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

Research Activities for the DORIS Contribution to the Next International Terrestrial Reference Frame

For the preparation of ITRF2008, the IDS processed data from 1993 to 2008, including data from TOPEX/Poseidon, the SPOT satellites and Envisat in the weekly solutions. Since the development of ITRF2008, the IDS has been engaged in a number of efforts to try and improve the reference frame solutions. These efforts include (i) assessing the contribution of the new DORIS satellites, Jason-2 and Cryosat2 (2008-2011), (ii) individually analyzing the DORIS satellite contributions to geocenter and scale, and (iii) improving orbit dynamics (atmospheric loading effects, satellite surface force modeling. . . ). We report on the preliminary results from these research activities, review the status of the IDS combination which is now routinely generated from the contributions of the IDS analysis centers, and discuss the prospects for continued improvement in the DORIS contribution to the next international reference frame

In-cell NMR characterization of the secondary structure populations of a disordered conformation of α-Synuclein within E. coli cells

α-Synuclein is a small protein strongly implicated in the pathogenesis of Parkinson’s disease and related neurodegenerative disorders. We report here the use of in-cell NMR spectroscopy to observe directly the structure and dynamics of this protein within E. coli cells. To improve the accuracy in the measurement of backbone chemical shifts within crowded in-cell NMR spectra, we have developed a deconvolution method to reduce inhomogeneous line broadening within cellular samples. The resulting chemical shift values were then used to evaluate the distribution of secondary structure populations which, in the absence of stable tertiary contacts, are a most effective way to describe the conformational fluctuations of disordered proteins. The results indicate that, at least within the bacterial cytosol, α-synuclein populates a highly dynamic state that, despite the highly crowded environment, has the same characteristics as the disordered monomeric form observed in aqueous solution

Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome–nascent chain complexes

The translational diffusion of macromolecules can be examined non-invasively by stimulated echo (STE) NMR experiments to accurately determine their molecular sizes. These measurements can be important probes of intermolecular interactions and protein folding and unfolding, and are crucial in monitoring the integrity of large macromolecular assemblies such as ribosome–nascent chain complexes (RNCs). However, NMR studies of these complexes can be severely constrained by their slow tumbling, low solubility (with maximum concentrations of up to 10 μM), and short lifetimes resulting in weak signal, and therefore continuing improvements in experimental sensitivity are essential. Here we explore the use of the paramagnetic longitudinal relaxation enhancement (PLRE) agent NiDO2A on the sensitivity of 15N XSTE and SORDID heteronuclear STE experiments, which can be used to monitor the integrity of these unstable complexes. We exploit the dependence of the PLRE effect on the gyromagnetic ratio and electronic relaxation time to accelerate recovery of 1H magnetization without adversely affecting storage on N z during diffusion delays or introducing significant transverse relaxation line broadening. By applying the longitudinal relaxation-optimized SORDID pulse sequence together with NiDO2A to 70S Escherichia coli ribosomes and RNCs, NMR diffusion sensitivity enhancements of up to 4.5-fold relative to XSTE are achieved, alongside ~1.9-fold improvements in two-dimensional NMR sensitivity, without compromising the sample integrity. We anticipate these results will significantly advance the use of NMR to probe dynamic regions of ribosomes and other large, unstable macromolecular assemblies