Membrane Anchorage Brings About Fusogenic Properties in a Short Synthetic Peptide

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Bacteriorhodopsin is a proton-transporting membrane protein in Halophilic archaea, and it is considered a prototype of membrane transporters and a model for G-protein-coupled receptors. Oligomerization of the protein has been reported, but it is unknown whether this feature is correlated with, for instance, light activation. Here, we have addressed this issue by reconstituting bacteriorhodopsin into giant unilamellar vesicles. The dynamics of the fully active protein was investigated using fluorescence correlation spectroscopy and freeze fracture electron microscopy. At low protein-tolipid ratios (<1:10 w/w), a decrease in mobility was observed upon protein photoactivation. This process occurred on a second time scale and was fully reversible, i.e. when the dark-adapted state was reestablished the lateral diffusion rate of the protein was returned to that prior to activation. A similar decrease in lateral mobility as observed upon photoactivation was obtained when bacteriorhodopsin was reconstituted at high protein-tolipid ratios (>1:10 w/w). We interpret the shifts in mobility during light adaptation as being caused by transient photoinduced oligomerization of bacteriorhodopsin. These observations are fully supported by freeze-fracture electron microscopy, and the size of the clusters during photoactivation was estimated to consist of two or three trimers.

Section: Methodsmentioning

confidence: 99%

Spatial Organization of Bacteriorhodopsin in Model Membranes

Kahya

Wiersma

Poolman

et al. 2002

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“…Fluorescence Assays for Fusion-Large unilamellar WAE-coupled vesicles (LP-WAE) (final peptide/lipid ratio, 1:40), consisting of PC and cholesterol (3.5:1.5) were prepared as described previously (14). Fusion was monitored by lipid mixing, using an assay based upon energy transfer (15).…”

Section: Methodsmentioning

confidence: 99%

“…The initial rates of lipid mixing were determined from the tangents drawn to the steepest parts of the fusion curves. Contents mixing was assayed between LP-WAE loaded with 2.5 mM TbCl 3 and target PS/PE vesicles loaded with 50 mM dipicolinic acid, as described earlier (14).…”

Section: Methodsmentioning

confidence: 99%

“…Evidence was obtained, indicating that lipids blocking fusion displayed a pronounced inhibitory effect when present into the target membrane and much less when present into the membrane that bears the fusogen. Molecular dissection of these intriguing observations was accomplished by using a simplified membrane system, which relies on a membrane-anchored peptide model that has been shown to trigger fusion with target membranes in a structure-dependent manner (10,14). When free in solution, the peptide, called WAE, 1 adopts mainly ␤-structure, and is fusion-incompetent.…”

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confidence: 99%

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Protein-induced Fusion Can Be Modulated by Target Membrane Lipids through a Structural Switch at the Level of the Fusion Peptide

Pécheur¹,

Martin²,

Bienvenüe³

et al. 2000

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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 ␣-helix to a nonfusogenic ␤-sheet. The "on/off" activation of this switch is governed by minor amounts of (lyso)-phospholipids in targets, causing a drop in ␣-helix and a dramatic increase in ␤-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.Membrane proteins located on the surfaces of viruses, sperm, and intracellular membranes are intimately involved in membrane fusion. The overall fusion machinery, including molecular factors for targeting, attachment, and regulation of these events, can be quite complex. For example, the SNARE (Soluble NSF Attachment protein REceptors) system, mediating intracellular fusion events, consists of specific membrane proteins, present on vesicle (v-SNARE) and target membrane (t-SNARE), and cytosolic ATPases, such as N-ethylmaleimide-

“…Two-step models for peptide interactions with membranes are not uncommon, as occurs with amphipathic cationic antimicrobial peptides that use electrostatic interactions to promote initial peptide-membrane interaction followed by membrane lysis or pore formation (55). Lipid anchors have also been used in contrived peptide systems to promote liposome aggregation and membrane fusion (47,56,57), although the fatty acid did not exert these enhancing effects by promoting peptide-liposome binding as occurs with the p15 ectodomain.…”

Section: Discussionmentioning

confidence: 99%

Cell-Cell Membrane Fusion Induced by p15 Fusion-associated Small Transmembrane (FAST) Protein Requires a Novel Fusion Peptide Motif Containing a Myristoylated Polyproline Type II Helix

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Dawe

et al. 2012

Background:The p15 FAST protein mediates cell-cell fusion using a 19-residue ectodomain. Results: The p15 ectodomain requires both an N-terminal myristate and a polyproline type II helix for membrane fusion activity. Conclusion:The p15 ectodomain functions as a novel fusion peptide motif. Significance: This novel fusion peptide provides new insights into the essential biological process of protein-mediated membrane fusion.