Calculation of Liquid-Disordered/Liquid-Ordered Line Tension from Pairwise Lipid Interactions.

The energy penalty for bilayer phase domain interfaces, line tension, is an important quantity for describing the phase domain size transition from the nanometer scale to the micrometer scale and larger. We connected pairwise lipid interaction energies in ternary lipid mixtures with experimentally measured line tensions by using the compositional differences between coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases known from phase diagrams. Using a mean-field theory model, we developed a computational procedure to map out Ld + Lo phase boundaries and thermodynamic tielines based on a set of pairwise interaction energies. We find that experimentally measured Ld/Lo line tension can be effectively modeled by the sum of pairwise interactions at the interface. This result indicates that pairwise lipid interactions make a major contribution to line tension.

Regulatory mechanisms of the acrosome reaction revealed by multiview microscopy of single starfish sperm.

The acrosome reaction in many animals is a coupled reaction involving an exocytotic step and a dramatic change in cell shape. It has been proposed that these morphological changes are regulated by intracellular ions such as Ca2+ and H+. We report here simultaneous visualization, under a multiview microscope, of intracellular free Ca2+ concentration ([Ca2+]i), intracellular pH (pHi), and morphological changes in a single starfish sperm (Asterina pectinifera). [Ca2+]i and pHi were monitored with the fluorescent probes indo-1 and SNARF-1, respectively. The acrosome reaction was induced with ionomycin. After the introduction of ionomycin in the medium, [Ca2+]i increased gradually and reached a plateau in approximately 30 s. The fusion of the acrosomal vacuole took place abruptly before the plateau, during the rising phase. Although the speed of the [Ca2+]i increase varied among the many sperm tested, exocytosis in all cases occurred at the same [Ca2+]i of approximately 2 microM (estimated using the dissociation constant of indo-1 for Ca2+ of 1.1 microM). This result suggests that the exocytotic mechanism in starfish sperm responds to [Ca2+]i rapidly, with a reaction time of the order of one second or less. Unlike the change in [Ca2+]i, an abrupt increase in pHi was observed immediately after exocytosis, suggesting the presence of a proton mobilizing system that is triggered by exocytosis. The rapid increase in pHi coincided with the formation of the acrosomal rod and the beginning of vigorous movement of the flagellum, both of which have been proposed to be pHi dependent. The exocytotic event itself was visualized with the fluorescent membrane probe RH292. The membrane of the acrosomal vacuole, concealed from the external medium in an unreacted sperm, was seen to fuse with the plasma membrane.

Reconstitution of M13 bacteriophage coat protein. A new strategy to analyze configuration of the protein in the membrane.

The configuration of M13 bacteriophage coat protein in a model membrane was analyzed using protease digestion followed by gel permeation chromatography on Fractogel TSK in formic acid/ethanol. Important information is contained in the chromatographic patterns of the membrane-bound fragments, as well as of the fragments released by the digestion. A new reconstitution was thereby developed which involves adding a small volume of a concentrated solution of cholate-solubilized coat protein to preformed vesicles (with the amount of detergent added being less than that required to solubilize the vesicles), freezing in liquid nitrogen, thawing, followed by dialysis to remove excess detergent. The coat protein is incorporated with high efficiency (95 percent) making subsequent fractionation unnecessary. In addition, the incorporated protein is not aggregated, and is incorporated with most molecules spanning the membrane, oriented in the same manner as in vivo (N-terminus outwards). Two previously described reconstitutions, using sonication or cholate dialysis, are analyzed and found to be less satisfactory.

Location and ion-binding of membrane-associated valinomycin, a proton nuclear magnetic resonance study.

Valinomycin, incorporated in small unilamellar vesicles of perdeuterated dimyristoylphosphatidylcholine, reveals several well-resolved 1H-NMR resonances. These resonances were used to examine the location, orientation and ion-binding of membrane-bound valinomycin. The order of affinity of membrane-bound valinomycin for cations is Rb+ greater than K+ greater than Cs+ greater than Ba2+, and binding is sensitive to surface change. The exchange between bound and free forms is fast on the NMR time scale. The intrinsic binding constants, extrapolated to zero anion concentration, are similar to those determined in aqueous solution. Rb+ and K+ show 1:1 binding to valinomycin, whereas the stoichiometry of Cs+ and Ba2+ is not certain. Paramagnetic chemical shift reagents and nitroxide spin label relaxation probes were used to study the location and orientation of valinomycin in the membrane. Despite relatively fast exchange of bound cations, the time average location of the cation-free form of valinomycin is deep within the bilayer under the conditions of these experiments. Upon complexation to K+, valinomycin moves closer to the interfacial region.

On the use of partition coefficients to characterize the distribution of fluorescent membrane probes between coexisting gel and fluid lipid phase: an analysis of the partition behavior of 1,6-diphenyl-1,3,5-hexatriene.

The distribution of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene between coexisting gel and fluid phospholipid phases in multilamellar vesicles has been examined using fluorescence quenching by spin-labeled phosphatidylcholine. For both thermally-induced and Ca2+-induced lipid phase separation, the ratio of probe concentration in the fluid liquid-crystal phase to that in the gel phase is found to be independent of either the probe concentration or the relative amounts of gel and fluid lipid phases, and hence is an equilibrium concentration ratio, or partition coefficient.

1H-NMR study of the location and motion of ubiquinones in perdeuterated phosphatidylcholine bilayers.

Ubiquinones (n = 1,2,3,4,7,9,10) and ubiquinols (n = 1,2,3,4,10) were incorporated into ordinary (protonated) or perdeuterated dimyristoyl phosphatidylcholine vesicles and were found to have significant local molecular motion. The motion of the quinone ring, as judged from the linewidth of the OCH3 proton resonances, decreased in longer-chain ubiquinones. Minimum values for the transverse mobility (flip-flop rates) of ubiquinones-1,2,3,4,10, measured with the aid of lanthanide shift reagents, suggest that they are all able to function in a protonmotive 'Q cycle' during electron transport. As the length of the side chain increases beyond 1 isoprenoid unit, the quinone/quinol ring tends to be deeper in the outer monolayer of small sonicated vesicles and in both monolayers of larger freeze-thaw vesicles, but little or no change in depth is observed in the inner monolayer of small vesicles. The ubiquinol rings are closer to the membrane surface than are the ubiquinone rings. For side chain n = 9 or 10, a second resonance from the OCH3 protons of ubiquinones and ubiquinols in vesicles appears in the 2H-NMR spectrum. This is due to the presence of two types of vesicles with different ubiquinone/phospholipid ratios.

A novel strategy for the preparation of liposomes: rapid solvent exchange.

During the preparation of multi-component model membranes, a primary consideration is that compositional homogeneity should prevail throughout the suspension. Some conventional sample preparation methods pass the lipid mixture through an intermediary, solvent-free state. This is an ordered, solid state and may favor the demixing of membrane components. A new preparative method has been developed which is specifically designed to avoid this intermediary state. This novel strategy is called rapid solvent exchange (RSE) and entails the direct transfer of lipid mixtures between organic solvent and aqueous buffer. RSE liposomes require no more than a minute to prepare and manifest considerable entrapment volumes with a high fraction of external surface area. In phospholipid/cholesterol mixtures of high cholesterol content, suspensions prepared by more conventional methods reveal evidence of artifactual demixing, whereas samples prepared by rapid solvent exchange do not. The principles which may lead to artifactual demixing during conventional sample preparation are discussed.

Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers.

In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures.

Effect of fluorophore linkage position of n-(9-anthroyloxy) fatty acids on probe distribution between coexisting gel and fluid phospholipid phases.

The quenching of probe fluorescence by spin-labeled phospholipid has been used to determine the distribution of a series of n-(9-anthroyloxy) fatty acids between coexisting gel and fluid liquid-crystal phases in multilamellar phospholipid vesicles. The phase distribution ratio in every case is found to favor the fluid lipid phase, but is much greater between fluid and Ca2+-induced gel than between fluid and thermal gel. For a given gel type, n-(9-anthroyloxy)stearic acids with n = 3, 6, 9 or 12 as well as 11-(9-anthroyloxy)undecanoic acid all exhibit similar behavior, favoring the fluid phase by about a factor of 4 over thermally-induced lipid gel phase and by 18 over Ca2+-induced gel phase. 16-(9-Anthroyloxy)palmitic acid, with the bulky probe at the terminus of the 16-carbon chain, favors the fluid phase less strongly, by a factor of 1.5 or 11 over thermally-induced or Ca2+-induced gel phase, respectively, indicating better packing of this probe in phospholipid gel phases.

Partitioning behavior of indocarbocyanine probes between coexisting gel and fluid phases in model membranes.

Gel-fluid partition coefficients, Kp, were measured for a series of indocarbocyanine dyes in multilamellar lipid vesicles. The dyes examined had alkyl chain lengths from 12 to 22 carbons. Fluorescence quenching by a spin-labeled phosphatidylcholine-enriched fluid phase created a large difference in quantum yield for indocarbocyanine fluorescence between fluid and gel phases, enabling reliable Kp determinations. The values range from Kp = 8 for the 12-carbon chain, favoring a fluid phase over a Ca2-phosphatidylserine rigid phase, to Kp = 0.02 for the 20-carbon chain dye, favoring a distearoylphosphatidylcholine-rich gel phase over the fluid phase.

NMR observation of gramicidin A' in phosphatidylcholine vesicles.

Dimyristoyl phosphatidylcholine was prepared with perdeuterated hydrocarbon chains and sonicated into bilayer vesicles together with gramicidin A'. The 1H NMR resonance from the tryptophan residues in the gramicidin has a linewidth of approximately 80 Hz, indicating significant local mobility for these residues. Paramagnetic lanthanides added to the aqueous medium cause a chemical shift of this signal indicating that some of the tryptophans may be located in the interfacial region of the bilayer.

Partitioning of gramicidin A' between coexisting fluid and gel phospholipid phases.

The partitioning behavior of gramicidin A' was investigated in four binary phospholipid mixtures with coexisting fluid and gel phases. The ratio of the equilibrium peptide concentration in the fluid phase to that in the gel phase (i.e., the partition coefficient, Kp) was determined by analysis of the quenching of gramicidin A' tryptophanyl fluorescence by a spin-labeled phosphatidylcholine. The partition coefficient was used as a measure of the relative solubility of gramicidin A' in the four types of gel phases analyzed. The composition of the gel phase was entirely Ca(dioleoylphosphatidylserine)2 (Ca(di18:1-PS)2), or was rich in either distearoylphosphatidylcholine (di18:0-PC), dipalmitoylphosphatidylcholine (di16:0-PC), or dimyristoylphosphatidylcholine (di14:0-PC). Except in the last case, the gel phase was depleted of gramicidin A': Kp approximately 30 when the gel phase was Ca(di18:1-PS)2 or di18:0-PC-rich, Kp approximately 10 when the gel phase was di16:0-PC-rich, and Kp approximately 1 when the gel phase was di14:0-PC-rich. The hydrophobic mismatch between the length of gramicidin A' and the length of the phospholipid acyl chains in the bulk gel phase is greatest with di18:1-PS and di18:0-PC, intermediate with di16:0-PC, and least with di14:0-PC. The Kp measurements presented here are consistent with increasing solubility of gramicidin A' in the gel phase with decreasing hydrophobic mismatch.

The interpretation of proton magnetic resonance linewidths for lecithin dispersions. Effect of particle size and chain packing.

Two previously reported theoretical treatments of the effect of sonication on the PMR spectrum of phospholipid bilayer membranes have led to divergent conclusions regarding the effects of sonication on the structure of the bilayer membrane. In this report these two theoretical treatments will be critically reviewed, and it will be shown that only the theory of Seiter and Chan (Seiter, C.H.A. and Chan, S.I. (1973) J. Am. Chem. Soc. 95, 7541-7553) yields predictions which are in agreement with experiment. Analysis of available and newly acquired NMR results for sonicated bilayer vesicles of different sizes, both above and below the thermal phase transition, indicates that sonication does disrupt the regular molecular packing of the phospholipid molecules in these systems.

Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes.

The mechanisms by which a cell uses and adapts its functional membrane organization are poorly understood and are the subject of ongoing investigation and discussion. Here, we study one proposed mechanism: the crosslinking of membrane components. In immune cell signaling (and other membrane-associated processes), a small change in the clustering of specific membrane proteins can lead to large-scale reorganizations that involve numerous other membrane components. We have investigated the large-scale physical effect of crosslinking a minor membrane component, the ganglioside GM1, in simple lipid models of the plasma membrane containing sphingomyelin, cholesterol, and phosphatidylcholine. We observe that crosslinking GM1 can cause uniform membranes to phase-separate into large, coexistent liquid ordered and liquid disordered membrane domains. We also find that this lipid separation causes a dramatic redistribution of a transmembrane peptide, consistent with a raft model of membrane organization. These experiments demonstrate a mechanism that could contribute to the effects of crosslinking observed in cellular processes: Domains induced by clustering a small number of proteins or lipids might rapidly reorganize many other membrane proteins.

On the nature of calcium ion binding between phosphatidylserine lamellae.

Ca2+ binding between phosphatidylserine (PS) lamellae gives rise to a phase with the composition Ca(PS)2. When aqueous Ca2+, hydrated PS, and Ca(PS)2 coexist at equilibrium, the aqueous Ca2+ concentration is invariant. At Ca2+ concentrations below this critical value, no binding of Ca2+ to PS is detected. Above this value, Ca2+ binds to PS to form Ca(PS)2. The invariant Ca2+ concentration is 0.14 microM for palmitoyloleoylphosphatidylserine (POPS) and 3.0 microM for dioleoylphosphatidylserine (DOPS). For the mixed acyl chain PS derived from bovine brain (BBPS) this Ca2+ concentration ranges from 0.25 to 0.7 microM. The observed phase behavior is described by the phase rule for the three-component system of water, Ca2+, and PS, with temperature and pressure constant. In order for Ca2+ to bind between PS lamellae to form the Ca(PS)2 phase, the aqueous Ca2+ concentration must be supersaturated. The equilibrium Ca2+ concentration is determined by dissolving Ca(PS)2 by use of Ca2+ chelators.

Partitioning of a fluorescent phospholipid between fluid bilayers: dependence on host lipid acyl chains.

The partition coefficient Kp was measured for a headgroup-labeled phospholipid (12:0,12:0)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PE (12-NBD-PE), equilibrated between LUV of a series of phosphatidylcholines (PC). Fluorescence resonance energy transfer between the 12-NBD-PE and a headgroup-rhodamine-labeled PE was used to find the equilibrium concentration of the 12-NBD-PE in the different LUV. Reliable equilibrium concentrations were obtained by monitoring the approach to equilibrium starting from a concentration below and from a concentration above the ultimate values. Using (16:0,18:1delta9)-PC as the reference lipid, Kp ranged from a high value of 1.65 favoring (16:0,18:1delta9)-PC over (16:1delta9,16:1delta9)-PC, to a low value of 0.90, favoring (22:1delta13,22:1delta13)-PC over (16:0,18:1delta9)-PC. The Kp values enabled calculation of the acyl chain contribution to the excess free energy of mixing for (12:0,12:0) acyl chains at infinite dilution in the L alpha phase of PC having acyl chains of (16:0,18:1delta9), (16:1delta9,16:1delta9), (18:1delta9,18:1delta9), (18:1delta6,18:1delta6), (20:1delta11,20:1delta11), and (22:1delta13,22:1delta13). (14:1delta9,14:1delta9)-PC was found to transfer so rapidly between LUV as to preclude reliable Kp measurement.

Calcium ion binding between lipid bilayers: the four-component system of phosphatidylserine, phosphatidylcholine, calcium chloride, and water.

Ca2+ binding between lamellae of phosphatidylserine (PS) and phosphatidylcholine (PC) gives rise to a rigid phase of Ca(PS)2. When aqueous Ca2+, hydrated PS/PC, and Ca(PS)2 coexist at equilibrium, the aqueous Ca2+ concentration is invariant and is characteristic of the PS/PC ratio. This characteristic Ca2+ concentration is 0.040 microM for palmitoyloleoylphosphatidylserine without PC and increases as the inverse square of the PS mole fraction at high PS concentration (Raoult's law) and as the inverse square of the PS mole fraction multiplied by a constant at low PS concentration (Henry's law). For example, for palmitoyloleoylphosphatidylserine/palmitoyloleoylphosphatidylcholi ne = 0.6/0.4 or 0.2/0.8, this characteristic Ca2+ concentration is about 0.1 or about 6 microM, respectively. These observations at constant temperature are summarized in a quaternary phase diagram for the four-component system CaCl2/PS/PC/water.

Polypeptide clearing in model membranes: an analysis of the partition of gramicidin a' between cadmium ion induced gel and liquid-crystalline phases in vesicles of phosphatidic acid and phosphatidylcholine.

Using a simple model for a biological membrane we examine cation-induced gel phase formation and the depletion of polypeptide from the gel phase. The model system consists of vesicles of phosphatidic acid and phosphatidylcholine which contain gramicidin A'. By use of electron spin resonance to monitor lipid phase behavior, Cd2+ is found to induce gel and liquid-crystal phase coexistence over a wide range of lipid composition. Quenching of gramicidin A' tryptophanyl fluorescence by spin-labeled phosphatidic acid or spin-labeled phosphatidylcholine is analyzed to obtain the partition coefficient, Kp, for gramicidin A' between gel and liquid-crystal phases. The value of Kp = 3 favoring the liquid-crystal phase indicates a partial clearing of the membrane-bound polypeptide from Cd2+-induced gel phase regions of the membrane.