Gadolinium-Based Paramagnetic Relaxation Enhancement Agent Enhances Sensitivity for NUS Multidimensional NMR-Based Metabolomics

Gadolinium is a paramagnetic relaxation enhancement (PRE) agent that accelerates the relaxation of metabolite nuclei. In this study, we noted the ability of gadolinium to improve the sensitivity of two-dimensional, non-uniform sampled NMR spectral data collected from metabolomics samples. In time-equivalent experiments, the addition of gadolinium increased the mean signal intensity measurement and the signal-to-noise ratio for metabolite resonances in both standard and plasma samples. Gadolinium led to highly linear intensity measurements that correlated with metabolite concentrations. In the presence of gadolinium, we were able to detect a broad array of metabolites with a lower limit of detection and quantification in the low micromolar range. We also observed an increase in the repeatability of intensity measurements upon the addition of gadolinium. The results of this study suggest that the addition of a gadolinium-based PRE agent to metabolite samples can improve NMR-based metabolomics.

Atomic view of cosolute-induced protein denaturation probed by NMR solvent paramagnetic relaxation enhancement

The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute–protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute–protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, 〈r−6〉norm, and an effective correlation time, τc. The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute–protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the 〈r−6〉norm values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.

Tuning the 1H NMR Paramagnetic Relaxation Enhancement and Local Order of [Aliquat]+-Based Systems Mixed with DMSO

Understanding the behavior of a chemical compound at a molecular level is fundamental, not only to explain its macroscopic properties, but also to enable the control and optimization of these properties. The present work aims to characterize a set of systems based on the ionic liquids [Aliquat][Cl] and [Aliquat][FeCl4] and on mixtures of these with different concentrations of DMSO by means of 1H NMR relaxometry, diffusometry and X-ray diffractometry. Without DMSO, the compounds reveal locally ordered domains, which are large enough to induce order fluctuation as a significant relaxation pathway, and present paramagnetic relaxation enhancement for the [Aliquat][Cl] and [Aliquat][FeCl4] mixture. The addition of DMSO provides a way of tuning both the local order of these systems and the relaxation enhancement produced by the tetrachloroferrate anion. Very small DMSO volume concentrations (at least up to 1%) lead to enhanced paramagnetic relaxation without compromising the locally ordered domains. Larger DMSO concentrations gradually destroy these domains and reduce the effect of paramagnetic relaxation, while solvating the ions present in the mixtures. The paramagnetic relaxation was explained as a correlated combination of inner and outer-sphere mechanisms, in line with the size and structure differences between cation and anion. This study presents a robust method of characterizing paramagnetic ionic systems and obtaining a consistent analysis for a large set of samples having different co-solvent concentrations.

Impact of the Hereditary P301L Mutation on the Correlated Conformational Dynamics of Human Tau Protein Revealed by the Paramagnetic Relaxation Enhancement NMR Experiments

Tau forms intracellular insoluble aggregates as a neuropathological hallmark of Alzheimer’s disease. Tau is largely unstructured, which complicates the characterization of the tau aggregation process. Recent studies have demonstrated that tau samples two distinct conformational ensembles, each of which contains the soluble and aggregation-prone states of tau. A shift to populate the aggregation-prone ensemble may promote tau fibrillization. However, the mechanism of this ensemble transition remains elusive. In this study, we explored the conformational dynamics of a tau fragment by using paramagnetic relaxation enhancement (PRE) and interference (PRI) NMR experiments. The PRE correlation map showed that tau is composed of segments consisting of residues in correlated motions. Intriguingly, residues forming the β-structures in the heparin-induced tau filament coincide with residues in these segments, suggesting that each segment behaves as a structural unit in fibrillization. PRI data demonstrated that the P301L mutation exclusively alters the transiently formed tau structures by changing the short- and long-range correlated motions among residues. The transient conformations of P301L tau expose the amyloid motif PHF6 to promote tau self-aggregation. We propose the correlated motions among residues within tau determine the population sizes of the conformational ensembles, and perturbing the correlated motions populates the aggregation-prone form.