Why is silk a tough fibre? Structure analysis of silk proteins elastomericity by small angle x-ray and neutron scattering
Lead Research Organisation:
University of Oxford
Department Name: Zoology
Abstract
Silks toughness and mechanical tunability does not depend solely on controlled processing but also on the self-organisation of the elastomeric precursor proteins of the silk. It appears that water hydration plays a major role for self-organisation and stability of silk proteins. Recent findings from our lab suggest that the elastomeric nature of silk protein in solution can be derived from a measure of their structural disorder and their relative content of glycine residues. In order to elucidate the mechanisms by which structural disorder and hydration are linked to promote elastomericity in silks we propose to visualise the processes as they occur using small angle scattering (Neutron and X-ray), polarised spectroscopy (FTIR/CD) and thermal analysis (DMA). This will allow us to examine experimentally and analyse in vitro (i) how silk proteins fold and assemble in solution; and, (ii) how hydration affects silk fibres mechanical performances. This project will provide the fundamentals to understand and quantify the dynamics of interactions between large structural proteins and their environment with application to artificial spinning of biopolymer fibres, and chemical control of biological aggregates, such as amyloids.
People |
ORCID iD |
| Fritz Vollrath (Principal Investigator) | |
| Ann Terry (Co-Investigator) |
Publications
Greving I
(2012)
Shear-induced self-assembly of native silk proteins into fibrils studied by atomic force microscopy.
in Biomacromolecules
Porter D
(2009)
Silk as a Biomimetic Ideal for Structural Polymers
in Advanced Materials
Koebley SR
(2015)
Silk Reconstitution Disrupts Fibroin Self-Assembly.
in Biomacromolecules
Greving I
(2010)
Small angle neutron scattering of native and reconstituted silk fibroin
in Soft Matter
Greving I
(2020)
Structural Diversity of Native Major Ampullate, Minor Ampullate, Cylindriform, and Flagelliform Silk Proteins in Solution.
in Biomacromolecules
| Description | Spiders and insects have independently achieved the controlled assembly of complex proteins from aqueous solutions into high performance fibres and adhesives, at ambient temperature and pressure. Inspired by this goal of silk-like material fabrication, under variable and sustainable conditions, we have to understand how assembly impacts the materials properties prior to and during processing. The structure of the protein, the building block of the fibre, before spinning in solution is still under debate. Whilst NMR suggests a random conformation of the protein, spectroscopic techniques indicate the presence of secondary structures. Addressing this contradiction we have demonstrated the suitability of small angle neutron scattering (SANS) and circular dichroism (CD) to investigate the morphology and structure of native silkworm fibroin at near in vivo conditions (Greving et al. 2010). Comparing native and artificial silk we observed significant differences in sizes, molecular weights, refolding and interactions. These observations question the validity of a presently widespread approach in silk analysis i.e. studying reconstituted silk with the goal to gain important insights into the initial structure and the mechanisms involved in the formation (storage and spinning) of native silks. Indeed we have shown that the two materials, native and regenerated silk are fundamentally different. Furthermore in collaboration with Prof. Schnniepps, College of William and Mary, Virginia, we have shown, using Atomic Force Microscopy (AFM), that native silk fibroin proteins exhibit a globular shape on a mica surface and not a rod-like shape as previously stated. Moreover we could visualise the assembly of the silk protein molecules under shear into a "bead of string like" assembly using spin coated silk samples (Greving et al. 2012). Interestingly we found two different fibrillar assemblies depending on protein concentration supporting our scattering findings. At very low protein concentrations the sheared proteins self assembled to very thin fibrils with diameters close to reported values from fibrils in silk fibres. Interestingly spiders spin several different types of silk fibres depending on their function in the web. Each of these silks consists of a different protein sequence and has very different mechanical properties. Therefore, spider silk is an ideal model system to study the structure function relationship in silks. Small angle scattering has again elucidated the shapes and sizes of the proteins in solutions, revealing key differences in structures between the different spider silks. A paper is in preparation. Underlying the formation of silk fibres is the beta-transition. It is accepted that to successfully form fibres with the relevant hierarchical assembly there is a need of a controlled transition to beta-sheet structures. The mechanism of this transition remains unclear. One reason for this lack of understanding stems from the non-equilibrium nature of this transition. It is therefore our aim to investigate the dynamics of the beta-transition in the picoseconds to milliseconds time regime to derive the motions responsible for beta-structures formation or inhibition. Two techniques have been implemented: Quasi Elastic Neutron Spectroscopy (QENS) and Cryo-Fourier Transform Infrared (cFTIR). Both techniques rely on temperature scans that sequentially activates motions and/or chemical vibrations. QENS informs on the ps to ns motion of molecules, whereas FTIR informs on the vibrational state of the molecules, ie structural content was of interest. To be able to compare the techniques, we used silk films. By gently casting silk solutions into films, the initial structure state of the solution is conserved with the added advantage that water content can now be controlled. A detailed investigation of the films from native silk fibroin and reconstituted silk fibroin suggests that water content affects the overall dynamics of silk proteins in the films and that the beta-transition is affected by the presence of "nano-second" water. Two papers are in preparation. |
| Exploitation Route | Through a thorough understanding of the shape, size and interactions of silk proteins in solution, the folding and assembly steps of the protein into a fibre can be considered. In our comparison of native and regenerated silk solutions, we have identified key variables in the solution scattering, for example aggregate size and polydispersity which directly affect the ability to spin a fibre and ultimately the fibre's mechanical performance. It is known that pH and ion changes as well as shear influence these structures. Small angle scattering applied to such systems allows the monitoring of a protein solution for its storage and spinnability. This has application not just for the spinning of silks but in the wider context of processing other naturally occurring polymers. Producing sustainable fibres with tailored mechanical performance would be the long term goal. The relating of the shapes, sizes and flexibilities of the fundamental building blocks to the final fibre's mechanical properties has always been a seductive goal for polymer scientists, ie the search for structure properties relationships. In our work on spider silk, the linking of mechanical properties e.g. extensibility/elasticity, to the shape of the protein in solution has wide applicability for the understanding of these fundamental relationships. Additionally, we have established a protocol for handling high molecular weight 'truly' native proteins without aggregation under two near in vivo conditions and measuring them by solution small angle scattering. This is a novel approach, since proteins are usually purified and treated chemically before the experiment. The ability to make and interpret such measurements allows this useful technique to be extended to the investigation of other native proteins in conditions as close to in vivo as possible. |
| Sectors | Other |
| URL | http://users.ox.ac.uk/~abrg/spider_site/ididdens.html |