Masters Theses

Date of Award

5-2005

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Ronald Wetzel

Committee Members

Chris Dealwis, Elizabeth Howell, Stephen Kennel

Abstract

Amyloid fibrils have been implicated in numerous human diseases including Alzheimer's disease (AD). The fibrils found in AD are formed by the misfolding and aggregation of the Aβ peptide. Amyloid fibrils are potentially important causative agents, but high-resolution structural information is not yet available. This research analyzes amyloid fibril structure via two different experimental routes. In one approach, ELISA techniques were utilized to study the binding of available anti-amyloid antibodies to single-residue proline and alanine mutant fibrils of Aβ. Binding to some cysteine and modified cysteine mutants was also tested. These data were compared to fibril stability, hydrophobicity change, ThT binding, and changes in fibril protection. There appeared to be no correlation to these, except for weak correlation to ThT binding. The results at specific residues did stand out as significant. WO1 and PGA1 binding appeared to be affected by both charge and structural changes at specific residues. For WO1, mutations at residues 16, 27, and 28 were shown to have the greatest impact on binding. For PGA1, mutations at residues 27, 28, 30, 31, and 36 were shown to do so. Upon closer examination, the enhanced binding to mutants at residues 16 and 36 appeared to be due to structural changes. Binding to mutants at residue 27 could be due to structural changes, but these results are not entirely clear. Binding to mutants at resides 28, 30, and 31 appeared to be due to charge differences. Binding of WO1 was found to be sensitive to salt and pH conditions suggesting electrostatic interactions are important to binding. Mutations in the Aβ sequence have a strong impact on binding. The basis of these results is still not entirely clear, but they could be important clues to the nature of fibril structure or the fibril epitopes recognized by the antibodies. Since the number of existing antibodies reacting with fibrils is limited, phage display techniques were employed to develop new binding reagents with a larger range of binding properties for fibrils. A library of antibody fragments was constructed in a phage display system and was selected for members with fibril-binding properties. Although the library was validated, to date, no suitable antibodies have been isolated.

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