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Real-Time Detection of SNARE Complex Assembly with FRET Using the Tetracysteine System

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Exocytosis and Endocytosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1174))

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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.

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Authors and Affiliations

  1. Division of Diabetes, Obesity, & Metabolism, Oregon National Primate Research Center, 505 NW 185th Ave, Mail Code L584, Beaverton, OR, 97006, USA

    Oleg Varlamov

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  1. Oleg Varlamov
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Correspondence to Oleg Varlamov .

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Editors and Affiliations

  1. Department of Human and Molecular Genetics, VCU Institute of Molecular Medicine, Virginia Commonwealth University, Richmond, Virginia, USA

    Andrei I. Ivanov

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Varlamov, O. (2014). Real-Time Detection of SNARE Complex Assembly with FRET Using the Tetracysteine System. In: Ivanov, A. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 1174. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0944-5_3

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  • DOI: https://doi.org/10.1007/978-1-4939-0944-5_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0943-8

  • Online ISBN: 978-1-4939-0944-5

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