Characterizing methyl-bearing side chain contacts and dynamics mediating amyloid β protofibril interactions using ¹³C(methyl)-DEST and lifetime line broadening

Abstract

Many details pertaining to the formation and interactions of protein aggregates associated with neurodegenerative diseases are invisible to conventional biophysical techniques. We recently introduced (15)N dark-state exchange saturation transfer (DEST) and (15)N lifetime line-broadening to study solution backbone dynamics and position-specific binding probabilities for amyloid β (Aβ) monomers in exchange with large (2-80 MDa) protofibrillar Aβ aggregates. Here we use (13)C(methyl)DEST and lifetime line-broadening to probe the interactions and dynamics of methyl-bearing side chains in the Aβ-protofibril-bound state. We show that all methyl groups of Aβ40 populate direct-contact bound states with a very fast effective transverse relaxation rate, indicative of side-chain-mediated direct binding to the protofibril surface. The data are consistent with position-specific enhancements of (13)C(methyl)-R₂(tethered) values in tethered states, providing further insights into the structural ensemble of the protofibril-bound state.

Keywords: NMR spectroscopy; amyloid β; high-molecular-weight assemblies; protein-protein interactions.

Figure 1

¹³C_methyl ΔR₂ and DEST data for Aβ40. A) Observed ¹³C_methyl-ΔR₂ (blue circles), the difference in effective R₂ of methyl group ¹³C spins in the presence (240 μM total Aβ) and absence (50 μM Aβ) of protofibrils, are well-matched by values calculated from best-fits to the equilibrium direct-contact/tethered state binding model (black diamonds; cf. Figure 2). Values for residues (Val and Leu) with two methyl groups are averaged. Error bars are 1 s.d. The amino acid sequence is displayed on top with hydrophobic residues colored in green. B) Examples of experimental (open circles) and best-fit (solid lines) ¹³C_methyl-DEST profiles at two radiofrequency field strengths, 763 (orange) and 1523 (blue) Hz, for methyl groups of alanine (A2 and A21), leucine (L17 and L34), and valine (V18 and V40). The best-fits obtained by nonlinear minimization and numerical integration of the McConnell equations for the direct-contact/tethered state model^[4] (cf. Figure 2) by optimization of a single global ${{}^{13}\mathrm{C}}_{\text{methyl}}-R_2^{\text{contact}}$ value and residue-specific ${{}^{13}\mathrm{C}}_{\text{methyl}}-R_2^{\text{tethered}}$ values and partition coefficients (K₃) between direct-contact and tethered states, with the global pseudo-first order association rate constant $k_{\text{on}}^{\text{app}}$ set to ${}^{15}\mathrm{N}-\mathrm{\Delta }{R}_2^{max}$ (2.5 s⁻¹; SI Figure S2), and the global dissociation rate constant k_off (51 s⁻¹) taken from Ref. [4]. The experimental data were recorded at a spectrometer ¹H frequency of 600 MHz.

Figure 2

Interaction of monomeric Aβ on the surface of Aβ protofibrils. A) Schematic of overall exchange process. B) The direct-contact/tethered bound-state model, which incorporates tethered and direct-contact states for each residue when bound to the protofibril surface, superimposed on a global two-state exchange model.

Figure 3

Best-fit values of A) the partition coefficients K₃ (blue) and B) ${{}^{13}\mathrm{C}}_{\text{methyl}}-R_2^{\text{tethered}}$ (blue). Values for the corresponding K₃ values obtained previously from ¹⁵N data^[4] (black) and experimental values of ${{}^{13}\mathrm{C}}_{\text{methyl}}-R_2^{\text{monomer}}$ for the monomeric state (black) are also shown in panels (A) and (B), respectively. Values for residues (Val and Leu) with two methyl groups are averaged. For reference, ${{}^{13}\mathrm{C}}_{\text{methyl}}-R_2^{\text{contact}}$, the best-fit value of the transverse relaxation rate, common to all methyl groups when in direct contact with the protofibril surface, is 1330±80 s⁻¹. Error bars are 1 s.d. The amino acid sequence is displayed on top with hydrophobic residues colored in green.

Figure 4

Correlation between A) experimental side-chain ¹³C_methyl-ΔR₂ and backbone ¹⁵N-ΔR₂ values, B) best-fit direct contact/tethered partition coefficients K₃ obtained from ¹³C_methyl and backbone ¹⁵N data; and C) best-fit ${{}^{13}\mathrm{C}}_{\text{methyl}}-R_2^{\text{tethered}}$ and ${}^{15}\mathrm{N}-R_2^{\text{tethered}}$ values for Aβ40. Values for residues (Val and Leu) with two methyl groups are averaged. Color coding: black, Ala; red, Ile (C_γmethyl); green, Leu; blue, Met; and grey, Val. ¹⁵N values could not be determined for Ala2 owing to water exchange and are represented here by values for Glu3. Error bars are 1 s.d.