Atomic force microscopy of self-assembled biomolecular structures and their interaction with metallic nanoparticles

We applied AFM to study biomolecular wires, both out of interest in thei r biological functions and in the framework of nanotechnology based fabr ication. We have focused on two different kinds of protein wires: Insuli n fibrils and microtubules. Microtubules are an important constituent of the cytoskeleton and fulfill multiple vital functions in the cell. Insu lin fibrils on the other hand are amyloid fibrils without a clear biolog ical role, but with intriguing polymerization properties that make them an interesting model system for the formation of amyloid fibrils. Both p rotein wires are formed by a self-assembly process and have robust struc tural properties, which make them interesting candidates to act as a tem plate for the formation of metal wires. A first part of this thesis deals with the characterization of the compl ex substructure of insulin fibrils. The multipathway polymerization proc ess of insulin results in fibrils that can be divided into two groups, e ach characterized by a different structural motif: Helical fibrils compo sed of protofilaments, and chains of rod-like segments. We observed both types of structures with AFM in great detail. The helical fibrils exist with a large variety of periodicities and heights, indicating that they can be formed by different numbers of protofilaments. These protofilame nts cannot directly be distinguished in the fibrils and their number has to be inferred from the height of the fibril. By studying defective hel ical fibrils that have frayed into their individual protofilaments, we w ere able to distinguish the individual protofilaments, confirming the fi bril substructure. This led to the observation that the protofilaments t hat constitute a helical fibril exhibit a periodicity themselves, which is not observed in protofilaments that are not involved in the formation of a helical fibril. The assembly of the fibrils was observed to occur in a hierarchical way: Two protofilaments twist around each other to for m a pair, and two pairs then twist around each other to form a 4-filamen t fibril. Another important assembly pathway leads to insulin fibrils consisting o f rod-like segments that are associated in a head to tail fashion. We we re able to elucidate this polymerization pathway with high-resolution AF M. Strikingly, all of the rod-like segments are uniform in size within o ne fibril, but differ greatly in size between different fibrils. This in dicates the importance of lateral interactions in