NMR - Publications

A primary goal of metabolomics is the characterization of a potentially very large number of metabolites that are part of complex mixtures. Application to biofluids and tissue samples offers insights into biochemical metabolic pathways and their role in health and disease. 1D ¹ H and 2D ^(13) C-¹ H HSQC NMR spectra are most commonly used for this purpose. They yield quantitative information about each proton of the mixture, but do not tell which protons belong to the same molecule....

Arginine kinase catalyzes reversible phosphoryl transfer between arginine and ATP. Crystal structures of arginine kinase in an open, substrate-free form and closed, transition state analog (TSA) complex indicate that the enzyme undergoes substantial domain and loop rearrangements required for substrate binding, catalysis, and product release. Nuclear magnetic resonance (NMR) has shown that substrate-free arginine kinase is rigid on the ps-ns timescale (average S²=0.84±0.08) yet quite dynamic on...

A quantum-chemistry based protocol, termed MOSS-DFT, is presented for the prediction of ^(13)C and ¹H NMR chemical shifts of a wide range of organic molecules in aqueous solution, including metabolites. Molecular motif-specific linear scaling parameters are reported for five different density functional theory (DFT) methods (B97-2/pcS-1, B97-2/pcS-2, B97-2/pcS-3, B3LYP/pcS-2, and BLYP/pcS-2), which were applied to a large set of 176 metabolite molecules. The chemical shift root-mean-square...

Many human proteins are predicted to contain intrinsically disordered regions (IDRs), yet their occurrence in enzymes is notably rare. Human pancreatic glucokinase (GCK) is one of a small, but growing number of enzymes shown to possess an IDR. In this commentary, we summarize the results of recent biophysical studies that provide evidence for a functionally significant disorder-order transition within the IDR of GCK during the enzyme's catalytic cycle. High-resolution NMR studies indicate that...

Standard three-dimensional Fourier transform (FT) NMR experiments of molecular systems often involve prolonged measurement times due to extensive sampling required along the indirect time domains to obtain adequate spectral resolution. In recent years, a wealth of alternative sampling methods has been proposed to ease this bottleneck. However, due to their algorithmic complexity, for a given sample and experiment it is often hard to determine the minimal sampling requirement, and hence the...

Metabolomics continues to make rapid progress through the development of new and better methods and their applications to gain insight into the metabolism of a wide range of different biological systems from a systems biology perspective. Customization of NMR databases and search tools allows the faster and more accurate identification of known metabolites, whereas the identification of unknowns, without a need for extensive purification, requires new strategies to integrate NMR with mass...

Three new rotenoids (1-3), two new isoflavonoids (4 and 5), and six known analogues (6-11) were isolated from an n-hexane partition of a methanol extract of the fruits of Millettia caerulea, with the structures of the new compounds elucidated by analysis of their spectroscopic data. The relative configurations of the rotenoids were determined by interpretation of their NMR spectroscopic data, and their absolute configurations were established using electronic circular dichroism spectra and...

It was recently discovered that the NRAS isoform 5 (20 amino acids) is expressed in melanoma and results in a more aggressive cell phenotype. This novel isoform is responsible for increased phosphorylation of downstream targets such as AKT, MEK, and ERK as well as increased cellular proliferation. This structure report describes the NMR solution structure of NRAS isoform 5 to be used as a starting point to understand its biophysical interactions. The isoform is highly flexible in aqueous...

A new environmental bacterial strain exhibited strong antimicrobial characteristics against methicillin-resistant Staphylococcus aureus, vancomycin-resistant strains of Enterococcus faecalis and Lactobacillus plantarum, and other Gram-positive bacteria. The producer strain, designated OSY-I1, was determined to be Brevibacillus laterosporusvia morphological, biochemical, and genetic analyses. The antimicrobial agent was extracted from cells of OSY-I1 with isopropanol, purified by high-performance...

This review discusses strategies for the identification of metabolites in complex biological mixtures, as encountered in metabolomics, which have emerged in the recent past. These include NMR database-assisted approaches for the identification of commonly known metabolites as well as novel combinations of NMR and MS analysis methods for the identification of unknown metabolites. The use of certain chemical additives to the NMR tube can permit identification of metabolites with specific physical...

The bromodomain and extraterminal domain (BET) protein family are promising therapeutic targets for a range of diseases linked to transcriptional activation, cancer, viral latency, and viral integration. Tandem bromodomains selectively tether BET proteins to chromatin by engaging cognate acetylated histone marks, and the extraterminal (ET) domain is the focal point for recruiting a range of cellular and viral proteins. BET proteins guide γ-retroviral integration to transcription start sites and...

The Na(+) /Ca(2+) exchanger provides a major Ca(2+) extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca(2+) concentrations. In Canis familiaris, Na(+) /Ca(2+) exchanger (NCX) activity is regulated by the binding of Ca(2+) to two cytosolic Ca(2+) -binding domains, CBD1 and CBD2, such that Ca(2+) -binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric...

Functional motions of (15)N-labeled proteins can be monitored by solution NMR spin relaxation experiments over a broad range of timescales. These experiments however typically take of the order of several days to a week per protein. Recently, NMR chemical exchange saturation transfer (CEST) experiments have emerged to probe slow millisecond motions complementing R1ρ and CPMG-type experiments. CEST also simultaneously reports on site-specific R1 and R2 parameters. It is shown here how...

Among human body fluids, serum plays a key role for diagnostic tests and, increasingly, for metabolomics analysis. However, the high protein content of serum poses significant challenges for nuclear magnetic resonance (NMR)-based metabolomics studies because it can strongly interfere with metabolite signal detection and quantitation. Although several methods for protein removal have been proposed, including ultrafiltration and organic-solvent-induced protein precipitation, there is currently no...

The flexible nature of protein loops and the time scales of their dynamics are critical for many biologically important events at the molecular level, such as protein interaction and recognition processes. In order to obtain a predictive understanding of the dynamic properties of loops, 500 ns molecular dynamics (MD) computer simulations of 38 different proteins were performed and validated using NMR chemical shifts. A total of 169 loops were analyzed and classified into three types, namely fast...

Functional protein motions covering a wide range of time scales can be studied, among other techniques, by NMR and by molecular dynamics (MD) computer simulations. MD simulations of proteins now routinely extend into the hundreds of nanoseconds time scale range exceeding the overall tumbling correlation times of proteins in solution by several orders of magnitude. This provides a unique opportunity to rigorously validate these simulations by quantitative comparison with model-free order...

A robust protocol for the treatment of NMR protein structures is presented that makes them amenable to long time scale molecular dynamics (MD) simulations that are stable. The protocol embeds an NMR structure in a native low energy region of the recently developed ff99SB_φψ(g24;CS) molecular mechanics force field. Extended MD trajectories that start from these structures show good consistency with proton-proton nuclear Overhauser effect data, and they reproduce NMR chemical shift data better...

We mined the most recent editions of the BioMagResDataBank and the protein data bank to parametrize a new empirical knowledge-based chemical shift predictor of protein backbone atoms using either a linear or an artificial neural network model. The resulting chemical shift predictor PPM_One accepts a single static 3D structure as input and emulates the effect of local protein dynamics via interatomic steric contacts. Furthermore, the chemical shift prediction was extended to most side-chain...

Metabolomics aims at a complete characterization of all metabolites in biological samples in terms of both their identities and concentrations. Because changes of metabolites and their concentrations are a direct reflection of cellular activity, it allows for a better understanding of cellular processes and function to be obtained. Although NMR spectroscopy is routinely applied to complex biological mixtures without purification, overlapping NMR peaks often pose a challenge for the comprehensive...

A novel metabolite identification strategy is presented for the combined NMR/MS analysis of complex metabolite mixtures. The approach first identifies metabolite candidates from 1D or 2D NMR spectra by NMR database query, which is followed by the determination of the masses (m/z) of their possible ions, adducts, fragments, and characteristic isotope distributions. The expected m/z ratios are then compared with the MS(1) spectrum for the direct assignment of those signals of the mass spectrum...