Dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of GNNQQNY nanocrystals and amyloid fibrils

Abstract

Dynamic nuclear polarization (DNP) utilizes the inherently larger polarization of electrons to enhance the sensitivity of conventional solid-state NMR experiments at low temperature. Recent advances in instrumentation development and sample preparation have transformed this field and have opened up new opportunities for its application to biological systems. Here, we present DNP-enhanced (13)C-(13)C and (15)N-(13)C correlation experiments on GNNQQNY nanocrystals and amyloid fibrils acquired at 9.4 T and 100 K and demonstrate that DNP can be used to obtain assignments and site-specific structural information very efficiently. We investigate the influence of temperature on the resolution, molecular conformation, structural integrity and dynamics in these two systems. In addition, we assess the low-temperature performance of two commonly used solid-state NMR experiments, proton-driven spin diffusion (PDSD) and transferred echo double resonance (TEDOR), and discuss their potential as tools for measurement of structurally relevant distances at low temperature in combination with DNP.

Fig. 1

¹³C CP spectra of [U–¹³C,¹⁵N GNNQ]QNY monoclinic crystals recorded with (top) and without (bottom) DNP. The enhancement for the signals arising from the crystals is ~20, while the enhancement for the glycerol peaks is ~40. The MW irradiation time is 6 s for both spectra.

Fig. 2

DNP-enhanced ¹³C–¹³C PDSD correlation spectrum of [U–¹³C,¹⁵N GNNQ]QNY monoclinic crystals obtained at 9.4 T, 105 K, ω_r/2σ = 9 kHz, and τ_mix = 5 ms.

Fig. 3

DNP-enhanced TEDOR spectra of [U–¹³C,¹⁵N GNNQ]QNY monoclinic crystals obtained at 9.4 T, 105 K, ω_r/2σ =9 kHz, and τ_mix = 1.8 ms (top), and 3.6 ms (bottom). Two intermolecular contacts are observed at τ_mix = 3.6 ms.

Fig. 4

Crystal lattice of monoclinic GNNQQNY (PDB ID 1YJP). Several possible intra- and intermolecular contacts between Q10 NE and the CA atoms of other labeled residues are indicated. The image was produced using the Chimera software.

Fig. 5

(a) ¹³C CP spectra of GNN[U–¹³C,¹⁵N QQN]Y fibrils obtained with DNP (top) and without DNP (bottom) at 400 MHz, ω_r/2σ = 9 kHz, 100 K, 16 scans, and MW irradiation time of 6.5 s. The enhancement for the fibrils and the glycerol peaks is 35. (b) ¹³C CP spectrum obtained with the same sample after the DNP experiments were performed. Spectrum was recorded at 750 MHz. (c) ¹³C CP spectrum of fibrils prepared without radical and glycerol, recorded at 700 MHz. Experimental parameters for (b) and (c) are ω_r/2σ = 9 kHz, 300 K, 128 scans, and scan delay of 3 s. Asterisks denote side bands.

Fig. 6

¹³C–¹³C correlations of GNN[U–¹³C,¹⁵N QQN]Y fibrils collected at (a) 750 MHz without DNP, PDSD τ_mix = 10 ms, ω_r/2σ = 9 kHz, 300 K, and (b) 400 MHz with DNP, PDSD τ_mix = 5 ms, ω_r/2σ = 9 kHz, 100 K. The asterisks in (a) denote side bands.

Fig. 7

¹⁵N–¹³C correlation experiments of GNN[U–¹³C,¹⁵N QQN]Y fibrils recorded with TEDOR mixing. (a) Spectrum recorded at 750 MHz, ω_r/2σ = 9 kHz, 300 K and τ_mix = 1.8 ms. (b) and (c) Spectra recorded with DNP at 400 MHz, ω_r/2σ = 9 kHz, 100 K and τ_mix = 1.8 ms and 3.6 ms respectively.

Fig. 8

1D TEDOR build-up curves of GNN[U–¹³C,¹⁵N QQN]Y fibrils obtained at (a) 400 MHz, 100 K with DNP and (b) 750 MHz, 300 K, without DNP.

Fig. 9

Comparison of the ¹³C and ¹⁵N chemical shifts obtained at 100 K with DNP and 298 K without DNP for [U–¹³C,¹⁵N GNNQ]QNY monoclinic crystals (top) and GNN[U–¹³C,¹⁵N QQN]Y fibrils (bottom).