Search

The exceptionally rich information content of nuclear magnetic resonance (NMR) spectra is routinely used to identify and characterize molecules and molecular interactions in a wide range of applications, including clinical biomarker discovery, drug discovery, environmental chemistry, and metabolomics. The set of peak positions and intensities from a reference NMR spectrum generally serves as the identifying signature for a compound Reference spectra normally are collected under specific conditions of pH, temperature, and magnetic field strength, because changes in conditions can distort the identifying signatures of compounds. A spin system matrix that parametrizes chemical shifts and coupling constants among spins provides a much richer feature set for a compound than a spectral signature based on peak positions and intensities. Spin system matrices expand the applicability of NMR spectral libraries beyond the specific conditions under which data were collected. In addition to being able to simulate spectra at any field strength, spin parameters can be adjusted to systematically explore alterations in chemical shift patterns due to variations in other experimental conditions, such as compound concentration, pH, or temperature. We present methodology and software for efficient interactive optimization of spin parameters against experimental ID-¹H NMR spectra of small molecules. We have used the software to generate spin system matrices for a set of key mammalian metabolites and are also using the software to parametrize spectra of small molecules used in NMR-based ligand screening. The software, along with optimized spin system matrix data for a growing number of compounds, is available from http://gissmo.nmrfam.wisc.edu/. [ABSTRACT FROM AUTHOR]

A natural bond orbital (NBO) analysis of unpaired electron spin density in metalloproteins is presented, which allows a fast and robust calculation of paramagnetic NMR parameters. Approximately 90% of the unpaired electron spin density occupies metal-ligand NBOs, allowing the majority of the density to be modeled by only a few NBOs that reflect the chemical bonding environment. We show that the paramagnetic relaxation rate of protons can be calculated accurately using only the metal-ligand NBOs and that these rates are in good agreement with corresponding rates measured experimentally. This holds, in particular, for protons of ligand residues where the point-dipole approximation breaks down. To describe the paramagnetic relaxation of heavy nuclei, also the electron spin density in the local orbitals must be taken into account. Geometric distance restraints for 15N can be derived from the paramagnetic relaxation enhancement and the Fermi contact shift when local NBOs are included in the analysis. Thus, the NBO approach allows us to include experimental paramagnetic NMR parameters of 15N nuclei as restraints in a structure optimization protocol. We performed a molecular dynamics simulation and structure determination of oxidized rubredoxin using the experimentally obtained paramagnetic NMR parameters of 15N. The corresponding structures obtained are in good agreement with the crystal structure of rubredoxin. Thus, the NBO approach allows an accurate description of the geometric structure and the dynamics of metalloproteins, when NMR parameters are available of nuclei in the immediate vicinity of the metal-site. [ABSTRACT FROM AUTHOR]

Quantitative one-dimensional (1D) 1H NMR spectroscopy is a useful tool for determining metabolite concentrations because of the direct proportionality of signal intensity to the quantity of analyte. However, severe signal overlap in 1D 1H NMR spectra of complex metabolite mixtures hinders accurate quantification. Extension of 1D 1H to 2D 1H-13C HSQC leads to the dispersion of peaks along the 13C dimension and greatly alleviates peak overlapping. Although peaks are better resolved in 2D 1H-13C HSQC than in 1D 1H NMR spectra, the simple proportionality of cross peaks to the quantity of individual metabolites is lost by resonance-specific signal attenuation during the coherence transfer periods. As a result, peaks for individual metabolites usually are quantified by reference to calibration data collected from samples of known concentration. We show here that data from a series of HSQC spectra acquired with incremented repetition times (the time between the end of the first 1H excitation pulse to the beginning of data acquisition) can be extrapolated back to zero time to yield a time-zero 2D 1H-13C HSQC spectrum (HSQC0) in which signal intensities are proportional to concentrations of individual metabolites. Relative concentrations determined from cross peak intensities can be converted to absolute concentrations by reference to an internal standard of known concentration. Clustering of the HSQC0 cross peaks by their normalized intensities identifies those corresponding to metabolites present at a given concentration, and this information can assist in assigning these peaks to specific compounds. The concentration measurement for an individual metabolite can be improved by averaging the intensities of multiple, nonoverlapping cross peaks assigned to that metabolite. [ABSTRACT FROM AUTHOR]

Although the majority of noncovalent interactions associated with hydrogen and heavy atoms in proteins and other biomolecules are classical hydrogen bonds between polar N-H or O-H moieties and O atoms or aromatic π electrons, high-resolution X-ray crystallographic models deposited in the Protein Data Bank show evidence for weaker C-H…O hydrogen bonds, including ones involving sp3-hybridized carbon atoms. Little evidence is available in proteins for the (even) weaker C-H… S interactions described in the crystallographic literature on small molecules. Here, we report experimental evidence and theoretical verification for the existence of nine aliphatic (sp3-hybridized) C-H…S 3-center-4-electron interactions in the protein Clostridium pasteurianutn rubre- doxin. Our evidence comes from the analysis of carbon-13 NMR chemical shifts assigned to atoms near the iron at the active site of this protein. We detected anomalous chemical shifts for these carbon-13 nuclei and explained their origin in terms of unpaired spin density from the iron atom being delocalized through interactions of the type: C-H… SFe, where S is the sulfur of one of the four cysteine side chains covalently bonded to the iron. These results suggest that polarized sulfur atoms in proteins can engage in multiple weak interactions with surrounding aliphatic groups. We analyze the strength and angular dependence of these interactions and conclude that they may contribute small, but significant, stabilization to the molecule. [ABSTRACT FROM AUTHOR]

The Rieske protein component of the cytochrome bccomplex contains a [2Fe-2S] cluster ligated by two cysteines and two histidines. We report here the PKa values of each of the imidazole rings of the two ligating histidines (His134 and His154) in the oxidized and reduced states of the Rieske protein from Therm us thermophilus (TtRp) as determined by NMR spectroscopy. Knowledge of these PKa values is of critical interest because of their pertinence to the mechanism of electron and proton transfer in the bifurcated O-cycle. Although we earlier had observed the pH dependence of a 15N NMR signal from each of the two ligand histidines in oxidized TtRp (Lin, I. J.; Chen, Y.; Fee, J. A.; Song, J.; Westler, W. M.; Markley, J. L. J. Am. Chem. Soc. 2006, 128, 10672-10673), the strong paramagnetism of the [2Fe-2S] cluster prevented the assignment of these signals by conventional methods. Our approach here was to take advantage of the unique histidine-leucine (Hisi 34-Leul 35) sequence and to use residue-selective labeling to establish a key sequence-specific assignment, which was then extended. Analysis of the pH dependence of assigned 13C, 13Cα, and 15Nϵ2 signals from the two histidine cluster ligands led to unambiguous assignment of the PKa values of oxidized and reduced TtRp. The results showed that the pKa of His134 changes from 9.1 in oxidized to ∼12.3 in reduced TtRp, whereas the pKa of His154 changes from 7.4 in oxidized to ∼12.6 in reduced TtRp. This establishes His154, which is close to the quinone when the Rieske protein is in the cytochrome b site, as the residue experiencing the remarkable redox-dependent pKa shift. Secondary structural analysis of oxidized and reduced TtRp based upon our extensive chemical shift assignments rules out a large conformational change between the oxidized and reduced states. Therefore, TtAp likely translocates between the cytochrome b and cytochrome c sites by passive diffusion. Our results are most consistent with a mechanism involving the coupled transfer of an electron and transfer of the proton across the hydrogen bond between the hydroquinone and His154 at the cytochrome b site. [ABSTRACT FROM AUTHOR]

Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2aPol) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic α-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a-membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2aPol accumulation. The second class of helix A mutations not only maintains efficient 1a-membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2aPol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action. [ABSTRACT FROM AUTHOR]

Paramagnetic NMR spectroscopy has been underutilized in the study of metalloproteins. One difficulty of the technique is that paramagnetic relaxation broadens signals from nuclei near paramagnetic centers. In systems with low electronic relaxation rates, this makes such signals difficult to observe or impossible to assign by traditional methods. We show how the challenges of detecting and assigning signals from nuclei near the metal center can be overcome through the combination of uniform and selective ²H, 13C, and 15N isotopic labeling with NMR experiments that utilize direct one-dimensional (²H, 13C, and 15N) and two-dimensional (13C-X) detection. We have developed methods for calculating NMR chemical shifts and relaxation rates by density functional theory (DFT) approaches. We use the correspondence between experimental NMR parameters and those calculated from structural models of ironsulfur clusters derived from X-ray crystallography to validate the computational approach and to investigate how structural differences are manifested in these values. We have applied this strategy to three iron-sulfur proteins: Clostridium pasteurianum rubredoxin, Anabaena [2Fe-2S] ferredoxin, and human [2Fe-2S] ferredoxin. Provided that an accurate structural model of the iron-sulfur cluster and surrounding residues is available from diffraction data, our results show that DFT calculations can return NMR observables with excellent accuracy. This suggests that it might be possible to use calculations to refine structures or to generate structural models of active sites when crystal structures are unavailable. The approach has yielded insights into the electronic structures of these iron-sulfur proteins. In rubredoxin, the results show that substantial unpaired electron spin is delocalized across NH…S hydrogen bonds and that the reduction potential can be changed by 77 mV simply by altering the strength of one of these hydrogen bonds. In reduced [2Fe-2S] ferredoxins, hyperfine shift data have provided quantitative information on the degree of valence trapping. The approach described here for iron-sulfur proteins offers new avenues for detailed studies of these and other metalloprotein systems. [ABSTRACT FROM AUTHOR]

NMR studies of paramagnetic proteins are hampered by the rapid relaxation of nuclei near the paramagnetic center, which prevents the application of conventional methods to investigations of the most interesting regions of such molecules. This problem is particularly acute in systems with slow electronic relaxation rates. We present a strategy that can be used with a protein with slow electronic relaxation to identify and assign resonances from nuclei near the paramagnetic center. Oxidized human [2Fe-2S] ferredoxin (adrenodoxin) was used to test the approach. The strategy involves six steps: (1) NMR signals from ¹H, 13C, and 15N nuclei unaffected or minimally affected by paramagnetic effects are assigned by standard multinuclear two- and three-dimensional (2D and 3D) spectroscopic methods with protein samples labeled uniformly with 13C and 15N. (2) The very broad, hyperfine-shifted signals from carbons in the residues that ligate the metal center are classified by amino acid and atom type by selective 13C labeling and one-dimensional (1D) 13C NMR spectroscopy. (3) Spin systems involving carbons near the paramagnetic center that are broadened but not hyperfine-shifted are elucidated by 13C{13C} constant time correlation spectroscopy (CT-COSY). (4) Signals from amide nitrogens affected by the paramagnetic center are assigned to amino acid type by selective 15N labeling and 1D 15N NMR spectroscopy. (5) Sequence-specific assignments of these carbon and nitrogen signals are determined by 1D 13C{15N} difference decoupling experiments. (6) Signals from ¹H nuclei in these spin systems are assigned by paramagnetic-optimized 2D and 3D ¹H{13C} experiments. For oxidized human ferredoxin, this strategy led to assignments (to amino acid and atom type) for 88% of the carbons in the [2Fe-2S] cluster-binding loops (residues 43-58 and 89-94). These included complete carbon spin-system assignments for eight of the 22 residues and partial assignments for each of the others. Sequence-specific assignments were determined for the backbone 15N signals from nine of the 22 residues and ambiguous assignments for five of the others. [ABSTRACT FROM AUTHOR]

Reports on the quantum chemical calculations on structural models of the catalytic site of chymotrypsin. Validation and calibration of calculated chemical shifts; Calculation of chemical and isotope shifts for molecules with short, strong hydrogen bonds; Potential energy curves for proton transfer.

Analyzes the Fermi contact portion of J-coupling using the natural J-coupling method. Application of finite perturbation theory to ab initio/density function theory wave functions; Calculation of individual and pairwise orbital contributions to the coupling; Classification of the computed coupling.

Presents information on a study which described experiments that reveal the D/H fractionation factors for His 57-H... in tetrahedral complexes between chymotrypsin and a series of four peptidyl-trifluormethylketones. Materials and methodology; Results; Discussion.

Investigates the D/H fractionation factors and the temperature dependence of the rate of hydrogen exchange at two sites in the catalytic triad of chymotrypsinogen. Consistency of results with the hypothesis that changes in hydrogen bonding strength serve to lower barriers along the reaction coordinate in the catalytic mechanism; Activation enthalpies.

The article present a study related to the detection and classification of hyperfine-shifted 1 hydrogen (H), 2H and 15 nitrogen resonances from the four cysteines that ligate iron in oxidized and reduced clostridium pasteurianum rubredoxin.

The flavoenzyme uridine 5'-diphosphate galactopyranose mutase (UGM or GIf) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. The latter is a key building block for cell wall construction in numerous pathogens, including Mycobacterium tuberculosis. Mechanistic studies of UGM suggested a novel role for the flavin, and we previously provided evidence that the catalytic mechanism proceeds through a covalent flavin—galactose imium. Here, we describe 2.3 and 2.5 Å resolution X-ray crystal structures of the substrate-bound enzyme in oxidized and reduced forms, respectively. In the latter, Cl of the substrate is 3.6 Å from the nucleophilic flavin N5 position. This orientation is consistent with covalent catalysis by flavin. [ABSTRACT FROM AUTHOR]

Investigates the strength of hydrogen bond in the catalytic dyad of transition-state analogue inhibitor complexes of chymotrypsin. List of transition state analogue inhibited complexes; Formation of low-barrier hydrogen bond; Use of tritium.

Examines the strategy for paramagnetic proteins with slow electronic relaxation rates. Determination of data using nuclear magnetic resonance for paramagnetic proteins; Correlation between nuclear and electronic relaxation; Connectivity of information for fast relaxing resonance.

Analyzes the hydrogen-bond in biomolecules. Analysis of the natural bond orbital; Nature of the hydrogen-bond; Production of wave function with J coupling information.

Introduces a pulse sequence called CBD-NOESY (complementary block-decoupled NOESY) which allows analysis of NOEs between protons in different spectral regions free of interference from magnetization transfer steps withiin each single region. Application of the sequence method to methylene groups in proteins; Effects on local structural parameters.

Looks at how spermine and spermidine are natural polyamines widely distributed in biological systems and have shown to be critical for cell growth and carcinogenesis. What the crystallographic X-ray analysis of yeast tRNAPhe reported; Identification of the binding site of spermine on tRNA; Use of carbon-bound methylene protons.

The metabolome, the collection of small molecules associated with an organism, is a growing subject of inquiry, with the data utilized for data-intensive systems biology, disease diagnostics, biomarker discovery, and the broader characterization of small molecules in mixtures. Owing to their close proximity to the functional endpoints that govern an organism's phenotype, metabolites are highly informative about functional states. The field of metabolomics identifies and quantifies endogenous and exogenous metabolites in biological samples. Information acquired from nuclear magnetic spectroscopy (NMR), mass spectrometry (MS), and the published literature, as processed by statistical approaches, are driving increasingly wider applications of metabolomics. This review focuses on the role of databases and software tools in advancing the rigor, robustness, reproducibility, and validation of metabolomics studies. [ABSTRACT FROM AUTHOR]

Abstract: Galactofuranose (Galf) residues are present in cell wall glycoconjugates of numerous pathogenic microbes. Uridine 5''-diphosphate (UDP) Galf, the biosynthetic precursor of Galf-containing glycoconjugates, is produced from UDP-galactopyranose (UDP-Galp) by the flavoenzyme UDP-galactopyranose mutase (UGM). The gene encoding UGM (glf) is essential for the viability of pathogens, including Mycobacterium tuberculosis, and this finding underscores the need to understand how UGM functions. Considerable effort has been devoted to elucidating the catalytic mechanism of UGM, but progress has been hindered by a lack of structural data for an enzyme–substrate complex. Such data could reveal not only substrate binding interactions but how UGM can act preferentially on two very different substrates, UDP-Galp and UDP-Galf, yet avoid other structurally related UDP sugars present in the cell. Herein, we describe the first structure of a UGM–ligand complex, which provides insight into the catalytic mechanism and molecular basis for substrate selectivity. The structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP-glucose (UDP-Glc) was solved by X-ray crystallographic methods and refined to 2.5 Å resolution. The ligand is proximal to the cofactor, a finding that is consistent with a proposed mechanism in which the reduced flavin engages in covalent catalysis. Despite this proximity, the glucose ring of the substrate analog is positioned such that it disfavors covalent catalysis. This orientation is consistent with data indicating that UDP-Glc is not a substrate for UGM. The relative binding orientations of UDP-Galp and UDP-Glc were compared using saturation transfer difference NMR. The results indicate that the uridine moiety occupies a similar location in both ligand complexes, and this relevant binding mode is defined by our structural data. In contrast, the orientations of the glucose and galactose sugar moieties differ. To understand the consequences of these differences, we derived a model for the productive UGM–substrate complex that highlights interactions that can contribute to catalysis and substrate discrimination. [Copyright &y& Elsevier]

The Ebola Virus is a causative agent of viral hemorrhagic fever outbreaks and a potential global health risk. The outbreak in West Africa (2013–2016) led to 11,000+ deaths and 30,000+ Ebola infected individuals. The current outbreak in the Democratic Republic of Congo (DRC) with 3000+ confirmed cases and 2000+ deaths attributed to Ebola virus infections provides a reminder that innovative countermeasures are still needed. Ebola virus encodes 7 open reading frames (ORFs). Of these, the nucleocapsid protein (eNP) encoded by the first ORF plays many significant roles, including a role in viral RNA synthesis. Here we describe efforts to target the C-terminal domain of eNP (eNP-CTD) that contains highly conserved residues 641–739 as a pan-Ebola antiviral target. Interactions of eNP-CTD with Ebola Viral Protein 30 (eVP30) and Viral Protein 40 (eVP40) have been shown to be crucial for viral RNA synthesis, virion formation, and virion transport. We used nuclear magnetic response (NMR)-based methods to screened the eNP-CTD against a fragment library. Perturbations of 1D 1H NMR spectra identified of 48 of the 439 compounds screened as potential eNP CTD interactors. Subsequent analysis of these compounds to measure chemical shift perturbations in 2D 1H,15N NMR spectra of 15N-labeled protein identified six with low millimolar affinities. All six perturbed an area consisting mainly of residues at or near the extreme C-terminus that we named "Site 1" while three other sites were perturbed by other compounds. Our findings here demonstrate the potential utility of eNP as a target, several fragment hits, and provide an experimental pipeline to validate viral-viral interactions as potential panfiloviral inhibitor targets. • Performed a high throughput screen (HTS) to identify binders of Ebola NP. • Identified several binders that target Ebola NP C-terminal domain. • Validated binding and propose structure-activity relationships. • Highlight the utility of Ebola NP C-terminus as a potential therapeutic target. [ABSTRACT FROM AUTHOR]

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as “membrane-distal” and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane. [ABSTRACT FROM AUTHOR]

Summary: PONDEROSA (Peak-picking Of Noe Data Enabled by Restriction of Shift Assignments) accepts input information consisting of a protein sequence, backbone and sidechain NMR resonance assignments, and 3D-NOESY (13C-edited and/or 15N-edited) spectra, and returns assignments of NOESY crosspeaks, distance and angle constraints, and a reliable NMR structure represented by a family of conformers. PONDEROSA incorporates and integrates external software packages (TALOS+, STRIDE and CYANA) to carry out different steps in the structure determination. PONDEROSA implements internal functions that identify and validate NOESY peak assignments and assess the quality of the calculated three-dimensional structure of the protein. The robustness of the analysis results from PONDEROSA's hierarchical processing steps that involve iterative interaction among the internal and external modules. PONDEROSA supports a variety of input formats: SPARKY assignment table (.shifts) and spectrum file formats (.ucsf), XEASY proton file format (.prot), and NMR-STAR format (.star). To demonstrate the utility of PONDEROSA, we used the package to determine 3D structures of two proteins: human ubiquitin and Escherichia coli iron-sulfur scaffold protein variant IscU(D39A). The automatically generated structural constraints and ensembles of conformers were as good as or better than those determined previously by much less automated means.Availability: The program, in the form of binary code along with tutorials and reference manuals, is available at http://ponderosa.nmrfam.wisc.edu/.Contact: whlee@nmrfam.wisc.edu; markley@nmrfam.wisc.eduSupplementary information: Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time. [ABSTRACT FROM AUTHOR]

Examines the isotope effects on the chemical shifts in Clostridium pasteurianum rubredoxin with the use of the fermi-contact effects relating to the hydrogen bonds. Contents of the rubredoxins; What the resolved splittings have enabled; Reports of isotope effects on hyperfine chemical shifts; Information on pairing signals.

Forazoline A, a novel antifungal polyketide with in vivo efficacy against Candida albicans, was discovered using LCMS-based metabolomics to investigate marine-invertebrate-associated bacteria. Forazoline A had a highly unusual and unprecedented skeleton. Acquisition of 13C-13C gCOSY and 13C-15N HMQC NMR data provided the direct carbon-carbon and carbon-nitrogen connectivity, respectively. This approach represents the first example of determining direct 13C-15N connectivity for a natural product. Using yeast chemical genomics, we propose that forazoline A operated through a new mechanism of action with a phenotypic outcome of disrupting membrane integrity. [ABSTRACT FROM AUTHOR]

Forazoline A, a novel antifungal polyketide with in vivo efficacy against Candida albicans, was discovered using LCMS-based metabolomics to investigate marine-invertebrateassociated bacteria. Forazoline A had a highly unusual and unprecedented skeleton. Acquisition of 13C-³C gCOSY and 13C-5N HMQC NMR data provided the direct carbon-carbon and carbon-nitrogen connectivity, respectively. This approach represents the first example of determining direct 13C-15N connectivity for a natural product. Using yeast chemical genomics, we propose that forazoline A operated through a new mechanism of action with a phenotypic outcome of disrupting membrane integrity. [ABSTRACT FROM AUTHOR]

We report nuclear magnetic resonance (NMR) data indicating that the Rieske protein from the cytochrome bc complex of Thermus thermophilus (TtRp) undergoes modest redox-state-dependent and ligand-dependent conformational changes. To test models concerning the mechanism by which TtRp transfers between different sites on the complex, we monitored ¹H, 15N, and 13C NMR signals as a function of the redox state and molar ratio of added ligand. Our studies of full-length TtRp were conducted in the presence of dodecyl phosphocholine micelles to solvate the membrane anchor of the protein and the hydrophobic tail of the ligand (hydroubiquinone). NMR data indicated that hydroubiquinone binds to TtRp and stabilizes an altered protein conformation. We utilized a truncated form of the Rieske protein lacking the membrane anchor (trunc-TtRp) to investigate redox-state-dependent conformational changes. Local chemical shift perturbations suggested possible conformational changes at prolyl residues. Detailed investigations showed that all observable prolyl residues of oxidized trunc-TtRp have trans peptide bond configurations but that two of these peptide bonds (Cys151-Pro152 and Gly169-Pro170 located near the iron–sulfur cluster) become cis in the reduced protein. Changes in the chemical shifts of backbone signals provided evidence of redox-state- and ligand-dependent conformational changes localized near the iron-sulfur cluster. These structural changes may alter interactions between the Rieske protein and the cytochrome b and c sites and provide part of the driving force for movement of the Rieske protein between these two sites. [ABSTRACT FROM AUTHOR]

Abstract: Vitamin D receptor (VDR) plays a crucial role in many cellular processes including calcium and phosphate homeostasis. Previous purification methods from prokaryotic and eukaryotic expression systems were challenged by low protein solubility accompanied by multi purification steps resulting in poor protein recovery. The full-length VDR and its ligand binding domain (LBD) were mostly (>90%) insoluble even when expressed at low temperatures in the bacterial system. We describe a one-step procedure that results in the purification of rat VDR and LBD proteins in high-yield from Escherichia coli inclusion bodies. The heterologously expressed protein constructs retained full function as demonstrated by ligand binding and DNA binding assays. Furthermore, we describe an efficient strategy for labeling these proteins with 2H, 13C, and 15N for structural and functional studies by nuclear magnetic resonance (NMR) spectroscopy. This efficient production system will facilitate future studies on the mechanism of vitamin D action including characterization of the large number of synthetic vitamin D analogs that have been developed. [Copyright &y& Elsevier]

Persistent infections are frequently caused by dormant and biofilm-associated bacteria, which often display characteristically slow growth. Antibiotics that require rapid cell growth may be ineffective against these organisms and thus fail to prevent reoccurring infections. In contrast to growth-based antimicrobial agents, membrane-targeting drugs effectively kill slow-growing bacteria, Herein we introduce 2-((3-(3,6-dichloro-9H-carbazol-9- yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane- 1,3-diol (DCAP), a potent broad-spectrum antibiotic that reduces the transmembrane potential of Gram-positive and Gram-negative bacteria and causes mislocalization of essential membrane-associated proteins, including MinD and FtsA. Importantly, DCAP kills nutrient-deprived microbes and sterilizes bacterial bioflims. DCAP is lethal against bacterial cells, has no effect on red blood cell membranes, and only decreases the viability of mammalian cells after ≥6 h. We conclude that membrane-active compounds are a promising solution for treating persistent infections. DCAP expands the limited number of compounds in this class of therapeutic small molecules and provides new opportunities for the development of potent broad-spectrum antimicrobial agents. [ABSTRACT FROM AUTHOR]

We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the "flap" structures (residues 37-61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations. We show that conformational isomerization in the flaps is correlated with structural reorganization of residues in the active site, and that it is preorganization of the active site that is a rate-limiting factor in catalysis. [ABSTRACT FROM AUTHOR]

Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogues, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogues cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a potent agonist analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D3 [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of coactivators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and -12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of coactivators. [ABSTRACT FROM AUTHOR]

Hsp12 (heat shock protein 12) belongs to the small heat shock protein family, partially characterized as a stress response, stationary phase entry, late embryonic abundant-like protein located at the plasma membrane to protect membrane from desiccation. Here, we report the structural characterization of Hsp12 by NMR and biophysical techniques. The protein was labeled uniformly with nitrogen-15 and carbon-13 so that its conformation could be determined in detail both in aqueous solution and in two membrane-mimetic environments, SDS and dodecylphosphocholine (DPC) micelles. Secondary structural elements determined from assigned chemical shifts indicated that Hsp12 is dynamically disordered in aqueous solution, whereas it gains four helical stretches in the presence of SDS micelles and a single helix in presence of DPC. These conclusions were reinforced by circular dichroism spectra of the protein in all three environments. The lack of long range interactions in NOESY spectra indicated that the helices present in SDS micelles do not pack together. R1 and R2, relaxation and heteronuclear NOE measurements showed that the protein is disordered in aqueous solution but becomes more ordered in presence of detergent micelles. NMR spectra collected in presence of paramagnetic spin relaxation agents (5DSA, 16DSA, and Gd(DTPA-BMA)) indicated that the amphipathic a-helices of Hsp12 in SDS micelles lie on the membrane surface. These observations are in agreement with studies suggesting that Hsp12 functions to protect the membrane from desiccation. [ABSTRACT FROM AUTHOR]

Isotope-based methods are commonly used for metabolic flux analysis and metabolite quantification in biological extracts. Nuclear magnetic resonance (NMR) spectroscopy isa powerful analytical tool for these studies because NMR can unambiguously identify compounds and accurately measure 13C enrichment. We have developed a new pulse sequence, isotope-edited total correlation spectroscopy (ITOCSY), that filters two-dimensional 1H-1H NMR spectra from 12C- and 13C-containing molecules into separate, quantitatively equivalent spectra. The ITOCSY spectra of labeled and unlabeled molecules are directly comparable and can be assigned using existing bioinformatics tools. In this study, we evaluate ITOCSY using synthetic mixtures of standards and extracts from Escherichia coil. We show that ITOCSY has low technical error (6.6% for metabolites ranging from 0.34 to 6.2 mM) and can detect molecules at concentrations less than 10 μM. We propose ITOCSY as a practical NMR strate' for metabolic flux analysis, isotope dilution experiments, and other methods that rely on carbon-13 labeling. [ABSTRACT FROM AUTHOR]

Stable isotope-labeling methods, coupled with novel techniques for detecting fast-relaxing NMR signals, now permit detailed investigations of paramagnetic centers of metalloproteins. We have utilized these advances to carry out comprehensive assignments of the hyperfine-shifted 13C and 15N signals of the rubredoxin from Clostridium pasteurianum (CpRd) in both its oxidized and reduced states. We used residue-specific labeling (by chemical synthesis) and residue-type-selective labeling (by biosynthesis) to assign signals detected by one-dimensional 15N NMR spectroscopy, to nitrogen atoms near the iron center. We refined and extended these 15N assignments to the adjacent carbonyl carbons by means of one-dimensional 13C[15N] decoupling difference experiments. We collected paramagnetic-optimized Su- perWEFT 13C[13C] constant time COSY (SW-CT-COSY) data to complete the assignment of 13C signals of reduced CpRd. By following these 13C signals as the protein was gradually oxidized, we transferred these assignments to carbons in the oxidized state. We have compared these assignments with hyperfine chemical shifts calculated from available X-ray structures of CpRd in its oxidized and reduced forms. The results allow the evaluation of the X-ray structural models as representative of the solution structure of the protein, and they provide a framework for future investigation of the active site of this protein. The methods developed here should be applicable to other proteins that contain a paramagnetic center with high spin and slow electron exchange. [ABSTRACT FROM AUTHOR]

IscU is a scaffold protein that functions in iron-sulfur cluster assembly and transfer. Its critical importance has been recently underscored by the finding that a single intronic mutation in the human iscu gene is associated with a myopathy resulting from deficient succinate dehydrogenase and aconitase [Mochel, F., Knight M. A., TOng W. H., Hernandez, D., Ayyad, K., Taivassalo, T., Andersen, P. M., Singleton, A., Rouault, T. A., Fischbeck, K. H., Haller, R. G. (2008) aAm. J. Hum. Genet.82, 652-660]. IscU functions through interactions with a chaperone protein HscA and a cochaperone protein HscB. To probe the molecular basis for these interactions, we have used NMR spectroscopy to invastigate the solution structure of IscU from Escherichia coli and its interaction with HscB from the same organism. We found that wild-type apo-IscU in solution exists as two distinct confrontation: one largely disordered and one largely ordered except for the metal binding residues. The two states interconvert on the millisecond time scale. The ordered conformation is stabilized by the addition of zinc or by the single-site IscU mutation, D39A. We used apo-IscU(D39A) as a surrogate for the folded state of wild-type IscU and assigned its NMR spectrum. These assignments made it possible to identify the region of IscU with the largest structural differences in the two conformational states. Subsequently, by following the NMR signals of apo-IscU (D39A) upon addition of HscB, we identified the most perturbed regions as the two N-terminal β-strands and the C-terminal α-helix. On the basis of these results and analysis of IscU sequences from multiple species, we have identified the surface region of IscU that interacts with HscB. We conclude that the IscU-HscB complex exists as two (or more) distinct states that interconvert at a rate much faster that the rate of dissociation of the complex and that HscB binds to and stabilizes the ordered state of apo-IscU. [ABSTRACT FROM AUTHOR]

One-dimensional (1D) 1H nuclear magnetic resonance (NMR) spectroscopy is used extensively for high-throughput analysis of metabolites in biological fluids and tissue extracts. Typically, such spectra are treated as multivariate statistical objects rather than as collections of quantifiable metabolites. We report here a two-dimensional (2D) 1H-13C NMR strategy (fast metabolite quantification, FMQ, by NMR) for identifying and quantifying the ∼40 most abundant metabolites in biological samples. To validate this technique, we prepared mixtures of synthetic compounds and extracts from Arabidopsis thaliana, Saccharomyces cerevisiae, and Medicago sativa. We show that accurate (technical error 2.7%) molar concentrations can be determined in 12 min using our quantitative 2D 1H-13C NMR strategy. In contrast, traditional 1D 1H NMR analysis resulted in 16.2% technical error under nearly ideal conditions. We propose FMQ by NMR as a practical alternative to 1D 1H NMR for metabolomics studies in which 50-mg (extract dry weight) samples can be obtained. [ABSTRACT FROM AUTHOR]

Complexation of the small serine protease inhibitor Schistocerca gregaria chymotrypsin inhibitor (SGCI), a member of the pacifastin inhibitor family, with bovine chymotrypsin was followed by NMR spectroscopy. 1H–15N correlation (HSQC) spectra of the inhibitor with increasing amounts of the enzyme reveal tight and specific binding in agreement with biochemical data. Unexpectedly, and unparalleled among canonical serine protease inhibitors, not only residues in the protease-binding loop of the inhibitor, but also some segments of it located spatially far from the substrate-binding cleft of the enzyme were affected by complexation. However, besides changes, some of the dynamical features of the free inhibitor are retained in the complex. Comparison of the free and complexed inhibitor structures revealed that most, but not all, of the observed chemical shift changes can be attributed to minor structural transitions. We suggest that the classical ‘scaffold + binding loop’ model of canonical inhibitors might not be fully valid for the inhibitor family studied. In our view, this feature allows for the emergence of both taxon-specific and nontaxon-specific inhibitors in this group of small proteins. [ABSTRACT FROM AUTHOR]

The rubredoxin from Ciostridium pasteurianum (CpRd) provides an excellent system for investigating how the protein sequence modulates the reduction potential of the active site in an iron-sulfur protein. 15N NMR spectroscopy has allowed us to determine with unprecedented accuracy the strengths of all six key hydrogen bonds between protein backbone amides and the sulfur atoms of the four cysteine residues that ligate the iron in the oxidized (FeII) and reduced (FeIII) forms of wild-type CpRd and nine mutants (V44G, V44A, V441, V441, V86, V8A, V81, V8L and V8G/V44G). The length (or strength) of each hydrogen bond was inferred from the magnitude of electron spin delocalized across the hydrogen bond from the iron atom onto the nitrogen. The aggregate lengths of these six hydrogen bonds are shorter in both oxidation states in variants with higher reduction potential than in those with lower reduction potential. Differences in aggregate hydrogen bonding upon reduction correlate linearly with the published reduction potentials for the 10 CpRd variants, which span 126 mV. Sequence effects on the reduction potential can be explained fully by their influence on hydrogen-bond strengths. [ABSTRACT FROM AUTHOR]

Toluene 4-monooxygenase, a four-protein complex from Pseudomonas mendocina KR1, catalyzes the NADH- and O2-dependent hydroxylation of toluene to form p-cresol. The solution structure of the 112-amino-acid Rieske ferredoxin component, T4moC, was determined from 2D and 3D 1H, 13C, and 15N NMR data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space with input from 1650 experimental restraints, including 1264 inter-proton distance restraints obtained from NOEs, 247 non-redundant intra-residue NOEs, 26 hydrogen bond restraints, and 113 dihedral angle ( φ, ψ) restraints. The 20 calculated conformers that best satisfied the input restraints were submitted to refinement in explicit solvent to improve the stereochemical quality. With exclusion of ill-defined N- and C-terminal segments (Ser2; His111–Ser112) and residues near to the [2Fe-2S] cluster, the atomic root mean square deviation for the 20 conformers with respect to the mean coordinates was 1.09 Å for the backbone and 1.60 Å for all non-hydrogen atoms. The T4moC structure consists of 10 β-strands arranged in the three anti-parallel β-sheet topology observed in all Rieske [2Fe-2S] domain proteins. The S γ of Cys45 and Cys64 and the N δ1 of His47 and His67 provide the ligands to the [2Fe-2S] cluster of T4moC. 1H–15N HSQC measurements show that both His47-N ε2 and His67-N ε2 are protonated at the pH of the NMR experiments. Comparisons are made between the present NMR structure, previous paramagnetic NMR studies of T4moC, and the X-ray structures of other members of the Rieske protein family. [ABSTRACT FROM AUTHOR]

The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to remain folded, one approach has been to encapsulate hydrated proteins in reverse micelles formed by a detergent and to dissolve the encapsulated protein in a low-viscosity fluid. The high volatility of suitable low-viscosity fluids requires that the samples be prepared and maintained under pressure. We describe a novel apparatus used for the preparation of such samples. The apparatus includes a chamber for mixing the detergent with the low-viscosity solvent, a second chamber for mixing this with hydrated protein, and a 5-mm (o.d.) zirconium oxide NMR sample tube with shut-off valves designed to contain pressures on the order of 10 bar, sufficient for liquid propane. Liquids are moved from one location to another by introducing minor pressure differentials between two pressurization vessels. We discuss the operation of this apparatus and illustrate this with data on a 30-kDa protein complex (chymotrypsin:turkey ovomucoid third domain) encapsulated in reverse micelles of the detergent, sodium bis (2-ethylhexyl) sulfosuccinate, aerosol-ot (AOT), dissolved in liquid propane. [Copyright &y& Elsevier]

The aryl hydrocarbon receptor (AHR) is a ligand-inducible transcription factor that is best known because it mediates the actions of polycyclic and halogenated aromatic hydrocarbon environmental toxicants such as 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. We report here the successful identification of an endogenous ligand for this receptor; ≈20 µg was isolated in pure form from 35 kg of porcine lung. Its structure was deduced as 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester from extensive physical measurements and quantum mechanical calculations. In a reporter gene assay, this ligand activates the AHR with a potency five times greater than that of β-naphthoflavone, a prototypical synthetic AHR ligand. 2-(l′H-indole-3′-carbonyl)thiazole-4-carboxylic acid methyl ester competes with 2,3,7,8[³H]tetrachlorodibenzo-p-dioxin for binding to human, murine, and fish AHRs, thus showing that AHR activation is caused by direct receptor binding, and that recognition of this endogenous ligand is conserved from early vertebrates (fish) to humans. [ABSTRACT FROM AUTHOR]

Presents a study which examined a peptide containing residues 36-59 of the human CD4 receptor, which includes most of the residues thought to be involved in binding the HIV surface glycoprotein, gp120. Synthesis of the peptide; Description of the human CD4; Characterization of CD4 peptides; Methodology employed in this study; Findings of the study.

Focuses on the nuclear magnetic resonance (NMR) investigations relating to the Clostridium pasteurianum rubredoxin. Representation of these rubredoxin; What the rubredoxin provides; How the correlation with experiment was improved.

A letter to the editor is presented in response to metabolite identification via the Madison metabolomics consortium database.

The article discusses the study that demonstrates the role of iron-ligated histidines in the pH dependence of the reduction potential. The researchers recorded a 15N NMR spectra of oxidized Rieke iron—sulfur protein from Thermus thermophilis as function of pH. Direct evidence for sequential dissociation of protons from N∈2 atoms of the two iron-coordinated histidine rings in the oxidized protein has been provided by the data.

Investigates the correlation between hydrogen bond lengths and reduction potentials in Clostridium pasteurianum Rubredoxin. Function of hydrogen bonding as determinant of reduction potential in rubredoxins; Mechanism by which a shorter hydrogen bond stabilizes the reduced state in CpRd; Capacity of electron delocalization.