Molecular assembly routes for biological materials

Biological materials present a wide variety of functional properties that provide inspiration for the development of new synthetic biomaterials. For example, spider silk has been extensively studied due to its exceptional toughness and adhesion properties. The exact pathway leading to the correct molecular assembly, which is the foundation of its extraordinary mechanical properties, is a complex process and not fully understood. This is one of the main reasons why the properties of synthetic silk materials are inferior to natural ones. This thesis attempts to answer the question of how recombinant spider silk proteins can be brought together in soluble form in a way that enables the interactions and microstructures that yield strong materials. The possible role of liquid-liquid phase separation, also known as coacervation, in the correct molecular assembly is of special interest. It has been suggested that many other natural organisms utilize coacervation as an intermediate step to produce functional materials, so gaining more information about its role on molecular assembly would be of great value to materials science. In publication I the coacervation of a recombinant spider silk-like protein, CBM-eADF3-CBM, was studied as an intermediate step for fiber assembly. Depending on the conditions, two kinds of coacervates were obtained: liquid-like and solid-like coacervates, but only the liquid-like ones enabled fiber formation and thus were more interesting for further studies. Publication II focused on the effect of molecular crowding on coacervation of CBM-eADF3-CBM. Dextran was used as an inert crowding agent, which enabled studying phase separation in lower protein concentrations, and in a controlled set-up. Furthermore, this set-up enabled thermodynamic studies of the system, which provided insight into the coacervation mechanism. Publication III presents the adhesion properties of CBM-eADF3-CBM in a system with delignified cellulose. Delignified cellulose proved to be a well-functioning model system to study silk as an adhesive, and CBM-eADF3-CBM formed a strong adhesive system with it. A method to produce native-sized recombinant silk proteins was developed in publication IV, and the effect of protein length on coacervation was studied. Post-translational in vitro ligation with the SpyCatcher2-SpyTag protein-peptide pair proved to be a robust way to produce native-sized silk proteins with high yield and solubility. Lengthening the protein lowered the critical concentration needed for coacervation and provided insight into the molecular interactions that lead to phase separation. To conclude, the results show that engineered recombinant silk proteins can be used to gain a deeper understanding of the molecular assembly behind the material formation process. Coacervation proved to be an important step in material formation and the methods to study it developed in this thesis increased the knowledge of molecular assembly processes.