Download or read book Single Molecule Studies of Enzymatic Dynamic Fluctuations written by Brian Patrick English and published by . This book was released on 2007 with total page 358 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Single Molecule Spectroscopy Studies of the Conformational Dynamics of Enzymes written by Maolin Lu and published by . This book was released on 2014 with total page 224 pages. Available in PDF, EPUB and Kindle. Book excerpt: Conformational motions of enzymes are highly dynamic and intrinsically stochastic. Obtaining molecular level insights into conformational dynamics of enzymes is critical for unraveling the complex intimate structure-to-function relationship. This dissertation describes the investigation of conformational dynamics of HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) and T4 lysozyme by single-molecule FRET (Forster/fluorescence resonance energy transfer) spectroscopy and photon stamping spectroscopy. This dissertation also demonstrates the developments of corresponding single-molecule spectroscopic approaches to serve scientifically experimental demands. This dissertation also demonstrates the developments of corresponding single-molecule spectroscopic approaches to serve scientifically experimental demands. Multiple conformational intermediate states and multi-dimensional conformational motions of T4 lysozyme have been observed. The Markov process has successfully reproduced the experimental observations, suggesting that T4 lysozyme hinge-bending open-close conformational changes follow multiple pathways involving multiple intermediate states. The combination of lifetime and anisotropy results presents a whole picture of multi-dimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending. The non-exponential features of both lifetime and anisotropy autocorrelation functions reveal dynamic and static inhomogeneity/complexity of multi-dimensional conformational fluctuations. The investigations of probing and manipulating HPPK conformational dynamics has been described. The consistency between the decay rate of donor lifetime and rising rate of acceptor lifetime gives a direct observation of FRET dynamic process at single-molecule level. The autocorrelation analysis of donor lifetimes have revealed intermittent conformational coherence of multiple HPPK Loop3-active site conformational states, regulated by substrate-enzyme interactions. Mechanically manipulating a targeted dye-labeled single HPPK in pinpoint nano-scale precision and simultaneously monitoring the conformational changes during the AFM pulling event has been achieved. The observed results of different lifetime fluctuations, distinct anisotropy fluctuations and various dynamic rates have suggested the existence of function-inert and function-active scenarios of HPPK Loop 3-active site conformational dynamic motions. The developments of single-molecule spectroscopic approaches have been demonstrated, including 1) single molecule photon stamping FRET spectroscopy, on the basis of only measuring the donor's lifetime trajectory; 2) single-molecule AFM-FRET nanoscopy, capable of effectively pinpointing and mechanically manipulating a targeted dye-labeled single protein in a large sampling area; and 3) single-molecule multi-parameter photon stamping spectroscopy system, integrating fluorescence anisotropy-FRET-lifetime and capable of observing single-molecule multi-dimensional conformational motions.
Download or read book The Fluctuating Enzyme written by G. Rickey Welch and published by Wiley-Interscience. This book was released on 1986 with total page 520 pages. Available in PDF, EPUB and Kindle. Book excerpt: There have emerged a number of theoretical models of enzyme action which maintain that classes of fluctuations in the protein structure provide means for generating high energy-free events at the active center. The purpose of the present volume is to review these theoretical pursuits.
Download or read book Single Molecule Optical Magnetic Tweezers Microscopy Studies of Protein Dynamics written by Qing Guo and published by . This book was released on 2015 with total page 156 pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation presents our research work aiming at conformational manipulation of single enzyme protein molecules, performed by single molecule magnetic tweezers correlated with optical fluorescence spectroscopy. To experimentally investigate the enzyme-substrate interactions and the related conformational fluctuations, we have developed a new approach to manipulate the enzymatic conformation and enzyme-substrate interaction at the single-molecule level by using a combined magnetic tweezers and simultaneous fluorescence resonance energy transfer (FRET) spectroscopic microscopy. By a repetitive pulling-releasing manipulation of a Cy3-Cy5 dye labeled 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) molecule under the conditions with and without enzymatic substrates, we have probed and analyzed the enzymatic conformational dynamics. Our results indicate that the enzymatic conformational flexibility can be regulated by enzyme-substrate interactions: (1) the enzyme at its conformation-perturbed state has less flexibility when binding substrates, and (2) substrate binding to the enzyme significantly changes the enzyme conformational flexibility, experimental evidence of so called entropy trapping in and enzyme-substrate reactive transition state. Furthermore, our results provide significant experimental analysis of folding-binding interactions of the enzyme-substrate interactions, and reveal the dynamic nature of the enzyme-substrate interactions. We also find supportive results from Steered Molecular Dynamics (SMD) Simulation, showing that in our studies, conformational manipulation by magnetic tweezers is able to distort the active domain of the enzyme molecules to an extent that significantly beyond thermal conformational fluctuations. Furthermore, we have also revealed the impact of partially unfolding the enzyme molecules on their activity by using single-molecule TIRF-magnetic tweezers spectroscopy to manipulate conformation of the enzyme molecules to a partially unfolded, yet not fully denatured condition. By conformationally distorting horseradish peroxidase (HRP) molecules via magnetic tweezers at the single molecule level, we successfully manipulated and examined the activity changes of the HRP catalyzed H2O2-Amplex Red reaction. We have observed significant tolerance of the enzyme activity to the enzyme conformation in its deformed or partially-unfolded states. We have identified that (1) enzymatic activity can be manipulated by our TIRF-magnetic tweezers at single molecule level; and (2) enzyme molecules in partially unfolded conformation are still capable of showing significant activity, although at a lower but measurable level, due to the enzymatic active site conformational fluctuation and substrate binding induced folding-binding conformational changes. We further provide our understanding of the enzyme behavior based on enzymatic conformational fluctuation, enzyme-substrate interactions, enzyme-substrate active complex formation, and protein folding-binding interactions.
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 2008-12-04 with total page 346 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.
Download or read book Single Molecule Studies of Enzyme Kinetics and Mechanism written by Jue Shi and published by . This book was released on 2006 with total page 272 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Single molecule Studies of Proteins written by Andres F. Oberhauser and published by Springer Science & Business Media. This book was released on 2012-11-09 with total page 283 pages. Available in PDF, EPUB and Kindle. Book excerpt: In Single Molecule Studies of Proteins, expert researchers discuss the successful application of single-molecule techniques to a wide range of biological events, such as the imaging and mapping of cell surface receptors, the analysis of the unfolding and folding pathways of single proteins, the analysis interaction forces between biomolecules, the study of enzyme catalysis or the visualization of molecular motors in action. The chapters are aimed at established investigators and post-doctoral researchers in the life sciences wanting to pursue research in the various areas in which single-molecule approaches are important; this volume also remains accessible to advanced graduate students seeking similar research goals.
Download or read book Single Molecules and Nanotechnology written by Rudolf Rigler and published by Springer Science & Business Media. This book was released on 2007-12-07 with total page 322 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book focuses on recent advances in the rapidly evolving field of single molecule research. These advances are of importance for the investigation of biopolymers and cellular biochemical reactions, and are essential to the development of quantitative biology. Written by leading experts in the field, the articles cover a broad range of topics, including quantum photonics of organic dyes and inorganic nanoparticles and monitoring of single molecule (enzymatic) reactions.
Download or read book Single molecule Spectroscopy Studies of Protein Conformational Dynamics in DNA Damage Recognition and Cell Signaling written by Sunidhi Jaiswal and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-molecule fluorescence spectroscopy has been developed as a powerful technique that provides details of protein-protein and protein-DNA interactions, enzyme reactions, and conformational dynamics. It is highly informative to study protein's conformational dynamics during their activity or under the enzymatic condition to understand their function. The real-time monitoring of the enzyme's active site conformational dynamics and simultaneously activity is informative towards understanding the mechanism of action. Studying how conformational fluctuation dynamics play role in protein or enzyme activity can shed light on the field of enzymology. Particularly in this dissertation, we have employed FoÌrster Resonance Energy Transfer (FRET) as a powerful tool to study the conformational dynamics of Calmodulin (CaM) during its interaction with an autoinhibitory domain (C28W peptide) of the Plasma Membrane Calcium ATPase (PMCA). FRET between donor dye-labeled N-domain of the calmodulin interacting with acceptor dye-labeled peptide reports the real-time conformational dynamics of this interaction which is essential for PMCA activation. Interestingly, by using a unique statistical method, the results provide a mechanistic understanding of CaM signaling interaction and activation of the Ca-ATPase through multiple-state binding to the C28W. The new single-molecule spectroscopic analyses demonstrated in this work can be applied for broad studies of protein functional conformation fluctuation and protein-protein interaction dynamics. In another study, the conformational dynamics of recognition proteins are studied to understand the mechanism of identification of DNA damage by two recognition proteins, Replication Protein A (RPA) and Xeroderma Pigmentosum Protein A (XPA). We use single-molecule fluorescence fluctuation measurements of a dye, labeled at a damaged position on DNA, to understand the interaction of the damage site with RPA14 and XPA. Our results suggest that interactive conformational dynamics of RPA14 with damaged DNA are inhomogeneous due to its low affinity for DNA, whereas binding of XPA with the already formed DNA- RPA14 complex may increase the specificity of damage recognition by controlling the conformational fluctuation dynamics of the complex. Further, we studied the activation of calmodulin by applying compressive force using the AFM technique. Significantly, we found that the picoNewton level compressive force can turn a calcium-free CaM molecule into an active binding form just like the calcium-activated CaM.
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.
Download or read book Unsolved Problems of Noise and Fluctuations written by Sergey M. Bezrukov and published by Springer Science & Business Media. This book was released on 2003-06-02 with total page 644 pages. Available in PDF, EPUB and Kindle. Book excerpt: All papers in this proceedings volume were peer reviewed. The purview of this third conference was shifted toward biology and medicine. Among the topics covered were: the constructive role of noise in the central nervous system, neuronal networks, and sensory transduction (hearing in humans, photo- and electroreception in marine animals), encoding of information into nerve pulse trains, single molecules and noise (including single molecule detection and characterization by nanopores - molecular "Coulter counting"), concepts of noise in neurophysiology (randomness and order in brain and heart electrical activities under normal conditions and in pathology), the role of noise in genetic regulation and gene expression, biosensors, etc.
Download or read book SINGLE MOLECULE MAGNETIC TWEEZERS DEVELOPMENT AND APPLICATION IN STUDIES OF ENZYME DYNAMICS AND CELL MANIPULATION written by Meiling Wu and published by . This book was released on 2020 with total page 170 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-molecule Magnetic tweezers have been developed as a powerful approach that provides detailed insight into single-molecule studies by mechanically manipulating and simultaneously monitoring the change of the photophysical properties. We have developed a few generations of magnetic tweezers and further utilized them for the studies of the mechanism of the product release during an enzymatic reaction, correlated studies of single-molecule enzymatic reaction dynamics and conformational dynamics, and live-cell motion manipulation. It is highly informative to manipulate the enzyme under the enzymatic conditions and simultaneously real-time monitor the conformational fluctuations of the enzymatic active site as well as the reaction activity change. Understanding how the enzyme dynamics of the conformational fluctuation relates to the enzyme activity can shed light on the field of enzymology. Motion manipulation of living cells and other biological entities is also important in the fields of biological research and tissue engineering. Particularly, in this dissertation, we have employed a Horseradish Peroxidase (HRP) catalyzed fluorogenic enzymatic reaction as a powerful probe to study the reaction and conformational dynamics of the enzymatic active site under mechanical force manipulation. The typical fluorogenic feature of this reaction makes the nascent-formed product molecule at its perfect fitted position at the enzymatic active site, serving as an in-situ probe to report the real-time active-site configuration and its fluctuations. Interestingly, the product releasing dynamics of HRP show the complex conformational behavior with multiple product-releasing pathways. However, under constant magnetic force manipulation, the complex nature of the multiple product- releasing pathways disappears, and more simplistic conformations of the active site are populated. Under oscillation force manipulation at different frequencies, we have observed that conformational dynamics can be significantly perturbed or altered. We have developed an integrated double-ring magnetic tweezer imaging microscope to actively manipulate the live-cell motions and selectively pick up the cell with an internalized paramagnetic bead. We have also demonstrated a lock-in amplifier coupled rotating magnetic tweezers approach, allowing synchronization of magnetic force response with the oscillation force.
Download or read book An Introduction to Single Molecule Biophysics written by Yuri L. Lyubchenko and published by CRC Press. This book was released on 2017-11-22 with total page 407 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Download or read book Single Molecule Enzymology Fluorescence Based and High Throughput Methods written by and published by Academic Press. This book was released on 2016-10-28 with total page 618 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in single-molecule enzymology Contains sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, nanopores, and tethered particle motion
Download or read book Single Particle Nanocatalysis written by Weilin Xu and published by John Wiley & Sons. This book was released on 2019-02-08 with total page 331 pages. Available in PDF, EPUB and Kindle. Book excerpt: Introduces the detailed basis and recent development of single molecule/particle nanocatalysis based on single molecule techniques This unique book introduces and summarizes the recent development of single molecule/particle nanocatalysis to provide both comprehensive coverage of fundamentals for different methods now in widespread use and the extensive applications in different catalytic systems. Chapters are mainly based on different detection methods, including single molecule fluorescence microscopy, surface plasmon resonance spectroscopy, X-ray microscopy, and surface enhanced Raman spectroscopy. The book also includes numerous basic principles of different methods and application examples and features illustrations that help clarify presentations. Single Particle Nanocatalysis: Fundamentals and Applications starts with the history and development of single molecule techniques for nanocatalysis. It then shows readers how single molecule fluorescence microscopy (SMFM) reveals catalytic kinetics and dynamics of individual nanocatalysts. Next, it examines traditional SMFM-based single molecule nanocatalysis without super-resolution (SR) imaging, before moving on to the topic of SMFM-based SR imaging in single molecule nanocatalysis. Following chapters cover scanning electrochemical microscopy for single particle nanocatalysis; surface plasmon resonance spectroscopy for single particle nanocatalysis/reactions; X-ray-based microscopy of single-particle nanocatalysis; and surface-enhanced Raman spectroscopy for single particle nanocatalysis. The book finishes by introducing some less-practiced techniques for single particle nanocatalysis/electrochemistry. -Presents a systematical and complete introduction to the subject of single particle nanocatalysis?covering all of its fundamentals and applications -Helps readers fully understand the basis, role, and recent development of single molecule nanocatalysis -Teaches researchers how to gain new knowledge to successfully conduct their own studies within this rapidly increasing new area of research Single Particle Nanocatalysis: Fundamentals and Applications is an excellent reference book for experts in this area as well as for general researchers who want to learn how to study nanocatalysis at single molecule/particle level.
Download or read book Time correlated single photon counting written by Desmond O'Connor and published by Academic Press. This book was released on 2012-12-02 with total page 299 pages. Available in PDF, EPUB and Kindle. Book excerpt: Time-correlated Single Photon Counting has been written in the hope that by relating the authors' experiences with a variety of different single photon counting systems, they may provide a useful service to users and potential users of this formidably sensitive technique. Of all the techniques available to obtain information on the rates of depopulation of excited electronic singlet states of molecular species, monitoring of fluorescence provides, in principle, the simplest and most direct measure of concentration. This volume comprises eight chapters, with the first focusing on the time dependence and applications of fluorescence. Succeeding chapters go on to discuss basic principles of the single photon counting lifetime measurement; light sources; photomultipliers; electronics; data analysis; nanosecond time-resolved emission spectroscopy; time dependence of fluorescence anisotropy. This book will be of interest to practitioners in the field of chemistry.
Download or read book Single Molecule Spectroscopy in Chemistry Physics and Biology written by Astrid Graslund and published by Springer Science & Business Media. This book was released on 2010-04-17 with total page 570 pages. Available in PDF, EPUB and Kindle. Book excerpt: Written by the leading experts in the field, this book describes the development and current state of the art in single molecule spectroscopy. The application of this technique, which started 1989, in physics, chemistry and biosciences is displayed.
