Sequence-specific assignment of methyl groups from the neuronal SNARE complex using lanthanide-induced pseudocontact shifts

Yun Zu Pan, Bradley Quade, Kyle D. Brewer, Monika Szabo, James D. Swarbrick, Bim Graham, Josep Rizo

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

Neurotransmitter release depends critically on the neuronal SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, as well as on other proteins such as Munc18-1, Munc13-1 and synaptotagmin-1. Although three-dimensional structures are available for these components, it is still unclear how they are assembled between the synaptic vesicle and plasma membranes to trigger fast, Ca2+-dependent membrane fusion. Methyl TROSY NMR experiments provide a powerful tool to study complexes between these proteins, but assignment of the methyl groups of the SNARE complex is hindered by its limited solubility. Here we report the assignment of the isoleucine, leucine, methionine and valine methyl groups of the four SNARE motifs of syntaxin-1, SNAP-25 and synaptobrevin within the SNARE complex based solely on measurements of lanthanide-induced pseudocontact shifts. Our results illustrate the power of this approach to assign protein resonances without the need of triple resonance experiments and provide an invaluable tool for future structural studies of how the SNARE complex binds to other components of the release machinery.

Original languageEnglish (US)
Pages (from-to)281-293
Number of pages13
JournalJournal of biomolecular NMR
Volume66
Issue number4
DOIs
StatePublished - Dec 1 2016

Keywords

  • Membrane fusion
  • Methyl assignment
  • Neurotransmitter release
  • Pseudocontact shifts
  • SNAREs

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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