Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods, Volume 582
1st Edition
Secure Checkout
Personal information is secured with SSL technology.Free Shipping
Free global shippingNo minimum order.
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
Description
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.
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
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists
Details
- No. of pages:
- 484
- Language:
- English
- Copyright:
- © Academic Press 2017
- Published:
- 12th January 2017
- Imprint:
- Academic Press
- Hardcover ISBN:
- 9780128093108
- eBook ISBN:
- 9780128095034
Reviews
Praise for the Series:
"Should be on the shelves of all libraries in the world as a whole collection." --Chemistry in Industry
"The work most often consulted in the lab." --Enzymologia
"The Methods in Enzymology series represents the gold-standard." --Neuroscience
Ratings and Reviews
About the Serial Volume Editors
Maria Spies
Graduate of Peter the Great St. Petersburg Polytechnic University, Russia (1996 MS diploma with honors (equivalent of cum laude) in physics/biophysics) and Osaka University, Japan (2000 PhD in biological sciences), Dr. Maria Spies is an Associate Professor of Biochemistry at the University of Iowa Carver College of Medicine. Spies’ research career has been focused on deciphering the intricate choreography of the molecular machines orchestrating the central steps in the homology directed DNA repair. Her doctoral research supported by the Japanese Government (MONBUSHO) Graduate Scholarship provided the first detailed biochemical characterization of archaeal recombinase RadA. In her postdoctoral work with Dr. Steve Kowalczykowski (UC Davis) supported by the American Cancer Society, Spies reconstituted at the single-molecule level the initial steps of bacterial recombination and helped to explain how this process is regulated. Spies’ laboratory at the University of Iowa emphasizes the molecular machinery of homologous recombination, how it is integrated into DNA replication, repair and recombination (the 3Rs of genome stability), and how it is misappropriated in the molecular pathways that process stalled DNA replication events and DNA breaks through highly mutagenic, genome destabilizing mechanisms. Her goal is to understand, reconstitute and manipulate an elaborate network of DNA recombination, replication and repair, and to harness this understanding for anticancer drug discovery. The Spies lab utilizes a broad spectrum of techniques from biochemical reconstitutions of the key biochemical reactions in DNA recombination, repair and replication, to structural and single-molecule analyses of the proteins and enzymes coordinating these reactions, to combined HTS/CADD campaigns targeting human DNA repair proteins. Work in Spies Lab has been funded by the American Cancer Society (ACS), Howard Hughes Medical Institute (HHMI), and is currently supported by the National Institutes of Health (NIH). She received several prestigious awards including HHMI Early Career Scientist Award and Margaret Oakley Dayhoff Award in Biophysics. She serves on the editorial board of the Journal of Biological Chemistry, and as an academic editor of the journal Plos-ONE. She is a permanent member and a chair of the American Cancer Society “DNA mechanisms in cancer” review panel.
Affiliations and Expertise
Carver College of Medicine, University of Iowa, USA
Yann Chemla
Associate Professor of Physics and Biophysics, Department of Physics, University of Illinois at Urbana-Champaign, USA
Affiliations and Expertise
Department of Physics, University of Illinois at Urbana-Champaign, USA
Request Quote
Tax Exemption
Elsevier.com visitor survey
We are always looking for ways to improve customer experience on Elsevier.com.
We would like to ask you for a moment of your time to fill in a short questionnaire, at the end of your visit.
If you decide to participate, a new browser tab will open so you can complete the survey after you have completed your visit to this website.
Thanks in advance for your time.