Single Molecule Enzymology: Methods and Protocols (Methods in Molecular Biology, 778, Band 778)

1. Single Enzyme Studies: A Historical Perspective

Alex E. Knight

2. Functional Surface-Attachment in a Sandwich Geometry of GFP-Labeled Motor Proteins

Volker Bormuth, Felix Zörgibel, Erik Schäffer, and Jonathon Howard

3. Studying Kinesin’s Enzymatic Cycle Using a Single-Motor Confocal Motility Assay, Employing Förster Resonance Energy Transfer

Zdenek Lansky and Erwin J.G. Peterman

4. Fluorescence Imaging with One Nanometer Accuracy: In Vitro and In Vivo Studies of Molecular Motors

Melinda Tonks Hoffman, Janet Sheung, and Paul R. Selvin

5. Snapshots of Kinesin Motors on Microtubule Tracks

Franck J. Fourniol and Carolyn A. Moores

6. Structural and Dynamic Characterization of Biochemical Processes by Atomic Force Microscopy

Frédéric Eghiaian and Iwan A.T. Schaap

7. Using Optical Tweezers to Study Fine Details of Myosin ATPase Mechanochemical Cycle

Christopher Batters and Claudia Veigel

8. Quantum Dot Labeling Strategies to Characterize Single Molecular Motors

Shane R. Nelson, M. Yusuf Ali, and David M. Warshaw

9. Imaging Individual Myosin Molecules Within Living Cells

Tatiana A. Nenasheva, Gregory I. Mashanov, Michelle Peckham, and Justin E. Molloy

10. Single Molecule Measurements Using Microneedles

Toshio Yanagida, Yoshiharu Ishii, and Akihiko Ishijima

11. Fluorescent Nucleoside Triphosphates for Single Molecule Enzymology

Christopher P. Toseland and Martin R. Webb

12. Probing the Mechanics of the Complete DNA Transcription Cycle in Real-Time Using Optical Tweezers

Christoph G. Baumann and Stephen J. Cross

13. A Single-Molecule Approach to Visualize the Unwinding Activity of DNA Helicases

Natalia Fili, Christopher P. Toseland, Mark S. Dillingham, Martin R. Webb, and Justin E. Molloy

14. Real-Time Single-Molecule Observation of Green Fluorescent Protein Synthesis by Immobilized Ribosomes

Ryo Iizuka, Takashi Funatsu, and Sotaro Uemura

15. Single-Molecule Measurements of Topoisomerase Activity with Magnetic Tweezers

Yeonee Seol and Keir C. Neuman

16. Assembly of Recombinant Nucleosomes on Nanofabricated DNA Curtains for Single Molecule Imaging

Ja Yil Lee and Eric C. Greene

17. Simultaneous Observation of Chemomechanical Coupling of a Molecular Motor

Takayuki Nishizaka*, Yuu Hashimoto, and Tomoko Masaike

18. Microsecond Resolution of Single Molecule Rotation Catalyzed by Molecular Motors

Tassilo Hornung, James Martin, David Spetzler, Robert Ishmukhametov, and Wayne D. Frasch

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.