Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods, Volume 582

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

Single-Molecule Enzymology, Part B, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in single-molecule enzymology, and includes sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, and nanopore and tethered particle motion.

• 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

Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists