Biophysical Investigations of the Aβ Aggregation Process

The presence in patient’s brain tissues of neuritic plaques containing Aβ aggregates is one of the pathological hallmarks of Alzheimer’s disease (AD), and Aβ aggregates have been implicated in the disease mechanism. These facts have inspired a large number of biophysical and structural studies on Aβ behavior over the last 25 years. Much remains to be learned, but there are a number of barriers to progress, including the challenges of making and manipulating these peptides and understanding their aggregation behavior. This thesis describes an improved method for the chemical synthesis of highly aggregation prone peptides like Aβ, insights into some previously unrealized limitations of a widely used “disaggregation” procedure for making high quality monomer solutions, and two fundamental studies on aspects of Aβ self-assembly. The improved synthesis method involves reversible addition of Lys residues to the C-terminus of the peptide during solid phase synthesis, which we show improves the synthetic yield and also improves the chromatographic behavior of the peptide during purification. The new knowledge about disaggregation reveals that a method involving sequential treatment of peptides with trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP), while very effective with Aβ40, can alter the self-assembly of Aβ42, compared with an alternative protocol, and introduce highly stable oligomers that may possess substantial toxicity. In one fundamental study, we show that the minor brain form, Aβ43, aggregates more slowly than Aβ42 to make amyloid fibrils that are highly inefficient at seeding Aβ42 monomers. In another study, we describe the surprising result that amyloid fibrils of D-Aβ40 can seed L-Aβ40 monomers, and vice versa, suggesting a curious lack of structural discrimination to the prion-like propagation of Aβ amyloid in vitro. The results add to our knowledge of Aβ amyloid assembly and how it can best be studied in the laboratory

Hydrogen/Deuterium Exchange Mass Spectrometry Provides Insights into the Role of Drosophila Testis-Specific Myosin VI Light Chain AndroCaM

In Drosophila testis, myosin VI plays a special role, distinct from its motor function, by anchoring components to the unusual actin-based structures (cones) that are required for spermatid individualization. For this, the two calmodulin (CaM) light-chain molecules of myosin VI are replaced by androcam (ACaM), a related protein with 67% identity to CaM. Although ACaM has a similar bi-lobed structure to CaM, with two EF hand-type Ca2+ binding sites per lobe, only one functional Ca2+ binding site operates in the amino-terminus. To understand this light chain substitution, we used hydrogen–deuterium exchange mass spectrometry (HDX-MS) to examine dynamic changes in ACaM and CaM upon Ca2+ binding and interaction with the two CaM binding motifs of myosin VI (insert2 and IQ motif). HDX-MS reveals that binding of Ca2+ to ACaM destabilizes its N-lobe but stabilizes the entire C-lobe, whereas for CaM, Ca2+ binding induces a pattern of alternating stabilization/destabilization throughout. The conformation of this stable holo-C-lobe of ACaM seems to be a “prefigured” version of the conformation adopted by the holo-C-lobe of CaM for binding to insert2 and the IQ motif of myosin VI. Strikingly, the interaction of holo-ACaM with either peptide converts the holo-N-lobe to its Ca2+-free, more stable, form. Thus, ACaM in vivo should bind the myosin VI light chain sites in an apo-N-lobe/holo-C-lobe state that cannot fulfill the Ca2+-related functions of holo-CaM required for myosin VI motor assembly and activity. These findings indicate that inhibition of myosin VI motor activity is a precondition for transition to an anchoring function