Effect of Different Crowding Agents on the Architectural Properties of The
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Eindhoven University of Technology MASTER Unraveling single-bond kinetics in tethered particle motion experiments using molecular dynamics simulations Merkus, K.E. Award date: 2015 Link to publication Disclaimer This document contains a student thesis (bachelor's or master's), as authored by a student at Eindhoven University of Technology. Student theses are made available in the TU/e repository upon obtaining the required degree. The grade received is not published on the document as presented in the repository. The required complexity or quality of research of student theses may vary by program, and the required minimum study period may vary in duration. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain Eindhoven University of Technology Department of Applied Physics Theory of polymers and soft matter Unraveling single-bond kinetics in tethered particle motion experiments using molecular dynamics simulations K.E. Merkus July 2015 Under supervision of dr. C. Storm Abstract Tethered particle motion (TPM), the motion of a micro- or nanobead tethered to a substrate by a macromolecule, is widely studied to understand various properties related to the tether. However, we propose a whole new way of looking at TPM: TPM as a probe for secondary bonds. -
Unpacking the Origins of In-Cell Crowding Kim A
COMMENTARY COMMENTARY Unpacking the origins of in-cell crowding Kim A. Sharpa,1 The living cell, the fundamental unit of biological meaning that a large excluded volume is a property of organization, was discovered in its apparent simplicity all macromolecules. An extensive review covers much by van Leeuwenhoek in the 17th century. Scientists of the subsequent literature (5). Of course, solutes can since then have continued to unpack nested levels cause nonideal behavior through a second mecha- of amazingly complex cellular structure and organiza- nism, strong intermolecular interactions such as elec- tion, right down to the atomic level. However, one of trostatic and hydrophobic effects. Although crowding the cell’s simplest properties is not yet understood, is now often used as a synonym for any effects of con- perhaps even misunderstood. The interior of the cell centrated solutes, for the purpose of this commentary I contains a high concentration of dissolved solutes, prin- will take crowding to refer to the excluded volume cipally ions, metabolites, proteins, and RNA. In a word, effects, as originally defined (3, 4), because disentan- it is crowded in there. Estimates range from 30% to 40% gling size effects and interaction effects is precisely by volume occupied by protein and RNA solutes, where the advances of Smith et al. (2) lie. I also discuss depending on the cell type and compartment (1). Bio- effects on equilibria only. Given our recently revised chemists and biophysicists have long been concerned understanding of equilibrium effects, it seems prema- that these high concentrations impart significantly non- ture to discuss the more complex effects of crowding ideal solution behavior. -
Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level
Int. J. Mol. Sci. 2013, 14, 3961-3992; doi:10.3390/ijms14023961 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level Carina Monico 1,*, Marco Capitanio 1,2, Gionata Belcastro 1, Francesco Vanzi 1,3 and Francesco S. Pavone 1,2,4,5 1 European Laboratory for Non-linear Spectroscopy, Via Nello Carrara 1, 50019 Sesto Fiorentino (FI), Italy; E-Mails: [email protected] (M.C.); [email protected] (G.B.); [email protected] (F.V.); [email protected] (F.S.P.) 2 Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto Fiorentino (FI), Italy 3 Department of Evolutionary Biology “Leo Pardi”, University of Florence, Via Romana 17, 50125 Florence, Italy 4 National Institute of Optics–National Research Council, Largo Fermi 6, 50125 Florence, Italy 5 International Center of Computational Neurophotonics, Via Nello Carrara 1, 50019 Sesto Fiorentino (FI), Italy * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +39-055-457-2475; Fax: +39-055-457-2451. Received: 15 December 2012; in revised form: 13 January 2013 / Accepted: 4 February 2013 / Published: 18 February 2013 Abstract: The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety of methods in biology. -
How Can Biochemical Reactions Within Cells Differ from Those in Test Tubes?
ß 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 000, 000–000 doi:10.1242/jcs.170183 CORRECTION How can biochemical reactions within cells differ from those in test tubes? Allen P. Minton There was an error published in J. Cell Sci. 119, 2863-2869. There is a typographical error in equation (2). The right-hand side of this equation should read ‘DFI,AB 2 (DFI,A+D FI,B)’. JCS and the author apologise for any confusion that this error might have caused. 1 Journal of Cell Science JCS170183.3d 22/2/15 09:24:29 The Charlesworth Group, Wakefield +44(0)1924 369598 - Rev 9.0.225/W (Oct 13 2006) Commentary 2863 How can biochemical reactions within cells differ from those in test tubes? Allen P. Minton Section on Physical Biochemistry, Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, USA e-mail: [email protected] Accepted 23 May 2006 Journal of Cell Science 119, 2863-2869 Published by The Company of Biologists 2006 doi:10.1242/jcs.03063 Summary Nonspecific interactions between individual macro- interactions tend to enhance the rate and extent of molecules and their immediate surroundings (‘background macromolecular associations in solution, whereas interactions’) within a medium as heterogeneous and predominately attractive background interactions tend to highly volume occupied as the interior of a living cell can enhance the tendency of macromolecules to associate on greatly influence the equilibria and rates of reactions in adsorbing surfaces. -
Eindhoven University of Technology BACHELOR Assay Optimization for Single DNA Tethered Particle Motion Hoeven, F.J.P
Eindhoven University of Technology BACHELOR Assay optimization for single DNA tethered particle motion Hoeven, F.J.P. Award date: 2014 Link to publication Disclaimer This document contains a student thesis (bachelor's or master's), as authored by a student at Eindhoven University of Technology. Student theses are made available in the TU/e repository upon obtaining the required degree. The grade received is not published on the document as presented in the repository. The required complexity or quality of research of student theses may vary by program, and the required minimum study period may vary in duration. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain Assay optimization for single DNA tethered particle motion Frank Hoeven MBx 2014-04 Supervisors: E.W.A. Visser and L.J. van IJzendoorn Eindhoven University of Technology Group Molecular Biosensors for Medical Diagnostics April-July 2014 TNO 3rd floor Abstract In this report research is described to investigate the possibility to apply Tethered Particle Motion (TPM) in an immunoassay. A magnetic particle binds a target molecule in a sandwich assay, where the magnetic particle and the target molecule bind via a biotin streptavidin bond and the target molecule binds to a surface with an antigen-antibody bond. -
Effects of Macromolecular Crowding on Gene Expression Studied In
Effects of macromolecular crowding on gene expression studied in protocell models The work described in this thesis was supported by a European Research Council (ERC), Advanced Grant (246812 Intercom) and a VICI grant from the Netherlands Organisation for Scientific Research (NWO) ISBN: 978-94-6295-151-8 Cover design: Denis Arslanov and Ekaterina Sokolova Printed & Lay Out by: Proefschriftmaken.nl || Uitgeverij BOXPress Published by: Uitgeverij BOXPress, ’s-Hertogenbosch Effects of macromolecular crowding on gene expression studied in protocell models Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. dr. Th.L.M. Engelen, volgens besluit van het college van decanen in het openbaar te verdedigen op dinsdag 12 mei 2015 om 14.30 uur precies door Ekaterina Alexandrovna Sokolova geboren op 30 augustus 1987 te Angren (Uzbekistan) Promotor: Prof. dr. W. T. S. Huck Manuscriptcommissie: Prof. dr. ir. J. C. M. van Hest (voorzitter) Prof. dr. H. N. W. Lekkerkerker (Universiteit Utrecht) Dr. V. Noireaux (University of Minnesota, VS) Effects of macromolecular crowding on gene expression studied in protocell models Doctoral Thesis to obtain the degree of doctor from Radboud University Nijmegen on the authority of the Rector Magnificus prof. dr. Th.L.M. Engelen, according to the decision of the Council of Deans to be defended in public on Tuesday, May 12, 2015 at 14.30 hours by Ekaterina Alexandrovna Sokolova Born on August 30, 1987 in Angren (Uzbekistan) Supervisor: Prof. dr. W. T. S. Huck Doctoral Thesis Committee: Prof. dr. ir. J. C. M. van Hest (chairman) Prof. -
Analysis of DNA Looping Kinetics in Tethered Particle Motion Experiments the Fragile X Mental Retardation-1 (Fmr1) Gene
418a Tuesday, February 5, 2013 to O1, O2, and O3 operators in the lac operon. Specifically, we demonstrated that the Brownian motion of beads tethered to a microscope cover slip. The Brow- LacI kept two supercoils within the 401 bp DNA-loop between O1 and O2 op- nian motion changes when a protein binds to and deforms the DNA ’leash’, for erators. Additionally, We carried out time course studies to determine the stabil- example in the formation of a DNA loop. A complicating factor in the interpre- ity of the topological barriers that are produced by the different LacI-operator tation of TPM data is that the number of observable states, corresponding to complexes. Our results showed that the stability of the topological barriers cor- different conformations of the DNA-protein complex, is often not known in ad- relates with the DNA-binding affinity of LacI to the different operators i.e., O1, vance. Moreover, conformational transitions that occur on time scales compa- O2, O3, and Os operators. Furthermore, we confirmed our previous observation rable to the diffusive motion of the bead are difficult to extract from the data. in which LacI is able to ‘‘keep’’ certain superhelical energy to stabilize LacI- We present an analysis method for TPM data that overcomes these limitations in lacO1 complexes. Our results can be explained by a model in which LacI be- existing approaches. Our method relies on variational Bayesian inference on a var- haves as a topological barrier in the lac operon to regulate the expression of lac- iantofthe Hidden Markov model. -
Calibration of Tethered Particle Motion Experiments
CALIBRATION OF TETHERED PARTICLE MOTION EXPERIMENTS LIN HAN∗, BERTRAND LUI∗†, SETH BLUMBERG∗‡, JOHN F. BEAUSANG§, PHILIP C. NELSON§ , AND ROB PHILLIPS∗¶ Abstract. The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA looping and tran- scription. TPM experiments exploit the Brownian motion of a DNA-tethered bead to probe biologically relevant conformational changes of the tether. In these experiments, a change in the extent of the bead’s random motion is used as a reporter of the underlying macromolecular dynamics and is often deemed sufficient for TPM analysis. However, a complete understanding of how the motion depends on the physical properties of the tethered particle complex would permit more quantitative and accurate evaluation of TPM data. For instance, such understanding can help extract details about a looped complex geometry (or multiple coexisting geometries) from TPM data. To better char- acterize the measurement capabilities of TPM experiments involving DNA tethers, we have carried out a detailed calibration of TPM magnitude as a function of DNA length and particle size. We also explore how experimental parameters such as acquisition time and exposure time affect the apparent motion of the tethered particle. We vary the DNA length from 200 bp to 2.6 kbp and consider particle diameters of 200, 490 and 970 nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation. Key words. Tethered particle; DNA; Brownian motion; calibration; single molecule AMS(MOS) subject classifications. -
Macromolecular Crowding and Protein Chemistry: Views from Inside and Outside Cells
Macromolecular Crowding and Protein Chemistry: Views from Inside and Outside Cells Yaqiang Wang A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry Chapel Hill 2012 Approved by: Nancy Allbritton, Ph.D. Christopher Fecko, Ph.D. Gary Pielak, Ph.D. Linda Spremulli, Ph.D. Nancy Thompson, Ph.D. © 2012 Yaqiang Wang ALL RIGHTS RESERVED ii Abstract YAQIANG WANG: Macromolecular Crowding and Protein Chemistry: Views from Inside and Outside Cells (Under the direction of Professor Gary J. Pielak, Ph.D.) The cytoplasm is crowded, and the concentration of macromolecules can reach ~ 300 g/L, an environment vastly different from the dilute, idealized conditions usually used in biophysical studies. Macromolecular crowding arise from two phenomena, excluded volume and nonspecific chemical interactions, until recently, only excluded volume effect has been considered. Theory predicts that this macromolecular crowding can have large effects. Most proteins, however, are studied outside cells in dilute solution with macromolecule concentrations of 10 g/L or less. In-cell NMR provides a means to assess protein biophysics at atomic resolution in living cells, but it remains in its infancy, and several potential challenges need to be addressed. One challenge is the inability to observe 15N-1H NMR spectra from many small globular proteins. 19F NMR was used to expand the application of in-cell NMR. This work suggests that high viscosity and weak interactions in the cytoplasm can make routine 15N enrichment a poor choice for in-cell NMR studies of globular proteins in Escherichia coli. -
1 Probing a Label-Free Local Bend in Dna by Single
PROBING A LABEL-FREE LOCAL BEND IN DNA BY SINGLE MOLECULE TETHERED PARTICLE MOTION 1,2,3,4,+ 3,4,+ 1,2 1,2 Annaël Brunet , Sébastien Chevalier , Nicolas Destainville , Manoel Manghi , Philippe Rousseau5,6, Maya Salhi5,6, Laurence Salomé3,4, Catherine Tardin3,4,*. 1 CNRS ; LPT (Laboratoire de Physique Théorique), UMR UPS-CNRS 5152, 118 route de Narbonne, F-31062 Toulouse, France. 2 Université de Toulouse, UPS; LPT, F-31062 Toulouse, France 3 CNRS; IPBS (Institut de Pharmacologie et de Biologie Structurale), UMR UPS-CNRS 5089, 205 route de Narbonne, F-31077 Toulouse, France 4 Université de Toulouse, UPS ; IPBS , F-31077 Toulouse, France 5 CNRS ; LMGM (Laboratoire de Microbiologie et Génétique Moléculaires), UMR CNRS-UPS 5100, 118 route de Narbonne, F-31062 Toulouse; France 6 Université de Toulouse ; UPS; LMGM; F-31062 Toulouse; France * To whom correspondence should be addressed. Tel: +33 561175468; Fax: +33 561175994; Email: [email protected] + The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. 1 ABSTRACT: Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. -
Biosensing Based on Tethered Particle Motion
Biosensing based on tethered particle motion Citation for published version (APA): Visser, E. W. A. (2017). Biosensing based on tethered particle motion. Technische Universiteit Eindhoven. Document status and date: Published: 19/04/2017 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us at: [email protected] providing details and we will investigate your claim. -
Investigating Molecular Crowding During Cell Division in Budding Yeast with FRET
Investigating molecular crowding during cell division in budding yeast with FRET Sarah Lecinski1,a, Jack W Shepherd1,a,b, Lewis Framec, Imogen Haytonb, Chris MacDonaldb, Mark C Leakea,b* 1These authors contributed equally a Department of Physics, University of York, York, YO10 5DD b Department of Biology, University of York, York, YO10 5DD c School of Natural Sciences, University of York, York, YO10 5DD * To whom correspondence should be addressed. Email [email protected] Abstract Cell division, aging, and stress recovery triggers spatial reorganization of cellular components in the cytoplasm, including membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model system to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a Förster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to cellular growth of both mother and daughter cells, in addition to osmotic stress, and reveal hot spots of crowding across the bud neck in the burgeoning daughter cell. This crowding might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding whilst simultaneously imaging a third color, which can be used as a marker that labels organelle membranes.