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Single molecule biophysics

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https://www.readbyqxmd.com/read/29345990/imaging-mrna-in-vivo-from-birth-to-death
#1
Evelina Tutucci, Nathan M Livingston, Robert H Singer, Bin Wu
RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred fromDNAto proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution...
January 18, 2018: Annual Review of Biophysics
https://www.readbyqxmd.com/read/29320683/changes-in-cholesterol-level-alter-integrin-sequestration-in-raft-mimicking-lipid-mixtures
#2
Yifan Ge, Jiayun Gao, Rainer Jordan, Christoph A Naumann
The influence of cholesterol (CHOL) level on integrin sequestration in raft-mimicking lipid mixtures forming coexisting liquid-ordered (lo) and liquid-disordered (ld) lipid domains is investigated using complementary, single-molecule-sensitive, confocal detection methods. Systematic analysis of membrane protein distribution in such a model membrane environment demonstrates that variation of CHOL level has a profound influence on lo-ld sequestration of integrins, thereby exhibiting overall ld preference in the absence of ligands and lo affinity upon vitronectin addition...
January 9, 2018: Biophysical Journal
https://www.readbyqxmd.com/read/29320680/k-ras4b-remains-monomeric-on-membranes-over-a%C3%A2-wide-range-of%C3%A2-surface-densities-and-lipid-compositions
#3
Jean K Chung, Young Kwang Lee, John-Paul Denson, William K Gillette, Steven Alvarez, Andrew G Stephen, Jay T Groves
Ras is a membrane-anchored signaling protein that serves as a hub for many signaling pathways and also plays a prominent role in cancer. The intrinsic behavior of Ras on the membrane has captivated the biophysics community in recent years, especially the possibility that it may form dimers. In this article, we describe results from a comprehensive series of experiments using fluorescence correlation spectroscopy and single-molecule tracking to probe the possible dimerization of natively expressed and fully processed K-Ras4B in supported lipid bilayer membranes...
January 9, 2018: Biophysical Journal
https://www.readbyqxmd.com/read/29320677/warhammers-for-peaceful-times
#4
Piotr E Marszalek
The Perkins group has recently developed a number of improved atomic force microscopy cantilevers using the focused ion beam technology. They compared the performance of these cantilevers in "real-life" biophysical single-molecule force spectroscopy measurements on protein unfolding, and the results of this comparison are reported in this issue of Biophysical Journal.
January 9, 2018: Biophysical Journal
https://www.readbyqxmd.com/read/29309633/structure-and-mechanism-of-a-bacterial-t6a-biosynthesis-system
#5
Amit Luthra, William Swinehart, Susan Bayooz, Phuc Phan, Boguslaw Stec, Dirk Iwata-Reuyl, Manal A Swairjo
The universal N(6)-threonylcarbamoyladenosine (t6A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood. Here, we report biochemical and biophysical studies of the t6A biosynthesis system from Thermotoga maritima. Small angle X-ray scattering analysis reveals a symmetric 2:2 stoichiometric complex of TsaB and TsaD (TsaB2D2), as well as 2:2:2 complex (TsaB2D2E2), in which TsaB acts as a dimerization module, similar to the role of Pcc1 in the archaeal system...
January 4, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29288412/closed-loop-doluisio-colon-small-intestine-and-single-pass-intestinal-perfusion-colon-jejunum-in-rat-biophysical-model-and-predictions-based-on-caco-2
#6
Isabel Lozoya-Agullo, Isabel Gonzalez-Alvarez, Moran Zur, Noa Fine-Shamir, Yael Cohen, Milica Markovic, Teresa M Garrigues, Arik Dahan, Marta Gonzalez-Alvarez, Matilde Merino-Sanjuán, Marival Bermejo, Alex Avdeef
PURPOSE: The effective rat intestinal permeability (P eff ) was deconvolved using a biophysical model based on parameterized paracellular, aqueous boundary layer, transcellular permeabilities, and the villus-fold surface area expansion factor. METHODS: Four types of rat intestinal perfusion data were considered: single-pass intestinal perfusion (SPIP) in the jejunum (n = 40), and colon (n = 15), closed-loop (Doluisio type) in the small intestine (n = 78), and colon (n = 74)...
December 29, 2017: Pharmaceutical Research
https://www.readbyqxmd.com/read/29259297/hyperstretching-dna
#7
Koen Schakenraad, Andreas S Biebricher, Maarten Sebregts, Brian Ten Bensel, Erwin J G Peterman, Gijs J L Wuite, Iddo Heller, Cornelis Storm, Paul van der Schoot
The three-dimensional structure of DNA is highly susceptible to changes by mechanical and biochemical cues in vivo and in vitro. In particular, large increases in base pair spacing compared to regular B-DNA are effected by mechanical (over)stretching and by intercalation of compounds that are widely used in biophysical/chemical assays and drug treatments. We present single-molecule experiments and a three-state statistical mechanical model that provide a quantitative understanding of the interplay between B-DNA, overstretched DNA and intercalated DNA...
December 19, 2017: Nature Communications
https://www.readbyqxmd.com/read/29232197/dna-based-construction-at-the-nanoscale-emerging-trends-and-applications
#8
Lourdu Xavier Paulraj, Arun Richard Chandrasekaran
The field of structural DNA nanotechnology has reached adolescence - starting with the creation of artificial immobile junctions leading to the recent DNA-protein hybrid nanoscale shapes - in a span of about 35 years. It is now possible to create complex DNA-based nanoscale shapes and assemblies with greater stability and predictability, thanks to the development of computational tools and advances in experimental techniques. Although started with the original goal of DNA-assisted structure determination of difficult-to-crystallize molecules, DNA nanotechnology has found its applications in a myriad of fields...
December 12, 2017: Nanotechnology
https://www.readbyqxmd.com/read/29217640/lipid-bilayer-mediates-ion-channel-cooperativity-in-a-model-of-hair-cell-mechanotransduction
#9
Francesco Gianoli, Thomas Risler, Andrei S Kozlov
Mechanoelectrical transduction in the inner ear is a biophysical process underlying the senses of hearing and balance. The key players involved in this process are mechanosensitive ion channels. They are located in the stereocilia of hair cells and opened by the tension in specialized molecular springs, the tip links, connecting adjacent stereocilia. When channels open, the tip links relax, reducing the hair-bundle stiffness. This gating compliance makes hair cells especially sensitive to small stimuli. The classical explanation for the gating compliance is that the conformational rearrangement of a single channel directly shortens the tip link...
December 19, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29215640/catching-dna-with-hoops-biophysical-approaches-to-clarify-the-mechanism-of-smc-proteins
#10
REVIEW
Jorine Eeftens, Cees Dekker
Structural maintenance of chromosome (SMC) complexes are central regulators of chromosome architecture that are essential in all domains of life. For decades, the structural biology field has been debating how these conserved protein complexes use their intricate ring-like structures to structurally organize DNA. Here, we review the contributions of single-molecule biophysical approaches to resolving the molecular mechanism of SMC protein function.
December 7, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/29211997/challenges-in-estimating-the-motility-parameters-of-single-processive-motor-proteins
#11
Felix Ruhnow, Linda Kloβ, Stefan Diez
Cytoskeletal motor proteins are essential to the function of a wide range of intracellular mechano-systems. The biophysical characterization of their movement along their filamentous tracks is therefore of large importance. Toward this end, single-molecule, in vitro stepping-motility assays are commonly used to determine motor velocity and run length. However, comparing results from such experiments has proved difficult due to influences from variations in the experimental conditions and the data analysis methods...
December 5, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/29190805/single-molecule-study-of-full-length-nachbac-by-planar-lipid-bilayer-recording
#12
Andrew Jo, Hiofan Hoi, Hang Zhou, Manisha Gupta, Carlo D Montemagno
Planar lipid bilayer device, alternatively known as BLM, is a powerful tool to study functional properties of conducting membrane proteins such as ion channels and porins. In this work, we used BLM to study the prokaryotic voltage-gated sodium channel (Nav) NaChBac in a well-defined membrane environment. Navs are an essential component for the generation and propagation of electric signals in excitable cells. The successes in the biochemical, biophysical and crystallographic studies on prokaryotic Navs in recent years has greatly promoted the understanding of the molecular mechanism that underlies these proteins and their eukaryotic counterparts...
2017: PloS One
https://www.readbyqxmd.com/read/29182873/mechanical-unfolding-of-macromolecules-coupled-to-bond-dissociation
#13
Ariane Nunes-Alves, Guilherme Menegon Arantes
Single molecule force spectroscopy has become a powerful tool to investigate molecular mechanisms in biophysics and materials science. In particular, the new field of polymer mechanochemistry has emerged to study how tension may induce chemical reactions in a macromolecule. A rich example is the mechanical unfolding of the metalloprotein rubredoxin coupled to dissociation of iron-sulfur bonds that has recently been studied in detail by atomic force microscopy. Here, we present a simple molecular model composed of a classical all-atom force field description, implicit solvation and steered molecular dynamics simulation to describe the mechanical properties and mechanism of forced unfolding coupled to covalent bond dissociation of macromolecules...
November 28, 2017: Journal of Chemical Theory and Computation
https://www.readbyqxmd.com/read/29175904/cohesin-sa2-is-a-sequence-independent-dna-binding-protein-that-recognizes-dna-replication-and-repair-intermediates
#14
Preston Countryman, Yanlin Fan, Aparna Gorthi, Hai Pan, Jack Strickland, Parminder Kaur, Xuechun Wang, Jianggguo Lin, Xiaoying Lei, Christian White, Changjiang You, Nicolas Wirth, Ingrid Tessmer, Jacob Pieler, Robert Riehn, Alexander J R Bishop, Yizhi Jane Tao, Hong Wang
Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA...
November 24, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29164072/a-new-strain-collection-for-improved-expression-of-outer-membrane-proteins
#15
Ina Meuskens, Marcin Michalik, Nandini Chauhan, Dirk Linke, Jack C Leo
Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging...
2017: Frontiers in Cellular and Infection Microbiology
https://www.readbyqxmd.com/read/29141138/differential-effects-of-strand-asymmetry-on-the-energetics-and-structural-flexibility-of-dna-internal-loops
#16
Thao Tran, Brian Cannon
Internal loops within structured nucleic acids disrupt local base-stacking and destabilize neighboring helical domains; however, these structural motifs also expand the conformational and functional capabilities for structured nucleic acids. Variations in size, distribution of loop nucleotides on opposing strands (strand asymmetry), and sequence alter their biophysical properties. Here, the thermodynamics and structural flexibility of poly(T)-rich DNA internal loops were systematically investigated in terms of loop size and strand asymmetry...
November 15, 2017: Biochemistry
https://www.readbyqxmd.com/read/29133841/nanopore-electric-snapshots-of-an-rna-tertiary-folding-pathway
#17
Xinyue Zhang, Dong Zhang, Chenhan Zhao, Kai Tian, Ruicheng Shi, Xiao Du, Andrew J Burcke, Jing Wang, Shi-Jie Chen, Li-Qun Gu
The chemical properties and biological mechanisms of RNAs are determined by their tertiary structures. Exploring the tertiary structure folding processes of RNA enables us to understand and control its biological functions. Here, we report a nanopore snapshot approach combined with coarse-grained molecular dynamics simulation and master equation analysis to elucidate the folding of an RNA pseudoknot structure. In this approach, single RNA molecules captured by the nanopore can freely fold from the unstructured state without constraint and can be programmed to terminate their folding process at different intermediates...
November 13, 2017: Nature Communications
https://www.readbyqxmd.com/read/29108427/what-is-all-this-fuss-about-tus-comparison-of-recent-findings-from-biophysical-and-biochemical-experiments
#18
Bojk A Berghuis, Vlad-Stefan Raducanu, Mohamed M Elshenawy, Slobodan Jergic, Martin Depken, Nicholas E Dixon, Samir M Hamdan, Nynke H Dekker
Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. In Escherichia coli, a "replication fork trap" - found within a chromosomal region where forks are allowed to enter but not leave - is set by the protein-DNA roadblock Tus-Ter. The exact sequence of events by which Tus-Ter blocks replisomes approaching from one direction but not the other has been the subject of controversy for many decades. Specific protein-protein interactions between the nonpermissive face of Tus and the approaching helicase were challenged by biochemical and structural studies...
November 6, 2017: Critical Reviews in Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/29068447/a-highly-occupied-single-cell-trapping-microarray-for-determination-of-cell-membrane-permeability
#19
Lindong Weng, Felix Ellett, Jon Edd, Keith H K Wong, Korkut Uygun, Daniel Irimia, Shannon L Stott, Mehmet Toner
Semi- and selective permeability is a fundamentally important characteristic of the cell membrane. Membrane permeability can be determined by monitoring the volumetric change of cells following exposure to a non-isotonic environment. For this purpose, several microfluidic perfusion chambers have been developed recently. However, these devices only allow the observation of one single cell or a group of cells that may interact with one another in an uncontrolled way. Some of these devices have integrated on-chip temperature control to investigate the temperature-dependence of membrane permeability, but they inevitably require sophisticated fabrication and assembly, and delicate temperature and pressure calibration...
November 21, 2017: Lab on a Chip
https://www.readbyqxmd.com/read/29054802/thermodynamic-study-of-aptamers-binding-to-their-target-proteins
#20
Taiichi Sakamoto, Eric Ennifar, Yoshikazu Nakamura
Aptamers are nucleic acids that bind to a target molecule with high affinity and specificity, which are selected from systematic evolution of ligands by exponential enrichment (SELEX). Aptamers feature high affinity and specificity to their target molecule and a large structural diversity; biophysical tools, together with structural studies, are essential to reveal the mechanism of aptamers recognition. Furthermore, understanding the mechanism of action would also contribute to their development for therapeutic applications...
October 18, 2017: Biochimie
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