
Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods
Description
Key Features
- Continues the legacy of this premier serial with quality chapters authored by leaders in the field
- Covers research methods in single-molecule enzymology
- Contains sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, and nanopore and tethered particle motion
Readership
Table of Contents
Chapter One: How to Measure Load-Dependent Kinetics of Individual Motor Molecules Without a Force-Clamp
- Abstract
- 1 Introduction
- 2 HFS: Basic Concept
- 3 Experimental Setup
- 4 Sample Preparations: Proteins, Reagents, and Buffers
- 5 Experimental Protocols
- 6 Trap Calibration
- 7 HFS: Theory and Data Analysis
- 8 Results and Discussion
- 9 Conclusion and Outlook
- Acknowledgments
Chapter Two: Studying the Mechanochemistry of Processive Cytoskeletal Motors With an Optical Trap
- Abstract
- 1 Introduction
- 2 Experimental Setup and Troubleshooting
- 3 Experimental Protocols
- 4 Conclusion
- Acknowledgments
Chapter Three: Single-Molecule Optical-Trapping Techniques to Study Molecular Mechanisms of a Replisome
- Abstract
- 1 Introduction
- 2 Instrument Design, Experimental Configuration, and Sample Preparation
- 3 Molecular Mechanisms of Individual Proteins in the Replisome Revealed by Optical-Trapping Techniques
- 4 Single-Molecule Studies of the Response of a Replisome to DNA Damage
- 5 Data Analysis
- 6 Unique Features of the Bacteriophage T7 Replisome Revealed by Single-Molecule Optical-Trapping Techniques
- 7 Conclusions
- Acknowledgments
Chapter Four: Recent Advances in Biological Single-Molecule Applications of Optical Tweezers and Fluorescence Microscopy
- Abstract
- 1 Introduction
- 2 Instrumentation
- 3 Applications
- 4 Experimental Protocol
- 5 Conclusion
- Acknowledgments
Chapter Five: Direct Visualization of Helicase Dynamics Using Fluorescence Localization and Optical Trapping
- Abstract
- 1 Introduction
- 2 Materials
- 3 Methods
- Acknowledgments
Chapter Six: High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy
- Abstract
- 1 Introduction
- 2 Optical Trapping and Single-Molecule Fluorescence
- 3 Instrument Design
- 4 Instrument Alignment
- 5 Combined Optical Trap/smFRET Assay
- Acknowledgments
Chapter Seven: Integrating Optical Tweezers, DNA Tightropes, and Single-Molecule Fluorescence Imaging: Pitfalls and Traps
- Abstract
- 1 Introduction
- 2 Elongating Bundled DNA for Imaging
- 3 Integrating Laser Tweezers Into Biological Experiments
- 4 Controlling and Detecting the Nanoprobe
- 5 Applying the Nanoprobe to Biological Study Systems
- 6 Conclusions and Outlook
Chapter Eight: Single-Stranded DNA Curtains for Studying Homologous Recombination
- Abstract
- 1 Introduction
- 2 Methods
- 3 Applications
- 4 Data Collection and Analysis
- 5 Conclusion and Future Directions
- Acknowledgments
Chapter Nine: Inserting Extrahelical Structures into Long DNA Substrates for Single-Molecule Studies of DNA Mismatch Repair
- Abstract
- 1 Introduction
- 2 Materials
- 3 Methods
- 4 Notes
- Acknowledgments
Chapter Ten: Single-Molecule Insight Into Target Recognition by CRISPR–Cas Complexes
- Abstract
- 1 Introduction
- 2 Single-Molecule Magnetic Tweezers Experiments: Technical Aspects
- 3 Studying CRISPR–Cas Systems of Streptococcus thermophilus
- 4 Studying E. coli Cascade
- 5 Perspectives and Conclusion
- Acknowledgments
Chapter Eleven: Preparation of DNA Substrates and Functionalized Glass Surfaces for Correlative Nanomanipulation and Colocalization (NanoCOSM) of Single Molecules
- Abstract
- 1 Introduction
- 2 Combining Single-Molecule Nanomanipulation and Fluorescence
- 3 Designing DNA Substrates
- 4 Overview of Experimental System
- 5 Streptavidin-Derivatized PEGylated Glass Surfaces
- 6 Antidigoxigenin-Derivatized Polystyrene-Coated Glass Surfaces
- 7 Preparation of DNA
- 8 Preparation of Antidigoxigenin-Functionalized Magnetic Beads
- 9 Assembly of Bead-DNA System and Loading of Reaction Chamber
- 10 General Considerations for Buffer Preparation
- 11 Conclusions and Perspectives
- Acknowledgments
Chapter Twelve: Measuring Force-Induced Dissociation Kinetics of Protein Complexes Using Single-Molecule Atomic Force Microscopy
- Abstract
- 1 Introduction
- 2 Models for the Mechanical Response of Receptor–Ligand Bonds
- 3 Measuring in vitro Force-Dependent Kinetics With an AFM
- 4 Using AFM Force Measurements to Characterize in vivo Unbinding Kinetics
- 5 Limitations of Current Technologies and Future Directions
- Acknowledgments
Chapter Thirteen: Improved Force Spectroscopy Using Focused-Ion-Beam-Modified Cantilevers
- Abstract
- 1 Introduction
- 2 Overview of Modification Process
- 3 Methods and Protocols
- 4 Improved Performance of FIB-Modified Cantilevers
- 5 Conclusions
- Acknowledgments
Chapter Fourteen: Single-Molecule Characterization of DNA–Protein Interactions Using Nanopore Biosensors
- Abstract
- 1 Introduction
- 2 The Basic Properties of Nanopore Translocation Measurements
- 3 Methods for Nanopore Fabrication and Assembly
- 4 Nanopores for Mapping the Binding Sites of Proteins Along Nucleic Acids
- 5 Nanopore Force Spectroscopy
- 6 Conclusions
- Acknowledgments
Chapter Fifteen: Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT
- Abstract
- 1 Why Are High-Resolution Real-Time Measurements on Enzymes Interesting?
- 2 Introduction to SPRNT
- 3 Nanopore Measurements
- 4 Nanopore Measurements Turned Into SPRNT
- 5 Application of SPRNT: Helicase Hel308
- 6 Capabilities of SPRNT
- 7 Comparison to Other Single-Molecule Techniques
- 8 Outlook
- 9 Summary
- Acknowledgments
Chapter Sixteen: Multiplexed, Tethered Particle Microscopy for Studies of DNA-Enzyme Dynamics
- Abstract
- 1 Introduction
- 2 Materials and Methods
- 3 Summary
- Acknowledgments
Product details
- No. of pages: 484
- Language: English
- Copyright: © Academic Press 2017
- Published: January 3, 2017
- Imprint: Academic Press
- Hardcover ISBN: 9780128093108
- eBook ISBN: 9780128095034
About the Serial Volume Editors
Maria Spies
Affiliations and Expertise
Yann Chemla
Affiliations and Expertise
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