An interdisciplinary study of the mechanical and dynamic properties of α-solenoid repeat proteins

Tandem-repeat proteins differ from globular proteins, both in their biophysical characteristics and in how they interact with their respective partners, yet they comprise nearly one third of the human proteome and are central to many cellular processes and disease phenotypes. Repeat proteins have been shown to behave like nano-sized biological springs: they are flexible, dynamic and elastic. Using coarse-grained models, I discuss how intrinsic flexibility may arise in repeat proteins and how it could be crucial for the biological function of two systems: PR65, the scaffold protein of the protein phosphatase 2A, and Rap proteins, which are involved in quorum sensing. To interrogate α-solenoids at physiologically relevant forces, I performed force spectroscopy experiments using a dumbbell optical tweezers set up for which it is necessary to attach the relevant protein to DNA. As PR65 is not amenable to current DNA-protein attachment methods, I developed a protocol that allows the cross-linking of DNA oligos to proteins using bio-orthogonal chemistry. I then explored the mechanics of the natural repeat protein, PR65, and a series of designed TPR proteins. I find that these proteins respond to forces in a novel manner which is significantly different to what has been previously reported. TPRs unfold and refold in quasi-equilibrium at constant force without energy loss. In contrast, PR65 unfolds in separate domains and refolds along an entirely different pathway. In conclusion, my doctoral studies explore the physical characteristics of repeat proteins in more detail. Using both experimental and computational techniques, I provide unique perspectives on different aspects of their mechanical and dynamic capabilities. This work provides the basis for future investigations of how such interesting mechanical behaviour relates to biological function. Are repeat proteins simply a molecular recognition platforms for their multitude of binding partners, or do their mechanics matter in a biological context?