Opportunities and challenges in the use of pressure-jump NMR to study Aβ and protein oligomerization.

Extracellular plaques comprised of amyloid-β (Aβ) are a defining hallmark of Alzheimer's disease. However, it remains debated whether these insoluble fibrils or the soluble oligomers that precede them are the toxic species responsible for pathology. Aβ oligomers are difficult to study by conventional biophysical techniques because of their transient nature and heterogeneous size. While Aβ40 oligomers are invisible to solution nuclear magnetic resonance (NMR) spectroscopy due to their slow tumbling, the intrinsically disordered monomeric form is readily detected by solution NMR. Under appropriate conditions, oligomers rapidly form at atmospheric pressure and can be dissociated by the application of ∼2.5 kbar hydrostatic pressure. By incorporating pressure jumps within NMR experiments, features of the NMR-invisible oligomers can be probed indirectly by detecting the NMR-visible monomer. This chapter discusses operation of a home-built pressure-jump apparatus and its application to measuring oligomeric relaxation times, chemical shifts, and 1H-1H NOE transfers. Aβ40 oligomers have particularly short 15N T2 relaxation times and long T1 relaxation times, especially for residues V18-A21 and I31-L34, indicating that these oligomers contain highly ordered regions that tumble slowly in solution. 1H-1H NOEs further reveal that Aβ40 oligomers contain hydrogen bonds between V18 and M35, between F20 and G33, and between E22 and I31. Zn2+ ions accelerate oligomer formation and promote homogeneous, rigid assemblies with reduced internal motion. Some of the technology development was carried out using Amelotin, an intrinsically disordered protein that also forms pressure-sensitive oligomers but is less prone to irreversible fibril formation and more amenable to large-scale perdeuterated expression.