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Measurement Of Molecular Conformations And Dynamics Using Single Molecule Fluorescence Techniques
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Book Synopsis Measurement of Molecular Conformations and Dynamics Using Single Molecule Fluorescence Techniques by : Huimin Chen
Download or read book Measurement of Molecular Conformations and Dynamics Using Single Molecule Fluorescence Techniques written by Huimin Chen and published by . This book was released on 2012 with total page 122 pages. Available in PDF, EPUB and Kindle. Book excerpt: The use of fluorescence spectroscopy to study biological problems has gained popularity over the past few decades. Beyond a spatial understanding provided by microscopy, fluorescence techniques like Fluorescence Correlation Spectroscopy (FCS) and fluorescence lifetime spectroscopy can also elucidate the important temporal dynamics of molecular conformations. We have applied FCS to study the conformational fluctuations in a model protein apomyoglobin. By pushing the technical limitations of FCS, we were able to observe conformational dynamics spanning two orders of magnitude in time (10[-]6 to 10[-]3 seconds). We found that the amplitude of fluctuations changes as the molecule becomes unfolded, with principal shifts of amplitudes and timescales occurring at the transition across the molten globule state. We also measured the diffusion of apomyoglobin, confirming theoretical predictions of less compaction of the molecule upon acid denaturation. We were able to observe the fluctuations in apomyoglobin using FCS due to the quenching of an N-terminal labeled Alexa488 fluorophore by contact with various amino acids. We showed that quenching can occur with up to four amino acids. We investigated the mechanisms of quenching using a combination of fluorescence intensity and lifetime measurements. We showed that quenching takes place through a combination of static and dynamic mechanisms. Our results demonstrate that care needs to be taken when making quan- titative measurements of fluorescently labeled proteins. Coupling single molecule functionality to fluorescence techniques allows researchers to discern subpopulations within ensembles. We used single molecule Förster Resonance Energy Transfer (smFRET) to measure the end-to-end distances in single stranded nucleic acids as a probe of the flexibility. We also measured the radius of gyration using small angle X-ray scattering, and extracted parameters of polymer properties that fit the data from both techniques. We observed clear differences between our model single stranded DNA (ssDNA) and RNA (ssRNA). We also observed a difference between the screening efficiency of monovalent and divalent cations. By characterizing the intrinsic differences in nucleic acids and its dependence on the ionic strength, we hope to improve our understanding of the mechanisms of RNA folding and the role of ions in the process.
Book Synopsis Make Life Visible by : Yoshiaki Toyama
Download or read book Make Life Visible written by Yoshiaki Toyama and published by Springer Nature. This book was released on 2019-10-02 with total page 292 pages. Available in PDF, EPUB and Kindle. Book excerpt: This open access book describes marked advances in imaging technology that have enabled the visualization of phenomena in ways formerly believed to be completelyimpossible. These technologies have made major contributions to the elucidation of the pathology of diseases as well as to their diagnosis and therapy. The volume presents various studies from molecular imaging to clinical imaging. It also focuses on innovative, creative, advanced research that gives full play to imaging technology inthe broad sense, while exploring cross-disciplinary areas in which individual research fields interact and pursuing the development of new techniques where they fuse together. The book is separated into three parts, the first of which addresses the topic of visualizing and controlling molecules for life. Th e second part is devoted to imaging of disease mechanisms, while the final part comprises studies on the application of imaging technologies to diagnosis and therapy. Th e book contains the proceedings of the 12th Uehara International Symposium 2017, “Make Life Visible” sponsored by the Uehara Memorial Foundation and held from June 12 to 14, 2017. It is written by leading scientists in the field and is an open access publication under a CC BY 4.0 license.
Book Synopsis Measuring Conformational Dynamics of Biological Molecules with Single-molecule Tracking and Fluorescence Correlation Spectroscopy by : Charles Limouse
Download or read book Measuring Conformational Dynamics of Biological Molecules with Single-molecule Tracking and Fluorescence Correlation Spectroscopy written by Charles Limouse and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The past two decades have seen great advances in single-molecule techniques capable of probing biomolecular systems with spatial resolution down to the nanometer level. However few techniques are specifically designed to measure larger scale organization between a few tens and a few hundreds of nanometers, especially when other figures of merit are considered, such as high time-resolution or the possibility to do measurements in bulk and without immobilization. Such features would be particularly useful in the study of molecular structures like chromatin or other nucleoprotein complexes, that exhibit organizational details and dynamics across a broad range of time and length scales. I present how the combination of fluorescence correlation spectroscopy (FCS) and single molecule tracking can be used to measure the conformational dynamics of molecules that diffuse freely in solution, at the scale of a few tens of nanometers. I discuss some aspects of the instrumentation as well as the parameters that define the spatial and temporal resolution. I describe other advantages of the technique such as the possibility to bypass a common difficulty associated with fluorescence correlation measurements and obtain a signal that is not convolved with the blinking dynamics of the fluorescent probe itself. Finally I present an experimental proof-of-principle where tracking-FCS was applied to measure the end-to-end Brownian dynamics of short DNA fragments in the semilflexible regime.
Book Synopsis Handbook of Single Molecule Fluorescence Spectroscopy by : Chris Gell
Download or read book Handbook of Single Molecule Fluorescence Spectroscopy written by Chris Gell and published by OUP Oxford. This book was released on 2006-08-17 with total page 278 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Single Molecule Spectroscopy by : R. Rigler
Download or read book Single Molecule Spectroscopy written by R. Rigler and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 375 pages. Available in PDF, EPUB and Kindle. Book excerpt: The topics range from single molecule experiments in quantum optics and solid-state physics to analogous investigations in physical chemistry and biophysics.
Book Synopsis Spectroscopy and Dynamics of Single Molecules by :
Download or read book Spectroscopy and Dynamics of Single Molecules written by and published by Elsevier. This book was released on 2019-08-14 with total page 402 pages. Available in PDF, EPUB and Kindle. Book excerpt: Spectroscopy and Dynamics of Single Molecules: Methods and Applications reviews the most recent developments in spectroscopic methods and applications. Spectroscopic techniques are the chief experimental methods for testing theoretical models and research in this area plays an important role in stimulating new theoretical developments in physical chemistry. This book provides an authoritative insight into the latest advances in the field, highlighting new techniques, current applications, and potential future developments An ideal reference for chemists and physicists alike, Spectroscopy and Dynamics of Single Molecules: Methods and Applications is a useful guide for all those working in the research, design, or application of spectroscopic tools and techniques across a wide range of fields. Includes the latest research on ultrafast vibrational and electronic dynamics, nonlinear spectroscopies, and single-molecule methods Makes the content accessible to researchers in chemistry, biophysics, and chemical physics through a rigorous multi-disciplinary approach Provides content edited by a world-renowned chemist with more than 30 years of experience in research and instruction
Book Synopsis Theory and Evaluation of Single-molecule Signals by : Eli Barkai
Download or read book Theory and Evaluation of Single-molecule Signals written by Eli Barkai and published by World Scientific. This book was released on 2008 with total page 416 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book reviews recently developed theoretical and numerical approaches to deal with optical and mechanical signals from individual molecules. The character of data generated by single molecules, and more generally by single nano-objects, qualitatively differs from those obtained in conventional experiments on large ensembles of molecules. Fluctuations, randomness and irreproducibility are central to single-molecule measurements, and the specific methods required to extract reliable and statistically relevant information from them are presented here. With contributions mainly from participants of the ?Theory, Modeling and Evaluation of Single-Molecule Measurements? workshop held in Leiden, the Netherlands, on April 16-20, 2007, this book is an authoritative compendium on the subject.
Book Synopsis Handbook of Fluorescence Spectroscopy and Imaging by : Markus Sauer
Download or read book Handbook of Fluorescence Spectroscopy and Imaging written by Markus Sauer and published by John Wiley & Sons. This book was released on 2010-12-23 with total page 425 pages. Available in PDF, EPUB and Kindle. Book excerpt: Providing much-needed information on fluorescence spectroscopy and microscopy, this ready reference covers detection techniques, data registration, and the use of spectroscopic tools, as well as new techniques for improving the resolution of optical microscopy below the resolution gap. Starting with the basic principles, the book goes on to treat fluorophores and labeling, single-molecule fluorescence spectroscopy and enzymatics, as well as excited state energy transfer, and super-resolution fluorescence imaging. Examples show how each technique can help in obtaining detailed and refined information from individual molecular systems.
Book Synopsis Protein Conformational Dynamics by : Ke-li Han
Download or read book Protein Conformational Dynamics written by Ke-li Han and published by Springer Science & Business Media. This book was released on 2014-01-20 with total page 488 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book discusses how biological molecules exert their function and regulate biological processes, with a clear focus on how conformational dynamics of proteins are critical in this respect. In the last decade, the advancements in computational biology, nuclear magnetic resonance including paramagnetic relaxation enhancement, and fluorescence-based ensemble/single-molecule techniques have shown that biological molecules (proteins, DNAs and RNAs) fluctuate under equilibrium conditions. The conformational and energetic spaces that these fluctuations explore likely contain active conformations that are critical for their function. More interestingly, these fluctuations can respond actively to external cues, which introduces layers of tight regulation on the biological processes that they dictate. A growing number of studies have suggested that conformational dynamics of proteins govern their role in regulating biological functions, examples of this regulation can be found in signal transduction, molecular recognition, apoptosis, protein / ion / other molecules translocation and gene expression. On the experimental side, the technical advances have offered deep insights into the conformational motions of a number of proteins. These studies greatly enrich our knowledge of the interplay between structure and function. On the theoretical side, novel approaches and detailed computational simulations have provided powerful tools in the study of enzyme catalysis, protein / drug design, protein / ion / other molecule translocation and protein folding/aggregation, to name but a few. This work contains detailed information, not only on the conformational motions of biological systems, but also on the potential governing forces of conformational dynamics (transient interactions, chemical and physical origins, thermodynamic properties). New developments in computational simulations will greatly enhance our understanding of how these molecules function in various biological events.
Book Synopsis Investigating Single-molecule Interactions and Photodynamics in Solution by Counteracting Brownian Motion by : Hsiang-Yu Yang
Download or read book Investigating Single-molecule Interactions and Photodynamics in Solution by Counteracting Brownian Motion written by Hsiang-Yu Yang and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-molecule fluorescence methods have become a mature and powerful tool to unravel the fundamental mechanisms of biological phenomena. These approaches enable researchers to investigate individual components on the nanoscale for heterogeneous mixtures and to monitor the dynamics of single biomolecules. Unfortunately, traditional burst-based methods are often limited by short observation time, and the immobilization of single biomolecules that enables long observation time is often undesirably perturbative. To address these limitations, a solution-based technique known as the Anti-Brownian ELectrokinetic (ABEL) trap has been developed. In the ABEL trap, high-speed feedback electronics sense position and compensate Brownian motion of a single molecule by applying electrokinetic forces, which keep the molecule in the trapping region for an extended period of time. This dissertation describes my work on the investigation of single-molecule interactions and photodynamics using the ABEL trap technology. First, I show how to measure and monitor single-molecule rotational diffusivity by combining the ABEL trap and maximum likelihood analysis of time-resolved fluorescence anisotropy based on the information inherent in each detected photon. The rotational diffusivity is extremely sensitive to the size of the molecule, and thus can be used to detect and monitor size-changing events, such as association and dissociation. We demonstrate this approach by resolving a mixture of single- and double-stranded fluorescently-labeled DNA molecules at equilibrium, freely rotating in a native solution environment. Next, we investigate photosynthetic photoprotection mechanisms at the level of individual antenna complexes (LHCII), the fundamental units which green plants use to harvest sunlight. We access the intrinsic conformational dynamics of individual LHCII complexes. In addition to an unquenched state, two partially quenched states of LHCII are observed. One of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in the LHCII complex. Last, we describe the theoretical basis of a new method to measure the diffusion coefficient of diffusing objects on the surface of individual nanoscale lipid vesicles. We discuss how to probe diffusion in these highly confined environments using FRET pairs and fluorescence intensity correlation, and illustrate that this method is essentially a fluorescence correlation spectroscopy (FCS) experiment with a nanometer-sized probing volume.
Book Synopsis Single Molecule Tools, Part A: Fluorescence Based Approaches by :
Download or read book Single Molecule Tools, Part A: Fluorescence Based Approaches written by and published by Academic Press. This book was released on 2010-08-17 with total page 559 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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
Book Synopsis Single Molecule Dynamics in Life Science by : Toshio Yanagida
Download or read book Single Molecule Dynamics in Life Science written by Toshio Yanagida and published by John Wiley & Sons. This book was released on 2008-11-24 with total page 360 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this first comprehensive resource to cover the application of single molecule techniques to biological measurements, the pioneers in the field show how to both set up and interpret a single molecule experiment. Following an introduction to single molecule measurements and enzymology, the expert authors consider molecular motors and mechanical properties before moving on to the applications themselves. Detailed discussions of studies on protein enzymes, ribozymes and nucleic acids are also included.
Book Synopsis Photon-counting Single-molecule Spectroscopy for Studying Conformational Dynamics and Macromolecular Interactions by :
Download or read book Photon-counting Single-molecule Spectroscopy for Studying Conformational Dynamics and Macromolecular Interactions written by and published by . This book was released on 2002 with total page 196 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-molecule methods have the potential to provide information about conformational dynamics and molecular interactions that cannot be obtained by other methods. Removal of ensemble averaging provides several benefits, including the ability to detect heterogeneous populations and the ability to observe asynchronous reactions. Single-molecule diffusion methodologies using fluorescence resonance energy transfer (FRET) are developed to monitor conformational dynamics while minimizing perturbations introduced by interactions between molecules and surfaces. These methods are used to perform studies of the folding of Chymotrypsin Inhibitor 2, a small, single-domain protein, and of single-stranded DNA (ssDNA) homopolymers. Confocal microscopy is used in combination with sensitive detectors to detect bursts of photons from fluorescently labeled biomolecules as they diffuse through the focal volume. These bursts are analyzed to extract fluorescence resonance energy transfer (FRET) efficiency. Advances in data acquisition and analysis techniques that are providing a more complete picture of the accessible molecular information are discussed. Photon Arrival-time Interval Distribution (PAID) analysis is a new method for monitoring macromolecular interactions by fluorescence detection with simultaneous determination of coincidence, brightness, diffusion time, and occupancy (proportional to concentration) of fluorescently-labeled molecules undergoing diffusion in a confocal detection volume. This method is based on recording the time of arrival of all detected photons, and then plotting the two-dimensional histogram of photon pairs, where one axis is the time interval between each pair of photons 1 and 2, and the second axis is the number of other photons detected in the time interval between photons 1 and 2. PAID is related to Fluorescence Correlation Spectroscopy (FCS) by a collapse of this histogram onto the time interval axis. PAID extends auto- and cross-correlation FCS by measuring the brightness of fluorescent species. A data-fitting model is developed, which is used to simultaneously determine coincidence, brightness, diffusion time, and occupancy from experiments performed on fluorophore-labeled dsDNA test samples. Using simulations, the performance of PAID is compared with existing methods. The statistical accuracy of the parameters extracted using PAID exceeds or matches the accuracy of the other methods, while providing additional information.
Book Synopsis Single Particle Tracking and Single Molecule Energy Transfer by : Christoph Bräuchle
Download or read book Single Particle Tracking and Single Molecule Energy Transfer written by Christoph Bräuchle and published by John Wiley & Sons. This book was released on 2009-10-30 with total page 359 pages. Available in PDF, EPUB and Kindle. Book excerpt: Closing a gap in the literature, this handbook gathers all the information on single particle tracking and single molecule energy transfer. It covers all aspects of this hot and modern topic, from detecting virus entry to membrane diffusion, and from protein folding using spFRET to coupled dye systems, as well recent achievements in the field. Throughout, the first-class editors and top international authors present content of the highest quality, making this a must-have for physical chemists, spectroscopists, molecular physicists and biochemists.
Book Synopsis Multimodal Measurements of Single-molecule Conformational Dynamics by : Ivan Enchev Ivanov
Download or read book Multimodal Measurements of Single-molecule Conformational Dynamics written by Ivan Enchev Ivanov and published by . This book was released on 2018 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-molecule methods can monitor the dynamics of biological macromolecules in real time. Here we develop FluoRBT, which provides multimodal measurements of structural transitions in nucleic acids and nucleoprotein complexes by combining magnetic tweezers manipulation, infrared darkfield imaging, and single-molecule FRET. We demonstrate the capabilities of FluoRBT by observing structural transitions in DNA under mechanical strain through concurrent changes in FRET and angle or extension. We also apply FluoRBT to study the dynamics of DNA interrogation by the RNA-guided CRISPR nuclease Cas9. We identify a structural intermediate corresponding to DNA:RNA hybridization within the target "seed" region, and characterize how the interplay between sequence mismatches and DNA superhelicity modulates access to states competent for stable binding and cleavage. FluoRBT can further be used to dissect the mechanism of mechanochemical coupling in DNA gyrase by correlating progress through the supercoiling reaction with rich dynamics of the N-gate ATPase domain observed in single-molecule FRET assays. By combining mechanical measurements of global deformations in nucleic acids with FRET measurements of local conformational changes, FluoRBT enables multidimensional investigations of systems ranging from DNA structures to large molecular machines.
Book Synopsis Extending Single-molecule Fluorescence Spectroscopy Methods to Study Protein Conformation and Dynamics of Complex Biological Systems by : Dominik Hänni
Download or read book Extending Single-molecule Fluorescence Spectroscopy Methods to Study Protein Conformation and Dynamics of Complex Biological Systems written by Dominik Hänni and published by . This book was released on 2012 with total page 189 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Fluorescence Anisotropy Measurements of Single Molecules to Probe Solution-phase Conformational Heterogeneity of Intrinsically Disordered Proteins by : Lydia Manger
Download or read book Fluorescence Anisotropy Measurements of Single Molecules to Probe Solution-phase Conformational Heterogeneity of Intrinsically Disordered Proteins written by Lydia Manger and published by . This book was released on 2018 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-molecule studies have opened a new avenue to pursue the heterogeneity of molecular systems that is obscured at the bulk level. Likewise, time-correlated single photon counting methods can provide characterization of evolving populations of photoexcited species that contribute to a bulk property such as photoluminescence. Tau protein is an intrinsically disordered protein (IDP) that is implicated in neurodegenerative diseases such as Alzheimer's disease. Characterizing and understanding the conformations that this protein adopts in solution is critical to understanding disease pathology. We examine the conformations adopted by tau protein in solution at the single-molecule level in order to elucidate the extent of structural heterogeneity. Traditional single-molecule fluorescence studies of proteins, however, rely on recording freely-diffusing molecules as they pass through a confocal volume - resulting in short observation time and unrestricted motion - or tethering the molecule to a surface - providing longer observation times but potentially introducing artifacts. Herein we describe the use of the anti-Brownian electrokinetic (ABEL) trap to prolong measurement of the anisotropy of individual proteins in solution to investigate the diversity of conformations sampled by the protein. In Chapter 2 we describe how tau protein exists in two families of conformations in solution, differentiated by their degree of compaction. Chapter 3 then provides an overview of different labeling positions that may be useful for further studying the conformational heterogeneity of tau protein. In Chapter 4, we describe a study of the recombination kinetics of a metal halide perovskite, methylammonium lead iodide (MAPbI3). Time-resolved photoluminescence is recorded using time-correlated single photon counting. These measurements provide insight into the time-varying populations of the photoexcited species present in the material that are obscured at the bulk level. These measurements are then used to define a set of rate equations to describe the evolving populations, leading us to invoke geminate recombination as an important method for recombination of electrons and holes.
