Diao, Jiajie Ishitsuka, Yuji Lee, Hanki Joo, Chirlmin Su, Zengliu Syed, Salman Shin, Yeon-Kyun Yoon, Tae-Young Ha, Taekjip 2024-01-20T15:03:25Z 2024-01-20T15:03:25Z 2021-09-05 2012-04 1754-2189 https://pubs.kist.re.kr/handle/201004/129381 SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3-6 d. English NATURE PUBLISHING GROUP MEMBRANE-FUSION LIPID VESICLES MOLECULE DRIVEN MACHINERY DOCKING COMPLEX SURFACE A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins Article 10.1038/nprot.2012.020 1 NATURE PROTOCOLS, v.7, no.5, pp.921 - 934 NATURE PROTOCOLS 7 5 921 934 scie scopus 000303359300010 2-s2.0-84871527218 Biochemical Research Methods Biochemistry & Molecular Biology Article MEMBRANE-FUSION LIPID VESICLES MOLECULE DRIVEN MACHINERY DOCKING COMPLEX SURFACE SNARE vesicle fusion assay single vesicle FRET Content-mixing assay lipid-mixing assay