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

    Research output: Contribution to journalArticle

    1 Scopus citations

    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 languageEnglish (US)
    Pages (from-to)49-55
    Number of pages7
    JournalMethods in Molecular Biology
    Volume1174
    DOIs
    StatePublished - Jan 1 2014

    Keywords

    • CSNAC
    • FRET
    • FlAsH
    • Folding
    • GFP
    • SNARE
    • Tetracysteine

    ASJC Scopus subject areas

    • Molecular Biology
    • Genetics

    Fingerprint Dive into the research topics of 'Real-time detection of snare complex assembly with fret using the tetracysteine system'. Together they form a unique fingerprint.

  • Cite this