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Faster magic-angle spinning leads to a resolution revolution in biological solid-state NMR

Periodic Reporting for period 2 - FASTER (Faster magic-angle spinning leads to a resolution revolution in biological solid-state NMR)

Reporting period: 2019-04-01 to 2020-09-30

Solid-state NMR is a relatively new method in structural biology that starts to make significant impact by providing atomic-resolution structures of previously uncharacterized proteins. A particularly relevant example is the Amyloid-beta (Aβ) peptide linked to Alzheimer’s disease where we determined the atomic-resolution structure of Aβ(1-42) and of the Osaka mutant of Aβ(1-40). The NMR data can also be combined with other results, e.g. from cryo-electron microscopy or electron paramagnetic resonance to obtain more complete pictures as demonstrated, for example, for alpha synuclein. Solid-state NMR is also ideally positioned to investigate the dynamics of proteins at physiological temperatures as it is sensitive to multiple timescales between picoseconds and seconds. This is an important feature as it becomes more and more clear that the dynamics of proteins and their complexes are intimately linked to the function.
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 to 150 kHz. A further significant improvement is envisioned during the second half of this project. Increasing the MAS frequency to 200-250 kHz will further improve the spectral quality as already experimentally observed for the step from 110 to 150 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. For example, it already has allowed us to combine solid-state NMR with cell-free expression of “difficult” proteins, e.g. the assembled envelopes of Hepatitis B viruses.
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.
WP1 Hardware development: The aim is to construct a spinning module for MAS frequencies above 200 kHz. We studied 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. It turns out that the dimensions of the air bearings (inner diameter, radius and number of jets are of critical importance for the achievable rotation frequency of these small and light rotors. This has been empirically tested by constructing a series of stator/Rotor assemblies and determining the achievable spinning rates in air, helium and under vacuum conditions in the chamber. A manuscript is currently under preparation. The spinning frequencies obtained are presently 110 kHz for systems in air but we are optimistic that considerable improvement will be realised. For WP 2 and 3 we presently use a 0.5 mm system obtained in collaboration with Ago Samoson (Tallinn) that can reach 160 kHz (under conditions of protein spectroscopy 150 kHz) Furthermore, an ultracentrifugation filling device for 0.5 mm rotors has been developed and produced for optimum filling with biomolecules in the ultracentrifuge.

WP2: We concluded a study ) using protein spectroscopy at MAS up to 150 kHz, the worldwide highest frequency for proteins. We found a significant advantage in terms of mass sensitivity and preparation of isotope-labelled protein samples. We detected a remarkable resolution improvement in the spectra of four fully protonated proteins and a small drug molecule, using MAS rotation up to a frequency of 150 kHz. We observe a reduction of the average homogeneous linewidth by a factor 1.5 when going from 100 to 150 kHz MAS and a decrease in the observed linewidth by a factor 1.25. We conclude that even faster MAS is highly attractive and increases mass sensitivity at a moderate price in overall sensitivity.
At the same time, we investigated different CP and J-based polarization-transfer schemes of the main chain spins as well as the ones in the sidechains. We have also been able to develop experiments to detect polarization transfer between sidechain protons and the 31P spins on the DNA/RNA allowing for a direct detection of DNA/protein contacts. (e.g. hPH 2D experiments) We have combine efficient schemes together to obtain a scheme for 5D-APSY spectroscopy for resonance assignment.

WP3: We have assigned the resonances of ASC, Cp149 and DnaB using 3D spectroscopy started with investigating the dynamical parameters of large proteins and protein complexes.
Because there is no fast-MAS probe presently that can guarantee the confinement of the probe material in the case of a rotor explosion, we have not yet worked with pathology-related fibrils but have favoured ASC, Cp149 and DnaB.
Dynamic parameters were mainly collected on the Cp149 viral capsidsThese applications were done at 80, 110 and 160 kHz. The data were interpreted in the context of the “detector” approach developed in our research group. We determined the homogeneous and inhomogeneous contributions to the 1H linewidth for a series of proteins. Thanks to the availability of a 1 microsecond MD trajectory for the Cp149, the capsid of the Hepatitis B vector, this system turned out to be of particular interest and we determined T2*, T1 and T1 relaxation times using 2D as well as 3D spectroscopy.
We expect to increase the spinning frequency beyond 200 kHz. For this project 0.4mm outer diameter rotors will be used.
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.