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https://www.readbyqxmd.com/read/29156676/uncoupling-torc2-from-agc-kinases-inhibits-tumour-growth
#1
Angus J M Cameron, Selvaraju Veeriah, Jacqueline J T Marshall, Elizabeth R Murray, Banafshé Larijani, Peter J Parker
Mammalian target of rapamycin (mTOR) is a central regulator of growth and metabolism. mTOR resides in two distinct multi-protein complexes - mTORC1 and mTORC2 - with distinct upstream regulators and downstream targets. While it is possible to specifically inhibit mTORC1 with rapamycin, or inhibit both mTOR complexes together with ATP pocket directed mTOR kinase inhibitors, it is not possible to assess the specific roles for mTORC2 pharmacologically. To overcome this, we have developed a novel, inducible, dominant negative system for disrupting substrate recruitment to mTORC2...
October 17, 2017: Oncotarget
https://www.readbyqxmd.com/read/29155761/advanced-confocal-microscopy-techniques-to-study-protein-protein-interactions-and-kinetics-at-dna-lesions
#2
Soňa Legartová, Jana Suchánková, Jana Krejčí, Alena Kovaříková, Eva Bártová
Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented...
November 12, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155588/determination-of-equilibrium-constant-and-relative-brightness-in-fret-fcs-by-including-the-third-order-correlations
#3
Lingyi Meng, Shanshan He, Xin Sheng Zhao
Fluorescence correlation spectroscopy (FCS) encodes the information of the equilibrium constant (K), the relative fluorescence brightness of fluorophore (Q), and the forward and backward reaction rate constants (k+ and k-) on a physical or chemical relaxation. However, it has been a long-standing problem to completely resolve the FCS data to get the thermodynamic and kinetic information. Recently, we have solved the problem for fluorescence auto-correlation spectroscopy (FACS). Here, we extend the method to fluorescence cross-correlation spectroscopy (FCCS), which appears when FCS is coupled with fluorescence resonance energy transfer (FRET)...
November 20, 2017: Journal of Physical Chemistry. B
https://www.readbyqxmd.com/read/29155567/ratiometric-fluorescent-detection-of-pb-2-by-fret-based-phthalocyanine-porphyrin-dyads
#4
Dongli Zhang, Mengliang Zhu, Luyang Zhao, Jinghui Zhang, Kang Wang, Dongdong Qi, Yang Zhou, Yongzhong Bian, Jianzhuang Jiang
Sensitive and selective detection of Pb(2+) is a very worthwhile endeavor in terms of both human health and environmental protection, as the heavy metal is fairly ubiquitous and highly toxic. In this study, we designed phthalocyanine-porphyrin (Pc-Por) heterodyads, namely, H2Pc-α-ZnPor (1) and H2Pc-β-ZnPor (2), by connecting a zinc(II) porphyrin moiety to the nonperipheral (α) or peripheral (β) position of a metal-free phthalocyanine moiety. Upon excitation at the porphyrin Soret region (420 nm), both of the dyads exhibited not only a porphyrin emission (605 nm) but also a phthalocyanine emission (ca...
November 20, 2017: Inorganic Chemistry
https://www.readbyqxmd.com/read/29153423/uracil-amino-acid-as-a-scaffold-for-%C3%AE-sheet-peptidomimetics-study-of-photophysics-and-interaction-with-bsa-protein
#5
Subhendu Sekhar Bag, Afsana Yashmeen
We report herein the uracil-di-aza-amino acid (U(r)AA) as a new family of molecular scaffold to induce β-hairpin structure with H-bonded β-sheet conformation in a short peptide. This has been demonstrated in two conceptual fluorescent pentapeptides wherein triazolylpyrenyl alanine and/or triazolylmethoxynapthyl alanine ((TPy)AlaDo and/or (TMNap)AlaDo) are embedded into two arms of the uracil-amino acid via an intervening leucine. Conformational analysis by CD, IR, variable temperature and 2D NMR spectroscopy reveals the β-hairpin structures for both the peptides...
November 10, 2017: Bioorganic & Medicinal Chemistry Letters
https://www.readbyqxmd.com/read/29152711/the-burst-phase-folding-intermediate-of-ribonuclease-h-changes-conformation-over-evolutionary-history
#6
Shion A Lim, Susan Marqusee
The amino acid sequence encodes the energy landscape of a protein. Therefore, we expect evolutionary mutations to change features of the protein energy landscape, including the conformations adopted by a polypeptide as it folds to its native state. Ribonucleases H (RNase H) from Escherichia coli and Thermus thermophilus both fold via a partially folded intermediate in which the core region of the protein (helices A-D and strands 4-5) is structured. Strand 1, however, uniquely contributes to the T. thermophilus RNase H folding intermediate (Icore+1 ), but not the E...
November 20, 2017: Biopolymers
https://www.readbyqxmd.com/read/29150443/kinetic-mechanism-of-controlled-fab-arm-exchange-for-the-formation-of-bispecific-immunoglobulin-g1-antibodies
#7
Dennis R Goulet, Steven J Orcutt, Adam Zwolak, Theo Rispens, Aran Labrijn, Rob N de Jong, William M Atkins, Mark L Chiu
Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Among the many platforms for creating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on minimal changes to native Ab structure and the simplicity with which bsAbs can be formed from two parental Abs. Despite a published protocol for cFAE and its widespread use in the pharmaceutical industry, the reaction mechanism has not been determined...
November 17, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29149694/a-high-brightness-probe-of-polymer-nanoparticles-for-biological-imaging
#8
Sirong Zhou, Jiarong Zhu, Yaping Li, Liheng Feng
Conjugated polymer nanoparticles (CPNs) with high brightness in long wavelength region were prepared by the nano-precipitation method. Based on fluorescence resonance energy transfer (FRET) mechanism, the high brightness property of the CPNs was realized by four different emission polymers. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) displayed that the CPNs possessed a spherical structure and an average diameter of ~75nm. Analysis assays showed that the CPNs had excellent biocompatibility, good photostability and low cytotoxicity...
November 11, 2017: Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
https://www.readbyqxmd.com/read/29148805/near-infrared-quantum-dot-emission-enhanced-by-stabilized-self-assembled-j-aggregate-antennas
#9
Francesca S Freyria, José M Cordero, Justin R Caram, Sandra Doria, Amro Dodin, Yue Chen, Adam P Willard, Moungi G Bawendi
Enhancing photoluminescent emission (PL) in the near-infrared-infrared (NIR-IR) spectral region has broad applications from solar energy conversion to biological imaging. We show that self-assembled molecular dye J-aggregates (light-harvesting nanotubes, LHNs) can increase the PL emission of NIR PbS quantum dots (QDs) in both liquid and solid media more than 8-fold, promoted primarily by a long-range antenna effect and efficient Förster resonance energy transfer (FRET) from donor to acceptor. To create this composite material and preserve the optical properties of the nanocrystals, we performed an in situ ligand substitution followed by a functionalization reaction using click-chemistry...
November 17, 2017: Nano Letters
https://www.readbyqxmd.com/read/29148735/stereoselective-stabilization-of-polymeric-vitamin-e-conjugate-micelles
#10
Min Gao, Jian Deng, Huiying Chu, Yu Tang, Zheng Wang, Yanjun Zhao, Guohui Li
Vitamin E (α-tocopherol) (TPGS) micelle is a robust nanocarrier in delivering hydrophobic active pharmaceutical ingredients, but is suffering from poor stability that is essential in terms of pharmaceutical and biomedical applications. Taking advantage of the chirality of vitamin E, this work reports the stereoselective stabilization of polymer-vitamin E conjugate micelles. Vitamin E was covalently linked to multivalent methoxy poly(ethylene glycol)-co-poly(glutamic acid), generating amphiphilic conjugates that could self-assemble into micelles...
November 17, 2017: Biomacromolecules
https://www.readbyqxmd.com/read/29148702/influence-of-particle-geometry-on-gastrointestinal-transit-and-absorption-following-oral-administration
#11
Dong Li, Jie Zhuang, Haisheng He, Sifan Jiang, Amrita Banerjee, Yi Lu, Wei Wu, Samir Mitragotri, Li Gan, Jianping Qi
Geometry has been considered as one of the important parameters in nanoparticle design since it affects cellular uptake, transport across the physiological barriers and in vivo distribution. However, only a few studies have been conducted to elucidate the influence of nanoparticle geometry in their in vivo fate after oral administration. This paper discloses the effect of nanoparticle shape on transport and absorption in gastrointestinal tract (GIT). Nanorods and nanospheres were prepared, and labeled using fluorescence resonance energy transfer (FRET) molecules to track the in vivo fate of intact nanoparticles accurately...
November 17, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/29144454/dynamics-of-p-type-atpase-transport-revealed-by-single-molecule-fret
#12
Mateusz Dyla, Daniel S Terry, Magnus Kjaergaard, Thomas L-M Sørensen, Jacob Lauwring Andersen, Jens Peter Andersen, Charlotte Rohde Knudsen, Roger B Altman, Poul Nissen, Scott C Blanchard
Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca(2+)-ATPase (LMCA1), an orthologue of eukaryotic Ca(2+)-ATPases...
November 16, 2017: Nature
https://www.readbyqxmd.com/read/29142589/flim-fret-analyzer-open-source-software-for-automation-of-lifetime-based-fret-analysis
#13
Jiho Kim, Yury Tsoy, Jan Persson, Regis Grailhe
Background: Despite the broad use of FRET techniques, available methods for analyzing protein-protein interaction are subject to high labor and lack of systematic analysis. We propose an open source software allowing the quantitative analysis of fluorescence lifetime imaging (FLIM) while integrating the steady-state fluorescence intensity information for protein-protein interaction studies. Findings: Our developed open source software is dedicated to fluorescence lifetime imaging microscopy (FLIM) data obtained from Becker & Hickl SPC-830...
2017: Source Code for Biology and Medicine
https://www.readbyqxmd.com/read/29142199/sensorfret-a-standardless-approach-to-measuring-pixel-based-spectral-bleed-through-and-fret-efficiency-using-spectral-imaging
#14
Paul T Arsenovic, Carl R Mayer, Daniel E Conway
Fluorescence microscopy of FRET-based biosensors allow nanoscale interactions to be probed in living cells. This paper describes a novel approach to spectrally resolved fluorescence microscopy, termed sensorFRET, that enables quantitative measurement of FRET efficiency. This approach is an improvement on existing methods (FLIM, sRET, luxFRET, pFRET), as it does not require single fluorophore standards to be measured with every experiment and the acquisition is intensity independent, allowing the laser power to be optimized for varying levels of fluorophore expression...
November 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29142072/salt-bridges-gate-alpha-catenin-activation-at-intercellular-junctions
#15
Samantha Barrick, Jing Li, Xinyu Kong, Alokananda Ray, Emad Tajkhorshid, Deborah Leckband
Cadherin complexes transduce force fluctuations at junctions to activate signals that reinforce stressed intercellular contacts. α-Catenin is an identified force transducer within cadherin complexes that is autoinhibited under low tension. Increased force triggers a conformational change that exposes a cryptic site for the actin-binding protein vinculin. This study tested predictions that salt bridges within the force-sensing core modulate α-catenin activation. Studies with a fluorescence resonance energy transfer (FRET)-based α-catenin conformation sensor demonstrated that the salt-bridge mutations R551A and D503N each enhance α-catenin activation in live cells, but R551A has a greater impact...
November 15, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29141138/differential-effects-of-strand-asymmetry-on-the-energetics-and-structural-flexibility-of-dna-internal-loops
#16
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/29138775/microsecond-resolved-single-molecule-fret-time-series-measurements-based-on-the-line-confocal-optical-system-combined-with-hybrid-photodetectors
#17
Hiroyuki Oikawa, Takumi Takahashi, Supawich Kamonprasertsuk, Satoshi Takahashi
Single-molecule (sm) fluorescence time series measurements based on the line confocal optical system are a powerful strategy for the investigation of the structure, dynamics, and heterogeneity of biological macromolecules. This method enables the detection of more than several thousands of fluorescence photons per millisecond from single fluorophores, implying that the potential time resolution for measurements of the fluorescence resonance energy transfer (FRET) efficiency is 10 μs. However, the necessity of using imaging photodetectors in the method limits the time resolution in the FRET efficiency measurements to approximately 100 μs...
November 15, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/29138065/comparison-of-lipid-dependent-bilayer-insertion-of-phlip-and-its-p20g-variant
#18
Victor Vasquez-Montes, Janessa Gerhart, Kelly E King, Damien Thévenin, Alexey S Ladokhin
The ability of the pH-Low Insertion Peptide (pHLIP) to insert into lipid membranes in a transbilayer conformation makes it an important tool for targeting acidic diseased tissues. pHLIP can also serve as a model template for thermodynamic studies of membrane insertion. We use intrinsic fluorescence and circular dichroism spectroscopy to examine the effect of replacing pHLIP's central proline on the pH-triggered lipid-dependent conformational switching of the peptide. We find that the P20G variant (pHLIP-P20G) has a higher helical propensity than the native pHLIP (pHLIP-WT), in both water:organic solvent mixtures and in the presence of lipid bilayers...
November 11, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29138008/g-quadruplex-structure-at-intron-2-of-tfe3-and-its-role-in-xp11-2-translocation-and-splicing
#19
Shiv Prakash Verma, Parimal Das
Transcription Factor E3 (TFE3) translocation is found in a group of different type of cancers and most of the translocations are located in the 5' region of TFE3 which may be considered as Breakpoint Region (BR). In our In silico study by QGRS mapper and non BdB web servers we found a Potential G-quadruplex forming Sequence (PQS) in the intron 2 of TFE3 gene. In vitro G-quadruplex formation was shown by native PAGE in presence of Pyridostatin(PDS), which with inter molecular secondary structure caused reduced mobility to migrate slower...
November 11, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29136199/site-specific-dual-color-labeling-of-long-rnas-for-single-molecule-spectroscopy
#20
Meng Zhao, Fabio D Steffen, Richard Börner, Michelle F Schaffer, Roland K O Sigel, Eva Freisinger
Labeling of long RNA molecules in a site-specific yet generally applicable manner is integral to many spectroscopic applications. Here we present a novel covalent labeling approach that is site-specific and scalable to long intricately folded RNAs. In this approach, a custom-designed DNA strand that hybridizes to the RNA guides a reactive group to target a preselected adenine residue. The functionalized nucleotide along with the concomitantly oxidized 3'-terminus can subsequently be conjugated to two different fluorophores via bio-orthogonal chemistry...
November 9, 2017: Nucleic Acids Research
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