Secondary structure and distribution of fusogenic LV-peptides in lipid membranes

LV-peptides were designed as membrane-spanning low-complexity model structures that mimic fusion protein transmembrane domains. These peptides harbor a hydrophobic core sequence that consists of helix-promoting and helix-destabilizing residues at different ratios. Previously, the fusogenicity of these peptides has been shown to increase with the conformational flexibility of their hydrophobic cores as determined in isotropic solution. Here, we examined the secondary structure, orientation, and distribution of LV-peptides in membranes. Our results reveal that the peptides are homogeneously distributed within the membranes of giant unilamellar liposomes and capable of fusing them. Increasing the valine content of the core up to the level of the β-branched residue content of SNARE TMDs (∼50%) enhances fusogenicity while maintaining a largely α-helical structure in liposomal membranes. A further increase in valine content or introduction of a glycine/proline pair favors β-sheet formation. In planar bilayers, the α-helices adopt oblique angles relative to the bilayer normal and the ratio of α-helix to β-sheet responds more sensitively to valine content. We propose that the fusogenic conformation of LV-peptides is likely to correspond to a membrane-spanning α-helix. β-Sheet formation in membranes may be considered a side-reaction whose extent reflects conformational flexibility of the core.     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp153 and Glu158, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His148 was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.     

The effect of interactions involving ionizable residues flanking membrane-inserted hydrophobic helices upon helix-helix interaction

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.     

Mode of membrane interaction and fusogenic properties of a de novo transmembrane model peptide depend on the length of the hydrophobic core

Model peptides composed of alanine and leucine residues are often used to mimic single helical transmembrane domains. Many studies have been carried out to determine how they interact with membranes. However, few studies have investigated their lipid-destabilizing effect. We designed three peptides designated KALRs containing a hydrophobic stretch of 14, 18, or 22 alanines/leucines surrounded by charged amino acids. Molecular modeling simulations in an implicit membrane model as well as attenuated total reflection-Fourier transform infrared analyses show that KALR is a good model of a transmembrane helix. However, tryptophan fluorescence and attenuated total reflection-Fourier transform infrared spectroscopy indicate that the extent of binding and insertion into lipids increases with the length of the peptide hydrophobic core. Although binding can be directly correlated to peptide hydrophobicity, we show that insertion of peptides into a membrane is determined by the length of the peptide hydrophobic core. Functional studies were performed by measuring the ability of peptides to induce lipid mixing and leakage of liposomes. The data reveal that whereas KALR14 does not destabilize liposomal membranes, KALR18 and KALR22 induce 40 and 50% of lipid-mixing, and 65 and 80% of leakage, respectively. These results indicate that a transmembrane model peptide can induce liposome fusion in vitro if it is long enough. The reasons for the link between length and fusogenicity are discussed in relation to studies of transmembrane domains of viral fusion proteins. We propose that fusogenicity depends not only on peptide insertion but also on the ability of peptides to destabilize the two leaflets of the liposome membrane.     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp¹⁵³ and Glu¹⁵⁸, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His¹⁴⁸ was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.     

The effects of hydrophobic mismatch between phosphatidylcholine bilayers and transmembrane alpha-helical peptides depend on the nature of interfacially exposed aromatic and charged residues

In this study, we investigated the extent to which different aromatic and positively charged side chains, which often flank transmembrane segments of proteins, can influence lipid-peptide interactions. Model systems consisting of phosphatidylcholine and hydrophobic alpha-helical peptides with different flanking residues were investigated. The peptides were incorporated in relatively thick and in relatively thin lipid bilayers to create a peptide-bilayer hydrophobic mismatch, and the compensating effects on lipid structure were analyzed. When relatively long with respect to the thickness of the bilayer, the peptides that are flanked by the aromatic side chains, Trp, Tyr, and Phe, all induce a significant ordering of the lipid acyl chains, while the peptides flanked by the charged residues Lys, Arg, and His do not. However, when the peptides are relatively short with respect to the thickness of the bilayer, their effect on lipid organization does not depend primarily on their aromatic or charged character. Peptides flanked by Trp, Tyr, Lys, or (at low pH) His residues are effective in inducing mismatch-relieving cubic and inverted hexagonal phases, while analogues flanked by Phe, Arg, or (at neutral pH) His residues cannot induce an inverted hexagonal phase. The different responses to mismatch might reflect the different interfacial affinities of the residues that were investigated.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylglycerol bilayers

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)), and members of the homologous series of anionic n-saturated diacyl phosphatidylglycerols (PGs). Analogues of L(24), in which the lysine residues were replaced by 2,3-diaminopropionic acid (L(24)DAP), or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (WL(22)W), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the host PG bilayers is altered by these peptides in a hydrophobic mismatch-dependent manner, as previously found with zwitterionic phosphatidylcholine (PC) bilayers. However, all three peptides reduce the phase transition temperature and enthalpy to a greater extent in anionic PG bilayers than in zwitterionic PC bilayers, with WL(22)W having the largest effect. All three peptides form very stable alpha-helices in PG bilayers, but small conformational changes are induced in response to a mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. Moreover, electrostatic and hydrogen-bonding interactions occur between the terminal lysine residues of L(24) and L(24)DAP and the polar headgroups of PG bilayers. However, such interactions were not observed in PG/WL(22)W bilayers, suggesting that the cation-pi interactions between the tryptophan and lysine residues predominate. These results indicate that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer, but also by the tryptophan-modulated electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the negatively charged polar headgroups of the PG bilayers.     

Effect of alteration of charged residues at the N termini of signal peptides on protein export in Bacillus subtilis.

The role of positively charged residues at the N termini of signal peptides in protein export has been studied in Bacillus subtilis. Bacillus signal peptides (alkaline protease [Apr] and neutral protease [Npr] from Bacillus amyloliquefaciens) were altered and fused to mature levansucrase (Lvs). The effects of the various alterations on the export of Lvs in B. subtilis were determined. The replacement of positively charged residues with neutral residues in both Apr and Npr signal peptides resulted in a slight defect in the export of Lvs from B. subtilis. Introduction of a negatively charged residue (aspartic acid) at the N terminus of Npr signal peptide blocked the export of Lvs. However, Apr signal peptide with a net charge of -3 (three aspartic acid residues) was still functional.     

Membrane fusion induced by mutual interaction of the two charge-reversed amphiphilic peptides at neutral pH

An anionic amphiphilic peptide and the charge-reversed cationic peptide are synthesized. They contain 20 amino acids with the same sequence except for 5 Glu residues for the anionic versus 5 Lys residues for the cationic peptides. Fusion of egg phosphatidylcholine large unilamellar vesicles is assayed with the fluorescent probes by the lipid mixing and the internal content mixing at neutral pH. The peptide mixture causes a rapid and efficient membrane fusion, in spite of no fusions with each peptide by itself. Each peptide takes nearly random coils with a small amount of helix, but the peptide mixture has an ordered helical structure. The equimolar peptide mixture forms a much more hydrophobic complex than those of different molar ratios of peptides and also that of each peptide itself. The equimolar peptide mixture causes the most efficient fusion. Preincubations of two peptides before addition to vesicles cause the slower rates of fusion. The fusion is greatly reduced at higher ionic strength and nearly zero at 800 mM NaCl and 40 mM sodium phosphate. Each peptide and the peptide mixture show the same alpha-helical structure, interact with vesicles, but do not induce fusion at higher ionic strengths. These results suggest that the two peptides interact mutually through the electrostatic Coulombic interaction between the charged groups. The electrically neutralized hydrophobic complex aggregates the separate vesicles together and interacts with the hydrocarbon region of lipid bilayers to cause fusion.     

Structural features of a chimeric peptide inducing cytotoxic T lymphocyte responses in saline.

Little information is available correlating the structural properties of peptides with their immunogenicity in terms of responses via cytotoxic T lymphocytes (CTLs). The TT-NP6 chimeric peptide, consisting of two copies of a promiscuous T-helper epitope (T: residues 288-302 from the fusion protein of the measles virus) linked to the NP6 T-cytotoxic epitope (NP6: residues 52-60 from the nucleoprotein of measles virus) was able to induce virus-specific CTL responses in the absence of any adjuvant and hydrophobic component. The present work was undertaken to gain insight into structural features of the TT-NP6 peptide that may be important in optimizing the CTL immunogenicity of the peptide. Circular dichroism data, obtained in a buffer of physiological ionic strength and pH, strongly suggest a self-associated state for the peptide, which was confirmed by a sedimentation velocity experiment. However, helix association is accompanied by loss of overall helical content. Thermal-dependence studies show that the unfolding of self-associated alpha-helices is significantly more pronounced than the unfolding of isolated alpha-helices. Circular dichroism data, together with tryptic limited proteolysis, suggest the presence of a charged amino acid within the hydrophobic core. This study should provide a basis for engineering more effective immunogenic peptides against the measles virus by increasing the stability of the TT-NP6 peptide.     

Adsorption equilibrium in immunoaffnity chromatography with antibodies to synthetic peptides

The effects of charged residues in peptide antigens on the binding characteristics of polyclonal antipeptide antibodies were studied using immunoadsorbents prepared by coupling the antibodies to CNBr-activated Sepharose 4B. Among the antipeptide antibodies, an antibody to the peptide without charged residues showed the most stable interaction with the peptide to the changes in pH. Conversely, the binding affinity of antibodies to the pep-tides with histidine residues having a unique pKa value of 6.0 decreased steeply with pH at around 6.0. The binding affinity of an antibody to the peptide with many charged residues decreased steeply with an increase in the ionic strength (adjusted by NaCl). Since circular dichroism (CD) spectrum measurements indicate that these peptides show disordered structures in the pH range of adsorption measurement, the dependence of peptide-antibody interaction on environmental conditions is attributed to the characteristics of side chains of the peptides. These results indicate that the dependence of the binding affinity of antipeptide antibodies on pH and the ionic strength is dominantly affected by the number and the pKa values of charged residues in the peptides.     

Design and characterization of anchoring amphiphilic peptides and their interactions with lipid vesicles.

In an effort to develop a polymer/peptide assembly for the immobilization of lipid vesicles, we have made and characterized four water-soluble amphiphilic peptides designed to associate spontaneously and strongly with lipid vesicles without causing significant leakage from anchored vesicles. These peptides have a primary amphiphilic structure with the following sequences: AAAAAAAAAAAAWKKKKKK, AALLLAAAAAAAAAAAAAAAAAAAWKKKKKK, and KKAALLLAAAAAAAAAAAAAAAAAAAWKKKKKK and its reversed homologue KKKKKKWAAAAA AAAAAAAAAAAAAALLLAAKK. Two of the four peptides have their hydrophobic segments capped at both termini with basic residues to stabilize the transmembrane orientation and to increase the affinity for negatively charged vesicles. We have studied the secondary structure and the membrane affinity of the peptides as well as the effect of the different peptides on the membrane permeability. The influence of the hydrophobic length and the role of lysine residues were clearly established. First, a hydrophobic segment of 24 amino acids, corresponding approximately to the thickness of a lipid bilayer, improves considerably the affinity to zwitterionic lipids compared to the shorter one of 12 amino acids. The shorter peptide has a low membrane affinity since it may not be long enough to adopt a stable conformation. Second, the presence of lysine residues is essential since the binding is dominated by electrostatic interactions, as illustrated by the enhanced binding with anionic lipids. The charges at both ends, however, prevent the peptide from inserting spontaneously in the bilayer since it would involve the translocation of a charged end through the apolar core of the bilayer. The direction of the amino acid sequence of the peptide has no significant influence on its behavior. None of these peptides perturbs membrane permeability even at an incubation lipid to peptide molar ratio of 0.5. Among the four peptides, AALLLAAAAAAAAAAAAAAAAAAAWKKKKKK is identified as the most suitable anchor for the immobilization of lipid vesicles.     

Position and ionization state of Asp in the core of membrane-inserted alpha helices control both the equilibrium between transmembrane and nontransmembrane helix topography and transmembrane helix positioning

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.     

Interaction between actin and the effector peptide of MARCKS-related protein. Identification of functional amino acid segments

It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. To clarify the mechanisms by which this interaction takes place, we have examined the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on both the dynamic and the structural properties of actin. We show that in the absence of cations the effector peptide polymerizes monomeric actin and causes the alignment of the formed filaments into bundle-like structures. Moreover, we document that binding of calmodulin or phosphorylation by protein kinase C both inhibit the actin polymerizing activity of the MRP effector peptide. Finally, several effector peptides were synthesized in which positively charged or hydrophobic segments were deleted or replaced by alanines. Our data suggest that a group of six positively charged amino acid residues at the N-terminus of the peptide is crucial for its interaction with actin. While its actin polymerizing activity critically depends on the presence of all three positively charged segments of the peptide, hydrophobic amino acid residues rather modulate the polymerization velocity.     

Conformational behavior of ionic self-complementary peptides

Several de novo designed ionic peptides that are able to undergo conformational change under the influence of temperature and pH were studied. These peptides have two distinct surfaces with regular repeats of alternating hydrophilic and hydrophobic side chains. This permits extensive ionic and hydrophobic interactions resulting in the formation of stable beta-sheet assemblies. The other defining characteristic of this type of peptide is a cluster of negatively charged aspartic or glutamic acid residues located toward the N-terminus and positively charged arginine or lysine residues located toward the C-terminus. This arrangement of charge balances the alpha-helical dipole moment (C --> N), resulting in a strong tendency to form stable alpha-helices as well. Therefore, these peptides can form both stable alpha-helices and beta-sheets. They are also able to undergo abrupt structural transformations between these structures induced by temperature and pH changes. The amino acid sequence of these peptides permits both stable beta-sheet and alpha-helix formation, resulting in a balance between these two forms as governed by the environment. Some segments in proteins may also undergo conformational changes in response to environmental changes. Analyzing the plasticity and dynamics of this type of peptide may provide insight into amyloid formation. Since these peptides have dynamic secondary structure, they will serve to refine our general understanding of protein structure.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylethanolamine Bilayers

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys2-Leu24-Lys2-amide (L24), and members of the homologous series of zwitterionic n-saturated diacyl phosphatidylethanolamines (PEs). Analogs of L24, in which the lysine residues were replaced by 2,3-diaminopropionic acid (acetyl-DAP2-Leu24-DAP2-amide (L24DAP)) or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (Ac-K2-W-L22-W-K2-amide (WL22W)), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the PE bilayers is altered by these peptides in a hydrophobic mismatch-independent manner, in contrast to the hydrophobic mismatch-dependent manner observed previously with zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) bilayers. Moreover, all three peptides reduce the phase transition temperature to a greater extent in PE bilayers than in PC and PG bilayers, indicating a greater disruption of PE gel-phase bilayer organization. Moreover, the lysine-anchored L24 reduces the phase transition temperature, enthalpy, and the cooperativity of PE bilayers to a much greater extent than DAP-anchored L24DAP, whereas replacement of the terminal leucines by tryptophan residues (Ac-K2-W-L22-W-K2-amide) only slightly attenuates the effects of this peptide on the chain-melting phase transition of the host PE bilayers. All three peptides form very stable alpha-helices in PE bilayers, but small conformational changes occur in response to mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. These results suggest that the lysine snorkeling plays a significant role in the peptide-PE interactions and that cation-pi-interactions between lysine and tryptophan residues may modulate these interactions. Altogether, these results suggest that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer but also by the electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the polar headgroups of PE bilayers.     

High-resolution solution structure of a designed peptide bound to lipopolysaccharide: transferred nuclear Overhauser effects, micelle selectivity, and anti-endotoxic activity

Designing peptides that would interact with lipopolysaccharides (LPS) and acquire a specific folded conformation can generate useful structural insights toward the development of anti-sepsis agents. In this work, we have constructed a 12-residue linear peptide, YW12, rich in aromatic and aliphatic amino acid residues with a centrally located stretch of four consecutive positively charged (KRKR) residues. In absence of LPS, YW12 is predominantly unstructured in aqueous solution. Using transferred nuclear Overhauser effect (Tr-NOE) spectroscopy, we demonstrate that YW12 adopts a well-folded structure as a complex with LPS. Structure calculations reveal that YW12 assumes an extended conformation at the N-terminus followed by two consecutive beta-turns at its C-terminus. A hydrophobic core is formed by extensive packing between number of aromatic and nonpolar residues, whereas the positively charged residues are segregated out to a separate region essentially stabilizing an amphipathic structure. In an in vitro LPS neutralization assay using NF-kappaB induction as the readout, YW12 shows moderate activity with an IC50 value of approximately 10 microM. As would be expected, tryptophan fluorescence studies demonstrate that YW12 shows selective interactions only with the negatively charged lipid micelles including sodium dodecyl sulfate (SDS), 1-palmitoyl-2-oleoylphosphatidyl-dl-glycerol (POPG), and LPS, and no significant interactions are detected with zwitterionic lipid micelles such as dodecyl-phosphocholine (DPC). Far-UV CD studies indicate the presence of beta-turns or beta-sheet-like conformations of the peptide in negatively charged micelles, whereas no structural transitions are apparent in DPC micelles. These results suggest that structural features of YW12 could be utilized to develop nontoxic antisepsis compounds.     

Mutagenesis and nuclear magnetic resonance analyses of the fusion peptide of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus F protein

The entry of enveloped viruses into cells is normally mediated by fusion between viral and cellular membranes, in which the fusion peptide plays a crucial role. The fusion peptides of group II nucleopolyhedrovirus (NPV) F proteins are quite conserved, with a hydrophobic region located at the N terminal of the F(1) fragment. For this report, we used mutagenesis and nuclear magnetic resonance (NMR) to study the structure and function of the fusion peptide of the Helicoverpa armigera single-nucleocapsid NPV (HearNPV) F protein (HaF). Five mutations in the fusion peptide of HaF, N(1)G, N(1)L, I(2)N, G(3)L, and D(11)L, were generated separately, and the mutated f genes were transformed into the f-null HearNPV bacmid. The mutations N(1)L, I(2)N, and D(11)L were found to completely abolish the ability of the recombinant bacmids to produce infectious budded virus, while the mutations N(1)G and G(3)L did not. The low-pH-induced envelope fusion assay demonstrated that the N(1)G substitution increased the fusogenicity of HaF, while the G(3)L substitution reduced its fusogenicity. NMR spectroscopy was used to determine the structure of a synthetic fusion peptide of HaF in the presence of sodium dodecyl sulfate micelles at pH 5.0. The fusion peptide appeared to be an amphiphilic structure composed of a flexible coil in the N terminus from N(1) to N(5), a 3(10)-helix from F(6) to G(8), a turn at S(9), and a regular alpha-helix from V(10) to D(19). The data provide the first NMR structure of a baculovirus fusion peptide and allow us to further understand the relationship of structure and function of the fusion peptide.     

A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides

Presecretory signal peptides of 39 proteins from diverse prokaryotic and eukaryotic sources have been compared. Although varying in length and amino acid composition, the labile peptides share a hydrophobic core of approximately 12 amino acids. A positively charged residue (Lys or Arg) usually precedes the hydrophobic core. Core termination is defined by the occurrence of a charged residue, a sequence of residues which may induce a beta-turn in a polypeptide, or an interruption in potential alpha-helix or beta-extended strand structure. The hydrophobic cores contain, by weight average, 37% Leu: 15% Ala: 10% Val: 10% Phe: 7% Ile plus 21% other hydrophobic amino acids arranged in a non-random sequence. Following the hydrophobic cores (aligned by their last residue) a highly non-random and localized distribution of Ala is apparent within the initial eight positions following the core: (formula; see text) Coincident with this observation, Ala-X-Ala is the most frequent sequence preceding signal peptidase cleavage. We propose the existence of a signal peptidase recognition sequence A-X-B with the preferred cleavage site located after the sixth amino acid following the core sequence. Twenty-two of the above 27 underlined Ala residues would participate as A or B in peptidase cleavage. Position A includes the larger aliphatic amino acids, Leu, Val and Ile, as well as the residues already found at B (principally Ala, Gly and Ser). Since a preferred cleavage site can be discerned from carboxyl and not amino terminal alignment of the hydrophobic cores it is proposed that the carboxyl ends are oriented inward toward the lumen of the endoplasmic reticulum where cleavage is thought to occur. This orientation coupled with the predicted beta-turn typically found between the core and the cleavage site implies reverse hairpin insertion of the signal sequence. The structural features which we describe should help identify signal peptides and cleavage sites in presumptive amino acid sequences derived from DNA sequences.     

Inhibition of virus-induced cell fusion by apolipoprotein A-I and its amphipathic peptide analogs.

Apolipoprotein A-I (apoA-I), the major protein component of serum high-density lipoproteins (HDL), was found to inhibit herpes simplex virus (HSV)-induced cell fusion at physiological (approximately 1 microM) concentrations, whereas HDL did not exert any inhibitory effect. Lipid-associating, synthetic amphipathic peptides corresponding to residues 1-33 (apoA-I[1-33]) or residues 66-120 (apoA-I[66-120]) of apoA-I, also inhibited HSV-induced cell fusion, whereas a peptide corresponding to residues 8-33 of apoA-I (apoA-I[8-33]), which fails to associate with lipids, did not exert any inhibitory effect. These results suggest that lipid binding may be a prerequisite for peptide-mediated fusion inhibition. Consistent with this idea, a series of lipid-binding 22-amino-acid-residue-long synthetic amphipathic peptides that correspond to the amphipathic helical domains of apoA-I (A-I consensus series), or 18-residue-long model amphipathic peptides (18A series), were found to exert variable levels of fusion-inhibitory activity. The extent of fusion-inhibitory activity did not correlate with hydrophobic moment, hydrophobicity of the nonpolar face, helix-forming ability, or lipid affinity of the different peptides. Peptides in which the nonpolar face was not interrupted by a charged residue displayed greater fusion-inhibitory activity. Also, the presence of positively charged residues at the polar-nonpolar interface was found to correlate with higher fusion-inhibitory activity.