Transmembrane peptides stabilize inverted cubic phases in a biphasic length-dependent manner: implications for protein-induced membrane fusion.

WALP peptides consist of repeating alanine-leucine sequences of different lengths, flanked with tryptophan "anchors" at each end. They form membrane-spanning alpha-helices in lipid membranes, and mimic protein transmembrane domains. WALP peptides of increasing length, from 19 to 31 amino acids, were incorporated into N-monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) at concentrations up to 0.5 mol % peptide. When pure DOPE-Me is heated slowly, the lamellar liquid crystalline (L(alpha)) phase first forms an inverted cubic (Q(II)) phase, and the inverted hexagonal (H(II)) phase at higher temperatures. Using time-resolved x-ray diffraction and slow temperature scans (1.5 degrees C/h), WALP peptides were shown to decrease the temperatures of Q(II) and H(II) phase formation (T(Q) and T(H), respectively) as a function of peptide concentration. The shortest and longest peptides reduced T(Q) the most, whereas intermediate lengths had weaker effects. These findings are relevant to membrane fusion because the first step in the L(alpha)/Q(II) phase transition is believed to be the formation of fusion pores between pure lipid membranes. These results imply that physiologically relevant concentrations of these peptides could increase the susceptibility of biomembrane lipids to fusion through an effect on lipid phase behavior, and may explain one role of the membrane-spanning domains in the proteins that mediate membrane fusion.

Interfacial positioning and stability of transmembrane peptides in lipid bilayers studied by combining hydrogen/deuterium exchange and mass spectrometry.

Nano-electrospray ionization mass spectrometry (ESI-MS) was used to analyze hydrogen/deuterium (H/D) exchange properties of transmembrane peptides with varying length and composition. Synthetic transmembrane peptides were used with a general acetyl-GW(2)(LA)(n)LW(2)A-ethanolamine sequence. These peptides were incorporated in large unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The vesicles were diluted in buffered deuterium oxide, and the H/D exchange after different incubation times was directly analyzed by means of ESI-MS. First, the influence of the length of the hydrophobic Leu-Ala sequence on exchange behavior was investigated. It was shown that longer peptide analogs are more protected from H/D exchange than expected on the basis of their length with respect to bilayer thickness. This is explained by an increased protection from the bilayer environment, because of stretching of the lipid acyl chains and/or tilting of the longer peptides. Next, the role of the flanking tryptophan residues was investigated. The length of the transmembrane part that shows very slow H/D exchange was found to depend on the exact position of the tryptophans in the peptide sequence, suggesting that tryptophan acts as a strong determinant for positioning of proteins at the membrane/water interface. Finally, the influence of putative helix breakers was studied. It was shown that the presence of Pro in the transmembrane segment results in much higher exchange rates as compared with Gly or Leu, suggesting a destabilization of the alpha-helix. Tandem MS measurements suggested that the increased exchange takes place over the entire transmembrane segment. The results show that ESI-MS is a convenient technique to gain detailed insight into properties of peptides in lipid bilayers by monitoring H/D exchange kinetics.

Desformylgramicidin: a model channel with an extremely high water permeability.

The water conductivity of desformylgramicidin exceeds the permeability of gramicidin A by two orders of magnitude. With respect to its single channel hydraulic permeability coefficient of 1.1.10(-12) cm(3) s(-1), desformylgramicidin may serve as a model for extremely permeable aquaporin water channel proteins (AQP4 and AQPZ). This osmotic permeability exceeds the conductivity that is predicted by the theory of single-file transport. It was derived from the concentration distributions of both pore-impermeable and -permeable cations that were simultaneously measured by double barreled microelectrodes in the immediate vicinity of a planar bilayer. From solvent drag experiments, approximately five water molecules were found to be transported by a single-file process along with one ion through the channel. The single channel proton, potassium, and sodium conductivities were determined to be equal to 17 pS (pH 2.5), 7 and 3 pS, respectively. Under any conditions, the desformyl-channel remains at least 10 times longer in its open state than gramicidin A.

Neighboring aliphatic/aromatic side chain interactions between residues 9 and 10 in gramicidin channels.

The interactions between an aliphatic or phenyl side chain and an indole ring in a phospholipid environment were investigated by synthesizing and characterizing gramicidins in which Trp(9) was ring-labeled and D-Leu(10) was replaced by D-Val, D-Ala, or D-Phe. All three analogues form conducting channels, with conductances that are lower than that of gramicidin A (gA) channels. The channel lifetimes vary by less than 50% from that of gA channels. Circular dichroism spectra and size-exclusion chromatography show that the conformation of each analogue in dimyristoylphosphatidylcholine (DMPC) vesicles is similar to the right-handed beta(6.3)-helical conformation that is observed for gA. (2)H NMR spectra of oriented samples in DMPC show large changes for the Trp(9) ring when residue 10 is modified, suggesting a steric interaction between D-Leu(10) and Trp(9), in agreement with previous acylation studies (R. E. Koeppe II et al. (1995) Biochemistry 34, 9299-9307). The outer quadrupolar splitting for Trp(9) is unchanged with D-Phe(10), at approximately 153 kHz, but increases by approximately 25 kHz with D-Val(10) and decreases by approximately 10 kHz with D-Ala(10). With D-Ala(10) or D-Val(10), the outer resonance splits into two in a temperature-dependent manner. The NMR spectra indicate that the side chain torsion angles chi1 and chi2 for Trp(9) change when residue 10 is substituted. The changes in chi1 are small, in all cases less than 10 degrees, as is Deltachi2 when D-Ala(10) is introduced, but with D-Val(10) and D-Phe(10) Deltachi2 is at least 25 degrees. We conclude that D-Leu(10) helps to stabilize an optimal orientation of Trp(9) in gA channels in lipid bilayers and that changes in Trp orientation alter channel conductance and lifetime without affecting the basic channel fold.

Tryptophan-anchored transmembrane peptides promote formation of nonlamellar phases in phosphatidylethanolamine model membranes in a mismatch-dependent manner.

To better understand the mutual interactions between lipids and membrane-spanning peptides, we investigated the effects of tryptophan-anchored hydrophobic peptides of various lengths on the phase behavior of 1,2-dielaidoylphosphatidylethanolamine (DEPE) dispersions, using (31)P nuclear magnetic resonance and small-angle X-ray diffraction. Designed alpha-helical transmembrane peptides (WALPn peptides, with n being the total number of amino acids) with a hydrophobic sequence of leucine and alanine of varying length, bordered at both ends by two tryptophan membrane anchors, were used as model peptides and were effective at low concentrations in DEPE. Incorporation of 2 mol % of relatively short peptides (WALP14-17) lowered the inverted hexagonal phase transition temperature (T(H)) of DEPE, with an efficiency that seemed to be independent of the extent of hydrophobic mismatch. However, the tube diameter of the H(II) phase induced by the peptides was clearly dependent on mismatch and decreased with shorter peptide length. Longer peptides (WALP19-27) induced a cubic phase, both below and above T(H). Incorporation of WALP27, which is significantly longer than the DEPE bilayer thickness, did not stabilize the bilayer. The longest peptide used, WALP31, hardly affected the lipid's phase behavior, and appeared not to incorporate into the bilayer. The consequences of hydrophobic mismatch between peptides and lipids are therefore more dramatic with shorter peptides. The data allow us to suggest a detailed molecular model of the mechanism by which these transmembrane peptides can affect lipid phase behavior.

Electrospray ionization mass spectrometry as a tool to analyze hydrogen/deuterium exchange kinetics of transmembrane peptides in lipid bilayers.

A method is described to study the precise positioning of transmembrane peptides in a phospholipid bilayer combining hydrogen/deuterium (H/D) exchange and nanoelectrospray ionization mass spectrometry. The method was tested by using model systems consisting of designed alpha-helical transmembrane peptides [acetylGW(2)(LA)(5)W(2)Aethanolamine (WALP16) and acetyl-(GA)(3)W(2)(LA)(5)W(2)(AG)(3)ethanolamine (WALP16(+10))] incorporated in large unilamellar vesicles of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine. Both peptides consist of an alternating leucine/alanine hydrophobic core sequence flanked by tryptophan residues as interfacial anchor residues. In the case of WALP16(+10), this sequence is extended at both ends by 5-aa glycine/alanine tails extending into the aqueous phase surrounding the bilayer. H/D exchange of labile hydrogens in these peptides was monitored in time after dilution of the vesicles in buffered deuterium oxide. It was found that the peptides can be measured by direct introduction of the proteoliposome suspension into the mass spectrometer. Several distinct H/D exchange rates were observed (corresponding to half-life values varying from

Steric interactions of valines 1, 5, and 7 in [valine 5, D-alanine 8] gramicidin A channels.

When the central valine residues 6, 7, and 8 of gramicidin A (gA) are shifted by one position, the resulting [Val(5), D-Ala(8)]gA forms right-handed channels with a single-channel conductance and average duration somewhat less than gA channels. The reduction in channel duration has been attributed to steric conflict between the side chains of Val(1) and Val(5) in opposing monomers (Koeppe, R. E. II, D. V. Greathouse, A. Jude, G. Saberwal, L. L. Providence, and O. S. Andersen. 1994. J. Biol. Chem. 269:12567-12576). To investigate the orientations and motions of valines in [Val(5), D-Ala(8)]gA, we have incorporated (2)H labels at Val 1, 5, or 7 and recorded (2)H-NMR spectra of oriented and nonoriented samples in hydrated dimyristoylphosphatidylcholine. Spectra of nonoriented samples at 4 degrees C reveal powder patterns that indicate rapid side chain "hopping" for Val(5), and an intermediate rate of hopping for Val(1) and Val(7) that is somewhat slower than in gA. Oriented samples of deuterated Val(1) and Val(7) show large changes in the methyl and C(beta)-(2)H quadrupolar splittings (Deltanu(q)) when Ala(5) in native gA is changed to Val(5). Three or more peaks for the Val(1) methyls with Deltanu(q) values that vary with the echo delay, together with an intermediate spectrum for nonoriented samples at 4 degrees C, suggest unusual side chain dynamics for Val(1) in [Val(5), D-Ala(8)]gA. These results are consistent with a steric conflict that has been introduced between the two opposing monomers. In contrast, the acylation of gA has little influence on the side chain dynamics of Val(1), regardless of the identity of residue 5.

Design and characterization of gramicidin channels with side chain or backbone mutations.

Mutations and chemical substitutions of amino acid side chains and backbone atoms have proved vital for understanding the folding, structure and function of gramicidin channels in phospholipid membranes. The channel's pore is lined by peptide backbone groups; their importance for channel structure and function is shown by a single amide-to-ester replacement within the backbone, which greatly reduces the resulting channel conductance and lifetime. The four tryptophans and the intervening leucines together govern the formation and dissociation of conducting channels from single-stranded subunits. Conducting double-stranded gramicidin conformations (channels) occur rarely in membranes--except when the sequence has been altered to permit special arrangements of tryptophans or (infrequently) in unusually thick membranes. The tryptophans anchor the single-stranded channels to the membrane/solution interface, and the indole dipoles promote cation transport through the channels. Removal of any indole dipole reduces ion conductance; whereas 5-fluorination of an indole, which increases its dipole moment, enhances ion conductance. Some sequence changes at the formyl-NH-terminus (in the membrane interior, away from the tryptophans), including fluorination of the formyl-NH-terminal valine, introduce voltage-dependent channel gating. Gramicidin channels are not just static conductors, but also dynamic entities whose structure and function can be manipulated by backbone and side chain modifications.

Peptide influences on lipids.

The extent to which the length of the membrane-spanning part of intrinsic membrane proteins matches the hydrophobic thickness of the lipid bilayer may be an important factor in determining membrane structure and function. To gain insight into the consequences of hydrophobic mismatch on a molecular level, we have carried out systematic studies on well-defined peptide-lipid complexes. As model peptides we have chosen gramicidin A and a series of artificial hydrophobic alpha-helical transmembrane peptides that resemble the gramicidin channel. These peptides consist of a hydrophobic stretch of alternating leucine and alanine residues with variable length, flanked by tryptophan residues. Using wide-line NMR techniques, we have investigated the interaction of these peptides with the bilayer-forming diacyl phosphatidylcholines and with phospholipids which by themselves have a tendency to form non-bilayer structures. We have shown that hydrophobic mismatch leads to systematic changes of the bilayer thickness and that it can even change the macroscopic organization of the lipids. The type of lipid organization induced by the peptides and the efficiency of the various processes depend on the properties of the lipids and on the precise extent of mismatch.

Different membrane anchoring positions of tryptophan and lysine in synthetic transmembrane alpha-helical peptides.

Specific interactions of membrane proteins with the membrane interfacial region potentially define protein position with respect to the lipid environment. We investigated the proposed roles of tryptophan and lysine side chains as "anchoring" residues of transmembrane proteins. Model systems were employed, consisting of phosphatidylcholine lipids and hydrophobic alpha-helical peptides, flanked either by tryptophans or lysines. Peptides were incorporated in bilayers of different thickness, and effects on lipid structure were analyzed. Induction of nonbilayer phases and also increases in bilayer thickness were observed that could be explained by a tendency of Trp as well as Lys residues to maintain interactions with the interfacial region. However, effects of the two peptides were remarkably different, indicating affinities of Trp and Lys for different sites at the interface. Our data support a model in which the Trp side chain has a specific affinity for a well defined site near the lipid carbonyl region, while the lysine side chain prefers to be located closer to the aqueous phase, near the lipid phosphate group. The information obtained in this study may further our understanding of the architecture of transmembrane proteins and may prove useful for refining prediction methods for transmembrane segments.

Modulation of gramicidin channel structure and function by the aliphatic "spacer" residues 10, 12, and 14 between the tryptophans.

In the linear gramicidins, the four aromatic residues at positions 9, 11, 13, and 15 are well-known to be important for the structure and function of membrane-spanning gramicidin channels. To investigate whether the "spacer" residues between the tryptophans in gramicidin A (gA) are important for channel structure and function, D-Leu-10, -12. and -14 of gA were replaced by Ala, Val, or Ile. (For practical reasons, the Ile substitutions were introduced into the enantiomeric gramicidin A-, gA-.) Circular dichroism spectra of [D-Ala10,12,14]gA, [D-Val10,12,14]gA, or [Ile10,12,14]gA- incorporated into sodium dodecyl sulfate micelles or 1, 2-dimyristoyl-sn-glycero-3-phosphocholine vesicles differ from the spectrum of the native [D-Leu10,12,14]gA. All the analogue spectra display reduced ellipticity at both 218 and 235 nm, indicating the presence of double-stranded conformers with the Ala analogue spectra showing the largest departure from the native gA spectra. Size-exclusion chromatograms of the Val and Ile analogues show both monomer and dimer peaks, accompanied by peak broadening; the chromatograms for the Ala analogue show broad, overlapping peaks and suggest the presence of higher oligomers and/or (rapidly) interconverting conformations. All three analogues form membrane-spanning channels, with the channel-forming potency of the Ala analogue being much less than that of gA or the other analogues. In 1.0 M CsCl, the conductance of each analogue channel is approximately 25% less than that of [D-Leu10,12,14]gA channels. The lifetimes of the analogue channels also are less than of [D-Leu10,12, 14]gA channels, with the largest (8-fold) reduction being for [D-Ala10,12,14]gA channels. Hybrid channel experiments show that the beta6.3-helical backbone folding pattern is retained in the channel-forming subunits and that the substitutions primarily influence ion entry. Both the bulk and the stereochemistry of the aliphatic residues between the tryptophans of gA are important for channel structure and function.

Design and characterization of gramicidin channels.

This article summarizes methods for the chemical synthesis and biophysical characterization of gramicidins with varying sequences and labels. The family of gramicidin channels has developed into a powerful model system for understanding fundamental properties, interactions, and dynamics of proteins and lipids generally, and ion channels specifically, in biological membranes.

Influence of lipid/peptide hydrophobic mismatch on the thickness of diacylphosphatidylcholine bilayers. A 2H NMR and ESR study using designed transmembrane alpha-helical peptides and gramicidin A.

We have investigated the effect of a series of hydrophobic polypeptides (WALP peptides) on the mean hydrophobic thickness of (chain-perdeuterated) phosphatidylcholines (PCs) with different acyl chain length, using 2H NMR and ESR techniques. The WALP peptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked, e.g., FmAW2(LA)nW2AEtn. 2H NMR measurements showed that the shortest peptide with a total length of 16 amino acids (WALP16) causes an increase of 0.6 A in bilayer thickness in di-C12-PC, a smaller increase in di-C14-PC, no effect in di-C16-PC, and a decrease of 0.4 A in di-C18-PC, which was the largest decrease observed in any of the peptide/lipid systems. The longest peptide, WALP19, in di-C12-PC caused the largest increase in thickness of the series (+1.4 A), which decreased again for longer lipids toward di-C18-PC, in which no effect was noticed. WALP17 displayed an influence intermediate between that of WALP16 and WALP19. Altogether, incorporation of the WALP peptides was found to result in small but very systematic changes in bilayer thickness and area per lipid molecule, depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. ESR measurements with spin-labeled lipid probes confirmed this result. Because thickness is expected to be influenced most at the lipids directly adjacent to the peptides, also the maximal adaptation of these first-shell lipids was estimated. The calculation was based on the assumption that there is little or no aggregation of the WALP peptides, as was supported by ESR, and that lipid exchange is rapid on the 2H NMR time scale. It was found that even the maximal possible changes in first-shell lipid length were relatively small and represented only a partial response to mismatch. The synthetic WALP peptides are structurally related to the gramicidin channel, which was therefore used for comparison. In most lipid systems, gramicidin proved to be a stronger perturber of bilayer thickness than WALP19, although its length should approximate that of the shorter WALP16. The effects of gramicidin and WALP peptides on bilayer thickness were evaluated with respect to previous 31P NMR studies on the effects of these peptides on macroscopic lipid phase behavior. Both approaches indicate that, in addition to the effective hydrophobic length, also the physical nature of the peptide surface is a modulator of lipid order.

Influence of membrane-spanning alpha-helical peptides on the phase behavior of the dioleoylphosphatidylcholine/water system.

The effect of solubilized hydrophobic peptides on the phase behavior of dioleoylphosphatidylcholine (DOPC)/water system was studied by 2H- and 31P-NMR spectroscopy and by x-ray diffraction, and partial phase diagrams were constructed. The utilized peptides were HCO-AWW(LA)5WWA-NHCH2CH2OH (WALP16), which is an artificial peptide designed to resemble a transmembrane part of a membrane protein; and VEYAGIALFFVAAVLTLWSMLQYLSAAR (Pgs peptide E), a peptide that is identical to one of the putative transmembrane segments of the membrane-associated protein phosphatidylglycerophosphate synthase (Pgs) in Escherichia coli. Circular dichroism spectroscopy suggests that both peptides are mostly alpha-helical in DOPC vesicles. The most striking features in the phase diagram of the WALP16/DOPC/water system are 1) a single lamellar liquid crystalline (L alpha) phase forms only at very low peptide concentrations. 2) At low water content and above a peptide/lipid molar ratio of approximately 1:75 a reversed hexagonal liquid crystalline (H[II]) phase coexists with an L alpha phase, while in excess water this phase forms at a peptide/lipid molar ratio of approximately 1:25. 3) At peptide/lipid ratios > or =1:6 a single H(II) phase is stable. Also, the Pgs peptide E strongly affects the phase behavior, and a single L alpha phase is only found at low peptide concentrations (peptide/lipid molar ratios <1:50), and water concentrations <45% (w/w). Higher peptide content results in coexistence of L alpha and isotropic phases. Generally, the fraction of the isotropic phase increases with increasing temperature and water concentration, and at 80% (w/w) water content only a single isotropic phase is stable at 55 degrees C. Thus, both peptides were found to be able to induce nonlamellar phases, although different in structure, in the DOPC/water system. The phase transitions, the extensions of the one-phase regions, and the phase structures observed for the two systems are discussed in terms of the molecular structure of the two peptides and the matching between the hydrophobic lengths of the peptides and the bilayer thickness of DOPC.

Gramicidin channels in phospholipid bilayers with unsaturated acyl chains.

In organic solvents gramicidin A (gA) occurs as a mixture of slowly interconverting double-stranded dimers. Membrane-spanning gA channels, in contrast, are almost exclusively single-stranded beta(6,3)-helical dimers. Based on spectroscopic evidence, it has previously been concluded that the conformational preference of gA in phospholipid bilayers varies as a function of the degree of unsaturation of the acyl chains. Double-stranded pi pi(5,6)-helical dimers predominate (over single-stranded beta(6,3)-helical dimers) in lipid bilayer membranes with polyunsaturated acyl chains. We therefore examined the characteristics of channels formed by gA in 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane, 1,2-dioleoylphosphatidylcholine/n-decane, and 1,2-dilinoleoylphosphatidylcholine/n-decane bilayers. We did not observe long-lived channels that could be conducting double-stranded pi pi(5,6)-helical dimers in any of these different membrane environments. We conclude that the single-stranded beta(6,3)-helical dimer is the only conducting species in these bilayers. Somewhat surprisingly, the average channel duration and channel-forming potency of gA are increased in dilinoleoylphosphatidylcholine/n-decane bilayers compared to 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane and dioleoylphosphatidylcholine/n-decane bilayers. To test for specific interactions between the aromatic side chains of gA and the acyl chains of the bilayer, we examined the properties of channels formed by gramicidin analogues in which the four tryptophan residues were replaced with naphthylalanine (gN), tyrosine (gT), and phenylalanine (gM). The results show that all of these analogue channels experience the same relative stabilization when going from dioleoylphosphatidylcholine to dilinoleoylphosphatidylcholine bilayers.

Gramicidin channels--a solvable membrane "protein" folding problem.

The linear gramicidins are peptide antibiotics that form cation-selective channels in lipid bilayers. Gramicidin channels have very well-defined functional characteristics, and the structure of membrane-spanning gramicidin A channels is known at atomic resolution. These features make the gramicidins well suited to study how the amino acid sequence encodes the structure and function of a membrane-spanning channel. We show how one can use electrophysiological measurements to obtain structural information about conducting channels and to quantify the conformational preferences of sequence-substituted gramicidin mutants.

Conformation of the acylation site of palmitoylgramicidin in lipid bilayers of dimyristoylphosphatidylcholine.

Gramicidin A(gA) can be palmitoylated by means of an ester linkage to the OH group of the terminal ethanolamine that sits at the membrane-water interface in the functional gA channel. We have investigated palmitoyl-gA as a model transmembrane acylprotein. Ethanolamine-d(4) (NH(2)CD(2)CD(2)OH) was incorporated into gA by total synthesis, and a portion of the labeled gA was palmitoylated. Solid-state (2)H-NMR spectra of acyl- and nonacyl-gA in hydrated dimyristoylphosphatidylcholine (DMPC) bilayers were compared. The spectra for both oriented and nonoriented samples at 4 and at 40 degrees C indicate that the ethanolamine of gA is highly mobile prior to acylation, but essentially immobile after palmitoylation. The (2)H quadrupolar splittings allow the conformation of the ethanolamine group in acyl-gA to be determined. By combining our data with the previously determined quadrupolar splittings for deuterium labels on the palmitoyl chain [Vogt, T.C.B., Killian, J.A., & de Kruijff, B. (1994) Biochemistry 33, 2063-2070], we also propose a model for the acyl chain. The ethanolamine group rotates over Leu(10) and toward the outside of the gA channel's cylinder upon acylation, so that the attached acyl chain passes between the side chains of Trp(9) and Leu(10). To accommodate the acyl chain, the six-membered portion of the indole ring of Trp(9) is displaced by about 0.9 angstroms, by means of 1-2 degree rotations in chi(1) and chi(2).