Methyl Resonances Research Articles

NMR measurement of bacteria death kinetics during heat stress

Modelling the thermal processing of food matrices incorporating microorganisms requires heat stress death kinetics. It is demonstrated that proton nuclear magnetic resonance (1H NMR) is a rapid means of quantifying the heat stress death kinetics of a human probiotic bacterium (Bifidobacterium animalis subsp. lactis DR10™ or strain HN019). It was found that the ratio of the methylene (CH2) resonance at 1.2 ppm to the methyl (CH3) resonance at 0.9 ppm signal intensity as observed by 1H NMR was directly proportional to the percentage of death of a population of cells during heat stress. The derived death kinetic rate constants for a series of heating temperatures showed excellent correlation to the Arrhenius equation. The method has several advantages over existing methods for measuring bacterial death kinetics, as it can be carried out in situ with minimal preparation and without the addition of chemical agents.

Functional Dynamics of Deuterated β2‐Adrenergic Receptor in Lipid Bilayers Revealed by NMR Spectroscopy

AbstractG‐protein‐coupled receptors (GPCRs) exist in conformational equilibrium between active and inactive states, and the former population determines the efficacy of signaling. However, the conformational equilibrium of GPCRs in lipid bilayers is unknown owing to the low sensitivities of their NMR signals. To increase the signal intensities, a deuteration method was developed for GPCRs expressed in an insect cell/baculovirus expression system. The NMR sensitivities of the methionine methyl resonances from the β2‐adrenergic receptor (β2AR) in lipid bilayers of reconstituted high‐density lipoprotein (rHDL) increased by approximately 5‐fold upon deuteration. NMR analyses revealed that the exchange rates for the conformational equilibrium of β2AR in rHDLs were remarkably different from those measured in detergents. The timescales of GPCR signaling, calculated from the exchange rates, are faster than those of receptor tyrosine kinases and thus enable rapid neurotransmission and sensory perception.

Functional dynamics of deuterated β2 -adrenergic receptor in lipid bilayers revealed by NMR spectroscopy.

G-protein-coupled receptors (GPCRs) exist in conformational equilibrium between active and inactive states, and the former population determines the efficacy of signaling. However, the conformational equilibrium of GPCRs in lipid bilayers is unknown owing to the low sensitivities of their NMR signals. To increase the signal intensities, a deuteration method was developed for GPCRs expressed in an insect cell/baculovirus expression system. The NMR sensitivities of the methionine methyl resonances from the β2 -adrenergic receptor (β2 AR) in lipid bilayers of reconstituted high-density lipoprotein (rHDL) increased by approximately 5-fold upon deuteration. NMR analyses revealed that the exchange rates for the conformational equilibrium of β2 AR in rHDLs were remarkably different from those measured in detergents. The timescales of GPCR signaling, calculated from the exchange rates, are faster than those of receptor tyrosine kinases and thus enable rapid neurotransmission and sensory perception.

Monitoring Cocrystal Formation via In Situ Solid-State NMR.

A detailed understanding of the mechanism of organic cocrystal formation remains elusive. Techniques that interrogate a reacting system in situ are preferred, though experimentally challenging. We report here the results of a solid-state in situ NMR study of the spontaneous formation of a cocrystal between a pharmaceutical mimic (caffeine) and a coformer (malonic acid). Using (13)C magic angle spinning NMR, we show that the formation of the cocrystal may be tracked in real time. We find no direct evidence for a short-lived, chemical shift-resolved amorphous solid intermediate. However, changes in the line width and line center of the malonic acid methylene resonance, in the course of the reaction, provide subtle clues to the mode of mass transfer that underlies cocrystal formation.

Peripheral Methyl Activation in η4-1,2,3,4-Tetramethylcyclobutadienylcobalt Complexes: Template Synthesis and Subsequent Reactivity of Triphosphamacrocycles

The cationic complex (η4-1,2,3,4-tetramethylcyclobutadienyl)cobalt(trisacetonitrile), [(η4-C4Me4)Co(NCCH3)3]+ (1), allows the stepwise introduction of suitable phosphine precursors to the [(η4-C4Me4)Co]+ fragment by replacement of the labile acetonitrile ligands. These reactions give rise to the piano-stool complexes [(η4-C4Me4)Co(dppe)(NCCH3)]+ (2), [(η4-C4Me4)Co(dppe)(PH2Ph)]+ (3), [(η4-C4Me4)Co(dfppb)(NCCH3)]+ (4), and [(η4-C4Me4)Co(dfppb)(PH2Ph)]+ (5), where dfppb = 1,2-bis{di(2-fluorophenyl)phosphino}benzene and dppe = 1,2-bis(diphenylphosphino)ethane. Complex 5 is a template for the synthesis of the P3 macrocycle complex [(η4-C4Me4)Co{1,4-bis(2-fluorophenyl)-7-phenyl[b,e,h]tribenzo-1,4,7-triphosphacyclononane}]+ (6), through base-promoted intramolecular macrocyclization. The hydrogens of two of the ring methyls of the tetramethylcyclobutadienyl ligand in the macrocycle complex 6 are sufficiently acidic to undergo deprotonation by KOtBu, promoting nucleophilic attack at the fluorine-bearing ortho-carbons of the 2-fluoroaryl groups on two of the phosphorus donors in 6. The resultant hemi-incarcerand complex [{η4,κP,κP,κP-Me2C4-[1,4-bis(2-CH2C6H4)-7-C6H5-[b,e,h]tribenzo-1,4,7-triphosphacyclononane]-1,2}Co]+ (cis-7) contains a hybrid phosphorus/carbon donor ligand where the P3 macrocycle is connected to the cyclobutadienyl function through two cis-2-methylphenyl links. The new complexes have been characterized fully by spectroscopic and analytical techniques including single-crystal X-ray structure determinations of 2, 3, 4, 5, 6, and cis-7.

Dual-phosphorylated human p38 alpha ILVM methyl resonance assignments in ADP-bound state

Dual-phosphorylated human p38 alpha ILVM methyl resonance assignments in ADP-bound state

Biosynthesis of the 4-Methyloxazoline-Containing Nonribosomal Peptides, JBIR-34 and -35, in Streptomyces sp. Sp080513GE-23

JBIR-34 and -35 produced by Streptomyces sp. Sp080513GE-23 are nonribosomal peptides that possess an unusual 4-methyloxazoline moiety. Through draft genome sequencing, cosmid cloning, and gene disruption, the JBIR-34 and -35 biosynthesis gene cluster (fmo cluster) was identified; it encodes 20 proteins including five nonribosomal peptide synthetases (NRPSs). Disruption of one of these NRPS genes (fmoA3) resulted in no JBIR-34 and -35 production and accumulation of 6-chloro-4-hydroxyindole-3-carboxylic acid. Stable isotope-feeding experiments indicated that the methyl group of the methyloxazoline ring is derived from alanine rather than methionine. A recombinant FmoH protein, aglycine/serine hydroxymethyltransferase homolog, catalyzed conversion of α-methyl-l-serine into d-alanine (the reverse reaction of α-methyl-l-serine synthesis catalyzed by FmoH invivo). Taken together, we concluded that α-methyl-l-serine synthesized from d-alanine is incorporated into JBIR-34 and -35 to form the 4-methyloxazoline moiety. We also propose the biosynthesis pathway of JBIR-34 and -35.

MO‐G‐18C‐07: Improving T2 Determination and Quantification of Lipid Methylene Protons in Proton Magnetic Resonance Spectroscopy at 3 T

Purpose:To improve proton magnetic resonance spectroscopy (MRS) transverse relaxation (T2) determination and quantification of lipid methylene chain (1.3 ppm) protons by rewinding their J‐coupling evolution.Methods:MRS experiments were performed on four lipid phantoms, namely, almond, corn, sunflower and oleic acid, using a 3 T Philips MRI scanner with a transmit/receive birdcage head coil. Two PRESS (Point RESolved Spectroscopy) pulse sequences were used. The first PRESS sequence employed standard bandwidth (BW) (∼550 Hz) RF (radiofrequency) refocussing pulses, while the second used refocussing pulses of narrow BW (∼50 Hz) designed to rewind J‐coupling evolution of the methylene protons in the voxel of interest. Signal was acquired with each sequence from a 5×5×5 mm3 voxel, with a repetition time (TR) of 3000 ms, and with echo times (TE) of 100 to 200 ms in steps of 20 ms. 2048 sample points were measured with a 2000 Hz sampling bandwidth. Additionally, 30 mm outer volume suppression slabs were used to suppress signal outside the voxel of interest. The frequency of the RF pulses was set to that of the methylene resonance. Methylene peak areas were calculated and fitted in MATLAB to a monexponentially decaying function of the form M0exp(‐TE/T2), where M0 is the extrapolated area when TE = 0 ms and yields a measure of concentration.Results:The determined values of M0 and T2 increased for all fatty acids when using the PRESS sequence with narrow BW refocussing pulses. M0 and T2 values increased by an average amount (over all the phantoms) of 31% and 14%, respectively.Conclusion:This investigation has demonstrated that J‐coupling interactions of lipid methylene protons causes non‐negligible signal losses which, if not accounted for, Result in underestimations of their levels and T2 values when performing MRS measurements.Funded by the Natural Sciences and Engineering Research Council of Canada and the Canadian Breast Cancer Foundation – Prairies.NWT

FLAMEnGO 2.0: An enhanced fuzzy logic algorithm for structure-based assignment of methyl group resonances

We present an enhanced version of the FLAMEnGO (Fuzzy Logic Assignment of Methyl Group) software, a structure-based method to assign methyl group resonances in large proteins. FLAMEnGO utilizes a fuzzy logic algorithm coupled with Monte Carlo sampling to obtain a probability-based assignment of the methyl group resonances. As an input, FLAMEnGO requires either the protein X-ray structure or an NMR structural ensemble including data such as methyl–methyl NOESY, paramagnetic relaxation enhancement (PRE), methine–methyl TOCSY data. Version 2.0 of this software (FLAMEnGO 2.0) has a user-friendly graphic interface and presents improved modules that enable the input of partial assignments and additional NMR restraints. We tested the performance of FLAMEnGO 2.0 on maltose binding protein (MBP) as well as the C-subunit of the cAMP-dependent protein kinase A (PKA-C). FLAMEnGO 2.0 can be used as a standalone method or to assist in the completion of partial resonance assignments and can be downloaded at www.chem.umn.edu/groups/veglia/forms/flamengo2-form.html.

Methyl-Beta-Cyclodextrins: The Role of Number and Types of Substituents in Solubilizing Power

Methylated cyclodextrins (CDs) are effective solubilizers of poorly soluble organic compounds. In this work, we compared various methylated β-CDs concerning their structure characterized by nuclear magnetic resonance spectroscopy, composition analyzed by HPLC and solubilizing capability by using model compounds such as cholesterol, fatty acids, furosemide, tamoxifen, and amiodarone. All the commercially available methylated β-CDs are mixtures of various isomers and homologues except trimethyl β-CD. The effects of the degree of methylation, the composition, as well as the influence of further derivatization with ionic groups were studied. The number of methyl groups in a CD ring should be around 14 to get the highest solubility for the included guest molecules. Although the distribution of isomers and related compounds has hardly any effect at constant degree of substitution, the introduction of amino and succinyl moieties on the CD ring adds ionic interactions to the hydrophobic interactions of the inclusion complex formation, which might result in synergic effect in solubilization.

Polypeptide backbone, C(β) and methyl group resonance assignments of the 24 kDa plectin repeat domain 6 from human protein plectin.

The 500kDa protein plectin is essential for the cytoskeletal organization of most mammalian cells and it is up-regulated in some types of cancer. Here, we report nearly complete sequence-specific polypeptide backbone, (13)C(β) and methyl group resonance assignments for 24kDa human plectin(4403-4606) containing the C-terminal plectin repeat domain 6.

Isolation and characterization of catechol derivatives from Semecarpus anacardium seeds and their antibacterial potential in in vitro

The aim of the present investigation was to isolate and identify the phytochemicals from Semecarpus anacardium seeds and evaluate their activity against Gram positive and Gram negative bacterial strains (Bacillus subtilis MTCC 441, Staphylococcus aureus MTCC 96, Escherichia coli MTCC 1689 and Proteus vulgaris MTCC 742 using well diffusion method). The phytochemicals were isolated using silica gel column chromatography and characterized as catechol derivatives by spectroscopic analyses. The antimicrobial activity of the isolated catechol derivatives I-IV varied according to the dose and bacterial strains tested. Among the four catechol derivatives, catechol derivatives I and IV showed prominent antibacterial activity against both the positive and negative human pathogenic organisms compared to catechol derivatives II, III and tetracycline – a reference antibiotic drug. From these findings, it can be concluded that catechol derivatives I and IV possessed methyl group (CH3) in aromatic ring and no double bond in the aliphatic side chain which was not present in catechol derivatives II and III and they possessed double bonds in the aliphatic side chain and no methyl group in the aromatic ring. These may be the possible reason for showing more inhibitory activity of catechol derivatives I and IV. These results showed the effectiveness of catechol derivatives against tested bacterial strains. Further work is necessary to explore these antimicrobial compounds, which are useful in clinical applications.

Neonatal case of classic maple syrup urine disease: Usefulness of 1H‐MRS in early diagnosis

We describe a male neonate with classic maple syrup urine disease (MSUD) in metabolic crisis. On day 7 of life, he was referred to hospital because of coma and metabolic acidosis with maple syrup odor. On day 4 after admission, brain magnetic resonance imaging findings were consistent with encephalopathy due to MSUD. Proton magnetic resonance spectroscopy ((1) H-MRS) showed a large methyl resonance peak at 0.9 p.p.m. The diagnosis of MSUD was confirmed on low branched-chain α-keto acid dehydrogenase complex activity in lymphocyte. (1) H-MR spectra were obtained in 10 min, while it took at least several days to obtain the results of other diagnostic examinations. In convalescence, the peak at 0.9 p.p.m. decreased. The large methyl resonance peak at 0.9 p.p.m. in brain (1) H-MRS would be one of the earliest clues to the diagnosis of classic MSUD in the neonatal period, especially in metabolic crisis.

Electrochemical Conversion of Micropollutants in Gray Water

Electrochemical conversion of micropollutants in real gray water effluent was studied for the first time. Six compounds that are frequently found in personal care and household products, namely methylparaben, propylparaben, bisphenol A, triclosan, galaxolide, and 4- methylbenzilidene camphor (4-MBC), were analyzed in the effluent of the aerobic gray water treatment system in full operation. The effluent was used for lab-scale experiments with an electrochemical cell operated in batch mode. Three different anodes and five different cathodes have been tested. Among the anodes, Ru/Ir mixed metal oxide showed the best performance. Ag and Pt cathodes worked slightly better than Ti and mixed metal oxide cathodes. The compounds that contain a phenolic ring (parabens, bisphenol A, and triclosan) were completely transformed on this anode at a specific electric charge Q = 0.03 Ah/L. The compounds, which contain a benzene ring and multiple side methyl methyl groups (galaxolide, 4-MBC) required high energy input (Q ≤ 0.6 Ah/L) for transformation. Concentrations of adsorbable organohalogens (AOX) in the gray water effluent increased significantly upon treatment for all electrode combinations tested. Oxidation of gray water on mixed metal oxide anodes could not be recommended as a post-treatment step for gray water treatment according to the results of this study. Possible solutions to overcome disadvantages revealed within this study are proposed.

Dependence of residual dipolar couplings on foot angle in 1H MR spectra from skeletal muscle

Foot dorsi and plantar flexion affects the pennation angle of skeletal muscle fibers and changes the fiber direction with respect to the main magnetic field, thereby affecting MR spectrum of the muscle. In order to analyze the effect that foot flexion has on the MR spectrum, tibialis anterior (TA) and soleus muscles were studied in humans and rats. Localized MRS was performed at different foot angles in clinical and pre-clinical settings using a 3T MRI/MRS GE Excite HD and 7T Bruker Clinscan scanner, respectively. In this study we show the effect of foot angle variation on total Creatine (tCr) resonance of 1H spectrum at 3.03 and 3.93ppm for TA and soleus muscles. In addition to this, we observe a 4-line splitting pattern for methylene resonance of tCr in the rat TA spectrum for a specific foot angle. This observation is attributed to the individual splitting of creatine and phosphocreatine of the tCr signal. Novel hydrogel application is demonstrated and used to support our in vivo observations and for the first time splitting of individual resonances of Cr and PCr has been shown in an in vitro set-up.

Backbone and ILV methyl resonance assignments of E. coli thymidylate synthase bound to cofactor and a nucleotide analogue

Backbone and ILV methyl resonance assignments of E. coli thymidylate synthase bound to cofactor and a nucleotide analogue

A NMR experiment for simultaneous correlations of valine and leucine/isoleucine methyls with carbonyl chemical shifts in proteins

A methyl-detected 'out-and-back' NMR experiment for obtaining simultaneous correlations of methyl resonances of valine and isoleucine/leucine residues with backbone carbonyl chemical shifts, SIM-HMCM(CGCBCA)CO, is described. The developed pulse-scheme serves the purpose of convenience in recording a single data set for all Ile(δ1), Leu(δ) and Val(γ) (ILV) methyl positions instead of acquiring two separate spectra selective for valine or leucine/isoleucine residues. The SIM-HMCM(CGCBCA)CO experiment can be used for ILV methyl assignments in moderately sized protein systems (up to ~100 kDa) where the backbone chemical shifts of (13)C(α), (13)Cβ and (13)CO are known from prior NMR studies and where some losses in sensitivity can be tolerated for the sake of an overall reduction in NMR acquisition time.

SYNTHESIS OF NITRO- AND AMINOINDOLES

Optimal conditions are proposed for the direct nitration of 2,3-dimethylindoles containing methyl and methoxy groups in the benzene ring. In contrast to 5- and 6-methylindoles and 5- and 6-methoxyindoles, nitration of 7-methyl- and 7-methoxy-2,3-dimethylindoles proceeds differently under the same conditions. A series of previously unreported aminoindoles was obtained by reduction of the nitroindoles. Authors: S. A. Yamashkin and M. A. Yurovskaya. English Translation in Chemistry of Heterocyclic Compounds , 1999, 35 (12), pp 1426-1432 http://link.springer.com/article/10.1007/BF02251816

High- and Mid-Valent Tantalum and Mono(peralkylcyclopentadienyl)tantalum Complexes of the Bicyclic Guanidinate Hexahydropyrimidopyrimidinate

Ta(hpp)Cl4, Ta(hpp)2Cl3, and (η-C5Me4R)Ta(hpp)Cl3 (R = Me, Et; hpp = anion of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine, hppH) are readily formed in good to excellent yields by addition of (hpp)SiMe3 to TaCl5, Ta(hpp)Cl4, or (η-C5Me4R)TaCl4, respectively, in dichloromethane. Ta(hpp)Cl4 in solution exhibits three CH2 multiplets in the proton NMR spectrum, consistent with a mirror plane that bisects the hpp ligand. The six-coordinate, yellow Ta(hpp)Cl4 possesses a distorted octahedral solid-state structure with chelating hpp and Ta–N distances of 2.038(1) and 2.059(1) Å. Ta(hpp)2Cl3 is fluxional in solution by proton NMR spectroscopy, with three CH2 multiplets at room temperature that split into additional multiplets at −50 °C, consistent with the six multiplets expected from the solid-state structure. In the solid state, bright yellow Ta(hpp)2Cl3 has an approximately pentagonal bipyramidal structure with both hpp chelates coordinated in the pentagonal plane. Ta–N distances are 2.114(3) and 2.110(3) Å for one hpp ligand and 2.112(3) and 2.117(3) Å for the other. The proton and carbon-13 solution NMR spectra of (η-C5Me4R)Ta(hpp)Cl3 are consistent with a static solution structure at room temperature, with the two coordinated nitrogens in different chemical environments as shown by six different methylene resonances. The solid-state structure of bright pinkish-red (η-C5Me5)Ta(hpp)Cl3 is a four-legged piano stool with axial ligation, with one hpp nitrogen coordinated in the square plane and one nitrogen in the axial position opposite the cyclopentadienyl centroid. Addition of (hpp)SiMe3 to the organoditantalum(III) complex (η-C5Me4R)2Ta2(μ-Cl)4 or Na/Hg reduction of (η-C5Me4R)Ta(hpp)Cl3 in benzene yields the first mid-valent tantalum hpp adduct, paramagnetic red (η-C5Me4R)Ta(hpp)Cl2. This Ta(IV) four-legged piano stool structure in the solid state has an hpp chelate with Ta–N distances of 2.118(3) and 2.091(3) Å. Intraligand bond distances and angles for the hpp ligands, proton and carbon-13 NMR solution spectra, EPR spectra, and UV–vis spectra for the new Ta(V) and Ta(IV) hpp complexes are discussed.

Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D2O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d10. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.