Silk fibroin

: a fundamental spin to biomaterials fabrication

Abstract

Silk fibroin (structural protein in silk fibres) exists in concentrated solutions within the insect, despite its general insolubility as a fibre. The transformation occurs when required. Incidentally, fibroin is polymorphic, with at least two isolable structures. Silk-I, found in the concentrated solution, thought to facilitate the transformation into Silk-II in the insoluble fibre. The first results chapter (Chapter 3) uses native-like silk fibroin (NLSF) as a surrogate for native fibroin to elucidate Silk-I structure and its assembly. It was found that fibroin heavy chain (FibH) from Bombyx Mori is multidomain with twelve β-solenoid domains linked by disordered domains. The β-solenoid structure fits various experimental data. Briefly, TEM showed a change from a globular appearance with globules (20 nm in diameter) to a fibrillar morphology (diameter below 5 nm) when pH decreased from 8 to 6—fitting the predicted solenoid. Equally, NMR analysis indicated that within the common tandem -GX- motif, every G residue shows dihedral angles (φ,ψ) of -135, 150 while other residue X (Ala, Ser, Tyr or Val) -100, 90. Matching very closely those from the acquired model. Docking in-silico experiments verified that N-terminal domain (NTD) drives assembly into supramolecular bottlebrush-like fibrillar structures upon reducing the pH below 7. Resulting in reversible sol-gel transformation, with the elastic component of the shear modulus (G’) going from about 2 Pa at pH 8 to about 200 Pa at pH 6, when concentrated at 7 wt%. Furthermore, it was found that the standard “regeneration” process hydrolyses the protein, particulaly NTD. The second results chapter (Chapter 4) exploits the understanding to fabricate biomimetic silk-like fibres. These, showed a transition from a hexagonal packing of solenoids -Silk-I- with a fibre X-ray reflection at ca. 17 Å, transforming to typical Silk-II as pulling stress increased. The fibres are fabricated by drawing a protein film formed at the water/air interface. The mechanical properties of the film are affected by pH, with the films showing an elastic component of the stiffness (k’) that increases from about 0.5 ± 0.2 N/m at pH 7 and 8 to about 1.4 ± 0.1 N/m at pH 6. The discovered method is versatile exemplified by producing mats and magnetic fibres. In the last results chapter (Chapter 5), biomorphic constructs are fabricated with NLSF for tissue engineering applications. It was found that there were limitations on the strength of composite materials fabricated with fibroin, given intrinsic softening in the presence of water. However, stronger materials are obtained from NLSF than RSF. Under similar fabrication, elastic moduli of 0.72 ± 0.08 and 0.10 ± 0.02 MPa at 10 % strain, respectively, for NLSF and RSF. Moreover, the pore architecture was controlled through controlling aggregation and ice-templating. Again, exploiting new understanding of the protein to postulate pathways involved in forming controlled gradations, replicating osteochondral tissues.

Date of Award	27 Sept 2022
Original language	English
Awarding Institution	University of Bristol
Sponsors	Orthox Ltd
Supervisor	Simon R Hall (Supervisor) & Sean A Davis (Supervisor)