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

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• 作者：Yun-Zu Pan ; Bradley Quade ; Kyle D. Brewer ; Monika Szabo…
• 关键词：Methyl assignment ; Pseudocontact shifts ; SNAREs ; Membrane fusion ; Neurotransmitter release
• 刊名：Journal of Biomolecular NMR
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：66
• 期：4
• 页码：281-293
• 全文大小：1170KB
• 刊物类别：Physics and Astronomy
• 刊物主题：Biological and Medical Physics, Biophysics; Biochemistry, general; Spectroscopy/Spectrometry;
• 出版者：Springer Netherlands
• ISSN：1573-5001
• 卷排序：66

文摘

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.