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Probing The Conformational Dynamics Of Membrane Associated Proteins Using Single Molecule Fluorescence
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Book Synopsis Probing the Conformational Dynamics of Membrane-associated Proteins Using Single Molecule Fluorescence by : Devdoot Majumdar
Download or read book Probing the Conformational Dynamics of Membrane-associated Proteins Using Single Molecule Fluorescence written by Devdoot Majumdar and published by . This book was released on 2009 with total page 224 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Single-Molecule Spectroscopy And Imaging Studies Of Protein Folding-Unfolding Conformational Dynamics by : Zijan Wang
Download or read book Single-Molecule Spectroscopy And Imaging Studies Of Protein Folding-Unfolding Conformational Dynamics written by Zijan Wang and published by . This book was released on 2016 with total page 132 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein conformational dynamics often plays a critical role in protein functions. We have characterized the spontaneous folding-unfolding conformational fluctuation dynamics of calmodulin (CaM) at thermodynamic equilibrium conditions by using single-molecule fluorescence resonance energy transfer (FRET) spectroscopy. We studied protein folding dynamics under simulated biological conditions to gain a deep, mechanistic understanding of this important biological process. We have identified multiple folding transition pathways and characterized the underlying energy landscape of the single-molecule protein conformational fluctuation trajectories. Our results suggest that the folding dynamics of CaM molecules involves a complex multiple-pathway multiple-state energy landscape, rather than an energy landscape of two-state dynamical process. Our probing single-molecule FRET fluctuation experiments demonstrate a new approach of studying spontaneous protein folding-unfolding conformational dynamics at the equilibrium that features recording long time single-molecule conformational fluctuation trajectories. This technique yields rich statistical and dynamical information far beyond traditional ensemble-averaged measurements. We characterize the conformational dynamics of single CaM interacting with C28W. The single CaM molecules are partially unfolded by GdmCl, and the folded and unfolded CaM molecules are approximately equally populated. Under this condition, the majority of the single protein CaM undergoes spontaneous folding-unfolding conformational fluctuations. Using single molecule FRET spectroscopy, we study each of the single protein’s conformational dynamics inthe presence of C28W-CaM interactions. The results show an interesting folding-upon-binding dynamic process, and a conformational selection mechanism is further confirmed. The effect of molecular crowding on protein folding process is a key issue in the understanding of protein folding dynamics in living cells. Due to the complexity and interplay between various interactions existing in an equally favored environment of protein folding and unfolding conformational dynamics, such simple reduced entropic enhancement model do not suffice in describing protein folding conformational dynamics. We observe, at higher concentration of crowding reagent Ficoll 70, single protein molecules spontaneously denature into unfolded proteins which involves a combined process of polymer-polymer interaction, entropic effects and solvation thermodynamics and dynamics. Such heterogeneous unfolding process can serve as a first step to a mechanistic understanding of living cell disease as a result of molecular crowding effect, protein aggregates and fibril formation.
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 High-resolution Single-molecule Spectroscopy to Probe Conformational Dynamics of Proteins by : Christian Gebhardt
Download or read book High-resolution Single-molecule Spectroscopy to Probe Conformational Dynamics of Proteins written by Christian Gebhardt and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Observing Protein Dynamics and Conformational Changes by Ensemble and Single-molecule Fluorescence Spectroscopy by : So Yeon Kim
Download or read book Observing Protein Dynamics and Conformational Changes by Ensemble and Single-molecule Fluorescence Spectroscopy written by So Yeon Kim and published by . This book was released on 2008 with total page 420 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Single-molecule Studies of Proteins by : Andres F. Oberhauser
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.
Book Synopsis Intrinsically Disordered Proteins by :
Download or read book Intrinsically Disordered Proteins written by and published by Academic Press. This book was released on 2018-11-21 with total page 756 pages. Available in PDF, EPUB and Kindle. Book excerpt: Intrinsically Disordered Proteins, Volume 611, the latest release in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on topics of interest, including the Characterization of Structure-Function relationships in the intrinsically disordered protein complexin, Distances, distance distributions, and ensembles of IDPs from single-molecule FRET, Biophysical characterization of disordered protein liquid phases, The Use of Mass Spectrometry to Examine IDPs – Unique Insights and Caveats, Fluorescence Depolarization Kinetics to Study Conformational Preference, Structural Plasticity and Membrane Binding of Intrinsically Disordered Proteins, Characterizing the Function of Intrinsically Disordered Proteins in the Circadian Clock, and more. Breadth of experimental approaches and systems that will be covered The expertise of the contributors writing the articles
Book Synopsis Single Proteins Under the Microscope by : Baoxu Liu
Download or read book Single Proteins Under the Microscope written by Baoxu Liu and published by . This book was released on 2013 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Single-Molecule Fluorescence Spectroscopy of the Folding of a Repeat Protein by : Sharona Cohen
Download or read book Single-Molecule Fluorescence Spectroscopy of the Folding of a Repeat Protein written by Sharona Cohen and published by Springer. This book was released on 2015-10-17 with total page 70 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this thesis single-molecule fluorescence resonance energy transfer (FRET) spectroscopy was used to study the folding of a protein that belongs to the large and important family of repeat proteins. Cohen shows that the dynamics of the expanded conformations is likely to be very fast, suggesting a spring-like motion of the whole chain. The findings shed new light on the elasticity of structure in repeat proteins, which is related to their function in binding multiple and disparate partners. This concise research summary provides useful insights for students beginning a PhD in this or a related area, and researchers entering this field.
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 Single-molecule Spectroscopy Studies of Protein Conformational Dynamics in DNA Damage Recognition and Cell Signaling by : Sunidhi Jaiswal
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.
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 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 Fluorescent Methods to Study Biological Membranes by : Yves Mely
Download or read book Fluorescent Methods to Study Biological Membranes written by Yves Mely and published by Springer Science & Business Media. This book was released on 2012-10-10 with total page 484 pages. Available in PDF, EPUB and Kindle. Book excerpt: Biological membranes play a central role in cell structure, shape and functions. However, investigating the membrane bilayer has proved to be difficult due to its highly dynamic and anisotropic structure, which generates steep gradients at the nanometer scale. Due to the decisive impact of recently developed fluorescence-based techniques, tremendous advances have been made in the last few years in our understanding of membrane characteristics and functions. In this context, the present book illustrates some of these major advances by collecting review articles written by highly respected experts. The book is organized in three parts, the first of which deals with membrane probes and model membranes. The second part describes the use of advanced quantitative and high-resolution techniques to explore the properties of biological membranes, illustrating the key progress made regarding membrane organization, dynamics and interactions. The third part is focused on the investigation of membrane proteins using the same techniques, and notably on the membrane receptors that play a central role in signaling pathways and therapeutic strategies. All chapters provide comprehensive information on membranes and their exploration for beginners in the field and advanced researchers alike.
Book Synopsis Single-Molecule Spectroscopy Studies of the Conformational Dynamics of Enzymes by : Maolin Lu
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.
Book Synopsis Sensing Conformational Dynamics of Single Trapped Proteins in Solution by : Samuel David Bockenhauer
Download or read book Sensing Conformational Dynamics of Single Trapped Proteins in Solution written by Samuel David Bockenhauer and published by . This book was released on 2013 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are macromolecular nanomachines that perform a wide variety of essential functions in living organisms. Distinct types of proteins in the cell serve as molecular sensors and signalers, sunlight-harvesting antennas and transducers, molecular motors for transport and metabolic energy storage, and oxidizers and reducers of physiological metal ions, to name a few. To achieve these complex functions, proteins are not static building blocks, but instead adopt multiple conformations in distinct functional states. These functions are inherently dynamic, meaning that methods capable of resolving dynamic behavior are necessary to fully elucidate protein function. Many bulk fluorescence methods have been used to study proteins by averaging together the signals from many copies of the same protein. However, such methods fail to capture distributions in protein conformations (e.g. mixtures of active and inactive states) and unsynchronized dynamics among these conformations (e.g. transition rates from inactive to active states under various conditions). Single-molecule fluorescence spectroscopy of proteins, on the other hand, allows direct observation of distributions and dynamics by watching only one protein at a time. In particular, a special device known as the Anti-Brownian ELectrokinetic (ABEL) trap can hold single fluorescently labeled proteins in solution for several seconds of spectroscopic observation without surface attachment, encapsulation, or the use of large beads. The ABEL trap combines fluorescence-based position estimation obtained by scanning a laser spot with the application of electrokinetic feedback forces to counter the Brownian motion of single proteins, one at a time. In this Dissertation I will describe my use of the ABEL trap technique to study dynamics of a variety of biomolecules, with emphasis on two proteins: the [beta]2-adrenergic receptor ([beta]2AR), an essential cellular signaling protein, and the peridinin-chlorophyll-protein (PCP), a sunlight-harvesting pigment-protein complex found in algae. In [beta]2AR, I observed a shift in protein conformation and in time scales of protein dynamics upon binding of an activating drug. In PCP, I observed two distinct classes of conformational change, indicating light-induced conformational flexibility, which may play a physiological role. Ongoing projects include resolving conformational substeps in FoF1 ATP synthase and measuring electron transfer kinetics of the multicopper oxidase Fet3p.
Book Synopsis Probing and Manipulating Protein Conformation Changes by Time-Resolved Single-Molecule Spectroscopy and Site-Specific Ultramicroscopy by :
Download or read book Probing and Manipulating Protein Conformation Changes by Time-Resolved Single-Molecule Spectroscopy and Site-Specific Ultramicroscopy written by and published by . This book was released on 2008 with total page 9 pages. Available in PDF, EPUB and Kindle. Book excerpt: This project was to develop and demonstrate a novel single-molecule spectroscopy and imaging technique to support the DARPA program on Control of Protein Conformations. The goal of our project is to advance, integrate, and apply cutting-edge atomic force microscopy (AFM) and single-molecule imaging techniques to control and analyze the protein conformational changes and according activity changes through AFM force manipulation and fluorescence resonant energy transfer (FRET) measurements as a function of local site geometry and molecular structure. In this project, we have focused our demonstration and study on controlling protein conformations to manipulate the enzyme reactivity, affinity, and selectivity. Our approach has been to use AFM tip to control protein conformational states and to apply single-molecule FRET imaging to measure the protein enzyme activity in real time. Our progress towards the demonstration of the new technology has provided an unprecedented advancement on a high spatially (nanometers) and temporally (micro's to seconds) resolved single-molecule spectroscopy of optically and mechanically controlling single-molecule protein conformational changes and activities.
