Phospholipid packing and conformation in small vesicles revealed by two-dimensional 1H nuclear magnetic resonance cross-relaxation spectroscopy.

Two-dimensional 1H-NMR spectroscopy has been used to examine cross-relaxation in sonicated phospholipid vesicle systems. The observed pattern of proton cross-relaxation reveals several important features of these vesicle systems. For example, cross-relaxation rates on each monolayer of the vesicle system can be resolved and reflect the expected geometric packing constraints of the vesicle system. Small but significant magnetization-exchange is also seen to develop between the headgroup N-methyl resonance and the terminal methyl resonance. Spectra taken with deuterated lipids indicate that this exchange is not mediated by spin-diffusion down the length of the alkyl chains. Since spin-diffusion is the only process that is expected to facilitate magnetization-exchange over distances of 15-20 A, a close proximity of headgroup and terminal methyl protons in a fraction of the membrane lipid is indicated by these results. This could occur by events such as lipid interdigitation or alkyl chain bends that terminate lipid alkyl chain ends near the membrane surface.     

Omega 1-decoupled 1H homonuclear shift-correlated nuclear magnetic resonance spectroscopy (COSYDEC) applied to steroids

Problems of cross-peak overlap in two-dimensional 1H homonuclear shift-correlated (COSY) spectra of steroids can often be avoided by use of the omega 1-decoupled COSY (COSYDEC) method. The selection of experimental parameters is discussed, and COSYDEC spectra are illustrated for 17a-oxa-D-homoandrost-4-ene-3,17-dione (testololactone), testosterone, and 17 alpha-hydroxyprogesterone. In a good case, a COSYDEC spectrum obtained at 250 MHz allows cross-peak recognition and assignment with facility comparable to that available only at 500 MHz for normal COSY spectra.     

Solution structure of neuromedin B by (1)H nuclear magnetic resonance spectroscopy.

The solution structure of neuromedin B (NMB) was investigated using two-dimensional nuclear magnetic resonance (NMR) spectroscopy in membrane-mimicking environments. NMB adopts a relaxed helical conformation from Trp(4) to Met(10) in 50% aqueous 2,2, 2-trifluoroethanol (TFE) solution and in 150 mM SDS micelles. Sidechain atoms of the three residues, Trp(4), His(8) and Phe(9) orient toward the same direction and these residues might play a key role on interacting with hydrophobic acyl chains of the phospholipids in the membrane. NOESY experiments performed on NMB in non-deuterated SDS micelle show that aromatic ring protons of Trp(4) and Phe(9) residues are in close contact with methylene protons of SDS micelles. In addition, proton longitudinal relaxation data proved that the interactions between NMB with SDS micelle are characterized as extrinsic interaction. Trp(4) and Phe(9) seem to be important in interaction with receptor and this agrees with the previous studies of structure-activity relationship (Howell, D.C. et al. (1996) Int. J. Pept. Protein Res. 48, 522-531). These conformational features might be helpful in understanding the molecular mechanism of the function of NMB and developing the efficient drugs.     

Use of relaxation-edited one-dimensional and two dimensional nuclear magnetic resonance spectroscopy to improve detection of small metabolites in blood plasma

The 1H nuclear magnetic resonance (NMR) spectra of biological samples, such as blood plasma and tissues, are information rich but data complex owing to superposition of the resonances from a multitude of different chemical entities in multiple-phase compartments, hampering detection and subsequent resonance assignments. To overcome these problems, several spectral-editing NMR experiments are described here, combining spin-relaxation filters (based on T(1), T(rho), and T(2)) with both one-dimensional and two-dimensional (2D) NMR spectroscopy. These techniques enable the separation of NMR resonances based on their relaxation times and allow simplification of the complex spectra. In this paper, the approach is exemplified using a control human blood plasma, which is a complex mixture of proteins, lipoproteins, and small-molecule metabolites. In the case of T(1rho)- and T(2)-edited 2D NMR experiments, a "flip-back" pulse was introduced after the relaxation editing to make the phase cycling of the "relaxation filter" and the 2D NMR part independent, thus enabling easy implementation of the phase-sensitive 2D NMR experiments. These methods also permit much higher receiver gains to be used to reduce digitization error, in particular, for the small resonances, which are sometimes vitally important for metabonomics studies. Both pulse sequences and experimental results are discussed for T(1)-, T(1rho)-, and T(2)-filtered COSY, T(2)-filtered phase-sensitive DQF-COSY, and T(1), T(1rho)-, and T(2)-filtered TOCSY NMR.     

Binding mode of Pyridoclax to myeloid cell leukemia-1 (Mcl-1) revealed by nuclear magnetic resonance spectroscopy,docking and molecular dynamics approaches

Abstract

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family proteins. Its amplification is one of the most frequent genetic aberrations found in human cancers. Pyridoclax, a promising BH3 mimetic inhibitor, interacts directly with Mcl-1 and induces massive apoptosis at a concentration of 15?µM in combination with anti-Bcl-x_L strategies in chemo-resistant ovarian cancer cell lines. In this study, a combined experimental and theoretical approach was used to investigate the binding mode of Pyridoclax to Mcl-1. The representative poses generated from dynamics simulations compared with NMR data revealed: (i) Pyridoclax bound to P1 and P2 pockets of Mcl-1 BH3 binding groove through its styryl and methyl groups establishing mainly hydrophobic contacts, (ii) one of the ending pyridines interacts through electrostatic interaction with K234 side chain, a negatively charged residue present only in this position in Mcl-1.

Communicated by Ramaswamy H. Sarma     

Use of 1H nuclear magnetic resonance to measure intracellular metabolite levels during growth and asexual sporulation in Neurospora crassa

Conidiation is an asexual sporulation pathway that is a response to adverse conditions and is the main mode of dispersal utilized by filamentous fungal pathogens for reestablishment in a more favorable environment. Heterotrimeric G proteins (consisting of α, β, and γ subunits) have been shown to regulate conidiation in diverse fungi. Previous work has demonstrated that all three of the Gα subunits in the filamentous fungus Neurospora crassa affect the accumulation of mass on poor carbon sources and that loss of gna-3 leads to the most dramatic effects on conidiation. In this study, we used (1)H nuclear magnetic resonance (NMR) to profile the metabolome of N. crassa in extracts isolated from vegetative hyphae and conidia from cultures grown under conditions of high or low sucrose. We compared wild-type and Δgna-3 strains to determine whether lack of gna-3 causes a significant difference in the global metabolite profile. The results demonstrate that the global metabolome of wild-type hyphae is influenced by carbon availability. The metabolome of the Δgna-3 strain cultured on both high and low sucrose is similar to that of the wild type grown on high sucrose, suggesting an overall defect in nutrient sensing in the mutant. However, analysis of individual metabolites revealed differences in wild-type and Δgna-3 strains cultured under conditions of low and high sucrose.     

Characterisation of germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy

Experiments were conducted to characterise the changes, especially of water status in germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. NMR relaxation time (T₂) measurements showed tri-phasic or bi-phasic characteristics during different stages of hydration, depending on the seed's ability to germinate. Component analysis of T₂ data revealed the existence of only two components, bound and bulk water, in dry seeds. In contrast, both the germinating and non-germinating wheat seeds had a three-component water proton system (bound, bulk and free water) in phase I of hydration. During the lag phase (phase II) of hydration, bulk water component of non-germinating seeds disappeared completely, resulting in a two component water proton system. Nevertheless, the three component water proton system was observed in the germinating seeds in phase II. Following phase II, rapid hydration (phase III) was observed in germinating seeds only. Water protons were re-organised and there were increases in bulk and free water but decreases in bound water concomitantly. Comparison of the physical state of water in these seeds by NMR spectroscopy with that of tissue leachate conductivity measurement suggests that the seed membrane system was affected more evidently in non-germinating seeds, leading to the disorganised cell structure. The present study provides evidence that the reorganisation of physical state of water in germinating wheat seeds during hydration is essential for its subsequent event of germination.     

Responses of the pea (Pisum sativum L.) leaf metabolome to drought stress assessed by nuclear magnetic resonance spectroscopy

While many compounds have been reported to change in laboratory based drought-stress experiments, little is known about how such compounds change, and are significant, under field conditions. The Pisum sativum L. (pea) leaf metabolome has been profiled, using 1D and 2D NMR spectroscopy, to monitor the changes induced by drought-stress, under both glasshouse and simulated field conditions. Significant changes in resonances were attributed to a range of compounds, identified as both primary and secondary metabolites, highlighting metabolic pathways that are stress-responsive. Importantly, these effects were largely consistent among different experiments with highly diverse conditions. The metabolites that were present at significantly higher concentrations in drought-stressed plants under all growth conditions included proline, valine, threonine, homoserine, myoinositol, γ-aminobutyrate (GABA) and trigonelline (nicotinic acid betaine). Metabolites that were altered in relative amounts in different experiments, but not specifically associated with drought-stress, were also identified. These included glutamate, asparagine and malate, with the last being present at up to 5-fold higher concentrations in plants grown in field experiments. Such changes may be expected to impact both on plant performance and crop end-use. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Solution conformation of leiurotoxin I (scyllatoxin) by H nuclear magnetic resonance Resonance assignment and secondary structure

A proton NMR study at 500 MHz of leiurotoxin I in water is presented. Nearly complete sequence-specific assignments of the individual backbone and side-chain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of this toxin is inferred from a combination of short-range nuclear Overhauser enhancements, scalar couplings and proton/deuteron exchange rates. Three disulflde bridges locate the N-terminal part (that is -helical from residue 6 to 16) on one side of a C-terminal two stranded antiparallel β sheet (from Leu¹⁸ to Val²⁹). The latter features a tight turn at Gly²³-Asp²⁴.     

Measurement of the helix opening rate in Z-DNA by 1H nuclear magnetic resonance relaxation spectroscopy

The exchange rate of the hydrogen-bonded guanine imino protons N(1) in the high-salt form of Poly(dG-dC) was measured by following the non-selective inversion-recovery of their 1H NMR signal at 360 MHz, in the temperature range between 77 degrees C and 90 degrees C. In a 4.5M NaCl solution, Poly(dG-dC) is believed to adapt the left-handed Z-conformation, and the results reported here represent the first quantitative measurements of this rate process for Z-DNA by Nuclear Magnetic Resonance, complementing previous measurements made by tritium exchange at 0 degrees C (Ramstein, J. and Leng, M. (1980) Nature 288, 413-414). The results confirm that this process is much slower in the Z-form, compared to the B-structure, and that this difference in rates results mainly from a large decrease in the entropy of activation for Z-DNA.     

The comparison of plasma deproteinization methods for the detection of low-molecular-weight metabolites by (1)H nuclear magnetic resonance spectroscopy

Blood plasma is the major vehicle by which metabolites are transported around the body in mammalian species, and chemical analysis of plasma can provide a wealth of information relating to the biochemical status of an individual and is important for diagnostic purposes. However, plasma is very complex in physicochemical terms because it is composed of a range of organic and inorganic constituents with a wide range of molecular weights and chemical classes and this makes analysis non-trivial. It is now well established that high-resolution (1)H NMR spectroscopy of blood plasma provides useful qualitative and quantitative biochemical information relating to metabolic disorders. However, one of the problems encountered in NMR spectroscopic analysis of blood plasma is the extensive peak overlap or presence of broad macromolecule peaks in the (1)H NMR spectrum, which can severely limit the amount of obtainable information. Even with spectroscopic editing, information relating to low-molecular-weight (MW) metabolites is frequently lost. Therefore, the efficiency of a range of conventional protein removal methods, in combination with the use of one- and two-dimensional NMR spectroscopic methods for evaluation, have been compared for the extraction of NMR-observable low-MW metabolites. It has been shown that these "deproteinization" methods vary considerably in recovery of low MW metabolites and a judicious choice is crucial for optimal extraction of a given analyte. The results presented here show that while ultrafiltration provides the "safest" method of plasma deproteinization, the signal-to-noise ratio of the resultant (1)H NMR spectra is poor. On the other hand, acetonitrile precipitation at physiological pH allows the detection of more low-MW metabolites and at higher concentrations than any other method and provides the further advantages of being a rapid and simple procedure.     

Acute biochemical effects of La(NO3)3 on liver and kidney tissues by magic-angle spinning 1H nuclear magnetic resonance spectroscopy and pattern recognition

High-resolution magic-angle spinning (MAS) 1H nuclear magnetic resonance (NMR) spectroscopic and pattern recognition (PR) based methods have been applied to studies on the acute biochemical effects of La(NO3)3 on rats. Male Wistar rats were treated with various doses of La(NO3)3 (2, 10, and 50 mg/kg body weight), and MAS 1H NMR spectra of their intact liver and kidney tissues were analyzed using principal components analysis to extract metabolic information. The biochemical effects of La(NO3)3 were characterized by the increase of triglyceride and bile acid and the decrease of glycogen in liver tissue, together with a slight elevation of triglyceride level in kidney tissue. The target lesion of La(NO3)3 to liver was found by MAS NMR-PR methods. This study illustrated the power of the combination of MAS 1H NMR and pattern recognition for the analysis of biochemical effects of rare earths.     

Determination of the three-dimensional structure of iberiotoxin in solution by 1H nuclear magnetic resonance spectroscopy.

The solution structure of chemically synthesized iberiotoxin, a scorpion toxin that blocks Ca(2+)-activated K+ channels, has been determined using 2D 1H NMR spectroscopy. Analysis of the NOEs, coupling constants, and HN-DN exchange rates indicates the structure consists of an antiparallel beta-sheet from residues 25 to 36, with a type 1 turn at residues 30-31, and a helix from residues 13 to 21. The carboxyl-terminal residues form a short, and distorted, third strand of the sheet. The NMR data are consistent with disulfide bonds from residues 7 to 28, 13 to 33, and 17 to 35. The disulfide bridging presents the same profile as in other scorpion toxins, where a Cys-X-Cys sequence in a strand of sheet forms two disulfide bonds to a Cys-X-X-X-Cys sequence in a helix. Three-dimensional structures were generated using the torsion angle space program PEGASUS. The best ten structures had an average rmsd over all pairwise comparisons of 1.49 A. The average rmsd to a calculated average structure is 1.0 A. The resulting structures appear very similar to those of charybdotoxin, a related scorpion toxin.     

Sequential1H and13C resonance assignments for an octa- and decasaccharide of theN-acetyllactosamine type by multiple-step relayed correlation and heteronuclear correlation nuclear magnetic resonance

400 MHz¹H-NMR and 100 MHz¹³C-NMR spectra of a neutral octasaccharide and of a disialyldecasaccharide of theN-acetyllactosamine type were studied. The resonance assignments were made by combining multiple-relayed coherence-transfer chemical-shift-correlated spectroscopy (multiple-RELAY-COSY) and¹H/¹³C-shift correlated 2D experiments. The complete analysis of the¹H and¹³C spectra was performed.     

Low- and high-resolution nuclear magnetic resonance (NMR) characterisation of hyaluronan-based native and sulfated hydrogels

Hyaluronan-based hydrogels were synthesised using different crosslinking agents, such as 1,3-diaminopropane (1,3-DAP) and 1,6-diaminohexane (1,6-DAE). The hydrogels were sulfated to provide materials (Hyal-1,3-DAP, Hyal-1,6-DAE, HyalS-1,3-DAP and HyalS-1,6-DAE) that were characterised by both high- and low-resolution nuclear magnetic resonance (NMR) spectroscopy. The (13)C NMR spectra of the materials were analysed to identify, characterise and study the crosslinking degree of the hydrogels. The crosslinking degree was also determined by potentiometric titration and the effectiveness of the two techniques was compared. Measurements of longitudinal relaxation times (spin-lattice) and of NOE enhancement were used to study the mobility of the hydrogels. Low-resolution NMR studies allowed the determination of the water transport properties in the hydrogels. In addition, the swelling degree for the various hydrogels was calculated as a function of the longitudinal and transversal relaxation times of the water molecules. Lastly, the self-diffusion coefficients of the water in interaction with the four polysaccharides were measured by the pulsed field gradient spin echo (PFGSE) sequence.     

The interaction of water molecules with purple membrane suspension using 2H double-quantum filter, 1H and 2H diffusion nuclear magnetic resonance

Bacteriorhodopsin is a membrane protein of the purple membrane (PM) of Halobacterium salinarum, which is isolated as sheets of highly organized two-dimensional hexagonal microcrystals and for which water molecules play a crucial role that affects its function as a proton pump. In this paper we used single- and double-quantum (2)H NMR as well as (1)H and (2)H diffusion NMR to characterize the interaction of water molecules with the PM in D(2)O suspensions. We found that, under the influence of a strong magnetic field on a concentrated PM sample (0.61 mM), the PM sheets affect the entire water population and a residual quadrupolar splitting (upsilon(q) approximately 5.5 Hz, 298 K, at 11.7 T) is observed for the D(2)O molecules. We found that the residual quadrupolar coupling, the creation time in which a maximal DQF signal was obtained (tau(max)), and the relative intensity of the (2)H DQF spectrum of the water molecules in the PM samples (referred to herein as NMR order parameters) are very sensitive to temperature, dilution, and chemical modifications of the PM. In concentrated PM samples in D(2)O, these NMR parameters seem to reflect the relative organization of the PM. Interestingly, we have observed that some of these parameters are sensitive to the efficiency of the trimer packing, as concluded from the apo-membrane behavior. The data for dionized blue membrane, partially delipidated sample, and detergent-treated PM show that these D(2)O NMR order parameters, which are magnetic field dependent, are sensitive to the structural integrity of the PM. In addition, we revealed that heating the PM sample inside or outside the NMR magnet has, after cooling, a different effect on the NMR characteristics of the water molecules in the concentrated PM suspensions. The difference in the D(2)O NMR order parameters for the PM samples, which were heated and cooled in the presence and in the absence of a strong magnetic field, corroborates the conclusions that the above D(2)O order parameters are indirect reflections of both microscopic and macroscopic order of the PM samples. In addition, (1)H NMR diffusion measurements showed that at least three distinct water populations could be identified, based on their diffusion coefficients. These water populations seem to correlate with different water populations previously reported for the PM system.     

Identification and description of alpha-helical regions in horse muscle acylphosphatase by 1H nuclear magnetic resonance spectroscopy

It has been proposed that combination of intraresidue, sequential and longer range nuclear Overhauser enhancements occurring in 1H nuclear magnetic resonance spectra of protein chains folded in a helix show a regular characteristic pattern. As a test case the spectra of horse muscle acylphosphatase were searched for this pattern together with other typical signs of a helical conformation (i.e. chemical shift, coupling constants and slow 2H-H exchange). Two amino acid sequences complying with these requirements were found. Just a few amino acid spin system assignments were then sufficient to locate the two segments within the primary structure (residues 22 to 35 and 55 to 66), thus providing the sequential assignment. The assignment of the side-chains was completed and a list of all nuclear magnetic resonance constraints within the two segments (126 intra- and 180 interresidue distances, 21 torsion angles phi and 19 hydrogen bonds) was produced. Distance geometry calculation shows that each segment forms an alpha-helix. The mutual orientation of the two helices was established subsequently.