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Single molecule biophysics

Carmine Di Rienzo, Paolo Annibale
The determination of the mode and rapidity of motion of individual molecules within a biological sample is becoming a more and more common analysis in biophysical investigations. Single molecule tracking (SMT) techniques allow reconstructing the trajectories of individual molecules within a movie, provided that the position from one frame to the other can be correctly linked. The outcomes, however, appear to depend on the specific method used, and most techniques display a limitation to capture fast modes of motion in a crowded environment...
October 1, 2016: Optics Letters
Tapas Paul, Subhas Chandra Bera, Nidhi Agnihotri, Padmaja P Mishra
A remarkable observation about adsorption and desorption mechanism of single-stranded oligo nucleotides and hybridization of double stranded DNA (ds-DNA) on graphene oxide (GO) surface has been monitored using ensemble and single molecule fluorescence methods. The probe and target DNA, both labeled individually with fluorescence resonance energy transfer (FRET) pair and having similar adsorption affinity towards the GO surface are used to provide the detailed insight into the hybridization mechanism. The single molecule-FRET (sm-FRET) results reveals the DNA hybridization mechanism to happen "in situ", i...
October 17, 2016: Journal of Physical Chemistry. B
Shalom D Goldberg, Rosa M F Cardoso, Tricia Lin, Tracy Spinka-Doms, Donna Klein, Steven A Jacobs, Vadim Dudkin, Gary Gilliland, Karyn T O'Neil
Targeted delivery of therapeutic payloads to specific tissues and cell types is an important component of modern pharmaceutical development. Antibodies or other scaffold proteins can provide the cellular address for delivering a covalently linked therapeutic via specific binding to cell-surface receptors. Optimization of the conjugation site on the targeting protein, linker chemistry and intracellular trafficking pathways can all influence the efficiency of delivery and potency of the drug candidate. In this study, we describe a comprehensive engineering experiment for an EGFR binding Centyrin, a highly stable fibronectin type III (FN3) domain, wherein all possible single-cysteine replacements were evaluated for expression, purification, conjugation efficiency, retention of target binding, biophysical properties and delivery of a cytotoxic small molecule payload...
October 13, 2016: Protein Engineering, Design & Selection: PEDS
Stefanie Hauck, Kerstin Hiesinger, Sabrina Khageh Hosseini, Janosch Achenbach, Ricardo M Biondi, Ewgenij Proschak, Martin Zörnig, Dalibor Odadzic
The transcriptional regulator FUSE binding protein 1 (FUBP1) is aberrantly upregulated in various malignancies, fulfilling its oncogenic role by the deregulation of critical genes involved in cell cycle control and apoptosis regulation. Thus, the pharmaceutical inhibition of this protein would represent an encouraging novel targeted chemotherapy. Here, we demonstrate the identification and initial optimization of a pyrazolo[1,5a]pyrimidine-based FUBP1 inhibitor derived from medium throughput screening, which interferes with the binding of FUBP1 to its single stranded target DNA FUSE...
September 14, 2016: Bioorganic & Medicinal Chemistry
Joshua Lequieu, Andrés Córdoba, David C Schwartz, Juan J de Pablo
Nucleosomes form the basic unit of compaction within eukaryotic genomes, and their locations represent an important, yet poorly understood, mechanism of genetic regulation. Quantifying the strength of interactions within the nucleosome is a central problem in biophysics and is critical to understanding how nucleosome positions influence gene expression. By comparing to single-molecule experiments, we demonstrate that a coarse-grained molecular model of the nucleosome can reproduce key aspects of nucleosome unwrapping...
September 28, 2016: ACS Central Science
Miguel A Soler, Ario de Marco, Sara Fortuna
Nanobodies (VHHs) have proved to be valuable substitutes of conventional antibodies for molecular recognition. Their small size represents a precious advantage for rational mutagenesis based on modelling. Here we address the problem of predicting how Camelidae nanobody sequences can tolerate mutations by developing a simulation protocol based on all-atom molecular dynamics and whole-molecule docking. The method was tested on two sets of nanobodies characterized experimentally for their biophysical features...
October 10, 2016: Scientific Reports
Jie Li, Gen He, Hiroshi Ueno, Chuancheng Jia, Hiroyuki Noji, Chuanmin Qi, Xuefeng Guo
We present an efficient strategy through surface functionalization to build a single silicon nanowire field-effect transistor-based biosensor that is capable of directly detecting protein adsorption/desorption at the single-event level. The step-wise signals in real-time detection of His-tag F1-ATPases demonstrate a promising electrical biosensing approach with single-molecule sensitivity, thus opening up new opportunities for studying single-molecule biophysics in broad biological systems.
September 15, 2016: Nanoscale
Marisela Vélez
In Nature, proteins perform functions that go well beyond controlled self-assembly at the nano scale. They are the principal components of diverse "biological machines" that can self-assemble into dynamic aggregates that achieve the cold conversion of chemical energy into motion to realize complex functions involved in cell division, cellular transport and cell motility. Nowadays, we have identified many of the proteins involved in these "molecular machines" and know much about their biochemistry, structure and biophysical behavior...
2016: Advances in Experimental Medicine and Biology
Lei Liu, Hai-Chen Wu
Nanopore sensing is an attractive, label-free approach that can measure single molecules. Although initially proposed for rapid and low-cost DNA sequencing, nanopore sensors have been successfully employed in the detection of a wide variety of substrates. Early successes were mostly achieved based on two main strategies by 1) creating sensing elements inside the nanopore through protein mutation and chemical modification or 2) using molecular adapters to enhance analyte recognition. Over the past five years, DNA molecules started to be used as probes for sensing rather than substrates for sequencing...
September 27, 2016: Angewandte Chemie
Philipp Consentius, Ulrich Gohlke, Bernhard Loll, Claudia Alings, Robert Müller, Udo Heinemann, Martin Kaupp, Markus Wahl, Thomas Risse
Proteins are dynamic molecules that can transiently adopt different conformational states. As the function of the system often depends critically on its conformational state a rigorous understanding of the correlation between structure, energetics and dynamics of the different accessible states is crucial. The biophysical characterization of such processes is, however, challenging as the excited states are often only marginally populated. We show that a combination of X-ray crystallography performed at 100 K as well as at room temperature and EPR spectroscopy on a spin-labeled single crystal allows to correlate the structures of the ground state and a thermally excited state with their thermodynamics using the variant 118R1 of T4 lysozyme as an example...
October 5, 2016: Journal of the American Chemical Society
Diana Di Paolo, Oshri Afanzar, Judith P Armitage, Richard M Berry
For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates...
November 5, 2016: Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences
Marija Iljina, Laura Tosatto, Minee L Choi, Jason C Sang, Yu Ye, Craig D Hughes, Clare E Bryant, Sonia Gandhi, David Klenerman
The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson's disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule Fӧrster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA...
September 27, 2016: Scientific Reports
Bengt Nordén
Einstein was wrong with his 1927 Solvay Conference claim that quantum mechanics is incomplete and incapable of describing diffraction of single particles. However, the Einstein-Podolsky-Rosen paradox of entangled pairs of particles remains lurking with its 'spooky action at a distance'. In molecules quantum entanglement can be viewed as basis of both chemical bonding and excitonic states. The latter are important in many biophysical contexts and involve coupling between subsystems in which virtual excitations lead to eigenstates of the total Hamiltonian, but not for the separate subsystems...
January 2016: Quarterly Reviews of Biophysics
Kathleen A Leamy, Sarah M Assmann, David H Mathews, Philip C Bevilacqua
Deciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functions in vivo...
January 2016: Quarterly Reviews of Biophysics
Sebastian Becker, Jens Boch
No abstract text is available yet for this article.
September 20, 2016: Nature Chemical Biology
Marjetka Podobnik, Nada Kraševec, Apolonija Bedina Zavec, Omar Naneh, Ajda Flašker, Simon Caserman, Vesna Hodnik, Gregor Anderluh
Physical and functional interactions between molecules in living systems are central to all biological processes. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Several powerful methodologies and techniques have been developed to study molecular interactions and thus help elucidate their nature and role in biology as well as potential ways how to interfere with them. All different techniques used in these studies have their strengths and weaknesses and since they are mostly employed in in vitro conditions, a single approach can hardly accurately reproduce interactions that happen under physiological conditions...
2016: Acta Chimica Slovenica
Jörg Schönfelder, David De Sancho, Raul Perez-Jimenez
One of the major challenges in modern biophysics is observing and understanding conformational changes during complex molecular processes, from the fundamental protein folding to the function of molecular machines. Single-molecule techniques have been one of the major driving forces of the huge progress attained in the last few years. Recent advances in resolution of the experimental setups, aided by theoretical developments and molecular dynamics simulations, have revealed a much higher degree of complexity inside these molecular processes than previously reported using traditional ensemble measurements...
October 23, 2016: Journal of Molecular Biology
Duncan G G McMillan, Rikiya Watanabe, Hiroshi Ueno, Gregory M Cook, Hiroyuki Noji
F1Fo ATP synthases are bidirectional molecular motors that translocate protons across the cell membrane by either synthesizing or hydrolyzing ATP. Alkaliphile ATP synthases are highly adapted, performing oxidative phosphorylation at high pH against an inverted pH gradient (acidin/alkalineout). Unlike mesophilic ATP synthases, alkaliphilic enzymes have tightly regulated ATP hydrolysis activity that can be relieved in the presence of LDAO. Here, we characterized the rotary dynamics of the Caldalkalibacillus thermarum TA2...
September 13, 2016: Journal of Biological Chemistry
Pu Guo, Ananya Paul, Arvind Kumar, Abdelbasset A Farahat, Dhiraj Kumar, Siming Wang, David W Boykin, W David Wilson
In spite of its importance in cell function, targeting DNA is under-represented in the design of small molecules. A barrier to progress in this area is the lack of a variety of modules that recognize G⋅C base pairs (bp) in DNA sequences. To overcome this barrier, an entirely new design concept for modules that can bind to mixed G⋅C and A⋅T sequences of DNA is reported herein. Because of their successes in biological applications, minor-groove-binding heterocyclic cations were selected as the platform for design...
October 17, 2016: Chemistry: a European Journal
Kristine Manibog, Kannan Sankar, Sun-Ae Kim, Yunxiang Zhang, Robert L Jernigan, Sanjeevi Sivasankar
Classical cadherin cell-cell adhesion proteins are essential for the formation and maintenance of tissue structures; their primary function is to physically couple neighboring cells and withstand mechanical force. Cadherins from opposing cells bind in two distinct trans conformations: strand-swap dimers and X-dimers. As cadherins convert between these conformations, they form ideal bonds (i.e., adhesive interactions that are insensitive to force). However, the biophysical mechanism for ideal bond formation is unknown...
September 27, 2016: Proceedings of the National Academy of Sciences of the United States of America
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