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

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 13 C and 15 N NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of *25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexes.

Is solid-state NMR enhanced by dynamic nuclear polarization?

“…studied by conventional ssNMR can be down to even $ 30 Hz (with specific isotopic labeling) [48], attributable to high crystallinity or fast conformational averaging. However, dissolution or suspension of the biomolecular analyte in frozen DNP matrices can increase this value to a few hundreds of Hz due to inhomogeneous broadening, leading to a decrease in resolution [49][50][51][52][53][54]. Just cooling the biological sample to low temperatures, in the absence of DNP matrices, can produce extensive inhomogeneous broadening as various conformers are trapped, especially in areas of high solvation level and mobility at RT [55].…”

Magic Angle Spinning NMR of Proteins: High-Frequency Dynamic Nuclear Polarization and 1H Detection

“…A N6-C6 (151). The enhancement (ε ∼ 25) and resolution enable the assignment of many residues at a cryogenic temperature of 100 K, for example, the threonine and alanine sites.…”

A Nanoparticle Catalyst for Heterogeneous Phase Para‐Hydrogen‐Induced Polarization in Water

“…Transmission electron microscopy (TEM) revealed that the nanoparticles have an average size of 2.0 nm (Figure 1 b). The capping of the nanoparticles is confirmed by 1 H NMR spectroscopy using the following experiment. When glutathione is dissolved in solution, narrow resonance signals attributable to the functional groups in the molecule are detected (Figure 1 c, top spectrum).…”

“…These substantial signal enhancements facilitate detection of molecules in low concentrations. Hyperpolarization implicates a wide range of applications, from structural biology [1][2][3] to quantum computation, [4] operando investigations of catalytic reactors, [5,6] molecular imaging, and in vivo diagnostics. [7][8][9][10][11][12][13] The para-hydrogen-induced polarization (PHIP) method utilizes the singlet spin order of para-hydrogen to create hyperpolarization by incorporating hydrogen from parahydrogen into a molecule of interest, or by a catalystmediated, reversible exchange process.…”

“…Herein, we demonstrate the use of Pt nanoparticles capped with glutathione to induce heterogeneous PHIP in water. The ligand-inhibited surface diffusion on the nanoparticles resulted in a 1 H polarization of P = 0.25 % for hydroxyethyl propionate, a known contrast agent for magnetic resonance angiography. Transferring the 1 H polarization to a 13 C nucleus using a para-hydrogen polarizer yielded a polarization of 0.013 %.…”