Successive Stages of Amyloid-β Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization

Abstract

Self-assembly of amyloid-β (Aβ) peptides in human brain tissue leads to neurodegeneration in Alzheimer's disease (AD). Amyloid fibrils, whose structures have been extensively characterized by solid state nuclear magnetic resonance (ssNMR) and other methods, are the thermodynamic end point of Aβ self-assembly. Oligomeric and protofibrillar assemblies, whose structures are less well understood, are also observed as intermediates in the assembly process in vitro and have been implicated as important neurotoxic species in AD. We report experiments in which the structural evolution of 40-residue Aβ (Aβ40) is monitored by ssNMR measurements on frozen solutions prepared at four successive stages of the self-assembly process. Measurements on transient intermediates are enabled by ssNMR signal enhancements from dynamic nuclear polarization (DNP) at temperatures below 30 K. DNP-enhanced ssNMR data reveal a monotonic increase in conformational order from an initial state comprised primarily of monomers and small oligomers in solution at high pH, to larger oligomers near neutral pH, to metastable protofibrils, and finally to fibrils. Surprisingly, the predominant molecular conformation, indicated by 13C NMR chemical shifts and by sidechain contacts between F19 and L34 residues, is qualitatively similar at all stages. However, the in-register parallel β-sheet supramolecular structure, indicated by intermolecular 13C spin polarization transfers, does not develop before the fibril stage. This work represents the first application of DNP-enhanced ssNMR to the characterization of peptide or protein self-assembly intermediates.

Low-temperature dynamic nuclear polarization with helium-cooled samples and nitrogen-driven magic-angle spinning

“…The intrinsic spin-lattice relaxation rate (i.e., in the absence of paramagnetic doping) is affected by the aggregation state of the solute and its content of methyl groups [11], and can be easily be 0.2 s −1 or greater below 30 K [21]. It should also be noted that, under MAS at low temperatures, ε typically overestimates the enhancement of nuclear spin polarizations relative to their true thermal equilibrium values, largely due to MAS-induced depolarization in the absence of microwave irradiation [30].…”

“…The recent surge in DNP-related activity in the solid state NMR community was originally driven by results from the Griffin group, including their demonstration of large NMR signal enhancements at high fields in paramagnetically doped frozen solutions using gyrotron microwave sources [6, 7], their demonstration of efficient DNP at high fields via the cross-effect mechanism [8, 9], and their introduction of nitroxide-based biradical dopants [10]. Applications of DNP in studies of biological [11–17] and non-biological [18–20] systems are now being actively pursued in many laboratories.…”

“…Our NMR probe design is unusual in that we use cold helium for sample cooling and warmer nitrogen gas for MAS bearings and drive. Although our original plan was to use this probe without DNP, taking advantage of the Curie-law dependence of nuclear spin polarizations on temperature to enhance NMR signals by factors greater than ten [21], we subsequently decided to combine this probe design with microwave irradiation in order to achieve further signal enhancements by DNP [11, 28–31]. …”

High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles

“…It has been shown that DNP can drastically increase the sensitivity in solid-state NMR (ssNMR) experiments (Ni et al 2013;Sauvee et al 2013;Song et al 2006) and it has been successfully applied to several biological systems (Becker-Baldus et al 2015;Debelouchina et al 2013;Fricke et al 2015;Gupta et al 2016;Kaplan et al 2015;Koers et al 2014;Potapov et al 2015). Unfortunately, those experiments need to be carried out at cryogenic temperatures as the enhancement factor decreases with increasing temperature (Rosay et al 2010;Sauvee et al 2013;Zagdoun et al 2013).…”

Distinct Membrane Disruption Pathways Are Induced by 40-Residue β-Amyloid Peptides

“…with the co-existence of both on-pathway and off-pathway intermediates. It might be possible to distinguish these intermediate species using novel high-resolution techniques such as dynamicnuclear-polarization-based solid-state NMR spectroscopy (24). However, the presence of multiple pathways will further increase complexity and make high-resolution studies infeasible.…”