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 titration calorimetry (ITC) show that the affinity of MUP for the pheromone 2-methoxy-3-isobutylpyrazine (IBMP) is mainly driven by enthalpy, with a small unfavourable 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 chemical shifts of carbonyl C′ and amide N nuclei. Correlated chemical shift modulations (CSM/CSM) in MUP have been determined 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 chemical shift anisotropies (CSA/CSA). The latter can be predicted from tensor parameters of C′ and N nuclei that have been determined in earlier work. The effects of complexation on slow time-scale protein dynamics can be determined by comparing the temperature dependence of the relaxation rates of APO-MUP (i.e., without ligand) and HOLO-MUP (i.e., with IBMP as a ligand

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)

Overview of the nomenclature and network of contributors to the development of bioreactors for human gut simulation using bibliometric tools: a fragmented landscape

The evolution of complex in vitro models of the human gastrointestinal system to interrogate the biochemical functionality of the gut microbiome has augmented our understanding of its role in human physiology and pathology. With 5718 authors from 52 countries, gut bioreactor research reflects the growing awareness of our need to understand the contribution of the gut microbiome to human health. Although a large body of knowledge has been generated from in vitro models, it is scattered and defined by application-specific terminologies. To better grasp the capacity of bioreactors and further our knowledge of the human gastrointestinal system, we have conducted a cross-field bibliometric search and mapped the evolution of human gastrointestinal in vitro research. We present reference material with the aim of identifying key authors and bioreactor types to enable researchers to make decisions regarding the choice of method for simulating the human gut in the context of microbiome functionality

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