Analytical ultracentrifugation as a tool to understand interactions in biomolecular materials

The modern world dictates new rules and requirements for materials used in various applications. The development of human society has reached a point where, in addition to the requirements for good functional properties, the requirements for safety and sustainability of the materials are also increasing. Bio-based materials satisfy all these criteria. They are functional, sustainable, renewable and have a good mechanical properties. One of the most promising representatives is spider silk, the fibers of which are formed on the basis of triblock proteins. Spider silk stands out for its excellent mechanical properties. However, to achieve the properties similar to native spider silk using artificial analogues, a deep understanding of processes and interactions at the molecular level is required. In overall this is the true for any materials. The lack of the knowledge about molecular interactions is critical and significantly complicates the material development. It can be compared to forging a sword when one knows nothing about metal processing. Theoretically, it is possible to forge a sword that looks like a sword. Applying a lot of efforts, one even can make it beautiful. But most likely it will be either fragile or too soft, since the person does not know how to process it correctly. Applying even more efforts, after many attempts, one can find the conditions under which the sword would have the desired properties, but what if after sword it is needed to forge a horseshoe, for example. There could be different requirements for properties and all the process of searching the best conditions will be repeated all over again. However, knowing the rules of hardening and alloying of iron, everything would be much simpler. Absolutely the same situation occurs with biomaterials and intermolecular interactions. This work devoted to certain gaps in the understanding of protein interactions at the molecular level, how they affect intermediate states such as liquid-liquid phase separation (LLPS), shows the importance of understanding the interactions, and also demonstrates the capabilities of Analytical Ultracentrifugation (AUC) in combination with other techniques for material science application. Publication 1 demonstrates the effect of terminal domains on LLPS of engineered three-block spider silk proteins. The discovered patterns will serve for creation of coacervates with controlled properties. Publication 2 is devoted to the stability of NT-2Rep-CT, studying the component composition of NT-2Rep-CT solution and demonstrating the influence of the history of the sample on its properties and the observed state. Publication 3 investigated the dimerization of the CBM molecule and tested various methods for AUC data analysis. In publication 4, the shape of molecule of the CBM-AQ12-CBM protein was investigated using a combination of AUC and MD simulation. A similar approach was used in publication 5, where the polypeptides PLL and PGA were the objects of study. Solution composition, molecular weights and shape were determined.