Mutsafi Prize: EVOLUTIONARY ORIGIN OF SILK MATERIAL HIERARCHY

Nature regularly assembles materials with exceptional physical properties and biological functionality, often including multi-scale hierarchical structures and specific molecular interactions. Silk fibers exemplify such material, exhibiting extraordinary mechanical properties, having high strength, breaking strain, and toughness1,2. Although silk fibers have been extensively studied, their formation and structural characteristics are still poorly understood. Apparently, this is why bio-inspired silk materials have failed to recreate the properties of native silk.

The natural formation of silk fibers ("spinning") is the process during which the silk protein (Fibroin) inside the silk gland transforms from its soluble random-coil state into solid beta-sheet-rich fibers. The transition results from shear stress, elongation flow, acidification, and metal ions chelation inside the gland3. Still, the evolution of silk consists of more complex and dynamic events, which are elusive and challenging to study.

The research aims to reveal the events in the dynamic process of the natural evolution of silk, the hierarchical structure of the fibers, and the role of metal ions in shaping this material. To address these challenges, we combined several advanced microscopy techniques to study the in vivo biological system in high-resolution imaging and spatial characterization.

First, several cryo-EM techniques were combined for studying the native process of silk spinning. The unperturbed in vivo process was examined by preserving silk glands using high-pressure freezing, followed by freeze-fracture and freeze-substitution, and imaging by cryo-SEM and TEM. The separated silk nano-fibrils were imaged by cryo-TEM after plunge-freezing extracted protein.

At the anterior part of the silk gland, the protein co-exists as a micelle-like form and as an amorphous bulk fluid. The micelle-like form helps to store the protein in its soluble unordered state, preventing premature and undesired fibrillation. This micelle-monomer equilibria shifts toward the monomeric state along with the whole anterior part of the silk gland.

As the fluid progresses at the anterior part towards the spinneret, shear stress and elongation flow force increase, which further triggers structural phase transitions in the protein. The first stage is evidenced by phase separation events, indicative of the protein disentangle and then aligned with the flow direction. Aligned proteins interact to form small nano-fibrils, inducing fibrillation of other nearby protein monomers. As the silk gland diameter reduces, the ever-increasing shear forces cause the fibrils to attach as small bundles, gradually forming from the gland`s outer diameter, corresponding to the shear stress difference.

In its final form, just before being extruded and spun into a micro-fiber, the protein dope is a matrix of numerous cross-linked nano-fibrils bundles. Each nano-fibril has a main trunk of 4-8 nm in diameter, branching out to connect other nano-fibrils assembled into "fish-bone" structures. Further work to better understand the single fibril structure is being done by TEM tomography.

The study of metal ions in the natural silk spinning and properties has been performed by combining SEM-EDS with other characterization techniques. Overall, metal ions concentration gradually increases from the posterior towards the anterior silk gland (except for Ca). These trends should induce the protein`s random-to-beta-sheet transition, which is crucial for the spinning process. However, the spatial elemental analysis done by SEM-EDS of both silk fibers and the anterior silk gland revealed an unexpected uneven distribution of elements. It was found that metal ions concentration is much higher in the coating sericin and the epithelial cells than in the silk dope. These substantial changes in ions concentration in the lumen is most probably caused by ion-transfer pumps of the epithelial cells and difference in proteins binding affinity. Thus, the final spinning steps requires the decrease of metal ions interacting with fibroin.

The changes and distributions of each metal ion along the silk gland are carfully tuned to suit the desired protein phase in each step of the spinning process, optimizing the conditions for storage, flow, or fibrillation.

In conclusion, the research reveals and visualizes the key steps and events in the natural process of silk fibers formation. The protein undergoes several structural transitions and phase separation events, forming multiple macromolecular and nano-structures during its flow along the anterior gland. This better understanding of the silk natural evolution helps unravel its secrets in the process of mimicking its superb material`s properties.

References

1. Koh, L. D. et al. Structures, mechanical properties and applications of silk fibroin materials. Prog. Polym. Sci. 46, 86–110 (2015).

2. Porter, D. & Vollrath, F. Silk as a biomimetic ideal for structural polymers. Adv. Mater. 21, 487–492 (2009).

3. Chen, X., Shao, Z. & Vollrath, F. The spinning processes for spider silk. Soft Matter 2, 448–451 (2006).