Infrared studies of fully hydrated unsaturated phosphatidylserine bilayers. Effect of Li+ and Ca2+

Infrared spectroscopy has been used to characterize the thermal-phase behavior of fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) as well as their interaction with Li+ and Ca2+. The order-disorder transition of POPS-NH4+ is at 17 degrees C; in the presence of Li+ a POPS-Li+ complex is formed, and the transition temperature of this complex is 40 degrees C. DOPS-NH4+ has an order-disorder transition at -11 degrees C, and unlike POPS the addition of Li+ has no effect on the thermal behavior of DOPS-NH4+. This indicates that the binding of Li+ to DOPS is negligible or very weak. Li+ binds to the phosphate and carboxylate groups of POPS, and as a result these groups lose their water of hydration. Li+ binding induces a conformational change, probably in the glycerol backbone of POPS; however, the conformation of the two P-O ester bonds remains gauche-gauche as in POPS-NH4+. Both POPS and DOPS form crystalline complexes with Ca2+. As a result of Ca2+ binding to the phosphate, this group loses its water of hydration and there is a conformational change in the P-O ester bonds from gauche-gauche to antiplanar-antiplanar. In contrast to the POPS-Li+ complex, the carboxylate group remains hydrated in the Ca2+ complexes. Furthermore, in these PS-Ca2+ complexes a new hydrogen bond is formed between one of the ester C=O groups and probably water. Such a situation is not found in the NH4+ and Li+ salts of phosphatidylserine.     

Structure and interactions of fully hydrated dioleoylphosphatidylcholine bilayers.

This study focuses on dioleoylphosphatidylcholine (DOPC) bilayers near full hydration. Volumetric data and high-resolution synchrotron x-ray data are used in a method that compares DOPC with well determined gel phase dipalmitoylphosphatidylcholine (DPPC). The key structural quantity obtained is fully hydrated area/lipid A0 = 72.2 +/- 1.1 A2 at 30 degrees C, from which other quantities such as thickness of the bilayer are obtained. Data for samples over osmotic pressures from 0 to 56 atmospheres give an estimate for the area compressibility of KA = 188 dyn/cm. Obtaining the continuous scattering transform and electron density profiles requires correction for liquid crystal fluctuations. Quantitation of these fluctuations opens an experimental window on the fluctuation pressure, the primary repulsive interaction near full hydration. The fluctuation pressure decays exponentially with water spacing, in agreement with analytical results for soft confinement. However, the ratio of decay length lambda(fl) = 5.8 A to hydration pressure decay length lambda = 2.2 A is significantly larger than the value of 2 predicted by analytical theory and close to the ratio obtained in recent simulations. We also obtain the traditional osmotic pressure versus water spacing data. Our analysis of these data shows that estimates of the Hamaker parameter H and the bending modulus Kc are strongly coupled.     

Infrared and 31P-NMR studies of the effect of Li+ and Ca2+ on phosphatidylserines

Infrared and 31P-NMR spectra of solid samples of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) have been recorded. Comparison of the spectra of the Na+ salts of these phospholipids with those of complexes formed with Li+ and Ca2+ ions allows the characterization of conformational changes induced by complexation with Li+ and Ca2+. Ca2+ forms tight, crystalline complexes with these phosphatidylserines (PS), irrespective of the degree of unsaturation in the hydrocarbon chains. In these PS-Ca2+ complexes the torsion angles of the two P-O ester bonds exhibit the antiplanar-antiplanar conformation which is significantly different from the standard gauche-gauche conformation commonly found in phosphodiesters. In contrast, complexation with Li+ does not induce this conformational change in the phosphodiester group. It is shown that the degree of unsaturation in the hydrocarbon chains, and related to it, the cross-sectional area of the phospholipid or the surface charge density, determine the affinity of the phosphatidylserine for the metal ion. In general, the affinity of phosphatidylserines for both Li+ and Ca2+ decreases with increasing unsaturation in the hydrocarbon chains or decreasing surface charge density; it is in the order DMPS greater than POPS greater than DOPS.     

X-ray structure determination of fully hydrated L alpha phase dipalmitoylphosphatidylcholine bilayers.

Bilayer form factors obtained from x-ray scattering data taken with high instrumental resolution are reported for multilamellar vesicles of L alpha phase lipid bilayers of dipalmitoylphosphatidylcholine at 50 degrees C under varying osmotic pressure. Artifacts in the magnitudes of the form factors due to liquid crystalline fluctuations have been eliminated by using modified Caillé theory. The Caillé fluctuation parameter eta 1 increases systematically with increasing lamellar D spacing and this explains why some higher order peaks are unobservable for the larger D spacings. The corrected form factors fall on one smooth continuous transform F(q); this shows that the bilayer does not change shape as D decreases from 67.2 A (fully hydrated) to 60.9 A. The distance between headgroup peaks is obtained from Fourier reconstruction of samples with four orders of diffraction and from electron density models that use 38 independent form factors. By combining these results with previous gel phase results, area AF per lipid molecule and other structural quantities are obtained for the fluid L alpha phase. Comparison with results that we derived from previous neutron diffraction data is excellent, and we conclude from diffraction studies that AF = 62.9 +/- 1.3 A2, which is in excellent agreement with a previous estimate from NMR data.     

Molecular studies of the gel to liquid-crystalline phase transition for fully hydrated DPPC and DPPE bilayers

Molecular dynamics simulations were used for a comprehensive study of the structural properties of saturated lipid bilayers, DPPC and DPPE, near the main phase transition. Though the chemical structure of DPPC and DPPE are largely similar (they only differ in the choline and ethanolamine groups), their transformation process from a gel to a liquid-crystalline state is contrasting. For DPPC, three distinct structures can be identified relative to the melting temperature (Tm): below Tm with "mixed" domains consisting of lipids that are tilted with partial overlap of the lipid tails between leaflet; near Tm with a slight increase in the average area per lipid, resulting in a rearrangement of the lipid tails and an increase in the bilayer thickness; and above Tm with unhindered lipid tails in random motion resulting in an increase in %gauche formed and increase in the level of interdigitation between lipid leaflets. For DPPE, the structures identified were below Tm with "ordered" domains consisting of slightly tilted lipid tails and non-overlapping lipid tails between leaflets, near Tm with minimal rearrangement of the lipids as the bilayer thickness reduces slightly with increasing temperature, and above Tm with unhindered lipid tails as that for DPPC. For DPPE, most of the lipid tails do not overlap as observed to DPPC, which is due to the tight packing of the DPPE molecules. The non-overlapping behavior of DPPE above Tm is confirmed from the density profile of the terminal carbon atoms in each leaflet, which shows a narrow distribution near the center of the bilayer core. This study also demonstrates that atomistic simulations are capable of capturing the phase transition behavior of lipid bilayers, providing a rich set of molecular and structural information at and near the transition state.     

Molecular studies of the gel to liquid-crystalline phase transition for fully hydrated DPPC and DPPE bilayers

Molecular dynamics simulations were used for a comprehensive study of the structural properties of saturated lipid bilayers, DPPC and DPPE, near the main phase transition. Though the chemical structure of DPPC and DPPE are largely similar (they only differ in the choline and ethanolamine groups), their transformation process from a gel to a liquid-crystalline state is contrasting. For DPPC, three distinct structures can be identified relative to the melting temperature (T_m): below T_m with “mixed” domains consisting of lipids that are tilted with partial overlap of the lipid tails between leaflet; near T_m with a slight increase in the average area per lipid, resulting in a rearrangement of the lipid tails and an increase in the bilayer thickness; and above T_m with unhindered lipid tails in random motion resulting in an increase in %gauche formed and increase in the level of interdigitation between lipid leaflets. For DPPE, the structures identified were below T_m with “ordered” domains consisting of slightly tilted lipid tails and non-overlapping lipid tails between leaflets, near T_m with minimal rearrangement of the lipids as the bilayer thickness reduces slightly with increasing temperature, and above T_m with unhindered lipid tails as that for DPPC. For DPPE, most of the lipid tails do not overlap as observed to DPPC, which is due to the tight packing of the DPPE molecules. The non-overlapping behavior of DPPE above T_m is confirmed from the density profile of the terminal carbon atoms in each leaflet, which shows a narrow distribution near the center of the bilayer core. This study also demonstrates that atomistic simulations are capable of capturing the phase transition behavior of lipid bilayers, providing a rich set of molecular and structural information at and near the transition state.     

Effect of hypoglycemic sulfonylureas on Ca2+ fluxes across lipid bilayers

Black lipid membranes and liposomes loaded with Ca2+ or 5,6-carboxyfluorescein were used for exploring the mechanism of action of insulin-releasing sulfonylureas. Unlike the Ca2+/H+ exchanging ionophore A-23187, tolbutamide did not stimulate the net efflux of Ca2+ from the liposomes. Glibenclamide caused a sustained release of Ca2+, but this effect could be attributed to labilization of the liposomal membrane as indicated by a quantitatively similar loss of the stability marker 5,6-carboxyfluorescein. Unlike the neutral ionophore nonactin or the channel forming quasi-ionophore gramicidin A, the sulfonylureas did not alter the conductance of black lipid membranes in medium containing Na+, K+, Ca2+, Mg2+, and Cl-. It is concluded that the sulfonylureas tested lack ionophore properties but that glibenclamide can labilize membranes.     

Structure of fully hydrated fluid phase lipid bilayers with monounsaturated chains

Quantitative structures are obtained at 30 degrees C for the fully hydrated fluid phases of palmitoyloleoylphosphatidylcholine (POPC), with a double bond on the sn-2 hydrocarbon chain, and for dierucoylphosphatidylcholine (di22:1PC), with a double bond on each hydrocarbon chain. The form factors F(qz) for both lipids are obtained using a combination of three methods. (1) Volumetric measurements provide F(0). (2) X-ray scattering from extruded unilamellar vesicles provides /F(qz)/ for low q(z). (3) Diffuse X-ray scattering from oriented stacks of bilayers provides /F(qz)/ for high q(z). Also, data using method (2) are added to our recent data for dioleoylphosphatidylcholine (DOPC) using methods (1) and (3); the new DOPC data agree very well with the recent data and with (4) our older data obtained using a liquid crystallographic X-ray method. We used hybrid electron density models to obtain structural results from these form factors. The result for area per lipid (A) for DOPC 72.4 +/- 0.5 A(2) agrees well with our earlier publications, and we find A = 69.3 +/- 0.5 A2 for di22:1PC and A = 68.3 +/- 1.5 A2 for POPC. We obtain the values for five different average thicknesses: hydrophobic, steric, head-head, phosphate-phosphate and Luzzati. Comparison of the results for these three lipids and for our recent dimyristoylphosphatidylcholine (DMPC) determination provides quantitative measures of the effect of unsaturation on bilayer structure. Our results suggest that lipids with one monounsaturated chain have quantitative bilayer structures closer to lipids with two monounsaturated chains than to lipids with two completely saturated chains.     

Closer look at structure of fully hydrated fluid phase DPPC bilayers

X-ray data are presented for the benchmark dipalmitoylphosphatidylcholine lipid bilayer in the most biologically relevant state in which the bilayers are fully hydrated and in the fluid (liquid-crystalline) phase. Form factors F(q(z)) are obtained from a combination of two sample preparations, oriented stacks of bilayers for q(z) extending to 0.85 A(-1) and unilamellar vesicles for smaller q(z). Modeling obtains the electron density profile and values for the area per molecule, for the locations of the component groups, and for the different types of thicknesses of the bilayer, such as the hydrocarbon thickness and the steric thickness.     

Area per molecule and distribution of water in fully hydrated dilauroylphosphatidylethanolamine bilayers

The area per lipid molecule for fully hydrated dilauroylphosphatidylethanolamine (DLPE) has been obtained in both the gel and liquid-crystalline states by combining wide-angle X-ray diffraction, electron density profiles, and previously published dilatometry results [Wilkinson, D. A., & Nagle, J. F. (1981) Biochemistry 20, 187-192]. The molecular area increases from 41.0 +/- 0.2 to 49.1 +/- 1.2 A2 upon melting from the gel to liquid-crystalline phase. The thickness of the bilayer, as measured from the electron density profiles, decreases about 4 A upon melting, from 45.2 +/- 0.3 to 41.0 +/- 0.6 A. A somewhat unexpected result is that the fluid layer between fully hydrated bilayers is the same in both gel and liquid-crystalline phases and is only about 5 A thick. From these data, plus the volume of the anhydrous DLPE molecule, it is possible to determine the number of water molecules per lipid and their approximate distribution relative to the lipid molecule. Our analysis shows that there are about 7 and 9 waters per DLPE molecule in the gel and liquid-crystalline phases, respectively. About half of the water is located in the fluid space between adjacent bilayers, and the remaining waters are intercalated into the bilayer, presumably in the head group region. There are significantly fewer water molecules in the fluid spaces between DLPE bilayers than in the fluid spaces in gel- or liquid-crystalline-phase phosphatidylcholine bilayers. This small fluid space in PE bilayers could arise from interbilayer hydrogen bond formation through the water molecules or electrostatic interactions between the amine and phosphate groups on apposing bilayers.     

Measurement of chain tilt angle in fully hydrated bilayers of gel phase lecithins.

The tilt angle theta tilt of the hydrocarbon chains has been determined for fully hydrated gel phase of a series of saturated lecithins. Oriented samples were prepared on glass substrates and hydrated with supersaturated water vapor. Evidence for full hydration was the same intensity pattern of the low angle lamellar peaks and the same lamellar repeat D as unoriented multilamellar vesicles. Tilting the sample permitted observation of all the wide angle arcs necessary to verify the theoretical diffraction pattern corresponding to tilting of the chains towards nearest neighbors. The length of the scattering unit corresponds to two hydrocarbon chains, requiring each bilayer to scatter coherently rather than each monolayer. For DPPC, theta tilt was determined to be 32.0 +/- 0.5 degrees at 19 degrees C, slightly larger than previous direct determinations and considerably smaller than the value required by recent gravimetric measurements. This new value allows more accurate determinations of a variety of structural parameters, such as area per lipid molecule, A = 47.2 +/- 0.5 A2, and number of water molecules of hydration, nw = 11.8 +/- 0.7. As the chain length n of the lipids was increased from 16 to 20 carbons, the parameters A and nw remained constant, suggesting that the headgroup packing is at its excluded volume limit for this range. However, theta tilt increased by 3 degrees and the chain area Ac decreased by 0.5 A2. This behavior is explained in terms of a competition between a bulk free energy term and a finite or end effect term.     

Effect of Ca2+ on thermotropic properties of saturated phosphatidylcholine liposomes

The effect of various concentrations of calcium ion (Ca²⁺) on dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and mixed DPPC/DSPC (1:1) liposomes was studied by differential scanning calorimetry. Ca²⁺ concentrations of 10 mM and above split the main transition peak of DPPC into two distinguishable components, and, at 30 mM and above, also resulted in the disappearance of a pre-transition peak. The effect of Ca²⁺ on DSPC liposomes was even more dramatic in that it induced a more definitive split in the main transition peak and caused the loss of the pretransition peak at only 10 mM concentration. The thermograms of the DPPC/DSPC mixed liposomes were unaltered in the presence of Ca²⁺, even at a concentration of 50 mM. Whether or not Ca²⁺ is able to alter thermograms of phosphatidylcholine liposomes appears to be dependent on the degree of molecular order of the bilayer prior to interaction with Ca²⁺.     

The translocation of Ca2+ across phospholipid bilayers induced by a synthetic neutral Ca2+ -ionophore.

The effect of a neutral synthetic Ca2+ -ligand, which induces selective Ca2+ transport in electrodialysis experiments in bulk membranes, on the Ca2+ permeability of phospholipid bilayers has been investigated. The ligand is able to promote the transport of Ca2+ across synthetic phospholipid bilayers and can therefore be classified as a Ca2+ -ionophore. Its activity is enhanced by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The efficiency of the neutral carrier-mediated Ca2+ transport is rather low as compared with that of the charged Ca2+ -ionophore X537A. The Ca2+ selectivity of the nuetral ionophore is decreased by its incorporation in the low dielectric ambient of the phospholipid bilayer.     

Effect of encapsulated Ca2+ on the surface properties of curved phosphatidylcholine bilayers

In this paper, the influence of Ca2+ on the adsorption properties of 1,8-anilinonaphthalenesulfonate (ANS) and analogous probes to sonicated vesicles of phosphatidylcholine was studied by means of spectrofluorometry. The fluorescence of ANS added to the vesicle dispersion increases with the Ca2+ concentration in the inner media but remains constant when Ca2+ concentration is changed in the outside solution. However, the fluorescence decreases when large anions such as ClO4- are present in the external solution. Ca2+ inside large liposomes promotes a similar behaviour to that found in sonicated vesicles when they are osmotically contracted in hypertonic media. The results can be interpreted in terms of Ca2+ adsorption on the inner interface and a cooperative interaction between the monolayers.     

Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade

The interaction of Ca2+ and Ba2+ with a Ca2+-activated K+ channel from rabbit skeletal muscle membranes is studied in planar lipid bilayers. At [Ca2+] greater than or equal to 100 microM in the cis side (the side to which the vesicles are added) and at positive voltages, the channel kinetics consisted of bursts of activity interrupted by long periods of quiescence. We found that the reciprocal of the mean burst time increases linearly with [Ca2+], whereas the mean time for the quiescent (closed) periods is independent of [Ca2+]. The number of quiescent periods is reduced by increasing [K+]. Micromolar amounts of cis Ba2+ do not activate the channel, but induce similar "slow" closings. Also, in this case, the mean burst time is inversely proportional to the [Ba2+] and the mean closed time is independent of [Ba2+]. Raising [K+] either symmetrically or only in the trans side relieved the Ba2+ effect. trans Ba2+ also induces changes in the slow kinetics, but in millimolar amounts. These results suggest that the quiescent periods correspond to a channel blocked by a Ba ion. The voltage dependence of the cis blockade indicates that the Ba2+ binding site is past the middle of the membrane field. The similarities in the slow kinetics induced by Ca2+ and Ba2+ suggest that Ca2+ blocks the channel by binding to the same site. However, binding of Ca2+ to the site is 10(5)- fold weaker.     

Phospholamban forms Ca2+-selective channels in lipid bilayers

Phospholamban is the major membrane protein of the heart phosphorylated in response to beta-adrenergic stimulation. A role for phospholamban in the control of Ca2+ transport by the sarcoplasmic reticulum has been postulated, but the mechanism is incompletely understood. Structural characterization of the purified protein suggests that it is capable of forming a membrane-spanning pore (Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). The experiments described here tested the hypothesis that canine cardiac phospholamban, isolated in the fully dephosphorylated state, forms ion channels in lipid bilayers. Phospholamban purified by two different methods formed channels that were permeable to cations, exhibited spontaneous openings and closings, and were selective for Ca2+ over K+. Dihydropyridine drugs and ryanodine did not affect channel activity. The putative membrane-spanning portion of the molecule, residues 26-52, also formed channels in the bilayer. The putative regulatory portion of the molecule, residues 2-25, did not. The results suggest that phospholamban may regulate sarcoplasmic reticulum Ca2+ flux by acting as a Ca2+ channel.     

Ca2+-induced fusion of large unilamellar phosphatidylserine/cholesterol vesicles

The effect of cholesterol on the Ca2+-induced aggregation and fusion of large unilamellar phosphatidylserine (PS) vesicles has been investigated. Mixing of aqueous vesicle contents was followed continuously with the Tb/dipicolinate assay, while the dissociation of pre-encapsulated Tb/dipicolinate complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure PS or PS/cholesterol mixtures at molar ratios of 4:1, 2:1 and 1:1 were employed at three different lipid concentrations, each at four different Ca2+ concentrations. The results could be well simulated in terms of a mass-action kinetic model, providing separately the rate constants of vesicle aggregation, c11, and of the fusion reaction itself, f11. In the analyses the possibility of deaggregation of aggregated vesicles was considered explicitly. Values of both c11 and f11 increase steeply with the Ca2+ concentration increasing from 2 to 5 mM. With increasing cholesterol content of the vesicles the value of c11 decreases, while the rate of the actual fusion reaction, f11, increases. Remarkably, the effect of cholesterol on both aggregation and fusion is quite moderate. The presence of cholesterol in the vesicle bilayer does not affect the leakage of vesicle contents during fusion.     

Combined monte carlo and molecular dynamics simulation of fully hydrated dioleyl and palmitoyl-oleyl phosphatidylcholine lipid bilayers

We have applied a new equilibration procedure for the atomic level simulation of a hydrated lipid bilayer to hydrated bilayers of dioleyl-phosphatidylcholine (DOPC) and palmitoyl-oleyl phosphatidylcholine (POPC). The procedure consists of alternating molecular dynamics trajectory calculations in a constant surface tension and temperature ensemble with configurational bias Monte Carlo moves to different regions of the configuration space of the bilayer in a constant volume and temperature ensemble. The procedure is applied to bilayers of 128 molecules of POPC with 4628 water molecules, and 128 molecules of DOPC with 4825 water molecules. Progress toward equilibration is almost three times as fast in central processing unit (CPU) time compared with a purely molecular dynamics (MD) simulation. Equilibration is complete, as judged by the lack of energy drift in 200-ps runs of continuous MD. After the equilibrium state was reached, as determined by agreement between the simulation volume per lipid molecule with experiment, continuous MD was run in an ensemble in which the lateral area was restrained to fluctuate about a mean value and a pressure of 1 atm applied normal to the bilayer surface. Three separate continuous MD runs, 200 ps in duration each, separated by 10,000 CBMC steps, were carried out for each system. Properties of the systems were calculated and averaged over the three separate runs. Results of the simulations are presented and compared with experimental data and with other recent simulations of POPC and DOPC. Analysis of the hydration environment in the headgroups supports a mechanism by which unsaturation contributes to reduced transition temperatures. In this view, the relatively horizontal orientation of the unsaturated bond increases the area per lipid, resulting in increased water penetration between the headgroups. As a result the headgroup-headgroup interactions are attenuated and shielded, and this contributes to the lowered transition temperature.