FIONA was employed to follow one motor domain of the dimeric myosin VI molecule. We found that myosin VI moves along actin using a "hand-over-hand" mechanism. SHREC was subsequently developed so that both motor domains of dimeric myosin V could be followed simultaneously. Myosin V was also observed to use a "hand-over-hand" mechanism. To study the kinetics of these dimeric myosins working concentrations approaching the Km of ATP binding to the myosin motor domain would be required (∼1 muM). Linear zero-mode waveguides (lZMW) were devised to reduce the background created by high concentrations of labeled ATP. The lZMW's reduce background by evanescent excitation, nanoconfinement, and limited fluorescence coupling out of the waveguide. Their linearity permits actin filaments to be included in the experimental design. As the collection of single molecule datasets employs specialized equipment and is time intensive, data analysis was optimized by statistical analysis to ensure that the most precise information was extracted.

This book focuses on the application of fluorescence to study motor proteins (myosins, kinesins, DNA helicases and RNA polymerases). It is intended for a large community of biochemists, biophysicists and cell biologists who study a diverse collection of motor proteins. It can be used by researchers to gain an insight into their first experiments, or by experienced researchers who are looking to expand their research to new areas. Each chapter provides valuable advice for executing the experiments, along with detailed background knowledge in order to develop own experiments.

Geared towards research scientists in structural and molecular biology, biochemistry, and biophysics, this manual will be useful to all who are interested in observing, manipulating and elucidating the molecular mechanisms and discrete properties of macromolecules.

Analytical measurements at the single molecule level under ambient conditions have become almost routine in the past few years. The application of this technology to fundamental studies of heterogeneity in biomolecular structure and dynamics, chemical and biological reaction kinetics, and photophysics provides a rich playground for molecular scientists. The potential use of single molecule detection for nanotechnology and quantum information processing is a new and almost unexplored area. This handbook is intended for those interested in a practical introduction to single molecule investigations using fluorescence techniques and places special emphasis on the practicalities of achieving single molecule resolution, analysing the resulting data and exploration of the applications in biophysics. It is ideal for graduate research students and others embarking on work in this exciting field.

This is a practical introduction to single molecule fluorescence experiments, the analysis of the data, and applications of the techniques to the study of biological structure and function.

The topics range from single molecule experiments in quantum optics and solid-state physics to analogous investigations in physical chemistry and biophysics.

This handbook describes experimental techniques to monitor and manipulate individual biomolecules, including fluorescence detection, atomic force microscopy, and optical and magnetic trapping. It includes single-molecule studies of physical properties of biomolecules such as folding, polymer physics of protein and DNA, enzymology and biochemistry, single molecules in the membrane, and single-molecule techniques in living cells.

This book gives an accessible, detailed overview on techniques of single molecule biophysics (SMB), showing how they are applied to numerous biological problems associated with understanding the molecular mechanisms of DNA replication, transcription, and translation, as well as functioning of molecular machines. It covers major single molecule imaging and probing techniques, highlighting key strengths and limitations of each method using recent examples. The chapters begin with a discussion of single molecule fluorescence techniques followed by an overview of the atomic force microscope and its use for direct time-lapse visualization of dynamics of molecular complexes at the nanoscale, as well as applications in measurements of interactions between molecules and mechanical properties of isolated molecules and their complexes. The next chapters address magnetic tweezers and optical tweezers, including instrumentation, fundamentals of operation, and applications. A final chapter turns to nanopore transport and nanopore-based DNA sequencing technology that will play a major role in next-generation genomics and healthcare applications.

Single molecule techniques, including single molecule fluorescence, optical tweezers, and scanning probe microscopy, allow for the manipulation and measurement of single biological molecules within a live cell or in culture. These approaches, amongst the most exciting tools available in biology today, offer powerful new ways to elucidate biological function, both in terms of revealing mechanisms of action on a molecular level as well as tracking the behaviour of molecules in living cells. This book provides the first complete and authoritative treatment of this rapidly emerging field, explicitly from a biological perspective.The contents are organized by biological system or molecule. Each chapter discusses insights that have been revealed about their mechanism, structure or function by single molecule techniques. Among the topics covered are enzymes, motor proteins, membrane channels, DNA, ribozymes, cytoskeletal proteins, and other key molecules of current interest. An introduction by the editor provides a concise review of key principles and an historical overview. The last section discusses applications in molecular diagnostics and drug discovery. - Organized by biological system or molecule - Each chapter discusses insights into mechanism of action, structure, and function - Covers enzymes, motor proteins, membrane channels, DNA, ribozymes, etc - Includes an introduction to key principles and an historical overview - Discusses applications in molecular diagnostics and drug discovery - Provides an expert's perspective on future development

Single molecule tools have begun to revolutionize the molecular sciences, from biophysics to chemistry to cell biology. They hold the promise to be able to directly observe previously unseen molecular heterogeneities, quantitatively dissect complex reaction kinetics, ultimately miniaturize enzyme assays, image components of spatially distributed samples, probe the mechanical properties of single molecules in their native environment, and "just look at the thing" as anticipated by the visionary Richard Feynman already half a century ago. Single Molecule Tools, Part A: Fluorescence Based Approaches captures a snapshot of this vibrant, rapidly expanding field, presenting articles from pioneers in the field intended to guide both the newcomer and the expert through the intricacies of getting single molecule tools. - Includes time-tested core methods and new innovations applicable to any researcher employing single molecule tools - Methods included are useful to both established researchers and newcomers to the field - Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines