Correlation between fusogenicity of synthetic modified peptides corresponding to the NH2-terminal extremity of simian immunodeficiency virus gp32 and their mode of insertion into the lipid bilayer: an infrared spectroscopy study.

The amino-terminal extremity of the simian immunodeficiency virus (SIV) transmembrane protein (gp32) has been shown to play a pivotal role in cell-virus fusion and syncytium formation. We provide here evidence of a correlation between the structure and orientation of the modified SIV fusion peptide after insertion into the lipid membrane and its fusogenic activity. The sequence of the wild-type SIV peptide has been modified in such a way that the calculated angles of insertion correspond to an oblique, parallel, or normal orientation with respect to the lipid-water interface. Fourier transform infrared spectroscopy was used to gain experimental informations about the structures and orientations, of the membrane-inserted peptides with respect to the lipid acyl chains. The peptides adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with large unilamellar liposomes, this beta sheet is partly converted into alpha helix. The ability of the modified peptides to promote lipid mixing was assessed by a fluorescence energy transfer assay. The data provide evidence that alpha-helix formation is not sufficient to induce lipid mixing and that the fusogenic activity of the peptide depends on its orientation in the lipid bilayer.     

Interaction of the 106-126 prion peptide with lipid membranes and potential implication for neurotoxicity

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation in the brain of an abnormally misfolded, protease-resistant, and beta-sheet rich pathogenic isoform (PrP(SC)) of the cellular prion protein (PrP(C)). In the present work, we were interested to study the mode of prion protein interaction with the membrane using the 106-126 peptide and small unilamellar lipid vesicles as model. As previously demonstrated, we showed by MTS assay that PrP 106-126 induces alterations in the human neuroblastoma SH-SY5Y cell line. We demonstrated for the first time by lipid-mixing assay and by the liposome vesicle leakage test that PrP 106-126, a non-tilted peptide, induces liposome fusion thus a potential cell membrane destabilization, as supported by membrane integrity assay (LDH). By circular dichroism (CD) analysis we showed that the fusogenic property of PrP 106-126 in the presence of liposome is associated with a predominantly beta-sheet structure. These data suggest that the fusogenic property associated with a predominant beta-sheet structure exhibited by the prion peptides contributes to the neurotoxicity of these peptides by destabilizing cellular membranes. The latter might be attached at the membrane surface in a parallel orientation as shown by molecular modeling.     

Study of the inhibition capacity of an 18-mer peptide domain of GBV-C virus on gp41-FP HIV-1 activity

The peptide sequence (175-192) RFPFHRCGAGPKLTKDLE (P59) of the E2 envelope protein of GB virus C (GBV-C) has been proved to decrease cellular membrane fusion and interfere with the HIV-1 infectivity in a dose-dependent manner. Based on these previous results, the main objective of this study was to deepen in the physicochemical aspects involved in this interaction. First, we analyzed the surface activity of P59 at the air-water interface as well as its interaction with zwitterionic or negatively charged lipid monolayers. Then we performed the same experiments with mixtures of P59/gp41-FP. Studies on lipid monolayers helped us to understand the lipid-peptide interaction and the influence of phospholipids on peptide penetration into lipid media. On another hand, studies with lipid bilayers showed that P59 decreased gp41-FP binding to anionic Large Unilamellar Vesicles. Results can be attributed to the differences in morphology of the peptides, as observed by Atomic Force Microscopy. When P59 and gp41-FP were incubated together, annular structures of about 200 nm in diameter appeared on the mica surface, thus indicating a peptide-peptide interaction. All these results confirm the gp41-FP-P59 interaction and thus support the hypothesis that gp41-FP is inhibited by P59.     

Structural and dynamical changes of the bindin B18 peptide upon binding to lipid membranes. A solid-state NMR study

Structural and dynamical features of the B18 peptide from the sea urchin sperm binding protein were determined in the crystalline state and in zwitterionic lipid bilayers at a peptide:lipid molar ratio of 1:12 using solid-state NMR spectroscopy. The study was focused on three (13)C and (15)N uniformly labeled leucine residues, which were introduced into three different B18 peptides at positions evenly distributed along the B18 primary structure. Isotropic (13)C and (15)N chemical shift measurements showed that while B18 possesses a nonhelical and non-sheet-like structure in the crystalline state, the peptide adopts an oligomeric beta-sheet structure in the membrane in the presence of Zn(2+) ions at high peptide:lipid ratio. Torsion angle measurements for the three leucine sites supported these results, with phi torsion angles between -80 degrees and -90 degrees in the crystalline state and between -110 degrees and -120 degrees in the membrane-bound form. These phi torsion angles determined for membrane-bound B18 are consistent with a parallel beta-sheet secondary structure. Analysis of motionally averaged dipolar coupling measurements established an increase of the mobility in the leucine side chains upon binding to the membrane, whereas the backbone mobility remained essentially unchanged, except in the binding site of Zn(2+) ions. This difference in mobility was related to the H-bond network in the parallel beta-sheet structure, which involves the backbone and excludes the side chains of leucine residues. The parallel beta-sheet structure of B18 in the membrane in the presence of Zn(2+) appears to be an active state for the fusion of zwitterionic membranes in the presence of Zn(2+). A fluorescence fusion assay indicated that high B18 concentrations are required to induce fusion in these systems. Therefore, it was hypothesized that the oligomeric beta-sheet secondary structure revealed in the study represents an active state of the peptide in a membrane environment during fusion.     

Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.     

Lipid membrane fusion induced by the human immunodeficiency virus type 1 gp41 N-terminal extremity is determined by its orientation in the lipid bilayer.

The amino-terminal extremity of the human immunodeficiency virus type 1 transmembrane protein (gp41) is thought to play a pivotal role in the fusion of virus membranes with the plasma membrane of the target cell and in syncytium formation. Peptides with sequences taken from the human immunodeficiency virus type 1 gp41 fusogenic (synthetic peptides SPwt and SP-2) and nonfusogenic (SP-3 and SP-4) glycoproteins adopt mainly a beta-sheet conformation in the absence of lipid, as determined by attenuated total reflection Fourier transform infrared spectroscopy, and after interaction with large unilamellar liposomes, the beta-sheet is partly converted into an alpha-helical conformation. Peptides SPwt and SP-2 but not SP-3 or SP-4 were able to promote lipid mixing as assessed by fluorescence energy transfer assay and dye leakage in a vesicle leakage assay. By using polarized attenuated total reflection Fourier transform infrared spectroscopy, SPwt and SP-2 were found to adopt an oblique orientation in the lipid membrane whereas SP-3 and SP-4 were oriented nearly parallel to the plane of the membrane. These findings confirm the correlation between the membrane orientation of the alpha-helix and the lipid mixing ability in vitro. Interestingly, the data provide a direct correlation with the fusogenic activity of the parent glycoproteins in vivo.     

Protein-induced fusion of phospholipid vesicles of heterogeneous sizes.

We have investigated the fusion of phospholipid vesicles induced by lysozyme and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vesicles were composed of dimyristoylphosphatidylcholine/dioleoylphosphatidylethanolamine/ cholesterol (DMPC:DOPE:Chol, 2:1:1). Small unilamellar vesicles (SUV, diameter ca. 30 nm) obtained by extensive sonication or large unilamellar vesicles (LUV, diameters ranged from 100 to 400 nm) obtained by extrusion methods were used. Fusion of LUV induced by lysozyme and GAPDH was drastically decreased when the diameter of the vesicles increased over a value of 100 nm. Lysozyme effect was stopped at the aggregation step while GAPDH effect was stopped at the fusion (lipid mixing) step. Fusion of heterogeneous vesicle populations (SUV with LUV) was observed only with GAPDH and this happened only when the lipids were in the liquid-crystalline state.     

Secondary structure, phospholipid membrane interactions, and fusion activity of two glutamate-rich analogs of influenza hemagglutinin fusion peptide

Two synthetic mutants of influenza HA2 fusion peptide (residues 1-25), containing Glu on the polar (residues 4,8-E5(4,8)) or the hydrophobic (residues 3,7-E5(3,7)) face of the amphipathic helix, were synthesized and labeled with NBD at the N-terminus. Introduction of Glu residues into the fusion peptide leads to increased sensitivity of various biochemical properties to pH compared to the wild type. The E5 peptides showed a decrease of alpha-helix content and increase of beta-sheet structure. Lipid binding was diminished, but not abolished even at high pH. The E5 analogs penetrate the lipid bilayer less deeply than the wild type, especially at high pH. The N-terminal half of the peptide showed significant variation of the depth of the penetration into the lipid bilayer. Both E5 peptides were fusion active. The properties of E5(3,7) were more affected by the Glu substitution and showed greater variation with pH than E5(4,8).     

Conformational consequences of coupling bullous pemphigoid antigenic peptides to glutathione-S-transferase and their diagnostic significance.

Recombinant epitopic peptides BP1 and BP2 representing the Bullous pemphigoid autoantigens of BP230 and BP180 bound to the fusion partner glutathione-S-transferase (pGEX-4T-2, Pharmacia) have been previously shown to increase the efficacy of diagnosis of the disease. Using glutathione-S-transferase-bound monomer peptides, the sensitivity of the immunological reaction exceeded that of the free synthetic epitopes and was further increased with the number of epitopic blocks in the multimer fusion products. This has been explained by the avidity effect of the fusion partner dimer formation and the high ligand affinity due to the tandem repetitions of epitopic sequences. However, a beneficial conformation of the bound epitopic peptides might also contribute to the above phenomenon. Circular dichroism (CD) and Fourier transform infrared (FTIR) absorption spectroscopic studies revealed the importance of glutathione-S-transferase to induce and stabilize ordered secondary structures of the epitopic peptides. The free monomer and multimer peptides in aqueous buffer were present as a mixture of unordered and beta-sheet conformation, while binding them to the fusion partner the proportion of ordered secondary structures increased in parallel with the number of antigenic epitopes. The most prominent changes in the conformational state of the monomers in the fusion form were the increase of alpha-helical and beta-sheet and the decrease of unordered conformation, while in the case of oligomeric peptides the adoption of a helical conformation was accompanied by the decrease of beta-sheet structure. An outstanding alpha-helix content (46%) was detected in the case of the trimeric BP1 in its recombinant fusion form.     

Effects of the peptide Magainin H2 on Supported Lipid Bilayers studied by different biophysical techniques

Given the increasing trend in bacterial antibiotic resistance, research on antimicrobial peptides and their mechanisms of action has become of huge relevance in the last years. Several studies have investigated the effects of a large variety of antimicrobial peptides directly on bacteria or on model lipid bilayers. In the case of model lipid bilayers, different systems are typically exploited; however, different results could be obtained due to the specific properties of the used system. Supported Lipid Bilayers and Giant Unilamellar Vesicles are among the most popular model systems. Here we used Atomic Force Microscopy and fluorescence microscopy to study the interaction of the antimicrobial peptide Magainin H2, an analog of Magainin 2 with increased hydrophobicity, on Supported Lipid Bilayers. We found that, for this kind of model bilayer, due to its strong interaction with the support, the lateral expansion of the membrane induced by the interaction with the peptides is initially inhibited and subsequently proceeds creating new bilayer regions with many defects. This scenario gives rise in Supported Lipid Bilayers to effects like initial increase of lateral pressure, formation of lipid tubes to release this increase, or development of bilayer regions with lower lipid density. Our results highlight that care should be given to the selected model system when studying and comparing the interaction of peptides with other lipid bilayer model systems.     

Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide

Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or Golgi membranes is strongly inhibited by minor amounts of (lyso)lipids when present in the target membrane but not when inserted into the viral or Golgi membrane itself. To investigate the underlying mechanism, we employ a membrane-anchored peptide system and show that fusion is similarly regulated by these lipids when inserted into the target but not when present in the peptide-containing membrane. Peptide-induced fusion is regulated by a reversible switch of secondary structure from a fusion-permissive alpha-helix to a nonfusogenic beta-sheet. The "on/off" activation of this switch is governed by minor amounts of (lyso)-phospholipids in targets, causing a drop in alpha-helix and a dramatic increase in beta-sheet contents. Concomitantly, fusion is inhibited, due to impaired peptide insertion into the target membrane. Our observations in biological fusion systems together with the model studies suggest that distinct lipids in target membranes provide a means for regulating membrane fusion by causing a reversible secondary structure switch of the fusion peptides.     

Identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a newly identified member of the family Coronaviridae and poses a serious public health threat. Recent studies indicated that the SARS-CoV viral spike glycoprotein is a class I viral fusion protein. A fusion peptide present at the N-terminal region of class I viral fusion proteins is believed to initiate viral and cell membrane interactions and subsequent fusion. Although the SARS-CoV fusion protein heptad repeats have been well characterized, the fusion peptide has yet to be identified. Based on the conserved features of known viral fusion peptides and using Wimley and White interfacial hydrophobicity plots, we have identified two putative fusion peptides (SARS(WW-I) and SARS(WW-II)) at the N terminus of the SARS-CoV S2 subunit. Both peptides are hydrophobic and rich in alanine, glycine, and/or phenylalanine residues and contain a canonical fusion tripeptide along with a central proline residue. Only the SARS(WW-I) peptide strongly partitioned into the membranes of large unilamellar vesicles (LUV), adopting a beta-sheet structure. Likewise, only SARS(WW-I) induced the fusion of LUV and caused membrane leakage of vesicle contents at peptide/lipid ratios of 1:50 and 1:100, respectively. The activity of this synthetic peptide appeared to be dependent on its amino acid (aa) sequence, as scrambling the peptide rendered it unable to partition into LUV, assume a defined secondary structure, or induce both fusion and leakage of LUV. Based on the activity of SARS(WW-I), we propose that the hydrophobic stretch of 19 aa corresponding to residues 770 to 788 is a fusion peptide of the SARS-CoV S2 subunit.     

Ultrastructural characterization of peptide-induced membrane fusion and peptide self-assembly in the lipid bilayer.

The peptide sequence B18, derived from the membrane-associated sea urchin sperm protein bindin, triggers fusion between lipid vesicles. It exhibits many similarities to viral fusion peptides and may have a corresponding function in fertilization. The lipid-peptide and peptide-peptide interactions of B18 are investigated here at the ultrastructural level by electron microscopy and x-ray diffraction. The histidine-rich peptide is shown to self-associate into two distinctly different supramolecular structures, depending on the presence of Zn(2+), which controls its fusogenic activity. In aqueous buffer the peptide per se assembles into beta-sheet amyloid fibrils, whereas in the presence of Zn(2+) it forms smooth globular clusters. When B18 per se is added to uncharged large unilamellar vesicles, they become visibly disrupted by the fibrils, but no genuine fusion is observed. Only in the presence of Zn(2+) does the peptide induce extensive fusion of vesicles, which is evident from their dramatic increase in size. Besides these morphological changes, we observed distinct fibrillar and particulate structures in the bilayer, which are attributed to B18 in either of its two self-assembled forms. We conclude that membrane fusion involves an alpha-helical peptide conformation, which can oligomerize further in the membrane. The role of Zn(2+) is to promote this local helical structure in B18 and to prevent its inactivation as beta-sheet fibrils.     

The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

Fourier transform infrared spectroscopy (FTIR) was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domain of the pro-apoptotic protein Bax (Bax-C) when incorporated into different lipid vesicles with or without negatively charged phospholipids. The infrared spectroscopy results showed that while the beta-sheet components are predominant in the membrane-free Bax-C secondary structure as well as in the presence of phosphatidylcholine vesicles, the peptide changes its secondary structure in the presence of negatively charged membranes, including phospholipids such as phosphatidylglycerol or phosphatidylinositol, depending on both the lipid composition and their molar ratio. The negative charges in the model membrane surface caused a marked change from beta-sheet to alpha-helix structure. Moreover, using attenuated total reflection infrared spectroscopy (ATR-FTIR), we investigated the orientation of Bax-C alpha-helical structures with respect to the normal to the internal reflection element. The orientation of Bax-C in membranes was also affected by negatively charged lipids, the presence of phosphatidylglycerol reduced the angle it forms with the normal to the germanium plate from 45 degrees in phosphatidylcholine to 27 degrees in phosphatidylglycerol vesicles. These results highlight the importance of lipid-protein interaction for the correct folding of membrane proteins and they suggest that the C-terminal domain of Bax will only span membranes with a net negative charge in their surface.     

Apoptosis induced in neuronal cells by C-terminal amyloid beta-fragments is correlated with their aggregation properties in phospholipid membranes

A number of findings suggest that lipophilic monomeric Abeta peptides can interact with the cellular lipid membranes. These interactions can affect the membrane integrity and result in the initiation of apoptotic cell death. The secondary structure of C-terminal Abeta peptides (29-40) and the longer (29-42) variant have been investigated in solution by circular dichroism measurements. The secondary structure of lipid bound Abeta (29-40) and (29-42) peptides prepared at different lipid/peptide ratio's, was investigated by ATR-FTIR spectroscopy. Finally, the changes in secondary structure (i.e. the transition of alpha-helix to beta-sheet) of the lipid bound peptides were correlated with the induction of neurotoxic and apoptotic effects in neuronal cells. The data suggest that the C-terminal fragments of the Abeta peptide induce a significant apoptotic cell death, as demonstrated by caspase-3 measurements and DNA laddering, with consistently a stronger effect of the longer Abeta (29-42) variant. Moreover, the induction of apoptotic death induced by these peptides can be correlated with the secondary structure of the lipid bound amyloid beta peptides. Based on these observations, it is proposed that membrane bound aggregated Abeta peptides (produced locally as the result of gamma-secretase cleavage) can accumulate and aggregate in the membrane. These membrane bound beta-sheet aggregated amyloid peptides induce neuronal apoptotic cell death.     

Modulation of the membrane orientation and secondary structure of the C-terminal domains of Bak and Bcl-2 by lipids

Infrared spectroscopy was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domains of the pro-apoptotic protein Bak (Bak-C) and the anti-apoptotic protein Bcl-2 (Bcl-2-C) when incorporated into different lipid vesicles. Whereas beta-pleated sheet was the predominant type of secondary structure of Bak-C in the absence of membranes, the same peptide adopted different structures depending on lipid composition when incorporated into membranes, with the predominance of the alpha-helical structure in the case of DMPC and other phospholipids, such as POPC and POPG. However, beta-pleated sheet was the predominant structure in other membranes containing phospholipids with longer fatty acyl chains and cholesterol, as well as in a mixture which imitates the composition of the outer mitochondrial membrane (OMM). Similarly, Bcl-2-C adopted a structure with a predominance of intermolecularly bound pleated beta-sheet in the absence of membranes, with alpha-helix as the main component in the presence of DMPC and POPG, but intermolecular beta-sheet in the presence of EYPC and cholesterol. Using ATR-IR, it was found that the orientation of the alpha-helical components of both domains was nearly perpendicular to the plane of the membrane in the presence of DMPC membranes, but not in EYPC or OMM membranes. (2)H NMR spectroscopy of DMPC-d(54) confirmed the transmembrane disposition of the domains, revealing that they broadened the phase transition temperature, although the order parameter of the C-D bonds was not affected, as might have been expected for intrinsic peptides. When all these results are taken together, it was concluded that the domains only form transmembrane helices in membranes of reduced thickness and that hydrophobic mismatching occurs in thicker membranes, as happens in the membrane imitating the composition of the OMM, where the peptides were partially located outside the membranes.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.     

Interactions of the HIV-1 fusion peptide with large unilamellar vesicles and monolayers. A cryo-TEM and spectroscopic study

We have examined the interaction of the human immunodeficiency virustype 1 fusion peptide (23 amino acid residues) and of a Trp-containing analog with vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and cholesterol (molar ratio, 1:1:1). Both the native and the Trp-substituted peptides bound the vesicles to the same extent and induced intervesicular lipid mixing with comparable efficiency. Infrared reflection-absorption spectroscopy data are compatible with the adoption by the peptide of a main beta-sheet structure in a cospread lipid/peptide monolayer. Cryo-transmission electron microscopy observations of peptide-treated vesicles reveal the existence of a peculiar morphology consisting of membrane tubular elongations protruding from single vesicles. Tryptophan fluorescence quenching by brominated phospholipids and by water-soluble acrylamide further indicated that the peptide penetrated into the acyl chain region closer to the interface rather than into the bilayer core. We conclude that the differential partition and shallow penetration of the fusion peptide into the outer monolayer of a surface-constrained bilayer may account for the detected morphological effects. Such single monolayer-restricted interaction and its structural consequences are compatible with specific predictions of current theories on viral fusion.     

Membrane-bound conformation and topology of the antimicrobial peptide tachyplesin I by solid-state NMR

The conformation and membrane topology of the disulfide-stabilized antimicrobial peptide tachyplesin I (TP) in lipid bilayers are determined by solid-state NMR spectroscopy. The backbone (phi and psi) torsion angles of Val(6) are found to be -133 degrees and 142 degrees , respectively, and the Val(6) CO-Phe(8) H(N) distance is 4.6 A. These constrain the middle of the N-terminal strand to a relatively ideal antiparallel beta-sheet conformation. In contrast, the phi angle of Gly(10) is +/-85 degrees , consistent with a beta-turn conformation. Thus, TP adopts a beta-hairpin conformation with straight strands, similar to its structure in aqueous solution but different from a recently reported structure in DPC micelles where bending of the two beta-strands was observed. The Val(6) and Gly(10) CO groups are both 6.8 A from the lipid (31)P, while the Val(6) side chain is in (1)H spin diffusion contact with the lipid acyl chains. These results suggest that TP is immersed in the glycerol backbone region of the membrane and is oriented roughly parallel to the plane of the membrane. This depth of insertion and orientation differs from those of the analogous beta-sheet antimicrobial peptide protegrin-1 and suggest the importance of structural amphiphilicity in determining the location and orientation of membrane peptides in lipid bilayers.     

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers.

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.