SNAREs pp 71-93 | Cite as

SNAREpin Assembly: Kinetic and Thermodynamic Approaches

  • Feng LiEmail author
  • Frederic PincetEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)


Proteins constantly interact and often form molecular complexes. The dynamics of most biological processes strongly rely on the kinetics and thermodynamics of assembly and disassembly of these complexes. Consequently an accurate characterization of these kinetics and thermodynamics that underlie them provides key information to better understand these processes. Here, we present two efficient techniques to quantify the assembly and disassembly of protein complexes: isothermal titration calorimetry and fluorescence anisotropy. As an example we focus on the formation of SNAREpins and also present how to prepare SNARE proteins to use in these experimental setups. We then show how to use these techniques to determine the critical factors that activate assembly kinetics.

Key words

Isothermal titration calorimetry Fluorescence polarization Fluorescence anisotropy SNARE VAMP2 Syntaxin SNAP25 Protein assembly Protein interaction 


  1. 1.
    Sollner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE (1993) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75:409–418CrossRefGoogle Scholar
  2. 2.
    Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman JE (1993) SNAP receptors implicated in vesicle targeting and fusion. Nature 362:318–324CrossRefGoogle Scholar
  3. 3.
    Rizo J, Rosenmund C (2008) Synaptic vesicle fusion. Nat Struct Mol Biol 15:665–674CrossRefGoogle Scholar
  4. 4.
    Sudhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323:474–477CrossRefGoogle Scholar
  5. 5.
    Jahn R, Fasshauer D (2012) Molecular machines governing exocytosis of synaptic vesicles. Nature 490:201–207CrossRefGoogle Scholar
  6. 6.
    Sudhof TC (2012) The presynaptic active zone. Neuron 75:11–25CrossRefGoogle Scholar
  7. 7.
    Sørensen JB (2009) Conflicting views on the membrane fusion machinery and the fusion pore. Annu Rev Cell Dev Biol 25:513–537CrossRefGoogle Scholar
  8. 8.
    Cho RW, Kummel D, Li F, Baguley SW, Coleman J, Rothman JE, Littleton JT (2014) Genetic analysis of the complexin trans-clamping model for cross-linking SNARE complexes in vivo. Proc Natl Acad Sci 111:10317–10322CrossRefGoogle Scholar
  9. 9.
    Sutton RB, Fasshauer D, Jahn R, Brunger AT (1998) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 angstrom resolution. Nature 395:347–353CrossRefGoogle Scholar
  10. 10.
    Fasshauer D, Sutton RB, Brunger AT, Jahn R (1998) Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc Natl Acad Sci U S A 95:15781–15786CrossRefGoogle Scholar
  11. 11.
    Li F, Pincet F, Perez E, Eng WS, Melia TJ, Rothman JE, Tareste D (2007) Energetics and dynamics of SNAREpin folding across lipid bilayers. Nat Struct Mol Biol 14:890–896CrossRefGoogle Scholar
  12. 12.
    Li F, Pincet F, Perez E, Giraudo CG, Tareste D, Rothman JE (2011) Complexin activates and clamps SNAREpins by a common mechanism involving an intermediate energetic state. Nat Struct Mol Biol 18:941–946CrossRefGoogle Scholar
  13. 13.
    Wang YJ, Li F, Rodriguez N, Lafosse X, Gourier C, Perez E, Pincet F (2016) Snapshot of sequential SNARE assembling states between membranes shows that N-terminal transient assembly initializes fusion. Proc Natl Acad Sci U S A. Scholar
  14. 14.
    Li F, Kummel D, Coleman J, Reinisch KM, Rothman JE, Pincet F (2014) A half-zippered SNARE complex represents a functional intermediate in membrane fusion. J Am Chem Soc 136:3456–3464CrossRefGoogle Scholar
  15. 15.
    Walter AM, Wiederhold K, Bruns D, Fasshauer D, Sorensen JB (2010) Synaptobrevin N-terminally bound to syntaxin-SNAP-25 defines the primed vesicle state in regulated exocytosis. J Cell Biol 188:401–413CrossRefGoogle Scholar
  16. 16.
    Sudhof TC (1995) The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375:645–653CrossRefGoogle Scholar
  17. 17.
    Pyle JL, Kavalali ET, Piedras-Renteria ES, Tsien RW (2000) Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28:221–231CrossRefGoogle Scholar
  18. 18.
    Dittman JS, Regehr WG (1998) Calcium dependence and recovery kinetics of presynaptic depression at the climbing fiber to Purkinje cell synapse. J Neurosci 18:6147–6162CrossRefGoogle Scholar
  19. 19.
    Crowley JJ, Carter AG, Regehr WG (2007) Fast vesicle replenishment and rapid recovery from desensitization at a single synaptic release site. J Neurosci 27:5448–5460CrossRefGoogle Scholar
  20. 20.
    Moser T, Beutner D (2000) Kinetics of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse of the mouse. Proc Natl Acad Sci U S A 97:883–888CrossRefGoogle Scholar
  21. 21.
    Hua SY, Charlton MP (1999) Activity-dependent changes in partial VAMP complexes during neurotransmitter release. Nat Neurosci 2:1078–1083CrossRefGoogle Scholar
  22. 22.
    Xu T, Rammner B, Margittai M, Artalejo AR, Neher E, Jahn R (1999) Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis. Cell 99:713–722CrossRefGoogle Scholar
  23. 23.
    Sorensen JB, Wiederhold K, Muller EM, Milosevic I, Nagy G, de Groot BL, Grubmuller H, Fasshauer D (2006) Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles. EMBO J 25:955–966CrossRefGoogle Scholar
  24. 24.
    Li F, Tiwari N, Rothman JE, Pincet F (2016) Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion. Proc Natl Acad Sci U S A 113:10536–10541CrossRefGoogle Scholar
  25. 25.
    Pierce MM, Raman CS, Nall BT (1999) Isothermal titration calorimetry of protein-protein interactions. Methods 19:213–221CrossRefGoogle Scholar
  26. 26.
    Morrison EA, DeKoster GT, Dutta S, Vafabakhsh R, Clarkson MW, Bahl A, Kern D, Ha T, Henzler-Wildman KA (2011) Antiparallel EmrE exports drugs by exchanging between asymmetric structures. Nature 481:45–50CrossRefGoogle Scholar
  27. 27.
    Weber G (1953) Rotational Brownian motion and polarization of the fluorescence of solutions. Adv Protein Chem 8:415–459CrossRefGoogle Scholar
  28. 28.
    Albrecht AC (1961) Polarizations and assignments of transitions - method of photoselection. J Mol Spectrosc 6:84–108CrossRefGoogle Scholar
  29. 29.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New YorkCrossRefGoogle Scholar
  30. 30.
    Jakhanwal S, Lee CT, Urlaub H, Jahn R (2017) An activated Q-SNARE/SM protein complex as a possible intermediate in SNARE assembly. EMBO J 36:1788–1802CrossRefGoogle Scholar
  31. 31.
    Kummel D, Krishnakumar SS, Radoff DT, Li F, Giraudo CG, Pincet F, Rothman JE, Reinisch KM (2011) Complexin cross-links prefusion SNAREs into a zigzag array. Nat Struct Mol Biol 18:927–933CrossRefGoogle Scholar
  32. 32.
    Cho RW, Buhl LK, Volfson D, Tran A, Li F, Akbergenova Y, Littleton JT (2015) Phosphorylation of complexin by PKA regulates activity-dependent spontaneous neurotransmitter release and structural synaptic plasticity. Neuron 88:749–761CrossRefGoogle Scholar
  33. 33.
    Heyduk T, Lee JC (1990) Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction. Proc Natl Acad Sci U S A 87:1744–1748CrossRefGoogle Scholar
  34. 34.
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefGoogle Scholar
  35. 35.
    Krohn RI (2011) The colorimetric detection and quantitation of total protein. Curr Protoc Cell Biol Appendix 3:3HPubMedGoogle Scholar
  36. 36.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar

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

Authors and Affiliations

  1. 1.Department of Cell Biology and Nanobiology Institute, School of MedicineYale UniversityNew HavenUSA
  2. 2.Laboratoire de Physique Statistique, Ecole Normale SupérieurePSL Research University, Université Paris Diderot Sorbonne Paris Cité, Sorbonne Universités UPMC Univ Paris 06, CNRSParisFrance

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