Single-Molecule Enzymology: Fluorescence-Based and High-Throughput Methods

1st Edition, Volume 581 - October 27, 2016
Imprint: Academic Press
Editors: Maria Spies, Yann R Chemla
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 8 0 9 2 6 7 - 5
eBook ISBN:
9 7 8 - 0 - 1 2 - 8 0 9 5 4 7 - 8

Single-Molecule Enzymology, Part A, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in… Read more

Single-Molecule Enzymology: Fluorescence-Based and High-Throughput Methods

Purchase options

BLOOM INTO SPRING SAVINGS

Save up to 25% on books and eBooks!

Shop now

Institutional subscription on ScienceDirect

Request a sales quote

Single-Molecule Enzymology, Part A, 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, nanopores, and tethered particle motion.

Preface
Chapter One: Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases
- Abstract
- 1 Introduction
- 2 Preparation of DNA Substrates
- 3 Fluorescent Labeling of Proteins
- 4 Instrument
- 5 Imaging DNA Unwinding by an Individual RecBCD Enzyme on a Single Molecule of DNA
- 6 Measuring DNA Unwinding by Imaging Formation of Fluorescent SSB–ssDNA Complexes
- 7 Data Analysis
- Acknowledgments
Chapter Two: Single-Molecule Imaging With One Color Fluorescence
- Abstract
- 1 Protein-Induced Fluorescence Enhancement
- 2 Experimental Preparation for PIFE
- 3 Static and Transient Protein Binding
- 4 Filament Formation and Protein Binding to Long DNA Strands
- 5 Protein Binding and Motility Kinetics
- 6 PIFE in the Presence of FRET
- 7 Conclusions
Chapter Three: Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy
- Abstract
- 1 Introduction
- 2 Preparation of Membrane Proteins for Fluorescent Studies
- 3 Preparation of Membranes for Equilibrium Measurements of Dimerization
- 4 TIRF Microscopy of Proteoliposomes
- 5 Calculation of Correction Factors for Determination of F_Dimer vs χ
- 6 Summary
Chapter Four: Fluorescent Labeling of Proteins in Whole Cell Extracts for Single-Molecule Imaging
- Abstract
- 1 Introduction
- 2 Genetically Encoded Tags for Fluorescent Labeling
- 3 Colocalization Single-Molecule Spectroscopy
- 4 Labeling SNAP_f-Tagged Proteins in Cell Extract
- 5 SNAP-SiMPull of the Yeast U1 snRNP
- 6 Common Protocols
- Acknowledgments
Chapter Five: Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy
- Abstract
- 1 Introduction
- 2 Observing Binary Macromolecular Interactions Using Single-Molecule TIRFM
- 3 Binary Interactions in the Presence of Inhibitors
- 4 Analysis of Binary and Ternary Complexes by Single-Molecule TIRFM
- 5 Conclusions and Future Outlook
- Acknowledgments
Chapter Six: How to Measure Separations and Angles Between Intramolecular Fluorescent Markers
- Abstract
- 1 Introduction
- 2 Methods
- 3 Results
- 4 Discussion
- 5 Conclusion and Perspectives
Chapter Seven: Precisely and Accurately Inferring Single-Molecule Rate Constants
- Abstract
- 1 Introduction
- 2 Single-Molecules and Stochastic Rate Constants
- 3 Calculating Stochastic Rate Constants from Signal Trajectories
- 4 Precision of Calculated Rate Constants
- 5 Accuracy of Calculated Stochastic Rate Constants
- 6 Conclusions
- Acknowledgments
Chapter Eight: Quantification of Functional Dynamics of Membrane Proteins Reconstituted in Nanodiscs Membranes by Single Turnover Functional Readout
- Abstract
- 1 Introduction and Outline
- 2 Lipid Membrane Systems for SM Functional Studies
- 3 Statistical Analysis of Trajectories of Individual Turnover Cycles
- 4 Summary
- Acknowledgments
Chapter Nine: Putting Humpty–Dumpty Together: Clustering the Functional Dynamics of Single Biomolecular Machines Such as the Spliceosome
- Abstract
- 1 Introduction
- 2 Experimental Methods and Data Analysis
- 3 Conclusions and Outlook
- Acknowledgments
Chapter Ten: Single-Molecule FRET to Measure Conformational Dynamics of DNA Mismatch Repair Proteins
- Abstract
- 1 Introduction
- 2 Methods of Acquiring FRET Signals from T. aquaticus DNA MMR Proteins
- 3 Data Analysis
- 4 Complementarity Between Single Molecule and Ensemble Kinetics: An MMR Case Study
- 5 Concluding Remarks
- Acknowledgments
Chapter Eleven: Single-Molecule Confocal FRET Microscopy to Dissect Conformational Changes in the Catalytic Cycle of DNA Topoisomerases
- Abstract
- 1 Introduction
- 2 Dissecting Conformational Changes in the Catalytic Cycle of Gyrase by Single-Molecule FRET
- 3 Conclusions and Outlook
- Acknowledgments
Chapter Twelve: Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET
- Abstract
- 1 Introduction
- 2 Single-Molecule Förster Resonance Energy Transfer
- 3 Purification and Site-Specific Labeling of Pol I (KF)
- 4 Monitoring the Conformational Landscape of Pol I (KF)
- 5 Analyzing the Conformational Landscapes for Nucleotide Selection of Pol I (KF)
- 6 Future Extensions of Single-Molecule FRET Analysis of DNA Polymerase Conformations
- Acknowledgments
Chapter Thirteen: Methods for Investigating DNA Accessibility with Single Nucleosomes
- Abstract
- 1 Introduction
- 2 Preparation of Fluorophore-Labeled Nucleosomes for Single-Molecule Fluorescence Measurements
- 3 Ensemble Fluorescence Measurements
- 4 Single-Molecule Total Internal Reflection Fluorescence Microscopy
- 5 Flow Cell Preparation and Single-Molecule Data Acquisition
- 6 Selection and Analysis of Data to Extract Lifetime Information
- 7 Conclusions
- Acknowledgments
Chapter Fourteen: Single-Molecule Fluorescence Studies of Fast Protein Folding
- Abstract
- 1 Introduction
- 2 SMF Techniques
- 3 Engineering Proteins for SM-FRET Applications
- 4 Optimizing Time-Resolution vs Conformational Dynamics
- 5 Analyzing Photon Trajectories From SM-FRET Experiments
- 6 A Case Example: One-State Downhill Folding
- 7 Concluding Remarks
- Acknowledgments
Chapter Fifteen: Single-Molecule Multicolor FRET Assay for Studying Structural Dynamics of Biomolecules
- Abstract
- 1 Introduction
- 2 Experimental Design
- 3 Data Analysis
- 4 Sample Preparation
- 5 Applications to Biological Systems
- 6 Conclusions
- Acknowledgments
Chapter Sixteen: A Multicolor Single-Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously
- Abstract
- 1 Introduction
- 2 Theoretical Background
- 3 Experimental Procedure
- 4 Data Processing
- 5 Data Analysis
- 6 Conclusion
- Acknowledgments
Chapter Seventeen: Interferometric Scattering Microscopy for the Study of Molecular Motors
- Abstract
- 1 Introduction
- 2 Interferometric Scattering Microscopy
- 3 Methods and Protocols
- 4 Applications
- 5 Outlook
- Acknowledgments
Chapter Eighteen: Enzyme Kinetics in Femtoliter Arrays
- Abstract
- 1 Introduction and Background
- 2 Preparation of Femtoliter Array
- 3 Sealing Methods
- 4 Surface Modification of Femtoliter Wells
- 5 Substrate Selection and Sample Preparation
- 6 Microscope Setup and Imaging
- 7 Image and Data Analysis
- 8 Summary and Conclusion
Author Index
Subject Index

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 R 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

Life Sciences

Physical Sciences & Engineering

Social Sciences & Humanities

Health