Real-time detection of snare complex assembly with fret using the tetracysteine system

Abstract

Small tetracysteine insertions are more suitable for fl uorescence resonance energy transfer (FRET) studies of protein folding and small complex assembly than bulky GFP-based fl uorophores. Here, we describe a procedure for expression, purifi cation, and fl uorescent 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 fl uorescent 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.