Evidence of Slow Motions by Cross-Correlated Chemical Shift Modulation in Deuterated and Protonated Proteins

Cross-correlated fluctuations of isotropic chemical shifts can provide evidence for slow motions in biomolecules. Slow side-chain dynamics have been investigated in 15N and 13C enriched ubiquitin by monitoring the relaxation of Cα-Cβ two-spin coherences (Frueh etal., 2001). This method, which had hitherto been demonstrated only for protonated ubiquitin, has now been applied to both protonated and deuterated proteins. Deuteration reduces the dipole-dipole contributions to the DD/DD cross-correlation, thus facilitating the observation of subtle effects due to cross-correlation of the fluctuations of the isotropic 13C chemical shifts. The decays of double- and zero-quantum coherences are significantly slower in the deuterated protein than in the protonated sample. Slow motions are found both in loops and in secondary structure element

“Ask Ernö”: a self-learning tool for assignment and prediction of nuclear magnetic resonance spectra

Background: We present "Ask Erno", a self-learning system for the automatic analysis of NMR spectra, consisting of integrated chemical shift assignment and prediction tools. The output of the automatic assignment component initializes and improves a database of assigned protons that is used by the chemical shift predictor. In turn, the predictions provided by the latter facilitate improvement of the assignment process. Iteration on these steps allows Ask Erno to improve its ability to assign and predict spectra without any prior knowledge or assistance from human experts. Results: This concept was tested by training such a system with a dataset of 2341 molecules and their H-1-NMR spectra, and evaluating the accuracy of chemical shift predictions on a test set of 298 partially assigned molecules (2007 assigned protons). After 10 iterations, Ask Erno was able to decrease its prediction error by 17 %, reaching an average error of 0.265 ppm. Over 60 % of the test chemical shifts were predicted within 0.2 ppm, while only 5 % still presented a prediction error of more than 1 ppm. Conclusions: Ask Erno introduces an innovative approach to automatic NMR analysis that constantly learns and improves when provided with new data. Furthermore, it completely avoids the need for manually assigned spectra. This system has the potential to be turned into a fully autonomous tool able to compete with the best alternatives currently available

Effects of Protein-pheromone Complexation on Correlated Chemical Shift Modulations

Major urinary protein (MUP) is a pheromone-carrying protein of the lipocalin family. Previous studies by isothermal titrn. calorimetry (ITC) show that the affinity of MUP for the pheromone 2-methoxy-3-isobutylpyrazine (IBMP) is mainly driven by enthalpy, with a small unfavorable entropic contribution. Entropic terms can be attributed in part to changes in internal motions of the protein upon binding. Slow internal motions can lead to correlated or anti-correlated modulations of the isotropic chem. shifts of carbonyl C' and amide N nuclei. Correlated chem. shift modulations (CSM/CSM) in MUP have been detd. by measuring differences of the transverse relaxation rates of zero- and double-quantum coherences ZQC{C'N} and DQC{C'N}, and by accounting for the effects of correlated fluctuations of dipole-dipole couplings (DD/DD) and chem. shift anisotropies (CSA/CSA). The latter can be predicted from tensor parameters of C' and N nuclei that have been detd. in earlier work. The effects of complexation on slow time-scale protein dynamics can be detd. by comparing the temp. dependence of the relaxation rates of APO-MUP (i.e., without ligand) and HOLO-MUP (i.e., with IBMP as a ligand). [on SciFinder (R)

Altered dietary behaviour during pregnancy impacts systemic metabolic phenotypes

RationaleEvidence suggests consumption of a Mediterranean diet (MD) can positively impact both maternal and offspring health, potentially mediated by a beneficial effect on inflammatory pathways. We aimed to apply metabolic profiling of serum and urine samples to assess differences between women who were stratified into high and low alignment to a MD throughout pregnancy and investigate the relationship of the diet to inflammatory markers.MethodsFrom the ORIGINS cohort, 51 pregnant women were stratified for persistent high and low alignment to a MD, based on validated MD questionnaires. 1H Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the urine and serum metabolite profiles of these women at 36 weeks of pregnancy. The relationship between diet, metabolite profile and inflammatory status was investigated.ResultsThere were clear differences in both the food choice and metabolic profiles of women who self-reported concordance to a high (HMDA) and low (LMDA) Mediterranean diet, indicating that alignment with the MD was associated with a specific metabolic phenotype during pregnancy. Reduced meat intake and higher vegetable intake in the HMDA group was supported by increased levels of urinary hippurate (p = 0.044) and lower creatine (p = 0.047) levels. Serum concentrations of the NMR spectroscopic inflammatory biomarkers GlycA (p = 0.020) and GlycB (p = 0.016) were significantly lower in the HDMA group and were negatively associated with serum acetate, histidine and isoleucine (p < 0.05) suggesting a greater level of plant-based nutrients in the diet. Serum branched chain and aromatic amino acids were positively associated with the HMDA group while both urinary and serum creatine, urine creatinine and dimethylamine were positively associated with the LMDA group.ConclusionMetabolic phenotypes of pregnant women who had a high alignment with the MD were significantly different from pregnant women who had a poor alignment with the MD. The metabolite profiles aligned with reported food intake. Differences were most significant biomarkers of systemic inflammation and selected gut-microbial metabolites. This research expands our understanding of the mechanisms driving health outcomes during the perinatal period and provides additional biomarkers for investigation in pregnant women to assess potential health risks

Slow dynamics in biomolecules studied by NMR spectroscopy

Since many biological processes occur on the μs to ms time scale, internal dynamics on that time scale may well be related to biological functionality. The characterization of internal dynamics is thus an important issue to improve our understanding of the biological activity of macromolecules, such as proteins, RNA and DNA fragments. Solution NMR spectroscopy, in particular relaxation measurements, is well suited for the deter tion of exchange rates on that slow time scale. In solution, relaxation of nuclear spins is caused mainly by Brownian tumbling and, to a lesser extent, by internal motions that modulate the effective field of the spins. Thus, these fluctuations depend on the electronic environment of each spin and on internal dynamics. On the one hand, the determination of structures of biomolecules suffers from local motions that lead to the determination of average structures. While, on the other hand, only little information is available concerning the time scale and the type of motions that may be responsible for important biological functions. To address this issue, new experiments have been designed to determine the contribution of slow internal motions (μs-ms) to relaxation of multiple-quantum coherences (MQC) involving carbonyl C' and nitrogen N nuclei. Local motions that are slower than the overall correlation time τc (ns) induce chemical shift modulations (CSM) of the spins involved in the motions. If correlated, such fluctuations can contribute to differential relaxation between zero- and double-quantum coherences. It is known that a train of π-pulses can reduce this contribution if the time scale of the modulations is comparable to the frequency at which refocusing pulses are applied. In the limit of slow pulse rates. both CSM/CSM (slow dynamics) and CSA/CSA (structure), the sum of this two terms being referred to as CS/CS, affect the differential relaxation of ZQC and DQC, whereas in the limit of fast pulse rates, only CSA/CSA affect the differential line broadening, allowing the discrimination between structural and dynamic contributions to relaxation. Related theoretical concepts and experimental methodology are extensively discussed in the text. In protonated and deuterated ubiquitin it was possible to confirm that the particularly large C'/N cross-correlated CS/CS relaxation rates observed for the Asparagine N25 residue were partly caused by a pronounced local mobility. This is in agreement with previous observations that indicate mobility in this part of the protein: the fact that the nitrogen R2 relaxation rate of N25 deviates from the N/NHN CSA/DD cross-correlated relaxation rates, the fact that the neighbouring Glutamic acid residue E24 is almost absent from the HSQC spectrum and that Isoleucine I23 shows a particularly large N/HN cross-correlated CS/CS relaxation rate

Structural Analysis from Classroom to Laboratory

The analysis of spectroscopic data to elucidate chemical structures requires intensive practice. A Web portal is described that allows uploading, storing, simulating, and sharing of spectroscopic data. The Web portal can also be used to create, embed, and solve exercises online. This tool and a demo course for Moodle are available online at no cost

Photocatalytic disinfection treatments: Viability, cultivability and metabolic changes of e. Coli using diferent mesurements methods

In this work, the oxidative stress was produced by the illumination of either fi xed TiO2 or suspended TiO2, and the relation between cultivability and viability under oxidative conditions was studied using a Direct Viable Count (DVC) and Fluorescence In Situ Hybridisation (FISH) methods. Under certain oxidative conditions simulating incomplete treatments, viable cells that had lost their cultivability were observed to recover all of their physiological functions when transferred to fresh nutrient media. Moreover, alterations in the nutrient assimilation processes were studied by 1H-NMR during and after the photocatalytic treatment. This allowed us to shed some light on how the damage chain propagates during the treatment. This work demonstrate that the Standard Plate Count (SPC) method commonly used to estimate the c ell populations during disinfection processes is ill adapted, whereas the FISH-DVC pair offers a reliable alternativeEn este trabajo, el estrés oxidativo producido con radiación en presencia TiO2 fijo o suspendido TiO2, y la relación entre cultivabilidad y viabilidad se han realizado, utilizando recuento directo de células viables (CDV) y fluorescencia de hibridación in situ (FISH). Se encontró que cuando el tratamiento es incompleto, las células viables que habían perdido su cultivabilidad pueden recuperar todas sus funciones fisiológicas, cuando son transferidas a un nuevo medio nutritivo. Por otra parte, las alteraciones en los procesos de asimilación de nutrientes fueron estudiados por 1H-RMN durante y después del tratamiento fotocatalítico. Esto permitió tener indicios sobre cómo se propaga la cadena de daños durante el tratamiento. Se demostró que el recuento en placa comúnmente usado para estimar la población de E coli en los procesos de desinfección no sería confiable, mientras que el método combinado FISH-DVC ofrece una alternativa fiabl

Fast and accurate algorithm for the simulation of NMR spectra of large spin systems

The computational cost for the simulation of NMR spectra grows exponentially with the number of nuclei. Today, the memory available to store the Hamiltonian limits the size of the system that can be studied. Modern computers enable to tackle systems containing up to 13 spins [1], which obviously does not allow to study most molecules of interest in research. This issue can be addressed by identifying groups of spins or fragments that are not or only weakly interacting together, i.e., that only share weakly coupled spin pairs. Such a fragmentation is only permitted in the weak coupling regime, i.e., when the coupling interaction is weak compared to the difference in chemical shift of the coupled spins. Here, we propose a procedure that removes weak coupling interactions in order to split the spin system efficiently and to correct a posteriori for the effect of the neglected couplings. This approach yields accurate spectra when the adequate interactions are removed, i.e., between spins only involved in weak coupling interactions, but fails otherwise. As a result, the computational time for the simulation of 1D spectra grows linearly with the size of the spin system. (C) 2010 Elsevier Inc. All rights reserved

Composition dependent transport diffusion in non-ideal mixtures from spatially resolved nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is a well-established technique for the measurement of intra-diffusion coefficients. Recently, such information has been used as a basis of predictive models to extrapolate to the Fick diffusion coefficient of liquid mixtures. The present work presents a new approach to directly access the Fick diffusion coefficient by spatially resolved NMR experiments. The Fick diffusion coefficient of the binary mixture TEA/H2O was determined at two temperatures, 283.2 K and 275.2 K. The results are consistent with values previously reported either from optical experiments or predictive Darken-type models developed for this system. The proposed methodology adds high-resolution NMR to the toolbox for the study of the transport diffusion of multicomponent mixtures. It is, however, still limited to mixtures with liquid–liquid equilibrium phase separation