De novo 100 kHz MAS structure determination of a viral nucleocapsid and insights from high field DNP

Kristaps Jaudzems (1) , Loren B Andreas (1) , Jan Stanek (1) , Sachin R Chaudhari (2) , Daniela Lalli (1) , Andrea Bertarello (1) , Tanguy Le Marchand (1) , Diane Cala - de Paepe (1) , Svetlana Kotelovica (3) , Inara Akopjana (3) , Lyndon Emsley (4) , Kaspars Tars (3) , Torsten Herrmann (5) , Anne Lesage (2) , Guido Pintacuda (1)

1 Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie
2 Solid-State NMR Methods for Materials - Méthodes de RMN à l'état solide pour les matériaux
3 Biomed Res & Study Ctr
4 ISIC - Institut des sciences et ingénierie chimiques
5 Computational Methods for Biomolecular Studies

The atomic-level characterization of large viral particles is one of the greatest challenges of modern structural biology, as well as a fundamental step for the design of effective antiviral treatments. Over the last decades, solid-state NMR (ssNMR) has developed into a powerful structural tool for studying structure and dynamics of solid biological samples at atomic resolution. However, the inherently low sensitivity and poor resolution of the technique has limited its applicability to small proteins that can be tightly packed at a high molar concentration, while large proteins or multi-domain assemblies were mostly inaccessible to site-specific ssNMR studies. This has been recently overcome by the introduction of faster spinning probes, which facilitate the use of proton-detected ssNMR experiments, as well as by dynamic nuclear polarization (DNP), which allows transfer of polarization from the unpaired electrons of a paramagnetic center to the surrounding nuclei, and can enhance the sensitivity of ssNMR experiments by several orders of magnitude. Here we demonstrate the effectiveness of the recently developed ssNMR methods employing proton detection at high field and 100 kHz MAS by structure determination of the 2.5 MDa icosahedral capsid of the AP205 bacteriophage. We show that at this spinning regime spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of 1H-1H proximities that define the dimeric capsid subunit structure. The subunit structure is then used in conjunction with a low resolution EM map to construct an atomic-level description of the global capsid architecture.Additionally, we show that high quality DNP NMR spectra of the AP205 nucleocapsid can be obtained by combining high magnetic field (800 MHz) and fast magic-angle spinning (40 kHz) .This enables assignment of aromatic resonances of the encapsidated RNA and the nucleoprotein, which are not observed at room temperature, opening up new possibilities for intermolecular interaction studies.

Domaines: Chimie analytique

Kristaps Jaudzems, Loren B Andreas, Jan Stanek, Sachin R Chaudhari, Daniela Lalli, et al.. De novo 100 kHz MAS structure determination of a viral nucleocapsid and insights from high field DNP. Journées Grand-Sud de RMN, May 2016, Montpellier, France. ⟨hal-01576228⟩