggests that the observed polymorphism originates from the assembly of protofilaments at the nanoscale but not from the molecular structure. The secondary structure and inter-residue proximities in the beta-LG(11-20) fibrils were probed using NMR experiments of the peptide with C-13- and N-15-labelled amino acid residues at selected positions. We can conclude that the peptides form parallel beta-sheets, but the NMR data was inconclusive regarding inter-sheet packing. Molecular dynamics simulations confirm the stability of parallel beta-sheets and suggest two preferred modes of packing. Comparison of molecular dynamics models with NMR data and calculated chemical shifts indicates that both packing models are possible.

Protein conformation and self-assembly dictates function and structure on different hierarchical scales. We herein explore confinement-induced assembly of whey protein nanofibrils (PNF) into microscale fibers, using microfocused synchrotron X-ray scattering. The solvent evaporation forces the PNFs to align in an anisotropic fiber and the process is followed in situ by scattering experiments in a droplet placed between two anchor points. Controlling the temperature allows for variation of the evaporation rate and exploration of how the kinetics of the alignment process affect the final structure. Our findings show that there is an optimal temperature at which the order of the protein fiber has a maximum. This suggests that the degree of order results from a balance between the time scales of the forced alignment by the droplet surface and the rotational diffusion of the fibrils. Moreover, we observe that the assembly process depends on the nanoscale morphology of the PNFs. Stiff PNFs with a persistence length in the micrometer scale are aligned at the air-water interface and the anisotropy gradually decrease towards the center of the droplet. Flexible fibrils with a short persistence length (< 100 nm) align uniformly throughout the droplet, possibly due to stronger local entanglements.

Biomaterials made of self-assembling protein building blocks are widely explored for biomedical applications, for example as drug carriers, tissue engineering scaffolds, and functionalized coatings. It has previously been shown that a recombinant spider silk protein functionalized with a cell binding motif from fibronectin, FN-4RepCT (FN-silk), self-assembles into fibrillar structures at interfaces, i.e. membranes, fibers, or foams at liquid/air interfaces, and fibrillar coatings at liquid/solid interfaces. Recently, we observed that FN-silk also assembles into microspheres in the bulk of a physiological buffer (PBS) solution. Herein, we investigate the self-assembly process of FN-silk into microspheres in the bulk, and how its progression is affected by the presence of hyaluronic acid (HA), both in solution and in a cross-linked HA hydrogel. Moreover, we characterize the size, morphology, mesostructure and protein secondary structure of the FN-silk microspheres prepared in PBS and HA. Finally, we examine how the FN-silk microspheres can be used to mediate cell adhesion and spreading of human mesenchymal stem cells (hMSCs) during cell culture. These investigations contribute to our fundamental understanding of the self-assembly of silk protein into materials and demonstrate the use of silk microspheres as additives for cell culture applications.