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Studying Munc18:Syntaxin Interactions Using Small-Angle Scattering

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SNAREs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1860))

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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.

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Author information

Authors and Affiliations

  1. Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW, Australia

    Andrew E. Whitten & Anthony P. Duff

  2. Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD, Australia

    Russell J. Jarrott, Shu-Hong Hu & Jennifer L. Martin

  3. Centre for Microscopy and Microanalysis, The University of Queensland, St Lucia, QLD, Australia

    Gordon J. King

  4. Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia

    Michelle P. Christie

Authors
  1. Andrew E. Whitten
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  2. Russell J. Jarrott
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  3. Shu-Hong Hu
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  4. Anthony P. Duff
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  5. Gordon J. King
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  6. Jennifer L. Martin
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  7. Michelle P. Christie
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Corresponding author

Correspondence to Michelle P. Christie .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Rutilio Fratti

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Whitten, A.E. et al. (2019). Studying Munc18:Syntaxin Interactions Using Small-Angle Scattering. In: Fratti, R. (eds) SNAREs. Methods in Molecular Biology, vol 1860. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8760-3_7

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  • DOI: https://doi.org/10.1007/978-1-4939-8760-3_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8759-7

  • Online ISBN: 978-1-4939-8760-3

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