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B. Norden - One of the best experts on this subject based on the ideXlab platform.
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Structure of human Rad51 Protein Filament from molecular modeling and site-specific linear dichroism spectroscopy
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: A. Reymer, K. Frykholm, K. Morimatsu, M. Takahashi, B. NordenAbstract:To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a Filament structure of a human Rad51 Protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 Protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA Filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleo-Protein complex. An inhomogeneously stretched double-stranded DNA fitted into the Filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-Protein Filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning Protein monomers along the DNA strand upon Filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our Filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.
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A Combined Approach for Structure Determination of a Human Rad51 Protein Filament: from Computer Modeling to Site-Specific Linear Dichroism
Biophysical Journal, 2009Co-Authors: A. Reymer, K. Frykholm, B. NordenAbstract:The human Rad51 Protein plays a crucial role in homologous recombination and DNA repair. The details of the recombination process, which is essential to all cells and has been evolutionarily conserved, are still to be revealed. Structural information on Rad51-DNA complexes in solution can contribute to mechanistic insight. We here combine molecular modeling of the Filamentous structure of human Rad51 Protein with experimental data for angular orientations of aromatic residues of a Rad51-DNA Filament in solution obtained by Site-Specific Linear Dichroism (SSLD), a spectroscopic technique in combination with Protein engineering. The resulting structural model is in fair agreement with a Filament structure previously deduced from electron microscopy. We show that the Filament has ability to house a DNA molecule and that putative DNA binding loops are strategically positioned for interactions with DNA.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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nucleotide cofactor dependent structural change of xenopus laevis rad51 Protein Filament detected by small angle neutron scattering measurements in solution
Biochemistry, 1997Co-Authors: Christine Ellouze, B. Norden, Kazuhiro Maeshima, Eimer Tuite, Katsumi Morimatsu, Toshihiro Horii, Kell Mortensen, Masayuki TakahashiAbstract:Rad51 Protein, a eukaryotic homologue of RecA Protein, forms a Filamentous complex with DNA and catalyzes homologous recombination. We have analyzed the structure of Xenopus Rad51 Protein (XRad51.1) in solution by small-angle neutron scattering (SANS). The measurements showed that XRad51.1 forms a helical Filament independently of DNA. The sizes of the cross-sectional and helical pitch of the Filament could be determined, respectively, from a Guinier plot and the position of the subsidiary maximum of SANS data. We observed that the helical structure is modified by nucleotide binding as in the case of RecA. Upon ATP binding under high-salt conditions (600 mM NaCl), the helical pitch of XRad51.1 Filament was increased from 8 to 10 nm and the cross-sectional diameter decreased from 7 to 6 nm. The pitch sizes of XRad51.1 are similar to, though slightly larger than, those of RecA Filament under corresponding conditions. A similar helical pitch size was observed by electron microscopy for budding yeast Rad51 [Ogawa, T., et al. (1993) Science 259, 1896-1899]. In contrast to the RecA Filament, the structure of XRad51.1 Filament with ADP is not significantly different from that with ATP. Thus, the hydrolysis of ATP to ADP does not modify the helical Filament of XRad51.1. Together with our recent observation that ADP does not weaken the XRad51.1/DNA interaction, the effect of ATP hydrolysis on XRad51.1 nucleoFilament should be very different from that on RecA.
A. Reymer - One of the best experts on this subject based on the ideXlab platform.
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Structure of human Rad51 Protein Filament from molecular modeling and site-specific linear dichroism spectroscopy
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: A. Reymer, K. Frykholm, K. Morimatsu, M. Takahashi, B. NordenAbstract:To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a Filament structure of a human Rad51 Protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 Protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA Filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleo-Protein complex. An inhomogeneously stretched double-stranded DNA fitted into the Filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-Protein Filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning Protein monomers along the DNA strand upon Filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our Filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.
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A Combined Approach for Structure Determination of a Human Rad51 Protein Filament: from Computer Modeling to Site-Specific Linear Dichroism
Biophysical Journal, 2009Co-Authors: A. Reymer, K. Frykholm, B. NordenAbstract:The human Rad51 Protein plays a crucial role in homologous recombination and DNA repair. The details of the recombination process, which is essential to all cells and has been evolutionarily conserved, are still to be revealed. Structural information on Rad51-DNA complexes in solution can contribute to mechanistic insight. We here combine molecular modeling of the Filamentous structure of human Rad51 Protein with experimental data for angular orientations of aromatic residues of a Rad51-DNA Filament in solution obtained by Site-Specific Linear Dichroism (SSLD), a spectroscopic technique in combination with Protein engineering. The resulting structural model is in fair agreement with a Filament structure previously deduced from electron microscopy. We show that the Filament has ability to house a DNA molecule and that putative DNA binding loops are strategically positioned for interactions with DNA.View Large Image | View Hi-Res Image | Download PowerPoint Slide
Tuomas P. J. Knowles - One of the best experts on this subject based on the ideXlab platform.
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Stochastic calculus of Protein Filament formation under spatial confinement
New Journal of Physics, 2018Co-Authors: Thomas C T Michaels, Alexander J Dear, Tuomas P. J. KnowlesAbstract:The growth of Filamentous aggregates from precursor Proteins is a process of central importance to both normal and aberrant biology, for instance as the driver of devastating human disorders such as Alzheimer's and Parkinson's diseases. The conventional theoretical framework for describing this class of phenomena in bulk is based upon the mean-field limit of the law of mass action, which implicitly assumes deterministic dynamics. However, Protein Filament formation processes under spatial confinement, such as in microdroplets or in the cellular environment, show intrinsic variability due to the molecular noise associated with small-volume effects. To account for this effect, in this paper we introduce a stochastic differential equation approach for investigating Protein Filament formation processes under spatial confinement. Using this framework, we study the statistical properties of stochastic aggregation curves, as well as the distribution of reaction lag-times. Moreover, we establish the gradual breakdown of the correlation between lag-time and normalized growth rate under spatial confinement. Our results establish the key role of spatial confinement in determining the onset of stochasticity in Protein Filament formation and offer a formalism for studying Protein aggregation kinetics in small volumes in terms of the kinetic parameters describing the aggregation dynamics in bulk.
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Physical principles of Filamentous Protein self-assembly kinetics.
Journal of physics. Condensed matter : an Institute of Physics journal, 2017Co-Authors: Thomas C T Michaels, Lucie X. Liu, Georg Meisl, Tuomas P. J. KnowlesAbstract:The polymerization of Proteins and peptides into Filamentous supramolecular structures is an elementary form of self-organization of key importance to the functioning biological systems, as in the case of actin bioFilaments that compose the cellular cytoskeleton. Aberrant Filamentous Protein self-assembly, however, is associated with undesired effects and severe clinical disorders, such as Alzheimer's and Parkinson's diseases, which, at the molecular level, are associated with the formation of certain forms of Filamentous Protein aggregates known as amyloids. Moreover, due to their unique physicochemical properties, Protein Filaments are finding extensive applications as biomaterials for nanotechnology. With all these different factors at play, the field of Filamentous Protein self-assembly has experienced tremendous activity in recent years. A key question in this area has been to elucidate the microscopic mechanisms through which Filamentous aggregates emerge from dispersed Proteins with the goal of uncovering the underlying physical principles. With the latest developments in the mathematical modeling of Protein aggregation kinetics as well as the improvement of the available experimental techniques it is now possible to tackle many of these complex systems and carry out detailed analyses of the underlying microscopic steps involved in Protein Filament formation. In this paper, we review some classical and modern kinetic theories of Protein Filament formation, highlighting their use as a general strategy for quantifying the molecular-level mechanisms and transition states involved in these processes.
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Scaling and dimensionality in the chemical kinetics of Protein Filament formation
International Reviews in Physical Chemistry, 2016Co-Authors: Thomas C T Michaels, Alexander J Dear, Tuomas P. J. KnowlesAbstract:The formation of elongated supra-molecular structures from Protein building blocks generates functional intracellular Filaments, but this process is also at the heart of many neurodegenerative conditions including Alzheimer’s and Parkinson’s diseases, where it occurs in an uncontrolled manner. When observed at appropriate concentration and time scales, the chemical kinetics of Filamentous Protein self-assembly exhibits the remarkable property of self-similarity: the dynamics appears similar as the observation scale changes. We discuss here how this property leads to crucial simplifications of the fundamental laws governing Protein Filament formation and the emergence of scaling laws that provide the basis for connecting microscopic events with macroscopic realisations of such processes. In particular, we review recent developments in the modelling of linear Protein self-assembly phenomena in the light of the concepts of dimensional analysis and physical self-similarity. We show how these tools and concept...
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fluctuations in the kinetics of linear Protein self assembly
Physical Review Letters, 2016Co-Authors: Thomas C T Michaels, David A Weitz, Alexander J Dear, Julius B Kirkegaard, Kadi L Saar, Tuomas P. J. KnowlesAbstract:Biological systems are characterized by compartmentalization from the subcellular to the tissue level, and thus reactions in small volumes are ubiquitous in living systems. Under such conditions, statistical number fluctuations, which are commonly negligible in bulk reactions, can become dominant and lead to stochastic behavior. We present here a stochastic model of Protein Filament formation in small volumes. We show that two principal regimes emerge for the system behavior, a small fluctuation regime close to bulk behavior and a large fluctuation regime characterized by single rare events. Our analysis shows that in both regimes the reaction lag-time scales inversely with the system volume, unlike in bulk. Finally, we use our stochastic model to connect data from small-volume microdroplet experiments of amyloid formation to bulk aggregation rates, and show that digital analysis of an ensemble of Protein aggregation reactions taking place under microconfinement provides an accurate measure of the rate of primary nucleation of Protein aggregates, a process that has been challenging to quantify from conventional bulk experiments.
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Hamiltonian Dynamics of Protein Filament Formation.
Physical review letters, 2016Co-Authors: Thomas C T Michaels, Samuel I. A. Cohen, Michele Vendruscolo, Christopher M. Dobson, Tuomas P. J. KnowlesAbstract:We establish the Hamiltonian structure of the rate equations describing the formation of Protein Filaments. We then show that this formalism provides a unified view of the behavior of a range of biological self-assembling systems as diverse as actin, prions, and amyloidogenic polypeptides. We further demonstrate that the time-translation symmetry of the resulting Hamiltonian leads to previously unsuggested conservation laws that connect the number and mass concentrations of fibrils and allow linear growth phenomena to be equated with autocatalytic growth processes. We finally show how these results reveal simple rate laws that provide the basis for interpreting experimental data in terms of specific mechanisms controlling the proliferation of fibrils.
K. Frykholm - One of the best experts on this subject based on the ideXlab platform.
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Structure of human Rad51 Protein Filament from molecular modeling and site-specific linear dichroism spectroscopy
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: A. Reymer, K. Frykholm, K. Morimatsu, M. Takahashi, B. NordenAbstract:To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a Filament structure of a human Rad51 Protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 Protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA Filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleo-Protein complex. An inhomogeneously stretched double-stranded DNA fitted into the Filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-Protein Filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning Protein monomers along the DNA strand upon Filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our Filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.
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A Combined Approach for Structure Determination of a Human Rad51 Protein Filament: from Computer Modeling to Site-Specific Linear Dichroism
Biophysical Journal, 2009Co-Authors: A. Reymer, K. Frykholm, B. NordenAbstract:The human Rad51 Protein plays a crucial role in homologous recombination and DNA repair. The details of the recombination process, which is essential to all cells and has been evolutionarily conserved, are still to be revealed. Structural information on Rad51-DNA complexes in solution can contribute to mechanistic insight. We here combine molecular modeling of the Filamentous structure of human Rad51 Protein with experimental data for angular orientations of aromatic residues of a Rad51-DNA Filament in solution obtained by Site-Specific Linear Dichroism (SSLD), a spectroscopic technique in combination with Protein engineering. The resulting structural model is in fair agreement with a Filament structure previously deduced from electron microscopy. We show that the Filament has ability to house a DNA molecule and that putative DNA binding loops are strategically positioned for interactions with DNA.View Large Image | View Hi-Res Image | Download PowerPoint Slide
Wesley Wong - One of the best experts on this subject based on the ideXlab platform.
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single molecule force spectroscopy reveals the dynamic strength of the hair cell tip link connection
Nature Communications, 2021Co-Authors: Eric M Mulhall, Andrew Ward, Darren Yang, Mounir A Koussa, David P Corey, Wesley WongAbstract:The conversion of auditory and vestibular stimuli into electrical signals is initiated by force transmitted to a mechanotransduction channel through the tip link, a double stranded Protein Filament held together by two adhesion bonds in the middle. Although thought to form a relatively static structure, the dynamics of the tip-link connection has not been measured. Here, we biophysically characterize the strength of the tip-link connection at single-molecule resolution. We show that a single tip-link bond is more mechanically stable relative to classic cadherins, and our data indicate that the double stranded tip-link connection is stabilized by single strand rebinding facilitated by strong cis-dimerization domains. The measured lifetime of seconds suggests the tip-link is far more dynamic than previously thought. We also show how Ca2+ alters tip-link lifetime through elastic modulation and reveal the mechanical phenotype of a hereditary deafness mutation. Together, these data show how the tip link is likely to function during mechanical stimuli. The conversion of auditory and vestibular stimuli into electrical signals is initiated by force transmitted to a mechanotransduction channel through the tip link. Here authors show that a single tip-link bond is more mechanically stable relative to classic cadherins, and that the double stranded tip-link connection is stabilized by single strand rebinding facilitated by strong cis-dimerization domains.
