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Covalent labeling

Carla Schmidt, Jamie A Macpherson, Andy M Lau, Ken Wei Tan, Franca Fraternali, Argyris Politis
Mass spectrometry (MS) has become an indispensable tool for investigating the architectures and dynamics of macromolecular assemblies. Here we show that covalent labeling of solvent accessible residues followed by their MS-based identification yields modeling restraints that allow mapping the location and orientation of subunits within protein assemblies. Together with complementary restraints derived from cross-linking and native MS, we built native-like models of four heterocomplexes with known subunit structures and compared them with available X-ray crystal structures...
February 7, 2017: Analytical Chemistry
Wouter W Kallemeijn, Saskia Scheij, Sascha Hoogendoorn, Martin D Witte, Daniela Herrera Moro Chao, Cindy P A A van Roomen, Roelof Ottenhoff, Herman S Overkleeft, Rolf G Boot, Johannes M F G Aerts
Deficiency of glucocerebrosidase (GBA) causes Gaucher disease (GD). In the common non-neuronopathic GD type I variant, glucosylceramide accumulates primarily in the lysosomes of visceral macrophages. Supplementing storage cells with lacking enzyme is accomplished via chronic intravenous administration of recombinant GBA containing mannose-terminated N-linked glycans, mediating the selective uptake by macrophages expressing mannose-binding lectin(s). Two recombinant GBA preparations with distinct N-linked glycans are registered in Europe for treatment of type I GD: imiglucerase (Genzyme), contains predominantly Man(3) glycans, and velaglucerase (Shire PLC) Man(9) glycans...
2017: PloS One
Hanane Issa, Emilie Huc-Claustre, Thamila Reddad, Nolwenn Bonadé Bottino, Maryelle Tropis, Christine Houssin, Mamadou Daffé, Nicolas Bayan, Nathalie Dautin
Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C...
2017: PloS One
Melissa M Budelier, Wayland W L Cheng, Lucie Bergdoll, Zi-Wei Chen, Jeff Abramson, Kathiresan Krishnan, Mingxing Qian, Douglas F Covey, James W Janetka, Alex S Evers
Identifying sites of protein-ligand interaction is important for structure-based drug discovery and understanding protein structure-function relationships. Mass spectrometry (MS) has emerged as a useful tool for identifying residues covalently modified by ligands. Current methods use database searches that are dependent on acquiring interpretable fragmentation spectra (MS2) of peptide-ligand adducts. This is problematic for identifying sites of hydrophobic ligand incorporation in integral membrane proteins (IMPs), where poor aqueous solubility and ionization of peptide-ligand adducts and collision-induced adduct loss hinder the acquisition of quality MS2 spectra...
February 9, 2017: Analytical Chemistry
Ye Zhou, Yue Wu, Mingdong Yao, Zheyi Liu, Jin Chen, Jun Chen, Lirong Tian, Guangye Han, Jian-Ren Shen, Fangjun Wang
Positively charged lysines are crucial to maintaining the native structures of proteins and protein complexes by forming hydrogen bonds and electrostatic interactions with their proximal amino acid residues. However, it is still a challenge to develop an efficient method for probing the active proximal microenvironments of lysines without changing their biochemical/physical properties. Herein, we developed an active covalent labeling strategy combined with mass spectrometry to systematically probe the lysine proximal microenvironments within membrane protein complexes (∼700 kDa) with high throughput...
December 20, 2016: Analytical Chemistry
Chen Cui, Ying Chen, Dechen Jiang, Jun-Jie Zhu, Hong-Yuan Chen
In this work, a self-electrochemiluminous graphene oxide-capped Au@L012 nanocomposite was prepared as the label at carcinoembryonic (CEA) antibody to detect attomole CEA antigen. To maximize the luminescence intensity, L012 molecules (luminol analogue) were linked with poly(diallyldimethylammonium chloride) (PDDA) to form positive charged PDDA&L012 pairs, which were modified on negative charged Au@nafion nanoparticles to construct a Au@nafion@PDDA&L012 (Au@L012) complex. Graphene oxide with carboxyl groups was capped at Au@L012 complex through electrostatic interaction to serve as an effective matrix for the covalent attachment of CEA antibody...
February 9, 2017: Analytical Chemistry
Peng Liu, Hengdi Zhang, Wenqing He, Hualin Li, Jieke Jiang, Meijin Liu, Hongyan Sun, Mingliang He, Jiaxi Cui, Lei Jiang, Xi Yao
Here, we describe a simple method to prepare oil-repellent surfaces with inherent reactivity. Liquid-like copolymers with pendant reactive groups are covalently immobilized onto substrates via a sequential layer-by-layer method. The stable and transparent nanocoatings showed oil repellency to a broad range of organic liquids even in the presence of reactive sites. Functional molecules could be covalently immobilized onto the oil-repellent surfaces. Moreover, the liquid repellency can be maintained or finely tailored after post-chemical modification via synergically tailoring the film thickness, selection of capping molecules, and labeling degree of the capping molecules...
February 16, 2017: ACS Nano
Tamar Shahal, Ori Green, Uri Hananel, Yael Michaeli, Doron Shabat, Yuval Ebenstein
The nucleobase 5-hydroxymethylcytosine (5-hmC), a modified form of cytosine, is an important epigenetic mark related to regulation of gene expression. 5-hmC levels are highly dynamic during early development and are modulated during the progression of neurodegenerative disease and cancer. We describe a spectroscopic method for the global quantification of 5-hmC in genomic DNA. This method relies on the enzymatic glucosylation of 5-hmC, followed by a glucose oxidation step that results in the formation of aldehyde moieties that are covalently linked to a fluorescent reporter by oxime ligation...
October 7, 2016: Methods and Applications in Fluorescence
Kanae Teruya, Gregory Rankin, Panagiotis Chrysanthopoulos, Kathryn Tonissen, Sally-Ann Poulsen
Chemical probes are small molecule reagents used by researchers for labeling and detection of biomolecules. We present the design, synthesis and characterisation of a panel of eleven structurally diverse photoaffinity labeling (PAL) probes as research tools for labeling the model enzyme carbonic anhydrase (CA) in challenging environments, including protein mixtures and cell lysates. We target ubiquitous CA II as well as the two cancer associated CAs (CA IX and CA XII), which are high priority as potential biomarkers of aggressive and/or multidrug resistant cancer...
February 8, 2017: Chembiochem: a European Journal of Chemical Biology
Laurent Adumeau, Coralie Genevois, Lydia Roudier, Christophe Schatz, Franck Couillaud, Stéphane Mornet
BACKGROUND: In the context of systematically administered nanomedicines, the physicochemistry of NP surfaces must be controlled as a prerequisite to improve blood circulation time, and passive and active targeting. In particular, there is a real need to develop NP stealth and labelling for both in vivo and microscopic fluorescence imaging in a mice model. METHODS: We have synthesized NIR/red dually fluorescent silica nanoparticles of 19nm covalently covered by a PEG layer of different grafting density in the brush conformational regime by using a reductive amination reaction...
February 4, 2017: Biochimica et Biophysica Acta
Nicholas B Borotto, Zhe Zhang, Jia Dong, Brittney Burant, Richard W Vachet
β-2-microglobulin (β2m) forms amyloid fibrils in the joints of patients undergoing dialysis treatment as a result of kidney failure. One of the ways in which β2m can be induced to form amyloid fibrils in vitro is via incubation with stoichiometric amounts of Cu(II). To better understand the structural changes caused by Cu(II) binding that allow β2m to form amyloid fibrils, we compared the effect of Ni(II) and Zn(II) binding, which are two similarly-sized divalent metal ions that do not induce β2m amyloid formation...
February 7, 2017: Biochemistry
E G Vlakh, E V Grachova, D D Zhukovsky, A V Hubina, A S Mikhailova, J R Shakirova, V V Sharoyko, S P Tunik, T B Tennikova
The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain...
February 3, 2017: Scientific Reports
Andreas Müller, Martin Neukam, Anna Ivanova, Anke Sönmez, Carla Münster, Susanne Kretschmar, Yannis Kalaidzidis, Thomas Kurth, Jean-Marc Verbavatz, Michele Solimena
Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections...
December 2017: Scientific Reports
Jonas Barandun, Fred F Damberger, Cyrille L Delley, Juerg Laederach, Frédéric H T Allain, Eilika Weber-Ban
BACKGROUND: The post-translational modification pathway referred to as pupylation marks proteins for proteasomal degradation in Mycobacterium tuberculosis and other actinobacteria by covalently attaching the small protein Pup (prokaryotic ubiquitin-like protein) to target lysine residues. In contrast to the functionally analogous eukaryotic ubiquitin, Pup is intrinsically disordered in its free form. Its unfolded state allows Pup to adopt different structures upon interaction with different binding partners like the Pup ligase PafA and the proteasomal ATPase Mpa...
February 1, 2017: BMC Structural Biology
Ahmet Aykaç, Magali Noiray, Milo Malanga, Valentina Agostoni, Juan Manuel Casas-Solvas, Éva Fenyvesi, Ruxandra Gref, Antonio Vargas-Berenguel
BACKGROUND: Metal-organic framework nanoparticles (nanoMOFs) are biodegradable highly porous materials with a remarkable ability to load therapeutic agents with a wide range of physico-chemical properties. Engineering the nanoMOFs surface may provide nanoparticles with higher stability, controlled release, and targeting abilities. Designing postsynthetic, non-covalent self-assembling shells for nanoMOFs is especially appealing due to their simplicity, versatility, absence of toxic byproducts and minimum impact on the original host-guest ability...
January 27, 2017: Biochimica et Biophysica Acta
J Wang, Y K Wong, J Zhang, Y-M Lee, Z-C Hua, H-M Shen, Q Lin
Identifying the cellular binding targets of drugs and other bioactive small molecules is a crucial step for understanding their molecular mechanisms of action as well as potential off-target effects. The field of chemical proteomics is an emerging discipline in chemical biology using synthetic chemistry and high-throughput detection techniques to study small molecule-protein interactions. In this chapter, we describe a quantitative chemical proteomics protocol combining bioorthogonal click chemistry and quantitation by isobaric tags for relative and absolute quantification (iTRAQ) to identify the specific binding targets of drugs and bioactive small molecules such as natural products...
2017: Methods in Enzymology
Jingyun Wang, Lei Zhang, Youju Huang, Anirban Dandapat, Liwei Dai, Ganggang Zhang, Xuefei Lu, Jiawei Zhang, Weihua Lai, Tao Chen
The probe materials play a significant role in improving the detection efficiency and sensitivity of lateral-flow immunochromatographic test strip (ICTS). Unlike conventional ICTS assay usually uses single-component, solid gold nanoparticles as labeled probes, in our present study, a bimetallic, hollow Au-Ag nanoparticles (NPs) labeled ICTS was successfully developed for the detection of clenbuterol (CLE). The hollow Au-Ag NPs with different Au/Ag mole ratio and tunable size were synthesized by varying the volume ratio of [HAuCl4]:[Ag NPs] via the galvanic replacement reaction...
January 30, 2017: Scientific Reports
Thu V Vuong, Bing Liu, Mats Sandgren, Emma R Master
Most existing methods for screening the activity of lytic polysaccharide mono-oxygenases (LPMOs) on polysaccharides are based on the detection of soluble oxidized sugars. This approach might underestimate the total performance of LPMOs since oxidation events that do not lead to oligosaccharide release are not detected. Using PcLPMO9D as a model enzyme, a microplate-based method has been developed to detect C1-oxidizing LPMO activity by covalently linking a water-soluble fluorophore to oxidized positions within the cellulose fiber...
January 26, 2017: Biomacromolecules
Markita Patricia Landry, Hiroki Ando, Allen Y Chen, Jicong Cao, Vishal Isaac Kottadiel, Linda Chio, Darwin Yang, Juyao Dong, Timothy K Lu, Michael S Strano
A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response...
January 23, 2017: Nature Nanotechnology
H R Sousa, R S Gaspar, E M L Sena, S A da Silva, J L de L Fontelles, T L S Araújo, M Mastrogiovanni, D M Fries, A P S Azevedo-Santos, F R M Laurindo, A Trostchansky, A M Paes
Protein disulfide isomerase (PDI) plays a major role in platelet aggregation and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized this peptide would also inhibit platelet aggregation by association to surface PDI. Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine...
January 21, 2017: Journal of Thrombosis and Haemostasis: JTH
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