Small-Molecule Contacts to NMR-Invisible Aβ40 Oligomers Revealed by Pressure-Jump NMR and t 1-Noise Suppression

Abstract Soluble amyloid-β (Aβ) oligomers are widely implicated as neurotoxic species in Alzheimer’s disease but remain difficult to characterize because their transient, heterogeneous assemblies are effectively invisible to conventional solution nuclear magnetic resonance (NMR) spectroscopy. Hydrostatic pressure (∼2.2 kbar) dissociates Aβ40 oligomers into monomers with sharp, intense resonances. Here, we exploit this pressure-dependent behavior to detect weak intermolecular contacts formed between small molecules and the NMR-invisible oligomers and read them out after pressure-induced dissociation. Using dimethyl sulfoxide (DMSO) as a simple small-molecule probe, we combine pressure-jump NMR with NOE difference spectroscopy to obtain residue-resolved DMSO-to-Aβ40-oligomer contacts. Because the resulting NOE signals are very weak, their detection requires substantial suppression of t1 noise that is shown to arise primarily from minute FID-to-FID phase and frequency instabilities. We therefore introduced RePAIR (Reference-based Phase And frequency Instability Reduction), a software package that uses an internal reference resonance to correct per-FID phase and frequency errors in gradient-encoded, phase-modulated data sets, suppressing t1 noise and revealing otherwise hidden intermolecular NOEs. The resulting contact profile shows preferential DMSO interactions with the ordered, hydrophobic β-sheet core of the Aβ40 oligomers. More broadly, this approach provides a framework for obtaining residue-resolved information on weak small-molecule interactions with transient protein assemblies that are otherwise inaccessible to conventional solution NMR.