Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods

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