Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy

Abstract

We present the protocol for the measurement and analysis of dark-state exchange saturation transfer (DEST), a novel solution NMR method for characterizing, at atomic resolution, the interaction between an NMR-'visible' free species and an NMR-'invisible' species transiently bound to a very high-molecular-weight (>1 MDa) macromolecular entity. The reduced rate of reorientational motion in the bound state that precludes characterization by traditional NMR methods permits the observation of DEST. (15)N-DEST profiles are measured on a sample comprising the dark state in exchange with an NMR-visible species; in addition, the difference (ΔR(2)) in (15)N transverse relaxation rates between this sample and a control sample comprising only the NMR-visible species is also obtained. The (15)N-DEST and ΔR(2) data for all residues are then fitted simultaneously to the McConnell equations for various exchange models describing the residue-specific dynamics in the bound state(s) and the interconversion rate constants. Although the length of the experiments depends strongly on sample conditions, approximately 1 week of NMR spectrometer time was sufficient for full characterization of samples of amyloid-β (Aβ) at concentrations of ~100 μM.

Figure 1

Summary of the kinetic models available in the DESTfit program. (a) Pseudo–two-state models in which the equilibrium between the NMR-visible and dark states is described by a single $k_{\text{on}}^{\text{app}}$ and k_off, connecting the free (M_NMR-visible) and dark (M_dark-state) states of the exchanging species, whereas the dark state is partitioned into residue-specific equilibria of ensembles of tethered (I_tethered) and direct-contact (I_contact) states for residue i, such that the concentration of I_NMR-visible = M_NMR-visible for all i (i.e., M refers to the entire molecule, whereas I provides a description on a residue basis). (b) A two-state model, described by a single $k_{\text{on}}^{\text{app}}$ and k_off. (c) Three-state models in which two thermodynamically distinct dark states are populated, including fully connected (Fit types 3 and 4), ‘off-pathway’ (Fit types 5 and 6) and ‘on-pathway’ (Fit type 7) models. (d) A two-state model incorporating the effect of dipolar coupling between spins of different chemical shifts, most useful for analysis of ¹H DEST data.

Figure 2

Screenshots of figures output by the DESTfit software. (a) Comparison of experimental (red) and calculated (blue) ¹⁵N-DEST profiles observed for Aβ40 at 260 μM for residues E3 (left), L17 (center) and N27 (right). (b) Comparison of experimental (red) and simulated (blue) ΔR₂ for Aβ40 at 260 μM relative to Aβ40 at 50 μM. (c,d) Best-fit residue-specific $R_2^{\text{tethered}}$ (c) and K₃ (d), in which the error bars represent confidence intervals for one s.d. Calculated experimental DEST and ΔR₂ profiles for the best-fit pseudo–two-state model with dark states comprising tethered and direct-contact ensembles, fit type 1 (blue solid lines) show significantly better agreement with experimental values compared with those for the best-fit simple two-state model, fit type 2 (blue dotted lines). Expt data, experimental data; freq, frequency.