Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
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 75:409–418
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 362:318–324
Rizo J, Rosenmund C (2008) Synaptic vesicle fusion. Nat Struct Mol Biol 15:665–674
Sudhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323:474–477
Jahn R, Fasshauer D (2012) Molecular machines governing exocytosis of synaptic vesicles. Nature 490:201–207
Sudhof TC (2012) The presynaptic active zone. Neuron 75:11–25
Sørensen JB (2009) Conflicting views on the membrane fusion machinery and the fusion pore. Annu Rev Cell Dev Biol 25:513–537
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 111:10317–10322
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 395:347–353
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 95:15781–15786
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 14:890–896
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 18:941–946
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. https://doi.org/10.1073/pnas.1518935113
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 136:3456–3464
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 188:401–413
Sudhof TC (1995) The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375:645–653
Pyle JL, Kavalali ET, Piedras-Renteria ES, Tsien RW (2000) Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28:221–231
Dittman JS, Regehr WG (1998) Calcium dependence and recovery kinetics of presynaptic depression at the climbing fiber to Purkinje cell synapse. J Neurosci 18:6147–6162
Crowley JJ, Carter AG, Regehr WG (2007) Fast vesicle replenishment and rapid recovery from desensitization at a single synaptic release site. J Neurosci 27:5448–5460
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 97:883–888
Hua SY, Charlton MP (1999) Activity-dependent changes in partial VAMP complexes during neurotransmitter release. Nat Neurosci 2:1078–1083
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 99:713–722
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 25:955–966
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 113:10536–10541
Pierce MM, Raman CS, Nall BT (1999) Isothermal titration calorimetry of protein-protein interactions. Methods 19:213–221
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 481:45–50
Weber G (1953) Rotational Brownian motion and polarization of the fluorescence of solutions. Adv Protein Chem 8:415–459
Albrecht AC (1961) Polarizations and assignments of transitions - method of photoselection. J Mol Spectrosc 6:84–108
Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New York
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 36:1788–1802
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 18:927–933
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 88:749–761
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 87:1744–1748
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 150:76–85
Krohn RI (2011) The colorimetric detection and quantitation of total protein. Curr Protoc Cell Biol Appendix 3:3H
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 72:248–254
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Li, F., Pincet, F. (2019). SNAREpin Assembly: Kinetic and Thermodynamic Approaches. 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_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8760-3_5
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8759-7
Online ISBN: 978-1-4939-8760-3
eBook Packages: Springer Protocols