Elsevier

Polymer

Volume 211, 21 December 2020, 123082
Polymer

Pressure- and humidity-induced structural transition of silk fibroin

https://doi.org/10.1016/j.polymer.2020.123082Get rights and content

Highlights

  • Effect of pressure and humidity on crystallization of spider and silkworm silk amorphous films was investigated.

  • Pressure did not induce crystallization of spider silk but promoted β-sheet formation of silkworm silk.

  • Water molecules induced crystallization of both spider and silkworm silk.

  • The formation of β-sheet enhanced breaking strength and modulus of silk.

Abstract

Spiders and silkworms convert silk fibroin solution into fibers by adjusting physiological conditions such as water content and internal pressure. Water molecules are known to plasticize molecular chains of silk fibroin, affording β-sheet structures. However, the effect of pressure on the crystallization of silk fibroin and the differences between pressure- and water-induced crystallization in spider and silkworm silk remain unexplained. Herein, the effect of pressure and humidity on the crystallization of spider and silkworm silk amorphous films is investigated. Despite not inducing irreversible crystallization of spider silk, pressure promotes β-sheet formation of silkworm silk. In contrast, water induces crystallization of both spider and silkworm silk. Pressure-induced crystallization results in larger crystallinity but smaller crystallite size in comparison to water-induced crystallization. The formation of β-sheet structures enhances breaking strength and modulus of the silk films, which paves the way toward silk-based structural materials.

Introduction

Silk is used as a structural material in nature such as a lifeline and framework of spiders and the silkworm cocoon to protect larvae from predators [1]. The silk fibers are lightweight and show excellent mechanical toughness, thus being a candidate of bio-based materials for next generation [2]. Although the silkworm silk is used as surgical sutures for decades, the bulk-scale production of the natural silk is still limited especially in the case of spider silk [3]. Hence the artificial silk production has been reported using recombinant protein expression and chemical synthesis [4,5]. Furthermore, the artificial spinning of regenerated silk fibroin solution has been used to modify the diameter and physical properties of resultant silk fibers by varying the reeling speed and coagulation bath [6]. However, the mechanical strength of the resultant silk fiber is often inferior compared with that of the natural counterpart [6]. One of the causes of the inferior strength is the lower molecular weight of the synthesized silk [7]. In addition, the lack of knowledge on the mechanism of structural transition from random coil/helix to β-sheet retards the development of the artificial silk production [8].

Spiders and silkworms are thought to precisely and simultaneously adjust physiological conditions such as water contents and internal pressure in their glands to secret silk fibers with excellent mechanical properties [9]. In order to understand the mechanism of silk fiber formation in detail, it is important to investigate effects of each factor on structural transition of silk fibroin. Water molecules inhibit the intermolecular hydrogen bonds between silk molecular chains and cause the plasticization of silk fibroin, inducing the β-sheet structures of silkworm silk [10,11]. Dynamic mechanical thermal analysis demonstrated that water molecules interacted with the disordered structure of the silk fibroin and lowered the glass transition temperature [12]. Spider silk fibers are known to shrink when immersed in water, which is known as supercontraction [13]. The supercontraction of spider silk is triggered by an entropic relaxation mainly occurred in an amorphous region of the spider silk [14]. Accordingly, the mechanical properties of spider and silkworm silk are largely dependent on the environmental humidity [15].

On the other hand, little is known about the effect of pressure on the structural transition of spider and silkworm silk. The effect of pressure on the natural spinning fiber formation process has been reported using the silkworm silk fibroin solution as a sample. They reported the pressure-dependent irreversible conversion from random coil to β-sheet of the silkworm silk fibroin solution. However, the approximate pressure value for the structural transition was not determined nor the pressure-dependent change of the mechanical properties [16]. On the other hand, the effects of pressure on the preformed fibers, in which crystalline β-sheets are already formed and the β-sheets predominantly oriented along the fiber axis, have also been demonstrated. Wide-angle X-ray scattering (WAXS) and Fourier-transform infrared (FT-IR) spectroscopy were employed to detect the shift of diffraction and vibration, respectively, of polyalanine in nanocrystals of spider silk fibers in response to the hydrostatic pressure [17,18]. The pressure-dependent WAXS and FT-IR spectroscopy of spider silk fibers demonstrated that the nanocrystals withstood hydrostatic pressures without the structural transition while the organization of amorphous phase increased reversibly [17,18]. The pressure-dependent structural change of silkworm silk fibers has also been recorded by WAXS and FT-IR [18]. The silkworm silk fibers underwent a reversible change of the lattice parameter under applied hydrostatic pressure [18]. Because the structural transition from random coil/helix to β-sheets occurs in the natural silk spinning system, the effect of pressure on structural transition should be monitored using the silk fibroin solution or film in an amorphous state.

In addition to water- and pressure-induced structural transition, elevating temperature also facilitate the β-sheet crystallization of silk molecular chains [19]. The nature of a crystallization process is governed by thermodynamic factors [20]. The temperature-dependent crystallization occurs because the entropy gain in the system by spatial randomization of the silk molecules has overcome the enthalpy change due to breaking and generating hydrogen bonds between silk molecular chains. In contrast, the pressure-dependent crystallization occurs because enthalpy gain originating from newly-formed hydrogen bonds between silk molecular chains compensates for the entropy loss due to the local alignment of silk molecular chains by compressibility.

In this study, we investigated the effect of pressure and water molecules on the structural transition of spider and silkworm silk film. The humidity-dependent structural and mechanical properties of silkworm silk film have been reported in previous studies [10,11], whereas those of native spider silk film have not been evaluated so far. We prepared the spider and silkworm silk film composed predominantly of amorphous region. The difference in reactivity to pressure and water molecules was investigated between spider and silkworm silk in terms of crystallization behavior. We also evaluated the effect of pressure on crystallization starting from a different conformational situation using hexafluoro-2-propanol (HFIP) that is known to arrange helical structure of spider and silkworm silk fibroin [19,[21], [22], [23], [24], [25]].

Section snippets

Materials

For these experiments, silkworm cocoons derived from Bombyx mori (B. mori) were bred at the experimental farm of Shinshu University. Spider dragline silk fibers were collected from Nephila clavata at a constant speed of 21 mm s−1, and were kept in lightproof boxes at a relative humidity (RH) ranging from 40% to 50% to prevent UV damage [26].

Preparation of silkworm silk films

Degummed B. mori silk films were prepared according to a previous report [10]. Briefly, silkworm silk cocoons were first degummed by boiling in a 0.02 M Na2

FT-IR and WAXS measurements of spider and silkworm silk films incubated at different pressure conditions

A hydrostatic pressure was applied to native spider and silkworm silk films to investigate the effect of pressure on the crystal structure of the silk fibroin. FT-IR spectroscopy has been used to monitor the characteristic stretching and bending vibrations associated with amide bonds derived from secondary structures of silk fibroin. On the other hand, WAXS measurement can detect the coherent interference of scattered X-rays passing through the crystal, enabling to monitor the crystal regions

Conclusions

The effect of pressure and humidity on the structural transition of silk fibroin was evaluated using spider and silkworm silk films consisting predominantly of amorphous regions. The crystal structure of silkworm silk films was found to be dependent on pressure, whereas the spider silk films did not show any irreversible structural change up to 980 MPa. The preformed helical structures induced by HFIP facilitated the formation of β-sheet structures of silkworm silk films. Meanwhile, water could

CRediT authorship contribution statement

Kenjiro Yazawa: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Supervision, Funding acquisition. Kosuke Hidaka: Formal analysis, Investigation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was financial support by JSPS Grants-in-Aid for Young Scientists Grant No. 18K14290. The WAXS measurement was performed under the approval of the Photon Factory Program Advisory Committee (Proposal No. 2019G002). The authors thank Dr. Jun Negishi of Shinshu University for hydrostatic pressure experiment.

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