Conformation of membrane fusion-active 20-residue peptides with or without lipid bilayers. Implication of .alpha.-helix formation for membrane fusion

Takahashi, Sho

doi:10.1021/bi00478a021

Cited by 96 publications

(89 citation statements)

References 26 publications

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“…The estimated f-structure found for the two omission analogues in the presence of micelles would therefore result in a direct interaction between phospholipids and the peptides, but would prevent the occurrence of a perturbation by these peptides of lipid bilayers. It should be noted that melittin analogues cannot assume an amphipathic character when they adopt a f-structure, as was also found for previously reported peptides (Epand et al, 1987;Takahashi, 1990;Lee et al, 1993). This, in turn, would be expected to result in a decrease in haemolytic activity.…”

Section: Discussionsupporting

confidence: 62%

“…Partial f-sheet structures are known to be highly stable if these peptides are on a phospholipid surface (Takahashi, 1990). The estimated f-structure found for the two omission analogues in the presence of micelles would therefore result in a direct interaction between phospholipids and the peptides, but would prevent the occurrence of a perturbation by these peptides of lipid bilayers.…”

Section: Discussionmentioning

confidence: 99%

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Determination of the secondary structure of selected melittin analogues with different haemolytic activities

Pérez‐Payá

Houghten

Blondelle

1994

Biochemical Journal

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In earlier studies, we have reported that minor modifications in the amino acid sequence of melittin result in dramatic changes in its biological activity. In the current study, we have investigated the secondary structure of melittin analogues with either increased or decreased haemolytic activity in order to further our understanding of the structural features involved in the binding and/or insertion of peptides into a phospholipid membrane from solution. This was accomplished by analysing the c.d. spectra of the analogues in solutions of various ionic strength and, separately, in the presence of micelles. These studies permit the assessment of the effect of small sequence modifications (i.e. single amino acid omission or substitution) on the self-association-induced secondary structure of melittin in aqueous solution, as well as its binding affinity to micelles. It was found that amphipathicity, as well as interchain distances and the orientation of hydrophobic residues, were involved in the induction of stabilized structures.

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Section: Discussionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Determination of the secondary structure of selected melittin analogues with different haemolytic activities

Pérez‐Payá

Houghten

Blondelle

1994

Biochemical Journal

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“…PC and PG isolated from egg yolk were purchased from Sigma. Peptides (HA, E5, and K5) were synthesized as described (16,17,21). E5 and K5 were dissolved in Milli-Q water (Nihon Millipore, Yonezawa, Japan) at a concentration of 2 mM, whereas HA was solubilized in dimethyl sulfoxide at a concentration of 10 mM.…”

mentioning

confidence: 99%

Microscopic observations reveal that fusogenic peptides induce liposome shrinkage prior to membrane fusion

Nomura

Inaba²,

Ishikawa³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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To study the mechanisms involved in membrane fusion, we visualized the fusion process of giant liposomes in real time by optical dark-field microscopy. To induce membrane fusion, we used (i) influenza hemagglutinin peptide (HA), a 20-aa peptide derived from the N-terminal fusion peptide region of the HA2 subunit, and (ii) two synthetic analogue peptides of HA, a negatively (E5) and positively (K5) charged analogue. We were able to visualize membrane fusion caused by E5 or by K5 alone, as well as by the mixture of these two peptides. The HA peptide however, did not induce membrane fusion, even at an acidic pH, which has been described as the optimal condition for the fusion of large unilamellar vesicles. Surprisingly, before membrane fusion, the shrinkage of liposomes was always observed. Our results suggest that a perturbation of lipid bilayers, which probably resulted from alterations in the bending folds of membranes, is a critical factor in fusion efficiency.giant liposome ͉ influenza hemagglutinin ͉ optical microscopy ͉ direct observation M embrane fusion plays an essential role in cellular activities such as exocytosis, endocytosis, and vesicle transport of various cellular organelles. Also, it is an essential step of infection by enveloped viruses. Because the fusion of biological membranes is so important, studies on the mechanism of membrane fusion have been performed. Many of those studies focused on membrane fusion driven by fusogenic peptides, for example, those derived from influenza hemagglutinin. Those studies demonstrated that there are several critical steps required for membrane fusion (1-4). To characterize membrane fusion, resonance-energy transfer and fluorescence-quenching methods have been commonly used (5, 6). The resonance-energy transfer method monitors the mixing of membrane lipids that occurs during fusion, whereas the f luorescence-quenching method monitors the mixing of vesicular contents. Light scattering to detect fusion kinetics, or electron microscopy to visualize the fusion process, has also been commonly used. Electron microscopy can provide snapshots of the changing membrane structures in detail (7), but it is not suitable for studying the time-dependent transition of the three-dimensional membrane morphology.For understanding the fusion mechanism, the real-time visualization of individual fusion processes is indispensable (6, 8). We used giant (1-20-m diameter) liposomes as a model for biological membrane vesicles, and we monitored their fusion process in real time by using optical high-intensity dark-field microscopy (8). Giant liposomes can be visualized by several optical microscopic methods (9-11). However, we have used dark-field microscopy because it gives the best high-contrast images of giant liposomes compared with other methods (8,(12)(13)(14)(15).To induce membrane fusion, we used three different peptides: HA, E5, and K5. HA is a 20-aa peptide derived from the N-terminal fusion peptide region of the influenza hemagglutinin HA2 subunit, whereas E5 and K5 are negativ...

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“…Upon binding to synthetic liposomes or lipids, the native fusion peptide and its analogs possess higher ␣-helical contents (18,21). The NMR solution structures of the native fusion peptide with a hostguest system of influenza hemagglutinin (22) and of the E5 peptide in dodecylphosphocholine (DPC) micelles (23) have been determined.…”

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confidence: 99%

Structural Characterizations of Fusion Peptide Analogs of Influenza Virus Hemagglutinin

Hsu

Wu²,

Chang

et al. 2002

Journal of Biological Chemistry

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Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L, and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher ␣-helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The threedimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu 2 -Glu 11 and Trp 14 -Ile 18 , form amphipathic helical conformations, with Gly 12 -Gly 13 forming a hinge. The hydrophobic residues in the N-and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp 14 -Ile 18 reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the Cterminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion peptide analog and explains how the analog interacts with the lipid bilayer at different pH values.Membrane fusion plays a vital role in a large and diverse number of essential biological processes. For example, infection by enveloped viruses involves fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. Hemagglutinin (HA) 1 is a homotrimeric surface glycoprotein of the influenza virus with a relative molecular mass of 220 kDa. Each monomer of HA consists of the receptor-binding HA1 domain (328 residues) and the membrane-interacting HA2 domain (221 residues) linked by a single disulfide bond. The membrane fusion activity of HA has been measured both in vivo and in vitro by a variety of techniques, including hemolysis (1), polykaryon formation (2), resonance energy transfer (3), liposome cell fusion (4), spin-labeled electron paramagnetic resonance (5), electron microscopy (6), flow cytometry (7), electron spin resonance, and Fourier transform infrared (8). The N-terminal 20 residues of the HA2 domain have been identified as the fusion peptide, which is capable of inserting into the target membrane and thereby plays a crucial role in triggering membrane fusion. The crystal structure of soluble trimers (BHA) at neut...

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Conformation of membrane fusion-active 20-residue peptides with or without lipid bilayers. Implication of .alpha.-helix formation for membrane fusion

Cited by 96 publications

References 26 publications

Determination of the secondary structure of selected melittin analogues with different haemolytic activities

Determination of the secondary structure of selected melittin analogues with different haemolytic activities

Microscopic observations reveal that fusogenic peptides induce liposome shrinkage prior to membrane fusion

Structural Characterizations of Fusion Peptide Analogs of Influenza Virus Hemagglutinin

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