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

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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
Externally publishedYes

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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