Probing of Amyloid Aβ (14-23) Trimers by Single-Molecule Force Spectroscopy.

Self-assembly and aggregation of amyloid peptides, such as Aβ(1-40) and Aβ(1-42), lead to the development of Alzheimer disease and similar neurodegenerative disorders associated with protein aggregation. The structures of large aggregates, specifically fibrils, are well characterized. However, our understanding about the structure of oligomeric forms of amyloids is incomplete and needs to be expanded, particularly given the finding that oligomeric rather than fibrillar amyloid morphologies are neurotoxic. This lack of knowledge is primarily due to the existence of transient oligomeric forms that require the use of non-traditional approaches capable of probing transiently existing amyloid forms. We have recently developed the Single-Molecule Force Spectroscopy (SMFS) approach enabling us to probe dimeric forms of amyloids. These studies suggest that the assembly of amyloid proteins into dimers leads to extremely stabilized amyloids in non-native, misfolded states [1]. Herein, we applied the SMFS approach to probe amyloid trimers. We used the Aβ(14-23) segment of Aβ42 protein that is responsible for full-size protein aggregation. The dimerization of this peptide was recently characterized [2]. The dimeric form of Aβ (14-23) was assembled by the use of a tandem Aβ(14-23)-YNGK-Aβ(14-23), in which the YNGK motif between the two Aβ(14-23) monomers makes a β turn to form a hairpin loop with an antiparallel arrangement of Aβ(14-23) monomers[3]. The Aβ(14-23) monomer was tethered to the AFM tip, and trimers were formed by approaching the tip to the mica surface on which the Aβ(14-23)-YNGK-Aβ(14-23) dimer was immobilized via a polyethylene glycol tether. We identified trimers by rupture forces that were considerably larger than those for dimers. Models for the trimer assembly process are discussed.