Detection of SNARE complexes with FRET using the tetracysteine system

    Research output: Contribution to journalArticle

    3 Citations (Scopus)

    Abstract

    The three proteins synaptosomal-associated protein 25 (SNAP-25), Syntaxin-1a and vesicle-associated membrane protein (VAMP-2) are collectively called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). By assembling into an exocytic complex, the three SNAREs help in catalyzing membrane fusion. Due to lack of probes that adequately reconstitute the intracellular behavior of endogenous SNAREs, the dynamics of SNARE complexes in living cells is poorly understood. Here we describe a new FRET-based probe, called Cerulean-SNAP-25-C4 (CSNAC), that can track the conformational changes undergone by SNAP-25 as exocytic complexes assemble. 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 and was attached to a short tetracysteine motif (C4) motif inserted into the so-called linker domain of SNAP-25. CSNAC reported successive FRET changes when first Syntaxin-1a and then VAMP-2 were added in vitro. Small tetracysteine insertions used as a FRET acceptor are expected to have less steric hindrance than previously used GFP-based fluorophores. We propose that genetically-encoded tetracysteine tags can be used to study regulated SNARE complex assembly in vivo.

    Original languageEnglish (US)
    Pages (from-to)103-108
    Number of pages6
    JournalBioTechniques
    Volume52
    Issue number2
    DOIs
    StatePublished - Feb 2012

    Fingerprint

    SNARE Proteins
    Synaptosomal-Associated Protein 25
    Vesicle-Associated Membrane Protein 2
    Syntaxin 1
    R-SNARE Proteins
    Membrane Fusion
    Fluorophores
    Proteins
    Coloring Agents
    Fusion reactions
    Cells
    Membranes

    Keywords

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

    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology(all)
    • Biotechnology

    Cite this

    Detection of SNARE complexes with FRET using the tetracysteine system. / Varlamov, Oleg.

    In: BioTechniques, Vol. 52, No. 2, 02.2012, p. 103-108.

    Research output: Contribution to journalArticle

    @article{5f8f0367eeca4f15b6602c02f0bde15e,
    title = "Detection of SNARE complexes with FRET using the tetracysteine system",
    abstract = "The three proteins synaptosomal-associated protein 25 (SNAP-25), Syntaxin-1a and vesicle-associated membrane protein (VAMP-2) are collectively called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). By assembling into an exocytic complex, the three SNAREs help in catalyzing membrane fusion. Due to lack of probes that adequately reconstitute the intracellular behavior of endogenous SNAREs, the dynamics of SNARE complexes in living cells is poorly understood. Here we describe a new FRET-based probe, called Cerulean-SNAP-25-C4 (CSNAC), that can track the conformational changes undergone by SNAP-25 as exocytic complexes assemble. 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 and was attached to a short tetracysteine motif (C4) motif inserted into the so-called linker domain of SNAP-25. CSNAC reported successive FRET changes when first Syntaxin-1a and then VAMP-2 were added in vitro. Small tetracysteine insertions used as a FRET acceptor are expected to have less steric hindrance than previously used GFP-based fluorophores. We propose that genetically-encoded tetracysteine tags can be used to study regulated SNARE complex assembly in vivo.",
    keywords = "FlAsH, Folding, FRET, GFP, SNARE, Tetracysteine",
    author = "Oleg Varlamov",
    year = "2012",
    month = "2",
    doi = "10.2144/000113811",
    language = "English (US)",
    volume = "52",
    pages = "103--108",
    journal = "BioTechniques",
    issn = "0736-6205",
    publisher = "Eaton Publishing Company",
    number = "2",

    }

    TY - JOUR

    T1 - Detection of SNARE complexes with FRET using the tetracysteine system

    AU - Varlamov, Oleg

    PY - 2012/2

    Y1 - 2012/2

    N2 - The three proteins synaptosomal-associated protein 25 (SNAP-25), Syntaxin-1a and vesicle-associated membrane protein (VAMP-2) are collectively called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). By assembling into an exocytic complex, the three SNAREs help in catalyzing membrane fusion. Due to lack of probes that adequately reconstitute the intracellular behavior of endogenous SNAREs, the dynamics of SNARE complexes in living cells is poorly understood. Here we describe a new FRET-based probe, called Cerulean-SNAP-25-C4 (CSNAC), that can track the conformational changes undergone by SNAP-25 as exocytic complexes assemble. 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 and was attached to a short tetracysteine motif (C4) motif inserted into the so-called linker domain of SNAP-25. CSNAC reported successive FRET changes when first Syntaxin-1a and then VAMP-2 were added in vitro. Small tetracysteine insertions used as a FRET acceptor are expected to have less steric hindrance than previously used GFP-based fluorophores. We propose that genetically-encoded tetracysteine tags can be used to study regulated SNARE complex assembly in vivo.

    AB - The three proteins synaptosomal-associated protein 25 (SNAP-25), Syntaxin-1a and vesicle-associated membrane protein (VAMP-2) are collectively called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). By assembling into an exocytic complex, the three SNAREs help in catalyzing membrane fusion. Due to lack of probes that adequately reconstitute the intracellular behavior of endogenous SNAREs, the dynamics of SNARE complexes in living cells is poorly understood. Here we describe a new FRET-based probe, called Cerulean-SNAP-25-C4 (CSNAC), that can track the conformational changes undergone by SNAP-25 as exocytic complexes assemble. 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 and was attached to a short tetracysteine motif (C4) motif inserted into the so-called linker domain of SNAP-25. CSNAC reported successive FRET changes when first Syntaxin-1a and then VAMP-2 were added in vitro. Small tetracysteine insertions used as a FRET acceptor are expected to have less steric hindrance than previously used GFP-based fluorophores. We propose that genetically-encoded tetracysteine tags can be used to study regulated SNARE complex assembly in vivo.

    KW - FlAsH

    KW - Folding

    KW - FRET

    KW - GFP

    KW - SNARE

    KW - Tetracysteine

    UR - http://www.scopus.com/inward/record.url?scp=84857322105&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=84857322105&partnerID=8YFLogxK

    U2 - 10.2144/000113811

    DO - 10.2144/000113811

    M3 - Article

    VL - 52

    SP - 103

    EP - 108

    JO - BioTechniques

    JF - BioTechniques

    SN - 0736-6205

    IS - 2

    ER -