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Single Molecule
Highly-conducting molecular circuits based on antiaromaticity
Jul 19, 2017   Nature Communications
Fujii S, Marqués-González S, Shin JY, Shinokubo H, Masuda T, Nishino T, Arasu NP, Vázquez H, Kiguchi M
Highly-conducting molecular circuits based on antiaromaticity
Jul 19, 2017
Nature Communications
Aromaticity is a fundamental concept in chemistry. It is described by Hückel's rule that states that a cyclic planar π-system is aromatic when it shares 4n+2 π-electrons and antiaromatic when it possesses 4n π-electrons. Antiaromatic compounds are predicted to exhibit remarkable charge transport properties and high redox activities. However, it has so far only been possible to measure compounds with reduced aromaticity but not antiaromatic species due to their energetic instability. Here, we address these issues by investigating the single-molecule charge transport properties of a genuinely antiaromatic compound, showing that antiaromaticity results in an order of magnitude increase in conductance compared with the aromatic counterpart. Single-molecule current-voltage measurements and ab initio transport calculations reveal that this results from a reduced energy gap and a frontier molecular resonance closer to the Fermi level in the antiaromatic species. The conductance of the antiaromatic complex is further modulated electrochemically, demonstrating its potential as a high-conductance transistor.
Impact of Metal Contacts to the Performance of Multilayer HfS2 Field-Effect Transistors
Jul 21, 2017   ACS Applied Materials & Interfaces
Nie XR, Sun BQ, Zhu H, Zhang M, Zhao DH, Chen L, Sun QQ, Zhang DW
Impact of Metal Contacts to the Performance of Multilayer HfS2 Field-Effect Transistors
Jul 21, 2017
ACS Applied Materials & Interfaces
HfS2 is one of the emerging transition metal dichalcogenides and is very promising for low-power nanoelectronics and high-sensitivity optoelectronic device applications. We studied the band structures of 1T-HfS2 with different thickness by first principles simulation, and the impact of different metal contacts to the HfS2 device performance has been experimentally studied. Back-gate and top-gate HfS2 field-effect transistors (FETs) were fabricated, and better electrical characteristics have been achieved with the FETs with Ti/Au contact as compared with the Pt-contacted FETs. Thin layers of Pt and Ti/Au films were deposited on HfS2 flakes to investigate the metal/HfS2 interface by using scanning electron microscopy, atomic force microscopy and Raman spectroscopy. Smoother Ti/Au film was formed on HfS2, resulting in higher carrier injection and transport efficiency. Phonon behavior dominated by the interface chemical bonding at the Ti/Au contact region has been confirmed with the more sensitive A1g phonon mode from bilayer HfS2.
Nonhelical heterometallic [Mo2M(npo)4(NCS)2] string complexes (M = Fe, Co, Ni) with high single-molecule conductance
Jul 24, 2017   Chemical Communications (Cambridge, England)
Chang WC, Chang CW, Sigrist M, Hua SA, Liu TJ, Lee GH, Jin BY, Chen CH, Peng SM
Nonhelical heterometallic [Mo2M(npo)4(NCS)2] string complexes (M = Fe, Co, Ni) with high single-molecule conductance
Jul 24, 2017
Chemical Communications (Cambridge, England)
Using the planar 1,8-naphthyridin-2(1H)-one (Hnpo) ligand, novel nonhelical HMSCs [Mo2M(npo)4(NCS)2] (M = Fe, Co, Ni) were synthesised and they exhibited high single-molecule conductance.
Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters
Jul 24, 2017   Molecular Cell
Krebs AR, Imanci D, Hoerner L, Gaidatzis D, Burger L, Schübeler D
Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters
Jul 24, 2017
Molecular Cell
Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Structural Biology outside the box-inside the cell
Jul 23, 2017   Current Opinion In Structural Biology
Plitzko JM, Schuler B, Selenko P
Structural Biology outside the box-inside the cell
Jul 23, 2017
Current Opinion In Structural Biology
Recent developments in cellular cryo-electron tomography, in-cell single-molecule Förster resonance energy transfer-spectroscopy, nuclear magnetic resonance-spectroscopy and electron paramagnetic resonance-spectroscopy delivered unprecedented insights into the inner workings of cells. Here, we review complementary aspects of these methods and provide an outlook toward joint applications in the future. Copyright © 2017. Published by Elsevier Ltd.
Development of new ganglioside probes and unraveling of raft domain structure by single-molecule imaging
Jul 23, 2017   Biochimica Et Biophysica Acta
Suzuki KGN, Ando H, Komura N, Fujiwara T, Kiso M, Kusumi A
Development of new ganglioside probes and unraveling of raft domain structure by single-molecule imaging
Jul 23, 2017
Biochimica Et Biophysica Acta
Gangliosides are involved in a variety of biological roles and are a component of lipid rafts found in cell plasma membranes (PMs). Gangliosides are especially abundant in neuronal PMs and are essential to their physiological functions. However, the dynamic behaviors of gangliosides have not been investigated in living cells due to a lack of fluorescent probes that behave like their parental molecules. We have recently developed, using an entirely chemical method, four new ganglioside probes (GM1, GM2, GM3, and GD1b) that act similarly to their parental molecules in terms of raft partitioning and binding affinity. Using single fluorescent-molecule imaging, we have found that ganglioside probes dynamically enter and leave rafts featuring CD59, a GPI-anchored protein. This occurs both before and after stimulation. The residency time of our ganglioside probes in rafts with CD59 oligomers was 48 ms, after stimulation. The residency times in CD59 homodimer and monomer rafts were 40 ms and 12 ms, respectively. In this review, we introduce an entirely chemical-based ganglioside analog synthesis method and describe its application in single-molecule imaging and for the study of the dynamic behavior of gangliosides in cell PMs. Finally, we discuss how raft domains are formed, both before and after receptor engagement. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa. Copyright © 2017. Published by Elsevier B.V.
Antibacterial properties of nano-silver coated PEEK prepared through magnetron sputtering
Jul 23, 2017   Dental Materials : Official Publication Of The Academy Of Dental Materials
Liu X, Gan K, Liu H, Song X, Chen T, Liu C
Antibacterial properties of nano-silver coated PEEK prepared through magnetron sputtering
Jul 23, 2017
Dental Materials : Official Publication Of The Academy Of Dental Materials
We aimed to investigate the cytotoxicity and antibacterial properties of nano-silver-coated polyetheretherketone (PEEK) produced through magnetron sputtering and provide a theoretical basis for its use in clinical applications. The surfaces of PEEKs were coated with nano-silver at varying thicknesses (3, 6, 9, and 12nm) through magnetron sputtering technology. The resulting coated PEEK samples were classified into the following groups according to the thickness of the nano-silver coating: PEEK-3 (3nm), PEEK-6 (6nm), PEEK-9 (9nm), PEEK-12 (12nm), and PEEK control group. The surface microstructure and composition of each sample were observed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and energy dispersive spectrum (EDS) analysis. The water contact angle of each sample was then measured by contact angle meters. A cell counting kit (CCK-8) was used to analyze the cytotoxicity of the mouse fibroblast cells (L929) in the coated groups (n=5) and group test samples (n=6), negative control (polyethylene, PE) (n=6), and positive control group (phenol) (n=6). The antibacterial properties of the samples were tested by co-culturing Streptococcus mutans and Straphylococcus aureus. The bacteria that adhered to the surface of samples were observed by SEM. The antibacterial adhesion ability of each sample was then evaluated. SEM and AFM analysis results showed that the surfaces of control group samples were smooth but compact. Homogeneous silver nano-particles (AgNPs) and nano-silver coating were uniformly distributed on the surface of the coated group samples. Compared with the control samples, the nano-silver coated samples had a significant increase in surface roughness (P
High-Resolution Imaging of a Single Gliding Protofilament of Tubulins by HS-AFM
Jul 22, 2017   Scientific Reports
Keya JJ, Inoue D, Suzuki Y, Kozai T, Ishikuro D, Kodera N, Uchihashi T, Kabir AMR, Endo M, Sada K, Kakugo A
High-Resolution Imaging of a Single Gliding Protofilament of Tubulins by HS-AFM
Jul 22, 2017
Scientific Reports
In vitro gliding assay of microtubules (MTs) on kinesins has provided us with valuable biophysical and chemo-mechanical insights of this biomolecular motor system. Visualization of MTs in an in vitro gliding assay has been mainly dependent on optical microscopes, limited resolution of which often render them insufficient sources of desired information. In this work, using high speed atomic force microscopy (HS-AFM), which allows imaging with higher resolution, we monitored MTs and protofilaments (PFs) of tubulins while gliding on kinesins. Moreover, under the HS-AFM, we also observed splitting of gliding MTs into single PFs at their leading ends. The split single PFs interacted with kinesins and exhibited translational motion, but with a slower velocity than the MTs. Our investigation at the molecular level, using the HS-AFM, would provide new insights to the mechanics of MTs in dynamic systems and their interaction with motor proteins.
Gap junction-mediated regulation of endothelial cellular stiffness
Jul 22, 2017   Scientific Reports
Okamoto T, Kawamoto E, Takagi Y, Akita N, Hayashi T, Park EJ, Suzuki K, Shimaoka M
Gap junction-mediated regulation of endothelial cellular stiffness
Jul 22, 2017
Scientific Reports
Endothelial monolayers have shown the ability to signal each other through gap junctions. Gap junction-mediated cell-cell interactions have been implicated in the modulation of endothelial cell functions during vascular inflammation. Inflammatory mediators alter the mechanical properties of endothelial cells, although the exact role of gap junctions in this process remains unclear. Here, we sought to study the role of gap junctions in the regulation of endothelial stiffness, an important physical feature that is associated with many vascular pathologies. The endothelial cellular stiffness of living endothelial cells was determined by using atomic force microscopy. We found that tumor necrosis factor-α transiently increased endothelial cellular stiffness, which is regulated by cytoskeletal rearrangement and cell-cell interactions. We explored the role of gap junctions in endothelial cellular stiffening by utilizing gap junction blockers, carbenoxolone, inhibitory anti-connexin 32 antibody or anti-connexin 43 antibody. Blockade of gap junctions induced the cellular stiffening associated with focal adhesion formation and cytoskeletal rearrangement, and prolonged tumor necrosis factor-α-induced endothelial cellular stiffening. These results suggest that gap junction-mediated cell-cell interactions play an important role in the regulation of endothelial cellular stiffness.
Nanoscale Investigation of Generation 1 PAMAM Dendrimers Interaction with a Protein Nanopore
Jul 22, 2017   Scientific Reports
Asandei A, Ciuca A, Apetrei A, Schiopu I, Mereuta L, Seo CH, Park Y, Luchian T
Nanoscale Investigation of Generation 1 PAMAM Dendrimers Interaction with a Protein Nanopore
Jul 22, 2017
Scientific Reports
Herein, we describe at uni-molecular level the interactions between poly(amidoamine) (PAMAM) dendrimers of generation 1 and the α-hemolysin protein nanopore, at acidic and neutral pH, and ionic strengths of 0.5 M and 1 M KCl, via single-molecule electrical recordings. The results indicate that kinetics of dendrimer-α-hemolysin reversible interactions is faster at neutral as compared to acidic pH, and we propose as a putative explanation the fine interplay among conformational and rigidity changes on the dendrimer structure, and the ionization state of the dendrimer and the α-hemolysin. From the analysis of the dendrimer's residence time inside the nanopore, we posit that the pH- and salt-dependent, long-range electrostatic interactions experienced by the dendrimer inside the ion-selective α-hemolysin, induce a non-Stokesian diffusive behavior of the analyte inside the nanopore. We also show that the ability of dendrimer molecules to adapt their structure to nanoscopic spaces, and control the flow of matter through the α-hemolysin nanopore, depends non-trivially on the pH- and salt-induced conformational changes of the dendrimer.
Single-Molecule Investigation of Energy Dynamics in a Coupled Plasmon-Exciton System
Jul 21, 2017   Physical Review Letters
Imada H, Miwa K, Imai-Imada M, Kawahara S, Kimura K, Kim Y
Single-Molecule Investigation of Energy Dynamics in a Coupled Plasmon-Exciton System
Jul 21, 2017
Physical Review Letters
We investigate the near-field interaction between an isolated free-base phthalocyanine molecule and a plasmon localized in the gap between an NaCl-covered Ag(111) surface and the tip apex of a scanning tunneling microscope. When the tip is located in the close proximity of the molecule, asymmetric dips emerge in the broad luminescence spectrum of the plasmon generated by the tunneling current. The origin of the dips is explained by energy transfer between the plasmon and molecular excitons and a quantum mechanical interference effect, where molecular vibrations provide additional degrees of freedom in the dynamic process.
Heterogeneity in Kinesin function
Jul 21, 2017   Traffic (Copenhagen, Denmark)
Reddy BJN, Tripathy S, Vershinin M, Tanenbaum M, Xu J, Mattson-Hoss M, Arabi K, Chapman D, Doolin T, Hyeon C, Gross SP
Heterogeneity in Kinesin function
Jul 21, 2017
Traffic (Copenhagen, Denmark)
The kinesin family proteins are often studied as prototypical molecular motors; a deeper understanding of them can illuminate regulation of intracellular transport. It is typically assumed that they function identically. Here we find that this assumption of homogeneous function appears incorrect: variation among motors' velocities in vivo and in vitro is larger than the stochastic variation expected for an ensemble of 'identical" motors. When moving on microtubules, slow and fast motors are persistently slow, and fast, respectively. We develop theory that provides quantitative criteria to determine whether the observed single-molecule variation is too large to be generated from an ensemble of identical molecules. To analyze such heterogeneity, we group traces into homogeneous sub-ensembles. Motility studies varying the temperature, pH, and glycerol concentration suggest at least two distinct functional states that are independently affected by external conditions. We end by investigating the functional ramifications of such heterogeneity through Monte-Carlo multi-motor simulations. This article is protected by copyright. All rights reserved.
Polycationic Probe-Guided Nanopore Single-Molecule Counter for Selective miRNA Detection
Jul 21, 2017   Methods In Molecular Biology (Clifton, N.J.)
Tian K, Shi R, Gu A, Pennella M, Gu LQ
Polycationic Probe-Guided Nanopore Single-Molecule Counter for Selective miRNA Detection
Jul 21, 2017
Methods In Molecular Biology (Clifton, N.J.)
MicroRNAs (miRNAs) are a class of noncoding RNAs that are being explored as a new type of disease biomarkers. The nanopore single-molecule sensor offers a potential noninvasive tool to detect miRNAs for diagnostics and prognosis applications. However, one of the challenges that limits its clinical applications is the presence of a large variety of nontarget nucleic acids in the biofluid extracts. Upon interacting with the nanopore, nontarget nucleic acids produce "contaminative" nanopore signals that interfere with target miRNA discrimination, thus severely lowering the accuracy in target miRNA detection. We have reported a novel method that utilizes a designed polycationic peptide-PNA probe to specifically guide the target miRNA migration toward the nanopore, whereas any nontarget nucleic acids without the probe bound is rejected by the nanopore. Consequently, nontarget species are driven away from the nanopore and only the target miRNA can be detected at low concentration. This method is also able to discriminate miRNAs with single-nucleotide difference by using PNA to capture miRNA. Considering the significance and impact of this substantial advance for the future miRNA detection in biofluid samples, we prepared this detailed protocol, by which the readers can view the experimental procedure, data analysis, and resulting explanation.
Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells
Jul 19, 2017   Nature Communications
Byrne A, Beaudin AE, Olsen HE, Jain M, Cole C, Palmer T, DuBois RM, Forsberg EC, Akeson M, Vollmers C
Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells
Jul 19, 2017
Nature Communications
Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single-cell level. Short-read RNAseq is limited in its ability to resolve complex isoforms because it fails to sequence full-length cDNA copies of RNA molecules. Here, we investigate whether RNAseq using the long-read single-molecule Oxford Nanopore MinION sequencer is able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. After benchmarking our approach, we analyse individual murine B1a cells using a custom multiplexing strategy. We identify thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. We also identify hundreds of genes expressed across B1a cells that display multiple complex isoforms, including several B cell-specific surface receptors. Our results show that we can identify and quantify complex isoforms at the single cell level.
Mechanically and Electrically Robust Self-Assembled Monolayers for Large-Area Tunneling Junctions
Jul 21, 2017   The Journal Of Physical Chemistry. C, Nanomaterials And Interfaces
Zhang Y, Qiu X, Gordiichuk P, Soni S, Krijger TL, Herrmann A, Chiechi RC
Mechanically and Electrically Robust Self-Assembled Monolayers for Large-Area Tunneling Junctions
Jul 21, 2017
The Journal Of Physical Chemistry. C, Nanomaterials And Interfaces
This paper examines the relationship between mechanical deformation and the electronic properties of self-assembled monolayers (SAMs) of the oligothiophene 4-([2,2':5',2″:5″,2‴-quaterthiophen]-5-yl)butane-1-thiol (T4C4) in tunneling junctions using conductive probe atomic force microscopy (CP-AFM) and eutectic Ga-In (EGaIn). We compared shifts in conductivity, transition voltages of T4C4 with increasing AFM tip loading force to alkanethiolates. While these shifts result from an increasing tilt angle from penetration of the SAM by the AFM tip for the latter, we ascribe them to distortions of the π system present in T4C4, which is more mechanically robust than alkanethiolates of comparable length; SAMs comprising T4C4 shows about five times higher Young's modulus than alkanethiolates. Density functional theory calculations confirm that mechanical deformations shift the barrier height due to changes in the frontier orbitals caused by small rearrangements to the conformation of the quaterthiophene moiety. The mechanical robustness of T4C4 manifests as an increased tolerance to high bias in large-area EGaIn junctions suggesting that electrostatic pressure plays a significant role in the shorting of molecular junctions at high bias.
Assembly of "3D" plasmonic clusters by "2D" AFM nanomanipulation of highly uniform and smooth gold nanospheres
Jul 21, 2017   Scientific Reports
Park KJ, Huh JH, Jung DW, Park JS, Choi GH, Lee G, Yoo PJ, Park HG, Yi GR, Lee S
Assembly of "3D" plasmonic clusters by "2D" AFM nanomanipulation of highly uniform and smooth gold nanospheres
Jul 21, 2017
Scientific Reports
Atomic force microscopy (AFM) nanomanipulation has been viewed as a deterministic method for the assembly of plasmonic metamolecules because it enables unprecedented engineering of clusters with exquisite control over particle number and geometry. Nevertheless, the dimensionality of plasmonic metamolecules via AFM nanomanipulation is limited to 2D, so as to restrict the design space of available artificial electromagnetisms. Here, we show that "2D" nanomanipulation of the AFM tip can be used to assemble "3D" plasmonic metamolecules in a versatile and deterministic way by dribbling highly spherical and smooth gold nanospheres (NSs) on a nanohole template rather than on a flat surface. Various 3D plasmonic clusters with controlled symmetry were successfully assembled with nanometer precision; the relevant 3D plasmonic modes (i.e., artificial magnetism and magnetic-based Fano resonance) were fully rationalized by both numerical calculation and dark-field spectroscopy. This templating strategy for advancing AFM nanomanipulation can be generalized to exploit the fundamental understanding of various electromagnetic 3D couplings and can serve as the basis for the design of metamolecules, metafluids, and metamaterials.
Spin-reversal energy barriers of 305 K for Fe2+ d6 ions with linear ligand coordination
Jul 20, 2017   Nanoscale
Xu L, Zangeneh Z, Yadav R, Avdoshenko S, van den Brink J, Jesche A, Hozoi L
Spin-reversal energy barriers of 305 K for Fe2+ d6 ions with linear ligand coordination
Jul 20, 2017
Nanoscale
A remarkably large magnetic anisotropy energy of 305 K is computed by quantum chemistry methods for divalent Fe2+ d6 substitutes at Li-ion sites with D6h point-group symmetry within the solid-state matrix of Li3N. This is similar to values calculated by the same approach and confirmed experimentally for linearly coordinated monovalent Fe1+ d7 species, among the largest so far in the research area of single-molecule magnets. Our ab initio results therefore mark a new exciting exploration path in the search for superior single-molecule magnets, rooted in the configuration of d6 transition-metal ions with linear or quasilinear nearest-neighbor coordination. This d6 axial anisotropy may be kept robust even for symmetries lower than D6h, provided the ligand and farther-neighbor environment is engineered such that the splitting remains large enough.
Conjugated polymer covalently modified graphene oxide quantum dots for ternary electronic memory devices
Jul 20, 2017   Nanoscale
Fan F, Zhang B, Cao Y, Yang X, Gu J, Chen Y
Conjugated polymer covalently modified graphene oxide quantum dots for ternary electronic memory devices
Jul 20, 2017
Nanoscale
Zero dimensional graphene oxide (GO) quantum dots (GOQDs) have been expected to play an important role in the development of new memory materials. When the size of GO was reduced to that of GOQDs, both the electron affinity and ionization potential of GO were found to be decreased, and this was followed by the elevation of lowest energy unoccupied molecular orbital (LUMO) energy level. This implies that the electron withdrawing ability of GOQDs is weaker than that of GO. In this work, a novel arylamine-based polyazomethine covalently functionalized graphene oxide quantum dots (TPAPAM-GOQDs), which was synthesized using an amidation reaction, was for the first time used to fabricate a ternary memory device with a configuration of gold/TPAPAM-GOQDs/indium tin oxide. The current ratio of OFF : ON-1 : ON-2 was found to be 1 : 60 : 3000. Its conductive nature was also revealed using an in situ conductive atomic force microscopy technique. This memory device could potentially increase the memory capacity of the device from the conventional 2n to 3n when compared to binary memory devices.
Strain-induced skeletal rearrangement of a polycyclic aromatic hydrocarbon on a copper surface
Jul 20, 2017   Nature Communications
Shiotari A, Nakae T, Iwata K, Mori S, Okujima T, Uno H, Sakaguchi H, Sugimoto Y
Strain-induced skeletal rearrangement of a polycyclic aromatic hydrocarbon on a copper surface
Jul 20, 2017
Nature Communications
Controlling the structural deformation of organic molecules can drive unique reactions that cannot be induced only by thermal, optical or electrochemical procedures. However, in conventional organic synthesis, including mechanochemical procedures, it is difficult to control skeletal rearrangement in polycyclic aromatic hydrocarbons (PAHs). Here, we demonstrate a reaction scheme for the skeletal rearrangement of PAHs on a metal surface using high-resolution noncontact atomic force microscopy. By a combination of organic synthesis and on-surface cyclodehydrogenation, we produce a well-designed PAH-diazuleno[1,2,3-cd:1',2',3'-fg]pyrene-adsorbed flatly onto Cu(001), in which two azuleno moieties are highly strained by their mutual proximity. This local strain drives the rearrangement of one of the azuleno moieties into a fulvaleno moiety, which has never been reported so far. Our proposed thermally driven, strain-induced synthesis on surfaces will pave the way for the production of a new class of nanocarbon materials that conventional synthetic techniques cannot attain.
Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines
Jul 20, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Olivares AO, Kotamarthi HC, Stein BJ, Sauer RT, Baker TA
Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines
Jul 20, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
AAA+ proteases and remodeling machines couple hydrolysis of ATP to mechanical unfolding and translocation of proteins following recognition of sequence tags called degrons. Here, we use single-molecule optical trapping to determine the mechanochemistry of two AAA+ proteases, Escherichia coli ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titinI27 domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons. We find that ClpXP and ClpAP unfold the wild-type titinI27 domain and a destabilized variant far more rapidly when pulling from the N terminus, whereas translocation speed is reduced only modestly in the N-to-C direction. These measurements establish the role of directionality in mechanical protein degradation, show that degron placement can change whether unfolding or translocation is rate limiting, and establish that one or a few power strokes are sufficient to unfold some protein domains.
Can single molecule localization microscopy be used to map closely spaced RGD nanodomains?
Jul 20, 2017   PloS One
Mollazade M, Tabarin T, Nicovich PR, Soeriyadi A, Nieves DJ, Gooding JJ, Gaus K
Can single molecule localization microscopy be used to map closely spaced RGD nanodomains?
Jul 20, 2017
PloS One
Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to detect nanodomains of the cell adhesion-stimulating tripeptide arginine-glycine-aspartic acid (RGD). These domains were formed by the phase separation of block copolymers with controllable spacing on the scale of tens of nanometers. We first determined the topology of the block copolymer with atomic force microscopy (AFM) and then imaged the localization of individual RGD peptides with direct stochastic optical reconstruction microscopy (dSTORM). To compare the data, we analyzed the dSTORM data with DBSCAN (density-based spatial clustering application with noise). The ligand distribution and polymer topology are not necessary identical since peptides may attach to the polymer outside the nanodomains and/or coupling and detection of peptides within the nanodomains is incomplete. We therefore performed simulations to explore the extent to which nanodomains could be mapped with dSTORM. We found that successful detection of nanodomains by dSTORM was influenced by the inter-domain spacing and the localization precision of individual fluorophores, and less by non-specific absorption of ligands to the substratum. For example, under our imaging conditions, DBSCAN identification of nanodomains spaced further than 50 nm apart was largely independent of background localisations, while nanodomains spaced closer than 50 nm required a localization precision of ~11 nm to correctly estimate the modal nearest neighbor distance (NDD) between nanodomains. We therefore conclude that SMLM is a promising technique to directly map the distribution and nanoscale organization of ligands and would benefit from an improved localization precision.
Discrimination Cascade Enabled Selective Detection of Single-Nucleotide Mutation
Jul 20, 2017   ACS Sensors
Li L, Xiao X, Ge J, Han M, Zhou X, Wang L, Su X, Yu C
Discrimination Cascade Enabled Selective Detection of Single-Nucleotide Mutation
Jul 20, 2017
ACS Sensors
Owing to the significance of single nucleotide mutation (SNM) for personalized medicine, the detection of SNM with high accuracy has recently attracted considerable interest. Here, we present a kinetic method for selective detection of SNM based on a discrimination cascade constructed by combining the toehold strand displacement (TSD) and endonuclease IV (Endo IV) catalyzed hydrolysis. The single-nucleotide specificity of the two DNA reactions allows highly selective detection of all types of single nucleotide changes (including single-nucleotide insertion and deletion), achieving a high discrimination factor with a median of 491 which is comparable with recently reported methods. For the first time, the enzyme assisted nucleic acid assay was characterized by single molecule analysis on total internal reflection fluorescence microscope (TIRFM) suggesting that the two steps do not work independently and the rate of TSD can be tuned by Endo IV facilitated conformation selection. The effective discrimination of the point mutation of BRAF gene in cancer and normal cell line suggests that this method can be a prominent post-PCR genotyping assay.
Salt Gradient Improving Signal-to-Noise Ratio in Solid-State Nanopore
Jul 20, 2017   ACS Sensors
Sha J, Shi H, Zhang Y, Chen C, Liu L, Chen Y
Salt Gradient Improving Signal-to-Noise Ratio in Solid-State Nanopore
Jul 20, 2017
ACS Sensors
As the single molecule detection tool, solid-state nanopores are being applied in more and more fields, such as medicine controlled delivery, ion conductance microscopes, nanosensors, and DNA sequencing. The critical information obtained from nanopores is the signal collected, which is the ionic block current caused by the molecules passing through the pores. However, the information collected is, in part, impeded by the relatively low signal-to-noise ratio of the current solid-state nanopore measurements. Here, we report that using a salt gradient across the nanopore could improve the signal-to-noise ratio when molecules translocate through Si3N4 nanopore. Furthermore, we demonstrate that the improved signal-to-noise ratio is connected with not only the value of surface charge but also that of a salt gradient between cis and trans sides of the nanopore.
Correction to "Observing Extremely Weak Protein-Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy"
Jul 20, 2017   Journal Of The American Chemical Society
Yoo J, Lee TS, Choi B, Shon MJ, Yoon TY
Interfacial Shear Strength and Adhesive Behavior of Silk Ionomer Surfaces
Jul 24, 2017   Biomacromolecules
Kim S, Geryak RD, Zhang S, Ma R, Calabrese R, Kaplan DL, Tsukruk VV
Interfacial Shear Strength and Adhesive Behavior of Silk Ionomer Surfaces
Jul 24, 2017
Biomacromolecules
The interfacial shear strength between different layers in multi-layered structures of layer-by-layer (LbL) microcapsules is a crucial mechanical property to ensure their robustness. In this work, we investigated the interfacial shear strength of modified silk fibroin ionomers utilized in LbL shells, an ionic-cationic pair with complimentary ionic pairing, (SF)-poly-L-glutamic acid (Glu) and SF- poly-L-lysine (Lys) and a complementary pair with partially screened Coulombic interactions due to the presence of poly(ethylene glycol) (PEG) segments, SF-Glu/SF-Lys[PEG] pair. Shearing and adhesive behavior between these silk ionomer surfaces in the swollen state were probed at different spatial scales and pressure ranges by using functionalized atomic force microscopy (AFM) tips as well as functionalized colloidal probes. The results show that both approaches were consistent in analyzing the interfacial shear strength of LbL silk ionomers at different spatial scales from a nanoscale to a fraction of a micron. Surprisingly, the interfacial shear strength between SF-Glu and SF-Lys[PEG] pair with partially screened ionic pairing was greater than the interfacial shear strength of the SF-Glu and SF-Lys pair with a high density of complementary ionic groups. The difference in interfacial shear strength and adhesive strength is suggested to be predominantly facilitated by the interlayer hydrogen bonding of complementary aminoacids and overlap of highly swollen PEG segments.

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