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https://www.readbyqxmd.com/read/28230529/single-molecule-fret-unveils-induced-fit-mechanism-for-substrate-selectivity-in-flap-endonuclease-1
#1
Fahad Rashid, Paul D Harris, Manal S Zaher, Mohamed A Sobhy, Luay I Joudeh, Chunli Yan, Hubert Piwonski, Susan E Tsutakawa, Ivaylo Ivanov, John A Tainer, Satoshi Habuchi, Samir M Hamdan
Human flap endonuclease 1 (FEN1) and related structure-specific 5'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage...
February 23, 2017: ELife
https://www.readbyqxmd.com/read/28230057/ensemble-and-single-molecule-dynamics-of-ift-dynein-in-caenorhabditis-elegans-cilia
#2
Jona Mijalkovic, Bram Prevo, Felix Oswald, Pierre Mangeol, Erwin J G Peterman
Cytoplasmic dyneins drive microtubule-based, minus-end directed transport in eukaryotic cells. Whereas cytoplasmic dynein 1 has been widely studied, IFT dynein has received far less attention. Here, we use fluorescence microscopy of labelled motors in living Caenorhabditis elegans to investigate IFT-dynein motility at the ensemble and single-molecule level. We find that while the kinesin composition of motor ensembles varies along the track, the amount of dynein remains relatively constant. Remarkably, this does not result in directionality changes of cargo along the track, as has been reported for other opposite-polarity, tug-of-war motility systems...
February 23, 2017: Nature Communications
https://www.readbyqxmd.com/read/28229597/molecular-seesaw-intricate-dynamics-and-versatile-chemistry-of-heteroaromatics-on-metal-surfaces
#3
Sergey N Filimonov, Wei Liu, Alexandre Tkatchenko
The design of novel elementary surface processes is important for applications in catalysis, single-molecule junctions, molecular sensors, switches and surface mounted molecular machines. Here we demonstrate by van der Waals inclusive density functional theory calculations that a small and relatively simple heteroaromatic compound s-triazine (C$_3$H$_3$N$_3$) unexpectedly possesses five metastable states when adsorbed on the Pt(111) surface. This diversity of the adsorption states stems from an interplay between versatile molecule/surface chemical bonding and van der Waals interactions and from ``softening'' of the aromatic ring by nitrogen substitution, which makes folding of the aromatic ring energetically much less demanding as compared to benzene...
February 23, 2017: Journal of Physical Chemistry Letters
https://www.readbyqxmd.com/read/28229510/gold-nanorod-enhanced-fluorescence-enables-single-molecule-electrochemistry-of-methylene-blue
#4
Weichun Zhang, Martín Caldarola, Biswajit Pradhan, Michel Orrit
Redox reactions are central to energy conversion and life metabolism. Herein we present electrochemical measurements with fluorescent readout of the redox-sensitive dye Methylene Blue (MB), at the single-molecule (SM) level. To overcome the low fluorescence quantum yield of MB we enhanced fluorescence by using individual gold nanorods to achieve the required sensitivity. By measuring the same molecule at different electrochemical potentials we determined the mid-point potential of each single molecule through its redox-induced fluorescence blinking dynamics...
February 23, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28229174/characterization-of-the-specific-interaction-between-the-dna-aptamer-sgc8c-and-protein-tyrosine-kinase-7-receptors-at-the-surface-of-t-cells-by-biosensing-afm
#5
Michael Leitner, Alexandra Poturnayova, Constanze Lamprecht, Sabine Weich, Maja Snejdarkova, Ivana Karpisova, Tibor Hianik, Andreas Ebner
We studied the interaction of the specific DNA aptamer sgc8c immobilized at the AFM tip with its corresponding receptor, the protein tyrosine kinase-7 (PTK7) embedded in the membrane of acute lymphoblastic leukemia (ALL) cells (Jurkat T-cells). Performing single molecule force spectroscopy (SMFS) experiments, we showed that the aptamer sgc8c bound with high probability (38.3 ± 7.48%) and high specificity to PTK7, as demonstrated by receptor blocking experiments and through comparison with the binding behavior of a nonspecific aptamer...
February 22, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28228376/xenopus-laevis-kif18a-is-a-highly-processive-kinesin-required-for-meiotic-spindle-integrity
#6
Martin M Möckel, Andreas Heim, Thomas Tischer, Thomas U Mayer
The assembly and functionality of the mitotic spindle depends on the coordinated activities of microtubule-associated motor-proteins of the dynein and kinesin superfamily. Our current understanding of the function of motor-proteins is significantly shaped by studies using Xenopus laevis egg extract as its open structure allows complex experimental manipulations hardly feasible in other model systems. Yet, the Kinesin-8 orthologue of human Kif18A has not been described in Xenopus laevis so far. Here, we report the cloning and characterization of Xenopus laevis (Xl) Kif18A...
February 22, 2017: Biology Open
https://www.readbyqxmd.com/read/28225608/time-dependent-markov-state-models-for-single-molecule-force-spectroscopy
#7
Susmita Ghosh, Abhijit Chatterjee, Swati Bhattacharya
This Letter demonstrates that using time-dependent Markov state models (TD-MSMs) one can obtain molecular-scale insights into force-extension curves for a variety of stretching experiments. A master-MSM constructed at a reference extension using a handful of molecular dynamics calculations forms the basis for generating the required TD-MSM. Changes in the network topology upon stretching is related through a thermodynamic quantity termed the mechanical disposition. Proof-of-principle is provided using a stretched alanine decapeptide under a time-varying pulling force...
February 22, 2017: Journal of Chemical Theory and Computation
https://www.readbyqxmd.com/read/28225108/an-integrated-perspective-on-rna-aptamer-ligand-recognition-models-clearing-muddy-waters
#8
K McCluskey, J Carlos Penedo
Riboswitches are short RNA motifs that sensitively and selectively bind cognate ligands to modulate gene expression. Like protein receptor-ligand pairs, their binding dynamics are traditionally categorized as following one of two paradigmatic mechanisms: conformational selection and induced fit. In conformational selection, ligand binding stabilizes a particular state already present in the receptor's dynamic ensemble. In induced fit, ligand-receptor interactions enable the system to overcome the energetic barrier into a previously inaccessible state...
February 22, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28225099/ultra-high-throughput-detection-1-million-droplets-per-second-of-fluorescent-droplets-using-a-cell-phone-camera-and-time-domain-encoded-optofluidics
#9
Venkata R Yelleswarapu, Heon-Ho Jeong, Sagar Yadavali, David Issadore
Droplet-based assays-in which ultra-sensitive molecular measurements are made by performing millions of parallel experiments in picoliter droplets-have generated enormous enthusiasm due to their single molecule resolution and robustness to reaction conditions. These assays have great untapped potential for point of care diagnostics but are currently confined to laboratory settings due to the instrumentation necessary to serially generate, control, and measure tens of millions of droplets. To address this challenge, we have developed the microdroplet megascale detector (μMD) that can generate and detect the fluorescence of millions of droplets per second (1000× faster than conventional approaches) using only a conventional cell phone camera...
February 22, 2017: Lab on a Chip
https://www.readbyqxmd.com/read/28224493/analyzing-single-molecule-fret-trajectories-using-hmm
#10
Kenji Okamoto
Structural dynamics of biomolecules, such as proteins, plays essential roles in many biological phenomena at molecular level. It is crucial to understand such dynamics in recent biology. The single-molecule Förster resonance energy transfer (smFRET) measurement is one of few methods that enable us to observe structural changes of biomolecules in realtime. Time series data of smFRET, however, typically contains significant fluctuation, making analysis difficult. On the other hand, one can often assume a Markov process behind such data so that the hidden Markov model (HMM) can be used to reproduce a state transition trajectory (STT)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28223518/integrated-view-of-internal-friction-in-unfolded-proteins-from-single-molecule-fret-contact-quenching-theory-and-simulations
#11
Andrea Soranno, Andrea Holla, Fabian Dingfelder, Daniel Nettels, Dmitrii E Makarov, Benjamin Schuler
Internal friction is an important contribution to protein dynamics at all stages along the folding reaction. Even in unfolded and intrinsically disordered proteins, internal friction has a large influence, as demonstrated with several experimental techniques and in simulations. However, these methods probe different facets of internal friction and have been applied to disparate molecular systems, raising questions regarding the compatibility of the results. To obtain an integrated view, we apply here the combination of two complementary experimental techniques, simulations, and theory to the same system: unfolded protein L...
February 21, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28223507/imaging-mrna-and-protein-interactions-within-neurons
#12
Carolina Eliscovich, Shailesh M Shenoy, Robert H Singer
RNA-protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance...
February 21, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28223461/identification-of-a-pseudomonas-aeruginosa-pao1-dna-methyltransferase-its-targets-and-physiological-roles
#13
Sebastian Doberenz, Denitsa Eckweiler, Olga Reichert, Vanessa Jensen, Boyke Bunk, Cathrin Spröer, Adrian Kordes, Emanuela Frangipani, Khai Luong, Jonas Korlach, Stephan Heeb, Jörg Overmann, Volkhard Kaever, Susanne Häussler
DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this study, we used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogen Pseudomonas aeruginosa PAO1...
February 21, 2017: MBio
https://www.readbyqxmd.com/read/28222010/optical-tweezers-studies-of-transcription-by-eukaryotic-rna-polymerases
#14
Ana Lisica, Stephan W Grill
Transcription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases...
February 21, 2017: Biomolecular Concepts
https://www.readbyqxmd.com/read/28221038/molecular-and-electronic-structures-of-m2o7-m-mn-tc-re
#15
Keith V Lawler, Bradley C Childs, Daniel S Mast, Kenneth R Czerwinski, Alfred P Sattelberger, Frederic Poineau, Paul M Forster
The molecular and electronic structures of the group 7 heptoxides were investigated by computational methods as both isolated molecules and in the solid-state. The metal-oxygen-metal bending angle of the single molecule increased with increasing atomic number, with Re2O7 preferring a linear structure. Natural bond orbital and localized orbital bonding analyses indicate that there is a three-center covalent bond between the metal atoms and the bridging oxygen, and the increasing ionic character of the bonds favors larger bond angles...
February 21, 2017: Inorganic Chemistry
https://www.readbyqxmd.com/read/28220651/elisa-type-assays-of-trace-biomarkers-using-microfluidic-methods
#16
REVIEW
Jinhua Dong, Hiroshi Ueda
Recently, great progress has been achieved for analytical technologies for biological substances. Traditionally, detection methods for analytes mainly rely on large instrumental analyses. These methods require costly equipment, skilled operators and long measurement time despite their generally low sensitivity. In contrast, immunoassays are becoming more and more popular for it is powerful, inexpensive, and convenient nature. Immunoassay has a range of applications, because it employs antibody, a protein produced by plasma cells in the acquired immune system to identify and neutralize diverse pathogens and other exogenous substances...
February 20, 2017: Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology
https://www.readbyqxmd.com/read/28218822/single-molecular-junction-study-on-h2o-c60-h2o-is-electrostatically-isolated
#17
Satoshi Kaneko, Yoshifumi Hashikawa, Shintaro Fujii, Yasujiro Murata, Manabu Kiguchi
A water molecule exhibits characteristic properties on the basis of hydrogen-bondings. In the past decade, a single water molecule placed in the non-hydrogen-bonding environment has attracted growing attention. To reveal fundamental properties of a single water molecule, endohedral fullerene H2O@C60 becomes an ideal and suitable model. We examined the electronic properties of H2O@C60 by applying the single-molecule measurement. The conductance of a single molecular junction based on H2O@C60 was found to be comparable with that of empty C60...
February 20, 2017: Chemphyschem: a European Journal of Chemical Physics and Physical Chemistry
https://www.readbyqxmd.com/read/28218040/comprehensive-profiling-and-quantitation-of-oncogenic-mutations-in-non-small-cell-lung-carcinoma-using-single-molecule-amplification-and-re-sequencing-technology
#18
Jian Shi, Meng Yuan, Zhan-Dong Wang, Xiao-Li Xu, Lei Hong, Shenglin Sun
The carcinogenesis of non-small cell lung carcinoma has been found to associate with activating and resistant mutations in the tyrosine kinase domain of specific oncogenes. Here, we assessed the type, frequency, and abundance of epithelial growth factor receptor, KRAS, BRAF, and ALK mutations in 154 non-small cell lung carcinoma specimens using single-molecule amplification and re-sequencing technology. We found that epithelial growth factor receptor mutations were the most prevalent (44.2%), followed by KRAS (18...
February 2017: Tumour Biology: the Journal of the International Society for Oncodevelopmental Biology and Medicine
https://www.readbyqxmd.com/read/28217990/telomerase-activity-detection-with-amplification-free-single-molecule-stochastic-binding-assay
#19
Xin Su, Zehao Li, Xinzhong Yan, Lei Wang, Xu Zhou, Lin Wei, Lehui Xiao, Changyuan Yu
Since the elongation of telomeres is associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification typically associated with poor reproducibility and high background which hamper their further applications particularly for real sample assay. Here, we demonstrated a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy (TIRFM)...
February 20, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28216791/molecular-processes-leading-to-necking-in-extensional-flow-of-polymer-solutions-using-microfluidics-and-single-dna-imaging
#20
Shaurya Sachdev, Aswin Muralidharan, Pouyan E Boukany
We study the necking and pinch-off dynamics of liquid droplets that contain a semidilute polymer solution of polyacrylamide close to overlap concentration by combining microfluidics and single DNA observation. Polymeric droplets are stretched by passing them through the stagnation point of a T-shaped microfluidic junction. In contrast with the sudden breakup of Newtonian droplets, a stable neck is formed between the separating ends of the droplet which delays the breakup process. Initially, polymeric filaments experience exponential thinning by forming a stable neck with extensional flow within the filament...
December 27, 2016: Macromolecules
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