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The concentration of cholesterol in cell membranes affects membrane fluidity and thickness, and might regulate different processes such as the formation of lipid rafts. Since interpreting experimental data from biological membranes is rather intricate, investigations on simple models with biological relevance are necessary to understand the natural systems. We study the effect of cholesterol on the molecular structure of multi-lamellar vesicles (MLVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a phospholipid ubiquitous in cell membranes, with compositions in the range 0-60 mol% cholesterol. Order parameters, |S(CH)|, are experimentally determined by using (1)H-(13)C solid-state nuclear magnetic resonance (NMR) spectroscopy with segmental detail for all parts of both the cholesterol and POPC molecules, namely the ring system and alkyl chain of the sterol, as well as the glycerol backbone, choline headgroup and the sn-1 and sn-2 acyl chains of POPC. With increasing cholesterol concentration the acyl chains gradually adopt a more extended conformation while the orientation and dynamics of the polar groups are rather unaffected. Additionally, we perform classical molecular dynamics simulations on virtual bilayers mimicking the POPC-cholesterol MLVs investigated by NMR. Good agreement between experiments and simulations is found for the cholesterol alignment in the bilayer and for the |S(CH)| profiles of acyl chains below 15 mol% cholesterol. Deviations occur for the choline headgroup and glycerol backbone parts of POPC, as well as for the phospholipid and cholesterol alkyl chains at higher cholesterol concentrations. The unprecedented detail of the NMR data enables a more complete comparison between simulations and experiments on POPC-cholesterol bilayers and may aid in developing more realistic model descriptions of biological membranes.

The interaction of small molecules, such as drugs or metabolites, with proteins and biomembranes is of fundamental importance for their bioavailability. The systematic characterization of the binding affinity for structurally related ligands may provide rules that allow its prediction for any other relevant molecule. In this work we have studied a homologous series of fluorescent fatty amines with the fluorescent moiety 7-nitrobenz-2-oxa-1,3-diazol-4-yl covalently bound to the amine group (NBD-C(n); n = 4, 6, 8, 10, 12, 14, and 16) in aqueous solution and associated with BSA or lipid bilayers. We have found a linear dependence with the length of the alkyl chain, up to NBD-C(10), for the Gibb's free energy of partition between the aqueous solution and 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers equal to ΔΔG = -2.5 ± 0.3 kJ/mol per methylene group. Additionally, the amphiphiles interacted efficiently with bovine serum albumin, and it was inhibited by fatty acids indicating that binding occurs to the fatty acids highest affinity binding site. The association of the amphiphiles with BSA and POPC bilayers was performed at different temperatures (15-35 °C) allowing for the calculation of the enthalpic and entropic contributions. A value of ΔH = -15 ± 4 kJ/mol was obtained for all amphiphiles and binding agents. The entropy contribution was always positive and increased with the length of the alkyl chain. The location of the ligand in the biological membrane is also of high relevance, namely because this will determine its effect on biomembrane properties at high ligand concentrations. With this goal, we have measured some photophysical properties of the amphiphiles inserted in POPC bilayers, and we found no significant variation along the series, indicating that the NBD group is located in a region with similar properties regardless of the length of the nonpolar group. An exception was noted for the case of NBD-C(14) whose parameters were somewhat different from the trend observed.

We studied the interaction of hematoporphyrin IX (HpIX) with bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) containing cholesterol at a molar fraction between 0 and 0.5. The membrane-associated fraction of HpIX decreases significantly over a period of hours, for porphyrin concentrations in the aqueous phase above 50 nM. This was attributed to self-aggregation of HpIX and was well described by a dimerization process. A model was developed to correct for aggregation and obtain the true partition coefficient which is dependent on the molar fraction of cholesterol with a maximum at 20 mol%. The chemical and physical effects on the lipid bilayer upon irradiation of HpIX were studied for lipid bilayers with POPC:Chol 1:1. Exposure of these bilayers to visible light in the presence of HpIX leads to several cholesterol oxidation products that were identified using GC-MS. A dramatic increase in the membrane leakiness was also observed, even for short irradiation times and small light intensities, as evaluated from the rate of pH equilibration and dithionite permeability. The relevance of these results for the mechanism of photodynamic therapy is discussed.

We present a detailed study of the translocation rate of two headgroup-labeled phospholipid derivatives, one with two acyl chains, NBD-DMPE, and the other with a single acyl chain, NBD-lysoMPE, in lipid bilayer membranes in the liquid-disordered state (POPC) and in the liquid-ordered states (POPC/cholesterol (Chol), molar ratio 1:1, and sphingomyelin (SpM)/Chol, molar ratio 6:4). The study was performed as a function of temperature and the thermodynamic parameters of the translocation process have been obtained. The most important findings are 1), the translocation of NBD-DMPE is significantly faster than the translocation of NBD-lysoMPE for all bilayer compositions and temperatures tested; and 2), for both phospholipid derivatives, the translocation in POPC bilayers is approximately 1 order of magnitude faster than in POPC/Chol (1:1) bilayers and approximately 2-3 orders of magnitude faster than in SpM/Chol (6:4) bilayers. The permeability of the lipid bilayers to dithionite has also been measured. In liquid disordered membranes, the permeability rate constant obtained is comparable to the translocation rate constant of NBD-DMPE. However, in liquid-ordered bilayers, the permeability of dithionite is significantly faster then the translocation of NBD-DMPE. The change in enthalpy and entropy associated with the formation of the activated state in the translocation and permeation processes has also been obtained.

The effects of solid-fluid phase separations on the kinetics of association of a single-chain fluorescent amphiphile were investigated in two different systems: pure DMPC (dimyristoylphosphatidylcholine) and a 1:1 mixture of DMPC and DSPC (distearoylphosphatidylcholine). In pure DMPC vesicles, solid (s) and fluid (l(d)) phases coexist at the phase transition temperature, T(m), whereas a 1:1 mixture of DMPC and DSPC shows a stable s-l(d) phase separation over a large temperature interval. We found that in single-component bilayers, within the main phase transition, the experimental kinetics of association are clearly not single-exponential, the deviation from that function becoming maximal at the T(m). This observation can be accounted for by a rate of desorption that is slower than desorption from either fluid or solid phases, leaving the rates of insertion unchanged, but a treatment in terms of stable fluid and solid domains may not be adequate for the analysis of the association of an amphiphile with pure DMPC vesicles at the T(m). In DMPC/DSPC mixtures with solid-fluid phase coexistence, association occurs overall faster than expected based on phase composition. The observed kinetics can be described by an increase in the rate of insertion, leaving the desorption rates unchanged. The fast kinetics of insertion of the amphiphile into two-phase bilayers in two-component vesicles is attributed to a more rapid insertion into defect-rich regions, which are most likely phase boundaries between solid and fluid domains. A two-component mixture of lipids that shows a stable phase separation between l(d)-s phases over a large temperature interval thus behaves very differently from a single-component bilayer at the T(m), with respect to insertion of amphiphiles.

The partition coefficients (K(P)) of a series of single-chain and double-chain fluorescent amphiphiles, between solid ordered (P(beta') and L(beta)) and liquid disordered (L(alpha) of the type l(d)) lipid phases coexisting in the same lipid bilayer, was studied using steady-state fluorescence emission anisotropy. The single-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-alkylamines, and the double-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-phosphatidylethanolamines with chain lengths of 12-18 carbon atoms. Saturated 18-carbon alkyl/acyl chain compounds were also compared with Delta(9)-cis unsaturated chains of the same chain length. The fluorescence anisotropy of the probes was examined in lipid bilayers (multilamellar vesicles) prepared from an equimolar mixture of dilauroylphosphatidylcholine and distearoylphosphatidylcholine and studied as a function of temperature through the entire temperature range of coexistence of ordered gel phases and a disordered fluid phase in this system. The unsaturated chain amphiphiles partitioned exclusively into the fluid phase whenever this phase was present, as did the saturated chain amphiphiles with the shortest chains (C(12:0)), while K(P) ranges between 1 and 2, in favor of the L(beta) solid phase, for the amphiphiles with long saturated (C(18:0)) alkyl/acyl chains, with intermediate behavior for the intermediate chain lengths. All probes appeared to be totally excluded from P(beta') solid (gel) phases. The technique was also used to determine partitioning of some of the probes between coexisting liquid ordered (cholesterol-containing) (l(o)) and liquid disordered (l(d)) L(alpha) phases. In this case the ratio of signal amplitude to noise allowed us to obtain a qualitative, but not quantitative, measure of the phase partitioning of the probes. We conclude that the partitioning behavior of the probes examined between coexisting l(o) and l(d) phases is qualitatively similar to that observed between solid ordered and liquid disordered phases.

Alpha-Hemolysin is an extracellular protein toxin (107 kDa) produced by some pathogenic strains of Escherichia coli. Although stable in aqueous medium, it can bind to lipid bilayers and produce membrane disruption in model and cell membranes. Previous studies had shown that toxin binding to the bilayer did not always lead to membrane lysis. In this paper, we find that alpha-hemolysin may bind the membranes in at least two ways, a reversible adsorption and an irreversible insertion. Reversibility is detected by the ability of liposome-bound toxin to induce hemolysis of added horse erythrocytes; insertion is accompanied by an increase in the protein intrinsic fluorescence. Toxin insertion does not necessarily lead to membrane lysis. Studies of alpha-hemolysin insertion into bilayers formed from a variety of single phospholipids, or binary mixtures of phospholipids, or of phospholipid and cholesterol, reveal that irreversible insertion is favored by fluid over gel states, by low over high cholesterol concentrations, by disordered liquid phases over gel or ordered liquid phases, and by gel over ordered liquid phases. These results are relevant to the mechanism of action of alpha-hemolysin and provide new insights into the membrane insertion of large proteins.

The lateral diffusion of a phospholipid probe is studied in bilayers of binary mixtures of dimyristoylphosphatidylcholine (DMPC)/cholesterol and distearoylphosphatidylcholine (DSPC)/cholesterol and in the ternary system DMPC/DSPC/cholesterol using fluorescence recovery after photobleaching. An approximate phase diagram for the ternary system, as a function of temperature and cholesterol concentration, was obtained using differential scanning calorimetry and the phase diagrams of the binary systems. This phase diagram is similar to those of the phospholipid/cholesterol binary mixtures. In bilayers where solid and liquid phases coexist, the diffusion results are interpreted in terms of phase percolation. The size of the liquid-phase domains is estimated using percolation theory. In the ternary system, addition of cholesterol up to approximately 20 mol% shifts the percolation threshold to lower area fractions of liquid, but the size of the liquid-phase domains does not change. Above approximately 20 mol% cholesterol, the liquid phase is always connected. The size of solid-phase domains clusters is estimated using a model recently developed (Almeida, P.F.F., W.L.C. Vaz, and T.E. Thompson. 1992. Biochemistry. 31:7198-7210). For cholesterol concentrations up to 20 mol%, the size of solid-phase domain units does not change. Beyond 20 mol%, cholesterol causes the size of the solid units to decrease.

In recent work [Vaz, W.L.C., Melo, E.C.C., & Thompson, T.E. (1989) Biophys. J. 56, 869-876] we have shown that translational diffusion studies using fluorescence recovery after photobleaching (FRAP) provide information concerning domain structures and fluid-phase connectivity in lipid bilayers in which solid and fluid phases coexist. In the present paper, translational diffusion of the fluid-phase-soluble, solid-phase-insoluble fluorescent lipid derivative N-(7-nitrobenzoxa-2,3-diazol-4-yl) dilauroyl-phosphatidylethanolamine and the fluid-phase connectivity are examined in lipid bilayers prepared from binary mixtures of 1-docosanoyl-2-dodecanoylphosphatidylcholine (C22:0C12:0PC) and 1,2-diheptadecanoylphosphatidylcholine (di-C17:0PC) by using FRAP. The phosphatidylcholine mixture used provides a eutectic system with a eutectic point at a composition of about 0.4 mole fraction of di-C17:0PC and a temperature of about 37 degrees C [Sisk, R.B., Wang, Z.Q., Lin, H.N., & Huang, C.H. (1990) Biophys. J. 58, 777-783]. Two regions in temperature and composition, respectively below and above 0.4 mole fraction of di-C17:0PC, where fluid and solid phases coexist in the same lipid bilayer, are available for examination of fluid-phase connectivity. In mixtures containing less than 0.4 mole fraction of di-C17:0PC the fluid phase coexists with a mixed interdigitated Lc gel phase composed mostly of C22:0C12:0PC, whereas in mixtures containing greater than 0.4 mole fraction of di-C17:0PC the fluid phase coexists with a P beta' gel phase mostly composed of di-C17:0PC. When the solid phase is a P beta' gel phase, the temperature of fluid-phase connectivity for the mixtures lies close to the fluidus, which means that a small (approximately 20%) mass fraction of solid phase can divide the large bulk of the bilayer that is fluid into nonconnected domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional fluid phase connectivity is examined in mixed bilayers of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine as a function of composition and temperature at constant pressure using fluorescence recovery after photobleaching (FRAP). These isomorphous phospholipid mixtures exhibit nearly ideal mixing behavior. Dilauroyl phosphatidylethanolamine covalently linked through its amino function to NBD is used as the fluorescent probe in this study. These studies show the line of connectivity to be coincident with the line connecting the midpoints of all tie lines in the two-phase region of the phase diagram.

The influence of sucrose, between 0 and 70% in the aqueous phase, upon multilamellar liposomes of dimyristoylphosphatidylcholine was examined by differential scanning calorimetry and X-ray diffraction analysis. Increasing concentrations of sucrose increase the temperatures of both the main transition and the pretransition of the lipid. The effect is greater on the pretransition than on the main transition. At 35 degrees C the interlamellar spacing in the multilamellar liposomes is reduced by increasing sucrose concentration in the aqueous phase and no significant effects are seen in the chain lattice of the bilayers. This result is interpreted as a dehydrating effect of sucrose upon the bilayer-water system at 35 degrees C. At 5 degrees C the interlamellar spacing is increased and this increase is, at high (70%) sucrose concentrations, attributable to an untilting of the lipid acyl chains with no change in the thickness of the aqueous layers in the multilamellae.

A systematic study of the translational diffusion of the phospholipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) has been undertaken in liquid crystalline phase phosphatidylcholine bilayers by using the fluorescence recovery after photobleaching technique. This work was done with the intention of comparing the experimental results with the predictions of theoretical models for diffusion in membranes. The following is shown. For NBD-PE, the dependence of the translational diffusion coefficient (Dt) upon the acyl chain length of the diffusant is not that predicted by continuum fluid hydrodynamic models for diffusion in membranes [Saffman, P.G., & Delbrueck, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113; Hughes, B. D., Pailthorpe, B. A., & White, L. R. (1981) J. Fluid Mech. 110, 349-372]. Plots of Dt vs. 1/T (Arrhenius plots) are nonlinear in dilauroyl-phosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers where the acyl chain composition of the NBD-PE is matched with that of the host bilayer lipid. This suggests that a "free volume" model may be appropriate for the description of lipid diffusion in lipid bilayers. In bilayers of phosphatidylcholines with saturated acyl chains at the same "reduced temperature", the magnitude of Dt follows the order distearoylphosphatidylcholine greater than DPPC greater than DMPC greater than DLPC. This is the inverse of what may be expected from the hydrodynamic model but is in agreement with the free volume in these bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

The technique of fluorescence recovery after photobleaching was used to investigate the lateral mobility of a fluorescein-labeled amphipathic apolipoprotein, ApoC-III, bound to multibilayers prepared from dipalmitoyl phosphatidylcholine, egg phosphatidylcholine, and a 1:1 (molar ratio) mixture of egg phosphatidylcholine and cholesterol. In dipalmitoyl phosphatidylcholine bilayers the lateral diffusion coefficient (D) for the protein is about 2 x 10(-9) cm(2) sec(-1) at 20 degrees C and about 9 x 10(-8) cm(2) sec(-1) at 45 degrees C. Plots of D versus temperature in this system show a transition between about 30 and 35 degrees C. Arrhenius activation energies for the diffusion in this case between 15 and 30 degrees C and between 35 and 45 degrees C are 28.5 and 7.0 kcal mol(-1), respectively (1 calorie = 4.18 joules). In egg phosphatidylcholine bilayers, D is about 3 x 10(-8) cm(2) sec(-1) at 20 degrees C and the Arrhenius activation energy for diffusion is 8.1 kcal mol(-1) between 15 and 35 degrees C in this system. In bilayers prepared from an equimolar mixture of egg phosphatidylcholine and cholesterol D at 20 degrees C is about 1.4 x 10(-9) cm(2) sec(-1) and the Arrhenius activation energy for the diffusion of the protein in this system between 15 and 35 degrees C is 15.1 kcal mol(-1). Light-scattering and fluorescence-polarization results indicate that binding of this protein does not affect the gel-to-liquid crystalline phase transition of bilayer membranes but does mediate a major, reversible aggregation of the vesicles at about 33 degrees C. These results lend support to the view that ApoC-III resides in the head-group region of the bilayer and suggest that its lateral diffusion coefficient represents an upper bound for integral membrane proteins.

The translational diffusion of the integral membrane sialoglycoprotein from erythrocyte membranes, glycophorin, incorporated into bilayer membranes of dimyristoyl-phosphatidylcholine at a protein/lipid molar ratio of 1:4500 was examined by using the fluorescence redistribution after photobleaching technique. A plot of the diffusion coefficient vs. temperature shows a sharp decrease in the rate of diffusion at about 15 degrees C. This sharp diffusion transition is at a temperature some 9 degrees C lower than the calorimetrically measured lipid gel-liquid crystalline phase transition temperature of the system. The difference between the diffusion transition temperature and the lipid phase transition temperature is attributed toi a localized fluidizing effect of the protein upon the gel phase lipid. The value of the diffusion coefficient above 15 degrees C was found to be (1-2) x 10(-8) cm(2)s(-1), and below 15 degrees C it was lower than about 5 x 10(-11)cm(2)s(-1). The fluorescence recovery in the bleached area as a consequence of diffusional redistribution appeared to be due to a single diffusing species at temperatures above 15 degrees C and due to more than one diffusing species below this temperature.

The two-dimensional connectivity is examined for mixed bilayers of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) as a function of composition and temperature at constant pressure using the fluorescence recovery after photobleaching (FRAP) method. These phospholipid mixtures exhibit peritectic behavior with a large region in which both gel and liquid crystalline phases coexist. Dilauroyl phosphatidylethanolamine covalently linked through the amino function in its head group to the fluorescent nitrobenzodiazolyl group (NBD-DLPE) was used as the fluorescent probe in this study, because it was found to partition almost exclusively in the liquid crystalline phase. The results of these studies show the line of connectivity to be close to the liquidus line on the phase diagram over a rather broad range of concentrations. In this range, a gel phase comprising approximately 20% of the system disconnects a liquid crystalline phase comprising 80% of the system. The implications of this result are discussed for domain shape and the organization of biological membrane components.

We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L alpha phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15 degrees and 45 degrees C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is approximately 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.