Boosting the resolution of low-field $$^{15}\hbox {N}$$ relaxation experiments on intrinsically disordered proteins with triple-resonance NMR

Abstract

Improving our understanding of nanosecond motions in disordered proteins requires the enhanced sampling of the spectral density function obtained from relaxation at low magnetic fields. High-resolution relaxometry and two-field NMR measurements of relaxation have, so far, only been based on the recording of one-or two-dimensional spectra, which provide insufficient resolution for challenging disordered proteins. Here, we introduce a 3D-HNCO-based two-field NMR experiment for measurements of protein backbone 15 N amide longitudinal relaxation rates. The experiment provides accurate longitudinal relaxation rates at low field (0.33 T in our case) preserving the resolution and sensitivity typical for high-field NMR spectroscopy. Radiofrequency pulses applied on 6 different radiofrequency channels are used to manipulate the spin system at both fields. The experiment was demonstrated on the C-terminal domain of δ subunit of RNA polymerase from Bacillus subtilis, a protein with highly repetitive amino-acid sequence and very low dispersion of backbone chemical shifts.

Detection of Metabolite-Protein Interactions in Complex Biological Samples by High-Resolution Relaxometry: Towards Interactomics by NMR

“…We have previously demonstrated that high-quality spectra could be obtained with HRR on small macromolecules. 59,72,88 Yet, recording spectra of small molecules in complex mixtures is even more demanding in terms of resolution. Nevertheless, we clearly identified 20 metabolites as suitable targets for HRR analysis on a 1 H NMR spectrum.…”

Theoretical and computational framework for the analysis of the relaxation properties of arbitrary spin systems. Application to high-resolution relaxometry

“…1 T down to ca. 100 µT; (4) A sample-shuttle apparatus can be used to combine relaxometry experiments with high-field NMR [14][15][16][17] to gain atomic resolution description of molecular dynamics; (5) This kind of device can also be used to investigate relaxation properties of spin terms that are only relevant at low fields [18]; (6) A sample shuttle may couple two magnetic centers in a two-field NMR spectrometer [19] to record multi-dimensional experiments where spins are manipulated at two vastly different fields [19][20][21][22]. Sample-shuttling experiments have been used to measure longitudinal relaxation rates over orders of magnitude of magnetic fields and characterize the dynamics of membrane vesicules [23], protein backbone [17,24] and side-chains [25].…”

Two-Field Transverse Relaxation-Optimized Spectroscopy for the Study of Large Biomolecules – an in Silico Investigation

“…A prototype of twofield NMR spectrometer 17 has already been used to overcome chemical exchange broadening, 18 obtain broad-band correlations throughout aliphatic and aromatic 13 C resonances 19 and measure accurate relaxation rates at low field. 20,21 We use numerical simulations to illustrate the concept of 2F-TROSY on aromatic 13 C- 19 F spin systems. We calculate the expected spectra of aromatic fluorinated protein side-chains in a twofield experiment where the 19 F coherence evolves at low field, while the 13 C-TROSY component is detected at high field, and compare them with the reported single-field 13 C-19 F TROSY experiment.…”