Polymer-cushioned bilayers. II. An investigation of interaction forces and fusion using the surface forces apparatus.

We have created phospholipid bilayers supported on soft polymer "cushions" which act as deformable substrates (see accompanying paper, Wong, J. Y., J. Majewski, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith. 1999. Biophys. J. 77:1445-1457). In contrast to "solid-supported" membranes, such "soft-supported" membranes can exhibit more natural (higher) fluidity. Our bilayer system was constructed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles onto polyethylenimine (PEI)-supported Langmuir-Blodgett lipid monolayers on mica. We used the surface forces apparatus (SFA) to investigate the long-range forces, adhesion, and fusion of two DMPC bilayers both above and below their main transition temperature (T(m) approximately 24 degrees C). Above T(m), hemi-fusion activation pressures of apposing bilayers were considerably smaller than for solid-supported bilayers, e.g., directly supported on mica. After separation, the bilayers naturally re-formed after short healing times. Also, for the first time, complete fusion of two fluid (liquid crystalline) phospholipid bilayers was observed in the SFA. Below T(m) (gel state), very high pressures were needed for hemi-fusion and the healing process became very slow. The presence of the polymer cushion significantly alters the interaction potential, e.g., long-range forces as well as fusion pressures, when compared to solid-supported systems. These fluid model membranes should allow the future study of integral membrane proteins under more physiological conditions.     

Interaction of substance P with phospholipid bilayers: A neutron diffraction study.

Neutron diffraction has been used to study the membrane-bound structure of substance P (SP), a member of the tachykinin family of neuropeptides. The depth of penetration of its C-terminus in zwitterionic and anionic phospholipid bilayers was probed by specific deuteration of leucine 10, the penultimate amino acid residue. The results show that the interaction of SP with bilayers, composed of either dioleoylphosphatidylcholine (DOPC), or a 50:50 mixture of DOPC and the anionic phospholipid dioleoylphosphatidylglycerol (DOPG), takes place at two locations. One requires insertion of the peptide into the hydrophobic region of the bilayer, the other is much more peripheral. The penetration of the peptide into the hydrophobic region of the bilayer is reflected in a marked difference in the water distribution profiles. SP is seen to insert into DOPC bilayers, but a larger proportion of the peptide is found at the surface when compared to the anionic bilayers. The positions of the two label populations show only minor differences between the two types of bilayer.     

A comparative study on the extraction of membrane-bound bilirubin from erythrocyte membranes using various methods.

In this study, we used three different methods for the extraction of membrane-bound bilirubin (EMB) from erythrocyte membranes. Use of 2.5% albumin, pH 7.4, for elution of EMB resulted in only 34% of the total EMB which was estimated after the solubilization of bilirubin-loaded erythrocyte membranes (BLEMs) with 1% SDS. On the other hand, incubation of BLEMs with 38 mM sodium carbonate solution containing 5 mM EDTA, pH 11.0, yielded 77% of the total EMB. Application of Fog's reaction for the estimation of EMB directly on the BLEMs resulted in the estimation of 75% of the total EMB. These results suggest that either of the above methods, i.e. use of albumin or high pH, or direct Fog's reaction cannot estimate the total EMB correctly. Increase in ionic strength from 0.15 to 0.45 did not release any EMB from erythrocyte membranes. Therefore, the best method for the estimation of total EMB is the solubilization of membrane with 1% SDS followed by Fog's reaction method.     

Effects of physiologically relevant pressures of helium on the structure of cholesterol-containing lipid bilayers. A neutron diffraction study.

We have used neutron diffraction to study the effects of helium gas (1-210 atm) on the structure of a lipid bilayer model of neuronal plasma membranes. We have recorded diffraction patterns from hydrated multilayers of dimyristoyl lecithin and 40% (molar) cholesterol to a resolution of approximately 6.5 A and have calculated scattering amplitude density distributions as a function of pressure. We find that there are no significant changes in the scattering density profiles at 95% confidence over the range of pressures investigated, suggesting that the physiological effects of high helium pressure are unlikely to be a consequence of changes in the structures of the lipid bilayer portions of membranes.     

Phospholipase A2 hydrolysis of supported phospholipid bilayers: a neutron reflectivity and ellipsometry study

We have investigated the phospholipase A(2) catalyzed hydrolysis of supported phospholipid bilayers using neutron reflection and ellipsometry. At the hydrophilic silica-water interface, hydrolysis of phosphatidylcholine bilayers by phospholipase A(2) from Naja mossambica mossambica venom is accompanied by destruction of the bilayer at an initial rate, which is comparable for DOPC and DPPC but is doubled for POPC. The extent of bilayer destruction at 25 degrees C decreases from DOPC to POPC and is dramatically reduced for DPPC. Neutron reflectivity measurements indicate that the enzyme penetrates into the bilayers in increasing order for DOPC, POPC, and DPPC, while the amount of enzyme adsorbed at the interface is smallest for DPPC and exhibits a maximum for POPC. Penetration into the hydrophobic chain region in the bilayer is further supported by the fact that the enzyme adsorbs strongly and irreversibly to a hydrophobic monolayer of octadecyltrichlorosilane. These results are rationalized in terms of the properties of the reaction products and the effect of their accumulation in the membrane on the kinetics of enzyme catalysis.     

A theoretical study of lipid-protein interactions in bilayers.

We present a theoretical study of the effect of different types of lipid-protein interactions on the thermodynamic properties of protein-containing lipid bilayers. The basis of this work is a theoretical model for pure lipid bilayer phase transitions developed earlier by Scott. Simple assumptions on the nature of the lipid conformations near a protein strongly affect the predicted properties of the model. Here we consider (a) random protein-lipid contacts, (b) enhanced contact between protein and lipid with a number of gauche bonds, and (c) enhanced contact between protein and all-trans but tilted lipid chains. Comparison of predicted results with experimental data seems to favor point c above but, by itself point c does not work well at larger protein concentrations. The results are discussed in the light of spectroscopic data, lipid-protein (plus annular lipid) miscibility, and interprotein forces.     

Interaction of long-chain n-alcohols with fluid DOPC bilayers: a neutron diffraction study

Lamellar phases composed of fluid dioleoylphosphatidylcholine (DOPC) bilayers containing alkan-1-ols (CnOH, n = 8, 10, 14, 16, 18 is the number of carbon atoms) at CnOH : DOPC = 0.3 molar ratio and hydrated with heavy water at 20.2 ≥ D2O : DOPC ≥ 14.4 molar ratio were studied by neutron diffraction. The bilayer thickness d(L) and the bilayer surface area A(L) per DOPC at the bilayer-water interface were obtained from the lamellar repeat period d using molecular volumes of DOPC, CnOH and D2O, and the Luzatti's method. Both the d(L) and A(L) increase with the CnOH chain length n at CnOH : DOPC = 0.3 molar ratio: d(L) = (3.888 ± 0.066) + (0.016 ± 0.005)·n (in nm), A(L) = (0.6711 ± 0.0107) + (0.0012 ± 0.0008)·n (in nm2).     

Cytomorphometry. A methodologic study of preparation techniques, selection methods and sample sizes

The influence of methodologic aspects on cytomorphometric features was studied using preparations of hepatoma and/or mastocytoma cells. First, two preparation techniques (smear and oese) were compared. Second, four methods of selecting cells for cytomorphometric analysis (two conventional and two stratified methods) were tested for reproducibility. Third, heterogeneous cell populations were used to estimate the required sample size using the running coefficient of variation (CV), and the results were compared with expected (theoretical) values of the required sample size calculated using the standard error of the mean. The results showed significantly lower CVs for the smear preparation technique. The stratified methods appeared to be superior to the conventional methods for selecting cells for measurement. The experimentally assessed sample sizes were considerably lower than the corresponding theoretical calculations. These findings suggest that morphometric assessments in cytologic smears should utilize a stratified cell selection method. While experimentally assessed sample sizes are relatively small and therefore better routinely applicable, they may yield less reliable results in some cases. The need to test a sample for its reproducibility as well as its discriminatory power is emphasized.     

Interaction of benzene with bilayers. Thermal and structural studies

The thermal and structural properties of saturated phosphatidylcholine liposomes are significantly altered by benzene. Upon the addition of benzene, the liposomes first swell and then disperse into small multilamellar vesicles. At 20 degrees C these vesicles contain striations or ripples in the plane of the bilayer. Major changes in the thermal behavior of DSPC-benzene liposomes occur near mole ratios of 2:1 and 1:1. At a 2:1 mole ratio, the area under the main endothermic peak, delta Hm, essentially disappears; however, the total heat absorbed, delta Hf, remains approximately equal to that of the control. This occurs because for benzene mole fractions 0.12 less than x less than 0.50, benzene increases the apparent molar heat capacity, Cp, of the gel phase to about 1.2 kcal/(mol . deg). We interpret this increase in heat capacity to be due to an increase in the concentration of defects (or disorder) in the gel phase. At mole fractions of benzene between 0.5 and 0.9, the transition temperature decreases by 20-30 degrees C, and broad, multiple transitions are observed. From 0.5 less than or equal to x less than or equal to 0.9, the apparent molar heat capacity of the liquid-crystal phase increases to that of the defected rippled gel phase. The value of delta Hf approaches the heat of fusion for 2 mol of n-octadecane, suggesting that benzene uncouples the liquid-crystalline acyl chains. The lipids affected by benzene or "boundary lipids" have higher heat capacity than nonperturbed lipids. The apparent molar specific heat, Cp, of 1,2-distearoyl-sn-glycero-3-phosphorylcholine (and 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine) multilamellar vesicles is 0.20 +/- 0.05 kcal/(mol. deg) in the L beta', P beta, and L alpha phases. Cp fluctuates about this value in all three phases upon repeated phase transitions in the same sample. However, the value of Cp in the P beta (rippled) phase exhibits much greater fluctuations in Cp than that in the L alpha phase. We attribute these fluctuations to crystal packing defects.     

High-pressure small-angle neutron scattering (SANS) study of 1,2-dielaidoyl-sn-glycero-3-phosphocholine bilayers

Small-angle neutron scattering of the trans-unsaturated DEPC has been investigated as a function of pressure at 12, 18.6 and 35 degrees C. A pressure-induced structural phase transition from a liquid-crystalline state to a gel state is observed at the temperatures studied. The critical pressure of this transition increases with increasing temperature with a delta P/delta T value of 51 bar/C degrees. The small-angle neutron scattering results indicate that the effect of the trans double bonds in DEPC is to enhance the conformational disorder in the hydrocarbon chains. In DEPC bilayers, a pressure-induced conformational ordering process is observed not only in the liquid-crystalline phase but also in the gel phase, which indicates that conformational disorder exists in the liquid-crystalline phase as well as in the gel phase.     

Interactions between ubiquinones and phospholipid bilayers. A spin-label study.

The effect of two ubiquinones of different side chain length (Q-3; Q-9), on the fluidity of phospholipid vesicles has been investigated using stearic acid spin labels. While both oxidized quinones have a disordering effect on the lipid bilayers, the reduced forms behave in an opposite way, in that Q-3 enhances and Q-9 decreases the order of the bilayer. The ordering effect of reduced Q-3 and the attendant decreased motional freedom in the bilayer might be the result of the insertion and stacking of the quinone between the phospholipid molecules in the bilayer. Such insertion might be related to the incapability of short-chain quinones in restoring NADH oxidation in Q-depleted mitochondria.     

Phase transitions in monoglyceride bilayers. A light scattering study.

Thermotropic phase transitions in single planar bilayers of glycerol mono-oleate have been investigated using quasi-elastic light scattering from thermally excited membrane fluctuations. In certain cases both spectroscopic and intensity information were derived from the observations. For solvent-free bilayers transitional changes were observed in several membrane parameters: in tension, viscosity and thickness, in a combination of lipid orientational order parameter and dielectric anisotropy, and in the lateral compression modulus. These changes, particularly those in membrane thickness and in the anisotropy/order combination, were clearly indicative of a chain-melting transition in the lipid molecules. The chain-melting transition temperature was identified as 16.6 +/- 0.03 degrees C (delta T 1/2 = 1.5 degrees C). The other changes tended to cluster around 12.5 and 16.6 degrees C, suggesting that a two-stage transition was involved. Analysis of pretransitional fluctuations in membrane viscosity, based on a Landau approach, suggested that at the transition the membrane was close to a critical point (T = 12.7 degrees C). Less information was accessible for membranes containing n-decane within their structure. In this case, the change in membrane tension was much smaller than in the solvent-free case and the transition was considerably broadened. These effects accord with an increase in 'interactive volume' within the bilayer due to solvent inclusion.     

Multipoint gene mapping using seriation. I. General methods.

Initial and accurate inference of locus order and estimates of interlocus distances and interference can be obtained using seriation techniques. The analysis requires a matrix of recombination values that can be estimated by standard pairwise linkage analysis. This allows combination of results from individual investigators without reanalysis of basic pedigree material. Seriation can be performed without the use of a computer.     

Nanoscale structural and mechanical effects of beta-amyloid (1-42) on polymer cushioned membranes: a combined study by neutron reflectometry and AFM Force Spectroscopy

The interaction of beta-amyloid peptides with lipid membranes is widely studied as trigger agents in Alzheimer's disease. Their mechanism of action at the molecular level is unknown and their interaction with the neural membrane is crucial to elucidate the onset of the disease. In this study we have investigated the interaction of water soluble forms of beta-amyloid Aβ(1-42) with lipid bilayers supported by polymer cushion. A reproducible protocol for the preparation of a supported phospholipid membrane with composition mimicking the neural membrane and in physiological condition (PBS buffer, pH=7.4) was refined by neutron reflectivity. The change in structure and local mechanical properties of the membrane in the presence of Aβ(1-42) was investigated by neutron reflectivity and Atomic Force Microscopy (AFM) Force Spectroscopy. Neutron reflectivity evidenced that Aβ(1-42) interacts strongly with the supported membrane, causing a change in the scattering length density profile of the lipid bilayer, and penetrates into the membrane. Concomitantly, the local mechanical properties of the bilayer are deeply modified by the interaction with the peptide as seen by AFM Force Spectroscopy. These results may be of great importance for the onset of the Alzheimer's disease, since a simultaneous change in the structural and mechanical properties of the lipid matrix could influence all membrane based signal cascades.     

A comparative study on interactions of alpha-aminoisobutyric acid containing antibiotic peptides, trichopolyn I and hypelcin A with phosphatidylcholine bilayers.

Interactions of alpha-aminoisobutyric acid containing antibiotic peptides, trichopolyn I and hypelcin A with phosphatidylcholine bilayers were investigated to obtain some basic information on their bioactive mechanisms. Trichopolyn I as well as hypelcin A induced the leakage of a fluorescent dye, calcein, entrapped in sonicated egg yolk L-alpha-phosphatidylcholine vesicles. A quantitative analysis revealed that both the binding affinity and the 'membrane-perturbing activity' of trichopolyn I to the vesicles are about one-third of those of hypelcin A. The conformations and the orientations of the peptide and lipid molecules in the membranes were studied using polarized Fourier transform infrared-attenuated total reflection spectroscopy, circular dichroism, and differential scanning calorimetry. In phosphatidylcholine bilayers, both peptides mainly conformed to helical structures irrespective of the membrane physical state (gel or liquid-crystalline). The helix axes, penetrating the hydrophobic region of the bilayers, were oriented neither parallel nor perpendicular to the membrane normal. The disruption in the lipid packing induced by the peptide insertion seems to be responsible for the leakage by these peptides.     

The structure of polyunsaturated lipid bilayers important for rhodopsin function: a neutron diffraction study

The structure of oriented 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine bilayers with perdeuterated stearoyl- or docosahexaenoyl hydrocarbon chains was investigated by neutron diffraction. Experiments were conducted at two different relative humidities, 66 and 86%. At both humidities we observed that the polyunsaturated docosahexaenoyl chain has a preference to reside near the lipid water interface. That leaves voids in the bilayer center that are occupied by saturated stearoyl chain segments. This uneven distribution of saturated- and polyunsaturated chain densities is likely to result in membrane elastic stress that modulates function of integral receptor proteins like rhodopsin.     

A solvent model for simulations of peptides in bilayers. I. Membrane-promoting alpha-helix formation

We describe an efficient solvation model for proteins. In this model atomic solvation parameters imitating the hydrocarbon core of a membrane, water, and weak polar solvent (octanol) were developed. An optimal number of solvation parameters was chosen based on analysis of atomic hydrophobicities and fitting experimental free energies of gas-cyclohexane, gas-water, and octanol-water transfer for amino acids. The solvation energy term incorporated into the ECEPP/2 potential energy function was tested in Monte Carlo simulations of a number of small peptides with known energies of bilayer-water and octanol-water transfer. The calculated properties were shown to agree reasonably well with the experimental data. Furthermore, the solvation model was used to assess membrane-promoting alpha-helix formation. To accomplish this, all-atom models of 20-residue homopolypeptides-poly-Leu, poly-Val, poly-Ile, and poly-Gly in initial random coil conformation-were subjected to nonrestrained Monte Carlo conformational search in vacuo and with the solvation terms mimicking the water and hydrophobic parts of the bilayer. All the peptides demonstrated their largest helix-forming tendencies in a nonpolar environment, where the lowest-energy conformers of poly-Leu, Val, Ile revealed 100, 95, and 80% of alpha-helical content, respectively. Energetic and conformational properties of Gly in all environments were shown to be different from those observed for residues with hydrophobic side chains. Applications of the solvation model to simulations of peptides and proteins in the presence of membrane, along with limitations of the approach, are discussed.