Silk Road Revealed: Mechanism of silk fibre formation inBombyx mori

Abstract

Silk fibroin (SF) is one of the most exploited and studied natural materials, yet the complex sequence of dynamic transitions required to transform from a liquid state to a solid fibre remain unresolved. We found that fibroin heavy chain (FibH) from Bombyx mori (B. mori), a multidomain protein with highly repetitive domains, folds to form β-solenoid structures. The single fibroin molecule undergoes conformational transformations from a globular form to an extended solenoid driven by pH reduction. Following this, the N-terminal domain (NTD) drives higher-order oligomerisation beyond the proposed biological tetrameric unit when in the extended state. The proposed supramolecular structures explain the complex rheological behaviour observed for silk and the liquid crystalline textures observed within the B. mori gland. Overall, our results demonstrate that silk assembly is a highly organised process, where a mild reduction in pH promotes molecular reorganisation and higher-order oligomerisation of the N-terminal domain into structures that precede fibre formation. Moreover, we suggest that NTD is at least partially hydrolysed and lost during the standard regeneration/reconstitution processing, explaining the historical differences between native and regenerated feedstocks. We believe our results expand our understanding of the liquid-to-solid transformation of fibroin molecules, as well as indicate to other researchers a clear strategy to determine the quality of their feedstock, which can be translated into improved materials fabrication.

Biomimetic Silk Fibre Assembly: Mimicking Nature's Pultrusion Process

“…Notably, the observed values agree with the RSF values observed at similar concentrations. 13 To some extent, these experiments verify the observations of DLS, 4 where the proteins had smaller diameters; hence, faster diffusion at higher pH values occurred. This indicates that the protein at pH 8 has a faster adsorption to the interface due to faster diffusion speeds under these conditions.…”

“…Once again, we propose that the switch-like behavior occurs at pH values just below 7. 4 At lower pH, the nature of the protein in solution is likely oligomeric, driven by NTD interactions; hence, the effective MW of the system increases, increasing the total cohesiveness, which translates to a higher modulus.…”

“…We interpret this re ection as coming from the hexagonal packing of hydrated solenoid units, which upon reaching a critical strain rate are disrupted, prompting extension of the backbone and collapse of the chains in β-sheets, consistent with the prediction in our previous work. 4 Following these experiments, we conducted single-bre tensile testing, and the results are presented in Fig. 5B-F.…”

“…2,3 Recent investigations have provided further insight into the molecular self-assembly mechanism of Lepidopteran silk broin, revealing its nano brillar structure in the Silk-I con guration and the intricate interactions driving its solidi cation. 4 At the macroscopic level, natural silk bres are not extruded but rather pulled into shape through a process akin to pultrusion. 5,6 This pultrusive mechanism, observed in silkworms and spiders, underscores the biomechanical sophistication of silk production in nature.…”

“…7 Reconstituted silk broin (RSF), although often used as a precursor in biomimetic silk material research, has several inherent limitations due to its reduced molecular weight and the lack of the N-terminal domain (NTD) responsible for pH-controlled supramolecular assembly. 4 As a result, RSF requires nonnative conditions, such as organic solvents, 8 coagulation baths, 9 or prepolymerized aggregates, 10 for bre formation. However, these methods impose environmental stress on the protein and deviate from the natural spinning process, where only minimal energy intake is required to fabricate the silk bre.…”