TY - JOUR TI - Mechanism of protein aggregation leading to amyloid formation DO - https://doi.org/doi:10.7282/T3MS3RS5 PY - 2012 AB - Amyloid deposition has been observed in more than 20 diseases. Each amyloid-related disease has a particular precursor protein or peptide that converts from itsnative state to insoluble cross-βamyloid assemblies. Understanding the amyloid aggre-gation mechanism is the basis for development of a rational strategy for preventing theaggregation cascade events. This dissertation contributes to the elucidation of amyloidaggregation mechanism by characterizing the aggregation intermediates with comple-mentary biophysical techniques and clarifying the fluorescence mechanism of a standardamyloid probe, thioflavin T (ThT). In the first part of this dissertation, aggregation state distribution was determinedat single-molecule level with atomic force microscopy (AFM), by development of aparticle size analysis package for AFM images. Combining the aggregation state dis-tribution from AFM and dynamic light scattering (DLS) measurements, a quantita-tive reservoir-nucleation model forβ-lactoglobulina(β-LGa) amyloid formation wasproposed based on kinetic simulation. The model successfully predicted the resultsof 1-anilino-8-naphthalene sulfonate (ANS) fluorescence and seeding experiments. Anaggregation free-energy landscape was constructed based on the simulation. A clas-sification scheme for oligomeric species was proposed to evaluate their roles during aggregation, based on their locations on the aggregation free-energy landscape. Dif-ferent types of oligomers were related to the amyloid cascade hypothesis and the toxicoligomer hypothesis for amyloid-related diseases. The second part of this dissertation focuses on fluorescence mechanism of ThT,which is a standard tool for amyloid fibril detection and aggregation kinetic studies.DLS was used to probe the aggregation state of ThT in solutions of different condi-tions. The micelle model was disproved by showing the absence of ThT micelles insolution. The exciton model was disproved by fit of the concentration dependence ofThT fluorescence intensity to a fluorescent monomer model. Enhanced quantum yieldwas observed for surface-bound ThT in both bulk and single-molecule fluorescence mea-surements, with substantial shift in the excitation spectra, compared with fibril-boundThT. The presence of surface-bound ThT fluorescence calls for re-evaluation of reportedThT photophysical properties in solutions of low viscosity, which are likely dominatedby the surface effect. Similar lifetime features were observed for surface-bound andfibril-bound ThT. These results revealed that ThT bound to amyloid fibrils rigidly asit bound to the surface. Interaction of ThT withα-Synuclein (αSyn) fibrils was studiedwith single-molecule fluorescence microscopy. The single-molecule fluorescence polar-ization images showed that ThT bound toαSyn fibrils with its long axis parallel to thefibril axis. KW - Chemistry and Chemical Biology KW - Amyloid beta-protein KW - Aggregation (Chemistry) KW - Amyloid LA - eng ER -