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Development of solid state NMR methods at 100 kHz magic angle spinning frequency for the study of internal protein dynamics and the application to membrane proteins

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Solid-state NMR reveals high-resolution protein dynamics on a microsecond scale

A rapidly growing number of techniques to study solid materials through nuclear magnetic resonance (NMR) spectroscopy provide unique views into the structure and dynamics of biological molecules. Employing solid-state NMR, EU-funded scientists conducted a detailed study of proteins embedded in biological membranes that are targets for modern medicinal drugs.

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Solid-state NMR allows insights that cannot be obtained with any other method, including liquid-state NMR. This method detects subtle changes in the magnetic behaviour of atomic nuclei resulting from interactions with neighbouring electrons and atoms. Most of these interactions are anisotropic, meaning that they depend on the orientation of the molecules in relation to the magnetic field. Protons are one of the most abundant elements in living organisms, a feature that renders 1H-detected NMR spectroscopy very attractive compared to 15N or 13C. However, the anisotropic interactions give rise to a strong network of proton dipolar couplings that can dramatically broaden the linewidth of any 1H signal. In such cases, use of increased magic angle spinning (MAS) has shown that it can surpass this obstacle. Depending on the sample, 1H dipolar couplings can be in the order of around 100 kHz. High MAS rates result in better resolved 1H signals (10-20 Hz) and allow the use of polarisation transfer using scalar couplings. In the EU-funded project SOLID_NMR_DYNAMICS (Development of solid state NMR methods at 100 kHz magic angle spinning frequency for the study of internal protein dynamics and the application to membrane proteins), scientists used solid-state NMR to measure relaxation of 1H and 15N in a time range between a few nanoseconds and 50 μs. Determining relaxation dispersion in such a time window is not possible with solution NMR. Measurements are important as they enable scientists to further investigate protein backbone dynamics. The team conducted a number of experiments to measure various 15N and 1H relaxation constants in [2H,15N,13C]-labelled microcrystalline ubiquitin where only the backbone amide group was fully protonated. Coherent effects such as those stemming from 1H-1H dipolar couplings and temperature effects were excluded. As a result, the team reported that the observed dependence of the relaxation rate constants of 15N R1ρ at different MAS frequencies can be attributed to the protein dynamics. According to the analysis, backbone protein dynamics occurred in a narrow time range at around 1 μs. Amplitude dynamics was found to be lower compared to what was concluded in previous solid-state NMR studies. The project study resolved the debate on the presence and amplitude of protein backbone dynamics in the nanometre-microsecond time range by experimentally showing in high resolution the microsecond-lasting dynamics in microcrystalline ubiquitin.


Solid-state NMR, protein dynamics, microsecond, magic angle spinning, SOLID_NMR_DYNAMICS

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