A theoretical model for the association of amphiphilic transmembrane peptides in lipid bilayers

A theoretical model is proposed for the association of trans-bilayer peptides in lipid bilayers. The model is based on a lattice model for the pure lipid bilayer, which accounts accurately for the most important conformational states of the lipids and their mutual interactions and statistics. Within the lattice formulation the bilayer is formed by two independent monolayers, each represented by a triangular lattice, on which sites the lipid chains are arrayed. The peptides are represented by regular objects, with no internal flexibility, and with a projected area on the bilayer plane corresponding to a hexagon with seven lattice sites. In addition, it is assumed that each peptide surface at the interface with the lipid chains is partially hydrophilic, and therefore interacts with the surrounding lipid matrix via selective anisotropic forces. The peptides would therefore assemble in order to shield their hydrophilic residues from the hydrophobic surroundings. The model describes the self-association of peptides in lipid bilayers via lateral and rotational diffusion, anisotropic lipid-peptide interactions, and peptide-peptide interactions involving the peptide hydrophilic regions. The intent of this model study is to analyse the conditions under which the association of trans-bilayer and partially hydrophilic peptides (or their dispersion in the lipid matrix) is lipid-mediated, and to what extent it is induced by direct interactions between the hydrophilic regions of the peptides. The model properties are calculated by a Monte Carlo computer simulation technique within the canonical ensemble. The results from the model study indicate that direct interactions between the hydrophilic regions of the peptides are necessary to induce peptide association in the lipid bilayer in the fluid phase. Furthermore, peptides within each aggregate are oriented in such a way as to shield their hydrophilic regions from the hydrophobic environment. The average number of peptides present in the aggregates formed depends on the degree of mismatch between the peptide hydrophobic length and the lipid bilayer hydrophobic thickness: The lower the degree of mismatch is the higher this number is. Received: 30 December 1996 / Accepted: 9 May 1997     

The transmembrane homotrimer of ADAM 1 in model lipid bilayers

Fertilin is a transmembrane protein heterodimer formed by the two subunits fertilin alpha and fertilin beta that plays an important role in sperm-egg fusion. Fertilin alpha and beta are members of the ADAM family, and contain each one transmembrane alpha-helix, and are termed ADAM 1 and ADAM 2, respectively. ADAM 1 is the subunit that contains a putative fusion peptide, and we have explored the possibility that the transmembrane alpha-helical domain of ADAM 1 forms homotrimers, in common with other viral fusion proteins. Although this peptide was found to form various homooligomers in SDS, the infrared dichroic data obtained with the isotopically labeled peptide at specific positions is consistent with the presence of only one species in DMPC or POPC lipid bilayers. Comparison of the experimental orientational data with molecular dynamics simulations performed with sequence homologues of ADAM 1 show that the species present in lipid bilayers is only consistent with an evolutionarily conserved homotrimeric model for which we provide a backbone structure. These results support a model where ADAM 1 forms homotrimers as a step to create a fusion active intermediate.     

Molecular organization in striated domains induced by transmembrane alpha-helical peptides in dipalmitoyl phosphatidylcholine bilayers

Transmembrane (TM) alpha-helical peptides with neutral flanking residues such as tryptophan form highly ordered striated domains when incorporated in gel-state 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers and inspected by atomic force microscopy (AFM) (1). In this study, we analyze the molecular organization of these striated domains using AFM, photo-cross-linking, fluorescence spectroscopy, nuclear magnetic resonance (NMR), and X-ray diffraction techniques on different functionalized TM peptides. The results demonstrate that the striated domains consist of linear arrays of single TM peptides with a dominantly antiparallel organization in which the peptides interact with each other and with lipids. The peptide arrays are regularly spaced by +/-8.5 nm and are separated by somewhat perturbed gel-state lipids with hexagonally organized acyl chains, which have lost their tilt. This system provides an example of how domains of peptides and lipids can be formed in membranes as a result of a combination of specific peptide-peptide and peptide-lipid interactions.     

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

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

Sphingolipid partitioning into ordered domains in cholesterol-free and cholesterol-containing lipid bilayers

We have used fluorescence-quenching measurements to characterize the partitioning of a variety of indolyl-labeled phospho- and sphingolipids between gel or liquid-ordered and liquid-disordered lipid domains in several types of lipid bilayers where such domains coexist. In both cholesterol-free and cholesterol-containing lipid mixtures, sphingolipids with diverse polar headgroups (ranging from sphingomyelin and monoglycosylceramides to ganglioside GM1) show a net preference for partitioning into ordered domains, which varies modestly in magnitude with varying headgroup structure. The affinities of different sphingolipids for ordered lipid domains do not vary in a consistent manner with the size or other simple structural properties of the polar headgroup, such that for example ganglioside GM1 partitions between ordered and disordered lipid domains in a manner very similar to sphingomyelin. Ceramide exhibits a dramatically higher affinity for ordered lipid domains in both cholesterol-free and cholesterol-containing bilayers than do other sphingolipids. Our findings suggest that sphingolipids with a variety of headgroup structures will be enriched by substantial factors in liquid-ordered versus liquid-disordered regions of membranes, in a manner that is only modestly dependent on the nature of the polar headgroup. Ceramide is predicted to show a very strong enrichment in such domains, supporting previous suggestions that ceramide-mediated signaling may be compartmentalized to liquid-ordered (raft and raft-related) domains in the plasma membrane.     

Tilt angles of transmembrane model peptides in oriented and non-oriented lipid bilayers as determined by 2H solid-state NMR

Solid-state NMR methods employing (2)H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane alpha-helical peptide acetyl-GWW(LA)(8)LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane alpha-helical conformation. A small tilt angle of 4.5 degrees was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2 degrees in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA)(6)LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.     

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

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

Different sphingolipids show differential partitioning into sphingolipid/cholesterol-rich domains in lipid bilayers

Two fluorescence-based approaches have been applied to examine the differential partitioning of fluorescent phospho- and sphingolipid molecules into sphingolipid-enriched domains modeling membrane "lipid rafts." Fluorescence-quenching measurements reveal that N-(diphenylhexatrienyl)propionyl- (DPH3:0-)-labeled gluco- and galactocerebroside partition into sphingolipid-enriched domains in sphingolipid/phosphatidylcholine/cholesterol bilayers with substantially higher affinity than do analogous sphingomyelin, ceramide, or phosphatidylcholine molecules. By contrast, the affinity of sphingomyelin and ceramide for such domains is only marginally greater than that of a phosphatidylcholine with similar hydrocarbon chains. By using direct measurements of molecular partitioning between vesicles of different compositions, we show that the relative affinities of different C(6)-NBD- and C(5)-Bodipy-labeled sphingolipids for sphingolipid-enriched domains are quantitatively, and in most circumstances even qualitatively, quite different from those found for species whose N-acyl chains more closely resemble the long saturated chains of cellular sphingolipids. These findings lend support in principle to previous suggestions that differential partitioning of different sphingolipids into "raft" domains could contribute to the differential trafficking of these species in eukaryotic cells. However, our findings also indicate that short-chain sphingolipid probes previously used to examine this phenomenon are in general ill-suited for such applications.     

Topological stability and self-association of a completely hydrophobic model transmembrane helix in lipid bilayers

Investigation of interactions between hydrophobic model peptides and lipid bilayers is perhaps the only way to elucidate the principles of the folding and stability of membrane proteins (White, S. H., and Wimley, W. C. (1998) Biochim. Biophys. Acta 1367, 339-352). We designed the completely hydrophobic "inert" peptide modeling a transmembrane (TM) helix without any of the specific side-chain interactions expected, X-(LALAAAA)(3)-NH(2) [X = Ac (I), 7-nitro-2-1,3-benzoxadiazol-4-yl (II), or 5(6)-carboxytetramethylrhodamine (III)]. Fourier transform infrared-polarized attenuated total reflection measurements revealed that I as well as II assume a TM helix in hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Dithionite quenching experiments detected no topological change (flip-flop) in the helix II for at least 24 h. Thus, the TM helix itself is a highly stable structure, even in the absence of flanking hydrophilic or aromatic amino acids which are suggested to play important roles in stable TM positioning. Helix self-association in lipid bilayers was detected by fluorescence resonance energy transfer between II and III. The peptide was in a monomer-antiparallel dimer equilibrium with an association free energy of approximately -13 kJ/mol. Electron spin resonance spectra of 1-palmitoyl-2-stearoyl-(14-doxyl)-sn-glycero-3-phosphocholine demonstrated the presence of a motionally restricted component at lower temperatures.     

General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides

Model compounds of modified hydrophobicity (H), hydrophobic moment (μ) and angle subtended by charged residues (Φ) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. H and μ influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters’ influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Φ, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of μ and Φ on both lipid bilayers and cell membranes.     

Perfringolysin O association with ordered lipid domains: implications for transmembrane protein raft affinity

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.     

Unraveling lipid/protein interaction in model lipid bilayers by Atomic Force Microscopy

The current view of the biological membrane is that in which lipids and proteins mutually interact to accomplish membrane functions. The lateral heterogeneity of the lipid bilayer can induce partitioning of membrane-associated proteins, favoring protein-protein interaction and influence signaling and trafficking. The Atomic Force Microscope allows to study the localization of membrane-associated proteins with respect to the lipid organization at the single molecule level and without the need for fluorescence staining. These features make AFM a technique of choice to study lipid/protein interactions in model systems or native membranes. Here we will review the technical aspects inherent to and the main results obtained by AFM in the study of protein partitioning in lipid domains concentrating in particular on GPI-anchored proteins, lipidated proteins, and transmembrane proteins. Whenever possible, we will also discuss the functional consequences of what has been imaged by Atomic Force Microscopy.     

Interactions of two transmembrane peptides in supported lipid bilayers studied by a P and N MAOSS NMR strategy

³¹P and ¹⁵N solid-state NMR with the magic angle-oriented sample spinning (MAOSS) strategy was used to investigate the effect of two model peptides on phospholipid bilayers mimicking biological membrane. One of the peptides, alamethicin, used as a reference of transmembrane alignment, has been shown to disrupt the lipid bilayer organisation, affecting the DMPC packaging. On the other hand, a α-helix alanine-rich peptide, K₃A₁₈K₃, with a ¹⁵N labelled alanine, did not present any effect in the DMPC bilayer organisation. The mean orientation of this peptide in the bilayer gave a transmembrane alignment of about 80%.     

Self-association of helical peptides in a lipid environment

The self-association of two model transmembrane helical peptides, differing in their surface topography, was compared in mixed micelles containing 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS) and dimyristoylphosphatidylcholine (DMPC). One peptide, Ac-KKL₂₄KK-amide (L₂₄), has large, rotationally mobile leucine side chains and a relatively rough surface. The other peptide, Ac-KKLLLLLLAALLALLAALLALLLLLLKK-amide (L₁₈A₆), has a patch of small alanines on one side of the helix that forms a smooth surface. The aggregation state of the peptides was sampled by chemical cross-linking with bis-sulfosuccinimidyl suberate (B53). A monomer-aggregate association constant was obtained from the cross-linking results in the range of 2 × 10⁵ M^–1 to 3 × 10⁵ M^–1 for both peptides. Kinetics of formation of cross-linked dimers indicated that the ratio of dimerization constants for L₁₈A₆ to L₂₄ was between 10 and 20. This suggests that the alanine patch contributes about 1.5 Kcal/mol more stabilization free energy to dimer formation of L₁₈A₆ compared to L₂₄.     

Initial structural and dynamic characterization of the M2 protein transmembrane and amphipathic helices in lipid bilayers

Amphipathic helices in membrane proteins that interact with the hydrophobic/hydrophilic interface of the lipid bilayer have been difficult to structurally characterize. Here, the backbone structure and orientation of an amphipathic helix in the full-length M2 protein from influenza A virus has been characterized. The protein has been studied in hydrated DMPC/DMPG lipid bilayers above the gel to liquid-crystalline phase transition temperature by solid-state NMR spectroscopy. Characteristic PISA (Polar Index Slant Angle) wheels reflecting helical wheels have been observed in uniformly aligned bilayer preparations of both uniformly 15N labeled and amino acid specific labeled M2 samples. Hydrogen/deuterium exchange studies have shown the very slow exchange of some residues in the amphipathic helix and more rapid exchange for the transmembrane helix. These latter results clearly suggest the presence of an aqueous pore. A variation in exchange rate about the transmembrane helical axis provides additional support for this claim and suggests that motions occur about the helical axes in this tetramer to expose the entire backbone to the pore.     

Plant metallothionein domains: functional insight into physiological metal binding and protein folding

Plant metallothioneins (MTs) differ from animal MTs by a peculiar sequence organization consisting of two short cysteine-rich terminal domains linked by a long cysteine-devoid spacer. The role of the plant MT domains in the protein structure and functionality is largely unknown. Here, we investigate the separate domain contribution to the in vivo binding of Zn and Cu and to confer metal tolerance to CUP1-null yeast cells of a plant type 2 MT (QsMT). For this purpose, we obtained three recombinant peptides that, respectively, correspond to the single N-terminal (N25) and C-terminal (C18) cysteine-rich domains of QsMT, and a chimera in which the spacer is replaced with a four-glycine bridge (N25-C18). The metal-peptide preparations recovered from Zn- or Cu-enriched cultures were characterized by ESI-MS, ICP-OES and CD and UV-vis spectroscopy and data compared to full length QsMT. Results are consistent with QsMT giving rise to homometallic Zn- or Cu-MT complexes according to a hairpin model in which the two Cys-rich domains interact to form a cluster. In this model the spacer region does not contribute to the metal coordination. However, our data from Zn-QsMT (but not from Cu-QsMT) support a fold of the spacer involving some interaction with the metal core. On the other hand, results from functional complementation assays in endogenous MT-defective yeast cells suggest that the spacer region may play a role in Cu-QsMT stability or subcellular localization. As a whole, our results provide the first insight into the structure/function relationship of plant MTs using the analysis of the separate domain abilities to bind physiological metals.     

General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides.

Model compounds of modified hydrophobicity (Eta), hydrophobic moment (mu) and angle subtended by charged residues (Phi) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. Eta and mu influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters' influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Phi, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of mu and Phi on both lipid bilayers and cell membranes.     

Structural biology of transmembrane domains: efficient production and characterization of transmembrane peptides by NMR

Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.