Single-molecule Spectroscopy Studies of Protein Conformational Dynamics in DNA Damage Recognition and Cell Signaling

Single-molecule Spectroscopy Studies of Protein Conformational Dynamics in DNA Damage Recognition and Cell Signaling
Author: Sunidhi Jaiswal
Publisher:
Total Pages: 0
Release: 2022
Genre: DNA
ISBN:


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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.

Protein Conformational Dynamics

Protein Conformational Dynamics
Author: Ke-li Han
Publisher: Springer
Total Pages: 0
Release: 2016-08-27
Genre: Medical
ISBN: 9783319353890


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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.

Single-Molecule Spectroscopy And Imaging Studies Of Protein Folding-Unfolding Conformational Dynamics

Single-Molecule Spectroscopy And Imaging Studies Of Protein Folding-Unfolding Conformational Dynamics
Author: Zijan Wang
Publisher:
Total Pages: 132
Release: 2016
Genre: Condensed matter
ISBN:


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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.

Measurement of Molecular Conformations and Dynamics Using Single Molecule Fluorescence Techniques

Measurement of Molecular Conformations and Dynamics Using Single Molecule Fluorescence Techniques
Author: Huimin Chen
Publisher:
Total Pages: 122
Release: 2012
Genre:
ISBN:


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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.

Fluorescence Fluctuation Spectroscopy Techniques to Quantify Molecular Interactions and Dynamics in Complex Biological Systems

Fluorescence Fluctuation Spectroscopy Techniques to Quantify Molecular Interactions and Dynamics in Complex Biological Systems
Author: Valentin Dunsing
Publisher:
Total Pages:
Release: 2020*
Genre:
ISBN:


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Living cells rely on transport and interaction of biomolecules to perform their diverse functions. A powerful toolbox to study these highly dynamic processes in the native environment is provided by fluorescence fluctuation spectroscopy (FFS) techniques. In more detail, FFS takes advantage of the inherent dynamics present in biological systems, such as diffusion, to infer molecular parameters from fluctuations of the signal emitted by an ensemble of fluorescently tagged molecules. In particular, two parameters are accessible: the concentration of molecules and their transit times through the observation volume. In addition, molecular interactions can be measured by analyzing the average signal emitted per molecule - the molecular brightness - and the cross-correlation of signals detected from differently tagged species. In the present work, several FFS techniques were implemented and applied in different biological contexts. In particular, scanning fluorescence correlation spectroscopy (sFCS) was performed to measure protein dynamics ...

Fluorescence Fluctuation Spectroscopy (FFS) Part B

Fluorescence Fluctuation Spectroscopy (FFS) Part B
Author:
Publisher: Academic Press
Total Pages: 395
Release: 2012-12-29
Genre: Science
ISBN: 0124058574


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This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers fluorescence fluctuation spectroscopy and includes chapters on such topics as Förster resonance energy transfer (fret) with fluctuation algorithms, protein corona on nanoparticles by FCS, and FFS approaches to the study of receptors in live cells. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers fluorescence fluctuation spectroscopy Contains chapters on such topics as Förster resonance energy transfer (fret) with fluctuation algorithms, protein corona on nanoparticles by FCS, and FFS approaches to the study of receptors in live cells