E-Book

Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods [electronic resource]

  • Chemla, Yann R. , Spies, Maria
  • Academic Press
  • 2017
Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods [electronic resource]
  • 자료유형
    단행본
  • 서명/저자사항
    Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods[electronic resource] / Maria Spies, Yann R. Chemla.
  • 발행사항
    [Cambridge, MA] : Academic Press, 2017.
  • 개인저자
    Chemla, Yann R., Spies, Maria
  • 형태사항
    1 online resource.
  • 총서명
    Methods in Enzymology ; 582
  • ISSN
    0076-6879
  • ISBN
    9780128093108
  • 언어
    영어

목차

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