Erwin London - Academia.edu (original) (raw)
Papers by Erwin London
Biochemistry, 2003
We examined the effect of ionizable residues at positions flanking the hydrophobic core of helix-... more We examined the effect of ionizable residues at positions flanking the hydrophobic core of helix-forming polyLeu peptides upon helix-helix interactions within model membrane vesicles composed of dioleoylphosphatidylcholine. The peptides studied were flanked on both the N and C termini either by two Lys (K 2 -flanked peptide), one Lys plus one Asp (DK-flanked peptide), or one Lys plus three Asp (KD 3 -flanked peptide). The fluorescence of a Trp residue positioned at the center of the hydrophobic sequence was used to evaluate peptide behavior. As judged by the concentration dependence of the maximum wavelength of Trp emission, there was significant oligomerization of the KD 3 -and DK-flanked peptides, but not the K 2 -flanked peptide, at neutral pH. At neutral pH mixtures of K 2 -and KD 3 -flanked peptides associated with each other, but mixtures of the K 2 -and DK-flanked peptides did not. Oligomerization by the DK-and KD 3 -flanked peptides decreased under low pH conditions in which the Asp residues were protonated. Additional experiments showed that at neutral pH the KD 3 -flanked peptide showed an increased tendency to oligomerize when as little as 10-15 mol % of an anionic lipid, phosphatidylglycerol, was present. The behavior of the other peptides was not strongly influenced by phosphatidylglycerol. These results can largely be explained by modulation of helix-helix interactions via electrostatic interactions involving the helix-flanking ionizable residues. Such interactions may influence membrane protein folding. The self-association of anionic KD 3 -flanked peptides suggests that additional interactions involving charged residues also can modulate helix-helix association. ; MALDI-TOF, matrixassisted laser desorption ionization time-of-flight mass spectrometry.
Position and Ionization State of Asp in the Core of Membrane-Inserted α Helices Control Both the Equilibrium between Transmembrane and Nontransmembrane Helix Topography and Transmembrane Helix Positioning †
Biochemistry, 2004
The behavior of model-membrane-inserted polyLeu-rich peptides containing Asp residues located at ... more The behavior of model-membrane-inserted polyLeu-rich peptides containing Asp residues located at various positions in their hydrophobic core was investigated. The topography of the bilayer-inserted alpha helices formed by these peptides was evaluated by measuring the emission lambda(max) and quenching the fluorescence of a Trp at the center of the peptide sequence. When Asp residues were protonated (at low pH), peptides that were incorporated into vesicles composed of dioleoylphosphatidylcholine (DOPC) adopted a topography in which the polyLeu sequence predominantly formed a normal transmembrane (TM) helix. When Asp residues were ionized (at neutral or high pH), topography was altered in a manner that would allow the charged Asp residues to reside near the bilayer surface. In DOPC vesicles, most peptides repositioned so that the longest segment of consecutive hydrophobic residues (12 residue minimum) formed a truncated/shifted TM structure. However, peptides with one or two charged Asp residues close to the center of the hydrophobic sequence and thus lacking even a 12-residue continuous hydrophobic segment, formed a helical non-TM state locating near the bilayer surface. At low pH, incorporation of the peptides into thicker bilayers composed of dierucoylphosphatidylcholine (DEuPC) resulted in the formation of a mixture of the normal TM state and the non-TM helical state located near the bilayer surface. In DEuPC vesicles at high pH, the non-TM state tended to predominate. How Asp-ionization-dependent shifts in helix topography may regulate the function of membrane proteins exposed to environments with differing pH in vivo (e.g., endosomes) is discussed.
Biochemistry, 2003
A novel fluorescence method for determining the depth of Trp residues in membrane-inserted polype... more A novel fluorescence method for determining the depth of Trp residues in membrane-inserted polypeptides is introduced. Quenching of Trp by acrylamide and 10-doxylnonadecane (10-DN) was used to measure Trp depth. Transmembrane helices with Trp residues at varying positions (and thus locating at different depths in lipid bilayers) were used to calibrate the method. It was found that acrylamide quenches Trp close to the bilayer surface more strongly than it quenches deeply buried Trp, while 10-DN quenches Trp close to the center of the bilayer more strongly than Trp close to the surface. The ratio of acrylamide quenching to that of 10-DN was found to be nearly linearly dependent on the depth of Trp in a membrane. It was also found that it was possible to detect coexisting shallowly and deeply inserted populations of Trp-containing polypeptides using these quenchers. In the presence of such mixed populations, acrylamide induced large blue shifts in fluorescence emission λ max whereas 10-DN induced large red shifts. In a more homogeneous population quencher-induced shifts were found to be minimal. Dual quencher analysis can be used to distinguish hydrophobic helices with a transmembrane orientation from those located close to the bilayer surface and, when applied to a number of different peptides, revealed novel aspects of hydrophobic helix behavior.
Raft-Like Membrane Domains in Pathogenic Microorganisms
Current Topics in Membranes, 2015
The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of li... more The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of lipid-protein microdomains. In eukaryotic cells, microdomains composed of sterols and sphingolipids, commonly known as lipid rafts, are believed to exist, and reports on the presence of sterol- or protein-mediated microdomains in bacterial cell membranes are also appearing. Despite increasing attention, little is known about microdomains in the plasma membrane of pathogenic microorganisms. This review attempts to provide an overview of the current state of knowledge of lipid rafts in pathogenic fungi and bacteria. The current literature on characterization of microdomains in pathogens is reviewed, and their potential role in growth, pathogenesis, and drug resistance is discussed. Better insight into the structure and function of membrane microdomains in pathogenic microorganisms might lead to a better understanding of their pathogenesis and development of raft-mediated approaches for therapy.
Journal of lipid research, 1979
Various detergents can be used to dissolve phospholipids, resulting in very narrow 31PNMR resonan... more Various detergents can be used to dissolve phospholipids, resulting in very narrow 31PNMR resonances. The resonances are well resolved, allowing identification and quantitative analysis of phospholipids in a mixture. The chemical shift depends strongly on pH, reflecting changes in the state of ionization of the phospholipid headgroup moieties. Samples of phospholipids dissolved in aqueous detergents are conveniently prepared and give narrower 31P resonances than do phospholipids dissolved in organic solvents.
Relationship between Sterol/Steroid Structure and Participation in Ordered Lipid Domains (Lipid Rafts): Implications for Lipid Raft Structure and Function
Biochemistry, 2004
The formation and stability of ordered lipid domains (rafts) in model membrane vesicles were stud... more The formation and stability of ordered lipid domains (rafts) in model membrane vesicles were studied using a series of sterols and steroids structurally similar to cholesterol. In one assay, insolubility in Triton X-100 was assessed in bilayers composed of sterol/steroid mixed with dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, or a 1:1 mixture of these phospholipids. In a second assay fluorescence quenching was used to determine the degree of ordered domain formation in bilayers containing sterol/steroid and a 1:1 mixture of DPPC and a quencher-carrying phosphatidylcholine. Both methods showed that several single modifications of the cholesterol structure weaken, but do not fully abolish, the ability of sterols and steroids to promote ordered domain formation when mixed with DPPC. Some of these modifications included a shift of the double bond from the 5-6 carbons (cholesterol) to 4-5 carbons (allocholesterol), derivatization of the 3-OH (cholesterol methyl ether, cholesteryl formate), and alteration of the 3-hydroxy to a keto group (cholestanone). An oxysterol involved in atherosclerosis, 7-ketocholesterol, formed domains with DPPC that were as thermally stable as those with cholesterol although not as tightly packed as judged by fluorescence anisotropy. It was also found that 7-ketocholesterol has fluorescence quenching properties making it a useful spectroscopic probe. Lathosterol, which has a 7-8 carbon double bond in place of the 5-6 double bond of cholesterol, formed rafts with DPPC that were at least as detergent-resistant as, and even more thermally stable than, rafts containing cholesterol. Because lathosterol is an intermediate in cholesterol biosynthesis, we conclude it is unlikely that sterol biosynthesis continues past lathosterol in order to create a raft-favoring lipid.
An amino acid “transmembrane tendency” scale that approaches the theoretical limit to accuracy for prediction of transmembrane helices: Relationship to biological hydrophobicity
Protein Science, 2006
Hydrophobicity analyses applied to databases of soluble and transmembrane (TM) proteins of known ... more Hydrophobicity analyses applied to databases of soluble and transmembrane (TM) proteins of known structure were used to resolve total genomic hydrophobicity profiles into (helical) TM sequences and mainly "subhydrophobic" soluble components. This information was used to define a refined "hydrophobicity"-type TM sequence prediction scale that should approach the theoretical limit of accuracy. The refinement procedure involved adjusting scale values to eliminate differences between the average amino acid composition of populations TM and soluble sequences of equal hydrophobicity, a required property of a scale having maximum accuracy. Application of this procedure to different hydrophobicity scales caused them to collapse to essentially a single TM tendency scale. As expected, when different scales were compared, the TM tendency scale was the most accurate at predicting TM sequences. It was especially highly correlated (r = 0.95) to the biological hydrophobicity scale, derived experimentally from the percent TM conformation formed by artificial sequences passing though the translocon. It was also found that resolution of total genomic sequence data into TM and soluble components could be used to define the percent probability that a sequence with a specific hydrophobicity value forms a TM segment. Application of the TM tendency scale to whole genomic data revealed an overlap of TM and soluble sequences in the "semihydrophobic" range. This raises the possibility that a significant number of proteins have sequences that can switch between TM and non-TM states. Such proteins may exist in moonlighting forms having properties very different from those of the predominant conformation.
Proceedings of the National Academy of Sciences, 1994
Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents b... more Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents because they associate with detergent-resistant membranes rich in cholesterol and sphingolipids. In this study, we demonstrated that cholesterol and sphingolipid-rich liposomes were incompletely solubilized by Triton X-100. GPI-anchored placental alkaline phosphatase incorporated in these liposomes was also not solubilized by cold Triton X-100. As sphingolipids have much higher melting temperatures (Tm) than cellular phospholipids, a property correlated with Tm might cause detergent inextractability. In support of this idea, we found that the low-T. lipid dioleoyl phosphatidylcholine (DOPC) was efficiently extracted from detergent-resistant liposomes by Triton X-100, whereas the high-Tm lipid dipalmitoyl phosphatidylcholine (DPPC) was not. The fluorescence polarization of liposome-incorporated diphenylhexatriene was measured to determine the "fluidity" of the detergent-resistant liposomes. We found that these liposomes were about as fluid as DPPC/cholesterol liposomes, which were present in the liquid-ordered phase, and much less fluid than DOPC or DOPC/cholesterol lposomes. These findings may explain the behavior of GPIanchored proteins, which often have saturated fatty acyl chains and should prefer a less-fluid membrane. Therefore, we pro-
Similarity of the conformation of diphtheria toxin at high temperature to that in the membrane-penetrating low-pH state
Proceedings of the National Academy of Sciences, 1986
At high temperature, nicked free monomers of diphtheria toxin undergo a transition to a thermally... more At high temperature, nicked free monomers of diphtheria toxin undergo a transition to a thermally denatured state, with a midpoint of 45-50 degrees C. In this report, the high-temperature (60 degrees C) conformation has been compared to the native (neutral pH) and low-pH (pH less than 5) conformations. The low-pH and high-temperature conformations are similar although not identical. As in the conformation at low pH, and unlike the toxin in its native conformation, the protein in its conformation at high temperature is hydrophobic, has low fluorescence intensity, and has increased exposure of tryptophan to aqueous solution. As at low pH, at high temperature the circular dichroism spectrum shows at most only partial unfolding of secondary structure. In contrast, the conformation of the toxin in guanidinium chloride is much closer to a random coil. The effects of high temperature and low pH interact in the sense that sensitivity of the native conformation to one is increased by the other. That is, the transition temperature between native and thermally denatured states is decreased as pH is decreased, and the transition pH between neutral-pH and low-pH states is increased as temperature is increased. This implies that there is some region on the protein where high temperature and low pH can disrupt conformation in a similar manner. Taken together, these results indicate that the low-pH and high-temperature conformations can both be defined as partially denatured states, even though unfolding may not be extensive at low pH. Similar behavior may occur in other proteins that undergo functionally important conformational disruption at low pH.
Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry
PLoS ONE, 2014
Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the mos... more Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes "artificial plasma membrane mimicking" ("PMm") vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.
Biophysical Journal, 2010
Upon interaction with cholesterol, perfringolysin O (PFO) inserts into membranes and forms a rigi... more Upon interaction with cholesterol, perfringolysin O (PFO) inserts into membranes and forms a rigid transmembrane (TM) b-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.
Protein-Lipid Interaction and Domain Formation in Asymmetric Membranes
Biophysical Journal, 2010
Biophysical Journal, 2011
We found that phospholipid bilayers are adhesive to each other at pH values lower than 5, while t... more We found that phospholipid bilayers are adhesive to each other at pH values lower than 5, while they are not adhesive at pH values higher than 6. This is significant to membrane fusion occurring at low pH, and to membrane experiments using lipid vesicles. We used the experimental method invented by Evans and collaborators in which one flaccid GUV was released to adhere to one tensed GUV. We developed a new analysis method to measure the adhesion energy per unit area. This new method is independent of how the adhesion state was reached. The order of magnitude of the adhesion energy is~0.01 tõ 0.02 erg/cm2 for SOPC. The addition of SOPE slightly decreases the adhesion energy of pure SOPC, while the addition of cholesterol has little effect. The same method of measurement was applied to a case where two lipid bilayers underwent the first step of membrane fusion, called hemifusion. Hemifusion was induced by injecting 5 wt % PEG8000 solution at pH 4. The PEG injection was used to produce a transient osmotic depletion attraction between the two GUVs. The energy of hemifusion is one order of magnitude larger than the adhesion energy, about~0.3 erg/cm2 for DOPC/DOPE/cholesterol (4:4:2). This is the first time the free energy of the membrane fusion intermediate state was experimentally measured.
Biophysical Journal, 2007
Despite the importance of lipid rafts, commonly defined as liquid-ordered domains rich in cholest... more 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 (T m ), the rules for raft formation in membranes are not completely understood. Here, a fluorescence-quenching strategy was used to define how lipids with low T m , which tend to form disordered fluid domains at physiological temperatures, can stabilize ordered domain formation by cholesterol and high-T m lipids (either sphingomyelin or dipalmitoylphosphatidylcholine). In bilayers containing mixtures of low-T m phosphatidylcholines, cholesterol, and high-T m lipid, the thermal stability of ordered domains decreased with the acyl-chain structure of low-T m lipids in the following order:
Measurement of Lipid Nanodomain (Raft) Formation and Size in Sphingomyelin/POPC/Cholesterol Vesicles Shows TX-100 and Transmembrane Helices Increase Domain Size by Coalescing Preexisting Nanodomains But Do Not Induce Domain Formation
Biophysical Journal, 2011
Mixtures of unsaturated lipids, sphingolipids, and cholesterol form coexisting liquid-disordered ... more 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 (= T(mid)) at ∼45°C. However, T(mid) was lower when detected using various fluorescence resonance energy transfer (FRET) pairs. Furthermore, the T(mid) 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 T(mid). However, FRET-detected T(mid) 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.
Folding changes in membrane-inserted diphtheria toxin that may play important roles in its translocation
Biochemistry, 1991
Diphtheria toxin membrane penetration is triggered by the low pH within the endosome lumen. Subse... more Diphtheria toxin membrane penetration is triggered by the low pH within the endosome lumen. Subsequent exposure to the neutral pH of the cytoplasm is believed to aid in translocation of the catalytic A domain of the toxin into the cytoplasm. To understand the effects of low pH and subsequent exposure to neutral pH on translocation, we studied toxin conformation in solution and in toxin inserted in model membranes. Two conformations were found at low pH. One form, L', predominates below 25-30 degrees C, and the other, L", predominates above 25-30 degrees C and is formed from the L' state by an unfolding event. Both forms are hydrophobic and penetrate deeply into membranes. After pH neutralization, the L' and L'' conformations give rise to two new conformations, R' and R'', respectively. The R' and R" conformations differ from each other in that in the R' state the A domain remains folded, whereas in the R" state the A domain is unfolded. This is confirmed by the finding that only the R' state possesses the capacity to bind and hydrolyze NAD+. It is also supported by the finding that the R'' state can also be formed by thermal unfolding of the R' state. The R conformations differ from the low-pH L conformations in that although they remain largely membrane-inserted, it appears that a large portion of the toxin is no longer in contact with the hydrophobic core of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)
Extension of the parallax analysis of membrane penetration depth to the polar region of model membranes: Use of fluorescence quenching by a spin-label attached to the phospholipid polar headgroup
Biochemistry, 1993
The parallax method is a method by which the depth of fluorescent molecules within a membrane is ... more The parallax method is a method by which the depth of fluorescent molecules within a membrane is calculated from the ratio of quenching induced by two spin-labeled phospholipids at different depths. In this report, the method is extended to measurements of depth in the polar headgroup region of the membrane through use of a lipid with a spin-label attached to the polar choline moiety. Quenching data indicate that the choline-attached nitroxide is close to 19.5 A from the bilayer center, in good agreement with the choline location previously determined by diffraction measurements. By using quenching results obtained with this polar headgroup-labeled phospholipid, depths more accurate than those measured previously can be obtained for fluorophores in the polar region of the membrane. It appears that the most reliable results are obtained when depth is calculated from the quenching of the two spin-labels that quench a specific fluorophore most strongly. Applying this approach to a series of anthroyloxy-labeled fatty acids indicates that the depth of the anthroyloxy group is almost linearly related to the number of carbon atoms between it and the carboxyl group. The fatty acid carboxyl group itself is close to 18.6 A from the bilayer center in the ionized form and 16 A from bilayer center in the protonated form. This is close to the depth of the carboxyl groups on phospholipid fatty acyl chains. More accurate depths have also been obtained for 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled phospholipids using the quenching of the choline-attached spin-label.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry, 1992
In this report, parallax analysis of fluorescence quenching (see the preceding paper in this issu... more In this report, parallax analysis of fluorescence quenching (see the preceding paper in this issue) was used to determine the location (depth) of anthroyloxy and carbazole probes attached to model membrane inserted fatty acids. A monotonic increase in depth was found as the number of carbon atoms between the attachment site of the probe and the fatty acyl carboxyl group is increased. It was also found that depth is sensitive to pH, with an increase in probe depth upon protonation of the fatty acid carboxyl group of around
Cumulative Effects of Amino Acid Substitutions and Hydrophobic Mismatch upon the Transmembrane Stability and Conformation of Hydrophobic α-Helices †
Biochemistry, 2003
The effects of amino acid substitutions upon the behavior of poly(Leu)-rich alpha-helices inserte... more The effects of amino acid substitutions upon the behavior of poly(Leu)-rich alpha-helices inserted into model membrane vesicles were investigated. One or two consecutive Leu residues in the hydrophobic core of the helix were substituted with A, F, G, S, D, K, H, P, GG, SS, PG, PP, KK, or DD residues. A Trp placed at the center of the sequence allowed assessment of peptide behavior via fluorescence emission lambda(max) and dual quenching analysis of Trp depth [Caputo, G. A., and London, E. (2003) Biochemistry 42, 3265-3274]. In vesicles composed of dioleoylphosphatidylcholine (DOPC), all of the peptides with single substitutions adopted a transmembrane (TM) state. Experiments were also performed in thicker bilayers composed of dierucoylphosphatidylcholine (DEuPC). In DEuPC vesicles TM states were destabilized by mismatch between helix length and bilayer thickness. Nevertheless, in DEuPC vesicles TM states were still prevalent for peptides with single substitutions, although less so for peptides with P, K, H, or D substitutions. In contrast to single substitutions, certain consecutive double substitutions strongly interfered with formation of TM states. In both DOPC and DEuPC vesicles DD and KK substitutions abolished the normal TM state, but GG and SS substitutions had little effect. In even wider bilayers, a SS substitution reduced the formation of a TM state. A peptide with a PP substitution maintained the TM state in DOPC vesicles, but in DEuPC vesicles the level of formation of the TM state was significantly reduced. Upon disruption of normal TM insertion peptides moved close to the bilayer surface, with the exception of the KK-substituted peptide in DOPC vesicles, which formed a truncated TM segment. These studies begin to provide a detailed relationship between sequence and the stability of TM insertion and show that the influence of insertion-destabilizing residues upon hydrophobic helices can be strongly modulated by properties such as mismatch. For certain helix-forming hydrophobic sequences, sensitivity to lipid structure may be sufficient to induce large conformational changes in vivo.
Biochemistry, 2003
We examined the effect of ionizable residues at positions flanking the hydrophobic core of helix-... more We examined the effect of ionizable residues at positions flanking the hydrophobic core of helix-forming polyLeu peptides upon helix-helix interactions within model membrane vesicles composed of dioleoylphosphatidylcholine. The peptides studied were flanked on both the N and C termini either by two Lys (K 2 -flanked peptide), one Lys plus one Asp (DK-flanked peptide), or one Lys plus three Asp (KD 3 -flanked peptide). The fluorescence of a Trp residue positioned at the center of the hydrophobic sequence was used to evaluate peptide behavior. As judged by the concentration dependence of the maximum wavelength of Trp emission, there was significant oligomerization of the KD 3 -and DK-flanked peptides, but not the K 2 -flanked peptide, at neutral pH. At neutral pH mixtures of K 2 -and KD 3 -flanked peptides associated with each other, but mixtures of the K 2 -and DK-flanked peptides did not. Oligomerization by the DK-and KD 3 -flanked peptides decreased under low pH conditions in which the Asp residues were protonated. Additional experiments showed that at neutral pH the KD 3 -flanked peptide showed an increased tendency to oligomerize when as little as 10-15 mol % of an anionic lipid, phosphatidylglycerol, was present. The behavior of the other peptides was not strongly influenced by phosphatidylglycerol. These results can largely be explained by modulation of helix-helix interactions via electrostatic interactions involving the helix-flanking ionizable residues. Such interactions may influence membrane protein folding. The self-association of anionic KD 3 -flanked peptides suggests that additional interactions involving charged residues also can modulate helix-helix association. ; MALDI-TOF, matrixassisted laser desorption ionization time-of-flight mass spectrometry.
Position and Ionization State of Asp in the Core of Membrane-Inserted α Helices Control Both the Equilibrium between Transmembrane and Nontransmembrane Helix Topography and Transmembrane Helix Positioning †
Biochemistry, 2004
The behavior of model-membrane-inserted polyLeu-rich peptides containing Asp residues located at ... more The behavior of model-membrane-inserted polyLeu-rich peptides containing Asp residues located at various positions in their hydrophobic core was investigated. The topography of the bilayer-inserted alpha helices formed by these peptides was evaluated by measuring the emission lambda(max) and quenching the fluorescence of a Trp at the center of the peptide sequence. When Asp residues were protonated (at low pH), peptides that were incorporated into vesicles composed of dioleoylphosphatidylcholine (DOPC) adopted a topography in which the polyLeu sequence predominantly formed a normal transmembrane (TM) helix. When Asp residues were ionized (at neutral or high pH), topography was altered in a manner that would allow the charged Asp residues to reside near the bilayer surface. In DOPC vesicles, most peptides repositioned so that the longest segment of consecutive hydrophobic residues (12 residue minimum) formed a truncated/shifted TM structure. However, peptides with one or two charged Asp residues close to the center of the hydrophobic sequence and thus lacking even a 12-residue continuous hydrophobic segment, formed a helical non-TM state locating near the bilayer surface. At low pH, incorporation of the peptides into thicker bilayers composed of dierucoylphosphatidylcholine (DEuPC) resulted in the formation of a mixture of the normal TM state and the non-TM helical state located near the bilayer surface. In DEuPC vesicles at high pH, the non-TM state tended to predominate. How Asp-ionization-dependent shifts in helix topography may regulate the function of membrane proteins exposed to environments with differing pH in vivo (e.g., endosomes) is discussed.
Biochemistry, 2003
A novel fluorescence method for determining the depth of Trp residues in membrane-inserted polype... more A novel fluorescence method for determining the depth of Trp residues in membrane-inserted polypeptides is introduced. Quenching of Trp by acrylamide and 10-doxylnonadecane (10-DN) was used to measure Trp depth. Transmembrane helices with Trp residues at varying positions (and thus locating at different depths in lipid bilayers) were used to calibrate the method. It was found that acrylamide quenches Trp close to the bilayer surface more strongly than it quenches deeply buried Trp, while 10-DN quenches Trp close to the center of the bilayer more strongly than Trp close to the surface. The ratio of acrylamide quenching to that of 10-DN was found to be nearly linearly dependent on the depth of Trp in a membrane. It was also found that it was possible to detect coexisting shallowly and deeply inserted populations of Trp-containing polypeptides using these quenchers. In the presence of such mixed populations, acrylamide induced large blue shifts in fluorescence emission λ max whereas 10-DN induced large red shifts. In a more homogeneous population quencher-induced shifts were found to be minimal. Dual quencher analysis can be used to distinguish hydrophobic helices with a transmembrane orientation from those located close to the bilayer surface and, when applied to a number of different peptides, revealed novel aspects of hydrophobic helix behavior.
Raft-Like Membrane Domains in Pathogenic Microorganisms
Current Topics in Membranes, 2015
The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of li... more The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of lipid-protein microdomains. In eukaryotic cells, microdomains composed of sterols and sphingolipids, commonly known as lipid rafts, are believed to exist, and reports on the presence of sterol- or protein-mediated microdomains in bacterial cell membranes are also appearing. Despite increasing attention, little is known about microdomains in the plasma membrane of pathogenic microorganisms. This review attempts to provide an overview of the current state of knowledge of lipid rafts in pathogenic fungi and bacteria. The current literature on characterization of microdomains in pathogens is reviewed, and their potential role in growth, pathogenesis, and drug resistance is discussed. Better insight into the structure and function of membrane microdomains in pathogenic microorganisms might lead to a better understanding of their pathogenesis and development of raft-mediated approaches for therapy.
Journal of lipid research, 1979
Various detergents can be used to dissolve phospholipids, resulting in very narrow 31PNMR resonan... more Various detergents can be used to dissolve phospholipids, resulting in very narrow 31PNMR resonances. The resonances are well resolved, allowing identification and quantitative analysis of phospholipids in a mixture. The chemical shift depends strongly on pH, reflecting changes in the state of ionization of the phospholipid headgroup moieties. Samples of phospholipids dissolved in aqueous detergents are conveniently prepared and give narrower 31P resonances than do phospholipids dissolved in organic solvents.
Relationship between Sterol/Steroid Structure and Participation in Ordered Lipid Domains (Lipid Rafts): Implications for Lipid Raft Structure and Function
Biochemistry, 2004
The formation and stability of ordered lipid domains (rafts) in model membrane vesicles were stud... more The formation and stability of ordered lipid domains (rafts) in model membrane vesicles were studied using a series of sterols and steroids structurally similar to cholesterol. In one assay, insolubility in Triton X-100 was assessed in bilayers composed of sterol/steroid mixed with dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, or a 1:1 mixture of these phospholipids. In a second assay fluorescence quenching was used to determine the degree of ordered domain formation in bilayers containing sterol/steroid and a 1:1 mixture of DPPC and a quencher-carrying phosphatidylcholine. Both methods showed that several single modifications of the cholesterol structure weaken, but do not fully abolish, the ability of sterols and steroids to promote ordered domain formation when mixed with DPPC. Some of these modifications included a shift of the double bond from the 5-6 carbons (cholesterol) to 4-5 carbons (allocholesterol), derivatization of the 3-OH (cholesterol methyl ether, cholesteryl formate), and alteration of the 3-hydroxy to a keto group (cholestanone). An oxysterol involved in atherosclerosis, 7-ketocholesterol, formed domains with DPPC that were as thermally stable as those with cholesterol although not as tightly packed as judged by fluorescence anisotropy. It was also found that 7-ketocholesterol has fluorescence quenching properties making it a useful spectroscopic probe. Lathosterol, which has a 7-8 carbon double bond in place of the 5-6 double bond of cholesterol, formed rafts with DPPC that were at least as detergent-resistant as, and even more thermally stable than, rafts containing cholesterol. Because lathosterol is an intermediate in cholesterol biosynthesis, we conclude it is unlikely that sterol biosynthesis continues past lathosterol in order to create a raft-favoring lipid.
An amino acid “transmembrane tendency” scale that approaches the theoretical limit to accuracy for prediction of transmembrane helices: Relationship to biological hydrophobicity
Protein Science, 2006
Hydrophobicity analyses applied to databases of soluble and transmembrane (TM) proteins of known ... more Hydrophobicity analyses applied to databases of soluble and transmembrane (TM) proteins of known structure were used to resolve total genomic hydrophobicity profiles into (helical) TM sequences and mainly "subhydrophobic" soluble components. This information was used to define a refined "hydrophobicity"-type TM sequence prediction scale that should approach the theoretical limit of accuracy. The refinement procedure involved adjusting scale values to eliminate differences between the average amino acid composition of populations TM and soluble sequences of equal hydrophobicity, a required property of a scale having maximum accuracy. Application of this procedure to different hydrophobicity scales caused them to collapse to essentially a single TM tendency scale. As expected, when different scales were compared, the TM tendency scale was the most accurate at predicting TM sequences. It was especially highly correlated (r = 0.95) to the biological hydrophobicity scale, derived experimentally from the percent TM conformation formed by artificial sequences passing though the translocon. It was also found that resolution of total genomic sequence data into TM and soluble components could be used to define the percent probability that a sequence with a specific hydrophobicity value forms a TM segment. Application of the TM tendency scale to whole genomic data revealed an overlap of TM and soluble sequences in the "semihydrophobic" range. This raises the possibility that a significant number of proteins have sequences that can switch between TM and non-TM states. Such proteins may exist in moonlighting forms having properties very different from those of the predominant conformation.
Proceedings of the National Academy of Sciences, 1994
Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents b... more Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents because they associate with detergent-resistant membranes rich in cholesterol and sphingolipids. In this study, we demonstrated that cholesterol and sphingolipid-rich liposomes were incompletely solubilized by Triton X-100. GPI-anchored placental alkaline phosphatase incorporated in these liposomes was also not solubilized by cold Triton X-100. As sphingolipids have much higher melting temperatures (Tm) than cellular phospholipids, a property correlated with Tm might cause detergent inextractability. In support of this idea, we found that the low-T. lipid dioleoyl phosphatidylcholine (DOPC) was efficiently extracted from detergent-resistant liposomes by Triton X-100, whereas the high-Tm lipid dipalmitoyl phosphatidylcholine (DPPC) was not. The fluorescence polarization of liposome-incorporated diphenylhexatriene was measured to determine the "fluidity" of the detergent-resistant liposomes. We found that these liposomes were about as fluid as DPPC/cholesterol liposomes, which were present in the liquid-ordered phase, and much less fluid than DOPC or DOPC/cholesterol lposomes. These findings may explain the behavior of GPIanchored proteins, which often have saturated fatty acyl chains and should prefer a less-fluid membrane. Therefore, we pro-
Similarity of the conformation of diphtheria toxin at high temperature to that in the membrane-penetrating low-pH state
Proceedings of the National Academy of Sciences, 1986
At high temperature, nicked free monomers of diphtheria toxin undergo a transition to a thermally... more At high temperature, nicked free monomers of diphtheria toxin undergo a transition to a thermally denatured state, with a midpoint of 45-50 degrees C. In this report, the high-temperature (60 degrees C) conformation has been compared to the native (neutral pH) and low-pH (pH less than 5) conformations. The low-pH and high-temperature conformations are similar although not identical. As in the conformation at low pH, and unlike the toxin in its native conformation, the protein in its conformation at high temperature is hydrophobic, has low fluorescence intensity, and has increased exposure of tryptophan to aqueous solution. As at low pH, at high temperature the circular dichroism spectrum shows at most only partial unfolding of secondary structure. In contrast, the conformation of the toxin in guanidinium chloride is much closer to a random coil. The effects of high temperature and low pH interact in the sense that sensitivity of the native conformation to one is increased by the other. That is, the transition temperature between native and thermally denatured states is decreased as pH is decreased, and the transition pH between neutral-pH and low-pH states is increased as temperature is increased. This implies that there is some region on the protein where high temperature and low pH can disrupt conformation in a similar manner. Taken together, these results indicate that the low-pH and high-temperature conformations can both be defined as partially denatured states, even though unfolding may not be extensive at low pH. Similar behavior may occur in other proteins that undergo functionally important conformational disruption at low pH.
Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry
PLoS ONE, 2014
Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the mos... more Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes "artificial plasma membrane mimicking" ("PMm") vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.
Biophysical Journal, 2010
Upon interaction with cholesterol, perfringolysin O (PFO) inserts into membranes and forms a rigi... more Upon interaction with cholesterol, perfringolysin O (PFO) inserts into membranes and forms a rigid transmembrane (TM) b-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.
Protein-Lipid Interaction and Domain Formation in Asymmetric Membranes
Biophysical Journal, 2010
Biophysical Journal, 2011
We found that phospholipid bilayers are adhesive to each other at pH values lower than 5, while t... more We found that phospholipid bilayers are adhesive to each other at pH values lower than 5, while they are not adhesive at pH values higher than 6. This is significant to membrane fusion occurring at low pH, and to membrane experiments using lipid vesicles. We used the experimental method invented by Evans and collaborators in which one flaccid GUV was released to adhere to one tensed GUV. We developed a new analysis method to measure the adhesion energy per unit area. This new method is independent of how the adhesion state was reached. The order of magnitude of the adhesion energy is~0.01 tõ 0.02 erg/cm2 for SOPC. The addition of SOPE slightly decreases the adhesion energy of pure SOPC, while the addition of cholesterol has little effect. The same method of measurement was applied to a case where two lipid bilayers underwent the first step of membrane fusion, called hemifusion. Hemifusion was induced by injecting 5 wt % PEG8000 solution at pH 4. The PEG injection was used to produce a transient osmotic depletion attraction between the two GUVs. The energy of hemifusion is one order of magnitude larger than the adhesion energy, about~0.3 erg/cm2 for DOPC/DOPE/cholesterol (4:4:2). This is the first time the free energy of the membrane fusion intermediate state was experimentally measured.
Biophysical Journal, 2007
Despite the importance of lipid rafts, commonly defined as liquid-ordered domains rich in cholest... more 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 (T m ), the rules for raft formation in membranes are not completely understood. Here, a fluorescence-quenching strategy was used to define how lipids with low T m , which tend to form disordered fluid domains at physiological temperatures, can stabilize ordered domain formation by cholesterol and high-T m lipids (either sphingomyelin or dipalmitoylphosphatidylcholine). In bilayers containing mixtures of low-T m phosphatidylcholines, cholesterol, and high-T m lipid, the thermal stability of ordered domains decreased with the acyl-chain structure of low-T m lipids in the following order:
Measurement of Lipid Nanodomain (Raft) Formation and Size in Sphingomyelin/POPC/Cholesterol Vesicles Shows TX-100 and Transmembrane Helices Increase Domain Size by Coalescing Preexisting Nanodomains But Do Not Induce Domain Formation
Biophysical Journal, 2011
Mixtures of unsaturated lipids, sphingolipids, and cholesterol form coexisting liquid-disordered ... more 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 (= T(mid)) at ∼45°C. However, T(mid) was lower when detected using various fluorescence resonance energy transfer (FRET) pairs. Furthermore, the T(mid) 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 T(mid). However, FRET-detected T(mid) 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.
Folding changes in membrane-inserted diphtheria toxin that may play important roles in its translocation
Biochemistry, 1991
Diphtheria toxin membrane penetration is triggered by the low pH within the endosome lumen. Subse... more Diphtheria toxin membrane penetration is triggered by the low pH within the endosome lumen. Subsequent exposure to the neutral pH of the cytoplasm is believed to aid in translocation of the catalytic A domain of the toxin into the cytoplasm. To understand the effects of low pH and subsequent exposure to neutral pH on translocation, we studied toxin conformation in solution and in toxin inserted in model membranes. Two conformations were found at low pH. One form, L', predominates below 25-30 degrees C, and the other, L", predominates above 25-30 degrees C and is formed from the L' state by an unfolding event. Both forms are hydrophobic and penetrate deeply into membranes. After pH neutralization, the L' and L'' conformations give rise to two new conformations, R' and R'', respectively. The R' and R" conformations differ from each other in that in the R' state the A domain remains folded, whereas in the R" state the A domain is unfolded. This is confirmed by the finding that only the R' state possesses the capacity to bind and hydrolyze NAD+. It is also supported by the finding that the R'' state can also be formed by thermal unfolding of the R' state. The R conformations differ from the low-pH L conformations in that although they remain largely membrane-inserted, it appears that a large portion of the toxin is no longer in contact with the hydrophobic core of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)
Extension of the parallax analysis of membrane penetration depth to the polar region of model membranes: Use of fluorescence quenching by a spin-label attached to the phospholipid polar headgroup
Biochemistry, 1993
The parallax method is a method by which the depth of fluorescent molecules within a membrane is ... more The parallax method is a method by which the depth of fluorescent molecules within a membrane is calculated from the ratio of quenching induced by two spin-labeled phospholipids at different depths. In this report, the method is extended to measurements of depth in the polar headgroup region of the membrane through use of a lipid with a spin-label attached to the polar choline moiety. Quenching data indicate that the choline-attached nitroxide is close to 19.5 A from the bilayer center, in good agreement with the choline location previously determined by diffraction measurements. By using quenching results obtained with this polar headgroup-labeled phospholipid, depths more accurate than those measured previously can be obtained for fluorophores in the polar region of the membrane. It appears that the most reliable results are obtained when depth is calculated from the quenching of the two spin-labels that quench a specific fluorophore most strongly. Applying this approach to a series of anthroyloxy-labeled fatty acids indicates that the depth of the anthroyloxy group is almost linearly related to the number of carbon atoms between it and the carboxyl group. The fatty acid carboxyl group itself is close to 18.6 A from the bilayer center in the ionized form and 16 A from bilayer center in the protonated form. This is close to the depth of the carboxyl groups on phospholipid fatty acyl chains. More accurate depths have also been obtained for 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled phospholipids using the quenching of the choline-attached spin-label.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry, 1992
In this report, parallax analysis of fluorescence quenching (see the preceding paper in this issu... more In this report, parallax analysis of fluorescence quenching (see the preceding paper in this issue) was used to determine the location (depth) of anthroyloxy and carbazole probes attached to model membrane inserted fatty acids. A monotonic increase in depth was found as the number of carbon atoms between the attachment site of the probe and the fatty acyl carboxyl group is increased. It was also found that depth is sensitive to pH, with an increase in probe depth upon protonation of the fatty acid carboxyl group of around
Cumulative Effects of Amino Acid Substitutions and Hydrophobic Mismatch upon the Transmembrane Stability and Conformation of Hydrophobic α-Helices †
Biochemistry, 2003
The effects of amino acid substitutions upon the behavior of poly(Leu)-rich alpha-helices inserte... more The effects of amino acid substitutions upon the behavior of poly(Leu)-rich alpha-helices inserted into model membrane vesicles were investigated. One or two consecutive Leu residues in the hydrophobic core of the helix were substituted with A, F, G, S, D, K, H, P, GG, SS, PG, PP, KK, or DD residues. A Trp placed at the center of the sequence allowed assessment of peptide behavior via fluorescence emission lambda(max) and dual quenching analysis of Trp depth [Caputo, G. A., and London, E. (2003) Biochemistry 42, 3265-3274]. In vesicles composed of dioleoylphosphatidylcholine (DOPC), all of the peptides with single substitutions adopted a transmembrane (TM) state. Experiments were also performed in thicker bilayers composed of dierucoylphosphatidylcholine (DEuPC). In DEuPC vesicles TM states were destabilized by mismatch between helix length and bilayer thickness. Nevertheless, in DEuPC vesicles TM states were still prevalent for peptides with single substitutions, although less so for peptides with P, K, H, or D substitutions. In contrast to single substitutions, certain consecutive double substitutions strongly interfered with formation of TM states. In both DOPC and DEuPC vesicles DD and KK substitutions abolished the normal TM state, but GG and SS substitutions had little effect. In even wider bilayers, a SS substitution reduced the formation of a TM state. A peptide with a PP substitution maintained the TM state in DOPC vesicles, but in DEuPC vesicles the level of formation of the TM state was significantly reduced. Upon disruption of normal TM insertion peptides moved close to the bilayer surface, with the exception of the KK-substituted peptide in DOPC vesicles, which formed a truncated TM segment. These studies begin to provide a detailed relationship between sequence and the stability of TM insertion and show that the influence of insertion-destabilizing residues upon hydrophobic helices can be strongly modulated by properties such as mismatch. For certain helix-forming hydrophobic sequences, sensitivity to lipid structure may be sufficient to induce large conformational changes in vivo.