Preferential location of lidocaine and etidocaine in lecithin bilayers as determined by EPR, fluorescence and 2H NMR

We have examined the effect of the uncharged species of lidocaine (LDC) and etidocaine (EDC) on the acyl chain moiety of egg phosphatidylcholine liposomes. Changes in membrane organization caused by both anesthetics were detected through the use of EPR spin labels (5, 7 and 12 doxyl stearic acid methyl ester) or fluorescence probes (4, 6, 10, 16 pyrene-fatty acids). The disturbance caused by the LA was greater when the probes were inserted in more external positions of the acyl chain and decreased towards the hydrophobic core of the membrane. The results indicate a preferential insertion of LDC at the polar interface of the bilayer and in the first half of the acyl chain, for EDC. Additionally, (2)H NMR spectra of multilamellar liposomes composed by acyl chain-perdeutero DMPC and EPC (1:4 mol%) allowed the determination of the segmental order (S(mol)) and dynamics (T(1)) of the acyl chain region. In accordance to the fluorescence and EPR results, changes in molecular orientation and dynamics are more prominent if the LA preferential location is more superficial, as for LDC while EDC seems to organize the acyl chain region between carbons 2-8, which is indicative of its positioning. We propose that the preferential location of LDC and EDC inside the bilayers creates a "transient site", which is related to the anesthetic potency since it could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel.     

Differential effects of uncharged aminoamide local anesthetics on phospholipid bilayers, as monitored by 1H-NMR measurements

We have collected evidences of a "transient site" for the local anesthetics (LA) lidocaine, etidocaine, bupivacaine and mepivacaine in sonicated egg phosphatidylcholine (EPC) vesicles. The effects of the uncharged anesthetic species at a fixed LA/EPC ratio inside the bilayer were measured by chemical shifts (C.S.) and longitudinal relaxation times (T(1)) of the lipid hydrogens. Two sort of changes were detected: (I) decrease, indicating specific orientation of the LA aromatic ring (measured as up-field C.S. changes by the short-range ring-current effect) and less rotational freedom (smaller T(1) values) for EPC hydrogens such as the two glycerol-CH(2) and the choline-CH(2) bound to the PO(4-) group, probably due to the nearby presence of the LA; (II) increase, indicating the aromatic ring is now perpendicular to the orientation observed before (causing down-field changes in C.S.) and larger T(1) values for all the choline and glycerol hydrogens, as a result of LA insertion behind these well-organized bilayer regions. The less hydrophobic, linear and nonlinear (lidocaine and mepivacaine, respectively) aminoamide analogs provide similar effects-described in I; their hydrophobic counterparts (etidocaine and bupivacaine) also produced comparable effects (depicted in II). The preferential positioning and orientation of each LA inside the bilayer is then determined by its hydrophobic and steric properties. We propose that this "transient site" in the lipid milieu exists also in biological membranes, where it can modulates the access of the uncharged LA species to its site(s) of action in the voltage-gated sodium channel.     

Lipid composition determines the effects of arbutin on the stability of membranes.

Arbutin (hydroquinone-beta-D-glucopyranoside) is an abundant solute in the leaves of many freezing- or desiccation-tolerant plants. Its physiological role in plants, however, is not known. Here we show that arbutin protects isolated spinach (Spinacia oleracea L.) thylakoid membranes from freeze-thaw damage. During freezing of liposomes, the presence of only 20 mM arbutin led to complete leakage of a soluble marker from egg PC (EPC) liposomes. When the nonbilayer-forming chloroplast lipid monogalactosyldiacylglycerol (MGDG) was included in the membranes, this leakage was prevented. Inclusion of more than 15% MGDG into the membranes led to a strong destabilization of liposomes during freezing. Under these conditions arbutin became a cryoprotectant, as only 5 mM arbutin reduced leakage from 75% to 20%. The nonbilayer lipid egg phosphatidylethanolamine (EPE) had an effect similar to that of MGDG, but was much less effective, even at concentrations up to 80% in EPC membranes. Arbutin-induced leakage during freezing was accompanied by massive bilayer fusion in EPC and EPC/EPE membranes. Twenty percent MGDG in EPC bilayers completely inhibited the fusogenic effect of arbutin. The membrane surface probes merocyanine 540 and 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosph ocholi ne (NBD-C(6)-HPC) revealed that arbutin reduced the ability of both probes to partition into the membranes. Steady-state anisotropy measurements with probes that localize at different positions in the membranes showed that headgroup mobility was increased in the presence of arbutin, whereas the mobility of the fatty acyl chains close to the glycerol backbone was reduced. This reduction, however, was not seen in membranes containing 20% MGDG. The effect of arbutin on lipid order was limited to the interfacial region of the membranes and was not evident in the hydrophobic core region. From these data we were able to derive a physical model of the perturbing or nonperturbing interactions of arbutin with lipid bilayers.     

Antithrombin III and its interaction with heparin. Comparison of the human, bovine, and porcine proteins by 1H NMR spectroscopy

1H NMR has been used to characterize and compare the structures of antithrombin III from human, bovine, and porcine plasma as well as to investigate the interactions of each of these proteins with heparin fragments of defined length. The amino acid compositions of the three proteins are very similar, which is reflected in the gross features of their 1H NMR spectra. In addition, aromatic and methyl proton resonances in upfield-shifted positions appear to be common to all three proteins and suggest similar tertiary structures. Human antithrombin III has five histidine residues, bovine has six, and porcine has five. The C(2) proton from each of these residues gives a narrow resonance and titrates with pH; the pKa's are in the range 5.15-7.25. It is concluded that all histidines in each protein are surface residues with considerable independent mobility. The carbohydrate chains in each protein also give sharp resonances consistent with a surface location and motional flexibility. The 1H spectra are sensitive to heparin binding. Although heparin resonances obscure protein resonances in the region 3.2-6.0 ppm, difference spectra between antithrombin III with and without heparin show clear perturbation of a small number of aromatic and aliphatic protein protons. These resonances include those of histidine C(2) and C(4) protons, of 10-20 other aromatic protons, of a methyl group, and also of protons with chemical shifts similar to those of lysine and/or arginine side chains. For human antithrombin III, it was shown that heparin fragments 8, 10, and 16 sugar residues in length result in almost identical perturbations to the protein. In contrast, tetrasaccharide results in fewer perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of local anesthetics with lipid bilayers investigated by (1)H MAS NMR spectroscopy

The membrane location of the local anesthetics (LA) lidocaine, dibucaine, tetracaine, and procaine hydrochloride as well as their influence on phospholipid bilayers were studied by (31)P and (1)H magic-angle spinning (MAS) NMR spectroscopy. The (31)P NMR spectra of the LA/lipid preparations confirmed that the overall bilayer structure of the membrane remained preserved. The relation between the molecular structure of the LAs and their membrane localization and orientation was investigated quantitatively using induced chemical shifts, nuclear Overhauser enhancement spectroscopy, and paramagnetic relaxation rates. All three methods revealed an average location of the aromatic rings of all LAs in the lipid-water interface of the membrane, with small differences between the individual LAs depending on their molecular properties. While lidocaine is placed in the upper chain/glycerol region of the membrane, for dibucaine and procaine the maximum of the distribution are slightly shifted into the glycerol region. Finally for tetracaine the aromatic ring is placed closest to the aqueous phase in the glycerol/headgroup region of the membrane. The hydrophobic side chains of the LA molecules dibucaine and tetracaine were located deeper in the membrane and showed an orientation towards the hydrocarbon core. In contrast the side chains of lidocaine and procaine are oriented towards the aqueous phase.     

Interaction of benzocaine with model membranes

We measured the absorption properties, water solubility and partition coefficients (P) between n-octanol, egg phosphatidylcholine (EPC) liposomes and erythrocyte ghosts/water for benzocaine (BZC), an ester-type always uncharged local anesthetic. The interaction of BZC with EPC liposomes was followed using Electron Paramagnetic Resonance, with spin labels at different positions in the acyl chain (5, 7, 12, 16-doxylstearic acid methyl ester). Changes in lipid organization upon BZC addition allowed the determination of P values, without phase separation. The effect of BZC in decreasing membrane organization (maximum of 11.6% at approx. 0.8:1 BZC:EPC) was compared to those caused by the local anesthetics tetracaine and lidocaine. Hemolytic tests revealed a biphasic (protective/inductive) concentration-dependent hemolytic effect for BZC upon rat erythrocytes, with an effective BZC:lipid molar ratio in the membrane for protection (RePROT), onset of hemolysis (ReSAT) and 100% membrane solubilization (ReSOL) of 1.0:1, 1.1:1 and 1.3:1, respectively. The results presented here reinforce the importance of considering hydrophobic interactions in the interpretation of the effects of anesthetics on membranes.     

Pressure-induced phase transitions of lipid bilayers observed by fluorescent probes Prodan and Laurdan

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and 6-dodecanoyl-2-(dimethylamino)naphthalene (Laurdan) in bilayer membranes of 1,2-distearoylphosphatidylcholine (DSPC) were observed as a function of pressure at constant temperature. The emission spectra of Prodan and Laurdan varied with the pressure-induced states of bilayer membranes. The maximum emission wavelength (lambda(max)) of Prodan characteristic of the liquid crystalline (L(alpha)), lamellar gel (L(beta)') and pressure-induced interdigitated gel (L(beta)I) phases of the DSPC bilayer was 480, 440 and 500 nm, respectively. On the other hand, the lambda(max) of Laurdan characteristic of the L(alpha) and L(beta)' phases was 480 and 440 nm in a similar manner as Prodan probe. However, no change in the lambda(max) was observed in spite of the occurrence of the interdigitation of bilayer. Since the lambda(max) reflects the solvent property around the probe molecules, we could speculate about the location of fluorescent probe in the bilayer membranes. In the L(alpha) phase the same chromophore group of Prodan and Laurdan probes distributes around phosphate group of lipid (i.e., polar region). The transformation of bilayer into the L(beta)' phase causes the Prodan and Laurdan molecules to move into the glycerol backbone (i.e., less polar) region. In the ripple gel (P(beta)') phase, the emission spectrum of Prodan shows a broad peak at about 480 nm and a shoulder around 440 nm, which means that the Prodan molecules are widespread over the wide range from the glycerol backbone to the hydrophilic part of bilayer. The P(beta)'/L(beta)I phase transition causes the Prodan molecule to squeeze out from the glycerol backbone region and to move the hydrophilic region near the bilayer surface. Contrarily, the Laurdan molecule was not squeezed out from the glycerol backbone region because the long acyl chain of Laurdan serves as an anchor in the hydrophobic core of bilayer. The ratio of fluorescence intensity of Prodan at 480 nm to that at 440 nm, F(480)/F(440), is available to observation of bilayer phase transitions. The plot of F(480)/F(440) versus pressure seems to be useful for the recognition of bilayer phase transition, especially the bilayer interdigitation.     

New BODIPY lipid probes for fluorescence studies of membranes

Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me(4)-BODIPY-8) at the end of C(3)-, C(5)-, C(7)-, or C(9)-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me(4)-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me(4)-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me(4)-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and approximately 506-515 nm) but also showed the absence of the 620-630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me(4)-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.     

Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations

Fluorescence solvent relaxation experiments are based on the characterization of time-dependent shifts in the fluorescence emission of a chromophore, yielding polarity and viscosity information about the chromophore’s immediate environment. A chromophore applied to a phospholipid bilayer at a well-defined location (with respect to the z-axis of the bilayer) allows monitoring of the hydration and mobility of the probed segment of the lipid molecules. Specifically, time-resolved fluorescence experiments, fluorescence quenching data and molecular dynamic (MD) simulations show that 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) probes the hydration and mobility of the sn-1 acyl groups in a phosphatidylcholine bilayer. The time-dependent fluorescence shift (TDFS) of Laurdan provides information on headgroup compression and expansion induced by the addition of different amounts of cationic lipids to phosphatidylcholine bilayers. Those changes were predicted by previous MD simulations. Addition of truncated oxidized phospholipids leads to increased mobility and hydration at the sn-1 acyl level. This experimental finding can be explained by MD simulations, which indicate that the truncated chains of the oxidized lipid molecules are looping back into aqueous phase, hence creating voids below the glycerol level. Fluorescence solvent relaxation experiments are also useful in understanding salt effects on the structure and dynamics of lipid bilayers. For example, such experiments demonstrate that large anions increase hydration and mobility at the sn-1 acyl level of phosphatidylcholine bilayers, an observation which could not be explained by standard MD simulations. If polarizability is introduced into the applied force field, however, MD simulations show that big soft polarizable anions are able to interact with the hydrophilic/hydrophobic interface of the lipid bilayer, penetrating to the level probed by Laurdan, and that they expand and destabilize the bilayer making it more hydrated and mobile.     

Interaction of local anesthetics with lipid bilayers investigated by 1H MAS NMR spectroscopy

The membrane location of the local anesthetics (LA) lidocaine, dibucaine, tetracaine, and procaine hydrochloride as well as their influence on phospholipid bilayers were studied by ³¹P and ¹H magic-angle spinning (MAS) NMR spectroscopy. The ³¹P NMR spectra of the LA/lipid preparations confirmed that the overall bilayer structure of the membrane remained preserved. The relation between the molecular structure of the LAs and their membrane localization and orientation was investigated quantitatively using induced chemical shifts, nuclear Overhauser enhancement spectroscopy, and paramagnetic relaxation rates. All three methods revealed an average location of the aromatic rings of all LAs in the lipid-water interface of the membrane, with small differences between the individual LAs depending on their molecular properties. While lidocaine is placed in the upper chain/glycerol region of the membrane, for dibucaine and procaine the maximum of the distribution are slightly shifted into the glycerol region. Finally for tetracaine the aromatic ring is placed closest to the aqueous phase in the glycerol/headgroup region of the membrane. The hydrophobic side chains of the LA molecules dibucaine and tetracaine were located deeper in the membrane and showed an orientation towards the hydrocarbon core. In contrast the side chains of lidocaine and procaine are oriented towards the aqueous phase.     

Profiling of dynamics in protein–lipid–water systems: a time-resolved fluorescence study of a model membrane protein with the label BADAN at specific membrane depths

Profiles of lipid-water bilayer dynamics were determined from picosecond time-resolved fluorescence spectra of membrane-embedded BADAN-labeled M13 coat protein. For this purpose, the protein was labeled at seven key positions. This places the label at well-defined locations from the water phase to the center of the hydrophobic acyl chain region of a phospholipid model membrane, providing us with a nanoscale ruler to map membranes. Analysis of the time-resolved fluorescence spectroscopic data provides the characteristic time constant for the twisting motion of the BADAN label, which is sensitive to the local flexibility of the protein–lipid environment. In addition, we obtain information about the mobility of water molecules at the membrane–water interface. The results provide an unprecedented nanoscale profiling of the dynamics and distribution of water in membrane systems. This information gives clear evidence that the actual barrier of membranes for ions and aqueous solvents is located at the region of carbonyl groups of the acyl chains.     

Lipid protein interactions in mitochondria. Spin and fluorescence probe studies on the effect of n-alkanols on phospholipid vesicles and mitochondrial membranes.

The effect of n-butanol on the mobility of phospholipids in phospholipid vesicles and beef heart mitochondrial membranes has been studied using three stearic acid spin labels having a paramagnetic doxyl group in positions 5,12, and 16, respectively, and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS). The mobility of the spin labels in the phospholipid aliphatic chains increases from the polar heads toward the methyl groups both in vesicles and in mitochondrial membranes; however, in the latter there is a higher constriction of rotational mobility observed at all levels in the lipid bilayer. Butanol determines a moderate increase in mobility of phospholipids in lipid vesicles, but the effect is more striking in the mitochondrial membranes, where the protein-induced constraint of mobility of the fatty acyl chains is removed at low concentrations of the alcohol. Butanol also enhances the mobility of tightly bound phospholipids residual in lipid-depleted mitochondrial preparations, although higher concentrations of butanol are required for this effect. The effect of the series of aliphatic n-alcohols is related to their hydrophobicity.Alcohols induce a decrease of the fluorescence of ANS bound to both lipid vesicles and mitochondrial membranes. The fluorescence decrease is not the result of a decreased partition of ANS from the aqueous medium to the bilayer, but depends upon a change in the chromophore environment. Since no shift of the emission maximum is observed after alcohol addition, such a change must be ascribed to increased mobility of the probe, in accord with the spin label data.As for the spin label data, the effect of the series of aliphatic n-alcohols is related to their hydrophobicity; at difference with the electron spin resonance results, however, the effects are maximal for pure phospholipid vesicles. It is calculated that alcohols affect both the long-range interactions between phospholipids and proteins in mitochondrial membranes (as detected by spin labels) and the order of phospholipid bilayers near the glycerol region (as detected by ANS). The differences between the two kinds of probes may be related to their differing localization in the lipid bilayer.     

400 MHz two-dimensional nuclear Overhauser spectroscopy on anesthetic interaction with lipid bilayer

Interaction between a volatile anesthetic, methoxyflurane, and dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was analyzed by nuclear Overhauser effect (NOE) difference spectroscopy and two-dimensional nuclear Overhauser spectroscopy (NOESY). The NOE difference spectra were obtained by selectively irradiating methoxy protons (hydrophobic end) of the anesthetic: a negative nuclear Overhauser effect of -2.94% was observed with the choline methyl protons of DPPC. The NOESY spectra revealed a cross-peak between the anesthetic methoxy protons and the choline methyl protons. A dipole-dipole interaction exists between the hydrophobic end of the anesthetic and the hydrophilic head group of DPPC. No other cross-peaks were observed. The anesthetic orients itself at the membrane/water interface by interacting with the hydrophilic surface of the DPPC membrane, leaving the hydrophilic end of the anesthetic molecule in the aqueous phase. The preferred residence site of dipolar volatile anesthetics is the membrane/water interface.     

Spin Trapping of Methyl Radicals by the Acyl Nitroso Compound PH-C (= O)NO Formed in the Photochemical Reaction Between Benzohydroxamic Acid, Dimethyl Sulfoxide and Hydrogen Peroxide. An Epr Study

By the use of EPR spectroscopy, it has been shown that acyl nitroso compounds can act as spin traps for short-lived radicals with the formation of acyl aminoxyl radicals. The reaction was studied for the system benzohydroxamicacid[Ph-C (= O)N(H)] - dimethyl sulfoxide - hydrogen peroxide. The acyl aminoxyl radicals appeared almost immediately when the reaction mixture was irradiated in situ in the EPR cavity with UV light. The trapping reaction involved two photochemical reactions, i.e. the oxidation of the hydroxamic acid to the acyl nitroso compound Ph-C (= O)NO, and the formation of methyl radicals from dimethyl sulfoxide. The EPR spectra are superpositions of the spectra of two species of acyl aminoxyl radicals, i.e. the radicals Ph-C (= O)N(O·)H formed by oxidation of the parent benzohydrox-amic acid, and the radical Ph-C (= O)N(O·)CH₃, formed by trapping of methyl radicals.     

蟾毒灵与脂膜相互作用的研究

应用＾１３Ｃ－ＣＰ／ＭＡＳ和ＤＳＣ方法研究蟾毒灵与磷脂膜相互作用的执致相变特性及动力学特性。ＤＳＣ曲线表明蟾毒灵使磷脂膜相变温度降低,吸热峰变宽。＾１３Ｃ－ＣＰ／ＭＡＳ谱表明磷脂膜的ＮＭＲ信号峰化学位移随温度稍有变化,提示磷脂膜在液晶态脂肪烃链有不同程序的反－旁式异构化。含蟾毒灵的ＥＰＣ脂双层ＮＭＲ谱,随温度升高有蟾毒灵信号峰出现,ＥＰＣ脂双层分子内各部分的信号峰强度和峰形变化明显,说明脂双层分子     

Locations and dynamical perturbations for lipids of cationic forms of procaine, tetracaine, and dibucaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy

Locations and dynamical perturbations for lipids of local anesthetics (procaine . HCl, tetracaine . HCl, and dibucaine . HCl) in sonicated egg yolk phosphatidylcholine (PC) vesicles have been studied by 1H-1H nuclear Overhauser effect (NOE) measurements. It was found that tetracaine and dibucaine bind much strongly to the neutral lipids than does procaine and that their mobilities are lowered to such an extent that spin diffusion is transmitted (i.e., omega 2 tau c2 much greater than 1). The intermolecular NOEs between drugs and PC were more effective in the case of dibucaine than with tetracaine, indicating that dibucaine binds to the lipids more strongly than tetracaine; this order agrees well with that of anesthetic potency. However, it was only tetracaine that gave any appreciable dynamical perturbation to the PC vesicles when they were monitored by the extent of transfer of the negative NOE from alpha-methylene protons to choline methyls, olefinic methines, acyl methylenes and terminal methyl protons. This finding was interpreted as being due to the differences in the locations of these drugs in small unilamellar vesicles: (1) procaine interacts with lipids very weakly at the outer surface of the vesicles; (2) tetracaine binds to the lipids both at the outer and inner halves of the bilayer, inserting its rod-like molecule in a forest of acyl chains of PC; (3) dibucaine binds tightly to the polar head-group of PC, which resides only at the outer half of the bilayer vesicles. It was concluded that the relative order of anesthetic potency within these drugs can be correlated not with the ability to affect membrane fluidity but with the ability to bind to lipids at the polar head-group of the bilayer vesicles.     

Multiequilibrium binding of a spin-labeled local anesthetic in phosphatidylcholine bilayers

The equilibria among spin-labeled amine local anesthetic species in dioleoylphosphatidylcholine liposomes at an anesthetic: lipid mole ratio of 1:100 are investigated. Electron spin resonance (ESR) spectra demonstrate that anesthetic mobility within the bilayer is charge-dependent, with the uncharged species the more mobile. Partition coefficient measurements confirm ESR evidence that changes in anesthetic mobility represent anesthetic-phospholipid interaction and not changes in bilayer fluidity. Spin-exchange attenuation experiments show that anesthetics within the bilayer are accessible to the aqueous medium. Dependence of tertiary-amine anesthetic pK on dielectric constant has been used to estimate the interfacial pK. We propose a model of equilibria among species of the tertiary amine anesthetic in the aqueous medium and those intercalated in the bilayer, including a species electrostatically bound to the lipid phosphate. Using experimentally determined equilibrium constants, the model provides the binding constant between the electrostatically bound and unbound cationic anesthetics within the bilayer. The model stimulates the pH dependence of the mobile fraction of total anesthetic population determined by subtraction techniques on experimental ESR spectra.     

Orientation of LamB signal peptides in bilayers: influence of lipid probes on peptide binding and interpretation of fluorescence quenching data.

The orientation in lipid bilayers of the signal sequence of the bacterial protein LamB was studied using binding, circular dichroism, and fluorescence quenching experiments. Measurements were made of binding modifications caused by the incorporation of lipid probes (brominated or nitroxide-labeled phospholipids) used in the parallax fluorescence quenching method of determining peptide penetration depth [Abrams, F. S., and London, E. (1992) Biochemistry 31, 5312-5322]. The signal peptide bound to a similar extent to neutral bilayers composed of either egg phosphatidylcholine (EPC) or phosphatidylcholines brominated at various positions on their acyl chains. The fluorescence of a tryptophan in either the 18 or 24 position of the peptide was quenched more by bromines in the 6 and 7 than in the 9 and 10 positions on the lipid hydrocarbon chain. Parallax calculations showed that tryptophan-18 was located only 4 A from the hydrocarbon-water interface, consistent with the peptide adopting a "hammock" configuration in the bilayer, with both termini exposed to the aqueous phase and the central alpha-helix located near the hydrocarbon-water interface. In contrast, the incorporation of 10% nitroxide-labeled lipids into EPC bilayers modified peptide binding in a manner dependent on the position of the nitroxide on the hydrocarbon chain; 7-Doxyl PC reduced the percent peptide bound by about one-half, whereas 12-Doxyl PC had little effect on binding. These binding differences modified tryptophan quenching by these probes, making parallax analysis problematical. In the presence of the positively charged LamB peptide, the incorporation of negatively charged phospholipids into EPC bilayers increased the level of peptide binding and modified tryptophan quenching by nitroxide probes. These results suggest that the nitroxide probe could be partially excluded from negatively charged lipid domains where the peptide preferentially bound. Quite different binding and quenching results were obtained with a negatively charged peptide analogue, showing that the charge on both the peptide and bilayer affects peptide-nitroxide probe interactions.     

Simultaneous observation of order and dynamics at several defined positions in a single acyl chain using 2H NMR of single acyl chain perdeuterated phosphatidylcholines

Deuterium nuclear magnetic resonance (2H NMR) spectra from aqueous dispersions of phosphatidylcholines in which perdeuterated palmitic acid is esterified at the sn-1 position have several very useful features. The powder spectra show six well-resolved 90 degree edges which correspond to the six positions closest to the methyl end of the acyl chain. The spectral overlap inherent in the multiple powder pattern line shape of these dispersions can be removed by using a "dePaking" procedure [Bloom, M., Davis, J.H., & Mackay, A. (1981) Chem. Phys. Lett. 80, 198-202] which calculates the spectra that would result if the lipid bilayers were oriented in the magnetic field. This procedure produces six well-resolved doublets whose NMR properties can be observed without interference from the resonances of other labeled positions. The presence of a single double bond in the sn-2 chain increases the order of the saturated 16:0 sn-1 chain at every position in the bilayer compared with a saturated sn-2 chain at the same reduced temperature. Surprisingly, addition of five more double bonds to the sn-2 chain only slightly reduces the order of the 16:0 sn-1 chain at many positions in the bilayer compared with the single double bond. Calculating oriented spectra from a spin-lattice (T1) relaxation series of powder spectra allows one to obtain the T1 relaxation times of six positions on the acyl chain simultaneously. As an example of the utility of these molecules, we demonstrate that the dependence of the spin-lattice (T1) relaxation rate as a function of orientational order for two unsaturated phospholipids differs significantly from the corresponding fully saturated analogue. Interpreting this difference using current models of acyl chain dynamics suggests that the bilayers containing either of the two unsaturated phospholipids are significantly more deformable than bilayers made from the fully saturated phospholipid.     

Membrane actions of water-soluble fusogens: Effect of dimethyl sulfoxide,glycerol and sucrose on lipid bilayer order and fluidity

The effect of three water-soluble fusogens: dimethyl sulfoxide (DMSO), glycerol and sucrose on the structural properties of model lipid membranes has been studied by electron spin resonance (ESR) using 5-doxylstearic acid as a spin probe and by fluorescence spectroscopy using pyrene as an excimer forming fluorescent probe. All three fusogens tested produce a marked increase in the order parameter of the region close to the polar surface of the lipid bilayer. The ordering effect of DMSO, but not of glycerol and sucrose, is much stronger with respect to membranes prepared from acidic than from neutral phospholipids. The membrane-perturbing action of glycerol and sucrose manifests itself also in the reduced lateral mobility of membrane incorporated pyrene, indicating thus a decreased fluidity of the bilayer hydrophobic region. The structural perturbations produced in model membranes by DMSO, glycerol and sucrose are discussed in relation to the mechanism by which these substances promote cell fusion.