Effects of Surfactant Addition on Dewatering of Alum Sludges

In Taiwan, surfactants are frequently used in the flotation process to aid in solid/liquid separation. Their effect on the dewatering of alum sludge was investigated. Various amounts of cationic and anionic surfactants were added to sludge samples, and the dewatering characteristics of the sludge and the water content of sludge cakes were evaluated. Both surfactants improved the dewatering of the sludge by lowering the specific resistance to filtration, decreasing the bound water content, and increasing the dewatering rate of the sludge. Different combinations of anionic and cationic surfactants and polyelectrolytes were also experimented on to study the effect of surfactant addition on the dewatering characteristics of polyelectrolyte-conditioned sludge. Experimental results indicated that both cationic and anionic surfactants adversely affected the dewatering of the conditioned sludge. The addition of surfactant to the oppositely charged polyelectrolyte proved to be most detrimental to sludge dewatering due to the precipitation between surfactant and polyelectrolyte. The addition of cationic surfactant to the cationic polyelectrolyte-conditioned sludge had the least effect.     

Cationic polymers for selectivity control in the capillary electrophoretic separation of inorganic anions

The capillary electrophoretic (CE) separation of the inorganic anions bromide, chloride, nitrate, nitrite, fluoride, sulfate and phosphate is described in 0.005 mol/L sodium chromate electrolyte in the presence of soluble polydisperse ionic polymers (polyelectrolytes). The cationic polyelectrolytes used were as follows: poly(1,1-dimethyl-3,5-dimethylenepiperidinium) chromate, hexadimethrine chromate, poly(1,1-dimethyl-3,5-dimethylenepyrrolidinium) chromate and ((diethylamino)ethyl)-dextran chromate in the concentration range 0.004-0.6% (w/v). These polyelectrolytes were shown to be capable of reversing the direction of the electroosmotic flow as well as inducing changes in analyte electrophoretic mobility, separation selectivity, and resolution. Changes in electrophoretic mobility by as much as 25% were observed for the sulfate anion, and the resolution of fluoride and phosphate was enhanced by a factor of 7.8. In the presence of 0.05-0.17% w/v poly(1,1-dimethyl-3,5-dimethylenepyrrolidinium) chromate at pH 8, separation currents were found to increase only slightly as compared to an electrolyte containing equivalent amounts of sodium chromate. Electroosmotic flow was also found to be fairly constant (+/- 16%) in the pH range 6.55-10.02 for 0.01% (w/v) poly(1,1-dimethyl-3,5-dimethylenepiperidinium) chromate at an ionic strength of 0.04, compared to a 400% change in the absence of the polyelectrolyte. The reproducibility of the electroosmotic mobility was between 0.36 and 6% RSD, and analyte electrophoretic mobility was between 0.01 and 1.6% RSD. Peak height reproducibility was 0.2-8.0% RSD. Separation efficiencies were between 258,000 and 780,000 theoretical plates, and detection limits were between 4.4 x 10(-7) and 9.1 x 10(-6) mol/L.     

Influence of annexin V on the structure and dynamics of phosphatidylcholine/phosphatidylserine bilayers: a fluorescence and NMR study

The consequences of the binding of annexin V on the structure and dynamics of PC/PS bilayers were studied by means of fluorescence polarization, 31P NMR, 2H NMR, and fluorescence recovery after photobleaching (FRAP). Even at complete coverage of the lipid bilayers by the protein, annexin V showed no influence on the lipid molecular packing and the acyl chain flexibility of both PC and PS. The fluorescence polarization of the probe DPH, the 31P NMR spectra, and deuterium quadrupolar splittings of P(d31)OPS remained unchanged. However, upon binding of annexin V, two distinct populations of PC were visible in 2H NMR, which were in slow exchange on the deuterium NMR time scale (microseconds). One component in the spectrum was identical to the protein-free sample, while a second, broad, component appeared. The presence of the protein induced a decrease in the transverse relaxation times (T2e), indicative of the appearance of slow motions (milliseconds to microseconds), in the P(d31)-OPS spectrum and in the P(d31)OPC broad component. FRAP experiments were carried out with the probes C12-NBD-PC and C12-NBD-PS: at saturation, annexin V reduced the lateral diffusion rate of PC by 40% and nearly blocked the diffusion of PS. These combined experiments are consistent with a model in which annexin V enters a proteolipidic complex in the form of an extended 2D network, stabilized by specific interactions with PS. As seen from the lateral diffusion rates and the acyl chains NMR spectral parameters, two separate lipid populations appear, presumably corresponding to those interacting with annexinV (PC and PS) and protein free domains (mainly PC).     

Effect of cholesteryl hemisuccinate on the interfacial properties of phosphatidylcholine bilayers

Cholesteryl hemisuccinate (CHEMS) is an amphipathic lipid that can regulate cell growth. A comparison of the effects of CHEMS and cholesterol on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers was investigated using fluorescence techniques. In liquid-crystalline phase POPC bilayers, CHEMS increased the interfacial surface charge, but was less effective than cholesterol in reducing acyl chain mobility and interfacial hydration. In liquid-crystalline phase DPPC bilayers, CHEMS and cholesterol were equally effective in reducing acyl chain mobility. Similar to the POPC matrix, CHEMS increased the interfacial surface charge and cholesterol decreased the surface hydration. The different effect of cholesterol and CHEMS on acyl chain mobility may be due to a preferential interaction of cholesterol with POPC. In gel phase DPPC bilayers, CHEMS and a succinylated pyrenyl cholesterol analog exhibited different effects on membrane physical-chemical properties than cholesterol. Succinylation also increased the rate of transfer of the pyrenyl cholesterol analog between single unilamellar vesicles approximately seven fold. This process demonstrated first-order kinetics which indicated that transbilayer migration was not a rate-limiting step. The succinylation of cholesterol places a carboxyl group at the lipid-water interface and the sterol ring deeper in the bilayer. For a structural model to explain its biological properties, CHEMS should be considered a bulky fatty acid.     

Globoside as a membrane receptor: a consideration of oligosaccharide communication with the hydrophobic domain

Recognition of macromolecules by glycosphingolipids is closely correlated with the nature of the glycolipid carbohydrate; however, it is also thought to be secondarily modulated by the structure of the single fatty acid. In the present work, we sought insight into what physical effect a change in this fatty acid has on the extramembranous portion of globosides at liposomal surfaces mimicking systems for which modulated receptor function has been recorded in the past. Protons of the exocyclic hydroxymethyl group on the terminal Gal residue of globotriaosylceramide (Gb3) were replaced with deuterium. In this location, the nonperturbing probe nuclei sampled cumulative conformational and orientational characteristics of the oligosaccharide chain at a sugar residue that is critical in specific binding of verotoxins. Deuterated Gb3 having 18:1 fatty acid was compared to the same species having 22:1 fatty acid, at 6.3 mol % in unsonicated bilayers of dipalmitoylphosphatidylcholine/cholesterol. Both produced narrow, apparently axially asymmetric 2H NMR spectra over a wide temperature range. Motional properties of the terminal sugar were measurably influenced by the fluidity of the host matrix; however, evidence was not found for conformational or orientational variation in this sugar brought about by the fatty acid alteration. In related experiments, acetate protons on the terminal N-acetyl galactosamine (GalNAc) residue of globotetraosylceramide (Gb4) were substituted with deuterium, and the natural fatty acid was replaced with 18:0 or 24:0 species deuterated at C2. Once again, species with short vs long fatty acid were examined for evidence of headgroup differences. Spectra of Gb4 were compared at 10 mol % in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine, and at 5 mol % in membranes containing 33 mol% cholesterol. Spectral splittings reflecting cumulative effects on conformation and order at the terminal deuterated sugar remained unchanged between species having 18:0 vs 24:0 fatty acid in POPC/cholesterol. In a pure POPC host matrix, there was clear evidence of a motional difference between the two--the longer chain Gb4 demonstrating spectral asymmetry--but the spectral width was unchanged. Transverse relaxation times, T2, were measured. Our findings appear to help correlate the conclusions of a number of workers dealing with the molecular basis of crypticity. We suggest that changes in glycolipid receptor function based on ceramide fatty acid variation have a major origin in the fatty acid's ability to determine the thermodynamics of interaction with the host matrix, as reflected in such parameters as glycolipid motional properties, local membrane curvature, and likely glycolipid time-dependent lateral associations. The result at low concentrations of glycolipid may often be only a subtly altered collective surface epitope, best detected by a specific recognition event.     

Membrane structure and dynamics as viewed by solid-state NMR spectroscopy

The purpose of the present study is the investigation of the structure and dynamics of biological membranes using solid-state nuclear magnetic resonance (NMR) spectroscopy. Two approaches are used in our laboratory. The first involves the measurement of high-resolution 13C and 1H spectra obtained by the magic angle spinning (MAS) technique while the second approach involves the measurement of 31P and 2H powder spectra in static samples. This paper will present some recent results obtained by high-resolution solid-state 1H NMR on the conformation of gramicidin A incorporated in a phosphatidylcholine bilayers. More specifically, we were able to observe changes in the gramicidin spectra as a function of the cosolubilization solvent initially used to prepare the samples. The interaction between lipid bilayers and an anticancer drug derived from chloroethylurea was also investigated using proton NMR spectroscopy. Finally, we have studied the interaction between cardiotoxin, a toxic protein extracted from snake venom, and negatively charged lipid bilayers using 31P solid-state NMR spectroscopy.     

Enzymatic reactions in liposomes using the detergent-induced liposome loading method

Microcompartmentalization is a crucial step in the origin of life. More than 30 years ago, Oparin et al. proposed models based on biochemical reactions taking place in so-called coacervates. Their intention was to develop systems with which semipermeable microcompartments could be established. In the present work we follow their intuition, but we use well-characterized bilayer structures instead of the poorly characterized coacervates. Liposomes from phospholipids can be used as microreactors but they exhibit only a modest permeability and, therefore, chemical reactions occurring inside these structures are depleted after a relatively short period. Here it is shown that even highly stable liposomes from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be used as semipermeable microreactors when treated with sodium cholate. Using this kind of mixed liposomes, we describe a biochemical reaction occurring inside the liposomes while the same reaction is prevented in the external medium. In addition, we show that this cholate-induced permeability of POPC bilayers can even be used to load macromolecules such as enzymes from the outside.     

Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes

The cellular uptake of liposomes is generally believed to be mediated by adsorption of liposomes onto the cell surface and subsequent endocytosis. This report examines the effect of liposome surface charge on liposomal binding and endocytosis in two different cell lines: a human ovarian carcinoma cell line (HeLa) and a murine derived mononuclear macrophage cell line (J774). The large unilamellar liposomes were composed of 1, 2-dioleolyl-sn-glycero-3-phosphatidylcholine with and without the addition of either a positively charged lipid, 1, 2-dioleoyl-3-dimethylammonium propanediol (DODAP), or a negatively charged lipid, 1,2-dioleolyl-sn-glycero-3-phosphatidylserine. In some experiments 5 mol % of the anionic PEG2000-PE or a neutral PEG lipid of the same molecular weight was added. HeLa cells were found to endocytose positively charged liposomes to a greater extent than either neutral or negatively charged liposomes. This preference was not lipid-specific since inclusion of a cationic cyanine dye, DiIC18(3), to impart positive charge in place of DODAP resulted in a similar extent of endocytosis. In contrast the extent of liposome interaction with J774 cells was greater for both cationic and anionic liposomes than for neutral liposomes. The greater uptake of positively charged liposomes by HeLa cells was also observed with sterically stabilized liposomes (PEG liposomes). Although the overall amount of endocytosis for all the PEG liposomes examined was attenuated relative to conventional liposomes, the extent of endocytosis was greatest for positively charged PEG liposomes, whereas negatively charged PEG2000-PE liposomes were hardly endocytosed by the HeLa cells. Incorporation of a neutral PEG lipid into liposomes permits the independent variation of liposome steric and electrostatic effects in a manner that may allow interactions with cells of the reticuloendothelial system to be minimized, yet permit strong interactions between liposomes and proliferating cells.     

The binding of phenol red to rabbit renal cortex

The binding of phenol red to the microsomal fraction of rabbit kidney cortex was rapid, reversible and consisted of two independent populations of binding sites: a high affinity and low capacity class which had an association constant of 11.29 - 10(3) M-1 and a binding capacity of 2.41 mumol phenol red bound per g of protein, and a low affinity binding population with an association constant of 0.80 - 10(3) M-1 and a maximal binding capacity of 55.06 mumol per g of protein. Probenecid (0.32 mM) competitively inhibited phenol red binding to only the high affinity binding site, whereas 2,4-dinitrophenol (0.77 mM) competitively inhibited phenol red binding to both the high and the low affinity population of binding sites. The binding of phenol red was highly sensitive to the cationic composition of the medium. The affinity of phenol red to the high and the low affinity binding populations was lowered by decreasing the sodium and potassium concentrations to 19 and 6 mequiv./l, respectively; however, the maximal binding capacity was unchanged. Calcium appeared to have no effect on the phenol red binding to the microsomes. All of these considerations suggest that the high affinity phenol red binding to the microsomal fraction may represent the interaction of phenol red with the physiological receptor necessary for organic acid transport at the peritubular membrane. Phenol red binding to the low affinity binding population may indicate an intracellular binding population which contributes to the intracellular accumulation of weak organic acids.     

1H- and 2H-NMR studies of a fragment of PMP1, a regulatory subunit associated with the yeast plasma membrane H(+)-ATPase. Conformational properties and lipid-peptide interactions

PMP1 is a 38-residue polypeptide associated with the yeast plasma membrane H(+)-ATPase, found to regulate the enzyme activity. To investigate the molecular basis of the PMP1 biological function, the conformational properties of a synthetic PMP1 fragment, A18-F38, comprising the predicted C-terminal cytoplasmic domain and a part of the transmembrane anchor have been studied by 1H- and 2H-NMR spectroscopies. High resolution 1H-NMR experiments showed that, in deuterated DPC micelles, the A18-G34 segment adopts a well defined helix conformation. Our data suggest that the whole PMP1 molecule forms a unique helix whose axis might be slightly tilted with respect to the bilayer normal. Protonated DPC, DMPC and DMPS were incorporated in deuterated micelles containing the PMP1 fragment for studying lipid-peptide interactions. Unusually strong and selective intermolecular NOEs between lipid chain and peptide side chain protons, especially those of the unique Trp residue, were observed. Solid state 2H-NMR experiments performed on pure deuterated POPC and mixed deuterated POPC:POPS (5:1) bilayers revealed that the PMP1 fragment specifically interacts with negatively charged PS lipids.     

Mitochondrial presequence inserts differently into membranes containing cardiolipin and phosphatidylglycerol

The interaction of the 25-residue presequence of yeast cytochrome oxidase subunit IV with lipid bilayers composed of phosphatidylglycerol, cardiolipin, or their (1:4) mixtures with phosphatidylcholine has been studied by spin-label ESR spectroscopy. Binding of the presequence progressively broadens the gel-to-fluid phase transition of dimyristoylphosphatidylglycerol bilayers, leading to abolition of the transition at a peptide/lipid ratio of > or = 1:5 mol/mol. The mobility of phosphatidylglycerol spin-labeled at the 5-position of the sn-2 chain is decreased in both gel and fluid phases on binding the presequence, with a progressively increasing ESR spectral anisotropy in the fluid phase. The ESR spectra of phosphatidylglycerol spin-labeled at the 14-position of the sn-2 chain contain a second motionally restricted component, in addition to the fluid bilayer spectral component, that arises from direct interaction of the bound presequence with the lipid chains. The proportion of this motionally restricted component is greater for dioleoylphosphatidylglycerol bilayers (corresponding to 2-3 lipids per peptide) than for cardiolipin bilayers (1-2 lipids/peptide), and this component is present also in the mixed bilayers containing 80% phosphatidylcholine. The ESR spectra of the presequence spin-labeled with a maleimide derivative at cysteine-19 evidence high mobility in solution and a very strong reduction in mobility on binding to bilayers containing negatively charged lipids. At low peptide to lipid ratios, the ESR spectra of the spin-labeled presequence sense the phase transition of dimyristoylphosphatidylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reduction in electroporation voltages by the addition of a surfactant to planar lipid bilayers

The effects of a nonionic surfactant, octaethyleneglycol mono n-dodecyl ether (C12E8), on the electroporation of planar bilayer lipid membranes made of the synthetic lipid 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 100-450 mV) rectangular voltage pulses were used to electroporate the bilayers, followed by a prolonged, low-amplitude ( approximately 65 mV) voltage clamp to monitor the ensuing changes in transmembrane conductance. The electroporation thresholds of the membranes were found for rectangular voltage pulses of given durations. The strength-duration relationship was determined over a range from 10 micros to 10 s. The addition of C12E8 at concentrations of 0.1, 1, and 10 microM to the bath surrounding the membranes decreased the electroporation threshold monotonically with concentration for all durations (p < 0.0001). The decrease from control values ranged from 10% to 40%, depending on surfactant concentration and pulse duration. For a 10-micros pulse, the transmembrane conductance 150 micros after electroporation (G150) increased monotonically with the surfactant concentration (p = 0.007 for 10 microM C12E8). These findings suggest that C12E8 incorporates into POPC bilayers, allowing electroporation at lower intensities and/or shorter durations, and demonstrate that surfactants can be used to manipulate the electroporation threshold of lipid bilayers.     

1H MAS and 1H[23Na] double resonance NMR studies on the modification of surface hydroxyl groups of gamma-alumina by sodium

The modification of surface hydroxyl groups with sodium in a series of Na2CO3-gamma-Al2O3 catalysts was investigated as a function of both the Na2CO3 loading and the calcination temperature by means of 1H magic angle spinning (MAS) and 1H(23Na) spin-echo double resonance NMR techniques. The 1H NMR experiments revealed that sodium ions are homogeneously distributed over the alumina surface and closely coordinated with the surface hydroxyl groups. In the catalysts calcined at 250 degrees C, the acidic hydroxyl groups (with a chemical shift of 2.0 ppm) are preferentially associated with sodium ions at low Na2CO3 coverages (5 and 10%), while both the acidic and the basic (0 ppm) hydroxyl groups are accessible for sodium ions at high coverages (15 and 20%). The coordination causes a low-field shift of about 2 ppm in the 1H MAS spectra, and a broad signal at 4.5 ppm appears. It is interesting that the 4.5 ppm signal is completely suppressed in the 1H(23Na) MAS experiments, providing direct evidence that a strong interaction exists between adsorbed sodium ions and the surface hydroxyl groups. Increasing the calcination temperature to 450 degrees C results in preferential removal of the acidic hydroxyl groups, and only the most basic hydroxyl groups remain when the calcination temperature is raised to 600 degrees C. This is attributed to the formation of the coordinated species. [formula: see text] which enhances the acidity of the surface hydroxyl groups and prompts their dehydroxylation, especially at high calcination temperature. Correlation of the 1H MAS NMR results and catalytic activity measurements indicates that the basic hydroxyl groups are essential for the carbonyl sulfide hydrolysis reaction.     

Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes

Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H', and F. These proteins share epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H' is 96%, between H and F 78%, and between H' and F 75%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH2-terminal amino acids, were constructed by polymerase chain reaction amplification and used for ribohomopolymer binding studies. Each qRRM repeat bound poly(rG), while only the NH2-terminal qRRM bound poly(rC) and poly(rU). None of the repeats bound detectable amounts of poly(rA). The expression levels of hnRNPs H and F were differentially regulated in pairs of normal and transformed fibroblasts and keratinocytes. In normal human keratinocytes, the expression level of H was unaffected by treatment with several substances tested including two second messengers and seven cytokines. Likewise the expression level of F was independent of these substances, although it was strikingly down-regulated by long term treatment with 4 beta-phorbol 12-myristate 13-acetate, indicating that the protein kinase C signaling pathway regulates its expression. No effect of 4 beta-phorbol 12-myristate 13-acetate was observed on the expression of hnRNP H. The genes coding for hnRNPs H, H', and F were chromosome-mapped to 5q35.3 (HNRPH1), 6q25.3-q26, and/or Xq22 (HNRPH2) and 10q11.21-q11.22 (HNRPF), respectively.     

On the domain structure of cytochrome P450 102 (BM-3): isolation and properties of a 45-kDa FAD/NADP domain

Cytochrome P450 102 is a catalytically self-sufficient monooxygenase isolated from barbiturate-induced Bacillus megaterium. The enzyme contains FAD, FMN, and heme in a single polypeptide chain of 1048 residues, and each of the cofactors is believed to be located in a separate domain. In the present study we have used exhaustive endogenous proteolysis to produce a 45 kDa fragment of the cytochrome. This fragment bound the 2',5'-adenosine diphosphate moiety of NADP(H) strongly, with approximately the same dissociation constant as in the native enzyme, and contained only FAD (0.93 equivalents per polypeptide, epsilon 453nm = 11,200 M-1cm-1). Reduction of the flavin by sodium dithionite proceeded quite slowly to yield FADH2, but no stable semiquinone species was produced upon air re-oxidation. In contrast, NADPH rapidly reduced this FAD/NADP(H) domain aerobically to produce the FADH. semiquinone radical. At a 75:1 molar ratio of the FAD/NADP(H) domain to the P450 102 heme domain, no laurate hydroxylase activity was observed. Gas-phase sequence analysis showed the presence of two major sequences beginning at Phe646 (403 residues, MW 45,033) and Asp652 (397 residues). These data are in agreement with the crystal structures of related enzymes and closely define the boundary of the FAD/NADP+ domain in P450 102.     

Sphingomyelinase induces lipid microdomain formation in a fluid phosphatidylcholine/sphingomyelin membrane

The behaviors of two chemically well-defined sphingolipids, N-palmitoyl-sphingomyelin (C16:0-SM) and the corresponding ceramide (C16:0-Cer), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) matrix were compared. Minor attenuation of lateral diffusion upon increasing the mole fraction of C16:0-SM (XSM, up to 0.25) was indicated by the slight decrement in the excimer/monomer intensity ratio (Ie/Im) for a trace amount (mole fraction X = 0.01) of a pyrene-labeled ceramide analogue (N-[(pyren)-1-yl]decanoyl-sphingosine, PDCer) in keeping with the miscibility of C16:0-SM in POPC. Increasing membrane order was revealed by the augmented polarization P for diphenylhexatriene (DPH). In contrast, when C16:0-Cer was substituted for C16:0-SM an approximately 1.6-fold increase in Ie/Im for PDCer was evident upon increasing Xcer, with parallel increment in DPH polarization. In agreement with our recent data on natural ceramides in dimyristoylphosphatidylcholine (DMPC) bilayers [Holopainen et al. (1997) Chem. Phys. Lipids 88, 1-13], we conclude that C16:0-Cer becomes enriched into microdomains in the fluid POPC membrane. Interestingly, enhanced formation of microdomains by ceramide was observed when the total sphingolipid content in tertiary alloys with POPC was maintained constant (Xcer + XSM = 0.25) and the SM/Cer stoichiometry was varied. Finally, when ceramide was generated enzymatically in POPC/C16:0-SM (3:1, molar fraction) LUVs by sphingomyelinase (SMase, Bacillus cereus), maximally approximately 85% of hydrolysis of sphingomyelin was measured within <3 min at 30 degreesC. The formation of ceramide was accompanied by a closely parallel increase in DPH polarization. There was also an increase in Ie/Im for PDCer; however, these changes in Ie/Im were significantly slower, requiring approximately 105 min to reach a steady state. These data show that the rapid enzymatic formation of ceramide under these conditions is followed by much slower reorganization process, resulting in the formation of microdomains enriched in this lipid.     

A conformational equilibrium in a protein fragment caused by two consecutive capping boxes: 1H-, 13C-NMR, and mutational analysis

The conformational properties of an 18 residues peptide spanning the entire sequence, L1KTPA5QFDAD10ELRAA15MKG, of the first helix (A-helix) of domain 2 of annexin I, were thoroughly investigated. This fragment exhibits several singular features, and in particular, two successive potential capping boxes, T3xxQ6 and D8xxE11. The former corresponds to the native hydrogen bond network stabilizing the alpha helix N-terminus in the protein; the latter is a non-native capping box able to break the helix at residue D8, and is observed in the domain 2 partially folded state. Using 2D-NMR techniques, we showed that two main populations of conformers coexist in aqueous solution. The first corresponds to a single helix extending from T3 to K17. The second corresponds to a broken helix at residue Ds. Four mutants, T3A, F7A, D8A, and E11A, were designed to further analyze the role of key amino acids in the equilibrium between the two ensembles of conformers. The sensitivity of NMR parameters to account for the variations in the populations of conformers was evaluated for each peptide. Our data show the delta13Calpha chemical shift to be the most relevant parameter. We used it to estimate the population ratio in the various peptides between the two main ensembles of conformers, the full helix and the broken helix. For the WT, E11A, and F7A peptides, these ratios are respectively 35/65, 60/40, 60/40. Our results were compared to the data obtained from helix/coil transition algorithms.     

Synthesis and analysis of the enantiomers of calmidazolium, and a 1H NMR demonstration of a chiral interaction with calmodulin

Calmidazolium [R24571, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4- dichlorophenyl)methoxy]ethyl]-1H-imidazolium chloride] is a potent calmodulin inhibitor. This paper describes the synthesis and properties of the enantiomers of calmidazolium from the enantiomers of miconazole [1(N)-(2-(2,4-dichlorobenzyloxy)-2-(2,4 dichlorophenyl))-ethyl imidazole], prepared from the racemate by chiral preparative scale high performance liquid chromatography. Overlap between ligand and protein resonances in the aromatic region of the 1H NMR spectrum of the calmidazolium-calmodulin complexes has been obviated by preparation of the protein with all of its nine phenylalanine rings deuterated (Phe-d5 calmodulin). This has been accomplished by the overexpression of calmodulin derived from Trypanosoma brucei rhodiesiense in E. coli in a medium supplemented with ring-deuterated phenylalanine. The kinetics of binding of each enantiomer are slow on the 1H NMR time scale as judged by the behaviour of the H2 resonance of Histidine-107, which is clearly visible under the sample conditions used. The aromatic spectral regions of the protein-bound (+) and (-) enantiomers contrast strikingly, reflecting differences in bound environment and/or conformation.     

Molecular motion and orientation distributions in melt-processed, fully aromatic liquid crystalline polyesters from 1H NMR

Fully-aromatic thermotropic liquid crystalline polymers (LCP) containing 4-hydroxybenzoic acid (HBA) and 6-hydroxy-2-naphthoic acid (HNA) were studied with 1H NMR. A two- or three-parameter nematic director distribution in molten or nearly molten samples was obtained via rigorous simulation of wideline spectral lineshapes. This methodology was further employed to yield the chain director distribution in macroscopic sections derived from a frozen contraction flow. In addition, the dynamic conformation of polymer chains through the melting transition was monitored via lineshape analysis of samples having (bulk) isotropic director distributions. Extension of rigorous 1H NMR spectral deconvolution to recently developed solid-state NMR imaging sequences is discussed.