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The effects of the transmembrane alpha-helical peptide Ac-K(2)(LA)(12)K(2)-amide [(LA)(12)] on the molecular organization and dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes were investigated using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels, and the results were compared with our earlier, similar study of Ac-K(2)L(24)K(2)-amide (L(24)) [Subczynski, W. K., Lewis, R. N. A. H., McElhaney, R. N., Hodges, R. S., Hyde, J. S., and Kusumi, A. (1998) Biochemistry 37, 3156-3164]. At peptide-to-POPC ratios between 1/10 and 1/40, both methods (covering a time scale of 100 ps-10 micros) detect the presence of a single homogeneous membrane environment for both peptides, suggesting that these peptides are both well dispersed and that POPC is exchanging rapidly between the boundary and the bulk domains. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane, studied by saturation-recovery EPR, decreases by a factor of about 2 by including 10 mol % (LA)(12) whereas incorporating L(24) has practically no effect. (LA)(12) increases the alkyl chain order of POPC more than L(24). L(24) increases hydrophobicity (decreases the degree of water penetration into the hydrophobic region of the membrane) more than does (LA)(12). We ascribe the much stronger effects of (LA)(12) on membrane order and dynamics to the increased roughness of its hydrophobic surface and also to the increased motional freedom of its leucine side chains. In L(24), the leucine side chains are packed tightly, giving a smooth hydrophobic surface. In (LA)(12), they are separated by the small methyl groups of the alanine side chains, giving them additional motional freedom and the ability to protrude between the phospholipid hydrocarbon chains. The frequency of gauche-trans isomerization of hydrocarbon chains and concentration of vacant pockets (voids) in the lipid bilayer are thus reduced, which decreases oxygen transport. This explanation was confirmed by calculating the orientational order of leucine side chains in (LA)(12) and L(24) from molecular dynamics simulation studies.

The molecular organization and dynamics have been investigated in membranes consisting of 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC) and various ratios of a transmembrane alpha-helical peptide, Ac-K2L24K2-amide (L24), in order to gain insights into how the transmembrane portions of membrane proteins are mixed with phospholipids and organized in biological membranes. Particular attention was paid to membranes with high peptide concentrations. The molecular organization and dynamics were studied in the ps-to-micros regime using various spin-labeling techniques. Conventional ESR spectra as well as saturation-recovery curves measured in both the presence and the absence of molecular oxygen showed that phosphatidylcholine spin-labels detect the existence of a single homogeneous environment, indicating that both L24 and POPC are likely to be undergoing fast translational diffusion in L24-POPC membranes of up to 9 mol % peptide. Since 16-18 molecules of phosphatidylcholine are required to surround a transmembrane alpha-helical peptide [Morrow, M. R., Huschilt, J. C., and Davis, J. H. (1985) Biochemistry 24, 5396-5406], L24 must form L24-rich regions at a P/L ratio of 1/10 instantaneously. However, these results suggest that the lipid exchange rates among the bulk, boundary, and L24-rich regions are fast, and that the L24-rich regions must be forming and dispersing rapidly in a time scale shorter than 0.1 micros, the conventional ESR spin-label time scale and the electron spin-lattice relaxation time scale in the presence of molecular oxygen. Although this does not exclude the possibility of the formation of small, stable oligomers of L24, it is unlikely because L24 lacks features that would favor their formation. L24 (9 mol %) increases the hydrophobicity of the central part of the POPC membrane from the level of 1-decanol to that of pure hexane and also increases the hydrophobicity near the membrane surface from the level of 2-propanol to that of 1-decanol. The effect of 9 mol % L24 on the order parameter profile is similar to that of decreasing the temperature by approximately 8 degrees C between 10 and 55 degrees C. It is concluded that L24 is highly miscible in POPC membranes even at high concentrations in the membrane.

Profiles of the local nitric oxide (.NO) diffusion-concentration product across the egg yolk phosphatidylcholine membrane in the absence and presence of 30 mol% cholesterol were obtained using line-broadening electron paramagnetic resonance (EPR) and lipid-soluble nitroxide spin labels. Membrane .NO permeability coefficients were calculated from these profiles. At 20 degrees C, values of 93 and 77 cm/s for membranes in the absence and presence of cholesterol were obtained, compared with 73 and 66 cm/s for water layers of the same thickness as the membranes. Fluid-phase membranes are not barriers to .NO transport. Cholesterol significantly increases .NO transport in the center of the lipid bilayer.

The binding and state of aggregation of cecropin in large unilamellar vesicles of different surface potential and varying acyl chain length were examined using a Cys-33 spin-labeled derivative of cecropin AD (CAD). Association isotherms of the peptide were measured for vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) containing 5, 15, and 30 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). The isotherms display a concentration-dependent positive cooperativity indicating the possible formation of cecropin aggregates in the lipid phase. The critical aqueous concentration for aggregation was dependent on the fraction of POPG, suggesting the involvement of acidic lipids in the formation and stabilization of the putative aggregate. Our data also indicate that cooperativity depends on the state of side-chain ionization of an acidic residue that titrates between pH 7 and 4.4. The binding of spin-labeled Cys-33 CAD was found to be influenced by the acyl chain length of the host lipid. The association isotherm of the peptide for dilaureoyl-sn-glycero-3-phosphatidylcholine vesicles containing 30 mol % dilauroyl-sn-glycero-3-phosphatidylglycerol (DLPG) differed significantly from that in POPC/POPG and could be interpreted in terms of a monomer-monomer partitioning between the aqueous and lipid phases. ESR line-shape analysis was consistent with peptide aggregation in dioleoyl-sn-glycero-3-phosphatidylglycerol vesicles but not in DLPG vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport and diffusion of molecular oxygen in phosphatidylcholine (PC)-cholesterol membranes and their molecular mechanism were investigated. A special attention was paid to the molecular interaction involving unsaturated alkyl chains and cholesterol. Oxygen transport was evaluated by monitoring the bimolecular collision rate of molecular oxygen and the lipid-type spin labels, tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid. The collision rate was determined by measuring the spin-lattice relaxation times (T1's) in the presence and absence of molecular oxygen with long-pulse saturation-recovery ESR techniques. In the absence of cholesterol, incorporation of either a cis or trans double bond at the C9-C10 position of the alkyl chain decreases oxygen transport at all locations in the membrane. The activation energy for the translational diffusion of molecular oxygen in the absence of cholesterol is 3.7-6.5 kcal/mol, which is comparable to the activation energy theoretically estimated for kink migration or C-C bond rotation of alkyl chains [Träuble, H. (1971) J. Membr. Biol. 4, 193-208; Pace, R. J., & Chan, S. I. (1982) J. Chem. Phys. 76, 4241-4247]. Intercalation of cholesterol in saturated PC membranes reduces oxygen transport in the headgroup region and the hydrophobic region near the membrane surface but little affects the transport in the central part of the bilayer. In unsaturated PC membranes, intercalation of cholesterol also reduces oxygen transport in and near the headgroup regions. In contrast, it increases oxygen transport in the middle of the bilayer. On the basis of these observations, a model for the mechanism of oxygen transport in the membrane is proposed in which oxygen molecules reside in vacant pockets created by gauche-trans isomerization of alkyl chains and the structural nonconformability of neighboring lipids, unsaturated PC and cholesterol in particular, and oxygen molecules jump from one pocket to the adjacent one or move along with the movement of the pocket itself. The presence of cholesterol decreases oxygen permeability across the membrane in all membranes used in this work in spite of the increase in oxygen transport in the central part of unsaturated PC-cholesterol membranes because cholesterol decreases oxygen transport in and near the headgroup regions, where the major barriers for oxygen permeability are located. Oxygen gradients across the membranes of the cells and the mitochondria are evaluated. Arguments are advanced that oxygen permeation across the protein-rich mitochondrial membranes can be a rate-limiting step for oxygen consumption under hypoxic conditions in vivo.

Saturated and unsaturated phosphatidylcholine (PC)-cholesterol membranes have been studied, with a special attention paid to fluid-phase immiscibility in cis-unsaturated phosphatidylcholine (PC)-cholesterol membranes as previously proposed and to the three-dimensional structure of the membrane. The investigation was carried out with dual probes: a membrane-soluble, square-planar copper complex, (3-ethoxy-2-oxobutyraldehyde bis(N4,N4-dimethylthiosemicarbazonato]copper(II) (CuKTSM2), and one of several nitroxide radical lipid-type spin-labels. Bimolecular collision rates between metal ion and spin-label were determined by measuring the nitroxide spin-lattice relaxation times (T1's) in the presence and absence of CuKTSM2 by use of saturation-recovery ESR techniques, and from these measured rates, translational diffusion coefficients of CuKTSM2 were estimated. Profiles of the collision rate across the membrane bilayer were obtained with Tempocholine phosphatidic acid ester, 5-doxylstearic acid, 16-doxylstearic acid, and cholesterol-type spin-labels as a function of cholesterol mole fraction, length and unsaturation of acyl chains, and temperature. In the liquid-crystalline phase of saturated PC membranes, incorporation of cholesterol decreases the collision rate at all depths in the membrane, and the effect of cholesterol is smallest in the middle of the bilayer. In trans-unsaturated PC membranes, a cholesterol-induced decrease of the collision rate was also observed, except in the head-group regions. In cis-unsaturated PC membranes, virtually no effect of cholesterol was observed on the collision rate, either with phospholipid-type spin-labels or with cholesterol-type spin-labels. This result is in clear contrast with our previous observation, in which the effect of cholesterol in cis-unsaturated PC membranes is small on the alkyl-chain motion of phospholipid-type spin-labels but large on the wobbling rotational diffusion of cholesterol-type spin-labels [Pasenkiewicz-Gierula, M., Subczynski, W. K., & Kusumi, A. (1990) Biochemistry 29, 4059-4069]. A model is proposed to explain these results in which the fluid-phase immiscibility is prevalent in cis-unsaturated PC-cholesterol membranes, but where cholesterol-rich (cholesterol oligomeric) domains are small (several lipids) and/or of short lifetime (10(-9) s to less than 10(-7) s). It is suggested that this microimmiscibility arises from the structural nonconformability between the rigid cholesterol ring structure and the rigid bend at the cis double bonds in PC alkyl chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Melittin spin-labeled specifically with a nitroxide at positions 7, 21, 23, or the amino terminus was bound to phospholipid membranes, and the exposure of the spin label to the aqueous phase was investigated by measurement of Heisenberg exchange with chromium oxalate in the solution. The exchange frequency was determined by saturation recovery electron paramagnetic resonance (EPR) using a loop-gap resonator. This method allows use of very low concentrations (less than 1 mM) of chromium oxalate compared with conventional measurements of EPR line broadening (typically 50 mM), thus avoiding problems associated with high metal ion concentration. Differences in exchange frequency between the various positions were also estimated by continuous wave power saturation methods. In either approach, the spin label at lysine 7 was found to be the most exposed to chromium oxalate whereas that at lysine 23 was found to be the least exposed. This is consistent with a model for the membrane bound peptide in which an amphiphilic helix lies with its axis parallel to the bilayer surface and the hydrophobic moment points toward the bilayer interior.

Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane.

The concentration of oxygen in the hydrocarbon region of lipid bilayer has been determined using a novel electron spin resonance (ESR) nitroxide-radical spin-probe method. For dimyristoylphosphatidylcholine (DMPC), the partition coefficient above the main transition temperature is approximately 3. Rapid decrease to 0.2 occurs below the pretransition temperature indicating exclusion of oxygen in the crystalline phase. The differences of molar free energy, enthalpy, and entropy of mixing between water and lipid have been determined for each phase.

Oxygen transport in phosphatidylcholine-cholesterol membranes has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed at various distances from the membrane surface using long-pulse saturation recovery ESR techniques. The collision rate was estimated for tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameter across the membrane were obtained as a function of cholesterol mol fraction and temperature in L-alpha-dimyristoylphosphatidylcholine ([ Myr2]PtdCho) and L-alpha-dioleoylphosphatidylcholine ([ Ole2]PtdCho) membranes. Membrane oxygen permeability coefficients were estimated from oxygen transport parameter profiles. At approximately 30 degrees C, the oxygen permeability coefficients in the presence and absence of 50 mol % cholesterol are 22.7 and 125.2 cm/s, respectively, for [Myr2]PtdCho membranes, and 54.7 and 114.2 cm/s, respectively, for [Ole2]PtdCho membranes (compared with 60-80 cm/s for water layers with the same thicknesses as the membranes). The major results in the liquid-crystalline phase are as follows: (i) In the absence of cholesterol, membranes are not barriers to oxygen transport. (ii) Addition of 50 mol % cholesterol decreases oxygen permeability by a factor of approximately 5 and approximately 2.5 in [Myr2]PtdCho and [Ole2]PtdCho membranes, respectively. The resistance to oxygen transport is located in and near the polar headgroup regions in the membrane. (iii) Cholesterol increases oxygen transport in the central regions of [Ole2]PtdCho membranes.

Lateral diffusion constants of the stearic acid nitroxide radical spin label 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolindinyl oxide in dispersions of dimyristoyl phosphatidylcholine have been measured. Electron-electron double resonance methods were used to determine the product of the bimolecular collision frequency and T1e, the electron spin-lattice relaxation time. T1e in turn was measured by the technique of saturation recovery. The theoretical model of Träuble and Sackmann was then used to relate the bimolecular collision frequencies to the diffusion constants. Results are in agreement with other methods. Lower spin-label concentrations than were used in previous electron paramagnetic resonance studies are needed (label-to-lipid ratio less than 0.5 mol%). Analysis of the data also yields values of the nitrogen nuclear spin-lattice relaxation time of the nitroxide moiety. These values are indicative of membrane fluidity.