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SNAREs pp 221-236 | Cite as

Using Nanodiscs to Probe Ca2+-Dependent Membrane Interaction of Synaptotagmin-1

  • Ekaterina Stroeva
  • Shyam S. Krishnakumar
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

In this chapter, we introduce a nanodisc-based experimental platform to study Ca2+-triggered membrane interaction of synaptotagmin-1. We describe and discuss in detail how to assemble this soluble mimetic of the docked vesicle–plasma membrane junction, with fluorescently labeled synaptotagmin-1 bound to trans SNAREpins assembled between nanodiscs and present the stopped-flow rapid mixing method used to monitor the conformational dynamics of Ca2+-activation process on a millisecond timescale.

Key words

Nanodiscs Soluble membrane-mimetic Synaptotagmin-1 SNARE proteins Calcium activation Membrane interaction Fluorescent labeling Rapid mixing stopped-flow 

Notes

Acknowledgments

This work was supported by National Institute of Health (NIH) grant DK027044. We thank Jeff Coleman for critical inputs and suggestions.

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Copyright information

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

Authors and Affiliations

  1. 1.Department of Cell BiologyYale University School of MedicineNew HavenUSA
  2. 2.Department of Clinical and Experimental Epilepsy, Institute of NeurologyUniversity College LondonLondonUK

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