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Single molecule biophysics

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https://www.readbyqxmd.com/read/29141138/differential-effects-of-strand-asymmetry-on-the-energetics-and-structural-flexibility-of-dna-internal-loops
#1
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
#2
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
#3
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
#4
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...
October 25, 2017: Lab on a Chip
https://www.readbyqxmd.com/read/29054802/thermodynamic-study-of-aptamers-binding-to-their-target-proteins
#5
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
https://www.readbyqxmd.com/read/29053695/targeting-cysteine-thiols-for-in-vitro-site-specific-glycosylation-of-recombinant-proteins
#6
Yoo Jung Choi, Jinhui Zhu, Steve Chung, Naveed Siddiqui, Qingping Feng, Peter B Stathopulos
Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca(2+)) entry which is the principal Ca(2+) signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca(2+) sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein...
October 4, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29045872/the-detection-of-nanoscale-membrane-bending-with-polarized-localization-microscopy
#7
Abir M Kabbani, Christopher V Kelly
The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. The initiation of membrane bending occurs at a length scale that is irresolvable by most superresolution optical microscopy methods. Here, we report the development of polarized localization microscopy (PLM), a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics and molecular sorting...
October 17, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/29024433/cation-induced-stabilization-and-denaturation-of-dna-origami-nanostructures-in-urea-and-guanidinium-chloride
#8
Saminathan Ramakrishnan, Georg Krainer, Guido Grundmeier, Michael Schlierf, Adrian Keller
The stability of DNA origami nanostructures under various environmental conditions constitutes an important issue in numerous applications, including drug delivery, molecular sensing, and single-molecule biophysics. Here, the effect of Na(+) and Mg(2+) concentrations on DNA origami stability is investigated in the presence of urea and guanidinium chloride (GdmCl), two strong denaturants commonly employed in protein folding studies. While increasing concentrations of both cations stabilize the DNA origami nanostructures against urea denaturation, they are found to promote DNA origami denaturation by GdmCl...
October 12, 2017: Small
https://www.readbyqxmd.com/read/29018324/design-and-characterization-of-a-human-monoclonal-antibody-that-modulates-mutant-connexin-26-hemichannels-implicated-in-deafness-and-skin-disorders
#9
Liang Xu, Andrea Carrer, Francesco Zonta, Zhihu Qu, Peixiang Ma, Sheng Li, Federico Ceriani, Damiano Buratto, Giulia Crispino, Veronica Zorzi, Gaia Ziraldo, Francesca Bruno, Chiara Nardin, Chiara Peres, Flavia Mazzarda, Anna M Salvatore, Marcello Raspa, Ferdinando Scavizzi, Youjun Chu, Sichun Xie, Xuemei Yang, Jun Liao, Xiao Liu, Wei Wang, Shanshan Wang, Guang Yang, Richard A Lerner, Fabio Mammano
Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions...
2017: Frontiers in Molecular Neuroscience
https://www.readbyqxmd.com/read/28993836/natural-and-unnatural-small-molecules-as-pharmacological-chaperones-and-inhibitors-in-cancer
#10
Isabel Betancor-Fernández, David J Timson, Eduardo Salido, Angel L Pey
Mutations causing single amino acid exchanges can dramatically affect protein stability and function, leading to disease. In this chapter, we will focus on several representative cases in which such mutations affect protein stability and function leading to cancer. Mutations in BRAF and p53 have been extensively characterized as paradigms of loss-of-function/gain-of-function mechanisms found in a remarkably large fraction of tumours. Loss of RB1 is strongly associated with cancer progression, although the molecular mechanisms by which missense mutations affect protein function and stability are not well known...
October 10, 2017: Handbook of Experimental Pharmacology
https://www.readbyqxmd.com/read/28975798/label-free-single-molecule-thermoscopy-using-a-laser-heated-nanopore
#11
Hirohito Yamazaki, Rui Hu, Robert Y Henley, Justin Halman, Kirill A Afonin, Dapeng Yu, Qing Zhao, Meni Wanunu
When light is used to excite electronic transitions in a material, nonradiative energy during relaxation is often released in the form of heat. In this work, we show that photoexcitation of a silicon nitride nanopore using a focused visible laser results in efficient localized photothermal heating, which reduces the nearby electrolyte viscosity and increases the ionic conductance. In addition, a strong localized thermal gradient in the pore vicinity is produced, evidenced by finite-element simulations and experimental observation of both ion and DNA thermophoresis...
October 10, 2017: Nano Letters
https://www.readbyqxmd.com/read/28972764/an-optical-tweezers-platform-for-single-molecule-force-spectroscopy-in-organic-solvents
#12
Jacob W Black, Maria Kamenetska, Ziad Ganim
Observation at the single molecule level has been a revolutionary tool for molecular biophysics and materials science, but single molecule studies of solution-phase chemistry are less widespread. In this work we develop an experimental platform for solution-phase single molecule force spectroscopy in organic solvents. This optical-tweezer-based platform was designed for broad chemical applicability and utilizes optically trapped core-shell microspheres, synthetic polymer tethers, and click chemistry linkages formed in situ...
October 17, 2017: Nano Letters
https://www.readbyqxmd.com/read/28972167/functional-analysis-of-an-unusual-porin-like-channel-that-imports-chitin-for-alternative-carbon-metabolism-in-escherichia-coli
#13
H Sasimali M Soysa, Albert Schulte, Wipa Suginta
Escherichia coli have the genetic potential to use chitin as a carbon source in the absence of glucose, importing it via the chitin-uptake channel EcChiP for processing by the glucosamine catabolic pathway. The chip gene is usually not expressed when E. coli are grown on glucose-enriched nutrients, providing a general regulatory mechanism for the pathway. EcChiP is unusual in that it is homologous to porins and monomeric instead of trimeric, the typical form of sugar-specific channels, making it unclear how this channel operates...
September 27, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28972148/time-course-negative-stain-electron-microscopy-based-analysis-for-investigating-protein-protein-interactions-at-the-single-molecule-level
#14
Bartek Nogal, Charles A Bowman, Andrew B Ward
Several biophysical approaches are available to study protein-protein interactions. Most approaches are conducted in bulk solution, and are therefore limited to an average measurement of the ensemble of molecular interactions. Here, we show how single-particle EM can enrich our understanding of protein-protein interactions at the single-molecule level and potentially capture states that are unobservable with ensemble methods because they are below the limit of detection or not conducted on an appropriate timescale...
September 29, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28968783/smlocalizer-a-gpu-accelerated-imagej-plugin-for-single-molecule-localization-microscopy
#15
Kristoffer Bernhem, Hjalmar Brismar
Summary: SMLocalizer combines the availability of ImageJ with the power of GPU processing for fast and accurate analysis of single molecule localization microscopy data. Analysis of 2D and 3D data in multiple channels is supported. Availability and Implementation: Plugin freely available for Fiji and ImageJ2.0 through https://sourceforge.net/projects/smlocalizer/ . Plugin also available for continuous updates through ImageJ update system, add http://sites.imagej...
September 4, 2017: Bioinformatics
https://www.readbyqxmd.com/read/28967738/structural-basis-for-g-protein-coupled-receptor-activation
#16
REVIEW
Aashish Manglik, Andrew C Kruse
G protein-coupled receptors (GPCRs) are critical regulators of human physiology and make up the largest single class of therapeutic drug targets. Although GPCRs regulate highly diverse physiology, they share a common signaling mechanism whereby extracellular stimuli induce conformational changes in the receptor that enable activation of heterotrimeric G proteins and other intracellular effectors. Advances in GPCR structural biology have made it possible to examine ligand-induced GPCR activation at an unprecedented level of detail...
October 24, 2017: Biochemistry
https://www.readbyqxmd.com/read/28961341/biophysical-insight-into-the-heparin-peptide-interaction-and-its-modulation-by-a-small-molecule
#17
Neha Tiwari, Ankit Srivastava, Bishwajit Kundu, Manoj Munde
The heparin-protein interaction plays a vital role in numerous physiological and pathological processes. Not only is the binding mechanism of these interactions poorly understood, studies concerning their therapeutic targeting are also limited. Here, we have studied the interaction of the heparin interacting peptide (HIP) from Tat (which plays important role in HIV infections) with heparin. Isothermal titration calorimetry binding exhibits distinct biphasic isotherm with two different affinities in the HIP-heparin complex formation...
September 29, 2017: Journal of Molecular Recognition: JMR
https://www.readbyqxmd.com/read/28949551/single-molecule-force-spectroscopy-and-molecular-dynamics-simulations-as-a-combined-platform-for-probing-protein-face-specific-binding
#18
Kartik Srinivasan, Suvrajit Banerjee, Siddharth Parimal, Lars Sejergaard, Ronen Berkovich, Blanca Barquera, Shekhar Garde, Steven M Cramer
Biomolecular interactions frequently occur in orientation-specific manner. For example, prior nuclear magnetic resonance spectroscopy experiments in our lab have suggested the presence of a group of strongly binding residues on a particular face of the protein ubiquitin for interactions with Capto MMC multimodal ligands ("Capto" ligands) (Srinivasan, K.; et al. Langmuir 2014, 30 (44), 13205-13216). We present a clear confirmation of those studies by performing single-molecule force spectroscopy (SMFS) measurements of unbinding complemented with molecular dynamics (MD) calculations of the adsorption free energy of ubiquitin in two distinct orientations with self-assembled monolayers (SAMs) functionalized with "Capto" ligands...
October 17, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
https://www.readbyqxmd.com/read/28945353/characterizing-protein-dynamics-with-integrative-use-of-bulk-and-single-molecule-techniques
#19
Zhu Liu, Zhou Gong, Yong Cao, Yue-He Ding, Meng-Qiu Dong, Yun-Bi Lu, Wei-Ping Zhang, Chun Tang
A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures that are more precise and accurate...
October 2, 2017: Biochemistry
https://www.readbyqxmd.com/read/28926278/planar-optical-nanoantennas-resolve-cholesterol-dependent-nanoscale-heterogeneities-in-the-plasma-membrane-of-living-cells
#20
Raju Regmi, Pamina M Winkler, Valentin Flauraud, Kyra J E Borgman, Carlo Manzo, Jürgen Brugger, Hervé Rigneault, Jérôme Wenger, María F García-Parajo
Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale...
October 11, 2017: Nano Letters
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