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SNAREs pp 115-144 | Cite as

Studying Munc18:Syntaxin Interactions Using Small-Angle Scattering

  • Andrew E. Whitten
  • Russell J. Jarrott
  • Shu-Hong Hu
  • Anthony P. Duff
  • Gordon J. King
  • Jennifer L. Martin
  • Michelle P. Christie
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

The interaction between the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (Sx) and regulatory partner Sec/Munc18 (SM) protein is a critical step in vesicle fusion. The exact role played by SM proteins, whether positive or negative, has been the topic of much debate. High-resolution structures of the SM:Sx complex have shown that SM proteins can bind syntaxin in a closed fusion incompetent state. However, in vitro and in vivo experiments also point to a positive regulatory role for SM proteins that is inconsistent with binding syntaxin in a closed conformation. Here we present protocols we used for the expression and purification of the SM proteins Munc18a and Munc18c and syntaxins 1 and 4 along with procedures used for small-angle X-ray and neutron scattering that showed that syntaxins can bind in an open conformation to SM proteins. We also describe methods for chemical cross-linking experiments and detail how this information can be combined with scattering data to obtain low-resolution structural models for SM:Sx protein complexes.

Key words

Small-angle X-ray scattering Small-angle neutron scattering Neutron contrast variation Munc18:syntaxin Protein complexes Cross-linking 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Andrew E. Whitten
    • 1
  • Russell J. Jarrott
    • 2
  • Shu-Hong Hu
    • 2
  • Anthony P. Duff
    • 1
  • Gordon J. King
    • 3
  • Jennifer L. Martin
    • 2
  • Michelle P. Christie
    • 4
  1. 1.Australian Nuclear Science and Technology OrganisationLucas HeightsNSWAustralia
  2. 2.Griffith Institute for Drug DiscoveryGriffith UniversityNathanAustralia
  3. 3.Centre for Microscopy and MicroanalysisThe University of QueenslandSt LuciaAustralia
  4. 4.Bio21 Molecular Science and Biotechnology InstituteThe University of MelbourneParkvilleAustralia

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