Periodic Reporting for period 4 - FASTER (Faster magic-angle spinning leads to a resolution revolution in biological solid-state NMR)
Reporting period: 2022-04-01 to 2023-01-31
A spectral resolution revolution is presently going on, and is a driver of the research in this project, that will enable solid-state NMR to address new frontiers in structural biology and medicine. We develop and test, on biologically relevant proteins, proton-detected experiments at high magnetic field an under faster and faster magic-angle spinning to efficiently average the 1H-1H dipolar interaction and obtain even higher spectral resolution. Accessible MAS frequencies for proteins have, during this project, been pushed for proteins, to 170 kHz. On the way to further improvements, we have investigated bearing and drive designs theoretically. Increasing the MAS frequency to 200-250 kHz will further improve the spectral quality as already experimentally observed for the step from 110 to 170 kHz. In addition, the amount of sample required is further reduced to approx. 50 μg, compared to the about 10 mg needed in 13C-detected experiments. This removes an important bottleneck in sample-preparation. We have shown that the combination with cell-free protein expression of “difficult” proteins is possible e.g. the assembled envelopes of Hepatitis B viruses or viral membrane proteins.
We have also demonstrated, for Hepatitis B virus (HBV), that the dynamical characterization of the entire viral capsid as well as its elusive, dynamic C-terminal is feasible with fast spinning technology.
The combination of high magnetic fields and faster spinning will open new horizons for solving urgent biological and medical questions. The importance of understanding disease on a molecular level has been recently been underlined in the Covid crisis.In this context, we have also applied the developed methods to investigate accessory proteins of SARS-CoV 2 .
WP2: We developed and optimized the experimental schemes and pulsequences for protein spectroscopy at MAS up to 160 kHz, the worldwide highest frequency for proteins. We found a significant advantage in terms of mass sensitivity and preparation of isotope-labelled protein samples. And we detected a remarkable resolution improvement in the spectra of. Published in (Callon et al., 2021; Schledorn et al., 2020)(manuscript in preparation)
Studies with a methodologic as well as an applied dimension include as two highlights new methods for characterizing the dynamics in Hepatitis B Virus (HBV), one for the rather rigid capsid and one for the highly dynamic C-ter: (i) Characterization of the dynamics of the Hepatitis B virus capsid and (ii) First-time observation of the dynamically disordered C-terminal domain for HBV.
Hydrogen bonding plays a central role in molecular recognition in chemistry and biology.
We show, on a model system, a reduction of the homogeneous linewidth by a factor 2, between 110 to 160 kHz MAS, for the side-chain protons. Combined with high magnetic field (1.2 GHz), it allows not only the reduction of the spectral linewidth but also increases the chemical-shift dispersion of these protons in the spectra. Applied to the core protein of the Hepatitis B virus capsid, we could assign 60 % of the aliphatic protons, achieved by a combination of hCCH-TOBSY and hNCH experiments.
Dynamic parameters were mainly collected on the Cp149 viral capsids. These applications were done at 80, 110 and 160 kHz. The data were interpreted in the context of the “detector” approach.
Fast Magic-Angle-Spinning NMR reveals the evasive HBV Capsid C-Terminal Domain.. Experimentally determined protein structures often feature missing domains. One example is the C-terminal domain (CTD) of the hepatitis B virus capsid protein, a functionall1y central part of this assembly, crucial in regulating nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR.
The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori..
NS5A is a protein of Hepatitis C virus that is anchored to the membrane by an alpha-helix and forms a symmetric dimer. Daclatasvir is a drug that binds its domain AHD1 in an unknown way. Here we present results from ssNMR experiments on AHD1 with Daclatasvir. The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. We present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes.
In terms of spectroscopic methods, we want to further increase the polarisation transfer efficiencies taking advantage of the increased T1 and T2' relaxation times. Together with the invrease in magnetic field from 20 to 28 Tesla (installed in May 2020 this should lead to new experiments for assignment, structure determination and analysis of the dynamics.