Download or read book Single Molecule Force Spectroscopy Studies of DNA Binding and Chaperone Proteins written by and published by . This book was released on 2008 with total page 155 pages. Available in PDF, EPUB and Kindle. Book excerpt: The nucleocapsid protein (NC) plays an important role in retroviral replication, in part, by facilitating numerous nucleic acid rearrangements throughout the reverse transcription process. The nucleic acid chaperone activity of the human immunodeficiency virus type-1 (HIV-1) NC has been extensively studied, and duplex destabilization, nucleic acid aggregation, and rapid protein binding kinetics have been identified as major components of the activity of this highly basic protein (pI ~10). The chaperone activity of other NC proteins is not well understood. We used single molecule DNA stretching to characterize the activity of HIV-1, RSV, and MMLV NC. We found distinct differences in the chaperone activities of each protein, which reflect the requirements for nucleic acid chaperone activity in each retroviral replication system. HTLV-1 NC exhibited overall poor nucleic acid chaperone acitivity. This result is explained by its poor aggregating activity and slow dissociation from single-stranded DNA. This NC protein is overall neutral at pH=7.5 and possesses a unique, acidic C-terminal domain. By studying different HTLV-1 NC mutants, the role of C-terminal domains to the chaperone activity was elucidated. The results suggest that the electrostatic interaction between HTLV-1 NC and nucleic acids is the major factor determining the kinetics. We also examine the nucleic acid interaction properties of the Apolipoprotein B mRNA editing enzyme, a catalytic polypeptide-like 3G (APOBEC3G/A3G) that is known to inhibit HIV-1 reverse transcription in absence of viral infectivity factor (Vif). Our stretching experiments suggested a novel mechanism for deaminase-independent inhibition of reverse transcription due to vital differences of nucleic acid binding kinetics between NC, A3G and reverse transcriptase (RT). Finally, Long interspersed nucleic elements (LINE) are highly repeated nucleic acids sequences in mammal genomes. Our single DNA molecule stretching experiments characterized the nucleic acid chaperone function of ORF1p in the mouse LINE-1 retrotransposon. We found that a single amino acid substitution altered retrotransposition efficiency by a factor of 15 due to a reduction in nucleic acid chaperone activity exhibited by ORF1p. For all of the studies presented here, we used single molecule methods to characterize the nucleic acid interactions of proteins involved in reverse transcription in retroviruses or retrotransposons. In each case, complementary bulk experiments were done by collaborators. The results are presented together in each chapter of the thesis.
Download or read book Single Molecule Science written by Krishnarao Appasani and published by Cambridge University Press. This book was released on 2022-05-26 with total page 171 pages. Available in PDF, EPUB and Kindle. Book excerpt: A comprehensive volume that brings together authoritative overviews of single molecule science techniques from a biological perspective.
Download or read book Regulation of DNA Metabolism by DNA Binding Proteins Probed by Single Molecule Spectroscopy written by and published by . This book was released on 2006 with total page 8 pages. Available in PDF, EPUB and Kindle. Book excerpt: Recent advances in single-molecule force spectroscopy of DNA make it possible to study the thermodynamics and kinetics of DNA binding proteins under a wide range of conditions. A biophysical model for the DNA binding T4 gene 32 protein has been developed to study the kinetics of DNA protein binding to transient single-stranded DNA regions due to thermal fluctuations. The model is used to analyze recent single-molecule spectroscopy data of this system.
Download or read book Single molecule Studies of Single stranded DNA Binding Proteins Involved in DNA Repair written by Jeffrey Mauritz Schaub and published by . This book was released on 2021 with total page 270 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Single Molecule Force Spectroscopy of Single Stranded DNA Binding Protein and Rep Helicase written by and published by . This book was released on 2012 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Single Molecule FRET Studies of DNA Binding and RNA Polymerizing Proteins written by Rahul Roy and published by . This book was released on 2007 with total page 298 pages. Available in PDF, EPUB and Kindle. Book excerpt: It has been proposed that the SSB tetramer can undergo migration on ssDNA (one-dimensional random walk) via a rolling mechanism by virtue of its four binding sites. DNA unwrapping necessary for such displacement was determined to be extremely fast and increases with increasing salt. Rapid translocation of SSB on ssDNA could also be tracked for the first time using single molecule methods. RecA protein mediated SSB removal was achieved using such rolling mechanism. Such efficient displacement of SSB and SSB's ability to disrupt DNA secondary structure stimulates RecA activity in the pre-synaptic stage of recombination.
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 Heterogeneity in Protein Folding Explored by Single Molecule Force Spectroscopy written by Dena Izadi and published by . This book was released on 2017 with total page 159 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book DNA Conforming Dynamics and Protein Binding written by and published by . This book was released on 2006 with total page 8 pages. Available in PDF, EPUB and Kindle. Book excerpt: Recent advances in single%molecule force spectroscopy of DNA make it possible to study the thermodynamics and kinetics of DNA binding proteins under a wide range of conditions. A biophysical model for the DNA binding T4 gene 32 protein has been developed to study the kinetics of DNA protein binding to transient single-stranded DNA regions due to thermal fluctuations. The model is used to analyze recent single-molecule spectroscopy data of this system.
Download or read book Overstretching Short DNA written by Niklas Bosaeus and published by . This book was released on 2013 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Single molecule and Ensemble Fluorescence Studies of Single stranded DNA and Replication Proteins written by Mary Cathleen McKinney and published by . This book was released on 2006 with total page 216 pages. Available in PDF, EPUB and Kindle. Book excerpt: DNA replication is a complex process involving many proteins that must be performed for a cell to proliferate. In this work, we will study three members of the replication system: single-stranded DNA, single-stranded DNA binding proteins, and the sliding clamp and clamp loader proteins. Fluorescence studies have been widely used to study the properties of biomolecules and here we will use both Fluorescence Polarization Anisotropy (FPA) and Fluorescence Resonance Energy Transfer (FRET) to study these biomolecules. First, we present a method by which single molecule FRET measurements can be used to determine the persistence length of single-stranded DNA. Our results indicate that the persistence length decreases from 3.0nm to 1.5nm as the salt concentration increases from 50mM NaCl to 2M NaCl. Furthermore, we use fluoresce anisotropy and FRET measurements in ensemble studies to examine properties of single stranded DNA binding proteins from archaeal organisms. Our results will show that the Archaea have exploited the single stranded DNA binding fold motif unlike the other domains of life to create a wide variety of proteins that have different binding properties leading to the probable conclusion that many of the proteins area a result of gene duplication and recombination events. Finally, we will examine the properties of the clamp loader protein as it loads a fluorescently labeled clamp to DNA on both the ensemble and single molecule measurements. Our findings show that loading the clamp is a multi-step process with different conformational states that can be observed for the first time at a single molecule level. We also will examine mutations of the clamp loader protein to bring further insight into the not fully understood Methanosarcina acetivorans clamp loader.
Download or read book Single Molecule Science written by Krishnarao Appasani and published by . This book was released on 2022-05-11 with total page 172 pages. Available in PDF, EPUB and Kindle. Book excerpt: A comprehensive volume that brings together authoritative overviews of single molecule science techniques from a biological perspective.
Download or read book Biophysics of DNA Protein Interactions written by Mark C. Williams and published by Springer Science & Business Media. This book was released on 2010-10-05 with total page 354 pages. Available in PDF, EPUB and Kindle. Book excerpt: Depite the rapid expansion of the field of biophysics, there are very few books that comprehensively treat specific topics in this area. Recently, the field of single molecule biophysics has developed very quickly, and a few books specifically treating single molecule methods are beginning to appear. However, the promise of single molecule biophysics is to contribute to the understanding of specific fields of biology using new methods. This book would focus on the specific topic of the biophysics of DNA-protein interactions, and would include the use of new approaches, including both bulk methods as well as single molecule methods. This would make the book attractive to anyone working in the general area of DNA-protein interactions, which is of course a much wider market than just single molecule biophysicists or even biophysicists. The subject of the book will be the biophysics of DNA-protein interactions, and will include new methods and results that describe the physical mechanism by which proteins interact with DNA. For example, there has been much recent work on the mechanism by which proteins search for specific binding sites on DNA. A few chapters will be devoted to experiments and theory that shed light on this important problem. We will also cover proteins that alter DNA properties to facilitate interactions important for transcription or replication. Another section of the book will cover the biophysical mechanism by which motor proteins interact with DNA. Finally, we will cover larger protein-DNA complexes, such as replication forks, recombination complexes, DNA repair interactions, and their chromatin context.
Download or read book Single molecule Fluorescence Studies of Protein Folding and Molecular Chaperones written by Martin Sikor and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Thermodynamics and Kinetics of DNA protein Interactions from Single Molecule Force Spectroscopy written by Leila Shokri and published by . This book was released on 2008 with total page 157 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Handbook of Single Molecule Biophysics written by Peter Hinterdorfer and published by Springer Science & Business Media. This book was released on 2009-12-24 with total page 634 pages. Available in PDF, EPUB and Kindle. Book excerpt: This handbook describes experimental techniques to monitor and manipulate individual biomolecules, including fluorescence detection, atomic force microscopy, and optical and magnetic trapping. It includes single-molecule studies of physical properties of biomolecules such as folding, polymer physics of protein and DNA, enzymology and biochemistry, single molecules in the membrane, and single-molecule techniques in living cells.
Download or read book Biophysics of DNA written by Alexander Vologodskii and published by Cambridge University Press. This book was released on 2015-03-12 with total page 279 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book surveys the last sixty years of research in the rapidly advancing field of DNA biophysics, addressing key questions and facilitating further research.
