A theory of the electric field-induced phase transition of phospholipid bilayers.

Improving the statistical mechanical model of Jacobs et al. (Jacobs, R.E., Hudson, B. and Andersen, H.C. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3993--3997) we have constructed a model which describes not only the temperature but also the external field dependence of the membrane structure of phospholipid bilayers. In addition to the interactions between head groups, between hydrocarbon chains, and the internal conformational energy of the chains (which were considered in Jacobs' model), our model includes the energy of deformation and the field energy as well. By the aid of this model we can explain the phenomenon of dielectric breakdown, the non-linearity of current-voltage characteristics, and the mechanism of membrane elasticity. The free energy of the membrane, the average number of the gauche conformations in the hydrocarbon interior and at the membrane surface, gauche distribution along the chain, the membrane thickness, area and volume are calculated at different temperatures and voltages. The calculation also gives the temperature dependence of Young's modulus and that of the linear thermal expansion coefficient.     

A theoretical model of the temperature- and pressure-induced phase transition of phospholipid bilayers

A statistical thermodynamic model of phospholipid bilayers is developed. In the model, a new concept of a closely packed system is applied, i.e., a system of hard cylinders of equal radii, the radius being a function of the average number of gauche rotations in a hydrocarbon chain. Using this concept of a closely packed system, reasonable values are obtained for the change in specific volume at the order-disorder transition of lecithin bilayers. In addition to interactions between the lipid matrix and water molecules, between the head groups themselves and between hydrocarbon chains, as well as the intramolecular energy associated with chain conformation, the Hamiltonian of the membrane also includes the energy of the pressure field. Thus, the phase transition of phospholipid membranes induced not only by temperature hut also by hydrostatic pressure is described by this model simultaneously. In accordance with the experimental results, a linear relationship is obtained between the phase transition temperature and phase transition pressure. The other calculated phase transition properties of lecithin homologues. e.g., changes in enthalpy, surface area. thickness and gauche number per chain are in agreement with the available experimental data. The ratio of kink to interstitial conduction of bilayers is also estimated.     

Chain ordering in liquid crystals: II. Structure of bilayer membranes

The ordering of the hydrocarbon chain interior of bilayer membranes has been calculated using the molecular field approximation developed in previous work on liquid crystals. Different statistical averages are evaluated by exact summation over all conformations of a single chain in the field due to neighboring molecules. The internal energy of each conformation, as well as contributions arising from interaction with the molecular field and from a lateral pressure on the chain have been included.The results describe properties of both lipid monolayers and bilayers. For monolayers, the calculated pressure-area relationships are in good agreement with experimental observations. The order parameter for hydrocarbon chains in bilayers (or monolayers) as a function of temperature, lateral pressure and position along the chain, is shown and compared with the available NMR data. Combining the results of calculation and NMR measurements we obtain the value for intrinsic lateral pressure within bilayer membranes, in excellent agreement with direct measurements on surface monolayers.The calculation also gives average length of hydrocarbon chains, thermal expansion coefficient and fraction of bonds in gauche conformations. The effect of cholesterol and proteins within the bilayer is qualitatively described, and the contribution of the bilayer interior to membrane elasticity is determined.     

Cooperative unit size in the gel-liquid crytalline phase transition of dipalmitoyl phosphatidylcholine-water multilayers: An estimate from raman spectroscopy

The temperature dependence of the Raman spectral transitions assigned to the acyl chain C-C stretching modes of dipalmitoyl phosphatidylcholine was determined for the gel, phase transition and liquid crystalline states of the lipid multilayers. The van't Hoff enthalpy differences $\text{ΔH}{}_{\mathrm{<mtext>VH</mtext>}}$ between trans and gauche rotational isomers were obtained from the Raman spectral data for the temperature region characteristics of each bilayer state. An average size for the cooperative unit undergoing the chain melting process during the phase transition was estimated from the ratio of the appropriate van't Hoff enthalpy to an adjusted calorimetric enthalpy.     

Action of surfactants on egg lecithin liposomes

The lytic action of several homologous series of surfactants including N-acyl derivatives of the Na-salt of amino acids on the egg lecithin multilamellar liposomes was examined. The affinity for the lipid membrane and the solubilising capacity of the agents were estimated. The contribution of a CH₂ group and that of the polar head group of surfactants to the free energy of the agent's binding to the membrane were evaluated. The results obtained indicate that the contribution of a CH₂ group to the free binding energy depends on the nature of the surfactants' head group. This dependence is attributed to either various localisation of the agent's molecules in the lipid bilayer or to different properties of the agent's hydrocarbon tails. The contributions of the head groups of the surfactants are assumed to reflect the affinity of these head groups for the lecithin polar head group at the membrane interface. The results obtained indicate some degree of specificity involved in the interactions of the head groups.     

Electric Field-Induced Changes in Lipids Investigated by Modulated Excitation FTIR Spectroscopy

The effect of electric fields on dry oriented multibilayers of dimyristoylphosphatidylcholine (DMPC) was investigated by transmission Fourier transform infrared electric field modulated excitation (E-ME) spectroscopy. A periodic rectangular electric potential (0–150 V, 1.25 Hz, 28.4°C ± 0.2°C) was applied across the sample. To discriminate electric field-induced effects from possible temperature-induced effects resulting from a current flow (<1 pA) across the sample, corresponding temperature-modulated excitation (T-ME) measurements within the temperature uncertainty limits of ±0.2°C at 28.4°C were performed. T-ME induced reversible gauche defects in the hydrocarbon chains, whereas E-ME resulted in reversible compression of dry DMPC bilayers. Periodic variation of the tilt angle of the hydrocarbon chains is suggested. The degree of absorbance modulation in the CH-stretching region was found to be in the order of 1:700, corresponding to a variation of the bilayer thickness of Δz = 0.0054 nm. Using a series connection of capacitors as equivalent circuit of the cell resulted in E = (1.2 ± 0.7) × 10⁷ V/m for the electric field in DMPC. Young's elasticity modulus of DMPC could be calculated to be E = 2.2 × 10⁶ Pa ± 1.8 × 10⁶ Pa, which is in good agreement with published data obtained by electric field-dependent capacitance measurements.     

Molecular conformational changes in the triglyceride and methyl ester of stearic acid as studied by temperature dependent carbon-13 chemical shifts

The temperature dependence of the ¹³C chemical shifts in tristearin and methyl stearate has been investigated in both the melt and solution phases. Intramolecular conformational changes dominate the observed behaviour and there is little evidence for intermolecular interactions even in the melt phase of tristearin. The terminal methyl carbons of methyl stearate and tristearin and the C17, C2, and glyceryl carbons of tristearin exhibit a temperature dependence consistent with there being only two rotamers of significant population. The calculated enthalpy difference between the terminal methyl and C17 rotamers is of the same order of magnitude as would be expected for tt and tg^± rotamers in hydrocarbon chains. For the glyceryl carbons the rotamer energy difference is very large and only one of the rotamers is significantly populated at room temperature. The remaining carbons (C16, C17, C15, C6, C4 and C3) show a general drift to high fields with increasing temperature but the observed temperature dependence requires the existence of more than two rotamers. In the absence of an acceptable mechanism for the chemical shifts of ¹³C nuclei in hydrocarbon chains it is not possible to use this data to investigate conformational changes along the hydrocarbon chain.     

Potentiometric estimation of charges in barnacle muscle fibers under internal perfusion

Summary A statistical mechanical treatment of the fluidity of lipid hydrocarbon chains in phospholipid bilayers is presented, which explicitly takes some account of interchain steric restrictions. With an effective energy separation of 750 cal/mole betweengauche andtrans conformations, it is found possible to account both for the chain dependence of the entropy and enthalpy change at the liquid crystalline transition of saturated lecithins, and also for intensity data in the laser raman spectra of dipalmitoyl lecithin. The method is used to calculate conformational probabilities in the lipid chains, in particular those for 2g 1 kinks. The calculated kink concentrations are found to be in agreement with the molecular permeability theory of H. Träuble (J Membrane Biol. 4:193, 1971).     

Intrinsic protein-lipid interactions. Infrared spectroscopic studies of gramicidin A, bacteriorhodopsin and Ca2+-ATPase in biomembranes and reconstituted systems

Infrared spectroscopy has been applied to the study of a number of aqueous systems of model and natural biomembranes. The absorption bands arising from water and buffer solutions were eliminated by means of an infrared spectrometer data station. Spectra were examined using H₂O and ²H₂O aqueous buffer systems. Pure lecithin-water systems, and various model biomembranes containing cholesterol, gramicidin A, bacteriorhodopsin or Ca²⁺-ATPase were examined. The infrared spectra of the reconstituted biomembranes were compared with those of the corresponding natural biomembranes, i.e. the purple membrane of Halobacterium halobium and also sarcoplasmic reticulum membranes, respectively.Changes in lipid chain conformation caused by the various intrinsic molecules incorporated within the model lipid bilayer structures were monitored by studying the shifts in frequency (cm^?1) of the CH₂ symmetric and asymmetric absorption bands arising from the lipid chains. The effect of gramicidin A and also the intrinsic proteins, as indicated by the shift of band frequencies, are quite different from that of cholesterol at temperatures above the main lipid transition temperature t_c. Cholesterol causes a reduction in gauche isomers which increases with concentration of cholesterol within the lipid bilayer. Whilst gramicidin A and the intrinsic proteins at low concentration cause a reduction of gauche isomers, at higher concentrations of these molecules, however, there is little difference in gauche isomer content when the intrinsic molecule is present compared with that of the fluid lipid alone. These results are considered and compared with previously published studies using deuterium nuclear magnetic resonance spectroscopy on similar model biomembrane systems. Below the lipid t_c value, all the intrinsic molecules produce an increase in gauche isomers presumably by disturbing the lipid chain packing in the crystalline lipid arrangement.Information about the polypeptide structure within gramicidin A. the reconstituted proteins and also the proteins in the natural biomembranes was obtained by examining the region of the infrared spectrum between 1600 and 1700 cm^?1 associated with the amide I and amide II bands. An examination of the infrared band frequencies of the different systems in this region leads to the conclusions: (1) that gramicidin A within a phospholipid bilayer structure probably has a single helix rather than a double helix structure; (2) that there are differences in band widths of the reconstituted Ca²⁺-ATPase and bacteriorhodopsin compared with the spectra of the corresponding sarcoplasmic reticulum and purple membrane; (3) different membrane proteins adopt different conformations as evinced by a comparison of the spectra of the sarcoplasmic reticulum and purple membrane; (4) the polypeptide arrangement in the purple membrane is mainly helical but the abnormal frequency of the amide I band suggests that some distortion of the helix occurs: and (5) the sarcoplasmic reticulum membrane contains unordered as well as α-helix polypeptide arrangements.     

The water permeability of the monoolein/triolein bilayer membrane

The water permeability of the lipid bilayer can be used as a probe of membrane structure. A simple model of the bilayer, the liquid hydrocarbon model, views the membrane as a thin slice of bulk hydrocarbon liquid. A previous study (Petersen, D. (1980) Biochim. Biophys. Acta 600, 666–677) showed that this model does not accurately predict the water permeability of the monoolein/n-hexadecane bilayer: the measured activation energy for water permeation is 50% above the predicted value. From this it was inferred that the hydrocarbon chains in the lipid bilayer are more ordered than in the bulk hydrocarbon liquid. The present study tests the liquid hydrocarbon model for the monoolein/triolein bilayer, which has been shown to contain very little triolein in the plane of the membrane (Waldbillig, R.C. and Szabo, G. (1979) Biochim. Biophys. Acta 557, 295–305). Measurements of the water permeability coefficient of the bilayer are compared with predictions of the liquid hydrocarbon model based on measurements of the water permeability coefficient of bulk 8-heptadecene. The predicted and measured values agree quite closely over the temperature range studied (15–35°C): the predicted activation energy is 11.1±0.2 kcal/mol, whereas the measured activation energy for the bilayer is 9.8±0.7 kcal/mol. This close agreement is in contrast with the monoolein/n-hexadecane results and suggests that, insofar as water permeation is concerned, the liquid hydrocarbon model quite closely represents the monoolein/triolein bilayer.     

Analysis of the bilayer phase transition temperatures of phosphatidylcholines with mixed chains

The analysis of the chain-length dependence of the chain-melting transition temperatures of bilayers composed of lipids with identical chains (Marsh, D. 1991. Biochim. Biophys. Acta. 1062: 1-6) is extended to include lipids with chains of unequal length. The bilayer transition temperatures of saturated asymmetrical phosphatidylcholines are interpreted by assuming that the transition enthalpy and transition entropy are linearly related to the absolute value of the difference in chain length between the sn-1 and sn-2 chains, with constant end contributions. Such an assumption is supported by calorimetric data on phosphatidylcholines of constant mean chainlength and varying chain asymmetry. In particular, a symmetrical linear dependence is observed on the chain asymmetry, Δn, which is centered around a value Δn° that corresponds to the conformational inequivalence of the sn-1 and sn-2 chains. The transition temperature then takes the form: T_t = T_t^∞(n - n_H - h′ | Δn + Δn° |)/(n - n_s - s′ | Δn + Δn°) where n_H, n_s are the end contributions, and h′, s′ are fractional deficits in the incremental transition enthalpy and entropy, respectively, arising from the overlapping regions of the longer chains. Optimization on the transition temperature data for the dependence on chain asymmetry of three series of phosphatidylcholines with constant mean chainlength, n, yields parameters that are capable of predicting the dependence of the transition temperatures on chain asymmetry for other mean chainlengths. The dependence of the transition temperature on mean chainlength for phosphatidylcholines in which the chain asymmetry is maintained constant, as well as the dependence on both mean chain length and chain asymmetry for phosphatidylcholines in which one of the two chains is maintained of constant length, are also described with high accuracy by using the same parameters.     

Water permeability and lipid composition of toad urinary bladder: The influence of temperature

Summary The water diffusional permeability, its activation energy and the lipid composition were studied in urinary bladders from toads adapted to different temperatures. It was observed that the unidirectional water flux greatly depends on the temperature at which the experiments are performed. This dependence is greater in the animals adapted to higher temperatures. Toads adapted to cold show strong reduction in the activation energy for water diffusion permeability (from 11.4±1.9 kcal·mol^–1 to 4.4±1.1 kcal·mol^–1) and an increase of 30% in the amount of total lipids from bladder epithelial cells. There were no significant changes in the phospholipid/cholesterol ratio, composition of the paraffinic chains or protein concentration between toads adapted to both temperatures. The possibility that water translocates through the mucosal border of the toad bladder by partitioning in the polar zone and diffusioning between the hydrocarbon chains of the membrane lipids and that cold adaptation would induce a stronger packing of lipids in the membrane is discussed.     

Forced-unfolding and force-quench refolding of RNA hairpins

Nanomanipulation of individual RNA molecules, using laser optical tweezers, has made it possible to infer the major features of their energy landscape. Time-dependent mechanical unfolding trajectories, measured at a constant stretching force (f_S) of simple RNA structures (hairpins and three-helix junctions) sandwiched between RNA/DNA hybrid handles show that they unfold in a reversible all-or-none manner. To provide a molecular interpretation of the experiments we use a general coarse-grained off-lattice Gō-like model, in which each nucleotide is represented using three interaction sites. Using the coarse-grained model we have explored forced-unfolding of RNA hairpin as a function of f_S and the loading rate (r_f). The simulations and theoretical analysis have been done both with and without the handles that are explicitly modeled by semiflexible polymer chains. The mechanisms and timescales for denaturation by temperature jump and mechanical unfolding are vastly different. The directed perturbation of the native state by f_S results in a sequential unfolding of the hairpin starting from their ends, whereas thermal denaturation occurs stochastically. From the dependence of the unfolding rates on r_f and f_S we show that the position of the unfolding transition state is not a constant but moves dramatically as either r_f or f_S is changed. The transition-state movements are interpreted by adopting the Hammond postulate for forced-unfolding. Forced-unfolding simulations of RNA, with handles attached to the two ends, show that the value of the unfolding force increases (especially at high pulling speeds) as the length of the handles increases. The pathways for refolding of RNA from stretched initial conformation, upon quenching f_S to the quench force f_Q, are highly heterogeneous. The refolding times, upon force-quench, are at least an order-of-magnitude greater than those obtained by temperature-quench. The long f_Q-dependent refolding times starting from fully stretched states are analyzed using a model that accounts for the microscopic steps in the rate-limiting step, which involves the trans to gauche transitions of the dihedral angles in the GAAA tetraloop. The simulations with explicit molecular model for the handles show that the dynamics of force-quench refolding is strongly dependent on the interplay of their contour length and persistence length and the RNA persistence length. Using the generality of our results, we also make a number of precise experimentally testable predictions.     

The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.     

Interaction between C18 fatty acids and DOPE PEG2000 in Langmuir monolayers: effect of degree of unsaturation

In this study, we address the effect of the cis-double bond in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamide-N-[methoxy(polyethylene glycol)-2000, DOPE PEG2000 (DP), on the Langmuir monolayer of C18 fatty acids—namely, stearic acid (SA), oleic acid (L1), linoleic acid (L2), and linolenic acid (L3)—with the same head group but different degrees of saturation on their hydrocarbon chains. Negative values of Gibbs free energy of mixing (ΔG _mix) were obtained throughout the investigated ranges of the unsaturated C18 fatty-acid (L1, L2 and L3) mixed systems, indicating that very strong attractions occurred between molecules in the monolayers. The bend and kink effects from the cis-double bond(s) in the hydrocarbon chain affected the membrane fluidity and molecular packing in the monolayers, which resulted in a greater interaction between unsaturated C18 fatty acids and DP. The most thermodynamically stable mole composition of unsaturated C18 fatty acids to DP was observed at 50:1; this ratio is suggested to be the best mole ratio and will be subsequently used to prepare DP–C18 fatty-acid nanoliposomes. The presence of cis-double bonds in both hydrocarbon chains of DOPE in DP also created an imperfection in the membrane structure of lipid-drug delivery systems, which is expected to enhance lipid-based systems for antibody conjugation and drug encapsulation.     

Electrostatically induced change of the conformational order of charged lipid membranes

Raman spectroscopy and X-ray diffraction are used to investigate the influence of surface charges on the structure of ionizable lipid membranes of dimyristoylmethylphosphatidic acid. The membrane surface charge density is regulated by varying the pH of the aqueous phase. Changes of the conformational order of the lipid chains are determined from the intensity of the CC stretch chain vibrations around 1100 cm^?1 in a lipid Raman spectrum. In going from an electrical neutral to a negatively charged membrane, the conformational order is reduced by 5% in the ordered and by 9% in the fluid membrane phase, corresponding to 0.6 and 0.8 CC bonds, respectively, which change from a trans to a gauche conformation. The electrostatically induced conformational change is mainly concentrated at the lipid chain ends as indicated by the spectral variations of the 890 cm^?1 CH₃ rocking band of the chain termini. The X-ray diffraction experiments show that increasing the surface charge density in the ordered membrane phase leads to a lateral expansion of the packing of the lipid polar groups, whereas the packing of the lipid chains in a plane perpendicular to the chain axes remains constant, indicating an increase of the tilt of the lipid chains from δ = 10° (pH 3) to δ = 27° (pH 9).     

Hydrocarbon chain dynamics in lipid bilayers

A model is proposed for hydrocarbon chain dynamics in lipid bilayers. In the upper and middle parts of the chain all motion occurs by concerted rotations around at least two carbon carbon bonds at a time, preserving a structural with kinks (that is gauche^±trans gauche^? conformations) as the only deviations from the all-trans chain. At the end, independent rotations around carboncarbon bonds play a larger and larger part. This gives a reasonable interpretation of deuterium NMR data.     

Phospholipid Reorientation at the Lipid/Water Interface Measured by High Resolution P Field Cycling NMR Spectroscopy

The magnetic field dependence of the ³¹P spin-lattice relaxation rate, R₁, of phospholipids can be used to differentiate motions for these molecules in a variety of unilamellar vesicles. In particular, internal motion with a 5- to 10-ns correlation time has been attributed to diffusion-in-a-cone of the phosphodiester region, analogous to motion of a cylinder in a liquid hydrocarbon. We use the temperature dependence of ³¹P R₁ at low field (0.03-0.08 T), which reflects this correlation time, to explore the energy barriers associated with this motion. Most phospholipids exhibit a similar energy barrier of 13.2 ± 1.9 kJ/mol at temperatures above that associated with their gel-to-liquid-crystalline transition (T_m); at temperatures below T_m, this barrier increases dramatically to 68.5 ± 7.3 kJ/mol. This temperature dependence is broadly interpreted as arising from diffusive motion of the lipid axis in a spatially rough potential energy landscape. The inclusion of cholesterol in these vesicles has only moderate effects for phospholipids at temperatures above their T_m, but significantly reduces the energy barrier (to 17 ± 4 kJ/mol) at temperatures below the T_m of the pure lipid. Very-low-field R₁ data indicate that cholesterol inclusion alters the averaged disposition of the phosphorus-to-glycerol-proton vector (both its average length and its average angle with respect to the membrane normal) that determines the ³¹P relaxation.     

Embedding Aβ42 in Heterogeneous Membranes Depends on Cholesterol Asymmetries

Using a coarse-grained lipid and peptide model, we show that the free energy stabilization of amyloid-β in heterogeneous lipid membranes is predicted to have a dependence on asymmetric distributions of cholesterol compositions across the membrane leaflets. We find that a highly asymmetric cholesterol distribution that is depleted on the exofacial leaflet but enhanced on the cytofacial leaflet of the model lipid membrane thermodynamically favors membrane retention of a fully embedded Aβ peptide. However, in the case of cholesterol redistribution that increases concentration of cholesterol on the exofacial layer, typical of aging or Alzheimer’s disease, the free energy favors peptide extrusion of the highly reactive N-terminus into the extracellular space that may be vulnerable to aggregation, oligomerization, or deleterious oxidative reactivity.     

Mode of Action of Nisin Z against Listeria monocytogenes Scott A Grown at High and Low Temperatures

Nisin Z, a natural nisin variant, was recently isolated from Lactococcus lactis subspecies lactis NIZO 22186. The gene for this lantibiotic, designated nisZ, has been cloned, and its nucleotide sequence was found to be identical to that of the precursor nisin gene with the exception of a single mutation resulting in the substitution of Asn-27 for His-27 in the mature polypeptide (J. W. M. Mulders, I. J. Boerrigter, H. S. Rollema, R. J. Siezen, and W. M. de Vos, Eur. J. Biochem. 201:581-584, 1991). A K⁺ electrode was used to investigate the effect of various environmental parameters on the action of nisin Z against Listeria monocytogenes. Addition of nisin Z resulted in immediate loss of cell K⁺, depolarization of the cytoplasmic membrane, inhibition of respiratory activity, and hydrolysis and partial efflux of cellular ATP. The action of nisin Z was optimal at pH 6.0 and was significantly reduced by di- and trivalent cations. The lanthanide gadolinium (Gd³⁺) was an efficient inhibitor and prevented nisin Z activity completely at a concentration of 0.2 mM. Nisin Z-induced loss of cell K⁺ was reduced at low temperatures, presumably as a result of the increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane. In cells grown at 30°C, the action of nisin Z was prevented below 7°C, whereas in cells grown at 4°C nisin Z was able to induce K⁺ leakage at this low temperature.