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Covalently cross linked

Nadia M Schoonhoven, Derek K O'Flaherty, Francis P McManus, Lauralicia Sacre, Anne M Noronha, M Judith Kornblatt, Christopher J Wilds
O⁶-Alkylguanine-DNA alkyltransferases (AGTs) are proteins responsible for the removal of mutagenic alkyl adducts at the O⁶-atom of guanine and O⁴-atom of thymine. In the current study we set out to understand the role of the Ser134 residue in the Escherichia coli AGT variant OGT on substrate discrimination. The S134P mutation in OGT increased the ability of the protein to repair both O⁶-adducts of guanine and O⁴-adducts of thymine. However, the S134P variant was unable, like wild-type OGT, to repair an interstrand cross-link (ICL) bridging two O⁶-atoms of guanine in a DNA duplex...
November 11, 2017: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Mari Takahara, Rie Wakabayashi, Kosuke Minamihata, Masahiro Goto, Noriho Kamiya
DNA-protein conjugates are promising biomolecules for use in areas ranging from therapeutics to analysis because of the dual functionalities of DNA and protein. Conjugation requires site-specific and efficient covalent bond formation without impairing the activity of both biomolecules. Herein, we have focused on the use of a microbial transglutaminase (MTG) that catalyzes the cross-linking reaction between a glutamine residue and a primary amine. In a model bioconjugation, a highly MTG-reactive Gln (Q)-donor peptide (FYPL<u>Q</u>MRG, FQ) was fused to enhanced green fluorescent protein (FQ-EGFP) and a primary amine-clustered DNA aptamer was enzymatically synthesized as a novel acyl-acceptor substrate of MTG, whose combination leads to efficient and convenient preparation of DNA-protein conjugates with high purity...
November 13, 2017: Bioconjugate Chemistry
Christopher R Sibley
RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. In doing so they help direct many essential roles in cellular physiology, while their perturbed activity can contribute to disease etiology. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. iCLIP achieves this by exploiting UV-induced covalent cross-links formed between RBPs and their target RNAs to both purify the RBP-RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct cross-link sites in the high throughput sequenced cDNA libraries...
2018: Methods in Molecular Biology
Raffaele Pugliese, Federico Fontana, Amanda Marchini, Fabrizio Gelain
Self-assembling peptides (SAP) have drawn an increasing interest in the tissue engineering community. They display unquestionable biomimetic properties, tailorability and promising biocompatibility. However their use has been hampered by poor mechanical properties making them fragile soft scaffolds. To increase SAP hydrogel stiffness we introduced a novel strategy based on multiple ramifications of (LDLK)3, a well-known linear SAP, connected with one or multiple "lysine knots". Differently branched SAPs were tested by increasing the number of (LDLK)3-like branches and by adding the neuro-regenerative functional motif BMHP1 as a single branch...
November 8, 2017: Acta Biomaterialia
Daniel F A R Dourado, Marcel Swart, Alexandra Teresa Pires Carvalho
A covalently bound flavin cofactor is predominant in the succinate:ubiquinone oxidoreductase (SQR, Complex II), an essential component of the aerobic electron transport, and in the menaquinol:fumarate oxidoreductase (QFR), the anaerobic counterpart, albeit being only present in ~10% of the known flavoenzymes. Here, we investigated the role of this 8α-N(3)-histidyl linkage between the flavin dinucleotide (FAD) cofactor and the respiratory Complex II. After parameterization with DFT we performed classical molecular dynamics simulations and quantum mechanics calculations of Complex II:FAD and Complex II:FADH2, covalently bound and unbound to His-A57...
November 10, 2017: Chemistry: a European Journal
William T Andrews, Susan B Skube, Amanda B Hummon
MALDI-TOF imaging mass spectrometry (IMS) is a common technique used for analyzing tissue samples, as it allows the user to detect multiple different analytes simultaneously. However, the detection and analysis of these analytes may sometimes be hampered due to the presence of contaminants in the tissue microenvironment, which leads to ion suppression. This challenge necessitates the development of an active extraction technique to selectively isolate analytes of interest without compromising their spatial localization within a tissue sample...
November 9, 2017: Analyst
Alisa Gricajeva, Simas Kazlauskas, Lilija Kalėdienė, Vida Bendikienė
Nowadays, for the industrial implementations, especially in the area of organic synthesis, immobilized enzymes are preferred over their soluble forms. Present study aimed to find fast, cost-efficient, and effective way of lipase immobilization for the use in organic media. Lipase from Aspergillus sp. (Resinase A 2X) was immobilized utilizing cross-linking of enzyme aggregates, covalent immobilization on magnetite particles and adsorption-immobilization using pyrolyzed sugar industry waste product as a novel type of carrier...
November 4, 2017: International Journal of Biological Macromolecules
Alfredo Castello, Christian K Frese, Bernd Fischer, Aino I Järvelin, Rastislav Horos, Anne-Marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W Hentze
This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection...
December 2017: Nature Protocols
Qiaohong Liu, Priyadarshini Singha, Hitesh Handa, Jason Locklin
Medical device coatings that resist protein adhesion and bacterial contamination are highly desirable in the healthcare industry. In this work, an antifouling zwitterionic terpolymer, 2-methacryloyloxyethyl phosphorylcholine-co-butyl methacrylate-co-benzophenone (BPMPC), is covalently grafted to a nitric oxide (NO) releasing antimicrobial biomedical grade copolymer of silicone-polycarbonate-urethane, CarboSil, to significantly enhance the biocompatibility, nonspecific protein repulsion and infection-resistant properties...
October 30, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
Carla Schmidt, Henning Urlaub
Determining the structures of, and gaining insight into, the function of large protein complexes at the molecular or atomic level has become a key part of modern structural biology. Electron cryo-microscopy (cryo-EM) can solve structures of highly dynamic macromolecular complexes that are not feasible with other structural techniques like X-ray of crystallized proteins (protein complexes) or nuclear magnetic resonance (NMR) spectroscopy of proteins (protein complexes) in solution. To resolve the regions that are less well defined in cryo-EM images, cross-linking coupled with mass spectrometry (CX-MS) provides valuable information on the proximity between amino-acid residues as distance constraints for homology or de novo modelling...
October 2017: Current Opinion in Structural Biology
Paula Nicolás, Verónica Lassalle, María Luján Ferreira
Magnetic biocatalysts offer enormous advantages over traditional ones. Their ability to be isolated by means of a magnet, in combination with their extensive reuse possibilities, makes them highly attractive and competitive from the commercial point of view. In this work, magnetic biocatalysts were prepared by immobilization of Candida antarctica Lipase B (E.C., CALB) on magnetite-lysine nanoparticles. Two methodologies were explored tending to find the optimal biocatalyst in terms of its practical implementation: I-physical adsorption of CALB followed by cross-linking, and II-covalent coupling of the lipase on the nanoparticles surface...
October 24, 2017: Bioprocess and Biosystems Engineering
Preeti Verma, Bibekananda Kar, Ritu Varshney, Partha Roy, Ashwani K Sharma
The 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) catalyzes final two steps of purine nucleotide de novo biosynthetic pathway. The present study reports the characterization of ATIC from Staphylococcus lugdunensis (SlugATIC). Apart from kinetic analysis and a detailed biophysical characterization of SlugATIC, the role of ATIC in cell proliferation has been demonstrated for the first time. The purified recombinant SlugATIC and its truncated domains exists mainly in dimeric form was revealed in gel filtration and glutaraldehyde cross linking studies...
October 24, 2017: FEBS Journal
Laetitia Sütterlin, Zainab Edoo, Jean-Emmanuel Hugonnet, Jean-Luc Mainardi, Michel Arthur
In most bacteria, the essential targets of β-lactam antibiotics are the D,D-transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4→3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3→3 cross-links generated by L,D-transpeptidases. To gain insight into the characteristics of C. difficile peptidoglycan cross-linking enzymes, we have purified the three putative C. difficile L,D-transpeptidases paralogues, LdtCd1, LdtCd2, and LdtCd3, which have been previously identified by sequence analysis...
October 23, 2017: Antimicrobial Agents and Chemotherapy
X Callies, C Véchambre, C Fonteneau, F Herbst, J-M Chenal, S Pensec, L Chazeau, W H Binder, L Bouteiller, C Creton
We investigate the nanostructure, the rheology and the adhesion of soft supramolecular materials elaborated by blending monofunctional and multifunctional poly(isobutene) (PIB) chains. Monofunctional PIB chains (PIBUT) are linear and unentangled polymer chains (Mn ≈ 3 kg mol(-1)) functionalized in the middle by a bis-urea interacting moiety, able to self-associate by four hydrogen bonds. Covalent coupling of monofunctional PIB allows us to synthesize longer chains bearing two or three interacting moieties...
November 8, 2017: Soft Matter
Ke Liu, Shuguang Chen, Hongbing Chen, Ping Tong, Jinyan Gao
Ovalbumin (OVA) is described as one of the major allergens in hen's egg, and it is the most abundant protein in egg white. Enzyme-mediated covalent cross-linking of food proteins, can influence their structure and allergenicity. The aim of this study was to investigate the potential of polyphenol oxidase from Agaricus bisporus to cross-link OVA (CL-OVA) in the presence or absence of caffeic acid, followed by characterizing the structure and allergenicity of CL-OVA. A single-factor experiment was designed to assess the optimum conditions for cross-linking of OVA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis...
October 14, 2017: International Journal of Biological Macromolecules
Ekaterina D Sormacheva, Peter S Sherin, Yuri P Tsentalovich
Photoinduced generation of radicals in the eye lens may play an important role in the modification of proteins leading to their coloration, aggregation, and insolubilization. The radicals can be formed via the reactions of photoexcited endogenous chromophores of the human lens with lens proteins, in particular with tryptophan residues. In the present work we studied the reactions induced by UV-A (315-400nm) light between kynurenic acid (KNA), an effective photosensitizer present in the human lens, and N-acetyl-L-tryptophan (NTrpH) under aerobic and anaerobic conditions...
October 9, 2017: Free Radical Biology & Medicine
Melinda Hauser, Chen Qian, Steven T King, Sarah Kauffman, Fred Naider, Robert L Hettich, Jeffrey M Becker
We are developing a rapid, time-resolved method using laser-activated cross-linking to capture protein-peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding-yeast mating pheromone (α-factor) and the decapeptide human gonadotropin-releasing hormone (GnRH). Cross-linking of α-factor, using a biotinylated, photoactivatable p-benzoyl-L-phenylalanine (Bpa)-modified analog, was energy-dependent and achieved within seconds of laser irradiation...
October 10, 2017: Journal of Molecular Recognition: JMR
Codjo Hountondji, Jean-Bernard Créchet, Jean-Pierre Le Caër, Véronique Lancelot, Jean A H Cognet, Soria Baouz
In this report, we have used periodate-oxidized tRNA (tRNAox) as an affinity laleling reagent to demonstrate that: (i) the bL12 protein contacts the CCA-arm of P-site bound tRNA on the E. coli 70S ribosomes; (ii) the stoichiometry of labeling is one molecule of tRNAox bound to one polypeptide chain of endogenous bL12; (iii) cross-linking in situ of bL12 with tRNAox on the ribosomes provokes the loss of activity; (iv) intact tRNA protects bL12 in the 70S ribosomes against cross-linking with tRNAox; (v) both tRNAox and pyridoxal 5'-phosphate (PLP) compete for the same or for proximal cross-linking site(s) on bL12 inside the ribosome; (vi) the stoichiometry of cross-linking of PLP to the recombinant E...
August 16, 2017: Journal of Biochemistry
Dibyangana Parbat, Sana Gaffar, Adil Majeed Rather, Aditi Gupta, Uttam Manna
The controlled modulation of both oil (under water) and water (in air) wettability is an emerging approach to develop several functional materials for various prospective applications including oil/water separation, anti-corrosive coatings, underwater robotics, protein crystallization, drug delivery, open microfluidics, water harvesting etc. Here, we report a 'reactive' and covalently cross-linked coating through a facile and robust Michael addition reaction, which is suitable for the controlled and extreme regulation of both water and oil wettability in air and under water respectively...
September 1, 2017: Chemical Science
Prosper Kanyong, Francis D Krampa, Yaw Aniweh, Gordon A Awandare
This review (with 35 references) summarizes the various strategies used in biosensors for galactose, and their analytical performance. A brief comparison of the enzyme immobilization methods employed and the analytical performance characteristics of a range of galactose biosensors are first summarized in tabular form and then described in detail. Selected examples have been included to demonstrate the various applications of these biosensors to real samples. Following an introduction into the field that covers the significance of sensing galactose in various fields, the review covers biosensors based on the use of galactose oxidase, with a discussion of methods for their immobilization (via cross-linking, adsorption, covalent bonding and entrapment)...
2017: Mikrochimica Acta
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