A31P NMR study of the interaction of amphibian antimicrobial peptides with the membranes of live bacteria

Amphibian skin is a rich source of peptides that are specificto pathogens and act by disrupting bacterial membranes. Threeantimicrobial peptides were isolated from the skin glands ofAustralian tree frogs, Litoria caerulea and Litoriagenimaculata. NMR spectroscopy was used to observe changesinduced by these peptides in the ³¹P resonances of bacterialmembranes in vivo. Caerin 1.1 and maculatin 1.1, both wide-spectrum antibiotics, disrupted the membranes ofBacillus cereus and Staphylococcus epidermidis (Gram-positive), leadingto an increase in the isotropic ³¹P NMR signal. Caerin 4.1, anarrow-spectrum antibiotic, however, did not affect the ³¹Pspectra of these organisms. The results demonstrate the use of³¹P NMR to study the effects of membrane-disrupting agents onthe membranes of live bacteria.     

31P and19F NMR studies of glycophorin-reconstituted membranes: Preferential interaction of glycophorin with phosphatidylserine

Summary Glycophorin A, a major glycoprotein of the erythrocyte membrane, has been incorporated into small unilamellar vesicles composed of a variety of pure and mixed phospholipids. Nuclear spin labels including³¹P and¹⁹F have been used at natural abundance or have been synthetically incorporated in lipids to act as probes of lipid-protein interaction. Interactions produce broadening of resonances in several cases and it can be used to demonstrate preferential interaction of certain lipids with glycophorin.³¹P and¹⁹F probes show a strong preferential interaction of glycophorin with phosphatidylserine over phosphatidylcholine. There is some evidence that interactions are more pronounced at the inner surface of the bilayer and these results are rationalized in terms of the asymmetric distribution of protein and lipid.     

A 31P NMR study of the internal pH of yeast peroxisomes

The internal pH of peroxisomes in the yeasts Hansenula polymorpha, Candida utilis and Trichosporon cutaneum X4 was estimated by ³¹P nuclear magnetic resonance (NMR) spectroscopy. ³¹P NMR spectra of suspensions of intact cells of these yeasts, grown under conditions of extensive peroxisomal proliferation, displayed two prominent P_i-peaks at different chemical shift positions. In control cells grown on glucose, which contain very few peroxisomes, only a single peak was observed. This latter peak, which was detected under all growth conditions, was assigned to cytosolic P_i at pH 7.1. The additional peak present in spectra of peroxisome-containing cells, reflected P_i at a considerably lower pH of approximately 5.8–6.0. Experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazon (CCCP) and the ionophores valinomycin and nigericin revealed that separation of the two P_i-peaks was caused by a pH-gradient across a membrane separating the two pools. Experiments with chloroquine confirmed the acidic nature of one of these pools. In a number of transfer experiments with the yeast H. polymorpha it was shown that the relative intensity of the P_i-signal at the low pH-position was correlated to the peroxisomal volume fraction. These results strongly suggest that this peak has to be assigned to P_i in peroxisomes, which therefore are acidic in nature. The presence of peroxisome-associated P_i was confirmed cytochemically.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazon - DCCD N,N-dicyclohexylcarbodiimide     

A CAUTIONARY NOTE ON THE USE OF THE 31P NMR SPECTROSCOPY IN STEREOCHEMICAL CORRELATION ANALYSIS

Stereoselectivity in condensation of protected ribonucleoside 3′-H-phosphonates with hydroxylic components was investigated using ³¹P NMR spectroscopy. The correlation between absolute configuration at the phosphorus center and the chemical shifts of the produced H-phosphonate diesters and the corresponding phosphorothioates, was studied.     

Rapid assignment of solution 31P NMR spectra of large proteins by solid-state spectroscopy

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein. For the complex of Ras with the GTP-analogue GppCH(2)p we show solid-state (31)P NMR methods to be more sensitive by almost one order of magnitude than liquid-state NMR. Thus, solid-state NMR seems to be the method of choice for obtaining the resonance assignment of the phosphorus signals of protein complexes in solution. Experiments on Ras.GDP complexes show that the microcrystalline sample can be substituted by a precipitate of the sample and that unexpectedly the two structural states observed earlier in solution are present in crystals as well.     

Interaction of the Water-Soluble Protein Aprotinin with Liposomes: Gel-Filtration,Turbidity Studies,and 31P NMR Studies

Abstract

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; ³¹P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV–aprotinin complexes was determined. Lipid–protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. ³¹P NMR spectroscopy of the MLV–aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.     

Locust flight metabolism studied in vivo by 31P NMR spectroscopy

Flight metabolism of locusts has been extensively studied, but biochemical and physiological methods have led to conflicting results. For this reason the non-invasive and non-destructive method of ³¹P NMR spectroscopy was used to study migratory locusts, Locusta migratoria, at rest and during flight.

Reevaluation of the phospholipid composition in membranes of adult human lenses by P NMR and MALDI MS

The phospholipid composition of adult human lens membranes differs dramatically from that of any other mammalian membrane. Due to minimal cell turnover, cells in the nucleus of the human lens may be considered as the longest lived cells in our body. This work reassesses previous assignments of phospholipid ³¹P NMR resonances in adult human lenses. The new assignments are based not only on chemical shifts but also on temperature coefficients. By addition of known phospholipids and examination by matrix-assisted laser desorption/ionization mass spectrometry, several misassigned resonances have been corrected. The revised composition reveals the possible presence of ceramide-1-phosphate and dihydroceramide-1-phosphate. Among glycerophospholipids, the most abundant one does not correspond to phosphatidylglycerol but may be due to the lysoform of alkyl-acyl analogs of phosphatidylethanolamine. Besides sphingophospholipids, adult human lens membranes contain significant amounts of ether (1-O-alkyl) glycerophospholipids and their corresponding lysoforms.     

Muscle pain after exercise is linked with an inorganic phosphate increase as shown by31P NMR

³¹P nuclear magnetic resonance (NMR) spectra were obtained from the forearm muscles of 5 subjects before and after performing a muscle stretching (eccentric) exercise routine. Spectra collected before and immediately after exercise showed normal resting phosphorylated metabolite levels and unchanged intracellular pH (pH_i). Measurements made on the day following exercise, when muscular pain was apparent, revealed an elevated inorganic phosphate level. No significant changes in other metabolites or pH_i were detected. This study gives the first indication of biochemical change following a form of exercise that is associated with considerable muscle pain and damage. The findings may help in understanding pathological processes resulting in pain and damage in muscle.     

Assessment of energy metabolism in the developing brain following aglycemic hypoxia by1H and31P NMR

The role played by external calcium and calcium channels in the recovery from aglycaemic hypoxia in cortical brain slices from 10-day old rats was investigated by¹H and³¹P NMR. 30 minutes of aglycaemic hypoxia significantly decreased the levels of phosphocreatine (PCr), ATP, lactate and intracellular pH (pH_i). After a 30 minute recovery period there was incomplete recovery of PCr and ATP with lactate increasing by 50% with pH_i normal. When the aglycemic hypoxia was carried out in media which had no added calcium (≈10 μM) the PCr and ATP recovery was significantly greater. Application of diltiazem or verapamil but not nifedipine significantly improved the recovery from the aglycemic hypoxia. These data suggest that calcium influx through L-type voltagegated calcium channels is involved in the ischemic damage in neonatal brain which manifests itself as a decrease in the energy state and an increase in lactate. Dedication This article is dedicated to our friend and colleague Herman Bachelard. We wish to thank him for his comradeship, advice and support over many years. Our hope for him is a long and fruiful retirement and that he will remain active in the neurosciences for many years, even though the establishment has blown for “full time”.     

Formation of new larger ring cyclic triphosphenium ions in solution - A P NMR study

In a notable extension of previous work, we have shown that several diphosphanes will form larger (7- or 8-membered ring) cyclic triphosphenium ions (CTIs) in solution by reaction with PX₃ (X = Cl, Br or I), sometimes in the presence of SnCl₂ or SnBr₂. We have also formed new CTIs from two ferrocene derivatives. The ions are readily identifiable by ³¹P NMR solution-state spectroscopy, having large ¹J_P-P values between the middle ‘bare’ phosphorus atom P_A and the outer phosphorus atoms P_B (and P_C where the outer groups are inequivalent). In addition, some data for known CTIs, but with different counter-ions, are presented. Three of the new CTIs have been successfully protonated, two by both AlCl₃/^tBuCl and triflic acid, and one via the triflic acid route only.     

<Superscript>31</Superscript>P NMR study of polyphosphate levels during different growth phases of <Emphasis Type="Italic">Phycomyces blakesleeanus</Emphasis>

The changes in relative polyphosphate content, estimated as the intensity ratio of core polyphosphate signal and intracellular inorganic phosphate signal from ³¹P NMR spectra, during the growth of Phycomyces blakesleeanus are reported. The ratio increases from 16 h to 28 h of growth, the minimum occurs at 32 h, followed by sharp increase up to 36 h, and a steady decrease afterwards. The changes in the biomass during mycelium growth showed steady increases, with a stagnation period between 32 h and 36 h during which a pronounced increase in the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal occurred. The reduction of growth temperature from 22°C to 18°C significantly decreased the rate and intensity of growth, but the pattern of polyphosphate changes remained unchanged. The changes of the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal are linked to characteristic stages of sporangiophore development. Analysis of core polyphosphates, intracellular inorganic phosphate and β-ATP signal intensities suggest the role of polyphosphates as an energy and/or a phosphate reserves during Phycomyces development.     

Principles of multiparametric optimization for phospholipidomics by 31P NMR spectroscopy

Phospholipids have long been known to be the principal constituents of the bilayer matrix of cell membranes. While the main function of cell membranes is to provide physical separation between intracellular and extracellular compartments, further biological and biochemical functions for phospholipids have been identified more recently, notably in cell signaling, cell recognition and cell–cell interaction, but also in cell growth, electrical insulation of neurons and many other processes. Therefore, accurate and efficient determination of tissue phospholipid composition is essential for our understanding of biological tissue function. ³¹P NMR spectroscopy is a quantitative and fast method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantified separately and reliably in ³¹P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. Until recently, little attention has been paid to the optimization of phospholipid ³¹P NMR spectra. This review surveys the basic physicochemical properties that determine the quality of phospholipid spectra, and describes an optimization strategy based on this assessment. Notably, the following experimental parameters need to be controlled for systematic optimization: (1) extract concentration, (2) concentration of chelating agent, (3) pH value of the aqueous component of the solvent system, and (4) temperature of the NMR measurement. We conclude that a multiparametric optimization approach is crucial to obtaining highly predictable and reproducible ³¹P NMR spectra of phospholipids.     

Influence of temperature on 31P NMR chemical shifts of phospholipids and their metabolites I. In chloroform-methanol-water

Spectral overlap of ³¹P NMR resonances and the lack of reproducibility in chemical shifts corresponding to phospholipids in organic solvents challenge the accuracy of band assignments and quantification. To alleviate these problems, the use of temperature coefficients is proposed. Changes in temperature enable the resolution of overlapped resonances and provide a facile approach for the computation of temperature coefficients. The coefficients were evaluated for various glycero- and sphingo-phospholipids. Their values suggest that differences in H-bonding between the phosphate and the head groups are responsible for the changes of chemical shift with temperature. Among parent phospholipids, and in addition to sphingomyelin, the smallest temperature coefficient values (closest to zero) were observed for phosphatidylcholine, phosphatidylglycerol, dihydrosphingomyelin, and cardiolipin. The highest values were exhibited by phospholipids with protonated head groups, such as phosphatidylserine and phosphatidylethanolamine. The lowest and, in fact, negative values were measured for phospholipids with an exposed phosphate group: phosphatidic acid, ceramide-1-phosphate, and dihydroceramide-1-phosphate. Diacyl, alkyl-acyl, and alkenyl-acyl phospholipids with the same head group exhibited comparable coefficients but differed slightly in chemical shifts. Compared to their parent glycerophospholipids, all lyso analogs had greater temperature coefficients, possibly due to the presence of an extra OH capable of forming a H-bond with the phosphate group.     

Interaction of the fusogenic peptide B18 in its amyloid-state with lipid membranes studied by solid state NMR

The interaction of the fusogenic polypeptide segment "B18" from the fertilization protein binding with lipid membranes was investigated by solid state 2H and 31P NMR, and by differential scanning calorimetry. B18 is known to adopt different conformations depending on peptide concentration, ionic conditions, pH and lipid environment. Here, the peptide was studied in its beta-stranded amyloid conformation. According to 31P NMR, the lamellar morphology of the DMPC bilayer remains intact in the presence of B18. In going from low (1:90) to high (1:10) peptide/lipid ratios, an increasing effect on several different 2H-labeled lipid segments was observed, reflecting changes in phase behavior and local dynamics. The strongest influence of B18 was detected at the acyl-chains, while no significant effect on the lipid headgroup conformation was observed. This suggests an insertion of B18 in its fibrillar state into the membrane driven by hydrophobic interactions, rather than a peripheral binding mediated by electrostatics.     

Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cultures of the cyanobacterium Microcystis firma show rhythmic uptake and release of ammonia under conditions of carbon limitation. The massive removal of ammonia from the medium during the first light phase has little impact on the intracellular pH: a pH shift of less than 0.2 U towards the alkaline can be measured by in vivo ³¹P NMR. Furthermore, the energy status of the cells remains regulated. In vivo ¹⁵N NMR of M. firma, cultivated either with labelled nitrate or ammonia as the sole nitrogen source, reveals only gradual differences in the pool of free amino acids. Additionally both cultivation types show -aminobutyric acid, acid amides and yet unassigned secondary metabolites as nitrogen storing compounds. Investigating the incorporation of nitrogen under carbon limitation, however, only the amide nitrogen of glutamine is found permanently labelled in situ. While transamination reactions are blocked, nitrate reduction to ammonia can still proceed. Cation exchange processes in the cell wall are considered regarding the ammonia disappearance in the first phase, and the control of ammonia uptake is discussed with respect to the avoidance of intracellular toxification.Abbreviations GABA -aminobutyric acid - GOGAT glutamate synthase - GS glutamine synthetase - MDP methylene diphosphonate - MOPSO 3-(N-morpholino)-2-hydroxy-propanesulfonic acid - NDPS nucieoside diphosphosugars - NOE nuclear Overhauser effect - NMR nuclear magnetic resonance For convenience, the term ammonia is used throughout to denote ammonia or ammonium ion when there is no good evidence as to which chemical species is involved     

31P Nuclear magnetic resonance studies of ethanol inhibition in Zymomonas mobilis

Ethanol inhibition of glucose catabolism in Zymomonas mobilis was investigated using ³¹P NMR spectroscopy in vivo and of perchloric acid extracts from cell suspensions incubated with 0, 5 and 10% (w/v) ethanol. In vivo ³¹P NMR experiments revealed slower glucose utilization and decreased levels of nucleoside triphosphates in the presence of 10% ethanol as compared to controls. Using ³¹P NMR spectroscopy of perchloric acid extracts, intracellular accumulation of 3.4 mM 3-phosphoglycerate was found when 10% ethanol was present in the medium. No accumulation of this metabolite occurred in cells incubated with 0 and 5% ethanol. Enzyme assays confirmed that phosphoglycerate-mutase and enolase were inhibited 31 and 40%, respectively, in the presence of 10% ethanol in the test system. Therefore, under the conditions used the decrease in the fermentative activity of Z. mobilis at high ethanol concentrations is due to inhibition of phosphoglycerate-mutase and enolase.Abbreviation KDPG 2-keto-3-deoxy-6-phosphogluconate     

31P Magnetization transfer in the phosphoglyceromutase-enolase coupled enzyme system

The rate of exchange of phosphoryl groups between 2-phosphoglycerate, 3-phosphoglycerate and phosphoenolpyruvate by the coupled phosphoglyceromutase-enolase enzyme system using one- and two-dimensional ³¹P NMR spectroscopy was measured. Magnetization exchange in one-dimensional experiments was achieved by saturation transfer with selective irradiation at both one and two sites in this three-site exchange system using the DANTE pulse sequence. The two-dimensional magnetization exchange experiment avoids the need to selectively saturate at one or more frequencies which may be difficult in complex exchange systems. Analysis of the two-dimensional exchange experiment by the back transformation method yielded exchange rate constants in good agreement with the saturation transfer method.     

Potentiometric and P NMR studies on inositol phosphates and their interaction with iron(III) ions

Potentiometric, conductometric and ³¹P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP₆ ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P–O–Fe–O–P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)–InsP₆ aggregates. ³¹P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T₁ relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP₆ molecule results in an unstable heterogeneous system.     

The effects of calcium deficiency on Cucurbita pepo L. hypocotyl cells: A 31P nuclear-magnetic-resonance study

Calcium deficiency in zucchini (Cucurbita pepo L.) is associated with reduced growth and a reduced ability to transport auxin (Allan and Rubery, 1991, Planta 183, 604–612). An investigation of the effects of calcium-deficiency on zucchini hypocotyl cells was made using weak-acid uptake and ³¹P-nuclear-magneticresonance (³¹P-NMR) spectroscopy in vivo and in tissue extracts. Calcium-deficient tissue had the same cytoplasmic and vacuolar pHs as normal tissue when extracellular pH was near neutral. At acidic external pH the vacuolar pH was lower in deficient tissue. Adenine nucleotides were present predominantly as ATP in both control and calcium-deficient tissues. Addition of calcium to calcium-deficient tissue, under conditions which cause recovery of auxin transport induced no changes in the ³¹P-NMR spectra of deficient tissue. The content of mobile, phosphorylated metabolites was reduced in calcium-deficient tissue in comparison to control tissue. However, a substantial increase in the content of phosphorylcholine occurs in calcium-deficient tissues compared with controls; this may reflect changes in lipid turnover in calcium-stressed cells. We wish to thank Drs. Terry Moore and Jamie Vandenberg for technical assistance and Professor Peter Morris for providing the gated oxygen device. A.C.A. thanks the Cambridge Commonwealth Trust for a Prince of Wales Scholarship and the O.R.S. Awards Scheme for an award.