In metabolomics, nuclear magnetic resonance (NMR) spectroscopy allows to identify and quantify compounds in biological samples. The sample preparation generally requires only few steps; however, an indispensable factor is the addition of a locking substance into the biofluid sample, such as deuterium oxide (D2O). While creatinine loss in pure D2O is well-described, the effects of different D2O concentrations on the signal profile of biological samples are unknown. In this work, we investigated the effect of D2O levels in the NMR buffer system in urine samples, in dependence on dwell time and temperature exposition. We reveal a decrease of the urinary creatinine peak area up to 35% after 24 h of dwell time at room temperature (RT) using 25% (v/v) D2O, but only 4% loss using 2.5% D2O. 1H, inverse-gated (IG) 13C, DEPT-HSQC NMR, and mass spectrometry (MS) experiments confirmed a proton-deuterium (H/D) exchange at the CH2. This leads to underestimation of creatinine levels and has an extensive effect when creatinine is used for normalization. This work offers a sample stability examination, depending on the D2O concentration, dwell time, and temperature and enables a method to correct for the successive loss. We propose an equation to correct the creatinine loss for samples prepared with various D2O concentrations and storage temperatures for dwell times up to 24 h. The correction function was validated against an external data set with n = 26 samples. To ensure sufficient creatinine stability in future studies, we suggest that a maximum of 10% D2O should be used at 4 °C or 2.5% D2O at RT, respectively.

Human excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate in the brain and are essential to maintain excitatory neurotransmission. Our understanding of the EAATs\' molecular mechanisms has been hampered by the lack of stability of purified protein samples for biophysical analyses. Here, we present approaches based on consensus mutagenesis to obtain thermostable EAAT1 variants that share up to ~95% amino acid identity with the wild type transporters, and remain natively folded and functional. Structural analyses of EAAT1 and the consensus designs using hydrogen-deuterium exchange linked to mass spectrometry show that small and highly cooperative unfolding events at the inter-subunit interface rate-limit their thermal denaturation, while the transport domain unfolds at a later stage in the unfolding pathway. Our findings provide structural insights into the kinetic stability of human glutamate transporters, and introduce general approaches to extend the lifetime of human membrane proteins for biophysical analyses.

The TG interacting factor-1 homeodomain (TGIF1-HD) binds with the consensus DNA motif 5\'-TGTCA-3\' in gene promoters through its three-amino acid loop extension (TALE) type homeodomain, and then recruits co-regulators to regulate gene expression. Although the solution NMR structure of human TGIF1-HD has been reported previously, little is known about its DNA binding mechanism. NMR titrations have been extensively used to study mechanisms of ligand binding to target proteins; however, an intermediate exchange occurred predominantly between TGIF1-HD in the free and bound states when titrated with the consensus DNA, which resulted in poor-quality NMR spectra and precluded further exploration of its interaction interface and conformational dynamics. Here, the helix α3 of TGIF1-HD was speculated as the specific DNA binding interface by hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments, and subsequently confirmed by chemical exchange saturation transfer (CEST) spectroscopy. In addition, simultaneous conformational changes in other regions, including α1 and α2, were induced by DNA binding, explaining the observation of chemical shift perturbations from extensive residues besides those located in α3. Further, low-populated DNA-bound TGIF1-HD undergoing a slow exchange at a rate of 130.2 ± 3.6 s-1 was derived from the analysis of the CEST data, and two residues, R220 and R221, located in the middle of α3 were identified to be crucial for DNA binding. Our study provides structural and dynamic insights into the mechanisms of TGIF1-HD recognition of extensive promoter DNA.