Solid-state NMR enhanced by dynamic nuclear polarization as a novel tool for ribosome structural biology

SolidDNPSpectrum

Overlay of a 13C–13C PDSD correlation spectrum (CO-CA region) of free (blue) and E30S-bound (red) His,Tyr-labeled IF1. The spectrum of the free form suffers from extensive overlap due to degenerate chemical shifts. Selective labeling of Tyr or both Tyr and His residues in IF1 allows differentiating between the two types of amino acids in the bound form. Comparison between the free and bound states suggests that two of the His residues are strongly affected by binding (HII and HIII), while of the two Tyr residues only one is not affected (Y)

Abstract

The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800 kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo- and heteronuclear 13C and 15N NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen

ribosome

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