Lateral diffusion of cholesterol and dimyristoylphosphatidylcholine in a lipid bilayer measured by pulsed field gradient NMR spectroscopy

The pulsed field gradient NMR method for measuring self-diffusion has been used for a direct determination of the lateral diffusion coefficient of cholesterol, fluorine labeled at the 6-position, for an oriented lamellar liquid-crystalline phase of dimyristoylphosphatidylcholine (DMPC)/cholesterol/water. It is found that the diffusion coefficients of DMPC and cholesterol are equal over a large temperature interval. The apparent energy of activation for the diffusion process (58 kJ/mol) is about the same as for a lamellar phase of DMPC/water, whereas the phospholipid lateral diffusion coefficient is approximately four times smaller in the presence of cholesterol.     

Membrane topology of a 14-mer model amphipathic peptide: a solid-state NMR spectroscopy study

We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31P, 2H, 15N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The 31P, 2H, and 15N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15N{31P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness.     

The effects of bilirubin on the thermal properties of phosphatidylcholine bilayers.

The thermotropic properties of multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC), as a function of the concentration of bilirubin in the range of 0.1 to 1 mol%, were measured. The exact effects of bilirubin depended on the chain length of the polymethylene chains. But the general effects of bilirubin were the same in all systems. At the lowest concentrations tested (0.1 mol bilirubin/100 mol phospholipid (0.1 mol%)), bilirubin broadened and shifted to higher temperatures the main phase transitions of all bilayers. For DPPC and DSPC, but not DMPC, this concentration of bilirubin was associated with a new transition at 25 degrees C (DPPC) or 34 degrees C (DSPC). Bilirubin at 0.2 mol% was required for the detection of a similar transition (at 13.7 degrees C) in DMPC. Higher concentrations of bilirubin (> 0.2 mol%) suppressed completely the main phase transitions in all bilayers but increased the enthalpy of the new transition. Maximal values of delta H for these transitions were reached at 0.5, 0.25, and 0.2 mol% bilirubin in DMPC, DPPC, and DSPC, respectively. Values of delta H and delta S for these transitions were far larger than for the corresponding gel-to-liquid crystal transitions in pure lipid bilayers but were equal to those expected for a transition between crystalline and liquid crystalline phases.     

Two-dimensional diffusion of amphiphiles in phospholipid monolayers at the air-water interface.

Steady-state and time-resolved fluorescence spectroscopy has been used to examine lateral diffusion in dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) and dimyristoyl-L-alpha-phosphatidylcholine (DMPC) monolayers at the air-water interface, by studying the fluorescence quenching of a pyrene-labeled phospholipid (pyrene-DPPE) by two amphiphilic quenchers. Steady-state fluorescence measurements revealed pyrene-DPPE to be homogeneously distributed in the DMPC lipid matrix for all measured surface pressures and only in the liquid-expanded (LE) phase of the DPPC monolayer. Time-resolved fluorescence decays for pyrene-DPPE in DMPC and DPPC (LE phase) in the absence of quencher were best described by a single-exponential function, also suggesting a homogeneous distribution of pyrene-DPPE within the monolayer films. Addition of quencher to the monolayer film produced nonexponential decay behavior, which is adequately described by the continuum theory of diffusion-controlled quenching in a two-dimensional environment. Steady-state fluorescence measurements yielded lateral diffusion coefficients significantly larger than those obtained from time-resolved data. The difference in these values was ascribed to the influence of static quenching in the case of the steady-state measurements. The lateral diffusion coefficients obtained in the DMPC monolayers were found to decrease with increasing surface pressure, reflecting a decrease in monolayer fluidity with compression.     

A proton NMR moment study of the gel and liquid-crystalline phases of dipalmitoyl phosphatidylcholine

Measurements of the proton second and fourth moments have been undertaken for multilamellar dispersions, in excess water, of protiated and chain-perdeuterated dipalmitoyl phosphatidylcholine (DPPC) in the temperature range -20 to 50 degrees C. The comparison of the measured moment with the rigid lattice M2 and the calculated second moment values in the presence of certain motions gives insight into the dynamic structure of the methylene chains of DPPC. This study demonstrates that at -15 degrees C there is still a significant amount of methylene chain motion or disorder in DPPC. At 35 degrees C the moment values indicate that the methylene chains are not in the fully extended all-trans conformation and they may also be rotationally disordered. At the pretransition there is a decrease in magnitude of the proton second moments, which can be accounted for by an increase in the lateral diffusion rate to a value greater than 10(-11) cm2/s. This work suggests that at temperatures just below the main transition the chain conformation is considerably different from the common model structure (P beta') in which the chains are fully extended. In the liquid-crystalline phase the proton moment data are in agreement with data from other techniques on the liquid-like nature of the methylene chains. It is demonstrated how the values of the moment ratio M4r/(M2r)2, which are relatively constant within each phase, can be used to calculate the molar fractions of coexisting liquid-crystalline and gel-phase phospholipid at temperatures near the main transition.     

Membrane electroporation--fast molecular exchange by electroosmosis.

Human and rabbit erythrocyte ghosts loaded with FITC-dextran (mol. mass = 10 kDa) and NBD-glucosamine (mol. mass = 342 Da) in buffers of different ionic strength and composition were subjected to electric pulses (intensity 0.7 kV/mm and decay half-time 1 ms) at 7-10 degrees C and 20-24 degrees C. The transfer of the fluorescent dyes from the interior of the ghosts through the electropores was observed by low light level video microscopy. The pulses caused the fluorescence to appear outside the membranes as a transient cylindrical cloud directed toward the negative electrode during the first video frame (17 ms). It was similar in both rabbit and human erythrocyte ghosts and at both temperatures but differs for the two dyes, the fluorescence cylinder is long and tall for the FITC-dextran and relatively short and thick for the NBD-glucosamine. The molecular exchange was 2-3 orders of magnitude faster within the first 17 ms after the pulse than the diffusional exchange. It decreased with increasing ionic strength. Formulae for the transfer of molecules by electroosmotic flow through the pores are in agreement with these observations. They allow estimation of the total area of pores with radii larger than that of the fluorescent dye during the pulse. The major conclusion is that electroosmosis is the dominating mechanism of molecular exchange in electroporation of erythrocyte ghosts.     

Lateral distribution of a pyrene-labeled phosphatidylcholine in phosphatidylcholine bilayers: fluorescence phase and modulation study

The lateral distribution of 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl]phosphatidylcholine (PyrPC) was studied in small unilamellar vesicles of 1,2-dipalmitoyl-, 1,2-dimyristoyl-, and 1-palmitoyl-2-oleoyl-phosphatidylcholine (DPPC, DMPC, and POPC, respectively) under anaerobic conditions. The DPPC and DMPC experiments were carried out over temperature ranges above and below the matrix phospholipid phase transition temperature (Tm). The excimer to monomer fluorescence intensity ratio (E/M) was determined as a function of temperature for the three PyrPC/lipid mixtures. Phase and modulation data were used to determine the temperature dependence of pyrene fluorescence rate parameters in gel and in liquid-crystalline bilayers. These parameters were then used to provide information about excited-state fluorescence in phospholipid bilayers, calculate the concentration of the probe within liquid-crystalline and gel domains in the phase transition region of PyrPC in DPPC, and simulate E/M vs. temperature curves for three systems whose phase diagrams are different. From the simulated curves we could determine the relationship between the shape of the three simulated E/M vs. temperature curves and the lateral distribution of the probe. This information was then used to interpret the three experimentally derived E/M vs. temperature curves. Our results indicate that PyrPC is randomly distributed in pure gel and fluid phosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnetic alignment and orientational order of dipalmitoylphosphatidylcholine bilayers containing palmitoyllysophosphatidylcholine

Mixed bilayers of 1-palmitoyl-sn-glycero-3-phosphocholine (palmitoyllysophosphatidylcholine; PaLPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dipalmitoyl phosphatidylcholine; DPPC) have been investigated by 2H-NMR and 31P-NMR spectroscopy. Binary phospholipid mixtures were studied in which the acyl chains of one or the other component were perdeuterated. At temperatures below the main order-disorder phase transition, the mixed PaLPC/DPPC bilayers appear to coexist with PaLPC micelles. The micelles disappear at temperatures above the phase transition, where mixed bilayers in the liquid-crystalline state are formed. The orientational order of the alkyl chains of the PaLPC component is essentially identical to that of the DPPC component in the mixed bilayers, both in the low temperature and liquid-crystalline phases. However, the presence of PaLPC perturbs the segmental ordering of DPPC as compared to the pure system. The order is increased in the low-temperature phase, where effective diffusion of the chains about their long axes occurs, but is decreased in the liquid-crystalline phase compared to pure DPPC bilayers. The mixed liquid-crystalline bilayers orient preferentially with their director axes perpendicular to the magnetic field. This alignment is easily observed in 31P- and 2H-NMR spectra, where the intensity of the perpendicular edges of the lineshapes is pronounced. One possible explanation of the magnetic alignment involves alteration of the curvature free energy of the DPPC bilayer due to incorporation of PaLPC in the mixed membranes.     

Hydrostatic pressure induces hydrocarbon chain interdigitation in single-component phospholipid bilayers

By use of neutron diffraction for structural analysis, the temperature-pressure phase diagrams of several fully hydrated single-component phospholipid bilayers have been explored up to hydrostatic pressures of 2 kbars. The gel to liquid-crystalline phase transition temperature Tm increases linearly with pressure over a 10(-3)-2 kbar range in accordance with the Clausius-Clapeyron relationship giving dTm/dP values of 23.0 degrees C/kbar for 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 28.0 degrees C/kbar for 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The so-called pretransition was not observed in the isothermal pressure experiments, suggesting that no appreciable volume change occurs at this transition. These results are in good agreement with those reported using other techniques. In addition, at pressures higher than the isothermal liquid-crystalline to gel transition pressure, a new pressure-induced phase transition was observed for DPPC and DSPC in which the hydrocarbon chains from apposing monolayers become interdigitated with the chains occupying a cross-sectional area approximately equal to 5% less than in the gel phase. The temperature-pressure phase diagrams show the gel-interdigitated phase boundaries to be highly curved and the minimum pressure at which interdigitation occurs to decrease with increasing hydrocarbon chain length.     

Phospholipid miscibility in ternary mixtures

The gel to liquid-crystalline phase transition of aqueous dispersions of phospholipid mixtures was investigated by means of the repartition of the spin label 2,2,6,6-tetramethylpiperidine-I-oxyl between aqueous space and lipid hydrocarbon region. The dimyristoylphosphatidylcholine (DMPC)/dibehenoylphosphatidylcholine (DBPC) and dipalmitoylphosphatidylcholine (DPPC)/DBPC phase diagrams indicate gel phase immiscibility, whereas the distearoylphosphatidylcholine (DSPC)/DBPC phase diagram indicates non-ideal gel phase miscibility at low DBPC molar fractions. Aqueous dispersions of DMPC/DPPC/DBPC ternary mixtures show two distinct phase transitions, the first associated with the melting of a DMPC/DPPC phase and the second with the melting of a DBPC phase. Aqueous dispersions of DMPC/DSPC/DBPC ternary mixtures show to phase transitions at low DSPC molar fractions; the first is probably associated with the melting of a DMPC/DSPC phase, and the second with the melting of a DBPC/DSPC phase. At high DSPC molar fractions, only one phase transition is observed; this suggests that all the lipids are mixed in gel state membranes.     

Dipyrenylphosphatidylcholines as membrane fluidity probes. Pressure and temperature dependence of the intramolecular excimer formation rate.

We have measured the pressure dependence of the intramolecular excimer formation rate, K(p), for di-(1'-pyrenedecanoyl)-phosphatidylcholine (dipy10PC) probes in single-component lipid multilamellar vesicles (MLV) as a function of temperature. Apparent volumes of activation (V(a)) for intramolecular excimer formation are obtained from the slopes of plots of log K(p) versus P. For liquid-crystalline saturated lipid MLV (DMPC and DPPC), these plots are linear and yield a unique V(a) at each temperature, whereas for unsaturated lipids (POPC and DOPC) they are curvilinear and V(a) appears to decrease with pressure. The isothermal pressure induced phase transition is marked by an abrupt drop in the values of K(p). The pressure to temperature equivalence values, dPm/dT, estimated from the midpoint of the transitions, are 47.0, 43.5, and 52.5 bar degree C-1 for DMPC, DPPC, and POPC, respectively. In liquid-crystalline DMPC, V(a) decreases linearly as a function of temperature, with a coefficient -dVa/dT = 0.65 +/- 0.11 ml degree C-1 mol-1. Using a modified free volume model of diffusion, we show that this value corresponds to the thermal expansivity of DMPC. Both the apparent energy and entropy of activation, Ea and delta Sa, increase with pressure in DMPC, whereas both decrease in POPC and DOPC. This difference is attributed to the sensitivity of the dynamics and/or packing of the dipy10PC probes to the location of the cis-double bonds in the chains of the unsaturated host phospholipids. Finally, the atmospheric pressure values of Ea and delta Sa for the four host MLV examined are shown to be linearly related. The relevance of this finding with respect to the structure of the excimers formed by the dipy10PC probes is briefly discussed.     

Phase transition behavior of single phosphatidylcholine bilayers on a solid spherical support studied by DSC, NMR and FT-IR

For the first time, the chain melting transition from the gel phase to the liquid crystalline phase of a single DPPC bilayer on a solid, spherical support (silica beads) is observed by differential scanning calorimetry (DSC). This transition is remarkably cooperative, exhibits a transition temperature T_m which is 2°C lower than usually found for DPPC multilamellar vesicles and its excess enthalpy is about 25% less than in DPPC multilayers. 31P- and 2H-NMR data as well as FT-IR data provide evidence that despite the highly asymmetric characteristic of the model system, the whole single bilayer undergoes the transition at T_m, i.e., there is no decoupling of the two monolayer leaflets at the main phase transition. Furthermore, our results show that the formation of the ripple (P_β') phase is inhibited in single bilayers on a solid support. This result confirms a conclusion which we reached previously on the basis of neutron scattering data obtained on planar supported bilayers. The most likely reason for this inhibition as well as for the above mentioned thermodynamic differences between multilamellar vesicles and supported membranes is a significantly higher lateral stress in the latter. Moreover, the exchange of lipids between two populations of spherical supported vesicles (DMPC and chain perdeuterated DMPC) is studied by DSC. It is shown that this exchange process is symmetric and its half-time is a factor of 3-4 higher than observed for small sonicated DMPC vesicles.     

Interaction of pulmonary surfactant protein SP-A with DPPC/egg-PG bilayers

In the mixture of lipids and proteins which comprise pulmonary surfactant, the dominant protein by mass is surfactant protein A (SP-A), a hydrophilic glycoprotein. SP-A forms octadecamers that interact with phospholipid bilayer surfaces in the presence of calcium. Deuterium NMR was used to characterize the perturbation by SP-A, in the presence of 5 mM Ca²⁺, of dipalmitoyl phosphatidylcholine (DPPC) properties in DPPC/egg-PG (7:3) bilayers. Effects of SP-A were uniformly distributed over the observed DPPC population. SP-A reduced DPPC chain orientational order significantly in the gel phase but only slightly in the liquid-crystalline phase. Quadrupole echo decay times for DPPC chain deuterons were sensitive to SP-A in the liquid-crystalline mixture but not in the gel phase. SP-A reduced quadrupole splittings of DPPC choline β-deuterons but had little effect on choline α-deuteron splittings. The observed effects of SP-A on DPPC/egg-PG bilayer properties differ from those of the hydrophobic surfactant proteins SP-B and SP-C. This is consistent with the expectation that SP-A interacts primarily at bilayer surfaces.     

Ion permeation across the bilayer of annealed phosphatidylcholine vesicles at elevated temperatures. Concentration dependence and the micelle-bilayer dynamic equilibrium

The relative stability of the lipid bilayer toward ions above the crystalline to liquid-crystalline phase transition temperature has been studied under isotonic conditions for small annealed vesicles of dilauroyl (DLPC), dimyristoyl (DMPC), diplamitoyl (DPPC), and distearoyl (DSPC) phosphatidylcholine by using lanthanide ions as a probe. The bilayer stability increased as the chain length of the lipid fatty acid increased, and a rapid translocation of ions across the bilayer started at about 60, 70, and 80° C for DMPC, DPPC, and DSPC vesicles, respectively. The bilayer of DLPC vesicles is apparently permeable for the tested ions even at room temperature. Two other important phenomena concomitant with the observed translocation of ions were found. Firstly, the ion leakage occurred in an “an-or-none” fashion, i.e. as soon as the vesicles start to become permeable toward ions, the concentration of ions in the intra-and extravesicular media are equalized within a short time. Secondly, the rate of the relative number of inward facing lipid molecules which become exposed to extravesicularly added paramagnetic lanthanide is a function of the inverse phosphatidylcholine concentration. This feature explicitly excludes the possibilities that the observed ion leakage occurs through a diffusion, pore formation, or through the rupture of vesicle walls induced by vesicle-vesicle collisions. We instead propose as the most probable mechanism that a dynamic equilibrium between the various states of the phosphatidylcholine molecules in water, such as monomers, micelles, vesicles, and multilamellar liposomes, is in fact responsible for the observed phenomena.     

Slow motions in lipid bilayers. Direct detection by two-dimensional solid-state deuterium nuclear magnetic resonance.

Two-dimensional solid-state 2H NMR spectroscopy of specifically deuteriated lipids is used to detect and to characterize the rate and mode of slow motions in two lipid bilayer systems. Lateral diffusion of lipid molecules over the curved surface of dipalmitoylphosphatidylcholine liposomes can be detected by two-dimensional exchange 2H NMR and it is shown that molecular orientational exchange is complete on the timescale of 100 ms. In contrast, it is shown that for the glycolipid 1,2-di-O-tetradecyl-3-O-Beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL), there is no evidence of a corresponding orientational exchange in the liquid-crystalline phase suggesting that this lipid forms relatively flat bilayers. In the gel phase of hydrated multibilayers of beta-DTGL, a slow (10(3) s(-1)) whole molecule axial motion is demonstrated at 40 degrees C. Comparison of the experimental and simulated 2D-NMR ridge patterns suggests that large angle jumps about the long molecular axis, rather than small step Brownian diffusion, can best account for the 2D-exchange spectra of beta-DTGL in the gel phase. The significance of this technique for the study of dynamics in other biological systems is discussed.     

Interactions of saturated diacylglycerols with phosphatidylcholine bilayers: A 2H NMR study.

The interactions of a series of saturated diacylglycerols (DAGs) with fatty acid side chain lengths of 6-14 carbons with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts were studied by 2H NMR spectrometry. We found that the perturbation induced by the DAGs into the bilayer structure strongly depends on the length of the DAG fatty acid side chain. Shorter chain 1,2-sn-dihexanoylglycerol and, to a larger degree, 1,2-sn-dioctanoylglycerol (diC8) induce transverse perturbation of the bilayer structure: the order parameters of the phospholipid side chains are increased by the intercalating DAG molecules in the region adjacent to the phospholipid headgroups and decreased toward the terminal methyls, corresponding to the bilayer interior. The longer chain DAGs (C greater than or equal to 12) studied in this and previous [De Boeck & Zidovetzki (1989) Biochemistry 28, 7439] work induce lateral phase separation of the lipids into DAG-enriched gellike domains and relatively DAG-free regions in the liquid-crystalline phase. Each of the DAGs studied induces a decrease in the area per phospholipid molecule, and a corresponding increase in the lateral surface pressure of the bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

Rapid diffusion of spectrin bound to a lipid surface.

Human erythrocyte spectrin was labelled with the probe 5, 5'-disulfato-1-(6-hexanoic acid N-hydroxysuccinimide ester)-1'-ethyl-3,3,3',3'-tetramethylindocarbocyanine (Cy3). Cy3-spectrin was bound to the outer surface of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and its diffusion measured by fluorescence recovery after photobleaching (FRAP). It was found that at 30 degrees C, above the lipid gel to liquid-crystalline phase transition of the lipids, Cy3-spectrin had an unexpectedly high diffusion coefficient D=(2.1+/-0.6)x10(-7)) cm2/s. At the phase transition, diffusion of Cy3-spectrin was only slightly lower; D=(1.3+/-0.3)x10(-7) cm2/s, whereas at 14 degrees C, well below the lipid phase transition, diffusion was found to be much slower with D=(3.1+/-0.12)x10(-9) cm2/s. The fast diffusion of Cy3-spectrin on the lipid surface implies that the individual bonds which bind spectrin to the lipid surface must rapidly be made and broken. In the light of these results, spectrin-lipid interactions alone appear unlikely to have any significant role in supporting the cell membrane. Probably, the interactions serve only to localise the spectrin at the inner lipid surface in order to facilitate formation of the cytoskeleton.     

Cholesterol dynamics in membranes.

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.     

Interaction between the 35 kDa apolipoprotein of pulmonary surfactant and saturated phosphatidylcholines. Effects of temperature

We studied the interaction between the 35 kDa apolipoprotein of canine pulmonary surfactant (SP 35) and five saturated phosphatidylcholines: distearoyl (DSPC), diheptadecanoyl (DHPC), dipalmitoyl (DPPC), dimyristoyl (DMPC), and dilauroyl (DLPC); and two monoenoic unsaturated phosphatidylcholines: dioleoyl (DOPC) and dielaidyl (DEPC), using temperatures at which all of the phospholipids except DOPC were in both the gel and liquid-crystalline states. The experiments were carried out in a buffer without Ca2+. The amount of apolipoprotein which was bound by both small unilamellar and multilayered vesicles of these lipids decreased as the temperature was increased. Moreover, near the temperatures of the phase transitions of all lipids except DLPC, there was an abrupt and marked reduction in binding of protein, in that over a 3-4 degree change in temperature there was an abrupt decrease in bound apolipoprotein. A similar change in binding occurred using DLPC, although the relatively large changes in bound protein occurred at about 10 and 20 degrees C, temperatures which are above the phase transition temperature of this lipid. Experiments using DOPC were limited to temperatures above the phase transition, and apolipoprotein binding was low. Experiments monitoring the intrinsic fluorescence of the protein, and the fluorescence of bis-1-anilino-8-naphthalene sulfonic acid bound to the protein, revealed a possible conformational change at about 40 degrees C. Measurement of intrinsic fluorescence provided the same result whether or not the protein was associated with lipid. DSC of the apolipoprotein indicated that this change was not associated with a measurable thermogenic process. We found that the interaction with DPPC was reversible at 42 degrees C, and we measured the thermodynamic parameters of the interaction at this temperature. These were: delta G0 = -8.0 kcal/mol apolipoprotein; delta H0 = -88 kcal/mol; delta S0 = -254 cal/Cdeg per mol. We conclude that the interaction between SP 35 and saturated phosphatidylcholines is temperature sensitive, and this probably reflects differences in the ability of gel and liquid-crystalline phospholipids to bind this protein. Both the delta H0 and delta S0 of the interaction are negative, and may reflect an immobilization of phospholipid around the apolipoprotein to form a boundary layer. This hypothesis is consistent with the findings obtained by DSC, in which the enthalpy of the phase transition of DMPC in lipid-apolipoprotein recombinants was found to be about 60% of that expected for a pure and unperturbed multilamellar dispersion.