Chih-Lung Shen, PhD 1995

Thesis Title: Aggregate structure and kinetics of fibrillogenesis of Alzheimer's beta-amyloid peptide

Aggregate structure and kinetics of fibrillogenesis of synthetic β-amyloid peptides (Aβ) were investigated. The predominant form of Aβ(1-28) was apparently tetramer. Aggregates containing amyloid fibrillar structures were observed at concentrations of approximately 0.3 mg/ml or greater. Concentrations of 0.5 mg/ml or greater were necessary for aggregation to be detectable by light scattering. The aggregation rate was much faster at physiological salt concentrations than at lower ionic strength. The data indicate that sample preparation method and sample history influence fibril size and number density. The size and flexibility of fibrils from Aβ(1-39) were determined on various pre-incubation conditions. The molecular weight and linear density of the fibrils increased with increasing pre-incubation time in 0.1% trifluoroacetate. The results show that the fibrils were semi-flexible chains, and that the fibril flexibility decreased with increasing pre-incubation time in 0.1% trifluoroacetate. Concomitant with the decrease in fibril flexibility, there was a change in phase behavior from precipitation to gelation. Solvent effects on Aβ(1-39) aggregation were investigated.Aβ(1-39) had no β-sheet content in pure dimethyl sulfoxide, but one-third β-sheet in 0.1% trifluoroacetate and two-thirds β-sheet in 35% acetonitrile/0.1% trifluoroacetate. Peptide stock solutions were diluted into phosphate-buffered saline, and fibril growth was followed by light scattering. The growth rate was substantially faster when the peptide was predissolved in 35% acetonitrile/0.1% trifluoroacetate than in 0.1% trifluoroacetate, 10% dimethyl sulfoxide, or 100% dimethylsulfoxide. Differences in growth rate were attributed to changes in the secondary structure of the peptide in the stock solvent. The kinetics of amyloid fibrils were proposed as consisting of dimerization of monomers to form intermolecular β-stranded structure, association of dimers to create multimeric nuclei, and aggregation of nuclei by stacking of βP-strands to form amyloid fibrils. The simulation indicates that initial conformation of the peptide may be a key step to reduce fibril production.