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:
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.
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 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.
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.
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.
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 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.
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:
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:
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
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.
Download or read book Single Molecule Biology written by Alexander E. Knight and published by Academic Press. This book was released on 2009-02-26 with total page 369 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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
Download or read book Single Molecular Spectroscopy and Atomic Force Manipulation of Protein Conformation and Dynamics written by Jin Cao and published by . This book was released on 2014 with total page 122 pages. Available in PDF, EPUB and Kindle. Book excerpt: The present thesis describes the study of protein conformation dynamics probed by single molecule techniques. Protein structural dynamic have been studied for many years. Interesting topics such as protein folding/unfolding, protein denature, and protein-protein interactions had been widely studied. Scientists are still interested in questions like: what exact forces contribute to the stability of protein secondary and tertiary structure; why different protein has different function based on their structure. Single molecular spectroscopy has the advantage to observe inhomogeneous behavior of protein molecule compared to ensemble measurement. The typical approach of single molecular spectroscopy is to record one single molecule at a time and repeat this process at one or multiple molecules that are chemically identical. Then use statistics way to analyze these data. The binding dynamics of CaM-C28W complex was studied using single molecule FRET experiment. The statistics results indicate the inhomogeneous nature of the process and the evidence of a slower process gauging the binding dynamics has been claimed. Furthermore, the responding mechanical dynamics of CaM molecule under AFM tip manipulation has been investigated by AFM-FRET correlated measurement. The repeated "collapsing" pattern in the force curve leads us to deeper study on the process. The nature of the process and the dynamics of the underlying conformation change have also been detailed described. Lastly, a similar AFM manipulation experiment has been performed on EGFR dimer complex that has more biological potential in terms of dimer formation and aggregation dynamics. In order to fit the nature of the molecule, we used two identical dyes to label either monomer. Instead of single molecule FRET, photo-stamping measurement has been used to build an AFM-optical correlated measurement. The statistics results shows the presents of multiple intermediate states. With the help of known crystal structure and the knowledge of EGFR dimer complex, we managed to find clue from our correlated results and assign possible conformation of each intermediate states. A possible conjugated state upon collapsing has been claimed.
Download or read book Computational Modeling of Biological Systems written by Nikolay V Dokholyan and published by Springer Science & Business Media. This book was released on 2012-02-12 with total page 360 pages. Available in PDF, EPUB and Kindle. Book excerpt: Computational modeling is emerging as a powerful new approach to study and manipulate biological systems. Multiple methods have been developed to model, visualize, and rationally alter systems at various length scales, starting from molecular modeling and design at atomic resolution to cellular pathways modeling and analysis. Higher time and length scale processes, such as molecular evolution, have also greatly benefited from new breeds of computational approaches. This book provides an overview of the established computational methods used for modeling biologically and medically relevant systems.
Download or read book Conformational Dynamics of Proteins by Fluorescence Fluctuation Spectroscopy written by Robel Birru Yirdaw and published by . This book was released on 2012 with total page 151 pages. Available in PDF, EPUB and Kindle. Book excerpt:
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.
