Main 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.

Contents:

Part I: Biophysics and Computational Analysis

1. Molecular Dynamics Simulations of the SNARE Complex

Maria Bykhovskaia

2. Mesoscale Computational Modelling of Protein-membrane Interactions based on Continuum Mean-field Theory

George Khelashvili

3. EPR Lineshape Analysis to IOnvestigate the SNARE Folding Intermediates

Ryan Khounlo, Brenden J.D. Hawk, and Yeon-Kyun Shin

4. Dynamic Light Scattering Analysis to Dissect Intermediates of SNARE-Mediated Membrane Fusion

Byoungjae Kong, Yoosoo Yang, and Dae-Hyuk Kweon

5. Kinetic and Thermodynamic Quantification of Protein Complex Assembly: The Example of SNAREpins

Feng Li and Frederic Pincet

6. Single-molecule Optical Tweezers Study of Regulated SNARE Assembly

Lu Ma, Junyi Jiao, and Yongli Zhang

7. 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, and Michelle P. Christie

8. Using Force Spectroscopy to Probe Coiled-Coil Assembly and Membrane Fusion

Hannes Witt and Andreas Janshoff

Part II: Biochemistry

9. SNAP25 S-Guanylation and SNARE Complex Formation

Yusuke Kishimoto, Takaaki Akaike, and Hideshi Ihara10. Analysis of the Role of Sec3 in SNARE Assembly and Membrane Fusion

Kunrong Mei, Peng Yue, and Wei Guo11. Use of Microscale Thermophoresis (MST) to Measure Binding Affinities of Components of the Fusion Machinery

Robert P. Sparks and Rutilio A. Fratti

12. Use of Surface Plasmon Resonance (SPR) to Determine Binding Affinities and Kinetic Parameters Between Components Important in Fusion Machinery

Robert P. Sparks, Jermaine Jenkins, and Rutilio A. Fratti

13. Determination of Sec18-Lipid Interactions by Liposome Binding Assay

Matthew L. Starr and Rutilio A. Fratti

14. Using Nanodiscs to Probe Ca2+-dependent Membrane Interaction of Synaptotagmin-1

Ekaterina Stroeva and Shyam S. Krishnakumar

15. Functional reconstitution of Intracellular Vesicle Fusion using Purified SNAREs and Sec1/Munc18 (SM) Proteins

Haijia Yu, Lauren Crisman, Michael H. B. Stowell, and Jingshi Shen

Part III: Functional Assays

16. Assay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles

Massimo D'Agostino and Andreas Mayer17. A Nanodisc-cell Fusion Assay with Single Pore Sensitivity and Sub-millisecond Time Resolution

Natasha R. Dudzinski, Zhenyong Wu, and Erdem Karatekin

18. An In Vitro Assay of trans-SNARE Complex Formation during Yeast Vacuole Fusion using Epitope Tag-free SNAREs

Youngsoo Jun

19. A Cell-Free Content Mixing Assay for SNARE-Mediated Multivesicular Body-Vacuole Membrane Fusion

Mahmoud Abdul Karim, Dieter Ronny Samyn, and Christopher Leonard Brett

20. Reconstituted Proteoliposome Fusion Mediated by yeast SNARE-family Proteins

Joji Mima

21. Real-Time Fluorescence Detection of Calcium Efflux during Vacuolar Membrane Fusion

Gregory E. Miner and Rutilio A. Fratti

Part IV: Microscopy

22. Single Molecule Fluorescence Measurement of SNARE-mediated Vesicle Fusion

Yachong Hu, Zhiqi Tian, and Jiajie Diao

23. Quantifying Intramolecular Protein Conformational Dynamics under Lipid Interaction using smFRET and FCCS

Pei Li, Yawei Dai, Markus Seeger, and Yan-Wen Tan

24. Visualization of SNARE-Mediated Organelle Membrane Hemifusion By Electron Microscopy

Sevan Mattie, Tom Kazmirchuk, Jeannie Mui, Hojatollah Vali, and Christopher Leonard Brett

25. Studies of the Secretory Machinery Dynamics by Total Internal Reflection Fluorescence Microscopy in Bovine Adrenal Chromaffin Cells

Jose Villanueva, Yolanda Gimenez-Molina, and Luis M. Gutierrez

26. Imaging SNAP-29 in Drosophila

Hao Xu and Bryan Stewart