Description / Table of Contents:
Molecular Dynamics Simulations of the SNARE Complex -- Mesoscale Computational Modelling of Protein-membrane Interactions based on Continuum Mean-field Theory -- EPR Lineshape Analysis to IOnvestigate the SNARE Folding Intermediates -- Dynamic Light Scattering Analysis to Dissect Intermediates of SNARE-Mediated Membrane Fusion -- Kinetic and Thermodynamic Quantification of Protein Complex Assembly: The Example of SNAREpins -- Single-molecule Optical Tweezers Study of Regulated SNARE Assembly -- Studying Munc18:Syntaxin Interactions using Small-angle Scattering -- Using Force Spectroscopy to Probe Coiled-Coil Assembly and Membrane Fusion -- SNAP25 S-Guanylation and SNARE Complex Formation -- Analysis of the Role of Sec3 in SNARE Assembly and Membrane Fusion -- Use of Microscale Thermophoresis (MST) to Measure Binding Affinities of Components of the Fusion Machinery -- Use of Surface Plasmon Resonance (SPR) to Determine Binding Affinities and Kinetic Parameters Between Components Important in Fusion Machinery -- Determination of Sec18-Lipid Interactions by Liposome Binding Assay -- Using Nanodiscs to Probe Ca2+-dependent Membrane Interaction of Synaptotagmin-1 -- Functional reconstitution of Intracellular Vesicle Fusion using Purified SNAREs and Sec1/Munc18 (SM) Proteins -- Assay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles -- A nanodisc-cell Fusion Assay with Single Pore Sensitivity and Sub-millisecond Time Resolution -- An In Vitro Assay of trans-SNARE Complex Formation during Yeast Vacuole Fusion using Epitope Tag-free SNAREs -- A Cell-Free Content Mixing Assay for SNARE-Mediated Multivesicular Body-Vacuole Membrane Fusion -- Reconstituted Proteoliposome Fusion Mediated by yeast SNARE-family Proteins -- Real-Time Fluorescence Detection of Calcium Efflux during Vacuolar Membrane Fusion -- Single Molecule Fluorescence Measurement of SNARE-mediated Vesicle Fusion -- Quantifying Intramolecular Protein Conformational Dynamics under Lipid Interaction using smFRET and FCCS -- Visualization of SNARE-Mediated Organelle Membrane Hemifusion By Electron Microscopy -- Studies of the Secretory Machinery Dynamics by Total Internal Reflection Fluorescence Microscopy in Bovine Adrenal Chromaffin Cells -- Imaging SNAP-29 in Drosophila
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
XIV, 407 p. 74 illus., 66 illus. in color. : online resource.