Single Molecule Enzymology

The last fifteen years have witnessed the birth and maturation of many original methods and the development of protocols specific to single molecule measurements and their analysis, including techniques involving optical imaging, electron microscopy, optical and magnetic trapping, and developments in atomic force microscopy. In Single Molecule Enzymology: Methods and Protocols , experts in the field provide procedures which enable the extraction of detailed information about enzyme work cycles, their static and kinetic properties, and information about their location and activity within cells. The detailed volume offers practical advice on many aspects of single molecule enzymology and includes strategic overviews of interconnected methods involved in sample preparation, single molecule measurements, and data analysis. Written in the highly successful Methods in Molecular Biology™ series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and up-to-date, Single Molecule Enzymology: Methods and Protocols is intended for use within the diverse community of molecular biologists, biochemists, and biophysicists studying enzymes in detail and can be used by researchers planning their first single molecule study or to aid more experienced researchers in further developing their existing studies.

Serves as an ideal guide for the diverse community of molecular biologists, biochemists, and biophysicists studying enzymes in detail Features cutting-edge, lab ready protocols Includes key tips and expert implementation advice to ensure successful results Includes supplementary material: sn.pub/extras

Inhalt

Single Enzyme Studies: A Historical Perspective.- Functional Surface-Attachment in a Sandwich Geometry of GFP-Labeled Motor Proteins.- Studying Kinesin's Enzymatic Cycle Using a Single-Motor Confocal Motility Assay, Employing Förster Resonance Energy Transfer.- Fluorescence Imaging with One Nanometer Accuracy: In Vitro and In Vivo Studies of Molecular Motors.- Snapshots of Kinesin Motors on Microtubule Tracks.- Structural and Dynamic Characterization of Biochemical Processes by Atomic Force Microscopy.- Using Optical Tweezers to Study Fine Details of Myosin ATPase Mechanochemical Cycle.- Quantum Dot Labeling Strategies to Characterize Single Molecular Motors.- Imaging Individual Myosin Molecules Within Living Cells.- Single Molecule Measurements Using Microneedles.- Fluorescent Nucleoside Triphosphates for Single Molecule Enzymology.- Probing the Mechanics of the Complete DNA Transcription Cycle in Real-Time Using Optical Tweezers.- A Single-Molecule Approach to Visualize the Unwinding Activity of DNA Helicases.- Real-Time Single-Molecule Observation of Green Fluorescent Protein Synthesis by Immobilized Ribosomes.- Single-Molecule Measurements of Topoisomerase Activity with Magnetic Tweezers.- Assembly of Recombinant Nucleosomes on Nanofabricated DNA Curtains for Single Molecule Imaging.- Simultaneous Observation of Chemomechanical Coupling of a Molecular Motor.- Microsecond Resolution of Single Molecule Rotation Catalyzed by Molecular Motors.