Dry-Spun Silk Produces Native-Like Fibroin Solutions

Silk's outstanding mechanical properties and energy efficient solidification mechanisms provide inspiration for biomaterial self-assembly as well as offering a diverse platform of materials suitable for many biotechnology applications. Experiments now reveal that the mulberry silkworm Bombyx mori secretes its silk in a practically "unspun" state that retains much of the solvent water and exhibits a surprisingly low degree of molecular order (β-sheet crystallinity) compared to the state found in a fully formed and matured fiber. These new observations challenge the general understanding of silk spinning and in particular the role of the spinning duct for structure development. Building on this discovery we report that silk spun in low humidity appears to arrest a molecular annealing process crucial for β-sheet formation. This, in turn, has significant positive implications, enabling the production of a high fidelity reconstituted silk fibroin with properties akin to the gold standard of unspun native silk

Structural diversity of native Major Ampullate, Minor Ampullate, Cylindriform, and Flagelliform silk proteins in solution

The foundations of silk spinning, the structure, storage, and activation of silk proteins, remain highly debated. By combining solution small-angle neutron and X-ray scattering (SANS and SAXS) alongside circular dichroism (CD), we reveal a shape anisotropy of the four principal native spider silk feedstocks from Nephila edulis. We show that these proteins behave in solution like elongated semiflexible polymers with locally rigid sections. We demonstrated that minor ampullate and cylindriform proteins adopt a monomeric conformation, while major ampullate and flagelliform proteins have a preference for dimerization. From an evolutionary perspective, we propose that such dimerization arose to help the processing of disordered silk proteins. Collectively, our results provide insights into the molecular-scale processing of silk, uncovering a degree of evolutionary convergence in protein structures and chemistry that supports the macroscale micellar/pseudo liquid crystalline spinning mechanisms proposed by the community

Why and how is silk spun? : integrating rheology with advanced spectroscopic techniques

This thesis investigates the mechanisms behind natural silk spinning by integrating rheology, spectroscopy and small angle scattering to better understand this process and to guide our efforts towards mimicking Nature’s ways of producing high performance fibres. As a result of natural selection, arthropods such as spiders and moths have evolved the ability to excrete silk proteins in a highly controlled manner. Spun from liquid feedstocks, silk fibres are used ex vivo to build structures with mechanical properties currently unmatched by industrial filaments. As yet, relatively little attention has been directed to the investigation of spinning under biologically relevant conditions. To better understand how and why silk is spun, this thesis bridges the gap between liquid silk flow properties and structure development. To directly connect the two, I have developed and deployed novel experimental platforms that combine infrared spectroscopy and small angle scattering with rheology. This approach has clarified long-standing ambiguities on the structural root of silk’s apparently complex flow properties. Small angle scattering revealed the length scales involved in the flow induced solidification under a range of spinning conditions. Mo reover, infrared spectroscopy offered a unique perspective into silk’s formation process immediately after excretion. In a similar manner to the post-extrusion tuning of the properties of partly solidified spider silk filaments, this thesis has revealed that silkworm silk fibres are far from completely formed once excreted. One might describe the filaments of mulberry silkworm as seeded molten polymers that form its hydrogen bonding network and crystallises slowly on site. Consequently, it enlightens that post-spinning conditions are equally paramount for silkworm silk, giving an explanation for the relatively poorer mechanical properties. The comparison of silks from a range of species, allowed this hypothesis to be extended to wild silkworm silk. My insights into spinning had the fortuitous repercussion of facilitating silk fibre solubilisation leading to the development of better artificial silk feedstocks flowing like native silks. With these findings, I believe we are now in an improved position to conceive artificial fibres with properties rivalling those of Nature.</p

Étude de la soie par spectroscopie infrarouge à réflexion totale atténuée

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2009-2010Les propriétés mécaniques de la soie sont supérieures à toute autre protéine fibreuse et n'ont jamais été surpassées par aucun matériau synthétique. Une meilleure compréhension de la structure moléculaire de la soie et des changements qui s'opèrent pendant, avant et après le filage est requise pour maîtriser sa conception. La spectroscopic infrarouge fut utilisée pour étudier la structure moléculaire des fibres microscopiques de soie. Afin d'éviter les problèmes liés à l'acquisition de spectres par transmission, la réflexion totale atténuée (ATR) a été utilisée. Avec un nouveau montage permetant la rotation de l'échantillon, l'orientation et la conformation d'une seule fibre de soie furent quantifiées pour la première fois par spectroscopic infrarouge. Des simulations spectrales ont été effectuées pour trouver les conditions expérimentales minimisant ces effets optiques nuisibles à l'interprétation des spectres. Les constantes optiques de la soie native du ver à soie ont été déterminées pour la première fois afin d'exécuter des simulations spectrales avec des paramètres variables. Ses simulations ont également permis d'estimer l'amplitude des effets optiques observés pour tout autre système

Why and how is silk spun? : integrating rheology with advanced spectroscopic techniques

This thesis investigates the mechanisms behind natural silk spinning by integrating rheology, spectroscopy and small angle scattering to better understand this process and to guide our efforts towards mimicking Nature’s ways of producing high performance fibres. As a result of natural selection, arthropods such as spiders and moths have evolved the ability to excrete silk proteins in a highly controlled manner. Spun from liquid feedstocks, silk fibres are used ex vivo to build structures with mechanical properties currently unmatched by industrial filaments. As yet, relatively little attention has been directed to the investigation of spinning under biologically relevant conditions. To better understand how and why silk is spun, this thesis bridges the gap between liquid silk flow properties and structure development. To directly connect the two, I have developed and deployed novel experimental platforms that combine infrared spectroscopy and small angle scattering with rheology. This approach has clarified long-standing ambiguities on the structural root of silk’s apparently complex flow properties. Small angle scattering revealed the length scales involved in the flow induced solidification under a range of spinning conditions. Mo reover, infrared spectroscopy offered a unique perspective into silk’s formation process immediately after excretion. In a similar manner to the post-extrusion tuning of the properties of partly solidified spider silk filaments, this thesis has revealed that silkworm silk fibres are far from completely formed once excreted. One might describe the filaments of mulberry silkworm as seeded molten polymers that form its hydrogen bonding network and crystallises slowly on site. Consequently, it enlightens that post-spinning conditions are equally paramount for silkworm silk, giving an explanation for the relatively poorer mechanical properties. The comparison of silks from a range of species, allowed this hypothesis to be extended to wild silkworm silk. My insights into spinning had the fortuitous repercussion of facilitating silk fibre solubilisation leading to the development of better artificial silk feedstocks flowing like native silks. With these findings, I believe we are now in an improved position to conceive artificial fibres with properties rivalling those of Nature.This thesis is not currently available in ORA

High-Throughput Thermal Stability Analysis of a Monoclonal Antibody by Attenuated Total Reflection FT-IR Spectroscopic Imaging

The use of biotherapeutics, such as monoclonal antibodies, has markedly increased in recent years. It is thus essential that biotherapeutic production pipelines are as efficient as possible. For the production process, one of the major concerns is the propensity of a biotherapeutic antibody to aggregate. In addition to reducing bioactive material recovery, protein aggregation can have major effects on drug potency and cause highly undesirable immunological effects. It is thus essential to identify processing conditions which maximize recovery while avoiding aggregation. Heat resistance is a proxy for long-term aggregation propensity. Thermal stability assays are routinely performed using various spectroscopic and scattering detection methods. Here, we evaluated the potential of macro attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopic imaging as a novel method for the high-throughput thermal stability assay of a monoclonal antibody. This chemically specific visualization method has the distinct advantage of being able to discriminate between monomeric and aggregated protein. Attenuated total reflection is particularly suitable for selectively probing the bottom of vessels, where precipitated aggregates accumulate. With focal plane array detection, we tested 12 different buffer conditions simultaneously to assess the effect of pH and ionic strength on protein thermal stability. Applying the Finke model to our imaging kinetics allowed us to determine the rate constants of nucleation and autocatalytic growth. This analysis demonstrated the greater stability of our immunoglobulin at higher pH and moderate ionic strength, revealing the key role of electrostatic interactions. The high-throughput approach presented here has significant potential for analyzing the stability of biotherapeutics as well as any other biological molecules prone to aggregation

High-Throughput Thermal Stability Analysis of a Monoclonal Antibody by Attenuated Total Reflection FT-IR Spectroscopic Imaging

The use of biotherapeutics, such as monoclonal antibodies, has markedly increased in recent years. It is thus essential that biotherapeutic production pipelines are as efficient as possible. For the production process, one of the major concerns is the propensity of a biotherapeutic antibody to aggregate. In addition to reducing bioactive material recovery, protein aggregation can have major effects on drug potency and cause highly undesirable immunological effects. It is thus essential to identify processing conditions which maximize recovery while avoiding aggregation. Heat resistance is a proxy for long-term aggregation propensity. Thermal stability assays are routinely performed using various spectroscopic and scattering detection methods. Here, we evaluated the potential of macro attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopic imaging as a novel method for the high-throughput thermal stability assay of a monoclonal antibody. This chemically specific visualization method has the distinct advantage of being able to discriminate between monomeric and aggregated protein. Attenuated total reflection is particularly suitable for selectively probing the bottom of vessels, where precipitated aggregates accumulate. With focal plane array detection, we tested 12 different buffer conditions simultaneously to assess the effect of pH and ionic strength on protein thermal stability. Applying the Finke model to our imaging kinetics allowed us to determine the rate constants of nucleation and autocatalytic growth. This analysis demonstrated the greater stability of our immunoglobulin at higher pH and moderate ionic strength, revealing the key role of electrostatic interactions. The high-throughput approach presented here has significant potential for analyzing the stability of biotherapeutics as well as any other biological molecules prone to aggregation