Hardware development: The aim was to construct a spinning module for MAS frequencies above 200 kHz. This was not achieved fully but we developed a much improved understanding of the relevant design parameter studing numerically the turbine shape and the bearing for the micro turbine design in order to estimate the torques and forces acting on the rotor. We developed a hydrodynamic description of the turbine as well as the bearing. We have built a number of 0.4 mm stator assemblies to test the predictions on actual devices. For WP 2 and 3 we used a 0.5 mm system obtained in collaboration with Ago Samoson (Tallinn) that can reach 170 kHz (under conditions of protein spectroscopy 160 kHz)
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.
