Metabolic changes in Atlantic salmon exposed to Aeromonas salmonicida detected by 1H-nuclear magnetic resonance spectroscopy of plasma

1H-NMR (nuclear magnetic resonance)-based chemometric methods have been applied for the first time to investigate changes in the plasma metabolite profiles of Atlantic salmon Salmo salar as a result of exposure to Aeromonas salmonicida subsp. salmonicida, a Gram-negative bacterium that is the etiological agent of furunculosis. Plasma samples were obtained from salmon that survived 21 d post exposure to A. salmonicida, and from a control group maintained under similar conditions. 1D 1H-NMR spectra were acquired and principal components analysis (PCA) was used to assess differences between the spectral profiles of plasma from salmon that survived an A. salmonicida challenge, and non-infected controls. PCA enables simultaneous comparison of spectra, presenting a simplified overview of the relationship between spectral data, where spectra cluster based on metabolite profile similarities and differences; information regarding the metabolite variations can therefore be readily deciphered. The major metabolite changes responsible for the spectral differences were related to modification in the lipoprotein profile and choline-based residues, with minor changes in carbohydrates, glycerol, trimethylamine-N-oxide and betaine. These changes indicated that exposure to A. salmonicida induced a characteristic biochemical response which could be used to determine the health status of salmon. This study suggests that with further development this metabolite profiling technique may be a useful tool for diagnosis of disease states in salmon and could provide a better understanding of the host-pathogen relationship which at present is poorly understood for A. salmonicida and Atlantic salmon.     

Anisotropic 2H-nuclear magnetic resonance spin-lattice relaxation in cerebroside- and phospholipid-cholesterol bilayer membranes.

The axially symmetric powder pattern 2H-nuclear magnetic resonance (NMR) lineshapes observed in the liquid crystalline phase of pure lipid or lipid/cholesterol bilayers are essentially invariant to temperature, or, equivalently, to variations in the correlation times characterizing C-2H bond reorientations. In either of these melted phases, where correlation times for C-2H bond motions are shorter than 10(-7) s, information on the molecular dynamics of the saturated hydrocarbon chain would be difficult to obtain using lineshape analyses alone, and one must resort to other methods, such as the measurement of 2H spin-lattice relaxation rates, in order to obtain dynamic information. In pure lipid bilayers, the full power of the spin-lattice relaxation technique has yet to be realized, since an important piece of information, namely the orientation dependence of the 2H spin-lattice relaxation rates is usually lost due to orientational averaging of T1 by rapid lateral diffusion. Under more favorable circumstances, such as those encountered in the lipid/cholesterol mixtures of this study, the effects of orientational averaging by lateral diffusion are nullified, due to either a marked reduction (by at least an order of magnitude) in the diffusion rate, or a marked increase in the radii of curvature of the liposomes. In either case, the angular dependence of 2H spin-lattice relaxation is accessible to experimental study, and can be used to test models of molecular dynamics in these systems. Simulations of the partially recovered lineshapes indicate that the observed T1 anisotropies are consistent with large amplitude molecular reorientation of the C-2H bond among a finite number of sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structural isomerisation of human-muscle adenylate kinase as studied by 1H-nuclear magnetic resonance

Human muscle adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3.) was studied by 1H-nuclear magnetic resonance spectroscopy. The C-2 and C-4 proton resonances of the active-center histidine His-36 could be identified; the pK of His-36 was determined as 6.1. The pK of His-189 is very low (4.9) although it is located at the surface of the protein. Other resonance lines are discussed in comparison with NMR spectra of porcine adenylate kinase [McDonald et al. (1975) J. Biol. Chem. 250, 6947-6954]. A pH-dependent structural isomerization as shown by X-ray crystallography in the pig enzyme [Pai et al. (1977) J. Mol. Biol. 114, 37-45] was not observed for human adenylate kinase in solution. However, the binding of adenosine(5')pentaphospho(5')adenosine (Ap5A), a bisubstrate inhibitor, to adenylate kinase causes an overall change of the NMR spectrum indicative of a large conformational change of the enzyme. The exchange rate (koff) for Ap5A was estimated as 10 s-1 and decreases by addition of Mg2+. On the basis of these values and the known dissociation constant it is likely that the binding of Ap5A is a diffusion-controlled process kon being 10(8) M-1 s-1. In conclusion, the system Ap5A/Mg2+/human adenylate kinase, which has been studied by NMR spectroscopy and X-ray diffraction in parallel, is suitable for analyzing the induced fit postulated by Jencks for all kinase-catalyzed reactions.     

1H-nuclear magnetic resonance studies of the neuropeptide head activator

The 1H-NMR spectrum of the neuropeptide head activator in aqueous solution has been completely assigned by two-dimensional NMR spectroscopy and selective deuteration. The apparent pseudo-first-order exchange rate, kex, of the backbone amide protons and the correspondent activation enthalpies, delta H not equal to, were determined. The exchange rates decrease and the activation enthalpies increase from the N-terminal to the C-terminal part of the peptide. The exchange rates vary from 21 to 0.3 s-1 at 274 K, the activation enthalpies from 60 to 75 kJ.mol-1. The pK values of the terminal carboxyl group and of the lysine amino group have been estimated as 3.3 and 10.3, respectively. The NMR results are in line with a dimeric structure in an antisymmetric arrangement of the subunits, forming an antiparallel beta-pleated sheet between C-terminal segments. The peptide bonds between pGlu-1, Pro-2 and Pro-3 are predominantly in trans-configuration, in fact no cis-isomers can be observed spectroscopically. The structure appears to be very stable; in the temperature and pH range studied, i.e., from 274 to 338 K and from pH 0.8 to pH 11.6, there are no spectroscopic indications for a global structural change.     

2H-nuclear magnetic resonance investigations on phospholipid acyl chain order and dynamics in the gramicidin-induced hexagonal HII phase.

The following results are reported in this paper: The interaction of gramicidin with [11,11-2H2]dioleoylphosphatidylcholine (DOPC) and [11,11-2H2]dioleoylphosphatidylethanolamine (DOPE) at different stages of hydration was studied by 2H- and 31P-nuclear magnetic resonance. In the L alpha phase in excess water the acyl chains of phosphatidylethanolamine (PE) are more ordered than phosphatidylcholine (PC) most likely as the result of the lower headgroup hydration of the former lipid. In excess water gramicidin incorporation above 5 mol % in DOPC causes a bilayer----hexagonal HII phase change. In the HII phase acyl chain order is virtually unaffected by gramicidin but the peptide restricts the fast chain motions. At low water content gramicidin cannot induce the HII phase but it markedly decreases chain order in the DOPC bilayer. Increasing water content results in separation between a gramicidin-poor and a gramicidin-rich L alpha phase with decreased order of the entire lipid molecule. Further increase in hydration reverts at low gramicidin contents the phase separation and at high gramicidin contents results in a direct change of the disordered lamellar to the hexagonal HII phase. Gramicidin also promotes HII phase formation in the PE system but interacts much less strongly with PE than with PC. The results support our hypothesis that gramicidin, by a combination of strong intermolecular attraction forces and its pronounced cone shape, both involving the four tryptophans at the COOH-terminus, has a strong tendency to organize, with the appropriate lipid, in intramembranous cylindrical structures such as is found in the HII phase.     

Characterisation of human embryonic stem cells conditioning media by 1H-nuclear magnetic resonance spectroscopy

Background

Cell culture media conditioned by human foreskin fibroblasts (HFFs) provide a complex supplement of protein and metabolic factors that support in vitro proliferation of human embryonic stem cells (hESCs). However, the conditioning process is variable with different media batches often exhibiting differing capacities to maintain hESCs in culture. While recent studies have examined the protein complement of conditioned culture media, detailed information regarding the metabolic component of this media is lacking.

Methodology/Principal Findings

Using a ¹H-Nuclear Magnetic Resonance (¹H-NMR) metabonomics approach, 32 metabolites and small compounds were identified and quantified in media conditioned by passage 11 HFFs (CMp11). A number of metabolites were secreted by HFFs with significantly higher concentration of lactate, alanine, and formate detected in CMp11 compared to non-conditioned media. In contrast, levels of tryptophan, folate and niacinamide were depleted in CMp11 indicating the utilisation of these metabolites by HFFs. Multivariate statistical analysis of the ¹H-NMR data revealed marked age-related differences in the metabolic profile of CMp11 collected from HFFs every 24 h over 72 h. Additionally, the metabolic profile of CMp11 was altered following freezing at −20°C for 2 weeks. CM derived from passage 18 HFFs (CMp18) was found to be ineffective at supporting hESCs in an undifferentiated state beyond 5 days culture. Multivariate statistical comparison of CMp11 and CMp18 metabolic profiles enabled rapid and clear discrimination between the two media with CMp18 containing lower concentrations of lactate and alanine as well as higher concentrations of glucose and glutamine.

Conclusions/Significance

¹H-NMR-based metabonomics offers a rapid and accurate method of characterising hESC conditioning media and is a valuable tool for monitoring, controlling and optimising hESC culture media preparation.     

Smoothed orientational order profile of lipid bilayers by 2H-nuclear magnetic resonance.

A new method has been developed to determine the complete orientational order profile of lipid bilayers using 2H-NMR. The profile is obtained from a single powder spectrum of a lipid which has a saturated chain fully deuteriated. The smoothed order profile is determined directly from the normalized dePaked spectrum assuming a monotonic decrease of the order along the acyl chain. The oscillatory variations of the order at the beginning of the chain are not described by this method. However the smoothed order profile reveals in a straightforward way the crucial features of the anisotropic order of the bilayer.     

Adenine nucleotide N-glycosidase activity of the A-chain of cinnamomin characterized by 1H-nuclear magnetic resonance

Plant ribosome-inactivating proteins specifically cleave an N-glycosidic bond of a unique adenosine in the largest ribosomal RNA, releasing an adenine from ribosomes of different sources. Here, 1H-nuclear magnetic resonance is used to analyze the enzymatic products of the A-chain of cinnamomin, a type-II ribosome-inactivating protein (RIP) acting on the nucleotides in situ. The enzymatic activities of the RIP on nine nucleotides are compared. Cinnamomin A-chain can cleave the N-glycosidic bond and release an adenine base from adenine nucleotides except 5'-ATP; however, it cannot act on 5'-GMP, 5'-CMP, and 5'-UMP. The A-chain in the mixture of cinnamomin A- and B-chain exhibits higher activity toward adenine nucleotides than the A-chain alone does, suggesting that the B-chain can conformationally stabilize the A-chain. Intact cinnamomin also exhibits lower activity toward adenine nucleotides. However, cinnamomin B-chain and heat-denatured intact cinnamomin cannot hydrolyze all the tested nucleotides. We conclude that hydrolysis of the N-C glycosidic bond of nucleotide compounds by cinnamomin A-chain has a base preference, and the negatively charged phosphate group(s) reduces the recognition ability of the A-chain to adenine nucleotide.     

Multiple forms of sulfmyoglobin as detected by 1H nuclear magnetic resonance spectroscopy

The proton nuclear magnetic resonance spectrum of sulfmyoglobin prepared in standard fashion reveals the presence of three forms, A, B, and C, with different chemical reactivity. Conditions for some interconversions of these forms are given. The 1H NMR spectra of the different forms show similar patterns. It appears that the differences between forms involve chemical modification on the porphyrin periphery. The altered heme can be extracted from FeIII(CN) sulfmyoglobin C to give a stable green substance.     

Conformational features of cell-bound drugs as detected by selective proton relaxation rate investigations

The nonselective and selective longitudinal relaxation rates were measured for procaine protons in the presence of model lipid membranes, biological membranes and whole cells. Unlike the nonselective relaxation rates, the selective rate was shown to be particularly sensitive in detecting binding interactions with macromolecular cell constituents. It was shown that the aromatic moiety of procaine is involved in binding the cell plasma membrane.     

Comparative study of conformational behaviour of leucine and methionine enkephalinamides by1H-nuclear magnetic resonance spectroscopy

The conformational proclivity of leucine and methionine enkephalinamides in deuterated dimethyl sulphoxide has been investigated using proton magnetic resonance at 500 MHz. The resonances from the spin system of the various amino acid residues have been assigned from the 2-dimensional correlated spectroscopy spectra. The temperature variation of the amide proton shifts indicates that none of the amide proton is intramolecularly hydrogen-bonded or solvent-shielded. The analysis of vicinal coupling constants,³J_{HN.C 2}H,along with temperature coefficients and the absence of characteristic nuclear Overhauser effect cross peaks between the NH protons reveal that there is no evidence of the chain folding in these molecules. However, the observation of nuclear Overhauser effect cross peaks between the NH and the C^αH of the preceding residue indicates preference for extended backbone conformation with preferred side chain orientations particularly of Tyr and Phe in both [Leu⁵]- and [Met⁵]-enkephalinamides.     

On the role of histidine and tyrosine residues in E. coli asparaginase. Chemical modification and 1H-nuclear magnetic resonance studies

The relative importance of tyrosine and histidine residues for the catalytic action of Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) was studied by chemical modification and 1H-NMR spectroscopy. We show that, under appropriate reaction conditions, N-bromosuccinimide (NBS) as well as diazonium-1H-tetrazole (DHT) inactivate by selectively modifying two tyrosine residues per asparaginase subunit without affecting histidyl moieties. We further show that diethyl pyrocarbonate (DEP), a reagent considered specific for histidine, also modifies tyrosine residues in asparaginase. Thus, inactivation of the enzyme by DEP is not indicative of histidine residues being involved in catalysis. In 1H-nuclear magnetic resonance (NMR) spectra of asparaginase signals from all three histidine residues were identified. By measuring the pH dependencies of these resonances, pKa values of 7.0 and 5.8 were derived for two of the histidines. Titration with aspartate which tightly binds to the enzyme at low pH strongly reduced the signal amplitude of the pKa 7 histidyl moiety as well as those of resonances of one or more tyrosine residues. This suggests that tyrosine and histidine are indeed constituents of the active site.     

Mobility of fluorescent probe molecules in lipid bilayer vesicles as studied by steady-state and time-dependent nuclear Overhauser effect measurements in 1H-nuclear magnetic resonance spectroscopy

In order to elucidate the mobilities of the fluorophores of fluorescent 2- and 16-(9-anthroyloxy)palmitic acids (16-AP and 2-AP, respectively) in lipid bilayer vesicles, the steady-state and time-dependent nuclear Overhauser effects in ¹H-NMR spectroscopy, but not the fluorescence depolarization in fluorescence spectroscopy, have been measured. The steady-state nuclear Overhauser effect measurements showed an appreciable magnitude of negative nuclear Overhauser effects between the resonances due to the fluorophores of the two fluorescent probes and lipids. These results definitely mean that in lipid bilayers, the fluorophores (anthroyloxy ring) of the fluorescent probes experience other types of motions with much longer correlation times than those detected by the fluorescence depolarization measurements, since at the correlation time showed by the fluorescent method (1–2 · 10⁻⁹ s or less), no such transfer of the negative nuclear Overhauser effects is expected to occur. The correlation times of the fluorophores, as calculated from the cross-relaxation rates of the anthroyl ring protons of 16-AP and 2-AP, were 3.8 · 10⁻⁸ and 1.1 · 10⁻⁷ s, respectively. These values, respectively, compare favorably with those of the terminal methyl of acyl chains and the choline methyl carbons which were estimated by ¹³C T₂ relaxation times. Thus, it is concluded that the fluorophores of both 16-AP and 2-AP have a slow form of motion which moves with a similar time scale to those of lipids in addition to the faster one that causes fluorescence depolarization.     

1H-nuclear magnetic resonance study of the association of the basic protein of central nervous system myelin with lysophosphatidylcholine

High-resolution 270 MHZ 1H-nuclear magnetic resonance spectroscopy has been used to follow the interaction of myristoyllysophosphatidylcholine with bovine myelin basic protein. At lipid/protein ratios up to 30:1 it proved possible to follow changes in the spectra of both the protein and the lipid. Lysophosphatidylcholine induced several changes in the protein spectrum. Foremost amongst these changes were downfield shifts of histidine C2 protons, and upfield shifts and broadening of the phenylalanine aromatic proteins. Several other resonances assigned to nonpolar amino acid side chains also broadened. But even at a lipid/protein molar ratio of 30:1 the majority of the protein appeared to remain in a loosely coiled conformation. In the presence of the protein the lipid acyl chain peaks were moved upfield and broadened, whereas the resonances associated with the head-group protons were unaffected. These changes were consistent with partial immobilization of the acyl chain of lysophosphatidylcholine on binding to the basic protein, with hydrophobic interactions providing the predominant attraction between this lipid and the basic protein.     

Structure of triphosphoryl nucleotide bound at the active site of yeast hexokinase: 1H-nuclear magnetic resonance study

Conformation of a nonhydrolyzable adenosine triphosphate (ATP) analogue, adenylyl-(,-methylene)-diphosphonate (AMPPCP) bound at the active site of yeast hexokinase-PII was determined by proton two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) and molecular dynamics simulations. The effect of the glucose-induced domain closure on the conformation of the nucleotide was evaluated by making measurements on two different complexes: PIIAMPPCPMg(II) and PIIGlcAMPPCPMg(II). TRNOE measurements were made at 500 MHz, 10°C, as a function of several mixing times varying in the range of 40 to 200 ms. Interproton distances derived from the analysis of NOE buildup curves were used as restraints in molecular dynamics simulations to determine the conformation of the enzyme bound nucleotide. The adenosine moiety was found to bind in high anti conformation with a glycosidic torsion angle = 48 ± 5 degrees in both complexes. However, significant differences in the conformations of the ribose and triphosphoryl chain of the nucleotide are observed between the two complexes. The phase angles of pseudorotation P in PIIAMPPCPMg(II) and PIIGlcAMPPCPMg(II) are 87 degrees and 77 degrees, describing a OE and OT₄ sugar pucker and the amplitudes of the sugar pucker () are 37 degrees and 61 degrees, respectively.     

Reversible trifluoroacetic acid-induced conformational changes in glycophorin as detected by proton nuclear magnetic resonance spectroscopy

High-field (270 MHz) ¹H-NMR has been employed to study the solution conformation of glycophorin A, a sialoglycoprotein which spans the human erythrocyte membrane. Glycophorin A is one of the most fully characterized integral membrane proteins known, making it an excellent model for the study of membrane-bound proteins. This protein consists of three distinct domains: a glycosylated extracellular N-terminus, a hydrophobic intramembranous segment, and a polar cytoplasmic C-terminus. These domains contain aromatic residues which serve as convenient ¹H-NMR conformational probes. The aromatic region of the NMR spectrum of glycophorin A in ²H₂O shows single, well-resolved His and Tyr resonances. No resonances are observed, however, for the Phe residues which are located in or near the hydrophobic domain. These observations suggest that considerable heterogeneity with respect to segmental motions exists within the protein. This is consistent with circular dichroism data showing the intramembranous segment to be completely helical with the extremities of the protein being predominantly random coils. The helix of the hydrophrobic domain is remarkably resistant to conventional denaturing conditions including variations in pH, and temperature, and treatment with guanidine hydrochloride. However, in trifluoroacetic acid, which strongly solvates peptide backbones, there is extensive reversible unfolding of the helical structure as evidenced by the appearance of Phe resonances. Solvent titration experiments indicate that approximately a 1 : 1 volume ratio of trifluoroacetic acid to ²H₂O is required to initiate unfolding of the helix.     

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.     

Noninvasive measurement of temperature and fractional dissociation of imidazole in human lower leg muscles using 1H-nuclear magnetic resonance spectroscopy.

The temperature change of the fractional dissociation of imidazole (alpha-imidazole) in resting human lower leg muscles was measured noninvasively using (1)H-nuclear magnetic resonance spectroscopy at 3.0 and 1.5 T on five normal male volunteers aged 30.6 +/- 10.4 yr (mean +/- SD). Using (1)H-nuclear magnetic resonance spectroscopy, water, carnosine, and creatine in the muscles could be simultaneously analyzed. Carnosine contains imidazole protons. The chemical shifts of water and carnosine imidazole protons relative to creatine could be used for estimating temperatures and alpha-imidazole, respectively. Using the chemical shift, the values of temperature in gastrocnemius (Gast) and soleus muscles at ambient temperature (21-25 degrees C) were estimated to be 35.5 +/- 0.5 and 37.4 +/- 0.6 degrees C (means +/- SE), respectively (significantly different; P < 0.01). The estimated values of alpha-imidazole in these muscles were 0.620 +/- 0.007 and 0.630 +/- 0.013 (means +/- SE), respectively (not significant). Alternation of the surface temperature of the lower leg from 40 to 10 degrees C significantly changed the temperature in Gast (P < 0.0001) from 38.1 +/- 0.5 to 28.0 +/- 1.2 degrees C, and the alpha-imidazole in Gast decreased from 0.631 +/- 0.003 to 0.580 +/- 0.011 (P < 0.05). However, the values of alpha-imidazole and the temperature in soleus muscles were not significantly affected by this maneuver. These results indicate that the alpha-imidazole in Gast changed significantly with alternation in muscle temperature (r = 0.877, P < 0.00001), and its change was estimated to be 0.0058/ degrees C.