The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Reinhard Jahn - One of the best experts on this subject based on the ideXlab platform.
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pna hybrid sequences as recognition units in SNARE Protein mimicking peptides
Angewandte Chemie, 2018Co-Authors: Barbara E. Hubrich, Pawan Kumar, Reinhard Jahn, Hermann Neitz, Matthias Grunwald, Tobias Grothe, Peter Walla, Ulf DiederichsenAbstract:Membrane fusion is an essential process in nature and is often accomplished by the specific interaction of SNARE Proteins. SNARE model systems, in which SNARE domains are replaced by small artificial units, represent valuable tools to study membrane fusion in vitro. The synthesis and analysis is presented of SNARE model peptides that exhibit a recognition motif composed of two different types of peptide nucleic acid (PNA) sequences. This novel recognition unit is designed to mimic the SNARE zippering mechanism that initiates SNARE-mediated fusion. It contains N-(2-aminoethyl)glycine-PNA (aeg-PNA) and alanyl-PNA, which both recognize the respective complementary strand but differ in duplex topology and duplex formation kinetics. The duplex formation of PNA hybrid oligomers as well as the fusogenicity of the model peptides in lipid-mixing assays were characterized and the peptides were found to induce liposome fusion. As an unexpected discovery, peptides with a recognition unit containing only five aeg-PNA nucleo amino acids were sufficient and most efficient to induce liposome fusion.
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role of the transmembrane domain in SNARE Protein mediated membrane fusion peptide nucleic acid peptide model systems
Molecular BioSystems, 2016Co-Authors: Jandirk Wehland, Pawan Kumar, Barbara E. Hubrich, Reinhard Jahn, Antonina S Lygina, Samit Guha, Ulf DiederichsenAbstract:Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor Proteins also known as SNAREs. The backbone of this essential process is the assembly of SNAREs from opposite membranes into tight four helix bundles forcing membranes in close proximity. With model systems resembling SNAREs with reduced complexity we aim to understand how these Proteins work at the molecular level. Here, peptide nucleic acids (PNAs) are used as excellent candidates for mimicking the SNARE recognition motif by forming well-characterized duplex structures. Hybridization between complementary PNA strands anchored in liposomes through native transmembrane domains (TMDs) induces the merger of the outer leaflets of the participating vesicles but not of the inner leaflets. A series of PNA/peptide hybrids differing in the length of TMDs and charges at the C-terminal end is presented. Interestingly, mixing of both outer and inner leaflets is seen for TMDs containing an amide in place of the natural carboxylic acid at the C-terminal end. Charged side chains at the C-terminal end of the TMDs are shown to have a negative impact on the mixing of liposomes. The length of the TMDs is vital for fusion as with the use of shortened TMDs, fusion was completely prevented.
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calcium promotes the formation of syntaxin 1 mesoscale domains through phosphatidylinositol 4 5 bisphosphate
Journal of Biological Chemistry, 2016Co-Authors: Dragomir Milovanovic, Ulf Diederichsen, Reinhard Jahn, Mitja Platen, Meike Junius, Iwan A T Schaap, Alf Honigmann, Geert Van Den BogaartAbstract:Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P2and syntaxin 1, a SNARE Protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca(2+)acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P2complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca(2+)concentrations is likely to be important for Ca(2+)-regulated secretion.
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Distance regulated vesicle fusion and docking mediated by β-peptide nucleic acid SNARE Protein analogues.
Chembiochem : a European journal of chemical biology, 2016Co-Authors: Muheeb Sadek, Daniel Berndt, Dragomir Milovanovic, Reinhard Jahn, Ulf DiederichsenAbstract:Artificial SNARE analogues derived from SNARE Proteins, which mediate synaptic membrane fusion, are of interest. They mimic the tetrameric α-helix bundle of the SNARE motif with various bio-oligomer recognition units. Interaction between complementary oligomers linked to the respective membrane by lipid or peptide anchors leads to proximity of vesicles and to fusion of lipid bilayers. β-Peptide nucleic acids were introduced as hybrid oligomers with the native SNARE Protein transmembrane/linker sequence, in order to evaluate a fusion system that allows distance tuning of approaching membranes. Formation of a four-base pair β-PNA double strand with 20 A length is sufficient for vesicle membrane fusion. Elongation of the recognition β-PNA duplex in the linker region yielded a 40 A β-peptide duplex and provided a vesicle–vesicle distance that only supported hemifusion of vesicle membranes.
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α snap interferes with the zippering of the SNARE Protein membrane fusion machinery
Journal of Biological Chemistry, 2014Co-Authors: Yongsoo Park, Wensi Vennekate, Peter Walla, Halenur Yavuz, Julia Preobraschenski, J M Hernandez, Dietmar Riedel, Reinhard JahnAbstract:Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment Protein receptor (SNARE) Proteins. Before fusion, SNARE Proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane.
Thomas Martin - One of the best experts on this subject based on the ideXlab platform.
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Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion
The Journal of biological chemistry, 2010Co-Authors: Neil J. Daily, Kristin L. Boswell, Declan J. James, Thomas MartinAbstract:CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE Proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE Protein isoforms and requires membrane integration of the SNARE Proteins. SNARE Protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three Proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both Proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE Proteins required for vesicle exocytosis.
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synaptotagmin 1 utilizes membrane bending and SNARE binding to drive fusion pore expansion
Molecular Biology of the Cell, 2008Co-Authors: Kara L Lynch, Roy Gerona, Dana M Kielar, Sascha Martens, Harvey T Mcmahon, Thomas MartinAbstract:In regulated vesicle exocytosis, SNARE Protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca2+ is mediated...
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g Protein βγ directly regulates SNARE Protein fusion machinery for secretory granule exocytosis
Nature Neuroscience, 2005Co-Authors: Trillium Blackmer, Eric C Larsen, Cheryl J Bartleson, Judith A Kowalchyk, Eun Ja Yoon, Anita M Preininger, Simon Alford, Heidi E Hamm, Thomas MartinAbstract:The activation of G Protein–coupled receptors (GPCRs) can result in an inhibition of Ca2+-dependent hormone and neurotransmitter secretion. This has been attributed in part to G Protein inhibition of Ca2+ influx. However, a frequently dominant inhibitory effect, of unknown mechanism, also occurs distal to Ca2+ entry. Here we characterize direct inhibitory actions of G Protein βγ (Gβγ) on Ca2+-triggered vesicle exocytosis in permeable PC12 cells. Gβγ inhibition was rapid (<1 s) and was attenuated by cleavage of synaptosome-associated Protein of 25 kD (SNAP25). Gβγ bound soluble N-ethylmaleimide-sensitive factor attachment Protein receptor (SNARE) complexes, and binding was reduced to SNARE complexes containing cleaved SNAP25 or by Ca2+-dependent synaptotagmin binding. Here we show inhibitory coupling between GPCRs and vesicle exocytosis mediated directly by Gβγ interactions with the Ca2+-dependent fusion machinery.
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the c terminus of snap25 is essential for ca2 dependent binding of synaptotagmin to SNARE complexes
Journal of Biological Chemistry, 2000Co-Authors: Roy Gerona, Judith A Kowalchyk, Eric C Larsen, Thomas MartinAbstract:The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment Protein receptor (SNARE) Proteins syntaxin and synaptosome-associated Protein of 25 kDa (SNAP25) and the vesicle SNARE Protein vesicle-associated membrane Protein (VAMP) are essential for a late Ca(2+)-dependent step in regulated exocytosis, but their precise roles and regulation by Ca(2+) are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg(180)-Ile(181), completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln(197)-Arg(198), is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca(2+) concentrations. BoNT A treatment essentially increased the Ca(2+) concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca(2+) regulation of exocytosis. Synaptotagmin, a proposed Ca(2+) sensor for exocytosis, was found to bind SNAP25 in a Ca(2+)-stimulated manner. Ca(2+)-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca(2+) concentration required for binding. The C terminus of SNAP25 was also essential for Ca(2+)-dependent synaptotagmin binding to SNAP25. syntaxin and SNAP25.syntaxin.VAMP SNARE complexes. These results clarify classical observations on the Ca(2+) reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca(2+)-dependent interactions between synaptotagmin and SNARE Protein complexes.
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n ethylmaleimide sensitive factor acts at a prefusion atp dependent step in ca2 activated exocytosis
Journal of Biological Chemistry, 1996Co-Authors: Abhijit Banerjee, Victoria A Barry, Bibhuti R Dasgupta, Thomas MartinAbstract:Abstract An ATP-dependent activity of NSF (N-ethylmaleimide-sensitive factor) that rearranges soluble NSF attachment Protein (SNAP) receptor (SNARE) Protein complexes was proposed to be the driving force for membrane fusion. The Ca2+-activated fusion of secretory vesicles with the plasma membrane in permeable PC12 cells requires ATP; however, the ATP requirement is for a priming step that precedes the Ca2+-triggered fusion reaction. While phosphoinositide phosphorylation is a key reaction required for priming, additional ATP-dependent reactions are also necessary. Here we report that the NSF-catalyzed rearrangement of SNARE Protein complexes occurs during ATP-dependent priming. NSF with α-SNAP (soluble NSF attachment Protein) were required for ATP-dependent priming but not Ca2+-triggered fusion, indicating that NSF acts at an ATP-dependent prefusion step rather than at fusion itself. NSF-catalyzed activation of SNARE Proteins may reorganize membranes to generate a vesicle-plasma membrane prefusion intermediate that is poised for conversion to full fusion by Ca2+-dependent mechanisms.
Erdem Karatekin - One of the best experts on this subject based on the ideXlab platform.
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regulation of exocytotic fusion pores by SNARE Protein transmembrane domains
Frontiers in Molecular Neuroscience, 2017Co-Authors: Sathish Thiyagarajan, Ben Oshaughnessy, Erdem KaratekinAbstract:Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment Protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble Proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE Proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically defined setting which have recently become available. Finally, computer simulations are valuable mechanistic tools because they have the power to access small length scales and very short times that are experimentally inaccessible.
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dilation of fusion pores by SNARE Protein crowding
Biophysical Journal, 2017Co-Authors: Oscar D Bello, Sathish Thiyagarajan, Sarah M Auclair, Wensi Vennekate, Shyam S Krishnakumar, Ben Oshaughnessy, Erdem KaratekinAbstract:Hormones and neurotransmitters are released through exocytotic fusion pores that can fluctuate in size and flicker open and shut multiple times. The kinetics and the amount of cargo released, and the mode of vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v- and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not known. Past approaches either monitored single exocytotic pores in live cells with unknown biochemistry, or used reconstitutions that lacked single pore sensitivity. Here, we probed dilation of single fusion pores using v-SNARE-reconstituted ∼23 nm diameter discoidal nanolipoProtein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, “flipped” t-SNAREs. A flipped t-SNARE cell is patch-clamped in the cell-attached configuration with the vNLPs included in the pipette solution. Fusion of a vNLP with the cell surface produces a pore connecting the cytosol with the pipette solution, through which direct-currents are measured under voltage clamp. The magnitude of the current reports pore size with sub-millisecond time resolution (Wu, Z. et al. Sci. Rep. 2016). We found that pore nucleation required a minimum of 2, and reached a maximum above ∼4 copies of v-SNAREs per NLP face. In contrast, the mean conductance of single pores increased as copy number was increased and was far from saturating at 15 copies, the NLP capacity. Thus, very different numbers of SNARE complexes cooperate at the distinct stages of fusion pore nucleation and pore dilation. We calculated pore size distributions and free energy profiles versus pore size. Combined with a mathematical model, these results suggest crowding of SNARE complexes at the pore waist drive pore expansion.
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nanodisc cell fusion control of fusion pore nucleation and lifetimes by SNARE Protein transmembrane domains
Scientific Reports, 2016Co-Authors: Sarah M Auclair, Oscar D Bello, Wensi Vennekate, Shyam S Krishnakumar, Natasha Dudzinski, Erdem KaratekinAbstract:The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle –the fusion pore– can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the Proteins contribute to pore stability.
Wensi Vennekate - One of the best experts on this subject based on the ideXlab platform.
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dilation of fusion pores by SNARE Protein crowding
Biophysical Journal, 2017Co-Authors: Oscar D Bello, Sathish Thiyagarajan, Sarah M Auclair, Wensi Vennekate, Shyam S Krishnakumar, Ben Oshaughnessy, Erdem KaratekinAbstract:Hormones and neurotransmitters are released through exocytotic fusion pores that can fluctuate in size and flicker open and shut multiple times. The kinetics and the amount of cargo released, and the mode of vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v- and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not known. Past approaches either monitored single exocytotic pores in live cells with unknown biochemistry, or used reconstitutions that lacked single pore sensitivity. Here, we probed dilation of single fusion pores using v-SNARE-reconstituted ∼23 nm diameter discoidal nanolipoProtein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, “flipped” t-SNAREs. A flipped t-SNARE cell is patch-clamped in the cell-attached configuration with the vNLPs included in the pipette solution. Fusion of a vNLP with the cell surface produces a pore connecting the cytosol with the pipette solution, through which direct-currents are measured under voltage clamp. The magnitude of the current reports pore size with sub-millisecond time resolution (Wu, Z. et al. Sci. Rep. 2016). We found that pore nucleation required a minimum of 2, and reached a maximum above ∼4 copies of v-SNAREs per NLP face. In contrast, the mean conductance of single pores increased as copy number was increased and was far from saturating at 15 copies, the NLP capacity. Thus, very different numbers of SNARE complexes cooperate at the distinct stages of fusion pore nucleation and pore dilation. We calculated pore size distributions and free energy profiles versus pore size. Combined with a mathematical model, these results suggest crowding of SNARE complexes at the pore waist drive pore expansion.
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nanodisc cell fusion control of fusion pore nucleation and lifetimes by SNARE Protein transmembrane domains
Scientific Reports, 2016Co-Authors: Sarah M Auclair, Oscar D Bello, Wensi Vennekate, Shyam S Krishnakumar, Natasha Dudzinski, Erdem KaratekinAbstract:The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle –the fusion pore– can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the Proteins contribute to pore stability.
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α snap interferes with the zippering of the SNARE Protein membrane fusion machinery
Journal of Biological Chemistry, 2014Co-Authors: Yongsoo Park, Wensi Vennekate, Peter Walla, Halenur Yavuz, Julia Preobraschenski, J M Hernandez, Dietmar Riedel, Reinhard JahnAbstract:Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment Protein receptor (SNARE) Proteins. Before fusion, SNARE Proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane.
Ulf Diederichsen - One of the best experts on this subject based on the ideXlab platform.
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pna hybrid sequences as recognition units in SNARE Protein mimicking peptides
Angewandte Chemie, 2018Co-Authors: Barbara E. Hubrich, Pawan Kumar, Reinhard Jahn, Hermann Neitz, Matthias Grunwald, Tobias Grothe, Peter Walla, Ulf DiederichsenAbstract:Membrane fusion is an essential process in nature and is often accomplished by the specific interaction of SNARE Proteins. SNARE model systems, in which SNARE domains are replaced by small artificial units, represent valuable tools to study membrane fusion in vitro. The synthesis and analysis is presented of SNARE model peptides that exhibit a recognition motif composed of two different types of peptide nucleic acid (PNA) sequences. This novel recognition unit is designed to mimic the SNARE zippering mechanism that initiates SNARE-mediated fusion. It contains N-(2-aminoethyl)glycine-PNA (aeg-PNA) and alanyl-PNA, which both recognize the respective complementary strand but differ in duplex topology and duplex formation kinetics. The duplex formation of PNA hybrid oligomers as well as the fusogenicity of the model peptides in lipid-mixing assays were characterized and the peptides were found to induce liposome fusion. As an unexpected discovery, peptides with a recognition unit containing only five aeg-PNA nucleo amino acids were sufficient and most efficient to induce liposome fusion.
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role of the transmembrane domain in SNARE Protein mediated membrane fusion peptide nucleic acid peptide model systems
Molecular BioSystems, 2016Co-Authors: Jandirk Wehland, Pawan Kumar, Barbara E. Hubrich, Reinhard Jahn, Antonina S Lygina, Samit Guha, Ulf DiederichsenAbstract:Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor Proteins also known as SNAREs. The backbone of this essential process is the assembly of SNAREs from opposite membranes into tight four helix bundles forcing membranes in close proximity. With model systems resembling SNAREs with reduced complexity we aim to understand how these Proteins work at the molecular level. Here, peptide nucleic acids (PNAs) are used as excellent candidates for mimicking the SNARE recognition motif by forming well-characterized duplex structures. Hybridization between complementary PNA strands anchored in liposomes through native transmembrane domains (TMDs) induces the merger of the outer leaflets of the participating vesicles but not of the inner leaflets. A series of PNA/peptide hybrids differing in the length of TMDs and charges at the C-terminal end is presented. Interestingly, mixing of both outer and inner leaflets is seen for TMDs containing an amide in place of the natural carboxylic acid at the C-terminal end. Charged side chains at the C-terminal end of the TMDs are shown to have a negative impact on the mixing of liposomes. The length of the TMDs is vital for fusion as with the use of shortened TMDs, fusion was completely prevented.
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calcium promotes the formation of syntaxin 1 mesoscale domains through phosphatidylinositol 4 5 bisphosphate
Journal of Biological Chemistry, 2016Co-Authors: Dragomir Milovanovic, Ulf Diederichsen, Reinhard Jahn, Mitja Platen, Meike Junius, Iwan A T Schaap, Alf Honigmann, Geert Van Den BogaartAbstract:Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P2and syntaxin 1, a SNARE Protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca(2+)acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P2complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca(2+)concentrations is likely to be important for Ca(2+)-regulated secretion.
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Distance regulated vesicle fusion and docking mediated by β-peptide nucleic acid SNARE Protein analogues.
Chembiochem : a European journal of chemical biology, 2016Co-Authors: Muheeb Sadek, Daniel Berndt, Dragomir Milovanovic, Reinhard Jahn, Ulf DiederichsenAbstract:Artificial SNARE analogues derived from SNARE Proteins, which mediate synaptic membrane fusion, are of interest. They mimic the tetrameric α-helix bundle of the SNARE motif with various bio-oligomer recognition units. Interaction between complementary oligomers linked to the respective membrane by lipid or peptide anchors leads to proximity of vesicles and to fusion of lipid bilayers. β-Peptide nucleic acids were introduced as hybrid oligomers with the native SNARE Protein transmembrane/linker sequence, in order to evaluate a fusion system that allows distance tuning of approaching membranes. Formation of a four-base pair β-PNA double strand with 20 A length is sufficient for vesicle membrane fusion. Elongation of the recognition β-PNA duplex in the linker region yielded a 40 A β-peptide duplex and provided a vesicle–vesicle distance that only supported hemifusion of vesicle membranes.