(162aw) Material and Structural Characteristics of Reversibly Self-Assembled Oxyntomodulin and Aib2-Oxyntomodulin Fibrils Revealed By AFM and Cryo-EM

The extended release therapeutic potential of fibrillar structures of oxyntomodulin (Oxm) has recently been demonstrated to be a promising strategy to design novel formulations to treat obesity and type-2 diabetes. Despite the fact that the parameters controlling the self-assembly process of such peptides have been identified, the fundamental physical interactions underpinning self-assembly and controlling stability of their fibrillar form remains to be resolved. A key element to completing our understanding of these physical interactions is the elucidation of the near-atomic level structure of the fibrils and the determination of their mechanical properties. In this work we report the detailed mechanical and structural characterization of the individual fibrils by atomic force microscopy (AFM) and cryogenic electron microscopy (cryo-EM). The reversibly self-assembled multi-filament fibrils, formed by free Oxm or Aib2-Oxm, were shown to have a mechanical stiffness, in terms of Young's modulus of elasticity, of 0.6 - 1.7 GPa, comparable to those of natural assemblies found within cells, such as actin and tubulin (0.3 - 1.2 GPa), which were previously found to be the reversible self-assembled fibrils, in vivo. This finding suggests the possibility of the controlled dissociation mechanism of Oxm and Aib2-Oxm fibrils, in vivo. Moreover, the comparisons of elastic moduli of Oxm and Aib2-Oxm fibrils with those of previously studied materials signify the fact that both dense hydrogen-bonding network and amphiphilic (i.e., hydrophobic and hydrophilic) interactions are comparably responsible for stability of Oxm and Aib2-Oxm fibrils. By combining data from both AFM and cryo-EM images, alongside modelling based on semiflexible polymer theory, we can conclude that the fibrils have a ribbon-like multifilament structure as opposed to a closely packed multifilament structure as previously speculated.