Dynamic Nuclear Polarization‐Enhanced Biomolecular NMR Spectroscopy at High Magnetic Field with Fast Magic‐Angle Spinning

Abstract

Dynamic nuclear polarization (DNP) is apowerful way to overcome the sensitivity limitation of magic-anglespinning (MAS) NMR experiments.However,the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures and in the presence of paramagnetic radicals.H igh-quality DNP-enhanced NMR spectra of the Acinetobacter phage 205 (AP205) nucleocapsid can be obtained by combining high magnetic field (800 MHz) and fast MAS (40 kHz). These conditions yield enhanced resolution and long coherence lifetimes allowing the acquisition of resolved 2D correlation spectra and of previously unfeasible scalar-based experiments.T his enables the assignment of aromatic resonances of the AP205 coat protein and its packaged RNA, as well as the detection of long-range contacts, which are not observed at room temperature,o pening new possibilities for structure determination.Dynamic nuclear polarization (DNP) allows transfer of polarization from the unpaired electrons of ap aramagnetic center to the surrounding nuclei, enhancing the magic-anglespinning (MAS) NMR signals by several orders of magnitude. [1] Thetechnique has been successfully applied in various fields from chemistry and materials to structural and cell biology.H owever,d espite convincing demonstrations on fibrils, [2] membrane-embedded proteins, [3] virus capsids, [4] and whole-cell assemblies, [5] thes ensitivity enhancement provided by DNP on biological systems is always associated with an umber of issues.W hile high-resolution solid-state MAS NMR spectra of proteins can be nowadays obtained at temperatures above 273 K, the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures (at about 100 K) and in the presence of paramagnetic radicals.F urthermore,D NP spectra of protein samples are currently almost exclusively acquired at moderate magnetic fields (usually 9.4 T) for maximum DNP enhancements and using 3.2 mm rotors with MAS rates up to 15 kHz. Under these experimental conditions,t he lines are broadened by both homogeneous broadening,o wing to incomplete averaging of nuclear and hyperfine interactions, and inhomogeneous broadening,a rising from protein conformational disorder. [6] Thelack of resolution associated with the DNP enhancements is therefore amajor obstacle for the detailed structural study of uniformly labeled biomolecular samples.This limitation has been previously addressed by using increased temperatures (180-200 K) [7] and higher magnetic field strengths [8] at the expense of lower DNP enhancements, as well as by exploring improved sample preparation methods. [9] Whilst these initial attempts have shown the potential for improving resolution in DNP spectra, af urther improvement is imperative to take full advantage of the signal amplification potentially provided by DNP.O ne factor that holds promise is the MAS rate.T he availability of prob...

Solid-state NMR of unlabeled plant cell walls: high-resolution structural analysis without isotopic enrichment

“…Multiple biradicals have been developed recently, such as the AsymPolPOK family that shortens the DNP build-up time [ 33 ] and the Tinypol and TEMTRIPol-I radicals that have shown improved performance over AMUPol on high-field (18.8 and 21.1 T) DNP instruments [ 31 , 59 , 60 ]. The resolution improvement at high-field MAS-DNP (e.g., 800 MHz/527 GHz) will allow us to better analyze biomolecular structures, at least for the molecules bearing structural order [ 61 , 62 ]. The rapidly evolving MAS-DNP technology has a great potential to bring biomass analysis to a new level of details.…”

Dynamic Nuclear Polarization Magic-Angle Spinning Nuclear Magnetic Resonance Combined with Molecular Dynamics Simulations Permits Detection of Order and Disorder in Viral Assemblies

“…13,44 Two factors have been shown previously to contribute to signal broadening and attenuation of intensities in DNP-enhanced measurements: conformational heterogeneity 34,41,[77][78] and paramagnetic relaxation enhancements (PREs) due to the presence of biradical. 39,41,79 The latter has been observed at ambient temperature for solvent exposed regions of K + channel integrated into membranes. 41 In our current study on tubular CA assemblies, relatively small increases in line widths were detected in 13 C- 13 C homonuclear experiments at ambient temperatures, 44 therefore, although the DNP spectra are recorded at a different temperature, PREs are not expected to be a significant source of signal broadening in the current DNP studies.…”

“…[35][36] On the other hand, the combination of cryogenic temperatures and high magnetic fields yields 2D lineshapes that are dominated by inhomogeneous contributions. [37][38][39][40] facilitating their analysis in terms of rapid fluctuations within a conformational distribution using MD simulations. 41 This approach is well suited to probe the degree of dynamical disorder in these viral assemblies, and the associated motions can be quantified through an integrated DNP MAS NMR/MD simulations approach.…”

Temperature-Dependent Nuclear Spin Relaxation Due to Paramagnetic Dopants Below 30 K: Relevance to DNP-Enhanced Magnetic Resonance Imaging

“…Short T 2 and T 1 ρ values due to fluctuating hyperfine fields from DNP dopants can also limit the effectiveness of various solid state NMR techniques that are used with magic-angle spinning (MAS) in structural studies of biopolymers, including 15 N- 13 C and 13 C- 13 C dipolar recoupling techniques for measuring structurally significant internuclear distances and various techniques for polarization transfers in multidimensional spectroscopy. The severity of these effects may depend on the external magnetic field strength, the MAS frequency, and the nature of the sample 50–51 , as well as dopant concentration and other factors. Results presented above suggest that T 2 and T 1 ρ can be lengthened by performing MAS DNP experiments at lower temperatures, especially if practical MAS NMR probes that operate below 10 K could be developed.…”