Atomic-resolution dynamics on the surface of amyloid-β protofibrils probed by solution NMR

Abstract

Exchange dynamics between molecules free in solution and bound to the surface of a large supramolecular structure, a polymer, a membrane or solid support are important in many phenomena in biology and materials science. Here we present a novel and generally applicable solution NMR technique, known as dark-state exchange saturation transfer (DEST), to probe such exchange phenomena with atomic resolution. This is illustrated by the exchange reaction between amyloid-β (Aβ) monomers and polydisperse, NMR-invisible ('dark') protofibrils, a process of significant interest because the accumulation of toxic, aggregated forms of Aβ, from small oligomers to very large assemblies, has been implicated in the aetiology of Alzheimer's disease. The (15)N-DEST experiment imprints with single-residue-resolution dynamic information on the protofibril-bound species in the form of (15)N transverse relaxation rates ((15)N-R(2)) and exchange kinetics between monomers and protofibrils onto the easily observed two-dimensional (1)H-(15)N correlation spectrum of the monomer. The exchanging species on the protofibril surface comprise an ensemble of sparsely populated states where each residue is either tethered to (through other residues) or in direct contact with the surface. The first eight residues exist predominantly in a mobile tethered state, whereas the largely hydrophobic central region and part of the carboxy (C)-terminal hydrophobic region are in direct contact with the protofibril surface for a significant proportion of the time. The C-terminal residues of both Aβ40 and Aβ42 display lower affinity for the protofibril surface, indicating that they are likely to be surface exposed rather than buried as in structures of Aβ fibrils, and might therefore comprise the critical nucleus for fibril formation. The values, however, are significantly larger for the C-terminal residues of Aβ42 than Aβ40, which might explain the former's higher propensity for rapid aggregation and fibril formation.

Figure 1. ¹⁵N Dark-state exchange saturation transfer (DEST) and ΔR₂ for Aβ40

Examples of normalized cross-peak intensities as a function of frequency offset from the ¹⁵N carrier (118.5 ppm): a, Glu-3 in the 50 µM Aβ40 sample (RF field strength of ¹⁵N saturation pulse = 170 Hz); b, c and d, Glu-3, Leu-17 and Asn-27, respectively, in the 270 µM Aβ40 sample with ¹⁵N saturation at RF field strengths of 170 (orange circles) and 350 (blue circles) Hz. The solid line in (a) is the calculated saturation profile for a ¹⁵N spin with the experimentally determined relaxation rates for monomeric Aβ40. The dashed and solid lines in (b)–(d) are the best-fits to an exchange model with a single protofibril-bound state (cf. Fig. 2a) and to a model incorporating residues tethered and in direct contact with the protofibril (cf. Fig. 2b), respectively. The s.d. of the DEST experimental data points is approximately equal to the size of the circles. e, Observed (black filled-in circles; error bars: 1 s.d.) versus calculated ΔR₂ values for the first (grey circles) and second (blue circles) models. The reduced χ² for the simultaneous best-fit to the DEST and ΔR₂ data is 9.0 for the first model and 1.8 for the second.

Figure 2. Kinetic schemes for monomer exchange on the surface of Aβ protofibrils

a, The protofibril-bound peptide (M_oligomer) exists in only a single state. b, The protofibril-bound peptide exists as a large ensemble of states such that each residue can be either tethered or in direct contact with the surface of the oligomer with $K_3(i)=k_2^{\mathit{\text{app}}}(i)/k_1^{\mathit{\text{app}}}(i)$. The circle in the diagrammatic representation of the states represents a single residue that is either tethered or in direct contact and for which three possible chain configurations are shown.

Figure 3. Comparison of residue-specific fitted parameters describing the ensemble of protofibril-bound states

a, Residue-specific equilibrium constant (K₃) for the relative partitioning of direct-contact and tethered states of the oligomer. b, ${}^{15}\mathrm{N}-R_2^{\mathit{\text{tethered}}}$ values for the tethered states. The values for Aβ40 and Aβ42 are displayed as blue and red circles, respectively. The proposed two β-strand regions in fibrils of Aβ40 and Aβ42 are indicated by arrows at the top of the figure. Error bars: 1 s.d.