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.
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Gerber SH, Sudhof TC (2002) Molecular determinants of regulated exocytosis. Diabetes 51(Suppl 1):S3–S11
Burgess TL, Kelly RB (1987) Constitutive and regulated secretion of proteins. Annu Rev Cell Biol 3:243–293
Sudhof TC (2013) Neurotransmitter release: the last millisecond in the life of a synaptic vesicle. Neuron 80:675–690
Sudhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323:474–477
Pang ZP, Sudhof TC (2010) Cell biology of Ca2+−triggered exocytosis. Curr Opin Cell Biol 22:496–505
Sudhof TC (2012) Calcium control of neurotransmitter release. Cold Spring Harb Perspect Biol 4:a011353
Krishnakumar SS, Kummel D, Jones SJ, Radoff DT, Reinisch KM, Rothman JE (2013) Conformational dynamics of calcium-triggered activation of fusion by Synaptotagmin-1. Biophys J 105:2507–2516
Ritchie TK, Grinkova YV, Bayburt TH, Denisov IG, Zolnerciks JK, Atkins WM, Sligar SG (2009) Chapter 11: Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol 464:211–231
Shi L, Howan K, Shen QT, Wang YJ, Rothman JE, Pincet F (2013) Preparation and characterization of SNARE-containing nanodiscs and direct study of cargo release through fusion pores. Nat Protoc 8:935–948
Shi L, Shen QT, Kiel A, Wang J, Wang HW, Melia TJ, Rothman JE, Pincet F (2012) SNARE proteins: one to fuse and three to keep the nascent fusion pore open. Science 335:1355–1359
Hui E, Gaffaney JD, Wang Z, Johnson CP, Evans CS, Chapman ER (2011) Mechanism and function of Synaptotagmin-1-mediated membrane apposition. Nat Struct Mol Biol 18:813–821
Bello OD, Auclair SM, Rothman JE, Krishnakumar SS (2016) Using ApoE Nanolipoprotein particles to analyze SNARE-induced fusion pores. Langmuir 32:3015–3023
Mahal LK, Sequeira SM, Gureasko JM, Sollner TH (2002) Calcium-independent stimulation of membrane fusion and SNAREpin formation by Synaptotagmin-1 I. J Cell Biol 158:273–282
Goldmann WH, Guttenberg Z, Ezzell RM, Isenberg G (1998) The study of fast reaction stopped flow method. In: Isenberg G (ed) Modern optics, electronics and high precision techniques in cell biology. Principles and practice. Springer, Berlin, Heidelberg
Hargrove MS (2005) Ligand binding with stopped-flow rapid mixing. In: Ulrich NG (ed) Protein-ligand interactions, Methods in molecular biology, vol 305. Humana Press, New York
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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Stroeva, E., Krishnakumar, S.S. (2019). Using Nanodiscs to Probe Ca2+-Dependent Membrane Interaction of Synaptotagmin-1. 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_14
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DOI: https://doi.org/10.1007/978-1-4939-8760-3_14
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