Hierarchical spidroin micellar nanoparticles as the fundamental precursors of spider silks

Abstract

Many natural silks produced by spiders and insects are unique materials in their exceptional toughness and tensile strength, while being lightweight and biodegradable–properties that are currently unparalleled in synthetic materials. Myriad approaches have been attempted to prepare artificial silks from recombinant spider silk spidroins but have each failed to achieve the advantageous properties of the natural material. This is because of an incomplete understanding of the in vivo spidroin-to-fiber spinning process and, particularly, because of a lack of knowledge of the true morphological nature of spidroin nanostructures in the precursor dope solution and the mechanisms by which these nanostructures transform into micrometer-scale silk fibers. Herein we determine the physical form of the natural spidroin precursor nanostructures stored within spider glands that seed the formation of their silks and reveal the fundamental structural transformations that occur during the initial stages of extrusion en route to fiber formation. Using a combination of solution phase diffusion NMR and cryogenic transmission electron microscopy (cryo-TEM), we reveal direct evidence that the concentrated spidroin proteins are stored in the silk glands of black widow spiders as complex, hierarchical nanoassemblies (∼300 nm diameter) that are composed of micellar subdomains, substructures that themselves are engaged in the initial nanoscale transformations that occur in response to shear. We find that the established micelle theory of silk fiber precursor storage is incomplete and that the first steps toward liquid crystalline organization during silk spinning involve the fibrillization of nanoscale hierarchical micelle subdomains.

Recombinant Silk Fiber Properties Correlate to Prefibrillar Self‐Assembly

“…This phenomenon was more frequently seen in HFIP dope, implying that nanoparticles in HFIP solvent are more dynamic and less stable, consistent with the broader size distribution observed in HFIP dope. The observed morphologies, including the potential for nanoparticle fusion, are also consistent with the recent cryo‐EM report of nanoparticle‐like assemblies comprising multiple micellar species in the MA gland of L. hesperus …”

“…Of particular importance, two primary and perhaps not mutually exclusive mechanisms for spidroin structural transition during passage through the gland and duct have been advanced: a liquid crystalline hypothesis and a micellar hypothesis . Recently, cryoelectron microscopy and diffusion NMR experiments have provided evidence for nanoparticle self‐assembly of spidroins in the gland setting, comprising micellar subdomains . In light of the requirement for self‐assembly, phase transition, and anisotropic orientation inherent in these fibrillogenesis mechanisms, favoring of such a process in mimicking the natural silk assembly process is likely to be beneficial.…”

Chemical Synthesis of Silk-Mimetic Polymers

“…The overall effect of these factors is to promote rapid β-sheet assembly and supramolecular aggregation. Several competing hypotheses exist for the specific mechanisms of silk fibroin structural transition in vivo, on which the reader is referred to relevant literature for further discussion [2,[71][72][73][74][75]. Importantly, the non-repetitive N and C-terminus domains have been implicated in controlling silk fibroin self-assembly [48][49][50].…”

Hydrothermal Effect on Mechanical Properties of Nephila pilipes Spidroin

“…Those hierarchically supra-molecular silk structures are greatly controlled by the sophisticated spinning process occurring in the spinning ducts of the spiders. Currently, two common hypotheses describe the hierarchical structures responsible for the spinning process that transforms silk protein solutions into solid filaments at the submicron and micron levels, namely the liquid crystalline model [9] and the micelles model [10][11][12]. The liquid crystalline model describes the transformation of liquid silk protein into fibers formed within the spinning duct of spiders, which in turn is accompanied by increasing shear forces that lead to the formation of intermolecular secondary structures.…”