Spider silk proteins

This is a collaborative project with the University of Bayreuth.

The bio-engineered spider silk protein eADF4(C16) is based on one component of the dragline silk fibers of the European garden spider, namely the consensus sequence of the core domain of Araneus diadematus fibroin 4 (ADF4). The protein eADF4(C16) is nontoxic, and materials made thereof are biodegradable, show a high tensile strength and good elasticity. Furthermore, they feature adjustable interactions with cells and tunable morphologies. The protein can be processes into fibrils, fibers, particles, capsules, films, and hydrogels. Based on this extraordinary versatility, it can be applied in numerous application fields. Consequently, we want to better understand the underlying mechanisms (as will be explained in the following) to extend the scope of application and to optimize already existing ones.

Since it is known that especially hydration water is of fundamental importance for the functionality of macromolecules, we are especially interested in these dynamics, for which quasi-elastic neutron scattering (QENS) is an ideal measurement technique. Due to the high neutron cross-section of hydrogen atoms, QENS is especially suited for the investigation of the diffusion of bulk and hydration water as well as protein dynamics (e.g., fast side chain relaxations/vibrations) and covers the relevant time and length scales. In a previous study, it has already been shown that QENS can be used to establish a link between the molecular mobility and macroscopic mechanical properties of spider silk proteins (although in a different spider silk system).

 

Figure 1: Schematic nucleation and self-assembly process of eADF4 proteins. A disordered eADF4 monomer (A) transforms into antiparallel ß-sheets (B,C), before forming a nucleus (D) and eventually fibrils (F). (Figure is taken from M. Humenik, M. Magdeburg, Th. Scheibel, ‘Influence of repeat numbers on self-assembly rates of repetitive recombinant spider silk proteins’, Volume 186, Issue 3, June 2014, Pages 431-437).

With the existing fundamental understanding of the structural properties of eADF4(C16) regarding nucleation and self-assembly, we now want to address the dynamical properties. In detail, we want to understand the role and the dynamics of water, from the hydration of eADF4(C16) to the bulk water during the self-assembly process. By heavy water substitution we plan to investigate also eADF4(C16) side chains/groups dynamics and their impact on nucleation and nanofibril formation.

If you are interested in investigating self-assembly process of spider silk proteins (for now in solution, but we want to extent our research to films and hydrogels) in order to understand their superior macroscopic properties, send an email to lucas.kreuzer@frm2.tum.de. Currently, there are open master and bachelor positions.

The macroscopic properties of the spider silk materials made of eADF4(C16) proteins are mainly governed by their self-assembly process, which was found to be based on a nucleation dependent mechanism. The eADF4(C16) proteins are disordered in solution and form nuclei, from which they self-assemble into ß-sheet rich nanofibrils (Figure 1). This structure formation is induced e.g., by exchanging 'salting-in' ions (e.g., Na+, Cl-) for 'salting-out' ions (Ca2+, PO43-). Increasing the temperature, accelerates nucleation and self-assembly by providing more kinetic energy for assembly. While the structural properties of the self-assembly process are relatively well understood, less is known about the dynamics of the protein and hydration water during the nuclei formation and self-assembly. So far, it is assumed that the internal protein and solvent dynamics play a major role during nucleation. It might be a process that thermodynamically freezes at a certain point. Therefore, it is important to understand how the dynamics are affected by the nuclei structure (and later on during the self-assembly process, the nanofibril structure) as well as by external parameters such as temperature.