Visualization of highly ordered striated domains induced by transmembrane peptides in supported phosphatidylcholine bilayers

We used atomic force microscopy (AFM) to study the lateral organization of transmembrane TmAW(2)(LA)(n)W(2)Etn peptides (WALP peptides) incorporated in phospholipid bilayers. These well-studied model peptides consist of a hydrophobic alanine-leucine stretch of variable length, flanked on each side by two tryptophans. They were incorporated in saturated phosphatidylcholine (PC) vesicles, which were deposited on a solid substrate via the vesicle fusion method, yielding hydrated gel-state supported bilayers. At low concentrations (1 mol %) WALP peptides induced primarily line-type depressions in the bilayer. In addition, striated lateral domains were observed, which increased in amount and size (from 25 nm up to 10 microm) upon increasing peptide concentration. At high peptide concentration (10 mol %), the bilayer consisted mainly of striated domains. The striated domains consist of line-type depressions and elevations with a repeat distance of 8 nm, which form an extremely ordered, predominantly hexagonal pattern. Overall, this pattern was independent of the length of the peptides (19-27 amino acids) and the length of the lipid acyl chains (16-18 carbon atoms). The striated domains could be pushed down reversibly by the AFM tip and are thermodynamically stable. This is the first direct visualization of alpha-helical transmembrane peptide-lipid domains in a bilayer. We propose that these striated domains consist of arrays of WALP peptides and fluidlike PC molecules, which appear as low lines. The presence of the peptides perturbs the bilayer organization, resulting in a decrease in the tilt of the lipids between the peptide arrays. These lipids therefore appear as high lines.     

Phase separation in dipalmitoyl phosphatidylcholine bilayers induced by ionophores and binary electrolytes

Thermotropic behavior of unsonicated aqueous dispersion of dipalmitoyl phosphatidylcholine (DPPC) has been studied by scanning microcalorimetry and fluorescent probe method. Phase separation in the lipid bilayers was observed for systems containing ionophores (valinomycin, dinactin) and 1 : 1 electrolytes (NaCl, KCl, RbCl, CsCl). The ratio of lipid phases coexisting in the systems appeared to be dependent on the concentration of the electrolytes. Changes in the thermotropic properties of the lipid phase induced by valinomycin were observed when K+ and Rb+ ions-forming complexes with the ionophore were present in the systems. The latter phenomenon was not found for the systems containing dinactin possessing a lower ability for complex formation with the cations.     

Strength of integration of transmembrane alpha-helical peptides in lipid bilayers as determined by atomic force spectroscopy

In this study we address the stability of integration of proteins in membranes. Using dynamic atomic force spectroscopy, we measured the strength of incorporation of peptides in lipid bilayers. The peptides model the transmembrane parts of alpha-helical proteins and were studied in both ordered peptide-rich and unordered peptide-poor bilayers. Using gold-coated AFM tips and thiolated peptides, we were able to observe force events which are related to the removal of single peptide molecules out of the bilayer. The data demonstrate that the peptides are very stably integrated into the bilayer and that single barriers within the investigated region of loading rates resist their removal. The distance between the ground state and the barrier for peptide removal was found to be 0.75 +/- 0.15 nm in different systems. This distance falls within the thickness of the interfacial layer of the bilayer. We conclude that the bilayer interface region plays an important role in stably anchoring transmembrane proteins into membranes.     

The effects of hydrophobic mismatch between phosphatidylcholine bilayers and transmembrane alpha-helical peptides depend on the nature of interfacially exposed aromatic and charged residues

In this study, we investigated the extent to which different aromatic and positively charged side chains, which often flank transmembrane segments of proteins, can influence lipid-peptide interactions. Model systems consisting of phosphatidylcholine and hydrophobic alpha-helical peptides with different flanking residues were investigated. The peptides were incorporated in relatively thick and in relatively thin lipid bilayers to create a peptide-bilayer hydrophobic mismatch, and the compensating effects on lipid structure were analyzed. When relatively long with respect to the thickness of the bilayer, the peptides that are flanked by the aromatic side chains, Trp, Tyr, and Phe, all induce a significant ordering of the lipid acyl chains, while the peptides flanked by the charged residues Lys, Arg, and His do not. However, when the peptides are relatively short with respect to the thickness of the bilayer, their effect on lipid organization does not depend primarily on their aromatic or charged character. Peptides flanked by Trp, Tyr, Lys, or (at low pH) His residues are effective in inducing mismatch-relieving cubic and inverted hexagonal phases, while analogues flanked by Phe, Arg, or (at neutral pH) His residues cannot induce an inverted hexagonal phase. The different responses to mismatch might reflect the different interfacial affinities of the residues that were investigated.     

Fatty-acid chain tilt angles and directions in dipalmitoyl phosphatidylcholine bilayers.

X-ray diffraction has been applied to determine the various tilt angles and directions (if any) which can be assumed by oriented gel phase multilayers of dipalmitoyl phosphatidylcholine (DPPC) as a function of hydration. We report for the first time that oriented DPPC multilayers with a repeat spacing (d-spacing) of 55.2A at 25 degrees C and 0% relative humidity (RH) have hydrocarbon chains tilted at an angle theta of 21.5 degrees with respect to the bilayer normal. In addition, the chains are tilted along one of the bisectors (omega = 0 degrees) of the hexagonal lattice (8 wide-angle maxima, 2 unique), a phase not previously reported in DPPC studies. At 100% RH, the chain tilt angle and d-spacing increased to approximately 29.0 degrees and 58.9A, respectively. Since at 100% RH only 4 wide-angle maxima are observed, we analyze the data on the assumption that the hydrocarbon chains may rotate independently of the hexagonal lattice (omega = 0-30 degrees), at a fixed chain tilt angle theta (Stamatoff, J.B., et al. 1979. Biophys. J. 25:253-262). The largest observed angle phi made by the wide-angle maxima with the equator is 29.5 degrees corresponding to a theta of approximately 32.6 degrees (omega avg. = 24 degrees) and the sample having a d-spacing of 64.0 A (excess water condition). Finally, theta remains relatively constant (approximately 21.5 degrees) up to a RH of approximately 45% and a d-spacing of 57.8A, after which, with increases in RH, theta increases to a maximum of 32.6 degrees.     

Domain formation in phosphatidylcholine bilayers containing transmembrane peptides: specific effects of flanking residues

Lateral segregation in biological membranes leads to the formation of domains. We have studied the lateral segregation in gel-state model membranes consisting of supported dipalmitoylphosphatidylcholine (DPPC) bilayers with various model peptides, using atomic force microscopy (AFM). The model peptides are derivatives of the Ac-GWWL(AL)(n)WWA-Etn peptides (the so-called WALP peptides) and have instead of tryptophans, other flanking residues. In a previous study, we found that WALP peptides induce the formation of extremely ordered, striated domains in supported DPPC bilayers. In this study, we show that WALP analogues with other uncharged residues (tyrosine, phenylalanine, or histidine at pH 9) can also induce the formation of striated domains, albeit in some cases with a slightly different pattern. The WALP analogues with positively charged residues (lysine or histidine at low pH) cannot induce striated domains and give rise to a completely different morphology: they induce irregularly shaped depressions in DPPC bilayers. The latter morphology is explained by the fact that the positively charged peptides repel each other and hence are not able to form striated domains in which they would have to be in close vicinity. They would reside in disordered, fluidlike lipid areas, appearing below the level of the ordered gel-state lipid domains, which would account for the irregularly shaped depressions.     

Differential scanning calorimetry and (2)H nuclear magnetic resonance and Fourier transform infrared spectroscopy studies of the effects of transmembrane alpha-helical peptides on the organization of phosphatidylcholine bilayers

We have studied the effects of the incorporation of the alpha-helical transmembrane peptides Ac-K(2)-L(24)-K(2)-amide (L(24)) and Ac-K(2)-(L-A)(12)-K(2)-amide ((LA)(12)) on the thermotropic phase behavior of 1,2-dipalmitoyl-d(62)-sn-glycero-3-phosphocholine (DPPC-d(62)) and 1-palmitoyl-d(31)-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d(31)) lipid bilayer model membranes by differential scanning calorimetry (DSC) and the conformational and orientational order of the phospholipid chains by Fourier transform infrared (FTIR) spectroscopy and (2)H nuclear magnetic resonance ((2)H-NMR) spectroscopy, respectively. Our DSC and FTIR spectroscopic studies indicate that the peptides L(24) and (LA)(12) both decrease the temperature and enthalpy of the gel/liquid-crystalline phase transition of DPPC-d(62) bilayers, with (LA)(12) having the greater effect in this regard. An examination of the frequencies of the CH(2) and CD(2) symmetric stretching bands of the infrared spectra of liquid-crystalline states of the peptide-free and peptide-containing DPPC-d(62) and POPC-d(31) samples, and a comparison with the orientational order as measured by (2)H-NMR spectroscopy as well as with the chain order as measured by electron spin resonance spectroscopy, lead us to conclude that the CH(2) (or CD(2)) stretching frequencies of lipid hydrocarbon chains are not a reliable measure of chain conformational order in lipid bilayers containing significant amounts of peptides or other lipophilic inclusions. In contrast, the results of our (2)H-NMR spectroscopic studies present a consistent picture in which both L(24) and (LA)(12) increased in a similar way the time-averaged orientational order of the lipid chains of their liquid-crystalline lipid bilayer hosts. The comparison of the effects L(24) and (LA)(12) on phosphatidylcholine bilayers indicates that the gel-to-liquid-crystalline phase transition appears to be more sensitive to small changes in transmembrane peptide surface topology than hydrocarbon carbon chain orientational order in the liquid-crystalline state.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylcholine bilayers

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of an alpha-helical transmembrane peptide, acetyl-Lys2-Leu24-Lys2-amide (L24), and odd-chain members of the homologous series of n-saturated diacylphosphatidylcholines. An analogue of L24, in which the lysine residues were all replaced by 2,3-diaminopropionic acid, and another, in which a leucine residue at each end of the polyLeu sequence was replaced by a tryptophan, were also studied. At low peptide concentrations, the DSC thermograms exhibited by these lipid/peptide mixtures are resolvable into two components. One of these components is fairly narrow, highly cooperative, and exhibits properties which are similar to but not identical with those of the pure lipid. In addition, the transition temperature and cooperativity of this component, and its fractional contribution to the total enthalpy change, decrease with an increase in peptide concentration, more or less independently of phospholipid acyl chain length. The other component is very broad and predominates at high peptide concentrations. These two components have been assigned to the chain-melting phase transitions of populations of peptide-poor and peptide-enriched lipid domains, respectively. Moreover, when the mean hydrophobic thickness of the PC bilayer is less than the peptide hydrophobic length, the peptide-associated lipid melts at higher temperatures than does the bulk lipid and vice versa. In addition, the chain-melting enthalpy of the broad endotherm does not decrease to zero even at high peptide concentrations, suggesting that these peptides reduce somewhat but do not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. Our DSC results indicate that the width of the broad phase transition observed at high peptide concentration is inversely but discontinuously related to hydrocarbon chain length. Our FTIR spectroscopic data indicate that these peptides form a very stable alpha-helix under all of our experimental conditions but that small distortions of their alpha-helical conformation are induced in response to mismatch between peptide hydrophobic length and gel-state bilayer hydrophobic thickness. We also present evidence that these distortions are localized to the N- and C-terminal regions of these peptides. Interestingly, replacing the terminal Lys residues of L24 by 2,3-diaminopropionic acid residues actually attenuates the hydrophobic mismatch effects of the peptide on the thermotropic phase behavior of the host PC bilayer, in contrast to the predictions of the snorkel hypothesis. We rationalize this attenuated hydrophobic mismatch effect by postulating that the 2,3-diaminopropionic acid residues are too short to engage in significant electrostatic and hydrogen-bonding interactions with the polar headgroups of the host phospholipid bilayer, even in the absence of any hydrophobic mismatch between incorporated peptide and the bilayer. Similarly, the reduced hydrophobic mismatch effect also observed when the two terminal Leu residues of L24 are replaced by Trp residues is rationalized by considering the lower energetic cost of exposing the Trp as opposed to the Leu residues to the aqueous phase in thin PC bilayers and the higher cost of inserting the Trp as opposed to the Leu residues into the hydrophobic cores of thick PC bilayers.     

Alterations in the organization of phosphatidylcholine/cholesterol bilayers by tetrahydrocannabinol

The interactions of delta 9-tetrahydrocannabinol (THC) with various phosphatidylcholines (PCs) was studied in model membranes by differential scanning calorimetry. THC present in PC bilayers above a certain concentration complexed stoichiometrically with phospholipids containing both saturated and unsaturated fatty acids. When the bilayer PCs were sufficiently dissimilar for phase separation to occur, THC preferentially associated with the lower melting point lipid. The presence of cholesterol below 20 mol% in dipalmitoylphosphatidylcholine bilayers enhanced THC X PC complex formation. Above 20 mol% cholesterol, there was no indication of THC X dipalmitoylphosphatidylcholine complex formation. This is in agreement with a phase rearrangement occurring in PC bilayers at concentrations of cholesterol of approximately 20 mol%. These studies suggest several possible mechanisms for the modulation of membrane activities by hydrophobic drugs such as THC.     

Molecular organization in hydroperoxidized POPC bilayers

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Binding and insertion of alpha-helical anti-microbial peptides in POPC bilayers studied by molecular dynamics simulations

We have performed molecular dynamics simulations of the interactions of two alpha-helical anti-microbial peptides, magainin2 and its synthetic analog of MSI-78, with palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayers. We used various initial positions and orientations of the peptide with respect to the lipid bilayer, including a surface-bound state parallel to the interface, a trans-membrane state, and a partially inserted state. Our 20 ns long simulations show that both magainin2 and MSI-78 are most stable in the lipid environment, with the peptide destabilized to different extents in both aqueous and lipid/water interfacial environments. We found that there are strong specific interactions between the lysine residues of the peptides and the lipid head-group regions. MSI-78, owing to its large number of lysines, shows better binding characteristics and overall stability when compared to magainin2. We also find that both peptides destabilize the bilayer environment, as observed by the increase in lipid tail disorder and the induction of local curvature on the lipid head-groups by the peptides. From all the simulations, we conclude that the hydrogen bonding interactions between the lysines of the peptides and the oxygens of the polar lipid head-groups are the strongest and determine the overall peptide binding characteristics to the lipids.     

Organization of model helical peptides in lipid bilayers: insight into the behavior of single-span protein transmembrane domains

Selectively deuterated transmembrane peptides comprising alternating leucine-alanine subunits were examined in fluid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy in an effort to gain insight into the behavior of membrane proteins. Two groups of peptides were studied: 21-mers having a 17-amino-acid hydrophobic domain calculated to be close in length to the hydrophobic thickness of 1-palmitoyl-2-oleoyl phosphatidylcholine and 26-mers having a 22-amino-acid hydrophobic domain calculated to exceed the membrane hydrophobic thickness. (2)H NMR spectral features similar to ones observed for transmembrane peptides from single-span receptors of higher animal cells were identified which apparently correspond to effectively monomeric peptide. Spectral observations suggested significant distortion of the transmembrane alpha-helix, and/or potential for restriction of rotation about the tilted helix long axis for even simple peptides. Quadrupole splittings arising from the 26-mer were consistent with greater peptide "tilt" than were those of the analogous 21-mer. Quadrupole splittings associated with monomeric peptide were relatively insensitive to concentration and temperature over the range studied, indicating stable average conformations, and a well-ordered rotation axis. At high peptide concentration (6 mol% relative to phospholipid) it appeared that the peptide predicted to be longer than the membrane thickness had a particular tendency toward reversible peptide-peptide interactions occurring on a timescale comparable with or faster than approximately 10(-5) s. This interaction may be direct or lipid-mediated and was manifest as line broadening. Peptide rotational diffusion rates within the membrane, calculated from quadrupolar relaxation times, T(2e), were consistent with such interactions. In the case of the peptide predicted to be equal to the membrane thickness, at low peptide concentration spectral lineshape indicated the additional presence of a population of peptide having rotational motion that was restricted on a timescale of 10(-5) s.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.     

Minimal peptide length for interaction of amphipathic alpha-helical peptides with phosphatidylcholine liposomes

The interactions of a series of amphipathic alpha-helical peptides containing from 6 to 18 amino acid residues with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied by optical and calorimetric methods. Several peptides rapidly decreased the turbidity of DMPC and DPPC liposomes when mixed at the phase transition temperatures of the lipids. The extent of the clearing depended upon the chain length of the peptides, with the most effective clearing attained with peptides 10-12 residues in length. An eight-residue peptide was somewhat less effective and a six-residue peptide had no effect on liposome structure. The peptides formed small micellar structures, as judged by gel filtration chromatography. The effects of the peptides on the phase transitions of the lipids were examined by differential scanning calorimetry. The peptides that were most effective in disrupting the liposomes and forming clear micelles were also most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of the lipid. The addition of DMPC or DPPC liposomes to the peptides increased the magnitude of the negative bonds at 208 and 222 nm in circular dichroism measurements, consistent with the expected formation of alpha-helical structure on binding to lipid. The extent of burial of the single tryptophan residue in the peptides was determined by fluorescence spectroscopy. In peptides that bound to lipid, the tryptophan was in a less solvent-exposed environment in the presence of lipid, as evidenced by a blue shift in the fluorescence emission maximum of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polylysine-induced 2H NMR-observable domains in phosphatidylserine/phosphatidylcholine lipid bilayers.

The interaction of three polylysines, Lys(5) (N = 5), Lys(30) (N = 30), and Lys(100) (N = 100), where N is the number of lysine residues per chain, with phosphatidylserine-containing lipid bilayer membranes was investigated using 2H NMR spectroscopy. Lys(30) and Lys(100) added to multilamellar vesicles composed of (70:30) (mol:mol) mixtures of choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) produced two resolvable 2H NMR spectral components under conditions of low ionic strength and for cases where the global anionic lipid charge was in excess over the global cationic polypeptide charge. The intensities and quadrupolar splittings of the two spectral components were consistent with the existence of polylysine-bound domains enriched in POPS, in coexistence with polylysine-free domains depleted in POPS. Lys(5), however, yielded no 2H NMR resolvable domains. Increasing ionic strength caused domains to become diffuse and eventually dissipate entirely. At physiological salt concentrations, only Lys(100) yielded 2H NMR-resolvable domains. Therefore, under physiological conditions of ionic strength, pH, and anionic lipid bilayer content, and in the absence of other, e.g., hydrophobic, contributions to the binding free energy, the minimum number of lysine residues sufficient to produce spectroscopically resolvable POPS-enriched domains on the 2H NMR millisecond timescale may be fewer than 100, but is certainly greater than 30.     

Kinetics of dye efflux and lipid flip-flop induced by delta-lysin in phosphatidylcholine vesicles and the mechanism of graded release by amphipathic, alpha-helical peptides

Delta-lysin is a 26-residue, amphipathic, alpha-helical peptide of bacterial origin. Its specificity is to some extent complementary to that of antimicrobial peptides. Therefore, understanding its mechanism is important for the more general goal of understanding the interaction of amphipathic peptides with membranes. In this article, we show that delta-lysin induces graded efflux of the contents of phosphatidylcholine vesicles. In view of this finding, carboxyfluorescein efflux kinetics were re-examined. In addition, peptide-induced lipid flip-flop was directly measured using fluorescence energy transfer between two lipid fluorophores initially placed on opposite leaflets of the bilayer. Carboxyfluorescein efflux and lipid flip-flop occur with essentially identical rate constants. On the basis of a detailed, quantitative analysis of the kinetics of peptide-vesicle interactions, we conclude that the peptide translocates across the bilayer as a small, transient aggregate, most likely a trimer. Dye efflux and lipid flip-flop occur concomitantly with the transient peptide-induced perturbation of the membrane. The experimental data are interpreted by comparing the predictions of the available models for the mechanism of action of amphipathic alpha-helical peptides. We demonstrate how the combination of the quantitative kinetic analysis, graded efflux, and reversibility of the peptide-vesicle interaction can be used to reject several models for this particular peptide. Two models are compatible with the data, the toroidal pore model and the sinking raft model. On the basis of the small aggregate size, a trimer, the latter appears to be more plausible. Some significant modifications are introduced in the sinking raft model to take into account the new finding of graded dye release. Furthermore, we present an explanation for the phenomenon of graded release in general, which, contrary to all-or-none efflux, has not been well-understood.     

Molecular dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine membranes containing transmembrane alpha-helical peptides with alternating leucine and alanine residues

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.