Professor Pavel Kadeřávek; Masaryk University; Head of Core Facility, Josef Dadok National NMR Centre Brno, Tschechien

"Improvement of investigation of backbone motions of IDPs using high-resolution relaxometry"

Intrinsically disordered proteins (IDPs) are characterised by their extensive flexibility, which provides them with unique features necessary for their various specific biological functions. Conventionally, relaxation experiments are utilised to probe the dynamics of proteins; however, the information content of these experiments is constrained by the choice of magnetic field employed in the NMR spectrometers used to conduct them. The aim of our work was to enhance the description of IDPs' dynamics using high-resolution relaxometry (HRR). To this aim, the applicability of HRR to challenging systems was assessed, including IDPs with low chemical shift dispersion and high requirements for the resolution of spectra to obtain residue-specific information.

The employment of high-resolution relaxometry permitted the measurement of relaxation at multiple fields using a single NMR spectrometer, and the acquisition of data at very low magnetic fields. This approach enabled the acquisition of spectra even at fields as low as 0.1 T, with sufficient sensitivity to reliably extract relaxation rates. While the sample was positioned at the high-field centre (at 14.1 T) for polarisation and detection it was moved to a low magnetic field for the relaxation delay. The HRR rates were measured using multidimensional spectra (with non-uniform sampling if necessary), providing the required resolution for the studied IDPs. The interpretation of the HRR rates necessitated consideration of cross-relaxation pathways active during the sample movement between various fields and during relaxation delays. The analysis protocol was validated using measurements of accurate relaxation rates at low field (0.33 T) using a two-field NMR spectrometer. It was demonstrated that HRR is fully compatible with standard relaxation experiments utilised to study the motions of IDPs although it requires a cartefull analysis. The HRR data has been shown to significantly improve the sampling of the spectral density functions, thereby providing an opportunity to reveal new details of the motions of biomolecules.