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SNARE derived peptide mimic inducing membrane fusion

SNARE derived peptide mimic inducing membrane fusion View Article Online / Journal Homepage / Table of Contents for this issue Dynamic Article Links ChemComm Cite this: Chem. Commun., 2011, 47, 9405–9407 www.rsc.org/chemcomm COMMUNICATION SNARE derived peptide mimic inducing membrane fusionw a a b b Karsten Meyenberg, Antonina S. Lygina, Geert van den Bogaart, Reinhard Jahn and Ulf Diederichsen* Received 16th May 2011, Accepted 28th June 2011 DOI: 10.1039/c1cc12879e SNARE proteins mediate membrane fusion between synaptic opens the chance to investigate the molecular requirements vesicles and the plasma membrane. A minimized peptide fulfilled by SNARE complex formation. SNARE model system with reduced complexity was introduced Several SNARE-mimicking model systems have been combining the native SNARE transmembrane (TMD) and linker reported with the aim of reducing complexity in membrane domains with artificial coiled-coil forming peptides. Specific fusion. Complementary artificial recognition motifs were linked 8,9 10–15 membrane fusion initiated by coiled-coil recognition was shown to cholesterol, phospholipids, or membrane penetrating 14,15 by lipid and content mixing vesicle assays. peptides and were subsequently embedded into the membrane. A variety of chemically diverse recognition motifs for linking the membranes has been reported such as the reversible reaction Membrane fusion is a central cellular process in eukaryotic 13 8,9,11,12 between boronic acid and diols, DNA double strands, cells. In the secretory pathway connecting organelles between and a vancomycin glycopeptide combined with a D-Ala-D-Ala the endoplasmic reticulum and the plasma membrane, fusion 14,15 moiety. Another reduced SNARE model has been introduced is mediated by sets of SNARE proteins (soluble N-ethyl- that is based on a coiled-coil forming a-peptide. Although in maleimide-sensitive factor attachment receptor). Comple- all of these cases specific membrane fusion was observed, they mentary sets of SNARE proteins are associated with the are structurally different from the SNAREs, and thus, do not respective membranes. Upon contact, the SNAREs form a thermodynamically stable four helix bundle connecting the allow for direct mechanistic extrapolation to SNARE-mediated fusion events. membranes (trans-complex) probably being generated in a 2,3 To overcome this limitation, we have recently introduced a zipper-like recognition process followed by helicalization. new SNARE-mimicking model using the native TMDs linked Thereby, the membranes are brought into proximity over- to peptide nucleic acid (PNA) recognition motifs and obtained coming the energy barrier for membrane fusion. efficient fusion that proceeds via hemifusion as intermediate. Neuronal exocytosis is catalyzed by the SNARE proteins We have now extended this approach by employing coiled-coil SNAP-25 (two a-helices), VAMP2 (synaptobrevin), and forming a-peptides. To this end, we have replaced the syntaxin-1A (each one a-helix), with the two latter each four SNARE helices (Fig. 1a) by smaller coiled-coil forming possessing a single transmembrane domain (TMD) at the 1,5 a-peptides (Fig. 1b and c). The hybrid between an a-peptide C-terminus (Fig. 1a). In vitro reconstitution of these recognition motif and the SNARE derived TMDs is advanta- SNAREs in liposomes ranging between 30–100 nm diameter results in SNARE-mediated membrane fusion. However, geous since it allows for studying the role of the native linkers and TMDs of synaptobrevin and syntaxin in the fusion reaction. both assembly and fusion kinetics in these systems are highly Here, we report about the synthesis of these molecules and complex; and it is difficult to differentiate between parameters affecting nucleation and zippering of the helix bundle and the downstream events of membrane contact, hemifusion, and fusion. In order to derive mechanistic insight into the fusion event, simpler model systems are needed. In particular, substitution of the SNARE complex by SNARE-mimicking peptides may shed light on the influence of the TMDs and the linker regions between the SNARE complex forming helices and the TMDs. In addition, investigation of various artificial recognition motifs Institut fu¨r Organische und Biomolekulare Chemie, Tammannstr. 2, Fig. 1 (a) Crystal structure of the synaptic ternary SNARE 37077 Go¨ttingen, Germany. E-mail: [email protected]; cis-complex (syntaxin1A—red, VAMP2—blue, SNAP25—grey; rattus Fax: 49 551-39 22944; Tel: 49 551 39 3221 norvegicus). (b) Sequence of the peptide SNARE analogues with Max-Planck-Institut fu¨r Biophysikalische Chemie, Am Faßberg 11, sequentially identical TMD domains. (c) Membrane incorporation 37077 Go¨ttingen, Germany and proposed interaction of the peptide/TMDs K3-syntaxin and w Electronic supplementary information (ESI) available. See DOI: 10.1039/c1cc12879e E3-VAMP2 embedded in opposing membranes. This journal is The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 9405–9407 9405 Downloaded by University of Goettingen on 13 March 2013 Published on 20 July 2011 on http://pubs.rsc.org | doi:10.1039/C1CC12879E View Article Online show that efficient fusion can be achieved by these SNARE- hybrid prior to the fusion experiment. Almost no change mimicking molecules. in fluorescence was observed in the mixing process with The peptide fragments H-WWG(KIAALKE) -OH (K3) E3-VAMP2 liposomes. Fluorescence anisotropy measure- and H-G(EIAALEK) -OH (E3) were selected as coiled-coil ments confirmed the binding of the soluble E3 peptide recognition unit. Both consist of three heptad repeats that to the membrane bound K3-syntaxin, thereby, inhibiting assemble by electrostatic and hydrophobic core interactions. recognition of the peptides bound in opposing membranes They form a stable heterodimer originally designed to serve as (supporting information Fig. S1). As a second control for the biosensor or affinity purification tag. The peptides interact with dependence of fusion on coiled-coil formation we incubated 7 1 1 ak of 10 mol L and require a free energy of 9.6 kcal mol DiD and OG labelled vesicles both containing E3-VAMP2. to unfold. Therefore, the free energy is qualitatively in good Almost no change in fluorescence was observed. Then, peptides agreement with the energy provided by the four helix bundle were synthesized in which the recognition units between the 1 19 of the neuronal SNARE complex (B20 kcal mol ). The VAMP and syntaxin fragments were swapped (K3-VAMP2 coiled-coil forming peptides were N-terminally linked to the and E3-syntaxin). Again, a robust increase of fluorescence was TMDs of the neuronal SNARE proteins VAMP2 (residues observed that was comparable to lipid mixing of vesicles 85–116) and syntaxin-1A (258–288). The SNARE hybrids mediated by K3-syntaxin and E3-VAMP2 (supporting infor- K3-syntaxin, E3-VAMP2 and respective reference peptides mation Fig. S2w). Together, these results show that replace- were synthesized by means of automated microwave assisted ment of the conserved four helix bundle by a simple binary solid phase peptide synthesis (SPPS) and characterized by ESI recognition motif that is fused to the linkers and transmembrane mass spectrometry (supporting informationw). domains of the neuronal SNAREs suffices to mediate membrane In order to investigate whether the peptides are able to fusion. Their specific recognition seems to be essential for induce membrane fusion, the peptides were reconstituted into membrane fusion, regardless to which transmembrane segments liposomes. Fusion was measured by a FRET assay for lipid the recognition motifs are linked. 0 0 0 mixing (Fig. 2) using the FRET pair 1,1 dioctadecyl-3,3,3 ,3 - To compare the kinetics of membrane fusion of our minimized tetramethylindodicarbocyanine perchlorate (DiD, acceptor) peptide fusion models with that of native SNAREs, two different 6 23 and oregon green (OG, donor). The peptides were incorporated and commonly used in vitro set-ups for native SNAREs were into liposomes in a 1/2000 peptide to lipid molar ratio using size employed. Firstly, syntaxin-1A (lacking its inhibitory Habc 21 24 exclusion chromatography. The membranes were composed domain; residue 183–288) containing vesicles were preincubated of a 5 : 2 : 2 : 1 molar ratio of phosphatidyl choline, phosphatidyl with SNAP25 yielding the syntaxin-1A/SNAP25 complex (2 : 1 ethanol amine, phosphatidyl serine (all from pig brain) and complex). Lipid mixing was measured with vesicles containing cholesterol as this mixture mimics the composition of synaptic this 2 : 1 complex with VAMP2 vesicles (Fig. 3). In this fusion membranes. Liposomes (80 mM total lipids) labelled with oregon assay, the fluorescence increase was about five times slower green 488 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine than that obtained with the SNARE-mimicking peptides. (OG-DHPE, 1.5 mol%) and containing K3-syntaxin were Secondly, we replaced the 2 : 1 complex with a stabilized mixed with DiD (1.5 mol%) labelled liposomes (80 mM) syntaxin/SNAP25 acceptor complex consisting of syntaxin-1A containing E3-VAMP2 in buffer solution at room temperature (183–288), SNAP-25, and a short fragment of VAMP2 (49–96; (Fig. 2). DN-complex) (Fig. 3). Contrary to the 2 : 1 complex, where Lipid mixing of both liposomes was indicated by an increase the VAMP2 binding site is blocked by the second copy of of the acceptor fluorescence over a period of 20 min due to syntaxin-1A, this DN-complex offers a free binding site for energy transfer from the OG to the DiD-fluorophor in the VAMP2. It, thereby, greatly facilitates SNARE nucleation resulting membrane. In order to prove that lipid mixing was and speeds up membrane docking. The dissociation of the initiated by coiled-coil recognition, a competition experiment VAMP2 (49–96) fragment is the rate limiting step for membrane was performed adding the soluble peptide H-G(EIAALEK) G-OH fusion and results in sigmoidal rather than exponential lipid (E3)(4 mM) to the liposomes containing the K3-syntaxin Fig. 2 Lipid mixing of liposomes containing SNARE-mimicking Fig. 3 Comparison of lipid mixing induced by SNARE-mimicking peptides using a fluorescence dequenching assay. E3-VAMP2 and peptides and neuronal SNAREs: E3-VAMP2 and K3-syntaxin (’), K3-syntaxin (’); addition of peptide E3 to K3-syntaxin liposomes 2 : 1 complex (b) and DN-complex (}) in a 1/1000 peptide/lipid ratio prior to fusion with E3-VAMP2 (K); vesicle populations both for all populations (for definition of the 2 : 1 complex and DN-complex containing E3-VAMP2 (n). see text). 9406 Chem. Commun., 2011, 47, 9405–9407 This journal is The Royal Society of Chemistry 2011 Downloaded by University of Goettingen on 13 March 2013 Published on 20 July 2011 on http://pubs.rsc.org | doi:10.1039/C1CC12879E View Article Online SNARE-mimicking peptides require only an E3/K3-recognition unit two times 21 amino acids. This constitutes a nine-fold smaller size and a substantially reduced complexity. Further investigations will concentrate on modifications in the linker region investigating various sequences and lengths regarding the membrane fusion ability. Generous support by the Deutsche Forschungsgemeinschaft (SFB 803) is gratefully acknowledged. Notes and references 1 R. Jahn and R. H. Scheller, Nat. Rev. Mol. Cell Biol., 2006, 7, 631. 2 P. I. Hanson, R. Roth, Morisaki, R. Jahn and J. E. Heuser, Cell, 1997, 90, 523. Fig. 4 Content mixing experiment with E3-VAMP liposomes filled 3 R. C. Lin and R. H. Scheller, Neuron, 1997, 19, 1087. with sulforhodamine B at fluorescence self-quenching concentration 4 T. H. Kloepper, C. N. Kienle and D. Fasshauer, Mol. Biol. Cell, (20 mM). Addition of unlabelled K3-syntaxin liposomes (’), liposomes 2007, 18, 3463. with E3-VAMP (K), liposomes lacking a fusion peptide (m). 5 T. Brunger, K. Weninger, M. Bowen and S. Chu, Annu. Rev. Biochem., 2009, 78, 903. mixing kinetics (Fig. 3). Nevertheless, because of the 6 T. Weber, B. V. Zemelman, J. A. McNew, B. Westermann, dramatic enhancement of the membrane docking efficiency, M. Gmachl, F. Parlati, T. H. So¨ llner and J. E. Rothman, Cell, 1998, 92, 759. the DN-complex mediates the fastest SNARE induced in vitro 23 7 H. R. Marsden, I. Tomatsu and A. Kros, Chem. Soc. Rev., 2011, fusion known. Since our minimal fusion peptides displayed 40, 1572. similar lipid mixing kinetics as the DN-complex, but do not 8 G. Stengel, R. Zahn and F. Ho¨ o¨ k, J. Am. Chem. Soc., 2007, require the (non-physiological) dissociation of a stabilizing 129, 9584. 9 G. Stengel, L. Simonsson, R. A. Campbell and F. Ho¨ o¨ k, J. Phys. peptide, we conclude that our K3-syntaxin/E3-VAMP2 mimics Chem. B, 2008, 112, 8264. are well suited for further investigations of SNARE mediated 10 H. R. Marsden, N. A. Elbers, P. Bomans, N. A. J. M. Sommerdijk membrane fusion. and A. Kros, Angew. Chem. Int. Ed., 2009, 48, 2330. The lipid mixing assay used in the experiments above does 11 Y. M. Chan, B. van Lengerich and S. G. Boxer, Biointerphases, 2008, 3, FA17. not allow distinguishing between full fusion and hemifusion 12 Y. H. Chan, B. van Lengerich and S. G. Boxer, Proc. Natl. Acad. where only the outer leaflet of the membrane bilayer is fused Sci. U. S. A., 2009, 106, 979. but the inner leaflet remains intact. Therefore, we carried out 13 A. Kashiwada, M. Tsuboi and K. Matsuda, Chem. Commun., 2009, 695. content mixing experiments using a self-quenching concentra- 14 Y. Gong, M. Ma, Y. Luo and D. Bong, J. Am. Chem. Soc., 2008, tion of 20 mM sulforhodamine B (SRB) encapsulated in the 130, 6196. 27–31 E3-VAMP2 liposomes. These liposomes were fused with 15 Y. Gong, Y. Luo and D. Bong, J. Am. Chem. Soc., 2006, 128, 1430. 16 A. S. Lygina, K. Meyenberg, R. Jahn and U. Diederichsen, Angew. K3-syntaxin liposomes similarly prepared but lacking the dye. Chem. Int. Ed., 2011, 50, DOI: 10.1002/ange.201101951. Full membrane fusion, but not hemifusion, results in dilution 17 A. Stein, G. Weber, M. C. Wahl and R. Jahn, Nature, 2009, of the encapsulated SRB. Dequenching can be detected by an 460, 525. increase in fluorescence. Upon addition of liposomes containing 18 J. R. Litowski and R. S. Hodges, J. Biol. Chem., 2002, 277, 37272. 19 F. Li, F. Pincet, E. Perez, W. S. Eng, T. J. Melia, J. E. Rothman K3-syntaxin (120 mM lipids) to SRB loaded E3-VAMP2 and D. Tareste, Nat. Struct. Mol. Biol., 2007, 14, 890. liposomes (40 mM lipids), a 30% increase of fluorescence 20 D. K. Struck, D. Hoekstra and R. E. Pagano, Biochemistry, 1981, was observed relative to the fluorescence at the start of the 20, 4093. experiment and the maximal fluorescence after Triton-X100 21 C. G. Schuette, K. Hatsuzawa, M. Margittai, A. Stein, D. Riedel, P. Ku¨ ster, M. Ko¨ nig, C. Seidel and R. Jahn, Proc. Natl. Acad. Sci. full membrane lysis (Fig. 4). As a control experiment, U. S. A., 2004, 101, 2858. SRB loaded E3-VAMP2 liposomes (40 mM) were mixed with 22 S. Takamori, M. Holt, K. Stenius, E. A. Lemke, M. Grønborg, liposomes (120 mM) lacking the fusion peptides or with D. Riedel, H. Urlaub, S. Schenck, B. Bru¨ gger, P. Ringler, S. A. Mu¨ ller, B. Rammner, F. Gra¨ ter, J. S. Hub, B. L. De Groot, E3-VAMP2 containing liposomes (120 mM). In both cases G. Mieskes, Y. Moriyama, J. Klingauf, H. Grubmu¨ ller, J. Heuser only minor changes in fluorescence were detected (Fig. 4). and F. Wieland, Cell, 2006, 127, 831. Therefore, it was concluded that coiled-coil formation of the 23 A. V. Pobbati, A. Stein and D. Fasshauer, Science, 2006, 313, 673. membrane bound K3-syntaxin/E3-VAMP2 peptides indeed 24 F. Parlati, T. Weber, J. A. McNew, B. Westermann, T. H. So¨ llner and J. E. Rothman, Proc. Natl. Acad. Sci. U. S. A., 1999, promotes full membrane fusion. 96, 12565. In summary, we report a novel SNARE-mimicking system 25 A. Stein, A. Radhakrishnan, D. Riedel, D. Fasshauer and R. Jahn, in which a recognition motif containing the coiled-coil forming Nat. Struct. Mol. Biol., 2007, 14, 904. 26 A. Cypionka, A. Stein, J. M. Hernandez, H. Hippchen, R. Jahn peptides E3/K3 was synthetically linked to the native TMDs of and P. J. Walla, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 18575. neuronal SNARE proteins. K3-syntaxin/E3-VAMP2 mediated 27 J. Wilschut, N. Duzgunes, R. Fraley and D. Papahadjopoulos, membrane fusion was very efficient in both lipid mixing and Biochemistry, 1980, 19, 6011. content mixing assays. Therefore, these peptide constructs 28 J. Wilschut and D. Papahadjopoulos, Nature, 1979, 281, 690. 29 J. N. Weinstein, S. Yoshikami, P. Henkart, R. Blumenthal and provide a simplified tool for studying the steps of membrane W. A. Hagins, Science, 1977, 195, 489. fusion of artificial membranes downstream of coiled-coil 30 B. J. Ravoo, W. D. Weringa and J. Engberts, Biophys. J., 1999, formation. Native SNAREs have a recognition motif of four 76, 374. domains, each about 90 amino acids in length. In contrast, the 31 R. El Jastimi and M. Lafleur, Biospectroscopy, 1999, 5, 133. This journal is The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 9405–9407 9407 Downloaded by University of Goettingen on 13 March 2013 Published on 20 July 2011 on http://pubs.rsc.org | doi:10.1039/C1CC12879E http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Chemical Communications Unpaywall

SNARE derived peptide mimic inducing membrane fusion

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View Article Online / Journal Homepage / Table of Contents for this issue Dynamic Article Links ChemComm Cite this: Chem. Commun., 2011, 47, 9405–9407 www.rsc.org/chemcomm COMMUNICATION SNARE derived peptide mimic inducing membrane fusionw a a b b Karsten Meyenberg, Antonina S. Lygina, Geert van den Bogaart, Reinhard Jahn and Ulf Diederichsen* Received 16th May 2011, Accepted 28th June 2011 DOI: 10.1039/c1cc12879e SNARE proteins mediate membrane fusion between synaptic opens the chance to investigate the molecular requirements vesicles and the plasma membrane. A minimized peptide fulfilled by SNARE complex formation. SNARE model system with reduced complexity was introduced Several SNARE-mimicking model systems have been combining the native SNARE transmembrane (TMD) and linker reported with the aim of reducing complexity in membrane domains with artificial coiled-coil forming peptides. Specific fusion. Complementary artificial recognition motifs were linked 8,9 10–15 membrane fusion initiated by coiled-coil recognition was shown to cholesterol, phospholipids, or membrane penetrating 14,15 by lipid and content mixing vesicle assays. peptides and were subsequently embedded into the membrane. A variety of chemically diverse recognition motifs for linking the membranes has been reported such as the reversible reaction Membrane fusion is a central cellular process in eukaryotic 13 8,9,11,12 between boronic acid and diols, DNA double strands, cells. In the secretory pathway connecting organelles between and a vancomycin glycopeptide combined with a D-Ala-D-Ala the endoplasmic reticulum and the plasma membrane, fusion 14,15 moiety. Another reduced SNARE model has been introduced is mediated by sets of SNARE proteins (soluble N-ethyl- that is based on a coiled-coil forming a-peptide. Although in maleimide-sensitive factor attachment receptor). Comple- all of these cases specific membrane fusion was observed, they mentary sets of SNARE proteins are associated with the are structurally different from the SNAREs, and thus, do not respective membranes. Upon contact, the SNAREs form a thermodynamically stable four helix bundle connecting the allow for direct mechanistic extrapolation to SNARE-mediated fusion events. membranes (trans-complex) probably being generated in a 2,3 To overcome this limitation, we have recently introduced a zipper-like recognition process followed by helicalization. new SNARE-mimicking model using the native TMDs linked Thereby, the membranes are brought into proximity over- to peptide nucleic acid (PNA) recognition motifs and obtained coming the energy barrier for membrane fusion. efficient fusion that proceeds via hemifusion as intermediate. Neuronal exocytosis is catalyzed by the SNARE proteins We have now extended this approach by employing coiled-coil SNAP-25 (two a-helices), VAMP2 (synaptobrevin), and forming a-peptides. To this end, we have replaced the syntaxin-1A (each one a-helix), with the two latter each four SNARE helices (Fig. 1a) by smaller coiled-coil forming possessing a single transmembrane domain (TMD) at the 1,5 a-peptides (Fig. 1b and c). The hybrid between an a-peptide C-terminus (Fig. 1a). In vitro reconstitution of these recognition motif and the SNARE derived TMDs is advanta- SNAREs in liposomes ranging between 30–100 nm diameter results in SNARE-mediated membrane fusion. However, geous since it allows for studying the role of the native linkers and TMDs of synaptobrevin and syntaxin in the fusion reaction. both assembly and fusion kinetics in these systems are highly Here, we report about the synthesis of these molecules and complex; and it is difficult to differentiate between parameters affecting nucleation and zippering of the helix bundle and the downstream events of membrane contact, hemifusion, and fusion. In order to derive mechanistic insight into the fusion event, simpler model systems are needed. In particular, substitution of the SNARE complex by SNARE-mimicking peptides may shed light on the influence of the TMDs and the linker regions between the SNARE complex forming helices and the TMDs. In addition, investigation of various artificial recognition motifs Institut fu¨r Organische und Biomolekulare Chemie, Tammannstr. 2, Fig. 1 (a) Crystal structure of the synaptic ternary SNARE 37077 Go¨ttingen, Germany. E-mail: [email protected]; cis-complex (syntaxin1A—red, VAMP2—blue, SNAP25—grey; rattus Fax: 49 551-39 22944; Tel: 49 551 39 3221 norvegicus). (b) Sequence of the peptide SNARE analogues with Max-Planck-Institut fu¨r Biophysikalische Chemie, Am Faßberg 11, sequentially identical TMD domains. (c) Membrane incorporation 37077 Go¨ttingen, Germany and proposed interaction of the peptide/TMDs K3-syntaxin and w Electronic supplementary information (ESI) available. See DOI: 10.1039/c1cc12879e E3-VAMP2 embedded in opposing membranes. This journal is The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 9405–9407 9405 Downloaded by University of Goettingen on 13 March 2013 Published on 20 July 2011 on http://pubs.rsc.org | doi:10.1039/C1CC12879E View Article Online show that efficient fusion can be achieved by these SNARE- hybrid prior to the fusion experiment. Almost no change mimicking molecules. in fluorescence was observed in the mixing process with The peptide fragments H-WWG(KIAALKE) -OH (K3) E3-VAMP2 liposomes. Fluorescence anisotropy measure- and H-G(EIAALEK) -OH (E3) were selected as coiled-coil ments confirmed the binding of the soluble E3 peptide recognition unit. Both consist of three heptad repeats that to the membrane bound K3-syntaxin, thereby, inhibiting assemble by electrostatic and hydrophobic core interactions. recognition of the peptides bound in opposing membranes They form a stable heterodimer originally designed to serve as (supporting information Fig. S1). As a second control for the biosensor or affinity purification tag. The peptides interact with dependence of fusion on coiled-coil formation we incubated 7 1 1 ak of 10 mol L and require a free energy of 9.6 kcal mol DiD and OG labelled vesicles both containing E3-VAMP2. to unfold. Therefore, the free energy is qualitatively in good Almost no change in fluorescence was observed. Then, peptides agreement with the energy provided by the four helix bundle were synthesized in which the recognition units between the 1 19 of the neuronal SNARE complex (B20 kcal mol ). The VAMP and syntaxin fragments were swapped (K3-VAMP2 coiled-coil forming peptides were N-terminally linked to the and E3-syntaxin). Again, a robust increase of fluorescence was TMDs of the neuronal SNARE proteins VAMP2 (residues observed that was comparable to lipid mixing of vesicles 85–116) and syntaxin-1A (258–288). The SNARE hybrids mediated by K3-syntaxin and E3-VAMP2 (supporting infor- K3-syntaxin, E3-VAMP2 and respective reference peptides mation Fig. S2w). Together, these results show that replace- were synthesized by means of automated microwave assisted ment of the conserved four helix bundle by a simple binary solid phase peptide synthesis (SPPS) and characterized by ESI recognition motif that is fused to the linkers and transmembrane mass spectrometry (supporting informationw). domains of the neuronal SNAREs suffices to mediate membrane In order to investigate whether the peptides are able to fusion. Their specific recognition seems to be essential for induce membrane fusion, the peptides were reconstituted into membrane fusion, regardless to which transmembrane segments liposomes. Fusion was measured by a FRET assay for lipid the recognition motifs are linked. 0 0 0 mixing (Fig. 2) using the FRET pair 1,1 dioctadecyl-3,3,3 ,3 - To compare the kinetics of membrane fusion of our minimized tetramethylindodicarbocyanine perchlorate (DiD, acceptor) peptide fusion models with that of native SNAREs, two different 6 23 and oregon green (OG, donor). The peptides were incorporated and commonly used in vitro set-ups for native SNAREs were into liposomes in a 1/2000 peptide to lipid molar ratio using size employed. Firstly, syntaxin-1A (lacking its inhibitory Habc 21 24 exclusion chromatography. The membranes were composed domain; residue 183–288) containing vesicles were preincubated of a 5 : 2 : 2 : 1 molar ratio of phosphatidyl choline, phosphatidyl with SNAP25 yielding the syntaxin-1A/SNAP25 complex (2 : 1 ethanol amine, phosphatidyl serine (all from pig brain) and complex). Lipid mixing was measured with vesicles containing cholesterol as this mixture mimics the composition of synaptic this 2 : 1 complex with VAMP2 vesicles (Fig. 3). In this fusion membranes. Liposomes (80 mM total lipids) labelled with oregon assay, the fluorescence increase was about five times slower green 488 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine than that obtained with the SNARE-mimicking peptides. (OG-DHPE, 1.5 mol%) and containing K3-syntaxin were Secondly, we replaced the 2 : 1 complex with a stabilized mixed with DiD (1.5 mol%) labelled liposomes (80 mM) syntaxin/SNAP25 acceptor complex consisting of syntaxin-1A containing E3-VAMP2 in buffer solution at room temperature (183–288), SNAP-25, and a short fragment of VAMP2 (49–96; (Fig. 2). DN-complex) (Fig. 3). Contrary to the 2 : 1 complex, where Lipid mixing of both liposomes was indicated by an increase the VAMP2 binding site is blocked by the second copy of of the acceptor fluorescence over a period of 20 min due to syntaxin-1A, this DN-complex offers a free binding site for energy transfer from the OG to the DiD-fluorophor in the VAMP2. It, thereby, greatly facilitates SNARE nucleation resulting membrane. In order to prove that lipid mixing was and speeds up membrane docking. The dissociation of the initiated by coiled-coil recognition, a competition experiment VAMP2 (49–96) fragment is the rate limiting step for membrane was performed adding the soluble peptide H-G(EIAALEK) G-OH fusion and results in sigmoidal rather than exponential lipid (E3)(4 mM) to the liposomes containing the K3-syntaxin Fig. 2 Lipid mixing of liposomes containing SNARE-mimicking Fig. 3 Comparison of lipid mixing induced by SNARE-mimicking peptides using a fluorescence dequenching assay. E3-VAMP2 and peptides and neuronal SNAREs: E3-VAMP2 and K3-syntaxin (’), K3-syntaxin (’); addition of peptide E3 to K3-syntaxin liposomes 2 : 1 complex (b) and DN-complex (}) in a 1/1000 peptide/lipid ratio prior to fusion with E3-VAMP2 (K); vesicle populations both for all populations (for definition of the 2 : 1 complex and DN-complex containing E3-VAMP2 (n). see text). 9406 Chem. Commun., 2011, 47, 9405–9407 This journal is The Royal Society of Chemistry 2011 Downloaded by University of Goettingen on 13 March 2013 Published on 20 July 2011 on http://pubs.rsc.org | doi:10.1039/C1CC12879E View Article Online SNARE-mimicking peptides require only an E3/K3-recognition unit two times 21 amino acids. This constitutes a nine-fold smaller size and a substantially reduced complexity. Further investigations will concentrate on modifications in the linker region investigating various sequences and lengths regarding the membrane fusion ability. Generous support by the Deutsche Forschungsgemeinschaft (SFB 803) is gratefully acknowledged. Notes and references 1 R. Jahn and R. H. Scheller, Nat. Rev. Mol. Cell Biol., 2006, 7, 631. 2 P. I. Hanson, R. Roth, Morisaki, R. Jahn and J. E. Heuser, Cell, 1997, 90, 523. Fig. 4 Content mixing experiment with E3-VAMP liposomes filled 3 R. C. Lin and R. H. Scheller, Neuron, 1997, 19, 1087. with sulforhodamine B at fluorescence self-quenching concentration 4 T. H. Kloepper, C. N. Kienle and D. Fasshauer, Mol. Biol. Cell, (20 mM). Addition of unlabelled K3-syntaxin liposomes (’), liposomes 2007, 18, 3463. with E3-VAMP (K), liposomes lacking a fusion peptide (m). 5 T. Brunger, K. Weninger, M. Bowen and S. Chu, Annu. Rev. Biochem., 2009, 78, 903. mixing kinetics (Fig. 3). Nevertheless, because of the 6 T. Weber, B. V. Zemelman, J. A. McNew, B. Westermann, dramatic enhancement of the membrane docking efficiency, M. Gmachl, F. Parlati, T. H. So¨ llner and J. E. Rothman, Cell, 1998, 92, 759. the DN-complex mediates the fastest SNARE induced in vitro 23 7 H. R. Marsden, I. Tomatsu and A. Kros, Chem. Soc. Rev., 2011, fusion known. Since our minimal fusion peptides displayed 40, 1572. similar lipid mixing kinetics as the DN-complex, but do not 8 G. Stengel, R. Zahn and F. Ho¨ o¨ k, J. Am. Chem. Soc., 2007, require the (non-physiological) dissociation of a stabilizing 129, 9584. 9 G. Stengel, L. Simonsson, R. A. Campbell and F. Ho¨ o¨ k, J. Phys. peptide, we conclude that our K3-syntaxin/E3-VAMP2 mimics Chem. B, 2008, 112, 8264. are well suited for further investigations of SNARE mediated 10 H. R. Marsden, N. A. Elbers, P. Bomans, N. A. J. M. Sommerdijk membrane fusion. and A. Kros, Angew. Chem. Int. Ed., 2009, 48, 2330. The lipid mixing assay used in the experiments above does 11 Y. M. Chan, B. van Lengerich and S. G. Boxer, Biointerphases, 2008, 3, FA17. not allow distinguishing between full fusion and hemifusion 12 Y. H. Chan, B. van Lengerich and S. G. Boxer, Proc. Natl. Acad. where only the outer leaflet of the membrane bilayer is fused Sci. U. S. A., 2009, 106, 979. but the inner leaflet remains intact. Therefore, we carried out 13 A. Kashiwada, M. Tsuboi and K. Matsuda, Chem. Commun., 2009, 695. content mixing experiments using a self-quenching concentra- 14 Y. Gong, M. Ma, Y. Luo and D. Bong, J. Am. Chem. Soc., 2008, tion of 20 mM sulforhodamine B (SRB) encapsulated in the 130, 6196. 27–31 E3-VAMP2 liposomes. These liposomes were fused with 15 Y. Gong, Y. Luo and D. Bong, J. Am. Chem. Soc., 2006, 128, 1430. 16 A. S. Lygina, K. Meyenberg, R. Jahn and U. Diederichsen, Angew. K3-syntaxin liposomes similarly prepared but lacking the dye. Chem. Int. Ed., 2011, 50, DOI: 10.1002/ange.201101951. Full membrane fusion, but not hemifusion, results in dilution 17 A. Stein, G. Weber, M. C. Wahl and R. Jahn, Nature, 2009, of the encapsulated SRB. Dequenching can be detected by an 460, 525. increase in fluorescence. Upon addition of liposomes containing 18 J. R. Litowski and R. S. Hodges, J. Biol. Chem., 2002, 277, 37272. 19 F. Li, F. Pincet, E. Perez, W. S. Eng, T. J. Melia, J. E. Rothman K3-syntaxin (120 mM lipids) to SRB loaded E3-VAMP2 and D. Tareste, Nat. Struct. Mol. Biol., 2007, 14, 890. liposomes (40 mM lipids), a 30% increase of fluorescence 20 D. K. Struck, D. Hoekstra and R. E. Pagano, Biochemistry, 1981, was observed relative to the fluorescence at the start of the 20, 4093. experiment and the maximal fluorescence after Triton-X100 21 C. G. Schuette, K. Hatsuzawa, M. Margittai, A. Stein, D. Riedel, P. Ku¨ ster, M. Ko¨ nig, C. Seidel and R. Jahn, Proc. Natl. Acad. Sci. full membrane lysis (Fig. 4). As a control experiment, U. S. A., 2004, 101, 2858. SRB loaded E3-VAMP2 liposomes (40 mM) were mixed with 22 S. Takamori, M. Holt, K. Stenius, E. A. Lemke, M. Grønborg, liposomes (120 mM) lacking the fusion peptides or with D. Riedel, H. Urlaub, S. Schenck, B. Bru¨ gger, P. Ringler, S. A. Mu¨ ller, B. Rammner, F. Gra¨ ter, J. S. Hub, B. L. De Groot, E3-VAMP2 containing liposomes (120 mM). In both cases G. Mieskes, Y. Moriyama, J. Klingauf, H. Grubmu¨ ller, J. Heuser only minor changes in fluorescence were detected (Fig. 4). and F. Wieland, Cell, 2006, 127, 831. Therefore, it was concluded that coiled-coil formation of the 23 A. V. Pobbati, A. Stein and D. Fasshauer, Science, 2006, 313, 673. membrane bound K3-syntaxin/E3-VAMP2 peptides indeed 24 F. Parlati, T. Weber, J. A. McNew, B. Westermann, T. H. So¨ llner and J. E. Rothman, Proc. Natl. Acad. Sci. U. S. A., 1999, promotes full membrane fusion. 96, 12565. In summary, we report a novel SNARE-mimicking system 25 A. Stein, A. Radhakrishnan, D. Riedel, D. Fasshauer and R. Jahn, in which a recognition motif containing the coiled-coil forming Nat. Struct. Mol. Biol., 2007, 14, 904. 26 A. Cypionka, A. Stein, J. M. Hernandez, H. Hippchen, R. Jahn peptides E3/K3 was synthetically linked to the native TMDs of and P. J. Walla, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 18575. neuronal SNARE proteins. K3-syntaxin/E3-VAMP2 mediated 27 J. Wilschut, N. Duzgunes, R. Fraley and D. Papahadjopoulos, membrane fusion was very efficient in both lipid mixing and Biochemistry, 1980, 19, 6011. content mixing assays. Therefore, these peptide constructs 28 J. Wilschut and D. Papahadjopoulos, Nature, 1979, 281, 690. 29 J. N. Weinstein, S. Yoshikami, P. Henkart, R. Blumenthal and provide a simplified tool for studying the steps of membrane W. A. Hagins, Science, 1977, 195, 489. fusion of artificial membranes downstream of coiled-coil 30 B. J. Ravoo, W. D. Weringa and J. Engberts, Biophys. J., 1999, formation. Native SNAREs have a recognition motif of four 76, 374. domains, each about 90 amino acids in length. In contrast, the 31 R. El Jastimi and M. Lafleur, Biospectroscopy, 1999, 5, 133. 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