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Single molecule biophysics

Bárbara Martín-García, Alejandro Martín-González, Carolina Carrasco, Ana M Hernández-Arriaga, Rubén Ruíz-Quero, Ramón Díaz-Orejas, Clara Aicart-Ramos, Fernando Moreno-Herrero, María A Oliva
In prokaryotes, the centromere is a specialized segment of DNA that promotes the assembly of the segrosome upon binding of the Centromere Binding Protein (CBP). The segrosome structure exposes a specific surface for the interaction of the CBP with the motor protein that mediates DNA movement during cell division. Additionally, the CBP usually controls the transcriptional regulation of the segregation system as a cell cycle checkpoint. Correct segrosome functioning is therefore indispensable for accurate DNA segregation...
May 14, 2018: Nucleic Acids Research
Allison M Gicking, Weihong Qiu, William O Hancock
Mitotic spindle assembly requires the collective action of multiple microtubule motors that coordinate their activities in ensembles. However, despite significant advances in our understanding of mitotic kinesins at the single-motor level, multi-motor systems are challenging to reconstitute in vitro and thus less well understood. Recent findings highlighted in this perspective demonstrate how various properties of kinesin-5 and -14 motors-diffusive searching, directional switching, and multivalent interactions-allow them to achieve their physiological roles of cross-linking parallel microtubules and sliding antiparallel ones during cell division...
May 15, 2018: Molecular Biology of the Cell
Michael Z Kamrath, Thomas R Rizzo
Ion mobility spectrometry (IMS) has become a valuable tool in biophysical and bioanalytical chemistry because of its ability to separate and characterize the structure of gas-phase biomolecular ions on the basis of their collisional cross section (CCS). Its importance has grown with the realization that in many cases, biomolecular ions retain important structural characteristics when produced in the gas phase by electrospray ionization (ESI). While a CCS can help distinguish between structures of radically different types, one cannot expect a single number to differentiate similar conformations of a complex molecule...
May 10, 2018: Accounts of Chemical Research
Alberto Sosa-Costa, Izabela K Piechocka, Lucia Gardini, Francesco S Pavone, Marco Capitanio, Maria F Garcia-Parajo, Carlo Manzo
Time traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted...
May 8, 2018: Biophysical Journal
Hila Nadler, Lihi Shaulov, Yossi Blitsman, Moran Mordechai, Jurgen Jopp, Neta Sal-Man, Ronen Berkovich
Bacterial type III secretion systems inject virulence factors by bacterial pathogens into host-cells during bacterial infection. In enteropathogenic Escherichia coli, this system contains an external filament, formed by a self-oligomerizing protein called E. coli secreted protein A (EspA). The EspA filament penetrates the thick viscous mucus layer to facilitate the attachment of the bacteria to the gut-epithelium. To do that, the EspA filament requires noteworthy mechanical endurance considering the mechanical shear stresses found within the intestinal tract...
May 4, 2018: Langmuir: the ACS Journal of Surfaces and Colloids
Javier Cervera, Alexis Pietak, Michael Levin, Salvador Mafe
We review the basic concepts involved in bioelectrically-coupled multicellular domains, focusing on the role of membrane potentials (Vmem ). In the first model, single-cell Vmem is modulated by two generic polarizing and depolarizing ion channels, while intercellular coupling is implemented via voltage-gated gap junctions. Biochemical and bioelectrical signals are integrated via a feedback loop between Vmem and the transcription and translation of a protein forming an ion channel. The effective rate constants depend on the single-cell Vmem because these potentials modulate the local concentrations of signaling molecules and ions...
April 21, 2018: Bioelectrochemistry
Krutika Bavishi, Darui Li, Stine Eiersholt, Emma N Hooley, Troels C Petersen, Birger Lindberg Møller, Nikos S Hatzakis, Tomas Laursen
Cytochrome P450 oxidoreductase (POR) is the primary electron donor in eukaryotic cytochrome P450 (CYP) containing systems. A wealth of ensemble biophysical studies of Cytochrome P450 oxidoreductase (POR) has reported a binary model of the conformational equilibrium directing its catalytic efficiency and biomolecular recognition. In this study, full length POR from the crop plant Sorghum bicolor was site-specifically labeled with Cy3 (donor) and Cy5 (acceptor) fluorophores and reconstituted in nanodiscs. Our single molecule fluorescence resonance energy transfer (smFRET) burst analyses of POR allowed the direct observation and quantification of at least three dominant conformational sub-populations, their distribution and occupancies...
May 1, 2018: Scientific Reports
Luuk Loeff, Stan J J Brouns, Chirlmin Joo
CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here, we employed single-molecule Förster resonance energy transfer (FRET) to probe the helicase activity with high spatiotemporal resolution...
April 17, 2018: Molecular Cell
Ilektra-Chara Giassa, Jan Rynes, Tomas Fessl, Silvie Foldynova-Trantirkova, Lukas Trantirek
Conventional biophysical and chemical biology approaches for delineating relationships between the structure and biological function of nucleic acids (NAs) abstract NAs from their native biological context. However, cumulative experimental observations have revealed that the structure, dynamics and interactions of NAs might be strongly influenced by a broad spectrum of specific and non-specific physical-chemical environmental factors. This consideration has recently sparked interest in the development of novel tools for structural characterization of NAs in the native cellular context...
April 21, 2018: FEBS Letters
Michael D Tomasini, Daniel S Johnson, Joshua S Mincer, Sanford M Simon
We report a computational model for the assembly of HIV-1 Gag into immature viral particles at the plasma membrane. To reproduce experimental structural and kinetic properties of assembly, a process occurring on the order of minutes, a coarse-grained representation consisting of a single particle per Gag molecule is developed. The model uses information relating the functional interfaces implicated in Gag assembly, results from cryo electron-tomography, and biophysical measurements from fluorescence microscopy, such as the dynamics of Gag assembly at single virions...
2018: PloS One
Anirban Kar, Nathan Jones, N Özlem Arat, Richard Fishel, Jack Griffith
Conformations adopted by long stretches of single stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo. This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ~150 nt, and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5'-TTAGGG-3')n as well as the interrogation of its structure by electron microscopy (EM) and single molecule magnetic tweezers (smMT)...
April 19, 2018: Journal of Biological Chemistry
Farhan Chowdhury, Sultan Doğanay, Benjamin J Leslie, Rishi Singh, Kshitij Amar, Bhavana Talluri, Seongjin Park, Ning Wang, Taekjip Ha
Recently, a robust mechanical method has been established to isolate a small subpopulation of highly tumorigenic tumor repopulating cells (TRCs) from parental melanoma cells. In order to characterize the molecular and mechanical properties of TRCs, we utilized the tension gauge tether (TGT) single-molecule platform and investigated force requirements during early cell spreading events. TRCs required the peak single molecular tension of around 40 pN through integrins for initial adhesion like the parental control cells, but unlike the control cells, they did not spread and formed very few mature focal adhesions (FAs)...
April 16, 2018: Biochemical and Biophysical Research Communications
K Tsafou, P B Tiwari, J D Forman-Kay, S J Metallo, J A Toretsky
Increased understanding of intrinsically disordered proteins (IDPs) and protein regions (IDRs) has revolutionized our view of the relationship between protein structure and function. Data now support that IDPs can be functional in the absence of a single, fixed, three-dimensional structure. Due to their dynamic morphology, IDPs have the ability to display a range of kinetics and affinity depending on what the system requires, as well as the potential for large-scale association. Although several studies have shed light on the functional properties of IDPs, the class of intrinsically disordered transcription factors (TFs) is still poorly characterized biophysically due to their combination of ordered and disordered sequences...
April 12, 2018: Journal of Molecular Biology
Kristin Støen Gunnarsen, Lene Støkken Høydahl, Ralf Stefan Neumann, Kaare Bjerregaard-Andersen, Nicolay Rustad Nilssen, Ludvig Magne Sollid, Inger Sandlie, Geir Åge Løset
There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles...
2018: PloS One
Ibrahim Kaya, Henrik Zetterberg, Kaj Blennow, Jörg Hanrieder
Senile plaques formed by aggregated amyloid β peptides are one of the major pathological hallmarks of Alzheimer's disease (AD) which have been suggested to be the primary influence triggering the AD pathogenesis and the rest of the disease process. However, neurotoxic Aβ aggregation and progression are associated with a wide range of enigmatic biochemical, biophysical and genetic processes. MALDI imaging mass spectrometry (IMS) is a label-free method to elucidate the spatial distribution patterns of intact molecules in biological tissue sections...
April 12, 2018: ACS Chemical Neuroscience
Cesar A Ramirez-Sarmiento, Elizabeth A Komives
Hydrogen-deuterium exchange mass spectrometry (HDXMS) has emerged as a powerful approach for revealing folding and allostery in protein-protein interactions. The advent of higher resolution mass spectrometers combined with ion mobility separation and ultra-high liquid chromatographic separations have allowed the complete coverage of large protein sequences and multi-protein complexes. Liquid-handling robots have improved the reproducibility and accurate temperature control of the sample preparation. Many researchers are also appreciating the power of combining biophysical approaches such as stopped-flow fluorescence, single molecule FRET, and molecular dynamics simulations with HDXMS...
April 5, 2018: Methods: a Companion to Methods in Enzymology
Dmitrii E Makarov, Benjamin Schuler
Single-molecule measurements are now almost routinely used to study biological systems and processes. The scope of this special topic emphasizes the physics side of single-molecule observations, with the goal of highlighting new developments in physical techniques as well as conceptual insights that single-molecule measurements bring to biophysics. This issue also comprises recent advances in theoretical physical models of single-molecule phenomena, interpretation of single-molecule signals, and fundamental areas of statistical mechanics that are related to single-molecule observations...
March 28, 2018: Journal of Chemical Physics
Viktorija Globyte, Sung Hyun Kim, Chirlmin Joo
Most everyday processes in life involve a necessity for an entity to locate its target. On a cellular level, many proteins have to find their target to perform their function. From gene-expression regulation to DNA repair to host defense, numerous nucleic acid-interacting proteins use distinct target search mechanisms. Several proteins achieve that with the help of short RNA strands known as guides. This review focuses on single-molecule advances studying the target search and recognition mechanism of Argonaute and CRISPR (clustered regularly interspaced short palindromic repeats) systems...
March 29, 2018: Annual Review of Biophysics
Paolo Cadinu, Giulia Campolo, Sergii Pud, Wayne Yang, Joshua B Edel, Cees Dekker, Aleksandar P Ivanov
The ability to control the motion of single biomolecules is key to improving a wide range of biophysical and diagnostic applications. Solid-state nanopores are a promising tool capable of solving this task. However, molecular control and the possibility of slow readouts of long polymer molecules are still limited due to fast analyte transport and low signal-to-noise ratios. Here, we report on a novel approach of actively controlling analyte transport by using a double-nanopore architecture where two nanopores are separated by only a ~20 nm gap...
March 23, 2018: Nano Letters
Anthony N Milin, Ashok A Deniz
Compartmentalization of biochemical components, interactions, and reactions is critical for the function of cells. While intracellular partitioning of molecules via membranes has been extensively studied, there has been an expanding focus in recent years on the critical cellular roles and biophysical mechanisms of action of membraneless organelles (MLOs) such as the nucleolus. In this context, a substantial body of recent work has demonstrated that liquid-liquid phase separation (LLPS) plays a key role in MLO formation...
March 23, 2018: Biochemistry
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