SNAREs pp 33-51 | Cite as

EPR Lineshape Analysis to Investigate the SNARE Folding Intermediates

  • Ryan Khounlo
  • Brenden J. D. Hawk
  • Yeon-Kyun ShinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)


SNARE complex formation, which is believed to drive intracellular membrane fusion, transits through multiple conformational states along the membrane fusion pathway. The SNARE intermediates are biologically important because they serve as targets for fusion regulators and clostridial neurotoxins. Spin-labeling EPR has contributed significantly to the understanding of the structures and the dynamics of SNARE intermediates. In particular, the EPR lineshape analysis, which is highly sensitive to protein conformational changes such as the local coil-to-helix transition, has revealed the sequential compacting steps leading to formation of the highly stable four-helix bundle.

Key words

SNAREs Nanodiscs Membrane fusion EPR Spin labels Exocytosis Neurotransmitter release 


  1. 1.
    Söllner 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. Scholar
  2. 2.
    Weber T et al (1998) SNAREpins: minimal machinery for membrane fusion. Cell 92:759–772CrossRefGoogle Scholar
  3. 3.
    Poirier MA, Xiao W, Macosko JC, Chan C, Shin YK, Bennett MK (1998) The synaptic SNARE complex is a parallel four-stranded helical bundle. Nat Struct Biol 5:765–769. Scholar
  4. 4.
    Stein A, Weber G, Wahl MC, Jahn R (2009) Helical extension of the neuronal SNARE complex into the membrane. Nature 460:525–528. Scholar
  5. 5.
    Sutton RB, Fasshauer D, Jahn R, Brunger AT (1998) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395:347–353. Scholar
  6. 6.
    Südhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323:474–477. Scholar
  7. 7.
    Lou XC, Shin YK (2016) SNARE zippering. Biosci Rep 36:7. Scholar
  8. 8.
    An J, Almers W (2004) Tracking SNARE complex formation in live endocrine cells. Science 306:1042–1046. Scholar
  9. 9.
    Khounlo R, Kim J, Yin L, Shin YK (2017) Botulinum toxins A and E inflict dynamic destabilization on t-SNARE to impair SNARE assembly and membrane fusion. Structure 25:1679–1686.e1675. Scholar
  10. 10.
    Sørensen JB et al (2006) Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles. EMBO J 25:955–966. Scholar
  11. 11.
    Gao Y et al (2012) Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science 337:1340–1343. Scholar
  12. 12.
    Min D, Kim K, Hyeon C, Cho YH, Shin YK, Yoon TY (2013) Mechanical unzipping and rezipping of a single SNARE complex reveals hysteresis as a force-generating mechanism. Nat Commun 4:1705. Scholar
  13. 13.
    Shin J, Lou X, Kweon DH, Shin YK (2014) Multiple conformations of a single SNAREpin between two nanodisc membranes reveal diverse pre-fusion states. Biochem J 459:95–102. Scholar
  14. 14.
    Hubbell WL, Gross A, Langen R, Lietzow MA (1998) Recent advances in site-directed spin labeling of proteins. Curr Opin Struct Biol 8:649–656CrossRefGoogle Scholar
  15. 15.
    Mchaourab HS, Steed PR, Kazmier K (2011) Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy. Structure 19:1549–1561. Scholar
  16. 16.
    Rabenstein MD, Shin YK (1995) Determination of the distance between two spin labels attached to a macromolecule. Proc Natl Acad Sci U S A 92:8239–8243CrossRefGoogle Scholar
  17. 17.
    Xiao W, Poirier MA, Bennett MK, Shin YK (2001) The neuronal t-SNARE complex is a parallel four-helix bundle. Nat Struct Biol 8:308–311. Scholar
  18. 18.
    Zhang F, Chen Y, Kweon DH, Kim CS, Shin YK (2002) The four-helix bundle of the neuronal target membrane SNARE complex is neither disordered in the middle nor uncoiled at the C-terminal region. J Biol Chem 277:24294–24298. Scholar
  19. 19.
    Tong J, Borbat PP, Freed JH, Shin YK (2009) A scissors mechanism for stimulation of SNARE-mediated lipid mixing by cholesterol. Proc Natl Acad Sci U S A 106:5141–5146. Scholar
  20. 20.
    Altenbach C, Greenhalgh DA, Khorana HG, Hubbell WL (1994) A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin. Proc Natl Acad Sci U S A 91:1667–1671CrossRefGoogle Scholar
  21. 21.
    Chen Y, Xu Y, Zhang F, Shin YK (2004) Constitutive versus regulated SNARE assembly: a structural basis. EMBO J 23:681–689. Scholar
  22. 22.
    Kim CS, Kweon DH, Shin YK (2002) Membrane topologies of neuronal SNARE folding intermediates. Biochemistry 41:10928–10933CrossRefGoogle Scholar
  23. 23.
    Kweon DH, Kim CS, Shin YK (2002) The membrane-dipped neuronal SNARE complex: a site-directed spin labeling electron paramagnetic resonance study. Biochemistry 41:9264–9268CrossRefGoogle Scholar
  24. 24.
    Kweon DH, Kim CS, Shin YK (2003) Insertion of the membrane-proximal region of the neuronal SNARE coiled coil into the membrane. J Biol Chem 278:12367–12373. Scholar
  25. 25.
    Xu Y, Zhang F, Su Z, McNew JA, Shin YK (2005) Hemifusion in SNARE-mediated membrane fusion. Nat Struct Mol Biol 12:417–422. Scholar
  26. 26.
    Columbus L, Hubbell WL (2002) A new spin on protein dynamics. Trends Biochem Sci 27:288–295CrossRefGoogle Scholar
  27. 27.
    Zhang Y, Su Z, Zhang F, Chen Y, Shin YK (2005) A partially zipped SNARE complex stabilized by the membrane. J Biol Chem 280:15595–15600. Scholar
  28. 28.
  29. 29.
    Bio-Rad: Handcasting Polyacrylamide Gels. Bio-Rad.
  30. 30.
  31. 31.
    QuikChange II Site-Directed Mutagenesis Kit: Instruction Manual. Agilent.
  32. 32.
    Kibbe WA (2007) OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res 35:W43–W46. Scholar
  33. 33.
    Addgene: Bacterial Transformation. (2017).

Copyright information

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

Authors and Affiliations

  • Ryan Khounlo
    • 1
  • Brenden J. D. Hawk
    • 1
  • Yeon-Kyun Shin
    • 1
    Email author
  1. 1.Roy J. Carver Department of Biochemistry, Biophysics and Molecular BiologyIowa State UniversityAmesUSA

Personalised recommendations