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Optical Fusion Assay Based on Membrane-Coated Beads in a 2D Assembly

dc.contributor.advisorJanshoff, Andreas Prof. Dr.
dc.contributor.authorBao, Chunxiao
dc.publisherNiedersächsische Staats- und Universitätsbibliothek Göttingende
dc.titleOptical Fusion Assay Based on Membrane-Coated Beads in a 2D Assemblyde
dc.contributor.refereeJanshoff, Andreas Prof. Dr.
dc.description.abstractengA novel assay of membrane fusion is established based on membrane-coated beads in a 2D assembly. This assay allows identifying the different stages of the fusion process. Membrane fusion driven by heterodimeric coiled coil formation as a proof of concept using fusogenic K- and E-peptides provided results comparable to those obtained with conventional liposome assays, but with additional information on docking efficiency. In this fusion assay, LBs and SBs are modified with i-K3Cys (LB-i-K3) and i-E3Cys (SB-i-E3) respectively and LBs are fluorescent labeled. The populations of fusogenic beads can be distinguished by size discrimination. When LB-i-K3and SB-i-E3 are mixed in water, the fusion process terminates in hemifusion. Interestingly, all fusion events including docking, hemifusion and full fusion are observed in the presence of Ca2+ in one experiment. This may due to the bridging effect of calcium ions binding to PC and non-reacted negative MCC-DOPE. Block or change the zipping conformational of coiled coil structure is necessary for the viral infection of host cells. In this bead-based assay, the introduction of inhibitors (i-E3Cys) efficiently decreases the hemifusion efficiency and abolishes full fusion (in the presence of Ca2+). Therefore, this approach is expected to be an invaluable tool to identify small-molecule inhibitors of viral fusion with unprecedented accuracy in prefusion state. Multiple fusion could be observed and distinguished, which takes place among more than two fusogenic membrane-coated beads. The orientations of coiled coil formation, including parallel and antiparallel orientations, are shown to have no effect on the fusion process in this bead-based assay. Most interestingly, size-dependent assays employed beads with increasing diameter scale, demonstrate that van der Waals interaction could provided energy for supported membrane detaching from the silica beads, hence promote membrane
dc.contributor.coRefereeVana, Philipp Prof. Dr.
dc.subject.engmembrane fusionde
dc.subject.engmembrane-coated beadsde
dc.affiliation.instituteFakultät für Chemiede
dc.subject.gokfullChemie  (PPN62138352X)de

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