Direct measurement of the force between two lipid bilayers and observation of their fusion

The force between two phosphatidylcholine bilayers is measured as a function of their separation, showing a strong hydration repulsion at short range, as previously reported by LeNeveu et al. (LeNeveu, D.M., Rand, R.P., Parsegian, V.A. and Gingell, D. (1977) (Biophys. J. 18, 209–230). The experimental technique also allows direct observation of the molecular process by which two bilayers fuse into one.     

Mechanical properties of double-stranded DNA biolayers immobilized on microcantilever under axial compression

In label-free biodetections based on microcantilever technology, double-stranded DNA (dsDNA) structures form through the linkage between probe single-stranded DNA (ssDNA) molecules immobilized on solid substrates and target ssDNA molecules in solutions. Mechanical/electrical properties of these biolayers are important factors for nanomechanical deflections of microcantilevers. In this paper, the biolayer immobilized on microcantilever is treated as a bar with a macroscopic elastic modulus on the basis of continuum mechanics viewpoints. In consideration of hydration force, screened electrostatic repulsion and conformational fluctuation in biolayers, load-deformation curves of dsDNA biolayers under axial compression are depicted with the help of the energy conservation law and a mesoscopic free energy presented by Strey et al. (1997, 1999) [Strey, H.H., Parsegian, V.A., Podgornik, R., 1997. Equation of state for DNA liquid crystals: fluctuation enhanced electrostatic double layer repulsion. Physical Review Letters 78, 895–898; Strey, H.H., Parsegian, V.A., Podgornik, R., 1999. Equation of state for polymer liquid crystals: theory and experiment. Physical Review E 59, 999–1008] from a liquid crystal theory. And the analytical relation between macroscopic Young's modulus of biolayers and nanoscopic geometrical properties of dsDNA, packing density, buffer salt solution concentration, etc. is also formulated.     

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. II. An experimental study.

The polymorphic phase behavior of unsaturated phosphatidylethanolamine (PE)/diacylglycerol (DG) binary lipid mixtures was investigated by the use of time-resolved fluorescence anisotropy. Using a fluorescent lipid, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenylethyl] carbonyl]3-sn-phosphatidyl-choline (DPH-PC), the orientational order and rotational dynamics of the above lipid mixtures in the liquid crystalline lamellar (L alpha) and inverted hexagonal (HII) phases were studied. By employing a one-exponential model (Cheng, K.H. 1989: Biophys. J. 55:1025-1031) to fit the anisotropy decay data, abrupt decreases in the normalized initial anisotropy decay slope and the residual anisotropy of DPH-PC were observed at approximately 6-8% DG, signifying a L alpha/HII phase transition. Using our new theoretical WOBHOP and P2P4HOP models as described in a preceding paper (Van Der Meer, B.W., K.H. Cheng, and S.Y. Chen. 1990. Biophys. J. 58:000-000), two or more rotational correlation times were required to describe the anisotropy decay behavior of DPH-PC in the HII phase. These rotation correlation times were further related to the second and fourth rank order parameters, and the wobbling and hopping diffusion constants of the fluorescent probe in the highly curved lipid cylindrical tubes of the HII phase. The hopping diffusion constant (DH) equals the lateral diffusion constant (DL) divided by R2 (R = radius of the lipid cylindrical tubes). The value of DL was estimated by measuring the excimer formation rate of 1-palmitoyl-2-[10-(1-pyrenl)decanoyl] phosphatidyl choline (py-PC) in the same PE/DG mixtures. Upon comparing the values of DH and DL, the value of R was determined to be approximately 10-15 A, and agreed with that derived from x-ray diffraction (Tate, M.W., and S.M. Gruner, 1989, Biochemistry. 28:4245-4253; Rand, R.P., N.L. Fuller, S.M. Gruner, and V.A. Parsegian. 1990. Biochemistry. 29:76-87).     

Reversible inhibition of the proton pump bacteriorhodopsin by modification of tyrosine 64

Five mol of lysine per mol of bacteriorhodopsin were modified with methylacetimidate. This treatment did not inactivate bacteriorhodopsin but prevented all lysines from subsequent reaction with diazotized sulfanilic acid. This reaction predominantly modified tyrosine 64 and light-induced proton translocation was abolished. Reduction of the mono(p-azobenzene sulfonic acid) tyrosine 64 to the corresponding 3-amino derivative with sodium dithionite led to complete reactivation of the proton translocation activity of bacteriorhodopsin. The relative location of tyrosines 26 and 64 and the COOH terminus on the two surfaces of the purple membrane was determined by incorporation into phospholipid vesicles, subsequent modification, and proteolytic treatment. The results obtained support the models proposed by Engelmann et al. (Engelman, D. M., Henderson, R. McLauchlan, A. D., and Wallace, B. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2023-2027) and by Ovchinnikov et al. (Ovchinnikov, Yu. A., Abdulaev, N. G., Feigina M. Yu., Kiselev A. V., and Lobanov, N. A. (1979) FEBS Lett. 100, 219-224). Tyrosine 64 is located on the extracellular side of the membrane, whereas tyrosine 26 and the COOH terminus are located on the cytoplasmic side. Because specific nitration of tyrosine 26 also leads to inactivation of bacteriorhodopsin (Lemke, H. D., and Oesterhelt, D. (1981) Eur. J. Biochem. 115, 595-604), the results obtained demonstrate that amino acid residues located on both surfaces of the purple membrane are involved in proton translocation.     

Membrane fusion promoters and inhibitors have contrasting effects on lipid bilayer structure and undulations

It has been established that the fusion of both biological membranes and phospholipid bilayers can be modulated by altering their lipid composition (Chernomordik et al., 1995 .J. Membr. Biol. 146:3). In particular, when added exogenously between apposing membranes, monomyristoylphosphatidylcholine (MMPC) inhibits membrane fusion, whereas glycerol monoleate (GMO), oleic acid (OA), and arachidonic acid (AA) promote fusion. This present study uses x-ray diffraction to investigate the effects of MMPC, GMO, OA, and AA on the bending and stability of lipid bilayers when bilayers are forced together with applied osmotic pressure. The addition of 10 and 30 mol% MMPC to egg phosphatidylcholine (EPC) bilayers maintains the bilayer structure, even when the interbilayer fluid spacing is reduced to approximately 3 A, and increases the repulsive pressure between bilayers so that the fluid spacing in excess water increases by 5 and 15 A, respectively. Thus MMPC increases the undulation pressure, implying that the addition of MMPC promotes out-of-plane bending and decreases the adhesion energy between bilayers. In contrast, the addition of GMO has minor effects on the undulation pressure; 10 and 50 mol% GMO increase the fluid spacing of EPC in excess water by 0 and 2 A, respectively. However, x-ray diffraction indicates that, at small interbilayer separations, GMO, OA, or AA converts the bilayer to a structure containing hexagonally packed scattering units approximately 50 A in diameter. Thus GMO, OA, or AA destabilizes bilayer structure as apposing bilayers are brought into contact, which could contribute to their role in promoting membrane fusion.     

Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy

The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 14185-14196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TLL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.     

Cell poking. Determination of the elastic area compressibility modulus of the erythrocyte membrane

Cell poking, a new method for measuring mechanical properties of single cells was used to determine the elastic area compressibility modulus of osmotically swollen human erythrocytes. With this method we determined the force required to indent cells attached to a glass coverslip (Petersen, N.O., W. B. McConnaughey , and E. L. Elson , 1982, Proc. Natl. Acad. Sci. USA, 79:5327. Forces on the order of one millidyne and indentations on the order of one micron were detected. An analysis of these data in terms of a simplified mechanical model yielded the elastic area compressibility modulus. This analysis used a variational approach to minimize the isothermal elastic potential energy density function given by E. A. Evans and R. Skalak (Mechanics and Thermodynamics of Biomembranes, 1980, CRC Press, Boca Raton , FL). Measurements on swollen erythrocytes gave a range of values, depending in part on the osmotic conditions, of 17.9 +/- 8.2 to 34.8 +/- 12.0 mdyn /micron for the elastic area compressibility modulus at 25 degrees C. Fractional area expansion greater than 2.6 +/- 0.8% produced rapid cell lysis. These values were not corrected for the reversible movement of water across the cell membrane in response to hydrostatic pressure gradients. Our results agree reasonably with those obtained by Evans et al. (Evans, E.A., R. Waugh , and L. Melnick , 1976, Biophys. J., 16:585-595.) using micropipette aspiration under similar conditions.     

Proton dissociation dynamics in the aqueous layer of multilamellar phospholipid vesicles

Summary The water layers interspacing between the phospholipid membranes of a multilamellar vesicle are 3–10 water layers across and their width is adjusted by osmotic pressure (Parsegian, V.A., et al., 1986.Methods Enzymol. 127:400–416).In these thin water layers we dissolved pyranine (8 hydroxypyrene 1,3,6 trisulfonate), a compound which, upon photo excitation, ejects it hydroxy proton with time constant of 100 psec. (Gutman, M. 1986.Methods Enzymol. 127:522–538).In the present study we investigated how the width of the aqueous layer, the density of phosphomoieties on the membrane's surface and the activity of water in the layer affect the capacity of protons to diffuse out from the electrostatic cage of the excited anion before it decays to the ground state.Using a combination of steady-state and subnanosecond time-resolved fluorescence measurements we determined the average number of proton excited-anion recombinations before the proton escapes from the Coulomb cage.The probability of recombination in thin water layer is significantly higher than in bulk. The factor contributing most to enhancement of recombination is the diminished water activity of the thin aqueous layer.The time frame for proton escape from an electrostatic trap as big as a membrane-bound protein is 3 orders of magnitude shorter than turnover time of membrane-bound enzymes. Thus the effects of local forces on proton diffusion, at the time scale of physiological processes, is negligible.     

Investigation of AC-DC magnetic field effects in planar phospholipid bilayers.

Observations recently reported by others indicate that a combination of a weak dc magnetic field and extremely-low-frequency ac magnetic field can produce resonant effects in biological systems. We report measurements of the effects of combined dc and ac magnetic fields on the dc current through channel-free planar phospholipid membranes. The combined dc-ac magnetic fields did affect the dc current through planar phospholipid membranes, but not in every membrane, and not consistently at the same values of magnetic flux density and frequency. None of our measurements showed resonant response akin to the cyclotron-like resonance reported in diatoms [Smith et al., 1987] and lymphocytes [Liboff et al., 1987].     

Vesiculation induced by hydrostatic pressure in human erythrocytes.

When human erythrocytes were subjected to hydrostatic pressure (1.1-2.0 kbar), it was found that membrane vesicles were released from the red cells above 1.4 kbar. As with hemolysis under high pressure, the amount of released vesicles was increased with increasing pressure but decreased by the cross-linking of membrane proteins with diamide. Vesicles obtained at 2.0 kbar were heterogeneous in size but similar to intact erythrocytes in phospholipid composition. Although it has been reported that spectrin-free vesicles are released by echinocytogenic agents, pressure-induced vesicles did contain considerable and similar amounts of spectrin irrespective of the difference in size. These results suggest that vesiculation by high pressure is associated with the disruption of the membrane skeleton, as previously seen in pressure-induced hemolysis [Yamaguchi et al. (1989) J. Biochem. 106, 1080-1085].     

Osmotic Equilibrium and Elastic Properties of Eel Intestinal Vesicles

Changes in volume of intestinal brush border membrane vesicles of the European eel Anguilla anguilla were measured as vesicles were exposed to media with different osmotic pressures. Preparing the vesicles in media of low osmotic pressure allowed the effects of a small hydrostatic pressure to become a significant factor in the osmotic equilibration. By applying LaPlace's law to relate pressure and volume and assuming a linear relation between membrane tension and area expansion, we estimate an initial membrane tension at 4.02 × 10⁻⁵ N cm⁻¹ and an area compressibility elastic modulus at 0.87 × 10⁻³ N cm⁻¹. The elastic modulus estimate falls in the low range of values reported for membranes from other tissues in other species. This lower modulus quantitatively accounts for why eel intestinal vesicles show measurable changes in volume in hypotonic media while rabbit kidney vesicles do not. Received: 28 January 1999/Revised: 15 June 1999     

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.     

Physical analysis of light-scattering changes in bovine photoreceptor membrane suspensions.

We have used electron microscopy and model calculations to analyze the physical basis of light-scattering signals from suspensions of photoreceptor membranes. These signals have previously been used to probe interactions between photoactivated rhodopsin (R*) and the peripheral membrane enzyme, GTP-binding protein (G) (Kühn et al., 1981, Proc. Natl. Acad. Sci. USA., 78:6873-6877). Although there is no unique physical interpretation of these signals, we have shown in this study that they were qualitatively unchanged when the rod outer segment fragments (containing stacked disks) were fragmented by sonication or osmotic shock to produce spherical disk membrane vesicles. An exact treatment of the scattering process for spherical vesicles enabled us to evaluate the effects of changing membrane thickness, refractive index, or vesicle diameter. We present a particular redistribution of mass upon R*-G interaction that fits the experimental data.     

Opsin is a phospholipid flippase

Polar lipids must flip-flop rapidly across biological membranes to sustain cellular life [1, 2], but flipping is energetically costly [3] and its intrinsic rate is low. To overcome this problem, cells have membrane proteins that function as lipid transporters (flippases) to accelerate flipping to a physiologically relevant rate. Flippases that operate at the plasma membrane of eukaryotes, coupling ATP hydrolysis to unidirectional lipid flipping, have been defined at a molecular level [2]. On the other hand, ATP-independent bidirectional flippases that translocate lipids in biogenic compartments, e.g., the endoplasmic reticulum, and specialized membranes, e.g., photoreceptor discs [4, 5], have not been identified even though their activity has been recognized for more than 30 years [1]. Here, we demonstrate that opsin is the ATP-independent phospholipid flippase of photoreceptor discs. We show that reconstitution of opsin into large unilamellar vesicles promotes rapid (τ<10 s) flipping of phospholipid probes across the vesicle membrane. This is the first molecular identification of an ATP-independent phospholipid flippase in any system. It reveals an unexpected activity for opsin and, in conjunction with recently available structural information on this G protein-coupled receptor [6, 7], significantly advances our understanding of the mechanism of ATP-independent lipid flip-flop.     

Interactions between the oligomycin sensitivity conferring protein (OSCP) and beef heart mitochondrial F1-ATPase. 1. Study of the binding parameters with a chemically radiolabeled OSCP

Upon treatment of beef heart mitochondrial oligomycin sensitivity conferring protein (OSCP) with [14C]-N-ethylmaleimide ( [14C]NEM) or dithiobis(nitro[14C] benzoate), 1 mol of either SH reagent was incorporated per mol of OSCP. Radiolabeling occurred at the level of the only cysteine residue, Cys-118, present in the OSCP sequence reported by Ovchinnikov et al. [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I. V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22]; it did not alter the biological activity of OSCP tested in a reconstituted F0-F1 system that catalyzed oligomycin-sensitive ATPase activity or ATP-Pi exchange. The parameters of [14C]NEM-OSCP binding to isolated beef heart mitochondrial F1 were assessed by equilibrium dialysis. Addition of trace amounts of Tween 20 prevented unspecific adsorption of OSCP. The binding curves showed that each F1 possesses a high-affinity OSCP binding site (Kd = 0.08 microM) and two low-affinity OSCP binding sites (Kd = 6-8 microM). Binding of OSCP to the high-affinity site on F1 is probably responsible for the ability of OSCP to confer oligomycin sensitivity to F1 in the ATPase complex.     

Labeling of phospholipids in vesicles and human erythrocytes by photoactivable fatty acid derivatives

The photoactivable glycolipid probes, 2-(4-azido-2-nitrophenoxy)palmitoyl[1-14C]glucosamine (compound A) and 12-(4-azido-2-nitrophenoxy)stearoyl[1-14C]glucosamine (compound B) were synthesized essentially as described before [Iwata, K. K. et al. (1978) Prog. Clin. Biol. Res. 22, 579-589]. These probes were used to label phospholipid vesicles and erythrocyte membranes. A chromatographic method was developed to quantify the individual probe-phospholipid adducts involving both phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. For both membranes as well as for both probes a phospholipid labeling pattern was obtained which appeared to reflect the relative content of fatty acid double bonds in each phospholipid class. The distinct labeling of phosphatidylserine in intact erythrocytes strongly suggested that the probes spontaneously and rapidly redistributed between the two halves of the membrane bilayer. In addition, both probes yielded an extensive labeling of the membrane proteins. Analysis by dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography has indicated that the protein labeling pattern was different, depending on whether the 'shallow' probe (compound A) or 'depth' probe (compound B) were used.     

Purification of a protein having pore forming activity from the rat liver mitochondrial outer membrane

A protein with pore-forming activity has been isolated from the outer membrane of rat liver mitochondria. The purification involves sucrose gradient centrifugation, differential centrifugation in the presence of Triton X-100, and DEAE-Sepharose and CM-Sepharose chromatography. The yield of the purified protein was approx. 2% of the total outer membrane proteins. The protein, when inserted into soya bean phospholipid vesicles, increases the [3H]sucrose permeability of the vesicles but had no effect on the permeability of high-molecular-weight [14C]dextran (Mr 70 000). The protein is very active, since as little as 3-4 micrograms of protein per mg of phospholipid is required for the complete release of [3H]sucrose from the vesicles. Sucrose diffusion channels could not be reconstituted with other membrane proteins such as rat liver cytochrome oxidase or cytochrome b5. Purified pore protein revealed a single band of apparent Mr 30000 when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This polypeptide could be further resolved by isoelectric focusing into a major (pI7.9) and two relatively minor (pI7.6 and 7.2) components. Proteolytic mapping with V8 proteinase from Staphylococcus aureus suggests that these probably represent a single component showing charge heterogeneity. The reason for the charge heterogeneity is not known. The amino acid composition of the protein revealed 47.8% polar amino acids with a relatively high lysine content.     

Helix packing in the lactose permease of Escherichia coli determined by site-directed thiol cross-linking: helix I is close to helices V and XI

Coexpression of lacY gene fragments encoding the first two transmembrane domains and the remaining 10 transmembrane domains complement in the membrane and catalyze active lactose transport [Wrubel, W., Stochaj, U., et al. (1990) J. Bacteriol. 172, 5374-5381]. Accordingly, a plasmid encoding contiguous, nonoverlapping permease fragments with a discontinuity in the cytoplasmic loop between helices II and III (loop II/III) was constructed (N2C10 permease). When Phe27 (helix I) is replaced with Cys, cross-linking is observed with two native Cys residues, Cys148 (helix V) and Cys355 (helix XI). Cross-linking of a Cys residue at position 27 to Cys148 occurs with N,N'-o-phenylenedimaleimide (o-PDM; rigid 6 A), with N,N'-p-phenylenedimaleimide (p-PDM; rigid 10 A), or with 1,6-bis(maleimido)hexane (BMH; flexible 16 A). On the other hand, with the Phe27-->Cys/Cys355 pair, cross-linking is observed with p-PDM or BMH but not o-PDM. In neither case is cross-linking observed with iodine. It is suggested that a Cys residue at position 27 is within 6-10 A from Cys148 and about 10 A from Cys355. The results provide evidence for proximity between helix I and helices V or XI in the tertiary structure of the permease. In addition, the findings are consistent with other results [Venkatesan, P., Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] indicating that Glu126 (helix IV) and Arg144 (helix V) are within the membrane, rather than at the membrane-water interface on the cytoplasmic face.     

Membrane destabilizing properties of cell-penetrating peptides

Although cell-penetrating peptides (CPPs), also denoted protein transduction domains (PTDs), have been widely used for intracellular delivery of large and hydrophilic molecules, the mechanism of uptake is still poorly understood. In a recent live cell study of the uptake of penetratin and tryptophan-containing analogues of Tat(48-60) and oligoarginine, denoted TatP59W, TatLysP59W and R(7)W, respectively, it was found that both endocytotic and non-endocytotic uptake pathways are involved [Thoren et al., Biochem. Biophys. Res. Commun. 307 (2003) 100-107]. Non-endocytotic uptake was only observed for the arginine-rich peptides TatP59W and R(7)W. In this paper, the interactions of penetratin, R(7)W, TatP59W and TatLysP59W with phospholipid vesicles are compared in the search for an understanding of the mechanisms for cellular uptake. While R(7)W, TatP59W and TatLysP59W are found to promote vesicle fusion, indicated by mixing of membrane components, penetratin merely induces vesicle aggregation. Studies of the leakage from dye-loaded vesicles indicate that none of the peptides forms membrane pores and that vesicle fusion is not accompanied by leakage of the aqueous contents of the vesicles. These observations are important for a proper interpretation of future experiments on the interactions of these peptides with model membranes. We suggest that the discovered variations in propensity to destabilize phospholipid bilayers between the peptides investigated, in some cases sufficient to induce fusion, may be related to their different cellular uptake properties.     

Hydration, structure, and molecular interactions in the headgroup region of dioleoylphosphatidylcholine bilayers: an electron spin resonance study

The relationship between bilayer hydration and the dynamic structure of headgroups and interbilayer water in multilamellar vesicles is investigated by electron spin resonance methods. Temperature variations of the order parameter of a headgroup spin label DPP-Tempo in DOPC in excess water and partially dehydrated (10 wt % water) show a cusp-like pattern around the main phase transition, Tc. This pattern is similar to those of temperature variations of the quadrupolar splitting of interbilayer D2O in PC and PE bilayers previously measured by 2H NMR, indicating that the ordering of the headgroup and the interbilayer water are correlated. The cusp-like pattern of these and other physical properties around Tc are suggestive of quasicritical fluctuations. Also, an increase (a decrease) in ordering of DPP-Tempo is correlated with water moving out of (into) interbilayer region into (from) the bulk water phase near the freezing point, Tf. Addition of cholesterol lowers Tf, which remains the point of increasing headgroup ordering. Using the small water-soluble spin probe 4-PT, it is shown that the ordering of interbilayer water increases with bilayer dehydration. It is suggested that increased ordering in the interbilayer region, implying a lowering of entropy, will itself lead to further dehydration of the interbilayer region until its lowered pressure resists further flow, i.e., an osmotic phenomenon.