title: Real-time detection of SNARE complex assembly with FRET using the tetracysteine system.
abstract: Small tetracysteine insertions are more suitable for fluorescence resonance energy transfer (FRET) studies of protein folding and small complex assembly than bulky GFP-based fluorophores. Here, we describe a procedure for expression, purification, and fluorescent labeling of a FRET-based probe, called CSNAC that can track the conformational changes undergone by SNAP-25 as it folds in the exocytic complex. The fluorescent protein Cerulean was attached to the N-terminus and served as a FRET donor. The biarsenical dye FlAsH, served as a FRET acceptor, was bound to a short tetracysteine motif positioned in the linker domain of SNAP-25. CSNAC can report real-time FRET changes when the Syntaxin soluble domain is added in vitro.
authors.affiliation: Division of Diabetes, Obesity, & Metabolism, Oregon National Primate Research Center, 505 NW 185th Ave, Mail Code L584, Beaverton, OR, 97006, USA, varlamov@ohsu.edu.
authors.forename: Oleg
authors.initials: O
authors.lastname: Varlamov
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015870:Gene Expression; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050600:SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D050600:SNARE Proteins