Understanding the Methyl-TROSY effect over a wide range of magnetic fields

The use of relaxation interference in the methyl Transverse Relaxation-Optimized SpectroscopY (TROSY) experiment has opened new avenues for the study of large proteins and protein assemblies in nuclear magnetic resonance. So far, the theoretical description of the methyl-TROSY experiment has been limited to the slow-tumbling approximation, which is correct for large proteins on high field spectrometers. In a recent paper, favorable relaxation interference was observed in the methyl groups of a small protein at a magnetic field as low as 0.33 T, well outside the slow-tumbling regime. Here, we present a model to describe relaxation interference in methyl groups over a broad range of magnetic fields, not limited to the slow-tumbling regime. We predict that the type of multiple-quantum transitions that show favorable relaxation properties change with the magnetic field. Under the condition of fast methyl-group rotation, methyl-TROSY experiments can be recorded over the entire range of magnetic fields from a fraction of 1 T up to 100 T

High-Resolution Two-Field Nuclear Magnetic Resonance Spectroscopy

International audienceNuclear Magnetic Resonance (NMR) is a ubiquitous branch of spectroscopy that can explore matter on the scale of the atom. Significant improvements in sensitivity and resolution have been driven by a steady increase of static magnetic field strengths. However, some properties of nuclei may be more favourable at low magnetic fields. For example, line-broadening due to chemical shift anisotropy increases sharply at higher magnetic fields. Here, we present a two-field NMR spectrometer that permits the application of rf-pulses and acquisition of NMR signals in two magnetic centres. Our prototype operates at 14.1 T and 0.33 T. The main features of this system are demonstrated by novel NMR experiments that correlate zero-quantum coherences at low magnetic field with single quantum coherences at high magnetic field, so that high resolution can be achieved in both dimensions, despite a ca. 10 ppm inhomogeneity of the low field centre. Two-field NMR spectroscopy offers the possibility to circumvent the limits of high magnetic fields, while benefiting from their exceptional sensitivity and resolution. This approach opens new avenues for NMR above 1 GHz

Determination of Protein ps-ns Motions by High-Resolution Relaxometry

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Kinetic isotope effects for fast deuterium and proton exchange rates

International audienceBy monitoring the effect of deuterium decoupling on the decay of transverse 15N magnetization in D–15N spin pairs during multiple-refocusing echo sequences, we have determined fast D–D exchange rates kD and compared them with fast H–H exchange rates kH in tryptophan to determine the kinetic isotope effect as a function of pH and temperature

Protein Dynamics from Accurate Low-Field Site-Specific Longitudinal and Transverse Nuclear Spin Relaxation

International audienceNuclear magnetic relaxation provides invaluable quantitative site-specific information on the dynamics of complex systems. Determining dynamics on nanosecond timescales requires relaxation measurements at low magnetic fields, incompatible with high-resolution NMR. Here, we use a two-field NMR spectrometer to measure carbon-13 transverse and longitudinal relaxation rates at a field as low as 0.33 T (proton Larmor frequency 14 MHz) in specifically labeled sidechains of the protein ubiquitin. The use of radiofrequency pulses enhances the accuracy of measurements as compared to high-resolution relaxometry approaches, where the sample is moved in the stray field of the superconducting magnet. Importantly, we demonstrate that accurate measurements at a single low magnetic field provide enough information to characterize complex motions on low nanosecond timescales, which opens a new window for the determination of site-specific nanosecond motions in complex systems such as proteins

Boosting the resolution of low-field 15 N relaxation experiments on intrinsically disordered proteins with triple-resonance NMR

International audienceImproving 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

Convergent vews on disordered protein dynamics from NMR and computational approaches

International audienceIntrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) is a class of biologically important proteins exhibiting specific biophysical characteristics. They lack a hydrophobic core, and their conformational behavior is strongly influenced by electrostatic interactions. IDPs and IDRs are highly dynamic, and a characterization of the motions of IDPs and IDRs is essential for their physically correct description. NMR together with molecular dynamics simulations are the methods best suited to such a task because they provide information about dynamics of proteins with atomistic resolution. Here, we present a study of motions of a disordered C-terminal domain of the delta subunit of RNA polymerase from Bacillus subtilis. Positively and negatively charged residues in the studied domain form transient electrostatic contacts critical for the biological function. Our study is focused on investigation of ps-ns dynamics of backbone of the delta subunit based on analysis of amide 15N NMR relaxation data and molecular dynamics simulations. In order to extend an informational content of NMR data to lower frequencies, which are more sensitive to slower motions, we combined standard (high-field) NMR relaxation experiments with high-resolution relaxometry. Altogether, we collected data reporting the relaxation at 12 different magnetic fields, resulting in an unprecedented data set. Our results document that the analysis of such data provides a consistent description of dynamics and confirms the validity of so far used protocols of the analysis of dynamics of IDPs also for a partially folded protein. In addition, the potential to access detailed description of motions at the timescale of tens of ns with the help of relaxometry data is discussed. Interestingly, in our case, it appears to be mostly relevant for a region involved in the formation of temporary contacts within the disordered region, which was previously proven to be biologically important

Time-resolved protein side-chain motions unraveled by high-resolution relaxometry and molecular dynamics simulations

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