| Amyloid β-protein (Aβ) is believed to cause neurodegeneration in Alzheimer's Disease. Aβ can aggregate into various assemblies including oligomers, protofibrils and fibrils. Aβ oligomers are the most toxic assemblies, suggesting that targeting inhibition of their formation is a potential therapeutic strategy. The oligomerization pathway and the oligomeric structures are poorly understood due to the metastable nature of Aβ; therefore, I seek to understand the oligomerization mechanism by probing the structures of Aβ oligomers using mass spectrometric techniques.;In order to isolate and study the individual oligomeric assemblies, photo-induced cross-linking of unmodified protein (PICUP) was used to stabilize the oligomeric structures. Since the smallest neurotoxic oligomer is dimeric in the AD brain, I investigated the structure of Aβ dimer. Cross-linking, radiolytic labeling and mass spectrometry were used to probe the structure of Aβ dimer. Aβ40 dimer was cross-linked at the Tyr10 region, probably through di-tyrosine interaction. In comparison to Aβ40 monomer, the dimer was more accessible to solvents at residues (29-40) and less accessible at (16-28) of Aβ40. These results suggest that Aβ40 dimerizes through interaction at Tyr10 region, and is stabilized by interaction in the (16-28) region.;MS can also be used to determine the site(s) of interaction between small molecules and proteins. A compound in the molecular tweezer (MT) class was found to protect against Aβ-induced toxicity. Activated ion electron capture dissociation (aiECD) with Fourier transform ion cyclotron resonance (FT-ICR) MS found three sites where MT interacts with Aβ. Because the two variants of MT (CLR01 and CLR02) have different affinities for lysine and arginine, their preferred binding sites differ: CLR01 prefers to bind to K16, and CLR02 prefers R5. MT's binding preferences reflect the relative accessibility of each site. K28 is the least accessible of all three binding sites because it is involved in an intra-molecular salt bridge interaction.;Ion mobility spectroscopy (IMS) combined with proton transfer reactions manipulate the charge states of the multiply charged ions in the gas phase. This allows comparison of the differences between the cross-sectional areas of the cross-linked dimers and the non-cross-linked dimers. The ability to observe the differences in IMS is the first step in differentiating between cross-linked dimers vs. non-cross-linked Aβ40. Charge transfer reactions could reduce multiply charged ions into lower charge states, where the collapse of the proteins can occur in gas phase. In the case of Aβ, the drift times of the cross-linked dimers and non-cross-linked dimers were found to be similar at 5+ charge states, but the differences in drift times became more apparent when the charge states were reduced to 3+ and 4+. Aβ non-cross-linked dimers have smaller cross-sectional areas than Aβ cross-linked dimers. This demonstrated the inability for Aβ40 cross-linked dimers to collapse, unlike the non-cross-linked dimers, because of the constraints from cross-links. |
