Abstract
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.
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References
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. https://doi.org/10.1038/362318a0
Weber T et al (1998) SNAREpins: minimal machinery for membrane fusion. Cell 92:759–772
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. https://doi.org/10.1038/1799
Stein A, Weber G, Wahl MC, Jahn R (2009) Helical extension of the neuronal SNARE complex into the membrane. Nature 460:525–528. https://doi.org/10.1038/nature08156
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. https://doi.org/10.1038/26412
Südhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323:474–477. https://doi.org/10.1126/science.1161748
Lou XC, Shin YK (2016) SNARE zippering. Biosci Rep 36:7. https://doi.org/10.1042/bsr20160004
An J, Almers W (2004) Tracking SNARE complex formation in live endocrine cells. Science 306:1042–1046. https://doi.org/10.1126/science.1102559
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. https://doi.org/10.1016/j.str.2017.09.004
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. https://doi.org/10.1038/sj.emboj.7601003
Gao Y et al (2012) Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science 337:1340–1343. https://doi.org/10.1126/science.1224492
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. https://doi.org/10.1038/ncomms2692
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. https://doi.org/10.1042/bj20131668
Hubbell WL, Gross A, Langen R, Lietzow MA (1998) Recent advances in site-directed spin labeling of proteins. Curr Opin Struct Biol 8:649–656
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. https://doi.org/10.1016/j.str.2011.10.009
Rabenstein MD, Shin YK (1995) Determination of the distance between two spin labels attached to a macromolecule. Proc Natl Acad Sci U S A 92:8239–8243
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. https://doi.org/10.1038/86174
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. https://doi.org/10.1074/jbc.M201200200
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. https://doi.org/10.1073/pnas.0813138106
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 91:1667–1671
Chen Y, Xu Y, Zhang F, Shin YK (2004) Constitutive versus regulated SNARE assembly: a structural basis. EMBO J 23:681–689. https://doi.org/10.1038/sj.emboj.7600083
Kim CS, Kweon DH, Shin YK (2002) Membrane topologies of neuronal SNARE folding intermediates. Biochemistry 41:10928–10933
Kweon DH, Kim CS, Shin YK (2002) The membrane-dipped neuronal SNARE complex: a site-directed spin labeling electron paramagnetic resonance study. Biochemistry 41:9264–9268
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. https://doi.org/10.1074/jbc.M211123200
Xu Y, Zhang F, Su Z, McNew JA, Shin YK (2005) Hemifusion in SNARE-mediated membrane fusion. Nat Struct Mol Biol 12:417–422. https://doi.org/10.1038/nsmb921
Columbus L, Hubbell WL (2002) A new spin on protein dynamics. Trends Biochem Sci 27:288–295
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. https://doi.org/10.1074/jbc.M500736200
QIAprep® Miniprep Handbook (2012) Qiagen. http://www.qiagen.com/us/resources/download.aspx?id=89bfa021-7310-4c0f-90e0-6a9c84f66cee〈=en
Bio-Rad: Handcasting Polyacrylamide Gels. Bio-Rad. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6201.pdf
Bio-Rad: RC DC Protein Assay. Bio - Rad. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110107A.pdf
QuikChange II Site-Directed Mutagenesis Kit: Instruction Manual. Agilent. https://www.genomics.agilent.com/files/Manual/200523.pdf
Kibbe WA (2007) OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res 35:W43–W46. https://doi.org/10.1093/nar/gkm234
Addgene: Bacterial Transformation. (2017). http://www.addgene.org/protocols/bacterial-transformation/
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Khounlo, R., Hawk, B.J.D., Shin, YK. (2019). EPR Lineshape Analysis to Investigate the SNARE Folding Intermediates. 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_3
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DOI: https://doi.org/10.1007/978-1-4939-8760-3_3
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