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Добірка наукової літератури з теми "Single-molecule biophysic"

Автор: Grafiati
Опубліковано: 10 березня 2023

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Статті в журналах з теми "Single-molecule biophysic"

1

Noji, Hroyuki. "SINGLE MOLECULE BIOPHYSICS OF F_1-ATPase motor protein." Proceedings of the Asian Pacific Conference on Biomechanics : emerging science and technology in biomechanics 2007.3 (2007): S1. http://dx.doi.org/10.1299/jsmeapbio.2007.3.s1.

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2

Weng, Zhuangfeng, Yuan Shang, Zeyang Ji, Fei Ye, Lin Lin, Rongguang Zhang, and Jinwei Zhu. "Structural Basis of Highly Specific Interaction between Nephrin and MAGI1 in Slit Diaphragm Assembly and Signaling." Journal of the American Society of Nephrology 29, no. 9 (July 13, 2018): 2362–71. http://dx.doi.org/10.1681/asn.2017121275.

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BackgroundThe slit diaphragm is a specialized adhesion junction between opposing podocytes, establishing the final filtration barrier that prevents passage of proteins from the capillary lumen into the urinary space. Nephrin, the key structural and signaling adhesion molecule expressed in the slit diaphragm, contains an evolutionally conserved, atypical PDZ-binding motif (PBM) reported to bind to a variety of proteins in the slit diaphragm. Several mutations in NPHS1 (the gene encoding nephrin) that result in nephrin lacking an intact PBM are associated with glomerular diseases. However, the molecular basis of nephrin-PBM–mediated protein complexes is still unclear.MethodsUsing a combination of biochemic, biophysic, and cell biologic approaches, we systematically investigated the interactions between nephrin-PBM and PDZ domain–containing proteins in the slit diaphragm.ResultsWe found that nephrin-PBM specifically binds to one member of the membrane-associated guanylate kinase family of scaffolding proteins, MAGI1, but not to another, MAGI2. The complex structure of MAGI1-PDZ3/nephrin-PBM reveals that the Gly at the −3 position of nephrin-PBM is the determining feature for MAGI1-PDZ3 recognition, which sharply contrasts with the typical PDZ/PBM binding mode. A single gain-of-function mutation within MAGI2 enabled nephrin-PBM binding. In addition, using our structural analysis, we developed a highly efficient inhibitory peptide capable of specifically blocking the nephrin/MAGI1 interaction.ConclusionsMAGI1 interacts with nephrin-PBM with exquisite specificity. A newly developed, potent inhibitory peptide that blocks this interaction may be useful for future functional investigations in vivo. Our findings also provide possible explanations for the diseases caused by NPHS1 mutations.
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3

LI, Chun-Biu, and Tamiki KOMATSUZAKI. "Handling Noisy Data from Single Molecule Experiments." Seibutsu Butsuri 54, no. 5 (2014): 257–58. http://dx.doi.org/10.2142/biophys.54.257.

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4

Joshi, Prakash, and Partha Pratim Mondal. "Single-Molecule Clustering for Super-Resolution Optical Fluorescence Microscopy." Photonics 9, no. 1 (December 24, 2021): 7. http://dx.doi.org/10.3390/photonics9010007.

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Molecular assembly in a complex cellular environment is vital for understanding underlying biological mechanisms. Biophysical parameters (such as single-molecule cluster density, cluster-area, pairwise distance, and number of molecules per cluster) related to molecular clusters directly associate with the physiological state (healthy/diseased) of a cell. Using super-resolution imaging along with powerful clustering methods (K-means, Gaussian mixture, and point clustering), we estimated these critical biophysical parameters associated with dense and sparse molecular clusters. We investigated Hemaglutinin (HA) molecules in an Influenza type A disease model. Subsequently, clustering parameters were estimated for transfected NIH3T3 cells. Investigations on test sample (randomly generated clusters) and NIH3T3 cells (expressing Dendra2-Hemaglutinin (Dendra2-HA) photoactivable molecules) show a significant disparity among the existing clustering techniques. It is observed that a single method is inadequate for estimating all relevant biophysical parameters accurately. Thus, a multimodel approach is necessary in order to characterize molecular clusters and determine critical parameters. The proposed study involving optical system development, photoactivable sample synthesis, and advanced clustering methods may facilitate a better understanding of single molecular clusters. Potential applications are in the emerging field of cell biology, biophysics, and fluorescence imaging.
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5

Kinz-Thompson, Colin D., Korak Kumar Ray, and Ruben L. Gonzalez. "Bayesian Inference: The Comprehensive Approach to Analyzing Single-Molecule Experiments." Annual Review of Biophysics 50, no. 1 (May 6, 2021): 191–208. http://dx.doi.org/10.1146/annurev-biophys-082120-103921.

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Biophysics experiments performed at single-molecule resolution provide exceptional insight into the structural details and dynamic behavior of biological systems. However, extracting this information from the corresponding experimental data unequivocally requires applying a biophysical model. In this review, we discuss how to use probability theory to apply these models to single-molecule data. Many current single-molecule data analysis methods apply parts of probability theory, sometimes unknowingly, and thus miss out on the full set of benefits provided by this self-consistent framework. The full application of probability theory involves a process called Bayesian inference that fully accounts for the uncertainties inherent to single-molecule experiments. Additionally, using Bayesian inference provides a scientifically rigorous method of incorporating information from multiple experiments into a single analysis and finding the best biophysical model for an experiment without the risk of overfitting the data. These benefits make the Bayesian approach ideal for analyzing any type of single-molecule experiment.
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Gopich, Irina V. "2SD0925 Theory of single-molecule photon trajectories and FRET efficiency distributions(2SD Bridging Single Molecule Biophysics and System Biology:New Experimental and Theoretical Challenges,The 48th Annual Meeting of the Biophysical Society of Japan)." Seibutsu Butsuri 50, supplement2 (2010): S12. http://dx.doi.org/10.2142/biophys.50.s12_2.

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Ritchie, Ken. "S01H3 Single molecule imaging of diffusion in E. Coll membranes(Systems Biology of Intracellular Signaling as Studied by Single-Molecule Imaging)." Seibutsu Butsuri 47, supplement (2007): S1. http://dx.doi.org/10.2142/biophys.47.s1_3.

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Cao, Jianshu. "1S5-5 Generic models for single molecule biological processes : Generic models for single molecule biological processes(1S5 Linking single molecule spectroscopy and energy landscape perspectives,The 46th Annual Meeting of the Biophysical Society of Japan)." Seibutsu Butsuri 48, supplement (2008): S5. http://dx.doi.org/10.2142/biophys.48.s5_1.

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Sei, Kazuto, Akinori Baba, Chun Biu Li, and Tamiki Komatsuzaki. "1P537 Randomness and Memory in Single Molecule Time Series(26. Single molecule biophysics,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)." Seibutsu Butsuri 46, supplement2 (2006): S281. http://dx.doi.org/10.2142/biophys.46.s281_1.

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Fernandez, Julio M. "S3B1 Protein mechanics studied with single molecule AFM techniques.(Single Molecure Dynamics and Reactions)." Seibutsu Butsuri 42, supplement2 (2002): S13. http://dx.doi.org/10.2142/biophys.42.s13_4.

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Більше джерел

Дисертації з теми "Single-molecule biophysic"

1

Mukund, Shreyas Ram. "Single molecule biophysics of homologous recombination." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708842.

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2

Jones, Nathan Jones. "Single Molecule Analysis of DNA Interactions." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1511959163350735.

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3

Le, Tung T. "Single-molecule biophysics of DNA bending: looping and unlooping." Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/53979.

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DNA bending plays a vital role in numerous cellular activities such as transcription, viral packaging, and nucleosome formation. Therefore, understanding the physics of DNA bending at the length scales relevant to these processes is one of the main keys to the quantitative description of life. However, previous studies provide a divided picture on how DNA should be modeled in strong bending condition relevant to biology. My thesis is devoted to answering how far an elastic rod model can be applied to DNA. We consider several subtle features that could potentially lead to the break-down of the worm-like chain model, such as local bendedness of the sequence and large bending angles. We used single-molecule Fluorescence Resonance Energy Transfer to track looping and unlooping of single DNA molecules in real time. We compared the measured looping and unlooping rates with theoretical predictions of the worm-like chain model. We found that the intrinsic curvature of the sequence affects the looping propensity of short DNA and an extended worm-like chain model including the helical parameters of individual base pairs could adequately explain our measurements. For DNA with random sequence and negligible curvature, we discovered that the worm-like chain model could explain the stability of small DNA loops only down to a critical loop size. Below the critical loop size, the bending stress stored in the DNA loop became less sensitive to loop size, indicative of softened dsDNA. The critical loop size is sensitive to salt condition, especially to magnesium at mM concentrations. This finding enabled us to explain several contrasting results in the past and shed new light on the energetics of DNA bending.
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4

Koussa, Mounir Ahmad. "The Biophysics of Vertebrate Hearing: A Single-Molecule Approach." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467499.

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Inner-ear mechanotransduction relies on tip links, fine protein filaments made of cadherin-23 and protocadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link cadherins are thought to form a heterotetrameric complex, with two cadherin-23 molecules forming the upper part of the filament and two protocadherin-15 molecules forming the lower end. The interaction between cadherin-23 and protocadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. We have developed molecular tools to perform single-molecule force spectroscopy on the tip-link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of cadherin-23 and protocadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip-link-functionalized nanoswitches are designed to provide a signature force-extension profile, which allows us to identify single-molecule rupture events that result from applying force. Using this system, we have been able to measure the cadherin-23-protocadherin-15 single-molecule force-dependent off rate, as well as the concentration-dependent on rate for a single pair of these proteins. The rates suggest that a single bond is inadequate to withstand physiological forces for physiological times, but we construct a new model for tip-link dynamics which greatly alters our understanding of tip-link function and explains the necessity for a two-filament tip link.
Medical Sciences
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5

Miller, Helen. "Novel super-resolution optical microscopy methods for single-molecule biophysics." Thesis, University of York, 2017. http://etheses.whiterose.ac.uk/18192/.

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Super-resolution microscopy is a relatively new and rapidly growing field. Development has been largely technology-driven, with high power lasers, higher resolution CCD cameras, and increasing computing power all enabling new biological questions to be explored. Single-molecule imaging is the tool of choice for studying systems where heterogeneity is present; ensemble methods can average away the interesting behaviour and lead to false conclusions. This thesis develops and optimises bespoke fluorescence microscopy for application to three biological questions, each pushing a limit of super-resolution imaging. Super-resolution imaging of lambda DNA labelled with the intercalating dye YOYO-1 and the minor groove binder SYTO-13 at localisation precisions of 40nm and 62nm respectively has been achieved in preparation for combined fluorescence imaging and magneto-optical tweezers experiments. The combination of these two methods is challenging as both operate with low tolerances.\ Single-molecule tracking was used to measure the diffusion coefficients of the chemokines CXCL13 and CCL19 at extremely high temporal resolution. Single-molecule imaging was found to have advantages over the ensemble techniques of FRAP and FCS for measuring the diffusion coefficient of the test molecule; Alexa Fluor 647 labelled bovine serum albumin. The diffusion coefficients of the two chemokines, CXCL13 and CCL19 were found by single particle tracking at sub-millisecond timescales in a collagen matrix to be 6.2±0.3µm2s-1 and 8.4±0.2µm2s-1. Further, CXCL13 was tracked in B cell follicle regions of ex vivo lymph node tissue sections at ~2 millisecond timescales, giving a diffusion coefficient of 6.6±0.4µm2s-1.\ Fluorescence microscopy was used to elucidate the stoichiometry of YOYO-1 on DNA origami tiles after treatment with low temperature plasma. Undamaged tiles were found to have a mean stoichiometry of 67.4±25.2 YOYO-1 molecules and a model of LTP damage to DNA origami tiles was proposed.
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6

Viader, Godoy Xavier. "Biophysical properties of single-stranded DNA studied with single-molecule force spectroscopy." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/670920.

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In this thesis, single-molecule experiments using LOT are employed to extract accurate information about the thermodynamics and kinetics of various molecular systems, with special emphasis on the elastic properties of single-stranded DNA (ssDNA). The thesis is divided in three parts. Part I provides a general description of the research field as well as the main theoretical framework for the basic concepts that will be developed in parts II and III. In Chapter 2 the miniTweezers and the experimental setup used throughout the thesis is described, as well as the physical basis of its working mechanisms, introducing the phenomenon of optical trapping. Chapter 3 contains a brief introduction of the biomolecules of study in this thesis, with an explanation of their historical discoveries, as well as their structure and function. The main focus of this chapter is on ssDNA, which is the main object of study of the thesis. Chapter 4 introduces the polymer models that are widely used in describing the elasticity of nucleic acids and proteins. Specifically, the Freely-Jointed Chain and Worm-Like Chain models are presented. Part II deals with the elasticity of single-stranded DNA. This is the main part of the thesis, and it includes chapters 5-7. Chapter 5 is about the study of the elasticity of ideal ssDNA chain, i.e. the one that can be modelled as ideal polymers (presented in Chapter 4). The study of the elasticity of different DNA sequences is presented. The blocking-splint oligo technique is described, a experimental technique developed for studying the elasticity of short (tens of bases) DNA molecules. This study shows the need of using extensible models to succesfully describe ssDNA elasticity over a large range of forces, which explains the previous discrepancies on the elastic parameters obtained in different studies. We also provide an explanation for the required extensibility of the model: a transition experienced at the nucleotide level: a change in DNA sugar pucker conformation. A simple two-states model is introduced and preeliminary results regarding its energetics are presented. The characterization of the ssDNA elasticity is central for the works developed in the following chapters. Chapter 6 studies the stacking-unstacking transition for ssDNA, previously observed for certain sequences (mainly purine-rich ones). Several molecules, with different degrees of stacking, are studied by obtaining their force extension curves (FECs). A cooperative helix-coil model including heterogeneity is developed and used to fit the obtained FECs, allowing to obtain elastic parameters to describe the stacked chain. The salt dependence of the unstacking transition is also measured by studying two of the sequences by varying the salt concentration over two decades. The free energy of formation of dsDNA duplexes depends on the salt concentration. The obtained salt dependence on the stacking free-energy of ssDNA provides a possible explanation for the salt dependence of duplex formation. Chapter 7 deals with the non-specific structures that arise at low forces and high salt concentration when pulling ssDNA molecules longer than $\sim 100$ bases. A helix-coil model with cooperativity is proposed and used to extract some mean-field characteristics of these structures. 8 different sequences are studied, characterizing their elasticity and deviation from the ideal elastic behaviour. The results for a $14$kb molecule for 3 decades of varying \ce{NaCl} and \ce{MgCl2} are also shown. All experimental FECs are fitted to the helix-coil model. The model can be used to predict the formation of secondary structures at zero force. A comparison between the predicted structures from the model and those obtained from Mfold is also investigated. Part III contains two studies which also need of the correct determination of ssDNA elasticity. In Chapter 8, we study the interaction between the RecQ helicase from E. coli and DNA, i.e. how the RecQ unwinds double-stranded DNA molecules, releasing single-stranded DNA. We obtain some of its kinematic properties as well as study the entropy production of the system using the Fluctuation Theorem. In Chapter 9, the effect of DNA mismatches, i.e. non complementary base pairing, on the stability of DNA is studied. To do so, two types of experiments on several DNA sequences are performed: stretching and releasing the molecule by moving the optical trap (pulling experiments) and monitoring the folding/unfolding of the molecule passively (hopping experiments).
En aquesta tesi hem realitzat experiments fent servir pinces òptiques per tal d’extreure informació precisa sobre les propietats termodinàmiques i cinètiques de diferents sistemes moleculars, posant especial èmfasi en les propietats elàstiques de la cadena simple d’ADN (ssDNA, pel seu acrònim en anglès). La tesi es troba dividida en tres parts. A la primera part s’introdueix de forma general el camp de recerca dels experiments de molècula única, així com s’expliquen els conceptes més bàsics que es desenvoluparan en les parts II i III. La configuració experimental emprada al llarg de tota la tesi, les pinces òptiques, s’introdueix al capítol 2. Per a fer-ho, s’expliquen els principis físics de funcionament de les pinces, que es basen en l’atrapament òptic. Breument, la focalització d’un feix de llum d’alta intensitat permet atrapar i exercir forces en micropartícules dielèctriques (pilotes fetes de plàstic de la mida d’un bacteri), que són recobertes químicament de manera que la molècula d’estudi pot estirar-se, de forma individual, repetides vegades. El capítol 3 conté una breu introducció a les biomolècules que apareixen en aquesta tesi, amb una breu explicació de la seva descoberta, així com la seva estructura i funció (íntimament relacionades). Ens centrem en la descripció de la ssDNA que és el principal objecte d’estudi de la tesi. Al capítol 4 s’introdueixen els models de polímers que s’empren habitualment per a descriure l’elasticitat d’àcids nucleics i proteïnes. En concret, es descriuen els models de la Freely-Jointed Chain i la Worm-Like Chain. La Part II tracta de l’elasticitat de la ssDNA, i inclou els capítols 5, 6 i 7. El capítol 5 es basa en la caracterització de l’elasticitat de la cadena ideal de ssDNA, és a dir, aquella que pot ser modelitzada pels polímers ideals introduïts en el capítol 4. S’estudia l’elasticitat de diferents seqüències de ssDNA, introduint un nou mètode experimental, blocking-splint oligo, per tal d’ampliar el rang de forces estudiat habitualment en molècules curtes (d’una longitud de desenes de bases) de ssDNA. L’estudi mostra la necessitat d’emprar models elàstics extensibles per a la correcte caracterització de l’elasticitat de ssDNA, que explica les discrepàncies existents entre els paràmetres elàstics trobats a la literatura. També hipotetitzem que l’extensibilitat del model pot ser explicada gràcies a la transició experimentada a nivell de nucleòtids: el canvi que experimenta la distància interfosfat de l’ADN es veu modificada segons quina sigui la configuració de l’anell de desoxiribosa. Tot i que és un fenomen molt més conegut en la cadena doble d’ADN, l’apilament-desapilament de bases també s’ha observat en certes seqüències de ssDNA (especialment les que són riques en contingut de purines). Al capítol 6 s’estudien quatre molècules amb un grau d’apilament diferent a partir de les seves corbes força-extensió (FECs). Es desenvolupa un model helix-coil (hèlix-cabdell) per tal d’ajustar les FECs, fet que permet d’obtenir, indirectament, les propietats elàstiques de la cadena apilada. També s’estudia la dependència d’aquesta transició variant la concentració de sal dels experiments en més de dos ordres de magnitud. A través d’aquests experiments, trobem una dependència amb la concentració de sal de l’energia lliure de formació de l’apilament de la ssDNA, fet que ens permet explicar, parcialment, la dependència que es troba en la literatura per la hibridació de la cadena doble d’ADN. El capítol 7 tracta de la formació d’estructures no específiques que apareixen a forces baixes i a concentració de sal alta per a molècules de ssDNA de més de ~100bases. Es proposa un model helix-coil amb cooperativitat per tal de caracteritzar propietats de camp mitjà de les estructures estudiades. S’estudien vuit seqüències diferents, entre 120 i ~14000 bases, i es caracteritza el seu desviament respecte de la corba elàstica ideal amb el model. També s’estudia la dependència de l’estructura secundària de la ssDNA en funció de la concentració de la sal. Analitzant experiments variant la concentració de MgCl2 i NaCl, aconseguim reproduir les FECs a partir de fer dependre els paràmetres del model amb la sal. Finalment, el model desenvolupat ens permet predir la formació d’estructura secundària a força zero (fet que no podem detectar directament a partir d’experiments d’espectroscopia de forces). Es comparen les previsions del model amb les trobades per Mfold, trobant una compatibilitat per als resultats per a molècules de de menys de 1000 bases. La darrera part se centra en col·laboracions que he fet durant a tesi i que necessiten una determinació precisa de les propietats elàstiques de la ssDNA. Al capítol 8 s’estudia la interacció entre l’helicasa del bacteri E. coli i l’ADN, que s’encarrega d’obrir la cadena doble d’ADN, alliberant ssDNA. S’extreuen les seves propietats cinètiques, com la velocitat de translocació – obtenim, independentment de la força aplicada, d’uns 50bp/s, d’acord amb la literatura –. També n’estudiem les seves propietats termodinàmiques, a partir del Teorema de Fluctuació. Finalment, al capítol 9 s’estudien els efectes de certs defectes en molècules d’ADN. A partir d’experiments fora de l’equilibri s’extrau la penalització que suposa per a la hibridització d’ADN la presència d’aquestes bases no complementàries (és a dir, que no són enllaços de A-T o G-C).
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Gryte, Kristofer. "Analysis methods for single molecule fluorescence spectroscopy." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:148969c6-78aa-49c2-8f0e-2d5e5018fd98.

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This thesis describes signal analysis methods for single-molecule fluorescence data. The primary factor motivating method development is the need to distinguish single-molecule FRET fluctuations due to conformational dynamics from fluctuations due to distance-independent FRET changes. Single-molecule Förster resonance energy transfer (FRET) promises a distinct advantage compared to alternative biochemical methods in its potential to relate biomolecular structure to function. Standard measurements assume that the mean transfer efficiency between two fluorescent probes, a donor and an acceptor, corresponds to the mean donor-acceptor distance, thus providing structural information. Accordingly, measurement analysis assumes that mean transfer efficiency fluctuations entail mean donor-acceptor distance fluctuations. Detecting such fluctuations is important in resolving molecular dynamics, as molecular function often necessitates structural changes. A problem arises, however, in that factors other than donor-acceptor distance changes may induce mean transfer efficiency fluctuations. We refer to these factors as distance-independent FRET changes. We present analysis methods to detect distance-independent photophysical dynamics and to determine their correlation with distance-dependent FRET dynamics. First, we review a theory of photon statistics and show how we can use the theory to detect FRET fluctuations. Second, we extend the theory to alternating laser excitation (ALEx) measurements and demonstrate how fluorophore stoichiometry, a measure of fluorophore brightness, reports on distance-independent photophysical dynamics. Next, we provide a measure to determine the extent to which stoichiometry fluctuations account for FRET dynamics. Finally, we use a framework similar to the preceding along with recent advances in the theory of total internal reflection fluorescence (TIRF) microscopy FRET measurements to detect TIRF FRET fluctuations which occur on a timescale faster than the measurement temporal resolution. We validate our methods with simulations and demonstrate their utility in delineating RNA polymerase open complex conformational dynamics.
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8

Dunn, James Albert. "Single Molecule Characterization of Peptide/Hematite Binding." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1494014864020062.

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9

Periz, Coloma Francisco Javier. "Single molecule fluorescence studies of viral transcription." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:d6e72aa8-060c-40fe-a07c-f695585dd43d.

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Rotaviruses are the single most common cause of fatal and severe childhood diarrhoeal illness worldwide (>125 million cases annually). Rotavirus shares structural and functional features with many viruses, such as the presence of segmented double-stranded RNA genomes selectively and tightly packed with a conserved number of transcription complexes in icosahedral capsids. Nascent transcripts exit the capsid through 12 channels, but it is unknown whether these channels specialise in specific transcripts or simply act as general exit conduits; a detailed description of this process is needed for understanding viral replication and genomic organisation. To test these opposing models, a novel single-molecule assay was developed for the capture and identification (CID) of newly synthesised specific RNA transcripts. CID combines the hybridisation of transcripts with biotinylated and FRET compatible labelled ssDNAs with the implementation of recent developments in single molecule fluorescence such as alternating laser excitation (ALEX) and total internal reflection fluorescence (TIRF) microscopy. CID identifies and quantifies specific transcripts of rotavirus based on a FRET/Stoichiometry (E*/S) value of the hybridised labelled probes. I used CID to pull down the capsid on the surface slide and identify partially extruded transcripts of three different segments 2, 6 and 11. The findings presented in this thesis support a model in which each channel specialises in extruding transcripts of a specific segment, that in turn is linked to a single transcription complex. The method can be extended to study other transcription systems including E.coli, and can be further developed as a potential diagnostic tool.
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Duchi, Llumigusin Diego Armando. "Single-molecule studies of transcription initiation." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:fa5d7117-4270-4362-95f4-ce1c870f2921.

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Single-molecule Förster resonance energy transfer (smFRET) has emerged as an important tool for studying biological reactions. This thesis describes smFRET investigations into the mechanism of bacterial transcription initiation. We developed protocols to immobilize RNAP-DNA initiation complexes using vesicles and antibodies. We used these techniques to show that the transcription bubble conformation in immobilized complexes exhibits inter-molecular heterogeneity. We observed large FRET changes that we attribute to transcription bubble opening and closing dynamics. We found that σ70 region 3.2 (σR3.2) influences the kinetics of the bubble dynamics, which supports proposals that σR3.2 interacts with the transcription bubble template strand. We extended our investigations to RNA synthesis and were able to observe abortive initiation cycles directly. We observed RNAP pausing and backtracking for the first time in transcription initiation. We obtained data suggesting that σR3.2 stabilises short RNAs at the active centre and forms a barrier to the extension of RNAs longer than 5-nt in length. We extended our abortive initiation assay to observe signal changes that we attribute to promoter escape. Our data revealed the number of abortive cycles that occur prior to escape, the kinetics of promoter escape, and pausing events that may have some regulatory function. We investigated the conformational dynamics of the RNAP β clamp and observed dynamic conformational changes between clamp-open and clamp-closed states. Our work confirms proposals that the clamp remains stably closed once the open complex (RPO) is formed. We investigated what affect the antibiotics Myxopyronin and Lipiarmycin have on the clamp conformation. Our results revealed that Myxopyronin traps the clamp in a closed conformation, while Lipiarmycin traps it in an open conformation. Overall, we made a number of novel observations that we believe advance our understanding of the mechanism of transcription. We hope that the discoveries reported here will direct future research efforts into RNAP function.
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Книги з теми "Single-molecule biophysic"

1

Komatsuzaki, Tamiki, Masaru Kawakami, Satoshi Takahashi, Haw Yang, and Robert J. Silbey, eds. Single-Molecule Biophysics. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.

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Single-molecule cellular biophysics. Cambridge: Cambridge University Press, 2012.

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3

Hinterdorfer, Peter, and Antoine Oijen, eds. Handbook of Single-Molecule Biophysics. New York, NY: Springer US, 2009. http://dx.doi.org/10.1007/978-0-387-76497-9.

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Handbook of single-molecule biophysics. Dordrecht: Springer, 2009.

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5

Lyubchenko, Yuri L., ed. An Introduction to Single Molecule Biophysics. Boca Raton : Taylor & Francis, 2017. | Series: Foundations of biochemistry and biophysics: CRC Press, 2017. http://dx.doi.org/10.1201/b22505.

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Theory and evaluation of single-molecule signals. Hackensack, NJ: World Scientific, 2008.

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7

Dinman, Jonathan D. Biophysical approaches to translational control of gene expression. New York, NY: Springer New York, 2013.

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8

Paul, Selvin. Single Molecule Biophysics. Taylor & Francis Group, 2011.

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9

Leake, Mark C. Single-Molecule Cellular Biophysics. Cambridge University Press, 2012.

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10

Leake, Mark C. Single-Molecule Cellular Biophysics. Cambridge University Press, 2013.

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Частини книг з теми "Single-molecule biophysic"

1

Yang, Haw. "Change-Point Localization and Wavelet Spectral Analysis of Single-Molecule Time Series." In Single-Molecule Biophysics, 217–43. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.ch9.

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Takahashi, Satoshi, and Kiyoto Kamagata. "Staring at a Protein: Ensemble and Single-Molecule Investigations on Protein-Folding Dynamics." In Single-Molecule Biophysics, 1–22. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.ch1.

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3

Gopich, Irina V., and Attila Szabo. "Theory of Single-Molecule FRET Efficiency Histograms." In Single-Molecule Biophysics, 245–97. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.ch10.

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Baba, Akinori, and Tamiki Komatsuzaki. "Multidimensional Energy Landscapes in Single-Molecule Biophysics." In Single-Molecule Biophysics, 299–327. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.ch11.

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Wu, Jianlan, and Jianshu Cao. "Generalized Michaelis-Menten Equation for Conformation-Modulated Monomeric Enzymes." In Single-Molecule Biophysics, 329–65. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.ch12.

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Flomenbom, Ophir. "Making it Possible: Constructing a Reliable Mechanism from a Finite Trajectory." In Single-Molecule Biophysics, 367–93. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.ch13.

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Yew, Zu Thur, Peter D. Olmsted, and Emanuele Paci. "Free Energy Landscapes of Proteins: Insights from Mechanical Probes." In Single-Molecule Biophysics, 395–417. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.ch14.

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Takagi, Hiroaki, and Masatoshi Nishikawa. "Mechanochemical Coupling Revealed by the Fluctuation Analysis of Different Biomolecular Motors." In Single-Molecule Biophysics, 419–35. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.ch15.

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9

Nettels, Daniel, and Benjamin Schuler. "Single-Molecule FRET of Protein-Folding Dynamics." In Single-Molecule Biophysics, 23–48. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.ch2.

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Okamoto, Kenji, and Masahide Terazima. "Quantitative Analysis of Single-Molecule FRET Signals and its Application to Telomere DNA." In Single-Molecule Biophysics, 49–70. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118131374.ch3.

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Тези доповідей конференцій з теми "Single-molecule biophysic"

1

Gregor, Ingo, Arindam Ghosh, Tao Chen, Sufi O. Raja, Alexey I. Chizhik, Christoph F. Schmidt, and Jörg Enderlein. "Studying membrane biophysics using Graphene-induced energy-transfer." In Single Molecule Spectroscopy and Superresolution Imaging XV, edited by Ingo Gregor, Rainer Erdmann, and Felix Koberling. SPIE, 2022. http://dx.doi.org/10.1117/12.2616863.

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Erickson, David. "Single-Molecule Biophysics with Optofluidic Trapping." In Frontiers in Optics. Washington, D.C.: OSA, 2011. http://dx.doi.org/10.1364/fio.2011.fma2.

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Shepard, Ken. "Solid-state electronics and single-molecule biophysics." In 2012 70th Annual Device Research Conference (DRC). IEEE, 2012. http://dx.doi.org/10.1109/drc.2012.6256965.

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Freedman, Kevin J., Maike Ju, Sally A. Peyman, Anmiv Prabhu, Per Jemth, Joshua Edel, and Min Jun Kim. "Single molecule protein biophysics using chemically modified nanopores." In 2010 Ninth IEEE Sensors Conference (SENSORS 2010). IEEE, 2010. http://dx.doi.org/10.1109/icsens.2010.5690733.

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Moerner, W. E. "Single-Molecule Biophysical Imaging, Superresolution, and Trapping." In Laser Science. Washington, D.C.: OSA, 2009. http://dx.doi.org/10.1364/ls.2009.lswa1.

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Moerner, W. E. "Single-Molecule Biophysical Imaging, Nanophotonics, and Trapping." In Frontiers in Optics. Washington, D.C.: OSA, 2007. http://dx.doi.org/10.1364/fio.2007.jmc1.

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7

Bao, Gang. "Single-Molecule Biomechanics: DNA and Protein Deformation." In ASME 2000 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2000. http://dx.doi.org/10.1115/imece2000-1918.

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Abstract With the advent of molecular biology and biophysics during the past decade, single-molecule biomechanics has emerged as a new field. Different techniques have been used to study the mechanical properties of DNA and protein molecules; various models have been developed to quantify the deformation of biomolecules under force. Here we review some of these advances, explore the connection between mechanics and biochemistry, and discuss the concepts, issues and challenges in developing molecular biomechanics.
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8

Schwille, Petra. "Fluorescence correlation spectroscopy and its impact on single molecule biophysics." In Laser Applications to Chemical and Environmental Analysis. Washington, D.C.: OSA, 2002. http://dx.doi.org/10.1364/lacea.2002.thc1.

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9

Block, Steven M. "Advances in Single Molecule Biophysics: Breaking the Nanometer Barrier with Optical Tweezers." In Frontiers in Optics. Washington, D.C.: OSA, 2007. http://dx.doi.org/10.1364/fio.2007.fwp1.

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Звіти організацій з теми "Single-molecule biophysic"

1

Ha, Ji Won. Single Molecule and Nanoparticle Imaging in Biophysical, Surface, and Photocatalysis Studies. Office of Scientific and Technical Information (OSTI), January 2013. http://dx.doi.org/10.2172/1116723.

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Tzfira, Tzvi, Michael Elbaum, and Sharon Wolf. DNA transfer by Agrobacterium: a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, December 2005. http://dx.doi.org/10.32747/2005.7695881.bard.

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Agrobacteriumtumefaciensmediates genetic transformation of plants. The possibility of exchanging the natural genes for other DNA has led to Agrobacterium’s emergence as the primary vector for genetic modification of plants. The similarity among eukaryotic mechanisms of nuclear import also suggests use of its active elements as media for non-viral genetic therapy in animals. These considerations motivate the present study of the process that carries DNA of bacterial origin into the host nucleus. The infective pathway of Agrobacterium involves excision of a single-stranded DNA molecule (T-strand) from the bacterial tumor-inducing plasmid. This transferred DNA (T-DNA) travels to the host cell cytoplasm along with two virulence proteins, VirD2 and VirE2, through a specific bacteriumplant channel(s). Little is known about the precise structure and composition of the resulting complex within the host cell and even less is known about the mechanism of its nuclear import and integration into the host cell genome. In the present proposal we combined the expertise of the US and Israeli labs and revealed many of the biophysical and biological properties of the genetic transformation process, thus enhancing our understanding of the processes leading to nuclear import and integration of the Agrobacterium T-DNA. Specifically, we sought to: I. Elucidate the interaction of the T-strand with its chaperones. II. Analyzing the three-dimensional structure of the T-complex and its chaperones in vitro. III. Analyze kinetics of T-complex formation and T-complex nuclear import. During the past three years we accomplished our goals and made the following major discoveries: (1) Resolved the VirE2-ssDNA three-dimensional structure. (2) Characterized VirE2-ssDNA assembly and aggregation, along with regulation by VirE1. (3) Studied VirE2-ssDNA nuclear import by electron tomography. (4) Showed that T-DNA integrates via double-stranded (ds) intermediates. (5) Identified that Arabidopsis Ku80 interacts with dsT-DNA intermediates and is essential for T-DNA integration. (6) Found a role of targeted proteolysis in T-DNA uncoating. Our research provide significant physical, molecular, and structural insights into the Tcomplex structure and composition, the effect of host receptors on its nuclear import, the mechanism of T-DNA nuclear import, proteolysis and integration in host cells. Understanding the mechanical and molecular basis for T-DNA nuclear import and integration is an essential key for the development of new strategies for genetic transformation of recalcitrant plant species. Thus, the knowledge gained in this study can potentially be applied to enhance the transformation process by interfering with key steps of the transformation process (i.e. nuclear import, proteolysis and integration). Finally, in addition to the study of Agrobacterium-host interaction, our research also revealed some fundamental insights into basic cellular mechanisms of nuclear import, targeted proteolysis, protein-DNA interactions and DNA repair.
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