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Covalent labeling

Dong-Eog Kim, Jeong-Yeon Kim, Su-Kyoung Lee, Ju Hee Ryu, Ick Chan Kwon, Cheol-Hee Ahn, Kwangmeyung Kim, Dawid Schellingerhout
Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker - a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation...
September 25, 2016: Journal of Visualized Experiments: JoVE
Christoph Göbl, Moritz Resch, Madeleine Strickland, Christoph Hartlmüller, Martin Viertler, Nico Tjandra, Tobias Madl
The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical-shift assignments, and data analysis that relies on well-separated resonances. We present a covalent paramagnetic lanthanide-binding tag (LBT) for increasing the chemical-shift dispersion and facilitating the chemical-shift assignment of challenging, repeat-containing IDPs. Linkage of the DOTA-based LBT to a cysteine residue induces pseudo-contact shifts (PCS) for resonances more than 20 residues from the spin-labeling site...
October 20, 2016: Angewandte Chemie
Berkay Ozcelik, Renxun Chen, Veronica Glattauer, Naresh Kumar, MarkD P Willcox, Helmut Thissen
Control over biointerfacial interactions on material surfaces is of significant interest in many biomedical applications and extends from the modulation of protein adsorption and cellular responses to the inhibition of bacterial attachment and biofilm formation. Effective control over biointerfaces is best achieved by reducing nonspecific interactions on the surface while also displaying specific bioactive signals. A poly(ethylene glycol) (PEG)-based multifunctional coating has been developed that provides effective reduction of protein fouling while enabling covalent immobilization of peptides in a one or two-step manner...
October 20, 2016: Macromolecular Bioscience
Woon Yong Sohn, Valérie Brenner, Eric Gloaguen, Michel Mons
Conformer-selective IR gas phase spectroscopy and high level quantum chemistry methods have been used to characterise the diversity of local NH-π interactions between the π ring of a phenylalanine aromatic residue and the nearby main chain amide groups. The study of model systems shows how the amide NH stretch vibrational features, in the 3410-3460 cm(-1) frequency range, can be used to monitor the strength of these local π H-bonds, which is found to depend on both the backbone conformation and the aromatic side chain orientation...
October 20, 2016: Physical Chemistry Chemical Physics: PCCP
Shyam K Konda, Celine Kelso, Jelena Medan, Brad E Sleebs, Don R Phillips, Suzanne M Cutts, J Grant Collins
The major covalent adduct formed between a (13)C-labelled formaldehyde activated bis-amino mitoxantrone analogue (WEHI-150) and the hexanucleotide d(CG(5Me)CGCG)2 has been isolated by HPLC chromatography and the structure determined by NMR spectroscopy. The results indicate that WEHI-150 forms one covalent bond through a primary amine to the N-2 of the G2 residue, with the polycyclic ring structure intercalated at the (5Me)C3pG4/G10p(5Me)C9 site. Furthermore, the WEHI-150 aromatic ring system is oriented approximately parallel to the long axis of the base pairs, with one aliphatic side-chain in the major groove and the other side-chain in the minor groove...
October 13, 2016: Organic & Biomolecular Chemistry
Haleh Hashemi Haeri, Philipp Spindler, Jörn Plackmeyer, Thomas Prisner
Carbon-centered radicals are interesting alternatives to otherwise commonly used nitroxide spin labels for dipolar spectroscopy techniques because of their narrow ESR linewidth. Herein, we present a novel BDPA biradical, where two BDPA (α,α,γ,γ-bisdiphenylene-β-phenylallyl) radicals are covalently tethered by a saturated biphenyl acetylene linker. The inter-spin distance between the two spin carrier fragments was measured using double quantum coherence (DQC) ESR methodology. The DQC experiment revealed a mean distance of only 1...
October 12, 2016: Physical Chemistry Chemical Physics: PCCP
Xiaohui Geng, Fanglin Zhang, Qiang Gao, Yingfeng Lei
A sensitive and disposable electrochemical impedance biosensor to detect Japanese encephalitis virus (JEV) was developed based on a gold nanoparticle (AuNP)-modified screen-printed carbon electrode (SPCE). A biosensor was fabricated through covalent grafting of a mixed self-assembled monolayer on AuNPs with a specific antibody. To detect JEV and achieve signal amplification, the horseradish peroxidase (HRP)-labeled second antibody was linked to the biosensor through a sandwich immunity reaction. HRP was used to catalyze 4-chloro-1-naphthol oxidation to produce an insoluble precipitate, which introduced a barrier to electron transfer on the electrode...
2016: Analytical Sciences: the International Journal of the Japan Society for Analytical Chemistry
Qingfeng Zhang, Juanmin Li, Yunyi Zhang, Zhenzhen Hu, Cong Yu
A black-hole quencher (BHQ-2) labeled DNA (Q-DNA) with a phosphorothioate backbone was covalently conjugated to the CdTe QDs during the QDs synthesis procedure. The hairpin structure of Q-DNA shortened the distance of the CdTe QDs and the BHQ-2 group, which resulted in fluorescence quenching of the QDs. The addition of target DNA or deoxyribonuclease I (DNase I) could move the BHQ-2 group away from the QDs. As a result, the fluorescence of the CdTe QDs recovered. This work provides a new way for target DNA and DNase I detection...
2016: Analytical Sciences: the International Journal of the Japan Society for Analytical Chemistry
Seema Singh, Pravin Jha, Vandana Singh, Kislay Sinha, Sahid Hussain, Manoj K Singh, Prolay Das
Non-targeted photosensitizers lack selectivity that undermines the potential use of photodynamic therapy (PDT). Herein, we report the DNA mediated assembly of a ZnSe/ZnS quantum dot (QD)-photosensitizer (PS)-Mucin 1(MUC1) aptamer conjugate for targeting the MUC1 cancer biomarker and simultaneous generation of reactive oxygen species (ROS). A photosensitizer, protoporphyrin IX (PpIX), was conjugated to a single stranded DNA and self-assembled to a complementary strand that was conjugated to a QD and harboring a MUC1 aptamer sequence...
October 10, 2016: Integrative Biology: Quantitative Biosciences From Nano to Macro
Jun Ando, Miwako Asanuma, Kosuke Dodo, Hiroyuki Yamakoshi, Satoshi Kawata, Katsumasa Fujita, Mikiko Sodeoka
Identification of small-molecule-binding sites in protein is important for drug discovery and analysis of protein function. Modified amino-acid residue(s) can be identified by proteolytic cleavage followed by liquid chromatography-mass spectrometry (LC-MS), but this is often hindered by the complexity of the peptide mixtures. We have developed alkyne-tag Raman screening (ATRaS) for identifying binding sites. In ATRaS, small molecules are tagged with alkyne and form covalent bond with proteins. After proteolysis and HPLC, fractions containing the labeled peptides with alkyne tags are detected by means of surface-enhanced Raman scattering (SERS) using silver nanoparticles and sent to MS/MS to identify the binding site...
October 10, 2016: Journal of the American Chemical Society
Christopher B Rodell, Christopher B Highley, Minna H Chen, Neville N Dusaj, Chao Wang, Lin Han, Jason A Burdick
Macromolecular interactions are used to form supramolecular assemblies, including through the interaction of guest-host chemical pairs. Microstructural heterogeneity has been observed within such physical hydrogels; yet, systematic investigation of the microstructure and its determining inputs are lacking. Herein, we investigated the hierarchical self-assembly of hyaluronic acid (HA) modified by the guest-host pair adamantane (Ad-HA, guest) and β-cyclodextrin (CD-HA, host), as well as with methacrylate groups to both tether fluorescent agents and to covalently stabilize the material structure...
September 20, 2016: Soft Matter
Aminat T Oki, Bernice Huang, Andrea R Beyer, Levi J May, Hilary K Truchan, Naomi J Walker, Nathan L Galloway, Dori L Borjesson, Jason A Carlyon
Anaplasma phagocytophilum, a member of the family Anaplasmataceae and the obligate intracellular bacterium that causes granulocytic anaplasmosis, resides in a host cell-derived vacuole. Bacterial proteins that localize to the A. phagocytophilum-occupied vacuole membrane (AVM) are critical host-pathogen interfaces. Of the few bacterial AVM proteins that have been identified, the domains responsible for AVM localization and the host cell pathways that they co-opt are poorly defined. APH0032 is an effector that is expressed and localizes to the AVM late during the infection cycle...
2016: Frontiers in Cellular and Infection Microbiology
Inge Leonie Van 't Veer, Nadia Leloup, Alexander Egan, Bert Janssen, Nathaniel Martin, Waldemar Vollmer, Eefjan Breukink
Surface Plasmon Resonance (SPR) is one of the most powerful label free methods to determine kinetic parameters of molecular interactions in real time and in a high sensitive way. Penicillin binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. The established protocols to analyze interactions of PBPs by SPR involves immobilization to an ampicillin coated chip surface, a β-lactam antibiotic mimicking its substrate, forming a covalent complex with the PBPs transpeptidase (TP) active site...
October 6, 2016: Chembiochem: a European Journal of Chemical Biology
Wai Ching Veronica Wong, Aurimas Narkevicius, Wing Ying Chow, David G Reid, Rakesh Rajan, Roger A Brooks, Maggie Green, Melinda J Duer
We have prepared mouse fur extensively (13)C,(15)N-labelled in all amino acid types enabling application of 2D solid state NMR techniques which establish covalent and spatial proximities within, and in favorable cases between, residues. (13)C double quantum-single quantum correlation and proton driven spin diffusion techniques are particularly useful for resolving certain amino acid types. Unlike 1D experiments on isotopically normal material, the 2D methods allow the chemical shifts of entire spin systems of numerous residue types to be determined, particularly those with one or more distinctively shifted atoms such as Gly, Ser, Thr, Tyr, Phe, Val, Leu, Ile and Pro...
October 3, 2016: Journal of Biomolecular NMR
Tonatiuh Molina-Villa, Valentín Mendoza, Fernando López-Casillas
By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-β receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype...
2016: Journal of Visualized Experiments: JoVE
Sarah A Head, Jun O Liu
Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods...
2016: Journal of Visualized Experiments: JoVE
Dae In Kim, Kyle J Roux
There are inherent limitations with traditional methods to study protein behavior or to determine the constituency of proteins in discrete subcellular compartments. In response to these limitations, several methods have recently been developed that use proximity-dependent labeling. By fusing proteins to enzymes that generate reactive molecules, most commonly biotin, proximate proteins are covalently labeled to enable their isolation and identification. In this review we describe current methods for proximity-dependent labeling in living cells and discuss their applications and future use in the study of protein behavior...
September 22, 2016: Trends in Cell Biology
Zhenyu Zhang, Meng Wang, Dongliang Gao, Da Luo, Qinghai Liu, Juan Yang, Yan Li
Graphene oxide (GO) is a reliable and multifunctional platform to perform cell imaging. In this work, a controllable Pt-seed-mediated method is used to prepare GO/gold nanoparticle (AuNP) hybrids, and after the covalent binding of folic acid (FA), GO/AuNP/FA hybrids are prepared. Selective labeling and Raman imaging of folate receptor (FR)-positive HeLa cells are realized using such GO-based hybrids. In this system, FA is the targeting agent, AuNPs work as surface-enhanced Raman scattering substrates, and GO takes the role of both supporting the AuNPs with FA and acting as a Raman probe...
October 11, 2016: Langmuir: the ACS Journal of Surfaces and Colloids
Douglas E Stack, Rachel Eastman
Regioselective labelling of arene rings via electrophilic exchange is often dictated by the electronic environment caused by substituents present on the aromatic system. Previously, we observed the presence of a t-butyl group, either covalently bond or added as an external reagent, could impart deuterium exchange to the unactivated, C1-position of estrone. Here, we provide nuclear magnetic resonance analysis of this exchange in a solvent system composed of 50:50 trifluoroacetic acid and D2 O with either 2-t-butylestrone or estrone in the presence of t-butyl alcohol has shed insights into the mechanism of this t-butyl-catalyzed exchange...
October 2016: Journal of Labelled Compounds & Radiopharmaceuticals
Susan O'Brien, Jeffrey A Jones, Steven E Coutre, Anthony R Mato, Peter Hillmen, Constantine Tam, Anders Österborg, Tanya Siddiqi, Michael J Thirman, Richard R Furman, Osman Ilhan, Michael J Keating, Timothy G Call, Jennifer R Brown, Michelle Stevens-Brogan, Yunfeng Li, Fong Clow, Danelle F James, Alvina D Chu, Michael Hallek, Stephan Stilgenbauer
BACKGROUND: The TP53 gene, encoding tumour suppressor protein p53, is located on the short arm of chromosome 17 (17p). Patients with 17p deletion (del17p) chronic lymphocytic leukaemia have poor responses and survival after chemoimmunotherapy. We assessed the activity and safety of ibrutinib, an oral covalent inhibitor of Bruton's tyrosine kinase, in relapsed or refractory patients with del17p chronic lymphocytic leukaemia or small lymphocytic lymphoma. METHODS: We did a multicentre, international, open-label, single-arm study at 40 sites in the USA, Canada, Europe, Australia, and New Zealand...
October 2016: Lancet Oncology
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