Amyloid formation by islet amyloid polypeptide (IAPP) contributes to β-cell dysfunction in type 2 diabetes. Perturbation of the β-cell membrane may contribute to IAPP-induced toxicity. We examine the effects of lipid composition, salt, and buffer on IAPP amyloid formation and on the ability of IAPP to induce leakage of model membranes. Even low levels of anionic lipids promote amyloid formation and membrane permeabilization. Increasing the percentage of the anionic lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) or 1,2-dioleoyl-sn-glycero-3-phospho(1′-rac-glycerol), enhances the rate of amyloid formation and increases the level of membrane permeabilization. The choice of zwitterionic lipid has no noticeable effect on membrane-catalyzed amyloid formation but in most cases affects leakage, which tends to decrease in the following order: 1,2-dioleoyl-sn-glycero-3-phosphocholine > 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine > sphingomyelin. Uncharged lipids that increase the level of membrane order weaken the ability of IAPP to induce leakage. Leakage is due predominately to pore formation rather than complete disruption of the vesicles under the conditions used in these studies. Cholesterol at or below physiological levels significantly reduces the rate of vesicle-catalyzed IAPP amyloid formation and decreases the susceptibility to IAPP-induced leakage. The effects of cholesterol on amyloid formation are masked by 25 mol % POPS. Overall, there is a strong inverse correlation between the time to form amyloid and the extent of vesicle leakage. NaCl reduces the rate of membrane-catalyzed amyloid formation by anionic vesicles, but accelerates amyloid formation in solution. The implications for IAPP membrane interactions are discussed, as is the possibility that the loss of phosphatidylserine asymmetry enhances IAPP amyloid formation and membrane damage in vivo via a positive feedback loop.

Sterols are important components of eukaryotic membranes, but rare in bacteria. Some bacteria obtain sterols from their host or environment. In some cases, these sterols form membrane domains analogous the lipid rafts proposed to exist in eukaryotic membranes. This review describes the properties and roles of sterols in Borrelia and Helicobacter.

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70–80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

Perfringolysin O (PFO) is a transmembrane (TM) β-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30–50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakage assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM β-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.

Methyl-β-cyclodextrin (MβCD) can be used to exchange membrane lipids between different vesicles in order to prepare model membrane vesicles with lipid asymmetry. To help define what factors influence lipid exchange, we studied how lipid interaction with cyclodextrins (CDs) was affected by lipid and CD structure. The decrease in light scattering upon CD-induced vesicle solubilization and the change in Förster resonance energy transfer of labeled lipids upon vesicle solubilization and lipid exchange were used to detect phospholipid–CD interaction. Of the CDs examined, MβCD, hydroxypropyl-α-cyclodextrin (HPαCD), and hydroxypropyl-β-cyclodextrin (HPβCD) were the three with the most suitable phospholipid interaction properties. Only MβCD was observed to dissolve lipid vesicles (at least at CD concentrations below 125 mM). Solubilization of lipid vesicles was half complete at 10–80 mM MβCD with progressively higher MβCD concentrations required as phospholipid acyl chain length increased from 14 to 22 carbons. Phospholipid acyl chain unsaturation and lipid headgroup structure also affected the amount of MβCD needed for solubilization. All three CDs studied were able to carry out phospholipid exchange. MβCD, which retained the ability to carry out lipid exchange below MβCD concentrations needed for solubilization, exchanged lipid more efficiently than HPαCD or HPβCD. However, the ability of HPαCD to exchange phospholipids, coupled with its inability to interact with cholesterol, indicates that it will be useful for preparing asymmetric vesicles with controlled amounts of cholesterol.

We show that anandamide (AEA) externally added to model membrane vesicles containing trapped fatty acid amide hydrolyase (FAAH) can be readily hydrolyzed, demonstrating facile, rapid anandamide movement across the lipid bilayer. The rate of hydrolysis is significantly facilitated by cholesterol and coprostanol, but not by cholesterol sulfate. The effects of sterol upon hydrolysis by FAAH bound to the outer surface of the bilayer were much smaller, although they followed the same pattern. We propose the facilitation of hydrolysis is a combination of the effects of sterol on accessibility of membrane-inserted endocannabinoids to surface protein, and on the rate of endocannabinod transport across the membrane bilayer.Keywords: Anandamide; cholesterol; FAAH; fatty acid amide hydrolase; liposome; LUV; transporter

Botulinum neurotoxins (BoNTs) undergo low pH-triggered membrane insertion, resulting in the translocation of their light (catalytic) chains into the cytoplasm. The T (translocation) domain of the BoNT heavy chain is believed to carry out translocation. Here, the behavior of isolated T domain from BoNT type A has been characterized, both in solution and when associated with model membranes. When BoNT T domain prepared in the detergent dodecylmaltoside was diluted into aqueous solution, it exhibited a low pH-dependent conformational change below pH 6. At low pH the T domain associated with, and formed pores within, model membrane vesicles composed of 30 mol% dioleoylphosphatidylglycerol/70 mol% dioleoylphosphatidylcholine. Although T domain interacted with vesicles at low (50 mM) and high (400 mM) NaCl concentrations, the interaction required much less lipid at low salt. However, even at high lipid concentrations pore formation was much more pronounced at low NaCl concentrations than at high NaCl concentration. Increasing salt concentration after insertion in the presence of 50 mM NaCl did not decrease pore formation. A similar effect of NaCl concentration upon pore formation was observed in vesicles composed solely of dioleoylphosphatidylcholine, showing that the effect of NaCl did not solely involve modulation of electrostatic interactions between protein and anionic lipids. These results indicate that some feature of membrane-bound T domain tertiary structure critical for pore formation is highly dependent upon salt concentration.

The diphtheria toxin T domain helps translocate the A chain of the toxin across membranes. To gain insight into translocation, the membrane topography of key residues in T domain attached to the A chain (AT protein) was compared to that in the isolated T domain using fluorescence techniques. This study demonstrates that residues in T domain hydrophobic helices (TH5−TH9) tended to be less exposed to aqueous solution in the AT protein than in the isolated T domain. Under conditions in which the loop connecting TH5 to TH6/7 is located stably on the cis (insertion) side of the membrane in the isolated T domain, it moves between the cis and trans sides of the membrane in the AT protein. This is indicative of the formation of a dynamic, transient transmembrane hairpin topography by TH5−TH7 in the AT protein. Since TH8 and TH9 also form a transmembrane hairpin, this means that TH5−TH9 may form a cluster of transmembrane helices. These helices have a nonpolar surface likely to face the lipid bilayer in a helix cluster and a surface rich in uncharged hydrophilic residues which in a helix cluster would likely be facing inward (and perhaps be pore-lining). This uncharged hydrophilic surface could play a crucial role in translocation, interacting transiently with the translocating A chain. A similar motif can be found in, and may be important for, other protein translocation systems.

Direct physical chemistry measurements of the hydrophobicity of amino acids or their derivatives have often been used to estimate the propensity of amino acids to participate in transmembrane helices. In this short note, it is found that there is a very high degree of correlation (r = 0.944–0.965) between an average physical chemistry hydrophobicity scale (an average of scales derived, e.g., from the solubility of amino acid derivatives in organic solvents versus water or their binding to hydrophobic particles) and the statistically based transmembrane tendency scale (derived from the relative abundance of residues in known transmembrane and soluble protein sequences (Zhao and London, Protein Sci 15:1987–2001, 2006)). This correlation indicates that, other than hydrophobicity, amino acid properties/interactions that promote or inhibit transmembrane helix formation in a specific membrane protein largely cancel out when averaged over all transmembrane sequences. In other words, other than hydrophobicity, there are no properties of a specific amino acid residue within a hydrophobic segment that have a strong systematic effect upon transmembrane helix formation independent of the remainder of the sequence in that hydrophobic segment. However, proline is an exception to this rule.

Diphtheria toxin T domain aids the membrane translocation of diphtheria toxin A chain. When the isolated T domain is deeply membrane-inserted, helices TH 8−9 form a transmembrane hairpin, while helices TH 5−7 form a deeply inserted nontransmembrane structure. Blocking deep insertion of TH 8−9 blocks deep insertion of TH 5−7 (Zhao, G., and London, E. (2005) Biochemistry44, 4488–4498). We now examine the effects of blocking the deep insertion of TH 5 and TH 6/7. An A282R/V283R dual substitution in TH 5 prevented its deep insertion, significantly decreased the deep insertion of TH 9, and to a lesser degree that of TH 6/7. Blocking deep insertion of TH 6/7 with a L307R mutation had no effect on the deep insertion of TH 5, similar to its previously characterized lack of effect on the deep insertion of TH 8−9. An I364K mutation in TH 9 blocked TH 8−9 deep insertion and greatly reduced pore formation by the T domain, consistent with the role of TH 8−9 in pore formation. The A282R/V283R mutations also reduced the extent of pore formation, but to a lesser degree, suggesting either that TH 5 is part of the pore or that interactions with TH 5 affect the ability of TH 8−9 to form pores. The L307R mutation enhanced the extent of pore formation, suggesting that deeply inserted TH 6/7 may act as a “cork” that partly blocks the pore. Combined, these results indicate that TH 5, 8, and 9 combine to form a deeply inserted scaffold of more strongly associated helices.

Ceramides are sphingolipids that greatly stabilize ordered membrane domains (lipid rafts), and displace cholesterol from them. Ceramide-rich rafts have been implicated in diverse biological processes. Because ceramide analogues have been useful for probing the biological function of ceramide, and may have biomedical applications, it is important to characterize how ceramide structure affects membrane properties, including lipid raft stability and composition. In this report, fluorescence quenching assays were used to evaluate the effect of analogues of ceramide with different N-acyl chains or different sphingoid backbones on raft stability and sterol content. The effect of replacing 18 mol% of sphingomyelin (SM) with ceramide in vesicles composed of a 1:1 (mol:mol) mixture of SM and dioleoylphosphatidylcholine (DOPC), with or without 25 mol% sterol, was examined. In the absence of sterol, the thermal stability of the SM-rich ordered domains increased with ceramide N-acyl chain length in the order C2:0 ∼ C6:0 ∼ C8:0 < no ceramide < C12:0 < C16:0. In vesicles containing 25 mol% cholesterol (1:1:0.66 sphingolipid:DOPC:cholesterol), the dependence of raft stability on ceramide N-acyl chain length increased in the order C8:0 ∼ C6:0 < C2:0 < C12:0 ∼ no ceramide < C16:0. We also studied the stability of lipid rafts in the presence of N-lauroyl- and N-palmitoylsphingosine analogues containing altered structures in or near the polar portion of the sphingoid base. In almost all cases, the analogues stabilized rafts to about the same degree as a normal ceramide containing the same acyl chain. The only exception was N-palmitoyl-4D-ribophytosphingosine, which was very strongly raft-stabilizing. We conclude that variations in sphingoid base structure induce only insignificant changes in raft properties. N-Lauroyl and N-palmitoylsphingosine and their analogues displaced sterol from rafts to a significant degree. Both C12:0 and C16:0 analogues of ceramide may be good mimics of natural ceramide, and useful for cellular studies in which maintenance of the normal physical properties of ceramide are important.

Asymmetry of inner and outer leaflet lipid composition is an important characteristic of eukaryotic plasma membranes. We previously described a technique in which methyl-β-cyclodextrin-induced lipid exchange is used to prepare biological membrane-like asymmetric small unilamellar vesicles (SUVs). Here, to mimic plasma membranes more closely, we used a lipid-exchange-based method to prepare asymmetric large unilamellar vesicles (LUVs), which have less membrane curvature than SUVs. Asymmetric LUVs in which sphingomyelin (SM) or SM + 1-palmitoyl-2-oleoyl-phosphatidylcholine was exchanged into the outer leaflet of vesicles composed of 1,2-dioleoyl-phosphatidylethanolamine (DOPE) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) were prepared with or without cholesterol. Approximately 80–100% replacement of outer leaflet DOPE and POPS was achieved. At room temperature, SM exchange into the outer leaflet increased the inner leaflet lipid order, suggesting significant interleaflet interaction. However, the SM-rich outer leaflet formed an ordered state, melting with a midpoint at ∼37°C. This was about the same value observed in pure SM vesicles, and was significantly higher than that observed in symmetric vesicles with the same SM content, which melted at ∼20°C. In other words, ordered state formation by outer-leaflet SM in asymmetric vesicles was not destabilized by an inner leaflet composed of DOPE and POPS. These properties suggest that the coupling between the physical states of the outer and inner leaflets in these asymmetric LUVs becomes very weak as the temperature approaches 37°C. Overall, the properties of asymmetric LUVs were very similar to those previously observed in asymmetric SUVs, indicating that they do not arise from the high membrane curvature of asymmetric SUVs.

Sphingolipid- and cholesterol-rich liquid-ordered (Lo) lipid domains (rafts) are thought to be important organizing elements in eukaryotic plasma membranes. How they form in the sphingolipid-poor cytosolic (inner) membrane leaflet is unclear. Here, we characterize how outer-leaflet Lo domains induce inner-leaflet-ordered domains, i.e., interleaflet coupling. Asymmetric vesicles studied contained physiologically relevant cholesterol levels (∼37 mol %), a mixture of SM (sphingomyelin) and DOPC (dioleoylphosphatidylcholine) in their outer leaflets, and DOPC in their inner leaflets. Lo domains were observed in both leaflets, and were in register, indicative of coupling between SM-rich outer-leaflet-ordered domains and inner-leaflet-ordered domains. For asymmetric vesicles with outer-leaflet egg SM or milk SM, a fluorescent lipid with unsaturated acyl chains (NBD-DOPE) was depleted in both the outer- and inner-leaflet-ordered domains. This suggests the inner-leaflet-ordered domains were depleted in unsaturated lipid (i.e., DOPC) and thus rich in cholesterol. For asymmetric vesicles containing egg SM, outer-leaflet Lo domains were also depleted in a saturated fluorescent lipid (NBD-DPPE), while inner-leaflet Lo domains were not. This indicates that inner- and outer-leaflet Lo domains can have significantly different physical properties. In contrast, in asymmetric vesicles containing outer-leaflet milk SM, which has long acyl chains capable of interdigitating into the inner leaflet, both outer- and inner-leaflet Lo domains were depleted, to a similar extent, in NBD-DPPE. This is indicative of interdigitation-enhanced coupling resulting in inner- and outer-leaflet Lo domains with similar physical properties.

We report a simple method to obtain stable asymmetric giant unilamellar vesicles (GUVs). Fluorescence correlation spectroscopy was used to quantitatively characterize vesicle properties. After brain sphingomyelin (bSM) was exchanged into dioleoylphosphatidylcholine (DOPC) GUVs, lateral diffusion in the bSM-containing outer leaflet decreased, whereas that in the DOPC-containing inner leaflet was largely unchanged, confirming asymmetry and a lack of coupling between the physical states of the inner and outer leaflets. In contrast, after bSM was exchanged into brain phosphatidylcholine vesicles, lateral diffusion decreased in both leaflets. Thus, asymmetric GUVs should be useful for investigating the molecular mechanisms behind interleaflet coupling.

A long-standing question about membrane structure and function is the degree to which the physical properties of the inner and outer leaflets of a bilayer are coupled to one another. Using our recently developed methods to prepare asymmetric vesicles, coupling was investigated for vesicles containing phosphatidylcholine (PC) in the inner leaflet and sphingomyelin (SM) in the outer leaflet. The coupling of both lateral diffusion and membrane order was monitored as a function of PC and SM acyl chain structure. The presence in the outer leaflet of brain SM, which decreased outer-leaflet lateral diffusion, had little effect upon lateral diffusion in inner leaflets composed of dioleoyl PC (i.e., diffusion was only weakly coupled in the two leaflets) but did greatly reduce lateral diffusion in inner leaflets composed of PC with one saturated and one oleoyl acyl chain (i.e., diffusion was strongly coupled in these cases). In addition, reduced outer-leaflet diffusion upon introduction of outer-leaflet milk SM or a synthetic C24:0 SM, both of which have long interdigitating acyl chains, also greatly reduce diffusion of inner leaflets composed of dioleoyl PC, indicative of strong coupling. Strikingly, several assays showed that the ordering of the outer leaflet induced by the presence of SM was not reflected in increased lipid order in the inner leaflet, i.e., there was no detectable coupling between inner and outer leaflet membrane order. We propose a model for how lateral diffusion can be coupled in opposite leaflets and discuss how this might impact membrane function.

The minimum hydrophobic length necessary to form a transmembrane (TM) helix in membranes was investigated using model membrane-inserted hydrophobic helices. The fluorescence of a Trp at the center of the sequence and its sensitivity to quenching were used to ascertain helix position within the membrane. Peptides with hydrophobic cores composed of poly(Leu) were compared to sequences containing a poly 1:1 Leu:Ala core (which have a hydrophobicity typical of natural TM helices). Studies varying bilayer width revealed that the poly(Leu) core peptides predominately formed a TM state when the bilayer width exceeded hydrophobic sequence length by (i.e. when negative mismatch was) up to ∼ 11-12 Å (e.g. the case of a 11-12 residue hydrophobic sequence in bilayers with a biologically relevant width, i.e. dioleoylphosphatidylcholine (DOPC) bilayers), while poly(LeuAla) core peptides formed predominantly TM state with negative mismatch of up to 9 Å (a 13 residue hydrophobic sequence in DOPC bilayers). This indicates that minimum length necessary to form a predominating amount of a TM state (minimum TM length) is only modestly hydrophobicity-dependent for the sequences studied here, and a formula that defines the minimum TM length as a function of hydrophobicity for moderately-to-highly hydrophobic sequences was derived. The minimum length able to form a stable TM helix for alternating LeuAla sequences, and that for sequences with a Leu block followed by an Ala block, was similar, suggesting that a hydrophobicity gradient along the sequence may not be an important factor in TM stability. TM stability was also similar for sequences flanked by different charged ionizable residues (Lys, His, Asp). However, ionizable flanking residues destabilized the TM configuration much more when charged than when uncharged. The ability of short hydrophobic sequences to form TM helices in membranes in the presence of substantial negative mismatch implies that lipid bilayers have a considerable ability to adjust to negative mismatch, and that short TM helices may be more common than generally believed. Factors that modulate the ability of bilayers to adjust to mismatch may strongly affect the configuration of short hydrophobic helices.

Upon interaction with cholesterol, perfringolysin O (PFO) inserts into membranes and forms a rigid transmembrane (TM) β-barrel. PFO is believed to interact with liquid ordered lipid domains (lipid rafts). Because the origin of TM protein affinity for rafts is poorly understood, we investigated PFO raft affinity in vesicles having coexisting ordered and disordered lipid domains. Fluorescence resonance energy transfer (FRET) from PFO Trp to domain-localized acceptors indicated that PFO generally has a raft affinity between that of LW peptide (low raft affinity) and cholera toxin B (high raft affinity) in vesicles containing ordered domains rich in brain sphingomyelin or distearoylphosphatidylcholine. FRET also showed that ceramide, which increases exposure of cholesterol to water and thus displaces it from rafts, does not displace PFO from ordered domains. This can be explained by shielding of PFO-bound cholesterol from water. Finally, FRET showed that PFO affinity for ordered domains was higher in its non-TM (prepore) form than in its TM form, demonstrating that the TM portion of PFO interacts unfavorably with rafts. Microscopy studies in giant unilamellar vesicles confirmed that PFO exhibits intermediate raft affinity, and showed that TM PFO (but not non-TM PFO) concentrated at the edges of liquid ordered domains. These studies suggest that a combination of binding to raft-associating molecules and having a rigid TM structure that is unable to pack well in a highly ordered lipid environment can control TM protein domain localization. To accommodate these constraints, raft-associated TM proteins in cells may tend to locate within liquid disordered shells encapsulated within ordered domains.

Mixtures of unsaturated lipids, sphingolipids, and cholesterol form coexisting liquid-disordered and sphingolipid and cholesterol-rich liquid-ordered (Lo) phases in water. The detergent Triton X-100 does not readily solubilize Lo domains, but does solubilize liquid-disordered domains, and is commonly used to prepare detergent-resistant membranes from cells and model membranes. However, it has been proposed that in membranes with mixtures of sphingomyelin (SM), 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), and cholesterol Triton X-100 may induce Lo domain formation, and therefore detergent-resistant membranes may not reflect the presence of preexisting domains. To examine this hypothesis, the effect of Triton on Lo domain formation was measured in SM/POPC/cholesterol vesicles. Nitroxide quenching methods that can detect ordered nanodomains with radii >12 Å showed that in the absence of Triton X-100 this mixture formed ordered state domains that melt with a midpoint (= Tmid) at ∼45°C. However, Tmid was lower when detected using various fluorescence resonance energy transfer (FRET) pairs. Furthermore, the Tmid value was Ro dependent, and decreased as Ro increased. Because FRET can only readily detect domains with radii >Ro, this result can be explained by domain radii that are close to Ro and decrease as temperature increases. An analysis of FRET and quenching data suggests that nanodomain radius gradually decreases from ≥150 Å to <40 Å as temperature increases from 10 to 45°C. Interestingly, the presence of Triton X-100 or a transmembrane-type peptide did not stabilize ordered state formation when detected by nitroxide quenching, i.e., did not increase Tmid. However, FRET-detected Tmid did increase in the presence of Triton X-100 or a transmembrane peptide, indicating that both increased domain size. Controls showed that the results could not be accounted for by probe-induced perturbations. Thus, SM/POPC/cholesterol, a mixture similar to that in the outer leaflet of plasma membranes, forms nanodomains at physiological temperatures, and TX-100 does not induce domain formation or increase the fraction of the bilayer in the ordered state, although it does increase domain size by coalescing preexisting domains.

Despite the importance of lipid rafts, commonly defined as liquid-ordered domains rich in cholesterol and in lipids with high gel-to-fluid melting temperatures (Tm), the rules for raft formation in membranes are not completely understood. Here, a fluorescence-quenching strategy was used to define how lipids with low Tm, which tend to form disordered fluid domains at physiological temperatures, can stabilize ordered domain formation by cholesterol and high-Tm lipids (either sphingomyelin or dipalmitoylphosphatidylcholine). In bilayers containing mixtures of low-Tm phosphatidylcholines, cholesterol, and high-Tm lipid, the thermal stability of ordered domains decreased with the acyl-chain structure of low-Tm lipids in the following order: diarachadonyl > diphytanoyl > 1-palmitoyl 2-docosahexenoyl = 1,2 dioleoyl = dimyristoleoyl = 1-palmitoyl, 2-oleoyl (PO). This shows that low-Tm lipids with two acyl chains having very poor tight-packing propensities can stabilize ordered domain formation by high-Tm lipids and cholesterol. The effect of headgroup structure was also studied. We found that even in the absence of high-Tm lipids, mixtures of cholesterol with PO phosphatidylethanolamine (POPE) and PO phosphatidylserine (POPS) or with brain PE and brain PS showed a (borderline) tendency to form ordered domains. Because these lipids are abundant in the inner (cytofacial) leaflet of mammalian membranes, this raises the possibility that PE and PS could participate in inner-leaflet raft formation or stabilization. In bilayers containing ternary mixtures of PO lipids, cholesterol, and high-Tm lipids, the thermal stability of ordered domains decreased with the polar headgroup structure of PO lipids in the order PE > PS > phosphatidylcholine (PC). Analogous experiments using diphytanoyl acyl chain lipids in place of PO acyl chain lipids showed that the stabilization of ordered lipid domains by acyl chain and headgroup structure was not additive. This implies that it is likely that there are two largely mutually exclusive mechanisms by which low-Tm lipids can stabilize ordered domain formation by high-Tm lipids and cholesterol: 1), by having structures resulting in immiscibility of low-Tm and high-Tm lipids, and 2), by having structures allowing them to pack tightly within ordered domains to a significant degree.

The ability of hydrophilic residues to shift the transverse position of transmembrane (TM) helices within bilayers was studied in model membrane vesicles. Transverse shifts were detected by fluorescence measurements of the membrane depth of a Trp residue at the center of a hydrophobic sequence. They were also estimated from the effective length of the TM-spanning sequence, derived from the stability of the TM configuration under conditions of negative hydrophobic mismatch. Hydrophilic residues (at the fifth position in a 21-residue hydrophobic sequence composed of alternating Leu and Ala residues and flanked on both ends by two Lys) induced transverse shifts that moved the hydrophilic residue closer to the membrane surface. At pH 7, the dependence of the extent of shift upon the identity of the hydrophilic residue increased in the order: L < G∼Y∼T < R∼H < S < P < K < E∼Q < N < D. By varying pH, shifts with ionizable residues fully charged or uncharged were measured, and the extent of shift increased in the order: L < G∼Y∼Ho∼T < Eo∼R < S < P < K+< Q∼Do∼H+ < N∼E– < D–. The dependence of transverse shifts upon hydrophilic residue identity was consistent with the hypothesis that shift magnitude is largely controlled by the combination of side chain hydrophilicity, ionization state, and ability to position polar groups near the bilayer surface (snorkeling). Additional experiments showed that shift was also modulated by the position of the hydrophilic residue in the sequence and the hydrophobicity of the sequence moved out of the bilayer core upon shifting. Combined, these studies show that the insertion boundaries of TM helices are very sensitive to sequence, and can be altered even by weakly hydrophilic residues. Thus, many TM helices may have the capacity to exist in more than one transverse position. Knowledge of the magnitudes of transverse shifts induced by different hydrophilic residues should be useful for design of mutagenesis studies measuring the effect of transverse TM helix position upon function.

To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine ∼ 6:4 POPC:cholesterol < POPC ∼ dioleoylphosphatidylcholine (DOPC) < 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG) ≤ 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] sodium salt (DOPS), indicating that the anionic lipids DOPG and DOPS most strongly stabilized the TM configuration. TM stabilization was near maximal at 20–30 mol% anionic lipid, which are physiologically relevant values. TM stabilization by anionic lipid was observed for hydrophobic sequences with a diverse set of sequences (including polyAla), diverse lengths (from 12 to 22 residues), and various cationic flanking residues (H, R, or K), but not when the flanking residues were uncharged. TM stabilization by anionic lipid was also dependent on the number of cationic residues flanking the hydrophobic sequence, but was still significant with only one cationic residue flanking each end of the peptide. These observations are consistent with TM-stabilizing effects being electrostatic in origin. However, Trp located more deeply in DOPS vesicles relative to DOPG vesicles, and peptides in DOPS vesicles showed increased helix formation relative to DOPG and all other lipid compositions. These observations fit a model in which DOPS anchors flanking residues near the membrane surface more strongly than does DOPG and/or increases the stability of the TM state to a greater degree than DOPG. We conclude that anionic lipids can have significant and headgroup structure-specific effects upon membrane protein topography.

The sequence of the transmembrane (TM) helix of ErbB2, a member of the epidermal growth factor receptor (ErbB) family, can influence its activity. In this report, the sequence and lipid dependence of the transverse position of a model-membrane-inserted peptides containing the ErbB2 TM helix and some of the juxtamembrane (JM) residues were studied. For the ErbB2 TM helix inserted into phosphatidylcholine vesicles, the activating V664E mutation was found to induce a transverse shift involving the movement of the E residue toward the membrane surface. This shortened the effective length of the TM-spanning portion of the sequence. The transverse shift was observed with the E664 residue in both the uncharged and charged states, but the extent of the shift was larger when the E residue was charged. When a series of hydrophilic residues was substituted for V664, the resulting transverse shifts at pH 7.0 decreased in the order D,H > E > Q > K > G > V. Except for His, this order is strongly correlated to that reported for the degree to which these substitutions induce cellular transformation when introduced into full-length ErbB2. To examine the effect of lipid on transverse shift, we studied the uncharged V664Q mutation. The presence of 20% of the anionic lipid DOPS (dioleoylphosphatidylserine) in the model membrane vesicles, which introduces a physiologically relevant level of anionic lipid, did not affect the degree of transverse shift. However, in the case of a peptide containing a V674Q substitution, in which the Q is closer to the C-terminus of the ErbB2 TM helix than the N-terminus, transverse shift was suppressed in vesicles containing 20% DOPS. This suggests that the interaction of the cationic JM residues flanking the C-terminus of the ErbB2 TM helix interact with anionic lipids to anchor the C-terminal end of the TM helix. This anchoring site may act as a pivot that amplifies transverse movements of the ErbB2 TM segment to induce a large swinging-type motion in the extracellular domain of the protein, affecting ErbB2 activity. Interactions interrupting C-terminal JM residue association with anionic lipid might partly impact ErbB2 activity by disrupting this pivoting.