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1.
Nucleic Acids Res ; 48(3): 1551-1571, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31956896

RESUMO

Chromatin compaction and gene accessibility are orchestrated by assembly and disassembly of nucleosomes. Although the disassembly process was widely studied, little is known about the structure and dynamics of the disordered histone tails, which play a pivotal role for nucleosome integrity. This is a gap filling experimental FRET study from the perspective of the histone H3 N-terminal tail (H3NtT) of reconstituted mononucleosomes. By systematic variation of the labeling positions we monitored the motions of the H3NtT relative to the dyad axis and linker DNA. Single-molecule FRET unveiled that H3NtTs do not diffuse freely but follow the DNA motions with multiple interaction modes with certain permitted dynamic transitions in the µs to ms time range. We also demonstrate that the H3NtT can allosterically sense charge-modifying mutations within the histone core (helix α3 of histone H2A (R81E/R88E)) resulting in increased dynamic transitions and lower rate constants. Those results complement our earlier model on the NaCl induced nucleosome disassembly as changes in H3NtT configurations coincide with two major steps: unwrapping of one linker DNA and weakening of the internal DNA - histone interactions on the other side. This emphasizes the contribution of the H3NtT to the fine-tuned equilibrium between overall nucleosome stability and DNA accessibility.


Assuntos
Cromatina/genética , DNA/ultraestrutura , Histonas/isolamento & purificação , Nucleossomos/genética , Animais , Montagem e Desmontagem da Cromatina , DNA/química , DNA/genética , Transferência Ressonante de Energia de Fluorescência , Histonas/química , Histonas/genética , Mutação/genética , Nanotecnologia , Conformação de Ácido Nucleico , Nucleossomos/química , Ligação Proteica/genética , Imagem Individual de Molécula , Xenopus laevis/genética
2.
Biophys J ; 114(10): 2465-2472, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29759373

RESUMO

Using fluorescence correlation spectroscopy in single-plane illumination microscopy, we investigated the dynamics of chromatin in interphase mouse adult fibroblast cell nuclei under the influence of the intermediate filament protein lamin A. We find that 1) lamin A-eGFP and histone H2A-mRFP show significant comobility, indicating that their motions are clearly interconnected in the nucleus, and 2) that the random motion of histones H2A within the chromatin network is subdiffusive, i.e., the effective diffusion coefficient decreases for slow timescales. Knocking out lamin A changes the diffusion back to normal. Thus, lamin A influences the dynamics of the entire chromatin network. Our conclusion is that lamin A plays a central role in determining the viscoelasticity of the chromatin network and helping to maintain local ordering of interphase chromosomes.


Assuntos
Cromatina/metabolismo , Lamina Tipo A/metabolismo , Movimento , Animais , Difusão , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos
3.
Biophys J ; 114(10): 2455-2464, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29753448

RESUMO

Photon-counting sensors based on standard complementary metal-oxide-semiconductor single-photon avalanche diodes (SPADs) represent an emerging class of imagers that enable the counting and/or timing of single photons at zero readout noise (better than high-speed electron-multiplying charge-coupling devices) and over large arrays. They have seen substantial progress over the last 15 years, increasing their spatial resolution, timing accuracy, and sensitivity while reducing spurious signals such as afterpulsing and dark counts. They are increasingly being applied for time-resolved applications with the added advantage of enabling real-time options such as autocorrelation. We report in this study on the use of such a state-of-the-art 512 × 128 SPAD array, capable of a time resolution of 10-5-10-6 s for full frames while retaining acceptable photosensitivity thanks to the use of dedicated microlenses, in a selective plane illumination-fluorescence correlation spectroscopy setup. The latter allows us to perform thousands of fluorescence-correlation spectroscopy measurements simultaneously in a two-dimensional slice of the sample. This high-speed SPAD imager enables the measurement of molecular motion of small fluorescent particles such as single chemical dye molecules. Inhomogeneities in the molecular detection efficiency were compensated for by means of a global fit of the auto- and cross-correlation curves, which also made a calibration-free measurement of various samples possible. The afterpulsing effect could also be mitigated, making the measurement of the diffusion of Alexa-488 possible, and the overall result quality was further improved by spatial binning. The particle concentrations in the focus tend to be overestimated by a factor of 1.7 compared to a confocal setup; a calibration is thus required if absolute concentrations need to be measured. The first high-speed selective plane illumination-fluorescence correlation spectroscopy in vivo measurements to our knowledge were also recorded: although two-component fit models could not be employed because of noise, the diffusion of eGFP oligomers in HeLa cells could be measured. Sensitivity and noise will be further improved in the next generation of SPAD-based widefield sensors, which are currently under testing.


Assuntos
Microscopia de Fluorescência/instrumentação , Fótons , Semicondutores , Células HeLa , Humanos , Fatores de Tempo
4.
Biophys J ; 114(10): 2386-2396, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29248151

RESUMO

Human mitochondrial transcription factor A (TFAM) distorts DNA into a U-turn, as shown by crystallographic studies. The relevance of this U-turn is associated with transcription initiation at the mitochondrial light strand promoter (LSP). However, it has not been yet discerned whether a tight U-turn or an alternative conformation, such as a V-shape, is formed in solution. Here, single-molecule FRET experiments on freely diffusing TFAM/LSP complexes containing different DNA lengths show that a DNA U-turn is induced by progressive and cooperative binding of the two TFAM HMG-box domains and the linker between them. SAXS studies further show compaction of the protein upon complex formation. Finally, molecular dynamics simulations reveal that TFAM/LSP complexes are dynamic entities, and the HMG boxes induce the U-turn against the tendency of the DNA to adopt a straighter conformation. This tension is resolved by reversible unfolding of the linker, which is a singular mechanism that allows a flexible protein to stabilize a tight bending of DNA.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Fenômenos Mecânicos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fenômenos Biomecânicos , Difusão , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Domínios Proteicos , Soluções
5.
Biophys J ; 114(10): 2408-2418, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29754715

RESUMO

Intermediate filaments (IFs) are principal components of the cytoskeleton, a dynamic integrated system of structural proteins that provides the functional architecture of metazoan cells. They are major contributors to the elasticity of cells and tissues due to their high mechanical stability and intrinsic flexibility. The basic building block for the assembly of IFs is a rod-like, 60-nm-long tetrameric complex made from two antiparallel, half-staggered coiled coils. In low ionic strength, tetramers form stable complexes that rapidly assemble into filaments upon raising the ionic strength. The first assembly products, "frozen" by instantaneous chemical fixation and viewed by electron microscopy, are 60-nm-long "unit-length" filaments (ULFs) that apparently form by lateral in-register association of tetramers. ULFs are the active elements of IF growth, undergoing longitudinal end-to-end annealing with one another and with growing filaments. Originally, we have employed quantitative time-lapse atomic force and electron microscopy to analyze the kinetics of vimentin-filament assembly starting from a few seconds to several hours. To obtain detailed quantitative insight into the productive reactions that drive ULF formation, we now introduce a "stopped-flow" approach in combination with static light-scattering measurements. Thereby, we determine the basic rate constants for lateral assembly of tetramers to ULFs. Processing of the recorded data by a global fitting procedure enables us to describe the hierarchical steps of IF formation. Specifically, we propose that tetramers are consumed within milliseconds to yield octamers that are obligatory intermediates toward ULF formation. Although the interaction of tetramers is diffusion controlled, it is strongly driven by their geometry to mediate effective subunit targeting. Importantly, our model conclusively reflects the previously described occurrence of polymorphic ULF and mature filaments in terms of their number of tetramers per cross section.


Assuntos
Filamentos Intermediários/metabolismo , Multimerização Proteica , Vimentina/química , Humanos , Cinética , Modelos Moleculares , Estrutura Quaternária de Proteína
6.
Methods ; 123: 3-10, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28648678

RESUMO

Single plane illumination microscopy (SPIM) is a new optical method that has become extremely important in recent years. It is based on the formation of a "light slice" in the specimen in which fluorescently tagged molecules are observed. The spatial resolution is close to that of confocal optics, but without the disadvantages inherent to scanning or high laser irradiation doses. A recent development is light sheet fluctuation microscopy, which exploits the dynamic information contained in the fluorescence intensity fluctuations of each image pixel. Here we review the principles of this method and show some recent applications to the dynamics of transcription factors and chromatin. We show that the dimerization of Fos and Jun proteins is directly linked to their binding to DNA; that nuclear receptor activation changes their intranuclear dynamics; and that the viscoelastic behavior of interphase chromatin strongly depends on the presence of lamin A.


Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Núcleo Celular/ultraestrutura , Cromatina/química , Cromatina/ultraestrutura , DNA/genética , DNA/metabolismo , Expressão Gênica , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/instrumentação , Imagem Molecular/instrumentação , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Proteína Vermelha Fluorescente
7.
Biophys J ; 112(3): 450-459, 2017 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-27931745

RESUMO

The N-terminal tail of histone H4 is an indispensable mediator for inter-nucleosome interaction, which is required for chromatin fiber condensation. H4K16 acetylation (H4K16Ac) activates gene transcription by influencing both chromatin structure and interplay with nonhistone proteins. To understand the influence of H4K16Ac on inter-nucleosome interaction, we performed a simulation study for the H4 tail in the context of two nucleosomes in neighboring unit cells in the crystal structure. The binding conformation of H4 tail with/without K16Ac was sampled by replica exchange with solute tempering, and the free energy landscape was explored by metadynamics. The results indicate two important features of H4K16: 1) it is the first button to anchor the H4 tail on the adjacent nucleosome; and 2) it is the only acetylation site interacting with the acidic patch. H4K16Ac disrupts the electrostatic interactions of K16, weakens H4 tail-acidic patch binding, and significantly increases H4 tail conformation diversity. Our study suggests that H4K16Ac directly reduces the inter-nucleosome interaction mediated by the H4 tail, which might further encourage the binding of nonhistone proteins on the acidic patch.


Assuntos
Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Nucleossomos/metabolismo , Acetilação , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína
8.
Nucleic Acids Res ; 43(3): 1433-43, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25589544

RESUMO

Using FRET in bulk and on single molecules, we assessed the structural role of histone acetylation in nucleosomes reconstituted on the 170 bp long Widom 601 sequence. We followed salt-induced nucleosome disassembly, using donor­acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on H2B histone for measuring H2A/H2B dimer exchange. This allowed us to distinguish the influence of acetylation on salt-induced DNA unwrapping at the entry­exit site from its effect on nucleosome core dissociation. The effect of lysine acetylation is not simply cumulative, but showed distinct histone-specificity. Both H3- and H4-acetylation enhance DNA unwrapping above physiological ionic strength; however, while H3-acetylation renders the nucleosome core more sensitive to salt-induced dissociation and to dimer exchange, H4-acetylation counteracts these effects. Thus, our data suggest, that H3- and H4-acetylation have partially opposing roles in regulating nucleosome architecture and that distinct aspects of nucleosome dynamics might be independently controlled by individual histones.


Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Acetilação , Dimerização , Ensaio de Desvio de Mobilidade Eletroforética , Transferência Ressonante de Energia de Fluorescência
9.
Nucleic Acids Res ; 42(12): 7561-76, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24829456

RESUMO

The TATA binding protein (TBP) is a critical transcription factor used for nucleating assembly of the RNA polymerase II machinery. TBP binds TATA box elements with high affinity and kinetic stability and in vivo is correlated with high levels of transcription activation. However, since most promoters use less stable TATA-less or TATA-like elements, while also competing with nucleosome occupancy, further mechanistic insight into TBP's DNA binding properties and ability to access chromatin is needed. Using bulk and single-molecule FRET, we find that TBP binds a minimal consensus TATA box as a two-state equilibrium process, showing no evidence for intermediate states. However, upon addition of flanking DNA sequence, we observe non-specific cooperative binding to multiple DNA sites that compete for TATA-box specificity. Thus, we conclude that TBP binding is defined by a branched pathway, wherein TBP initially binds with little sequence specificity and is thermodynamically positioned by its kinetic stability to the TATA box. Furthermore, we observed the real-time access of TBP binding to TATA box DNA located within the DNA entry-exit site of the nucleosome. From these data, we determined salt-dependent changes in the nucleosome conformation regulate TBP's access to the TATA box, where access is highly constrained under physiological conditions, but is alleviated by histone acetylation and TFIIA.


Assuntos
Nucleossomos/química , Nucleossomos/metabolismo , TATA Box , Proteína de Ligação a TATA-Box/metabolismo , Acetilação , Sequência de Bases , Sítios de Ligação , DNA/química , DNA/metabolismo , Histonas/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Fator de Transcrição TFIIA/metabolismo
10.
Biophys J ; 107(12): 2911-2922, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25517156

RESUMO

Histone tails play an important role in gene transcription and expression. We present here a systematic computational study of the role of histone tails in the nucleosome, using replica exchange molecular dynamics simulations with an implicit solvent model and different well-established force fields. We performed simulations for all four histone tails, H4, H3, H2A, and H2B, isolated and with inclusion of the nucleosome. The results confirm predictions of previous theoretical studies for the secondary structure of the isolated tails but show a strong dependence on the force field used. In the presence of the entire nucleosome for all force fields, the secondary structure of the histone tails is destabilized. Specific contacts are found between charged lysine and arginine residues and DNA phosphate groups and other binding sites in the minor and major DNA grooves. Using cluster analysis, we found a single dominant configuration of binding to DNA for the H4 and H2A histone tails, whereas H3 and H2B show multiple binding configurations with an equal probability. The leading stabilizing contribution for those binding configurations is the attractive interaction between the positively charged lysine and arginine residues and the negatively charged phosphate groups, and thus the resulting charge neutralization. Finally, we present results of molecular dynamics simulations in explicit solvent to confirm our conclusions. Results from both implicit and explicit solvent models show that large portions of the histone tails are not bound to DNA, supporting the complex role of these tails in gene transcription and expression and making them possible candidates for binding sites of transcription factors, enzymes, and other proteins.


Assuntos
DNA/química , Histonas/química , Simulação de Dinâmica Molecular , Nucleossomos/química , Sequência de Aminoácidos , Sequência de Bases , DNA/metabolismo , Histonas/metabolismo , Dados de Sequência Molecular , Ligação Proteica
11.
Opt Express ; 22(3): 2358-75, 2014 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-24663528

RESUMO

Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, flow velocities and concentrations in an imaging mode. Here we extend this technique to two-color fluorescence cross-correlation spectroscopy (SPIM-FCCS), which allows to measure molecular interactions in an imaging mode. We present a theoretical framework for SPIM-FCCS fitting models, which is subsequently used to evaluate several test measurements of in-vitro (labeled microspheres, several DNAs and small unilamellar vesicles) and in-vivo samples (dimeric and monomeric dual-color fluorescent proteins, as well as membrane bound proteins). Our method yields the same quantitative results as the well-established confocal FCCS, but in addition provides unmatched statistics and true imaging capabilities.


Assuntos
Aumento da Imagem/instrumentação , Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Espectrometria de Fluorescência/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento
12.
Cytometry A ; 83(9): 839-46, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23843180

RESUMO

Opening of the nucleosome structure is essential for accessing genomic DNA. To study the mechanism of this process, we monitor the distance between various fluorescently labeled positions on mononucleosomes by single-molecule Förster resonance energy transfer (FRET). Here, we compare nucleosomes reconstituted from recombinant mouse, Xenopus, and yeast histones. As DNA sequences we compared, the effect of 5S rDNA, MMTV-B sequence, and Widom 601 DNA. The stability, as measured by the salt concentration at the opening transition midpoint, is lowest for yeast, followed by Xenopus and mouse. The 601 DNA sequence builds much more stable nucleosomes and the distribution of FRET efficiencies is narrower than for those reconstituted on 5S rDNA or MMTV-B sequences. The opening pathway through an intermediate state, as found for Xenopus histones, could be verified for the mouse and yeast systems and for the different DNA sequences, suggesting a general mechanism for accessing nucleosomal DNA.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Histonas/química , Nucleossomos/química , Animais , Sequência de Bases , Cromatina/genética , DNA Ribossômico/genética , Corantes Fluorescentes , Histonas/genética , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Nucleossomos/genética , RNA Ribossômico 5S/genética , Análise de Sequência de DNA , Xenopus laevis , Leveduras/genética
13.
Opt Express ; 21(7): 8652-68, 2013 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-23571955

RESUMO

Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, transport, flow velocities and concentrations in an imaging mode. SPIM-FCS records correlation functions over a whole plane in a sample, which requires array detectors for recording the fluorescence signal. Several types of image sensors are suitable for FCS. They differ in properties such as effective area per pixel, quantum efficiency, noise level and read-out speed. Here we compare the performance of several low light array detectors based on three different technologies: (1) Single-photon avalanche diode (SPAD) arrays, (2) passive-pixel electron multiplying charge coupled device (EMCCD) and (3) active-pixel scientific-grade complementary metal oxide semiconductor cameras (sCMOS). We discuss the influence of the detector characteristics on the effective FCS observation volume, and demonstrate that light sheet based SPIM-FCS provides absolute diffusion coefficients. This is verified by parallel measurements with confocal FCS, single particle tracking (SPT), and the determination of concentration gradients in space and time. While EMCCD cameras have a temporal resolution in the millisecond range, sCMOS cameras and SPAD arrays can extend the time resolution of SPIM-FCS down to 10 µs or lower.


Assuntos
Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência/instrumentação , Semicondutores , Espectrometria de Fluorescência/instrumentação , Transdutores , Desenho de Equipamento , Análise de Falha de Equipamento , Luz
14.
Int J Med Sci ; 10(9): 1136-48, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23869190

RESUMO

The highly organized DNA architecture inside of the nuclei of cells is accepted in the scientific world. In the human genome about 3 billion nucleotides are organized as chromatin in the cell nucleus. In general, they are involved in gene regulation and transcription by histone modification. Small chromosomes are localized in a central nuclear position whereas the large chromosomes are peripherally positioned. In our experiments we inserted fusion proteins consisting of a component of the nuclear lamina (lamin B1) and also histone H2A, both combined with the light inducible fluorescence protein KillerRed (KRED). After activation, KRED generates reactive oxygen species (ROS) producing toxic effects and may cause cell death. We analyzed the spatial damage distribution in the chromatin after illumination of the cells with visible light. The extent of DNA damage was strongly dependent on its localization inside of nuclei. The ROS activity allowed to gain information about the location of genes and their functions via sequencing and data base analysis of the double strand breaks of the isolated DNA. A connection between the damaged gene sequences and some diseases was found.


Assuntos
Fragmentação do DNA/efeitos da radiação , Histonas/metabolismo , Luz , Linhagem Celular Tumoral , Humanos , Lamina Tipo B/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
Nucleic Acids Res ; 39(8): 3093-102, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21177647

RESUMO

Nucleosomes are multi-component macromolecular assemblies which present a formidable obstacle to enzymatic activities that require access to the DNA, e.g. DNA and RNA polymerases. The mechanism and pathway(s) by which nucleosomes disassemble to allow DNA access are not well understood. Here we present evidence from single molecule FRET experiments for a previously uncharacterized intermediate structural state before H2A-H2B dimer release, which is characterized by an increased distance between H2B and the nucleosomal dyad. This suggests that the first step in nucleosome disassembly is the opening of the (H3-H4)(2) tetramer/(H2A-H2B) dimer interface, followed by H2A-H2B dimer release from the DNA and, lastly, (H3-H4)(2) tetramer removal. We estimate that the open intermediate state is populated at 0.2-3% under physiological conditions. This finding could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery.


Assuntos
Montagem e Desmontagem da Cromatina , Histonas/química , Nucleossomos/química , Transferência Ressonante de Energia de Fluorescência , Histonas/metabolismo , Modelos Moleculares , Nucleossomos/metabolismo , Multimerização Proteica , Cloreto de Sódio/química , Espectrometria de Fluorescência
16.
Biophys J ; 102(4): 849-58, 2012 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-22385856

RESUMO

To permit access to DNA-binding proteins involved in the control and expression of the genome, the nucleosome undergoes structural remodeling including unwrapping of nucleosomal DNA segments from the nucleosome core. Here we examine the mechanism of DNA dissociation from the nucleosome using microsecond timescale coarse-grained molecular dynamics simulations. The simulations exhibit short-lived, reversible DNA detachments from the nucleosome and long-lived DNA detachments not reversible on the timescale of the simulation. During the short-lived DNA detachments, 9 bp dissociate at one extremity of the nucleosome core and the H3 tail occupies the space freed by the detached DNA. The long-lived DNA detachments are characterized by structural rearrangements of the H3 tail including the formation of a turn-like structure at the base of the tail that sterically impedes the rewrapping of DNA on the nucleosome surface. Removal of the H3 tails causes the long-lived detachments to disappear. The physical consistency of the CG long-lived open state was verified by mapping a CG structure representative of this state back to atomic resolution and performing molecular dynamics as well as by comparing conformation-dependent free energies. Our results suggest that the H3 tail may stabilize the nucleosome in the open state during the initial stages of the nucleosome remodeling process.


Assuntos
DNA/química , DNA/metabolismo , Simulação de Dinâmica Molecular , Nucleossomos/química , Nucleossomos/metabolismo , Histonas/química , Histonas/metabolismo , Conformação de Ácido Nucleico , Conformação Proteica , Solventes/química , Fatores de Tempo
17.
Opt Express ; 20(16): 17767-82, 2012 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-23038328

RESUMO

With the evolving technology in CMOS integration, new classes of 2D-imaging detectors have recently become available. In particular, single photon avalanche diode (SPAD) arrays allow detection of single photons at high acquisition rates (≥ 100 kfps), which is about two orders of magnitude higher than with currently available cameras. Here we demonstrate the use of a SPAD array for imaging fluorescence correlation spectroscopy (imFCS), a tool to create 2D maps of the dynamics of fluorescent molecules inside living cells. Time-dependent fluorescence fluctuations, due to fluorophores entering and leaving the observed pixels, are evaluated by means of autocorrelation analysis. The multi-τ correlation algorithm is an appropriate choice, as it does not rely on the full data set to be held in memory. Thus, this algorithm can be efficiently implemented in custom logic. We describe a new implementation for massively parallel multi-τ correlation hardware. Our current implementation can calculate 1024 correlation functions at a resolution of 10 µs in real-time and therefore correlate real-time image streams from high speed single photon cameras with thousands of pixels.


Assuntos
Eletrônica/instrumentação , Eletrônica/métodos , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Algoritmos , Simulação por Computador , Fótons , Espectrometria de Fluorescência
18.
PLoS Comput Biol ; 7(12): e1002279, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22207822

RESUMO

Histone tails play an important role in nucleosome structure and dynamics. Here we investigate the effect of truncation of histone tails H3, H4, H2A and H2B on nucleosome structure with 100 ns all-atom molecular dynamics simulations. Tail domains of H3 and H2B show propensity of α-helics formation during the intact nucleosome simulation. On truncation of H4 or H2B tails no structural change occurs in histones. However, H3 or H2A tail truncation results in structural alterations in the histone core domain, and in both the cases the structural change occurs in the H2Aα3 domain. We also find that the contacts between the histone H2A C terminal docking domain and surrounding residues are destabilized upon H3 tail truncation. The relation between the present observations and corresponding experiments is discussed.


Assuntos
Histonas/química , Histonas/fisiologia , Nucleossomos/química , Nucleossomos/fisiologia , Histonas/ultraestrutura , Modelos Moleculares , Simulação de Dinâmica Molecular , Nucleossomos/ultraestrutura , Conformação Proteica , Relação Estrutura-Atividade
19.
Chromosome Res ; 19(1): 63-81, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21116704

RESUMO

Using Monte Carlo simulations, we have investigated the decondensation of chromosomes during interphase and the diffusive transport of spherical probe particles in the chromatin network. The chromatin fibers are modeled as semiflexible polymer chains on a fixed three-dimensional grid, taking into account their flexibility and eventual chain crossing by the aid of topoisomerases. The network thus created will obstruct the diffusion of macromolecules. A result of our simulations is that crowding of diffusing molecules leaves the dynamics of the chromosomes and the behavior of other diffusing molecules qualitatively unaffected. Furthermore, the capability of the simulated chromatin network to trap diffusing molecules over long times is lower than that measured in microrheological experiments. Microrheology is a technique that allows to determine the viscoelastic properties of a material by the motion of embedded tracer particles. Long-time trapping requires a stiff network, as only such a network quickly responds to the diffusive fluctuations of tracers and prevents them from squeezing through meshes. A high degree of crosslinking amplifies this effect. The presence of a flexible and uncrosslinked polymer simply increases the effective viscosity sensed by tracer particles. The diffusion of tracers in our simulations reveals rather viscous than elastic chromatin networks, suggesting that chromatin alone cannot account for the high elasticity of the cell nucleus.


Assuntos
Cromossomos/química , Simulação por Computador , Interfase , Simulação de Dinâmica Molecular , Cromatina/ultraestrutura , Difusão , Elasticidade , Modelos Biológicos , Método de Monte Carlo , Conformação Proteica , Viscosidade
20.
J Chem Phys ; 137(6): 064114, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22897262

RESUMO

We propose a kinetic Monte Carlo method for the simulation of subdiffusive random walks on a cartesian lattice. The random walkers are subject to viscoelastic forces which we compute from their individual trajectories via the fractional Langevin equation. At every step the walkers move by one lattice unit, which makes them differ essentially from continuous time random walks, where the subdiffusive behavior is induced by random waiting. To enable computationally inexpensive simulations with n-step memories, we use an approximation of the memory and the memory kernel functions with a complexity O(log n). Eventual discretization and approximation artifacts are compensated with numerical adjustments of the memory kernel functions. We verify with a number of analyses that this new method provides binary fractional random walks that are fully consistent with the theory of fractional brownian motion.


Assuntos
Simulação por Computador , Método de Monte Carlo , Viscosidade , Difusão , Íons/química , Cinética
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