Download Molecular Mechanisms of Fluorescent Dye Interaction with Flemish

Molecular Mechanisms of Fluorescent Dye Interaction with Flemish Mutation in Amyloid Beta
Precursor Protein
Stephanie Phuong Le
Mentors: James Nowick, Kevin Chen
Point mutations in amyloid beta precursor protein (APP) such as the Flemish mutation cause early-onset
Alzheimer’s disease. Previous studies found that fibril-like assemblies of amyloid beta proteins can be
visualized and quantified using fluorescent dyes such as Thioflavin-T (ThT) and Congo Red. These dyes are
widely used, but their molecular mechanisms are unknown. Previous x-ray crystallographic study in the
Nowick lab have shown that a macrocyclic peptide model of amyloid-beta’s Flemish mutation (A21G) forms
fibril-like assemblies in a crystal lattice; therefore, co-crystallization with ThT and Congo Red may reveal dye
binding mechanisms. The macrocyclic peptide model of Flemish mutation was synthesized using solid phase
peptide synthesis. Following synthesis and purification, crystals were grown in previously established
conditions with ThT and Congo Red as additives. Co-crystallization with Congo Red produced crystals with
space group C2221 at 2.4 Å resolution, differing from the Flemish mutation without additive (P212121). ThT
co-crystallization had the same space group as the Flemish (P212121 at 2.0 Å resolution). Electron density
maps revealed no interaction of the Flemish mutation macrocyclic peptide with ThT. Additionally, partial
electron density near the tripeptide mimic, Hao, was observed in the electron density map of the Flemish
mutation macrocyclic peptide with Congo Red, indicating hydrophobic interactions. However, due to the
poor electron density, the presence of Congo Red in the crystal lattice cannot be concluded. Future studies
include crystal optimization to generate better x-ray diffracting crystals.