SNAREs: Methods and Protocols

Description

This book details multiple ways that soluble N-ethylmaleimide-sensitive factor attachment protein receptors( SNAREs) and their function are examined in the laboratory. The methods described in each chapter are described in detail so that novice as well as experienced researchers can explore the mechanisms of SNARE-mediated membrane fusion. Chapters guide readers through an overview of the basic properties of SNAREs, distribution and interaction with regulators of membrane fusion, activation of SNAREs in the priming stage by NSF/Sec18 and a-SNAP/Sec17, examining the structures and interactions of SNAREs, measuring the interactions of SNAREs, interactions of SNAREs, and post-translational modifications of SNAREs and how they affect function. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, SNAREs: Methods and Protocols aims to be a valuable tool for all investigators interested in the field. Front Matter ....Pages i-xiv Front Matter ....Pages 1-1 Molecular Dynamics Simulations of the SNARE Complex (Maria Bykhovskaia)....Pages 3-13 Mesoscale Computational Modeling of Protein-Membrane Interactions Based on Continuum Mean-Field Theory (George Khelashvili)....Pages 15-31 EPR Lineshape Analysis to Investigate the SNARE Folding Intermediates (Ryan Khounlo, Brenden J. D. Hawk, Yeon-Kyun Shin)....Pages 33-51 Dynamic Light Scattering Analysis to Dissect Intermediates of SNARE-Mediated Membrane Fusion (Byoungjae Kong, Yoosoo Yang, Dae-Hyuk Kweon)....Pages 53-69 SNAREpin Assembly: Kinetic and Thermodynamic Approaches (Feng Li, Frederic Pincet)....Pages 71-93 Single-Molecule Optical Tweezers Study of Regulated SNARE Assembly (Lu Ma, Junyi Jiao, Yongli Zhang)....Pages 95-114 Studying Munc18:Syntaxin Interactions Using Small-Angle Scattering (Andrew E. Whitten, Russell J. Jarrott, Shu-Hong Hu, Anthony P. Duff, Gordon J. King, Jennifer L. Martin et al.)....Pages 115-144 Using Force Spectroscopy to Probe Coiled-Coil Assembly and Membrane Fusion (Hannes Witt, Andreas Janshoff)....Pages 145-159 Front Matter ....Pages 161-161 SNAP-25 S-Guanylation and SNARE Complex Formation (Yusuke Kishimoto, Takaaki Akaike, Hideshi Ihara)....Pages 163-173 Analysis of the Role of Sec3 in SNARE Assembly and Membrane Fusion (Kunrong Mei, Peng Yue, Wei Guo)....Pages 175-189 Use of Microscale Thermophoresis (MST) to Measure Binding Affinities of Components of the Fusion Machinery (Robert P. Sparks, Rutilio Fratti)....Pages 191-198 Use of Surface Plasmon Resonance (SPR) to Determine Binding Affinities and Kinetic Parameters Between Components Important in Fusion Machinery (Robert P. Sparks, Jermaine L. Jenkins, Rutilio Fratti)....Pages 199-210 Determination of Sec18-Lipid Interactions by Liposome-Binding Assay (Matthew L. Starr, Rutilio Fratti)....Pages 211-220 Using Nanodiscs to Probe Ca2+-Dependent Membrane Interaction of Synaptotagmin-1 (Ekaterina Stroeva, Shyam S. Krishnakumar)....Pages 221-236 Functional Reconstitution of Intracellular Vesicle Fusion Using Purified SNAREs and Sec1/Munc18 (SM) Proteins (Haijia Yu, Lauren Crisman, Michael H. B. Stowell, Jingshi Shen)....Pages 237-249 Front Matter ....Pages 251-251 Assay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles (Massimo D’Agostino, Andreas Mayer)....Pages 253-262 A Nanodisc-Cell Fusion Assay with Single-Pore Sensitivity and Sub-millisecond Time Resolution (Natasha R. Dudzinski, Zhenyong Wu, Erdem Karatekin)....Pages 263-275 An In Vitro Assay of Trans-SNARE Complex Formation During Yeast Vacuole Fusion Using Epitope Tag-Free SNAREs (Youngsoo Jun)....Pages 277-288 A Cell-Free Content Mixing Assay for SNARE-Mediated Multivesicular Body-Vacuole Membrane Fusion (Mahmoud Abdul Karim, Dieter Ronny Samyn, Christopher Leonard Brett)....Pages 289-301 Reconstituted Proteoliposome Fusion Mediated by Yeast SNARE-Family Proteins (Joji Mima)....Pages 303-322 Real-Time Fluorescence Detection of Calcium Efflux During Vacuolar Membrane Fusion (Gregory E. Miner, Rutilio Fratti)....Pages 323-331 Front Matter ....Pages 333-333 Single-Molecule Fluorescence Measurement of SNARE-Mediated Vesicle Fusion (Yachong Hu, Zhiqi Tian, Jiajie Diao)....Pages 335-344 Quantifying Intramolecular Protein Conformational Dynamics Under Lipid Interaction Using smFRET and FCCS (Pei Li, Yawei Dai, Markus Seeger, Yan-Wen Tan)....Pages 345-359 Visualization of SNARE-Mediated Organelle Membrane Hemifusion by Electron Microscopy (Sevan Mattie, Tom Kazmirchuk, Jeannie Mui, Hojatollah Vali, Christopher Leonard Brett)....Pages 361-377 Studies of the Secretory Machinery Dynamics by Total Internal Reflection Fluorescence Microscopy in Bovine Adrenal Chromaffin Cells (José Villanueva, Yolanda Gimenez-Molina, Luis M. Gutiérrez)....Pages 379-389 Imaging SNAP-29 in Drosophila (Hao Xu, Bryan Stewart)....Pages 391-401 Back Matter ....Pages 403-407