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.
Original language | English (US) |
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Pages (from-to) | 49-55 |
Number of pages | 7 |
Journal | Methods in Molecular Biology |
Volume | 1174 |
DOIs | |
State | Published - 2014 |
Keywords
- CSNAC
- FRET
- FlAsH
- Folding
- GFP
- SNARE
- Tetracysteine
ASJC Scopus subject areas
- Molecular Biology
- Genetics