The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to β-sheet transition upon fiber formation and highlight the helix No4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix No4 has the ability of pH-induced conversion into β-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly.

Proteins found in nature offer a vast range of exceptional materials, including high-performancebiopolymers such as spider silks and whey protein nanofibrils. Fibrous proteins possess immensepotential for developing novel materials suited for various applications, such as medicalbiomaterials or industrial products. This thesis uses an interdisciplinary approach based onexperimental and computational methods to present insights into the fundamental aspects ofprotein fibers, exploring details on the molecular level and their self-assembly.Spider silk threads exhibit strength, elasticity, and the ability to withstand high-energy loads.Additionally, silk is naturally degradable, and compatible with cell growth, and non-immunogenic.This thesis examines the molecular assembly of spider silk, particularly the secondary structurelevel, which contributes greatly to its properties. We unveil the structural details of therecombinant spidroins 4RepCT and the CT domain over hydrophobic/hydrophilic surfaces,describing their periodic oriented macrostructure and stability. Furthermore, it is reported that theCT domain form β-nanocrystalline components, revealing a specific segment (helix No4) that canself-assemble into nanofibrils in a pH-sensitive manner. In addition, we describe the method ofsortase-mediated transpeptidation reaction used to catalyze the covalent coupling of the spidroins4Rep and CT, resulting in partially isotopically labeled fibers suitable for solid-state NMRspectroscopy analyses.β-lactoglobulin is an emerging protein source used to create advanced biomaterials because of itshigh availability and ability to assemble to protein nanofibrils (PNFs). Recombinant and syntheticβ-lactoglobulin PNFs with isotopic labelling are generated and analyzed using solid-state NMRspectroscopy and atomic force microscopy. The fibrils of both species present congruenciesregarding morphology with unbranched conformation and a height of approximately 6 nm. At thesame time, their NMR spectra demonstrate accordance with their hydrophobic residues (i.e., Ala,Val, Ile, and Leu) as β-sheets. In addition, distinct inter-residue cross-peaks of Ser-Thr and LeuIle provide insights into the molecular structure of β-lactoglobulin PNF.This thesis presents new knowledge about the hierarchy of protein fibrils and the structure ofprotein-based materials at the molecular level. This knowledge can unlock the design anddevelopment of innovative protein-based materials for various applications.

Proteiner som finns i naturen möjliggör ett brett utbud av exceptionella material, inklusivehögpresterande biopolymerer som spindelsilke och nanofibriller av vassleprotein. Fibrösaproteiner har en enorm potential för att utveckla nya material lämpliga för olika tillämpningar,såsom medicinska biomaterial eller industriella produkter. Denna avhandling använder entvärvetenskaplig ansats baserad på experimentella och beräkningsmetoder för att presenterainsikter om de grundläggande aspekterna av proteinfibrer, utforska detaljer på molekylär nivå ochderas förmåga att gå samman till material.Spindelsilke uppvisar styrka, elasticitet och förmåga att motstå höga energibelastningar. Dessutomär det naturligt nedbrytbart, kompatibelt med celltillväxt, och icke-immunogent. Denna avhandlingundersöker den molekylära sammansättningen av spindelsilke, särskilt sekundärstrukturen, vilketi hög grad bidrar till dess egenskaper. Vi undersöker de strukturella detaljerna för de rekombinantaspidroinerna 4RepCT och CT-domänen över hydrofoba/hydrofila ytor, och beskriva derasperiodiskt orienterade makrostruktur och stabilitet. Dessutom rapporteras det att CT-domänenbildar β-nanokristallina komponenter, vilket avslöjar ett specifikt segment (helix No4) som kanbilda nanofibriller på ett pH-beroende sätt. Vi beskriver också en metod för sortas-medieradtranspeptideringsreaktion som används för att katalysera den kovalenta kopplingen av spidroinerna4Rep och CT, vilket resulterar i delvis isotopmärkta fibrer som är lämpliga för anlys med fastfasNMR-spektroskopi.β-laktoglobulin är en proteinkälla av ökande intresse för att skapa avancerade biomaterial på grundav dess goda tillgänglighet och förmåga att bilda protein-nanofibriller (PNF). Rekombinanta ochsyntetiskta β-laktoglobulin-PNF med isotopinmärkning genereras och analyseras med hjälp av fastfas NMR-spektroskopi och atomkraftsmikroskopi. Fibrillerna hos båda varianterna uppvisaröverensstämmelse med avseende på morfologi med ogrenad konformation och en höjd av cirka 6nm. Samtidigt visar deras NMR-spektra överensstämmelse med deras hydrofoba aminosyrarester(dvs. Ala, Val, Ile och Leu) som beta flak. Dessutom ger distinkta korstoppar med Ser-Thr ochLeu-Ile insikter i den molekylära strukturen av β-laktoglobulin PNF.Denna avhandling presenterar ny kunskap om hierarkin av proteinfibriller och strukturen hosproteinbaserade material på molekylär nivå. Denna kunskap kan underlätta design och utvecklingav innovativa proteinbaserade material för olika tillämpningar. 

A detailed insight about the molecular organization behind spider silk assembly is valuable for the decoding of the unique properties of silk. The recombinant partial spider silk protein 4RepCT contains four poly-alanine/glycine-rich repeats followed by an amphiphilic C-terminal domain and has shown the capacity to self-assemble into fibrils on hydrophobic surfaces. We herein use molecular dynamic simulations to address the structure of 4RepCT and its different parts on hydrophobic versus hydrophilic surfaces. When 4RepCT is placed in a wing arrangement model and periodically repeated on a hydrophobic surface, fi-sheet structures of the poly-alanine repeats are preserved, while the CT part is settled on top, presenting a fibril with a height of similar to 7 nm and a width of similar to 11 nm. Both atomic force microscopy and cryo-electron microscopy imaging support this model as a possible fibril formation on hydrophobic surfaces. These results contribute to the understanding of silk assembly and alignment mechanism onto hydrophobic surfaces.