A covalent modification for graphene by adamantane groups through two-step chlorination-Grignard reactions

NASA Astrophysics Data System (ADS)

Sun, Xuzhuo; Li, Bo; Lu, Mingxia

2017-07-01

Chemical modification of graphene is a promising approach to manipulate its properties for its end applications. Herein we designed a two-step route through chlorination-Grignard reactions to covalently decorate the surface of graphene with adamantane groups. The chemically modified graphene was characterized by Raman spectroscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Chlorination of graphene occurred rapidly, and the substitution of chlorine atoms on chlorinated graphene by adamantane Grignard reagent afforded adamantane graphene in almost quantitative yield. Adamantane groups were found to be covalently bonded to the graphene carbons. The present two-step procedure may provide an effective and facile route for graphene modification with varieties of organic functional groups.

Non-covalent attachment of silver nanoclusters onto single-walled carbon nanotubes with human serum albumin as linking molecule

NASA Astrophysics Data System (ADS)

Rodríguez-Galván, Andrés; Heredia, Alejandro; Amelines-Sarria, Oscar; Rivera, Margarita; Medina, Luis A.; Basiuk, Vladimir A.

2015-03-01

The attachment of silver nanoclusters (AgNCs) onto single-walled carbon nanotubes (SWNTs) for the formation of integrated fluorescence sites has attracted much attention due their potential applications as biological probes and nanovectors in theragnosis. Here, we report the preparation through assembly of fluorescent quasi 1-D nanomaterial based on SWNTs and silver nanoclusters (AgNCs) non-covalently attached to human serum albumin as biological linker. The fluorescent SWNT-AgNCs-HSA conjugates were characterized by atomic force microscopy, high-resolution transmission electron microscopy (HRTEM), high angle annular dark field scanning TEM (HAADF-STEM), fluorescent and UV-vis spectroscopy. The above techniques confirmed that AgNCs were non-covalently attached onto the external surface of SWNTs. In addition, it was observed that the modification did not affect the optical properties of the synthesized AgNCs since the absorption spectra and fluorescence under UV irradiation (λ = 365 nm) remain the same. The effect of the functionalized systems was tested on mammal red blood cells (RBCs) and it was found that their structural integrity was compromised by the conjugates, limiting their biological and medical applications.

Derivatization of single-walled carbon nanotubes with redox mediator for biocatalytic oxygen electrodes.

PubMed

Sadowska, K; Stolarczyk, K; Biernat, J F; Roberts, K P; Rogalski, J; Bilewicz, R

2010-11-01

Single-walled carbon nanotubes (SWCNTs) were covalently modified with a redox mediator derived from 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), and implemented in the construction of electrodes for biocatalytic oxygen reduction. The procedure is based on: covalent bonding of mediator to nanotubes, placing the nanotubes directly on the carbon electrode surface and covering the nanostructured electrode with a Nafion film containing laccase as the biocatalyst. The modified electrode is stable and the problem of mediator (ABTS) leaking from the film is eliminated by binding it covalently to the nanotubes. Three different synthetic approaches were used to obtain ABTS-modified carbon nanotubes. Nanotubes were modified at ends/defect sites or on the nanotube sidewalls and characterized by Raman spectroscopy, TGA and electrochemistry. The accessibility of differently located ABTS units by the laccase active center and mediation of electron transfer were studied by cyclic voltammetry. The surface concentrations of ABTS groups electrically connected with the electrode were compared for each of the electrodes based on the charges of the voltammetric peaks recorded in the deaerated solution. The nanotube modification procedure giving the best parameters of the catalytic process was selected. Copyright © 2010 Elsevier B.V. All rights reserved.

Atomic Covalent Functionalization of Graphene

PubMed Central

Johns, James E.; Hersam, Mark C.

2012-01-01

Conspectus Although graphene’s physical structure is a single atom thick, two-dimensional, hexagonal crystal of sp2 bonded carbon, this simple description belies the myriad interesting and complex physical properties attributed to this fascinating material. Because of its unusual electronic structure and superlative properties, graphene serves as a leading candidate for many next generation technologies including high frequency electronics, broadband photodetectors, biological and gas sensors, and transparent conductive coatings. Despite this promise, researchers could apply graphene more routinely in real-world technologies if they could chemically adjust graphene’s electronic properties. For example, the covalent modification of graphene to create a band gap comparable to silicon (~1 eV) would enable its use in digital electronics, and larger band gaps would provide new opportunities for graphene-based photonics. Towards this end, researchers have focused considerable effort on the chemical functionalization of graphene. Due to its high thermodynamic stability and chemical inertness, new methods and techniques are required to create covalent bonds without promoting undesirable side reactions or irreversible damage to the underlying carbon lattice. In this Account, we review and discuss recent theoretical and experimental work studying covalent modifications to graphene using gas phase atomic radicals. Atomic radicals have sufficient energy to overcome the kinetic and thermodynamic barriers associated with covalent reactions on the basal plane of graphene but lack the energy required to break the C-C sigma bonds that would destroy the carbon lattice. Furthermore, because they are atomic species, radicals substantially reduce the likelihood of unwanted side reactions that confound other covalent chemistries. Overall, these methods based on atomic radicals show promise for the homogeneous functionalization of graphene and the production of new classes of two-dimensional materials with fundamentally different electronic and physical properties. Specifically, we focus on recent studies of the addition of atomic hydrogen, fluorine, and oxygen to the basal plane of graphene. In each of these reactions a high energy, activating step initiates the process, breaking the local π structure and distorting the surrounding lattice. Scanning tunneling microscopy experiments reveal that substrate mediated interactions often dominate when the initial binding event occurs. We then compare these substrate effects with the results of theoretical studies that typically assume a vacuum environment. As the surface coverage increases, clusters often form around the initial distortion, and the stoichiometric composition of the saturated end product depends strongly on both the substrate and reactant species. In addition to these chemical and structural observations, we review how covalent modification can extend the range of physical properties that are achievable in two-dimensional materials. PMID:23030800

Monolayer of Hydrazine Facilitates the Direct Covalent Attachment of C60 Fullerene to a Silicon Surface.

PubMed

Gao, Fei; Teplyakov, Andrew V

2017-09-05

The development of oxygen-free organic-inorganic interfaces has led to new schemes for the functionalization of silicon surfaces with nitrogen-based chemical groups. However, building layers of large structures directly on this functionalized surface has remained elusive. This work confirms the path to form a stable interface between silicon and buckminsterfullerene C 60 based on covalent chemical bonds. The starting point for this modification is the hydrazine-reacted Si(111) surface with the diamine functionality, which is further reacted directly with the C 60 molecules. The chemistry of this process is confirmed spectroscopically and microscopically and can be used to form organic-inorganic interfaces separated by a single layer of nitrogen.

Post‐Graphene 2D Chemistry: The Emerging Field of Molybdenum Disulfide and Black Phosphorus Functionalization

PubMed Central

Hauke, Frank

2018-01-01

Abstract The current state of the chemical functionalization of three types of single sheet 2D materials, namely, graphene, molybdenum disulfide (MoS2), and black phosphorus (BP) is summarized. Such 2D sheet polymers are currently an emerging field at the interface of synthetic chemistry, physics, and materials science. Both covalent and non‐covalent functionalization of sheet architectures allows a systematic modification of their properties, that is, an improvement of solubility and processability, the prevention of re‐aggregation, or band‐gap tuning. Next to successful functionalization concepts, fundamental challenges are also addressed. These include the insolubility and polydispersity of most 2D sheet polymers, the development of suitable characterization tools, the identification of effective binding strategies, the chemical activation of the usually rather unreactive basal planes for covalent addend binding, and the regioselectivity of plane addition reactions. Although a number of these questions remain elusive in this Review, the first promising concepts to overcome such hurdles are presented. PMID:29024321

Modeling covalent-modifier drugs.

PubMed

Awoonor-Williams, Ernest; Walsh, Andrew G; Rowley, Christopher N

2017-11-01

In this review, we present a summary of how computer modeling has been used in the development of covalent-modifier drugs. Covalent-modifier drugs bind by forming a chemical bond with their target. This covalent binding can improve the selectivity of the drug for a target with complementary reactivity and result in increased binding affinities due to the strength of the covalent bond formed. In some cases, this results in irreversible inhibition of the target, but some targeted covalent inhibitor (TCI) drugs bind covalently but reversibly. Computer modeling is widely used in drug discovery, but different computational methods must be used to model covalent modifiers because of the chemical bonds formed. Structural and bioinformatic analysis has identified sites of modification that could yield selectivity for a chosen target. Docking methods, which are used to rank binding poses of large sets of inhibitors, have been augmented to support the formation of protein-ligand bonds and are now capable of predicting the binding pose of covalent modifiers accurately. The pK a 's of amino acids can be calculated in order to assess their reactivity towards electrophiles. QM/MM methods have been used to model the reaction mechanisms of covalent modification. The continued development of these tools will allow computation to aid in the development of new covalent-modifier drugs. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

PubMed

Punthasee, Puminan; Laciak, Adrian R; Cummings, Andrea H; Ruddraraju, Kasi Viswanatharaju; Lewis, Sarah M; Hillebrand, Roman; Singh, Harkewal; Tanner, John J; Gates, Kent S

2017-04-11

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10 2 M -1 min -1 . Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Effects of covalent modification by 4-hydroxy-2-nonenal on the noncovalent oligomerization of ubiquitin.

PubMed

Grasso, Giuseppe; Axelsen, Paul H

2017-01-01

When lipid membranes containing ω-6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4-hydroxy-2-nonenal (HNE)-a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non-covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

PubMed Central

Mendoza, Vanessa Leah; Vachet, Richard W.

2009-01-01

For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

Mechanism-based strategies for protein thermostabilization.

PubMed

Mozhaev, V V

1993-03-01

Strategies for stabilizing enzymes can be derived from a two-step model of irreversible inactivation that involves preliminary reversible unfolding, followed by an irreversible step. Reversible unfolding is best prevented by covalent immobilization, whereas methods such as covalent modification of amino acid residues or 'medium engineering' (by the addition of low-molecular-weight compounds) are effective against irreversible 'incorrect' refolding. Genetic modification of the protein sequence is the most effective approach for preventing chemical deterioration.

Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry

PubMed Central

Souda, Puneet; Ryan, Christopher M.; Cramer, William A.; Whitelegge, Julian

2011-01-01

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein’s native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electroncapture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation. PMID:21982782

Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry.

PubMed

Souda, Puneet; Ryan, Christopher M; Cramer, William A; Whitelegge, Julian

2011-12-01

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electron-capture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation. Copyright © 2011 Elsevier Inc. All rights reserved.

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

PubMed Central

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications. PMID:25403473

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

PubMed

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

RNA-modifying proteins as anticancer drug targets.

PubMed

Boriack-Sjodin, P Ann; Ribich, Scott; Copeland, Robert A

2018-06-01

All major biological macromolecules (DNA, RNA, proteins and lipids) undergo enzyme-catalysed covalent modifications that impact their structure, function and stability. A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA stability and translation to proteins; these mechanisms of translational control have been termed epitranscriptomics. Emerging data suggest that some epitranscriptomic mechanisms are altered in human cancers as well as other human diseases. In this Review, we examine the current understanding of RNA modifications with a focus on mRNA methylation, highlight their possible roles in specific cancer indications and discuss the emerging potential of RNA-modifying proteins as therapeutic targets.

Probing the Intermediacy of Covalent RNA Enzyme Complexes in RNA Modification Enzymes

PubMed Central

Chervin, Stephanie M.; Kittendorf, Jeffrey D.; Garcia, George A.

2009-01-01

Within the large and diverse group of RNA-modifying enzymes, a number of enzymes seem to form stable covalent linkages to their respective RNA substrates. A complete understanding of the chemical and kinetic mechanisms of these enzymes, some of which have identified pathological roles, is lacking. As part of our ongoing work studying the posttranscriptional modification of tRNA with queuine, we wish to understand fully the chemical and kinetic mechanisms involved in this key transglycosylation reaction. In our previous investigations, we have used a gel mobility-shift assay to characterize an apparent covalent enzyme-RNA intermediate believed to be operative in the catalytic pathway. However, the simple observation of a covalent complex is not sufficient to prove intermediacy. To be a true intermediate, the complex must be both chemically and kinetically competent. As a case study for the proof of intermediacy, we report the use of this gel-shift assay under mildly denaturing conditions to probe the kinetic competency of the covalent association between RNA and the tRNA modifying enzyme tRNA-guanine transglycosylase (TGT). PMID:17673081

Irreversible covalent modification of type I dehydroquinase with a stable Schiff base.

PubMed

Tizón, Lorena; Maneiro, María; Peón, Antonio; Otero, José M; Lence, Emilio; Poza, Sergio; van Raaij, Mark J; Thompson, Paul; Hawkins, Alastair R; González-Bello, Concepción

2015-01-21

The irreversible inhibition of type I dehydroquinase (DHQ1), the third enzyme of the shikimic acid pathway, is investigated by structural, biochemical and computational studies. Two epoxides, which are mimetics of the natural substrate, were designed as irreversible inhibitors of the DHQ1 enzyme and to study the binding requirements of the linkage to the enzyme. The epoxide with the S configuration caused the covalent modification of the protein whereas no reaction was obtained with its epimer. The first crystal structure of DHQ1 from Salmonella typhi covalently modified by the S epoxide, which is reported at 1.4 Å, revealed that the modified ligand is surprisingly covalently attached to the essential Lys170 by the formation of a stable Schiff base. The experimental and molecular dynamics simulation studies reported here highlight the huge importance of the conformation of the C3 carbon of the ligand for covalent linkage to this type of aldolase I enzyme, revealed the key role played by the essential His143 as a Lewis acid in this process and show the need for a neatly closed active site for catalysis.

NPPB structure-specifically activates TRPA1 channels.

PubMed

Liu, Kun; Samuel, Manoj; Ho, Melisa; Harrison, Richard K; Paslay, Jeff W

2010-07-01

TRPA1 channels have been found to play an important role in mammalian pain sensation, especially when the pain is caused by chemicals on site of inflammation. A large number of structurally diverse chemicals are found to activate TRPA1 channels, implicating a potential chemosensor in neuronal nociception. Identification of the channel activation by cysteine modification through covalent chemical reaction provides arguments for the diversity of the agonist structures. However, it is largely unknown how nonreactive compounds activate TRPA1 channels. Here, we report that NPPB, a classic Cl(-) channel blocker, potently activated human TRPA1 channels overexpressed in mammalian HEK-293 cells. This effect was confirmed in Ca(2+) imaging assay, patch clamp whole cell and single channel recordings. The NPPB response was quick, fully reversible and replicable, contrary to the effect of covalent modification by AITC. The mutagenesis studies revealed a refreshed look at several mutations known to be critical for the actions of AITC and menthol. The blocking profile of NPPB on these mutants showed that the NPPB activation was similar to that of FTS and different from AITC and menthol. The results indicated a possible close interaction between S5 and N-terminal domains of the channel. We also tested a group of NPPB analogs on TRPA1 channel activities. The results demonstrated that NPPB activation was tightly associated with chemical structure. None of the single chemical group was sufficient to activate the channel, indicating that NPPB activated TRPA1 through a structure-specific mechanism. (c) 2010 Elsevier Inc. All rights reserved.

A study of the reactivity of S(VI)-F containing warheads with nucleophilic amino-acid side chains under physiological conditions.

PubMed

Mukherjee, H; Debreczeni, J; Breed, J; Tentarelli, S; Aquila, B; Dowling, J E; Whitty, A; Grimster, N P

2017-11-22

Sulfonyl fluorides (SFs) have recently emerged as a promising warhead for the targeted covalent modification of proteins. Despite numerous examples of the successful deployment of SFs as covalent probe compounds, a detailed exploration of the factors influencing the stability and reactivity of SFs has not yet appeared. In this work we present an extensive study on the influence of steric and electronic factors on the reactivity and stability of the SF and related S VI -F groups. While SFs react rapidly with N-acetylcysteine, the resulting adducts were found to be unstable, rendering SFs inappropriate for the durable covalent inhibition of cysteine residues. In contrast, SFs afforded stable adducts with both N-acetyltyrosine and N-acetyllysine; furthermore, we show that the reactivity of arylsulfonyl fluorides towards these nucleophilic amino acids can be predictably modulated by adjusting the electronic properties of the warhead. These trends were largely conserved when the covalent reaction occurred within a protein binding pocket. We have also obtained a crystal structure depicting covalent modification of the catalytic lysine of a tyrosine kinase (FGFR1) by the ATP analog 5'-O-3-((fluorosulfonyl)benzoyl)adenosine (m-FSBA). Highly reactive warheads were demonstrated to be unstable with respect to hydrolysis in buffered aqueous solutions, indicating that warhead reactivity must be carefully tuned to provide optimal rates of protein modification. Our results demonstrate that the reactivity of SFs complements that of more commonly studied acrylamides, and we hope that this work spurs the rational design of novel SF-containing covalent probe compounds and inhibitors, particularly in cases where a suitably positioned cysteine residue is not present.

Efficient Site-Specific Labeling of Proteins via Cysteines

PubMed Central

Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

2011-01-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

Efficient site-specific labeling of proteins via cysteines.

PubMed

Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

2008-03-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

Electronegativity effects and single covalent bond lengths of molecules in the gas phase.

PubMed

Lang, Peter F; Smith, Barry C

2014-06-07

This paper discusses in detail the calculation of internuclear distances of heteronuclear single bond covalent molecules in the gaseous state. It reviews briefly the effect of electronegativity in covalent bond length. A set of single bond covalent radii and electronegativity values are proposed. Covalent bond lengths calculated by an adapted form of a simple expression (which calculated internuclear separation of different Group 1 and Group 2 crystalline salts to a remarkable degree of accuracy) show very good agreement with observed values. A small number of bond lengths with double bonds as well as bond lengths in the crystalline state are calculated using the same expression and when compared with observed values also give good agreement. This work shows that covalent radii are not additive and that radii in the crystalline state are different from those in the gaseous state. The results also show that electronegativity is a major influence on covalent bond lengths and the set of electronegativity scale and covalent radii proposed in this work can be used to calculate covalent bond lengths in different environments that have not yet been experimentally measured.

Rapid Covalent Modification of Silicon Oxide Surfaces through Microwave-Assisted Reactions with Alcohols.

PubMed

Lee, Austin W H; Gates, Byron D

2016-07-26

We demonstrate the method of a rapid covalent modification of silicon oxide surfaces with alcohol-containing compounds with assistance by microwave reactions. Alcohol-containing compounds are prevalent reagents in the laboratory, which are also relatively easy to handle because of their stability against exposure to atmospheric moisture. The condensation of these alcohols with the surfaces of silicon oxides is often hindered by slow reaction kinetics. Microwave radiation effectively accelerates this condensation reaction by heating the substrates and/or solvents. A variety of substrates were modified in this demonstration, such as silicon oxide films of various thicknesses, glass substrates such as microscope slides (soda lime), and quartz. The monolayers prepared through this strategy demonstrated the successful formation of covalent surface modifications of silicon oxides with water contact angles of up to 110° and typical hysteresis values of 2° or less. An evaluation of the hydrolytic stability of these monolayers demonstrated their excellent stability under acidic conditions. The techniques introduced in this article were successfully applied to tune the surface chemistry of silicon oxides to achieve hydrophobic, oleophobic, and/or charged surfaces.

Solid-state mAbs and ADCs subjected to heat-stress stability conditions can be covalently modified with buffer and excipient molecules.

PubMed

Valliere-Douglass, John F; Lewis, Patsy; Salas-Solano, Oscar; Jiang, Shan

2015-02-01

We report that a unique type of chemical modification occurs on lyophilized proteins. Freeze-dried mAbs and antibody-drug conjugates (ADCs) can be covalently modified with buffer and excipient molecules on the side chains of Glu, Asp, Thr, and Ser amino acids when subjected to temperature stress. The reaction occurs primarily via condensation of common buffers and excipients such as histidine, tris, trehalose and sucrose, with Glu and Asp carboxylates in the primary sequence of proteins. The reaction was also found to proceed through condensation of carboxylate containing buffers such as citrate, with Thr and Ser hydroxyls in the primary sequence of proteins. Based on the mass of the covalent adducts observed on mAbs and ADCs, it is apparent that the reaction produces water as a product and is thus favored in a low moisture environments such as a lyophilized protein cake. Herein, we present the evidence for the covalent modification of proteins drawn from case studies of in-depth characterization of heat-stressed mAbs and ADCs in the solid state. We also demonstrate how common charge variant assays such as imaged capillary isoelectric focusing and mass spectrometry can be used to monitor this specific class of protein modification. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Epigenetics in myeloid derived suppressor cells: a sheathed sword towards cancer

PubMed Central

Zhang, Chao; Wang, Shuo; Liu, Yufeng; Yang, Cheng

2016-01-01

Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells composed of progenitors and precursors to myeloid cells, are deemed to participate in the development of tumor-favoring immunosuppressive microenvironment. Thus, the regulatory strategies targeting MDSCs' expansion, differentiation, accumulation and function could possibly be effective “weapons” in anti-tumor immunotherapies. Epigenetic mechanisms, which involve DNA modification, covalent histone modification and RNA interference, result in the heritable down-regulation or silencing of gene expression without a change in DNA sequences. Epigenetic modification of MDSC's functional plasticity leads to the remodeling of its characteristics, therefore reframing the microenvironment towards countering tumor growth and metastasis. This review summarized the pertinent findings on the DNA methylation, covalent histone modification, microRNAs and small interfering RNAs targeting MDSC in cancer genesis, progression and metastasis. The potentials as well as possible obstacles in translating into anti-cancer therapeutics were also discussed. PMID:27458169

"siRNA traffic lights": arabino-configured 2'-anchors for fluorescent dyes are key for dual color readout in cell imaging.

PubMed

Steinmeyer, Jeannine; Walter, Heidi-Kristin; Bichelberger, Mathilde A; Schneider, Violetta; Kubař, Tomáš; Rönicke, Franziska; Olshausen, Bettina; Nienhaus, Karin; Nienhaus, Gerd Ulrich; Schepers, Ute; Elstner, Marcus; Wagenknecht, Hans-Achim

2018-05-23

Two fluorescent dyes covalently attached in diagonal interstrand orientation to siRNA undergo energy transfer and thereby enable a dual color fluorescence readout (red/green) for hybridization. Three different structural variations were carried out and compared by their optical properties, including (i) the base surrogate approach with an acyclic linker as a substitute of the 2-deoxyriboside between the phosphodiester bridges, (ii) the 2'-modification of conventional ribofuranosides and (iii) the arabino-configured 2'-modification. The double stranded siRNA with the latter type of modification delivered the best energy transfer efficiency, which was explained by molecular dynamics simulations that showed that the two dyes are more flexible at the arabino-configured sugars compared to the completely stacked situation at the ribo-configured ones. Single molecule fluorescence lifetime measurements indicate their application in fluorescence cell imaging, which reveals a red/green fluorescence contrast in particular for the arabino-configured 2'-modification by the two dyes, which is key for tracking of siRNA transport into HeLa cells.

Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization.

PubMed

Cowan, Don A; Fernandez-Lafuente, Roberto

2011-09-10

The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process. Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications. Copyright © 2011 Elsevier Inc. All rights reserved.

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lovrinovic, Marina; Niemeyer, Christof M.

2005-09-30

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter weremore » ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.« less

Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.

PubMed

Olorunniji, Femi J; McPherson, Arlene L; Pavlou, Hania J; McIlwraith, Michael J; Brazier, John A; Cosstick, Richard; Stark, W Marshall

2015-07-13

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase-DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine

PubMed Central

Li, Bin; Schopfer, Lawrence M.; Grigoryan, Hasmik; Thompson, Charles M.; Hinrichs, Steven H.; Masson, Patrick; Lockridge, Oksana

2009-01-01

The expectation from the literature is that organophosphorus (OP) agents bind to proteins that have an active site serine. However, transferrin, a protein with no active site serine, was covalently modified in vitro by 0.5mM 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecanamide, chlorpyrifos oxon, diisopropylfluorophosphate, dichlorvos, sarin, and soman. The site of covalent attachment was identified by analyzing tryptic peptides in the mass spectrometer. Tyr 238 and Tyr 574 in human transferrin and Tyr 238, Tyr 319, Tyr 429, Tyr 491, and Tyr 518 in mouse transferrin were labeled by OP. Tyrosine in the small synthetic peptide ArgTyrThrArg made a covalent bond with diisopropylfluorophosphate, chlorpyrifos oxon, and dichlorvos at pH 8.3. These results, together with our previous demonstration that albumin and tubulin bind OP on tyrosine, lead to the conclusion that OP bind covalently to tyrosine, and that OP binding to tyrosine is a new OP-binding residue. The OP-reactive tyrosines are activated by interaction with Arg or Lys. It is suggested that many proteins in addition to those already identified may be modified by OP on tyrosine. The extent to which tyrosine modification by OP can occur in vivo and the toxicological implications of such modifications require further investigation. PMID:18930948

Supramolecular PEGylation of biopharmaceuticals

PubMed Central

Webber, Matthew J.; Vinciguerra, Brittany; Cortinas, Abel B.; Thapa, Lavanya S.; Jhunjhunwala, Siddharth; Isaacs, Lyle; Langer, Robert; Anderson, Daniel G.

2016-01-01

The covalent modification of therapeutic biomolecules has been broadly explored, leading to a number of clinically approved modified protein drugs. These modifications are typically intended to address challenges arising in biopharmaceutical practice by promoting improved stability and shelf life of therapeutic proteins in formulation, or modifying pharmacokinetics in the body. Toward these objectives, covalent modification with poly(ethylene glycol) (PEG) has been a common direction. Here, a platform approach to biopharmaceutical modification is described that relies on noncovalent, supramolecular host–guest interactions to endow proteins with prosthetic functionality. Specifically, a series of cucurbit[7]uril (CB[7])–PEG conjugates are shown to substantially increase the stability of three distinct protein drugs in formulation. Leveraging the known and high-affinity interaction between CB[7] and an N-terminal aromatic residue on one specific protein drug, insulin, further results in altering of its pharmacological properties in vivo by extending activity in a manner dependent on molecular weight of the attached PEG chain. Supramolecular modification of therapeutic proteins affords a noncovalent route to modify its properties, improving protein stability and activity as a formulation excipient. Furthermore, this offers a modular approach to append functionality to biopharmaceuticals by noncovalent modification with other molecules or polymers, for applications in formulation or therapy. PMID:27911829

Fabrication and functionalization of single asymmetric nanochannels for electrostatic/hydrophobic association of protein molecules

NASA Astrophysics Data System (ADS)

Ali, Mubarak; Bayer, Veronika; Schiedt, Birgitta; Neumann, Reinhard; Ensinger, Wolfgang

2008-12-01

We have developed a facile and reproducible method for surfactant-controlled track-etching and chemical functionalization of single asymmetric nanochannels in PET (polyethylene terephthalate) membranes. Carboxyl groups present on the channel surface were converted into pentafluorophenyl esters using EDC/PFP (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/pentafluorophenol) coupling chemistry. The resulting amine-reactive esters were further covalently coupled with ethylenediamine or propylamine in order to manipulate the charge polarity and hydrophilicity of the nanochannels, respectively. Characterization of the modified channels was done by measuring their current-voltage (I-V) curves as well as their permselectivity before and after the chemical modification. The electrostatic/hydrophobic association of bovine serum albumin on the channel surface was observed through the change in rectification behaviour upon the variation of pH values.

Nanoporous Anodic Alumina Surface Modification by Electrostatic, Covalent, and Immune Complexation Binding Investigated by Capillary Filling.

PubMed

Eckstein, Chris; Acosta, Laura K; Pol, Laura; Xifré-Pérez, Elisabet; Pallares, Josep; Ferré-Borrull, Josep; Marsal, Lluis F

2018-03-28

The fluid imbibition-coupled laser interferometry (FICLI) technique has been applied to detect and quantify surface changes and pore dimension variations in nanoporous anodic alumina (NAA) structures. FICLI is a noninvasive optical technique that permits the determination of the NAA average pore radius with high accuracy. In this work, the technique is applied after each step of different surface modification paths of the NAA pores: (i) electrostatic immobilization of bovine serum albumin (BSA), (ii) covalent attachment of streptavidin via (3-aminipropyl)-triethoxysilane and glutaraldehyde grafting, and (iii) immune complexation. Results show that BSA attachment can be detected as a reduction in estimated radius from FICLI with high accuracy and reproducibility. In the case of the covalent attachment of streptavidin, FICLI is able to recognize a multilayer formation of the silane and the protein. For immune complexation, the technique is able to detect different antibody-antigen bindings and distinguish different dynamics among different immune species.

Beyond cysteine: recent developments in the area of targeted covalent inhibition.

PubMed

Mukherjee, Herschel; Grimster, Neil P

2018-05-29

Over the past decade targeted covalent inhibitors have undergone a renaissance due to the clinical validation and regulatory approval of several small molecule therapeutics that are designed to irreversibly modify their target protein. Invariably, these compounds rely on the serendipitous placement of a cysteine residue proximal to the small molecule binding site; while this strategy has afforded numerous successes, it necessarily limits the number of proteins that can be targeted by this approach. This drawback has led several research groups to develop novel methodologies that target non-cysteine residues for covalent modification. Herein, we survey the current literature of warheads that covalently modify non-cysteine amino acids in proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

Chemical Modification of Papain and Subtilisin: An Active Site Comparison

ERIC Educational Resources Information Center

St-Vincent, Mireille; Dickman, Michael

2004-01-01

An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

The tomato UV-damaged DNA binding protein 1 (DDB1) plays a role in organ size control via an epigenetic manner

USDA-ARS?s Scientific Manuscript database

Epigenetic regulation, including various covalent modifications of histone proteins and methylation of cytosine bases in DNA, participates broadly in many fundamentally physiological and developmental processes. The repressed or active states of transcription resulted from epigenetic modifications a...

A New Pathway for Protein Haptenation by β-Lactams.

PubMed

Pérez-Ruíz, Raúl; Lence, Emilio; Andreu, Inmaculada; Limones-Herrero, Daniel; González-Bello, Concepción; Miranda, Miguel A; Jiménez, M Consuelo

2017-10-09

The covalent binding of β-lactams to proteins upon photochemical activation has been demonstrated by using an integrated approach that combines photochemical, proteomic and computational studies, selecting human serum albumin (HSA) as a target protein and ezetimibe (1) as a probe. The results have revealed a novel protein haptenation pathway for this family of drugs that is an alternative to the known nucleophilic ring opening of β-lactams by the free amino group of lysine residues. Thus, photochemical ring splitting of the β-lactam ring, following a formal retro-Staudinger reaction, gives a highly reactive ketene intermediate that is trapped by the neighbouring lysine residues, leading to an amide adduct. For the investigated 1/HSA system, covalent modification of residues Lys414 and Lys525, which are located in sub-domains IIIA and IIIB, respectively, occurs. The observed photobinding may constitute the key step in the sequence of events leading to photoallergy. Docking and molecular dynamics simulation studies provide an insight into the molecular basis of the selectivity of 1 for these HSA sub-domains and the covalent modification mechanism. Computational studies also reveal positive cooperative binding of sub-domain IIIB that explains the experimentally observed modification of Lys414, which is located in a barely accessible pocket (sub-domain IIIA). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discrimination Between Peptide O-Sulfo- and O-Phosphotyrosine Residues by Negative Ion Mode Electrospray Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Edelson-Averbukh, Marina; Shevchenko, Andrej; Pipkorn, Rüdiger; Lehmann, Wolf D.

2011-12-01

Unambiguous differentiation between isobaric sulfated and phosphorylated tyrosine residues (sTyr and pTyr) of proteins by mass spectrometry is challenging, even using high resolution mass spectrometers. Here we show that upon negative ion mode collision-induced dissociation (CID), pTyr- and sTyr-containing peptides exhibit entirely different modification-specific fragmentation patterns leading to a rapid discrimination between the isobaric covalent modifications using the tandem mass spectral data. This study reveals that the ratio between the relative abundances of [M-H-80]- and [M-H-98]- fragment ions in ion-trap CID and higher energy collision dissociation (HCD) spectra of singly deprotonated +80 Da Tyr-peptides can be used as a reliable indication of the Tyr modification group nature. For multiply deprotonated +80 Da Tyr-peptides, CID spectra of sTyr- and pTyr-containing sequences can be readily distinguished based on the presence/absence of the [M-nH-79](n-1)- and [M-nH-79-NL]( n-1)- ( n = 2, 3) fragment ions (NL = neutral loss).

The effect of covalently linked RGD peptide on the conformation of polysaccharides in aqueous solutions.

PubMed

Bernstein-Levi, Ortal; Ochbaum, Guy; Bitton, Ronit

2016-01-01

Covalently modified polysaccharides are routinely used in tissue engineering due to their tailored biofunctionality. Understanding the effect of single-chain level modification on the solution conformation of the single chain, and more importantly on the self-assembly and aggregation of the ensemble of chains is expected to improve our ability to control network topology and the properties of the resulting gels. Attaching an RGD peptide to a polysaccharide backbone is a common procedure used to promote cell adhesion in hydrogel scaffolds. Recently it has been shown that the spatial presentation of the RGD sequences affects the cell behavior; thus, understanding the effects of grafted RGD on the conformational properties of the solvated polysaccharide chains is a prerequisite for rational design of polysaccharide-peptide based biomaterials. Here we investigate the effect of covalently linked G4RGDS on the conformational state of the individual chain and chain assemblies of alginate, chitosan, and hyaluronic acid (HA) in aqueous solutions. Two peptide fractions were studied using small-angle X-ray scattering (SAXS) and rheology. In all cases, upon peptide conjugation structural differences were observed. Analysis of the scattering data shows evidence of clustering for a higher fraction of bound peptide. Moreover for all three polysaccharides the typical shear thinning behavior of the natural polysaccharide solutions is replaced by a Newtonian fluid behavior for the lower fraction conjugated peptide while a more pronounced shear thinning behavior is observed for the higher fraction. These results indicate that the fraction of the bounded peptide, determines the behavior of a polysaccharide-peptide conjugates in solution, regardless of the specific nature of the polysaccharide. Copyright © 2015 Elsevier B.V. All rights reserved.

Generation of novel covalent RNA-protein complexes in cells by ultraviolet B irradiation: implications for autoimmunity.

PubMed

Andrade, Felipe; Casciola-Rosen, Livia A; Rosen, Antony

2005-04-01

To determine whether ultraviolet B (UVB) irradiation induces novel modifications in autoantigens targeted during experimental photoinduced epidermal damage. To search for novel UVB-induced autoantigen modifications, lysates made from UVB-irradiated human keratinocytes or HeLa cells were immunoblotted using human autoantibodies that recognize ribonucleoprotein autoantigens. Novel autoantigen structures identified were further characterized using nucleases and RNA hybridization. Human sera that recognize U1-70 kd (U1-70K) and La by immunoblotting also recognized multiple novel species when they were used to immunoblot lysates of UVB-irradiated keratinocytes or HeLa cells. These species were not present in control cells and were not observed when apoptosis was induced by Fas ligation or cytotoxic lymphocyte granule contents. Biochemical analysis using multiple assays revealed that these novel UVB-induced molecular species result from the covalent crosslinking between the U1 RNA and the hYRNA molecules with their associated proteins, including U1-70K, La, and likely components of the Sm particle. These data demonstrate that UVB irradiation of live cells can directly induce covalent RNA-protein complexes, which are recognized by human autoantibodies. As previously described for other autoantigens, these covalent complexes of RNA and proteins may have important consequences in terms of antigen capture and processing.

Functional analysis of proteins and protein species using shotgun proteomics and linear mathematics.

PubMed

Hoehenwarter, Wolfgang; Chen, Yanmei; Recuenco-Munoz, Luis; Wienkoop, Stefanie; Weckwerth, Wolfram

2011-07-01

Covalent post-translational modification of proteins is the primary modulator of protein function in the cell. It greatly expands the functional potential of the proteome compared to the genome. In the past few years shotgun proteomics-based research, where the proteome is digested into peptides prior to mass spectrometric analysis has been prolific in this area. It has determined the kinetics of tens of thousands of sites of covalent modification on an equally large number of proteins under various biological conditions and uncovered a transiently active regulatory network that extends into diverse branches of cellular physiology. In this review, we discuss this work in light of the concept of protein speciation, which emphasizes the entire post-translationally modified molecule and its interactions and not just the modification site as the functional entity. Sometimes, particularly when considering complex multisite modification, all of the modified molecular species involved in the investigated condition, the protein species must be completely resolved for full understanding. We present a mathematical technique that delivers a good approximation for shotgun proteomics data.

Covalent modifications of the amyloid beta peptide by hydroxynonenal: Effects on metal ion binding by monomers and insights into the fibril topology.

PubMed

Grasso, G; Komatsu, H; Axelsen, P H

2017-09-01

Amyloid β peptides (Aβ) and metal ions are associated with oxidative stress in Alzheimer's disease (AD). Oxidative stress, acting on ω-6 polyunsaturated fatty acyl chains, produces diverse products, including 4-hydroxy-2-nonenal (HNE), which can covalently modify the Aβ that helped to produce it. To examine possible feedback mechanisms involving Aβ, metal ions and HNE production, the effects of HNE modification and fibril formation on metal ion binding was investigated. Results indicate that copper(II) generally inhibits the modification of His side chains in Aβ by HNE, but that once modified, copper(II) still binds to Aβ with high affinity. Fibril formation protects only one of the three His residues in Aβ from HNE modification, and this protection is consistent with proposed models of fibril structure. These results provide insight into a network of biochemical reactions that may be operating as a consequence of oxidative stress in AD, or as part of the pathogenic process. Copyright © 2016. Published by Elsevier Inc.

Direct covalent modification as a strategy to inhibit nuclear factor-kappa B.

PubMed

Pande, Vineet; Sousa, Sérgio F; Ramos, Maria João

2009-01-01

Nuclear Factor-KkappaB (NF-kappaB) is a transcription factor whose inappropriate activation may result in the development of a number of diseases including cancer, inflammation, neurodegeneration and AIDS. Recent studies on NF-kappaB mediated pathologies, made therapeutic interventions leading to its inhibition an emerging theme in pharmaceutical research. NF-kappaB resides in the cytoplasm and is activated by several time-dependent factors, leading to proteasome-dependent degradation of its inhibitory protein (IkappaB), resulting in free NF-kappaB (p50 and p65 subunits, involved in disease states), which binds to target DNA sites, further resulting in enhanced transcription of several disease associated proteins. The complex pathway of NF-kappaB, finally leading to its DNA binding, has attracted several approaches interfering with this pathway. One such approach is that of a direct covalent modification of NF-kappaB. In this article, we present a critical review on the pharmacological agents that have been studied as inhibitors of NF-kappaB by covalently modifying redox-regulated cysteine residues in its subunits, ultimately resulting in the inhibition of kappaB DNA recognition and binding. Beginning with a general overview of NF-kappaB pathway and several possibilities of chemical interventions, the significance of redox-regulation in NF-kappaB activation and DNA binding is presented. Further, protein S-thiolation, S-nitrosylation and irreversible covalent modification are described as regular biochemical events in the cell, having provided a guideline for the development of NF-kappaB inhibitors discussed further. Although just a handful of inhibitors, with most of them being alkylating agents have been studied in the present context, this approach presents potential for the development of a new class of NF-kappaB-inhibitors.

Capillary electrophoresis of covalently functionalized single-chirality carbon nanotubes.

PubMed

He, Pingli; Meany, Brendan; Wang, Chunyan; Piao, Yanmei; Kwon, Hyejin; Deng, Shunliu; Wang, YuHuang

2017-07-01

We demonstrate the separation of chirality-enriched single-walled carbon nanotubes (SWCNTs) by degree of surface functionalization using high-performance CE. Controlled amounts of negatively charged and positively charged functional groups were attached to the sidewall of chirality-enriched SWCNTs through covalent functionalization using 4-carboxybenzenediazonium tetrafluoroborate or 4-diazo-N,N-diethylaniline tetrafluoroborate, respectively. Surfactant- and pH-dependent studies confirmed that under conditions that minimized ionic screening effects, separation of these functionalized SWCNTs was strongly dependent on the surface charge density introduced through covalent surface chemistry. For both heterogeneous mixtures and single-chirality-enriched samples, covalently functionalized SWCNTs showed substantially increased peak width in electropherogram spectra compared to nonfunctionalized SWCNTs, which can be attributed to a distribution of surface charges along the functionalized nanotubes. Successful separation of functionalized single-chirality SWCNTs by functional density was confirmed with UV-Vis-NIR absorption and Raman scattering spectroscopies of fraction collected samples. These results suggest a high degree of structural heterogeneity in covalently functionalized SWCNTs, even for chirality-enriched samples, and show the feasibility of applying CE for high-performance separation of nanomaterials based on differences in surface functional density. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A photochemical approach for a fast and self-limited covalent modification of surface supported graphene with photoactive dyes

NASA Astrophysics Data System (ADS)

Sergeeva, Natalia N.; Chaika, Alexander N.; Walls, Brian; Murphy, Barry E.; Walshe, Killian; Martin, David P.; Richards, Billy D. O.; Jose, Gin; Fleischer, Karsten; Aristov, Victor Yu; Molodtsova, Olga V.; Shvets, Igor V.; Krasnikov, Sergey A.

2018-07-01

Herein, we report a simple method for a covalent modification of surface supported graphene with photoactive dyes. Graphene was fabricated on cubic-SiC/Si(001) wafers due to their low cost and suitability for mass-production of continuous graphene fit for electronic applications on millimetre scale. Functionalisation of the graphene surface was carried out in solution via white light induced photochemical generation of phenazine radicals from phenazine diazonium salt. The resulting covalently bonded phenazine-graphene hybrid structure was characterised by scanning tunnelling microscopy (STM) and spectroscopy (STS), Raman spectroscopy and density functional theory (DFT) calculations. It was found that phenazine molecules form an overlayer, which exhibit a short range order with a rectangular unit cell on the graphene surface. DFT calculations based on STM results reveal that molecules are standing up in the overlayer with the maximum coverage of 0.25 molecules per graphene unit cell. Raman spectroscopy and STM results show that the growth is limited to one monolayer of standing molecules. STS reveals that the phenazine-graphene hybrid structure has a band gap of 0.8 eV.

Synthesis, surface modifications, and size-sorting of mixed nickel-zinc ferrite colloidal magnetic nanoparticles.

PubMed

Majewski, P; Krysiński, P

2008-01-01

We report on the spontaneous covalent growth of monomolecular adlayers on mixed nickel-zinc nanoferrite colloidal suspensions (ferrofluids). Synthesized nanoparticles were subjected to surface modification by means of acid chloride chemistry, leading to the formation of covalent bonds between the hydroxy groups at the nanoparticle surface and the acid chloride molecules. This procedure can be easily tailored to allow for the formation of adlayers containing both hydrophobic and hydrophilic regions stacked at predetermined distances from the magnetic core, and also providing the nanoferrites with functional carboxy groups capable of further modifications with, for example, drug molecules. Here, fluorophore aminopyrene molecules were bound to such modified nanoferrites through amide bonds. We also used the same chemistry to modify the surface with covalently bound long-chain palmitoyl moieties, and for comparison we also modified the nanoferrite surface by simple adsorption of oleic acid. Both procedures made the surface highly hydrophobic. These hydrophobic colloids were subsequently spread on an aqueous surface to form Langmuir monolayers with different characteristics. Moreover, since uniformity of size is crucial in a number of applications, we propose an efficient way of sorting the magnetic nanoparticles by size in their colloidal suspension. The suspension is centrifuged at increasing rotational speed and the fractions are collected after each run. The mean size of nanoferrite in each fraction was measured by the powder X-ray diffraction (PXRD) technique.

Protein and DNA Modifications: Evolutionary Imprints of Bacterial Biochemical Diversification and Geochemistry on the Provenance of Eukaryotic Epigenetics

PubMed Central

Aravind, L.; Burroughs, A. Maxwell; Zhang, Dapeng; Iyer, Lakshminarayan M.

2014-01-01

Epigenetic information, which plays a major role in eukaryotic biology, is transmitted by covalent modifications of nuclear proteins (e.g., histones) and DNA, along with poorly understood processes involving cytoplasmic/secreted proteins and RNAs. The origin of eukaryotes was accompanied by emergence of a highly developed biochemical apparatus for encoding, resetting, and reading covalent epigenetic marks in proteins such as histones and tubulins. The provenance of this apparatus remained unclear until recently. Developments in comparative genomics show that key components of eukaryotic epigenetics emerged as part of the extensive biochemical innovation of secondary metabolism and intergenomic/interorganismal conflict systems in prokaryotes, particularly bacteria. These supplied not only enzymatic components for encoding and removing epigenetic modifications, but also readers of some of these marks. Diversification of these prokaryotic systems and subsequently eukaryotic epigenetics appear to have been considerably influenced by the great oxygenation event in the Earth’s history. PMID:24984775

Protein and DNA modifications: evolutionary imprints of bacterial biochemical diversification and geochemistry on the provenance of eukaryotic epigenetics.

PubMed

Aravind, L; Burroughs, A Maxwell; Zhang, Dapeng; Iyer, Lakshminarayan M

2014-07-01

Epigenetic information, which plays a major role in eukaryotic biology, is transmitted by covalent modifications of nuclear proteins (e.g., histones) and DNA, along with poorly understood processes involving cytoplasmic/secreted proteins and RNAs. The origin of eukaryotes was accompanied by emergence of a highly developed biochemical apparatus for encoding, resetting, and reading covalent epigenetic marks in proteins such as histones and tubulins. The provenance of this apparatus remained unclear until recently. Developments in comparative genomics show that key components of eukaryotic epigenetics emerged as part of the extensive biochemical innovation of secondary metabolism and intergenomic/interorganismal conflict systems in prokaryotes, particularly bacteria. These supplied not only enzymatic components for encoding and removing epigenetic modifications, but also readers of some of these marks. Diversification of these prokaryotic systems and subsequently eukaryotic epigenetics appear to have been considerably influenced by the great oxygenation event in the Earth's history. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Functionalization of multiwalled carbon nanotubes by microwave irradiation for lysozyme attachment: comparison of covalent and adsorption methods by kinetics of thermal inactivation

NASA Astrophysics Data System (ADS)

Puentes-Camacho, Daniel; Velázquez, Enrique F.; Rodríguez-Félix, Dora E.; Castillo-Ortega, Mónica; Sotelo-Mundo, Rogerio R.; del Castillo-Castro, Teresa

2017-12-01

Proteins suffer changes in their tertiary structure when they are immobilized, and enzymatic activity is affected due to the low biocompatibility of some supporting materials. In this work immobilization of lysozyme on carbon nanotubes previously functionalized by microwave irradiation was studied. The effectiveness of the microwave-assisted acid treatment of carbon nanotubes was evaluated by XPS, TEM, Raman and FTIR spectroscopy. The carboxylic modification of nanotube surfaces by this fast, simple and feasible method allowed the physical adsorption and covalent linking of active lysozyme onto the carbonaceous material. Thermal inactivation kinetics, thermodynamic parameters and storage stability were studied for adsorbed and covalent enzyme complexes. A major stability was found for lysozyme immobilized by the covalent method, the activation energy for inactivation of the enzyme was higher for the covalent method and it was stable after 50 d of storage at 4 °C. The current study highlights the effect of protein immobilization method on the biotechnological potential of nanostructured biocatalysts.

Synthesis, characterization, and interactions of single-walled carbon nanotubes modified with doxorubicin with Langmuir-Blodgett biomimetic membranes.

PubMed

Matyszewska, Dorota; Napora, Ewelina; Żelechowska, Kamila; Biernat, Jan F; Bilewicz, Renata

2018-01-01

The synthesis, characterization, and the influence of single-walled carbon nanotubes (SWCNTs) modified with an anticancer drug doxorubicin (DOx) on the properties of model biological membrane as well as the comparison of the two modes of modification has been presented. The drug was covalently attached to the nanotubes either preferentially on the sides or at the ends of the nanotubes by the formation of hydrazone bond. The efficiency of the modification was proved by the results of FTIR, Raman, and thermogravimetric analysis. In order to characterize the influence of SWCNT-DOx conjugates on model biological membranes, Langmuir technique has been employed. The mixed monolayers composed of 1,2-dipalmitoyl- sn -glycero-3-phosphothioethanol (DPPTE) and SWCNT-DOx with different weight ratio have been prepared. It has been shown that changes in the isotherm characteristics depend on the SWCNTs content. While smaller amounts of SWCNTs do not exert significant differences, the introduction of the prevailing content of the nanotubes increases area per molecule and decreases the maximum value of compression modulus, leading to more fluid monolayer. However, upon increasing the surface pressure, the aggregation of carbon nanotubes within the thiolipid matrix has been observed. Mixed layers of DPPTE/SWCNT-DOx were also transferred onto gold electrodes by means of LB method. Cyclic voltammetry showed that SWCNT-DOx conjugates remain adsorbed at the electrode surface and are stable in time. Additionally, higher values of peak current and DOx surface concentration obtained for side modification prove that side modification allows for more efficient conjugation of the drug to carbon nanotubes. Graphical abstractᅟ.

Synthesis, characterization, and interactions of single-walled carbon nanotubes modified with doxorubicin with Langmuir-Blodgett biomimetic membranes

NASA Astrophysics Data System (ADS)

Matyszewska, Dorota; Napora, Ewelina; Żelechowska, Kamila; Biernat, Jan F.; Bilewicz, Renata

2018-05-01

The synthesis, characterization, and the influence of single-walled carbon nanotubes (SWCNTs) modified with an anticancer drug doxorubicin (DOx) on the properties of model biological membrane as well as the comparison of the two modes of modification has been presented. The drug was covalently attached to the nanotubes either preferentially on the sides or at the ends of the nanotubes by the formation of hydrazone bond. The efficiency of the modification was proved by the results of FTIR, Raman, and thermogravimetric analysis. In order to characterize the influence of SWCNT-DOx conjugates on model biological membranes, Langmuir technique has been employed. The mixed monolayers composed of 1,2-dipalmitoyl- sn-glycero-3-phosphothioethanol (DPPTE) and SWCNT-DOx with different weight ratio have been prepared. It has been shown that changes in the isotherm characteristics depend on the SWCNTs content. While smaller amounts of SWCNTs do not exert significant differences, the introduction of the prevailing content of the nanotubes increases area per molecule and decreases the maximum value of compression modulus, leading to more fluid monolayer. However, upon increasing the surface pressure, the aggregation of carbon nanotubes within the thiolipid matrix has been observed. Mixed layers of DPPTE/SWCNT-DOx were also transferred onto gold electrodes by means of LB method. Cyclic voltammetry showed that SWCNT-DOx conjugates remain adsorbed at the electrode surface and are stable in time. Additionally, higher values of peak current and DOx surface concentration obtained for side modification prove that side modification allows for more efficient conjugation of the drug to carbon nanotubes. [Figure not available: see fulltext.

Covalent bond force profile and cleavage in a single polymer chain

NASA Astrophysics Data System (ADS)

Garnier, Lionel; Gauthier-Manuel, Bernard; van der Vegte, Eric W.; Snijders, Jaap; Hadziioannou, Georges

2000-08-01

We present here the measurement of the single-polymer entropic elasticity and the single covalent bond force profile, probed with two types of atomic force microscopes (AFM) on a synthetic polymer molecule: polymethacrylic acid in water. The conventional AFM allowed us to distinguish two types of interactions present in this system when doing force spectroscopic measurements: the first interaction is associated with adsorption sites of the polymer chains onto a bare gold surface, the second interaction is directly correlated to the rupture process of a single covalent bond. All these bridging interactions allowed us to stretch the single polymer chain and to determine the various factors playing a role in the elasticity of these molecules. To obtain a closer insight into the bond rupture process, we moved to a force sensor stable in position when measuring attractive forces. By optimizing the polymer length so as to fulfill the elastic stability conditions, we were able for the first time to map out the entire force profile associated with the cleavage of a single covalent bond. Experimental data coupled with molecular quantum mechanical calculations strongly suggest that the breaking bond is located at one end of the polymer chain.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu Shanshan; Zhang Hong; Matunis, Michael J.

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMOmore » substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.« less

Elements of the cellular metabolic structure

PubMed Central

De la Fuente, Ildefonso M.

2015-01-01

A large number of studies have demonstrated the existence of metabolic covalent modifications in different molecular structures, which are able to store biochemical information that is not encoded by DNA. Some of these covalent mark patterns can be transmitted across generations (epigenetic changes). Recently, the emergence of Hopfield-like attractor dynamics has been observed in self-organized enzymatic networks, which have the capacity to store functional catalytic patterns that can be correctly recovered by specific input stimuli. Hopfield-like metabolic dynamics are stable and can be maintained as a long-term biochemical memory. In addition, specific molecular information can be transferred from the functional dynamics of the metabolic networks to the enzymatic activity involved in covalent post-translational modulation, so that determined functional memory can be embedded in multiple stable molecular marks. The metabolic dynamics governed by Hopfield-type attractors (functional processes), as well as the enzymatic covalent modifications of specific molecules (structural dynamic processes) seem to represent the two stages of the dynamical memory of cellular metabolism (metabolic memory). Epigenetic processes appear to be the structural manifestation of this cellular metabolic memory. Here, a new framework for molecular information storage in the cell is presented, which is characterized by two functionally and molecularly interrelated systems: a dynamic, flexible and adaptive system (metabolic memory) and an essentially conservative system (genetic memory). The molecular information of both systems seems to coordinate the physiological development of the whole cell. PMID:25988183

Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents.

PubMed

Mohr, S; Stamler, J S; Brüne, B

1994-07-18

Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO.-induced [32P]NAD(+)-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO. release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS-NO. Peroxynitrite (ONOO-) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO2+) through the intermediacy of RS-NO. However, the efficiency of this reaction is limited by ONOO- -induced oxidation of protein SH groups at the active site. ONOO- generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS-NO are likely to support the NO group transfer to thiol-containing proteins.

Cp2 TiX Complexes for Sustainable Catalysis in Single-Electron Steps.

PubMed

Richrath, Ruben B; Olyschläger, Theresa; Hildebrandt, Sven; Enny, Daniel G; Fianu, Godfred D; Flowers, Robert A; Gansäuer, Andreas

2018-04-25

We present a combined electrochemical, kinetic, and synthetic study with a novel and easily accessible class of titanocene catalysts for catalysis in single-electron steps. The tailoring of the electronic properties of our Cp 2 TiX-catalysts that are prepared in situ from readily available Cp 2 TiX 2 is achieved by varying the anionic ligand X. Of the complexes investigated, Cp 2 TiOMs proved to be either equal or substantially superior to the best catalysts developed earlier. The kinetic and thermodynamic properties pertinent to catalysis have been determined. They allow a mechanistic understanding of the subtle interplay of properties required for an efficient oxidative addition and reduction. Therefore, our study highlights that efficient catalysts do not require the elaborate covalent modification of the cyclopentadienyl ligands. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Polymer-wrapped single-walled carbon nanotubes: a transformation toward better applications in healthcare.

PubMed

Chik, Mazzura Wan; Hussain, Zahid; Zulkefeli, Mohd; Tripathy, Minaketan; Kumar, Sunil; Majeed, Abu Bakar Abdul; Byrappa, K

2018-03-28

Carbon nanotubes (CNTs) possess outstanding properties that could be useful in several technological, drug delivery, and diagnostic applications. However, their unique physical and chemical properties are hindered due to their poor solubility. This article review's the different ways and means of solubility enhancement of single-wall carbon nanotubes (SWNTs). The advantages of SWNTs over the multi-walled carbon nanotubes (MWNTs) and the method of non-covalent modification for solubility enhancement has been the key interest in this review. The review also highlights a few examples of dispersant design. The review includes some interesting utility of SWNTs being wrapped with polymer especially in biological media that could mediate proper drug delivery to target cells. Further, the use of wrapped SWNTs with phospholipids, nucleic acid, and amphiphillic polymers as biosensors is of research interest. The review aims at summarizing the developments relating to wrapped SWNTs to generate further research prospects in healthcare.

Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection

NASA Astrophysics Data System (ADS)

Andrew Mackay, J.; Chen, Mingnan; McDaniel, Jonathan R.; Liu, Wenge; Simnick, Andrew J.; Chilkoti, Ashutosh

2009-12-01

New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100-nm-sized, near-monodisperse nanoparticles on conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics, leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a fourfold higher maximum tolerated dose than free drug, and induce nearly complete tumour regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents and targeting moieties into multifunctional nanomedicines.

Room temperature single photon generation at 1. 5 μ m from covalent dopant states of carbon nanotubes

NASA Astrophysics Data System (ADS)

Htoonb, Han; He, Xiaowei; Hartmann, Nicolai; Ma, Xuedan; Doorn, Stephen; CenterIntegrated Nanotechnologies, Los Alamos National Laboratory Team

Recent demonstration that oxygen dopant states covalently attached to the single-walled carbon nanotubes (SWCNTs) are capable of emitting single photons at room-T (RT) opens the possibility of building room-T electrically-driven single photon sources for quantum communication applications. The RT single photon generation was not observed only at wavelength beyond 1.3 μ m. Here in this work we demonstrate RT single photon generation at 1. 5 μ m from diazonium dopant states of (10,3) nanotubes.

Dynamic covalent polymers

PubMed Central

García, Fátima

2016-01-01

ABSTRACT This Highlight presents an overview of the rapidly growing field of dynamic covalent polymers. This class of polymers combines intrinsic reversibility with the robustness of covalent bonds, thus enabling formation of mechanically stable, polymer‐based materials that are responsive to external stimuli. It will be discussed how the inherent dynamic nature of the dynamic covalent bonds on the molecular level can be translated to the macroscopic level of the polymer, giving access to a range of applications, such as stimuli‐responsive or self‐healing materials. A primary distinction will be made based on the type of dynamic covalent bond employed, while a secondary distinction will be based on the consideration whether the dynamic covalent bond is used in the main chain of the polymer or whether it is used to allow side chain modification of the polymer. Emphasis will be on the chemistry of the dynamic covalent bonds present in the polymer, in particular in relation to how the specific (dynamic) features of the bond impart functionality to the polymer material, and to the conditions under which this dynamic behavior is manifested. © 2016 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016, 54, 3551–3577. PMID:27917019

Preparation and properties of electro-conductive fabrics based on polypyrrole: covalent vs. non-covalent attachment

NASA Astrophysics Data System (ADS)

David, N. C.; Anavi, D.; Milanovich, M.; Popowski, Y.; Frid, L.; Amir, E.

2017-10-01

Electro-conductive fabrics were prepared via in situ oxidative polymerization of pyrrole (Py) in the presence of unmodified and chemically modified cotton fabrics. Chemical modification of cotton fabric was achieved by covalent attachment of a bifunctional linker molecule to the surface of the fabric, followed by incorporation of a monomer unit onto the linker. The fabrics were characterized using Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron spectroscopy, and thermal analysis. Furthermore, the effect of Py concentration on the degree of polypyrrole (PPy) grafting, surface morphology, electrical resistivity, and laundering durability were studied for both types of cotton fabrics. Reductions of several orders of magnitude in surface and volume electrical resistivities were observed for both non-covalently and covalently linked cotton-PPy systems, whereas the effect of covalent pre-treatment of the fabric was stronger at low Py concentration. On the other hand, at higher monomer concentration, the electrical properties and laundering durability of the fabrics we comparable for both unmodified and chemically pre-treated cotton fabrics, indicating that only a small fraction of PPy chains were chemically grafted onto the fabric surface with the majority of the polymer being connected to the fabric through hydrogen bonds.

In Situ Synthesis of Lipid Membranes

NASA Astrophysics Data System (ADS)

Devaraj, N. K.

2017-07-01

We have a strong interest in applying covalent coupling reactions to the formation and modification of lipid membranes. We have utilized chemoselective reactions, such as copper-catalyzed triazole formation or the native chemical ligation.

[Native, modified, and immobilized chymotrypsin in chaotropic media. Stabilization limits].

PubMed

Panova, A A; Levitskiĭ, V Iu; Mozhaev, V V

1994-07-01

To stabilize alpha-chymotrypsin against irreversible thermal inactivation at high temperatures, methods of covalent modification and multi-point immobilization in combination with the addition of salting-in compounds were used. The upper limit of the protein stability proved to be the same for a combination of the modification and salting-in media and for each of these methods separately. The limit of stabilization reached by means of covalent immobilization is higher than the limit of stabilization reached by two other methods. The greatest stabilization of immobilized alpha-chymotrypsin by the salting-in media (a 10000 fold increase in the native enzyme's stability level) takes place only in the case of the protein with the minimum number of bonds with the support. Stabilization of the enzyme by these methods is explained in terms of the suppression of the conformational inactivation processes.

Dual-Functional Hydrazide-Reactive and Anhydride-Containing Oligomeric Hydrogel Building Blocks.

PubMed

Kascholke, Christian; Loth, Tina; Kohn-Polster, Caroline; Möller, Stephanie; Bellstedt, Peter; Schulz-Siegmund, Michaela; Schnabelrauch, Matthias; Hacker, Michael C

2017-03-13

Biomimetic hydrogels are advanced biomaterials that have been developed following different synthetic routes. Covalent postfabrication functionalization is a promising strategy to achieve efficient matrix modification decoupled of general material properties. To this end, dual-functional macromers were synthesized by free radical polymerization of maleic anhydride with diacetone acrylamide (N-(1,1-dimethyl-3-oxobutyl)acrylamide) and pentaerythritol diacrylate monostearate. Amphiphilic oligomers (M n < 7.5 kDa) with anhydride contents of 7-20% offered cross-linking reactivity to yield rigid hydrogels with gelatinous peptides (E = 4-13 kPa) and good cell adhesion properties. Mildly reactive methyl ketones as second functionality remained intact during hydrogel formation and potential of covalent matrix modification was shown using hydrazide and hydrazine model compounds. Successful secondary dihydrazide cross-linking was demonstrated by an increase of hydrogel stiffness (>40%). Efficient hydrazide/hydrazine immobilization depending on solution pH, hydrogel ketone content as well as ligand concentration for bioconjugation was shown and reversibility of hydrazone formation was indicated by physiologically relevant hydrazide release over 7 days. Proof-of-concept experiments with hydrazido-functionalized hyaluronan demonstrated potential for covalent aECM immobilization. The presented dual-functional macromers have perspective as reactive hydrogel building blocks for various biomedical applications.

Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization

PubMed Central

Brune, Karl D.; Leneghan, Darren B.; Brian, Iona J.; Ishizuka, Andrew S.; Bachmann, Martin F.; Draper, Simon J.; Biswas, Sumi; Howarth, Mark

2016-01-01

Virus-like particles (VLPs) are non-infectious self-assembling nanoparticles, useful in medicine and nanotechnology. Their repetitive molecularly-defined architecture is attractive for engineering multivalency, notably for vaccination. However, decorating VLPs with target-antigens by genetic fusion or chemical modification is time-consuming and often leads to capsid misassembly or antigen misfolding, hindering generation of protective immunity. Here we establish a platform for irreversibly decorating VLPs simply by mixing with protein antigen. SpyCatcher is a genetically-encoded protein designed to spontaneously form a covalent bond to its peptide-partner SpyTag. We expressed in E. coli VLPs from the bacteriophage AP205 genetically fused to SpyCatcher. We demonstrated quantitative covalent coupling to SpyCatcher-VLPs after mixing with SpyTag-linked to malaria antigens, including CIDR and Pfs25. In addition, we showed coupling to the VLPs for peptides relevant to cancer from epidermal growth factor receptor and telomerase. Injecting SpyCatcher-VLPs decorated with a malarial antigen efficiently induced antibody responses after only a single immunization. This simple, efficient and modular decoration of nanoparticles should accelerate vaccine development, as well as other applications of nanoparticle devices. PMID:26781591

Covalent Immobilization of (-)-Riboflavin on Polymer Functionalized Silica Particles: Application in the Photocatalytic E→Z Isomerization of Polarized Alkenes.

PubMed

Metternich, Jan B; Sagebiel, Sven; Lückener, Anne; Lamping, Sebastian; Ravoo, Bart Jan; Gilmour, Ryan

2018-03-20

The covalent immobilization of the biomimetic, photo-organocatalyst (-)-riboflavin on silica micro- and nanoparticles via atom transfer radical polymerization (ATRP) is disclosed. Given the effectiveness of (-)-riboflavin as a versatile, environmentally benign photocatalyst, an immobilization strategy based on acrylate-linker modification of the catalyst core and controlled polymerization on initiator pre-functionalized silica particles has been developed. Validation of this approach is demonstrated in the E→Z isomerization of a benchmark cinnamonitrile (Z/E up to 88:12) with 0.97 mol % catalyst loading. Characterization of the immobilized photocatalyst supports covalent embedding of the catalyst in the polymeric brushes on the silica particle surface. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of Heterogeneous Metal-Organic Framework Organocatalysts Prepared by Postsynthetic Modification

PubMed Central

Garibay, Sergio J.; Wang, Zhenqiang; Cohen, Seth M.

2010-01-01

A metal-organic framework (MOF) containing 2-amino-1,4-benzenedicarboxylate (NH2-BDC) as a building block is shown to undergo chemical modification with a set of cyclic anhydrides. The modification of the aluminum-based MOF known as MIL-53(Al)-NH2 (MIL = Matérial Institut Lavoisier) by these reagents is demonstrated by using a variety of methods, including NMR and ESI-MS, and the structural integrity of the modified MOFs has been confirmed by TGA, PXRD, and gas sorption analysis. Reaction with these cyclic anhydrides produces MOFs that display carboxylic acid functional groups within their pores. Furthermore, it is shown that maleic acid functionalized MIL-53(Al)-AMMal can act as a Brønsted acid catalyst and facilitate the methanolysis of several small epoxides. Experiments show that MIL-53(Al)-AMMal acts in a heterogeneous manner and is recyclable with consistent activity over at least three catalytic cycles. The findings presented here demonstrate several important features of covalent postsynthetic modification (PSM) on MOFs, including: 1) facile introduction of catalytic functionality using simple organic reagents (e.g. anhydrides); 2) the ability to utilize and recycle organocatalytic MOFs; 3) control of catalytic activity through choice of functional group. The findings clearly illustrate that covalent postsynthetic modification represents a powerful means to access new MOF compounds that serve as organocatalytic materials. PMID:20698561

Single step signal group-imidazole labeling of organic phosphate groups under aqueous conditions

DOEpatents

Giese, Roger W.; Wang, Poguang

1996-01-01

Compounds and methods for single step, covalent labeling of the phosphate group of an organic substance under aqueous conditions are described. The labeling compound includes any kind of detectable signal group covalently bound to an imidazole moiety, which can be imidazole or a substituted imidazole. A preferred labeling compound has the formula ##STR1##

Covalent modification of proteins by ligands of steroid hormone receptors.

PubMed

Takahashi, N; Breitman, T R

1992-11-15

Retinoylation, acylation with retinoic acid (RA), is a covalent modification of proteins occurring in a variety of eukaryotic cell lines. In this study, we found that proteins in HL-60 cells were labeled by 17 beta-[3H]estradiol (E2), [3H]progesterone (Pg), 1 alpha,25-dihydroxy[3H]vitamin D3 [1,25(OH)2D3], [125I]triiodothyronine (T3), [125I]thyroxine (T4), and [3H]prostaglandin E2 (PGE2). All of these hormones, except PGE2, are ligands of the steroid hormone receptor family. Addition to the growth medium of 5 microM ketoconazole, an inhibitor of cytochrome P450-dependent enzymes, increased about 2-fold the labeling of proteins by T3, T4, 1,25(OH)2D3, and PGE2. In contrast, ketoconazole did not change markedly the extent of labeling by RA, E2, or Pg. Alkaline methanolysis, which cleaves ester bonds, released variable percentages of the radioactive ligands bound to protein. These values were about 80% for RA and PGE2; 50% for T3, T4, and Pg; and 20% for E2 and 1,25(OH)2D3. Treatment with thioether-cleavage reagents, iodomethane or Raney nickel catalyst, released < 2% of the covalently bound ligands. Two-dimensional polyacrylamide gel electrophoresis patterns of labeled proteins were unique for each ligand. Proteins of M(r) 47,000 and 51,000 were labeled by RA, E2, T3, and T4. These proteins had the same mobilities as RI and RII, the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases. 1,25(OH)2D3 also bound to proteins of M(r) 47,000 and 51,000. However, these proteins had pI values different from those of RI or RII. These results suggest that some activities of ligands of the steroid hormone receptor family and of PGE2 may be mediated by their covalent modification of proteins.

Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro

PubMed Central

Talhaoui, Ibtissam; Lebedeva, Natalia A.; Zarkovic, Gabriella; Saint-Pierre, Christine; Kutuzov, Mikhail M.; Sukhanova, Maria V.; Matkarimov, Bakhyt T.; Gasparutto, Didier; Saparbaev, Murat K.; Lavrik, Olga I.; Ishchenko, Alexander A.

2016-01-01

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3′-cordycepin, 5′- and 3′-phosphate and also to 5′-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5′-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2′-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2′,1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1′ of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs. PMID:27471034

Trafficking and function of the cystic fibrosis transmembrane conductance regulator: a complex network of posttranslational modifications

PubMed Central

McClure, Michelle L.; Barnes, Stephen; Brodsky, Jeffrey L.

2016-01-01

Posttranslational modifications add diversity to protein function. Throughout its life cycle, the cystic fibrosis transmembrane conductance regulator (CFTR) undergoes numerous covalent posttranslational modifications (PTMs), including glycosylation, ubiquitination, sumoylation, phosphorylation, and palmitoylation. These modifications regulate key steps during protein biogenesis, such as protein folding, trafficking, stability, function, and association with protein partners and therefore may serve as targets for therapeutic manipulation. More generally, an improved understanding of molecular mechanisms that underlie CFTR PTMs may suggest novel treatment strategies for CF and perhaps other protein conformational diseases. This review provides a comprehensive summary of co- and posttranslational CFTR modifications and their significance with regard to protein biogenesis. PMID:27474090

Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

PubMed Central

Mahajan, Rohit; Gerace, Larry; Melchior, Frauke

1998-01-01

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role. PMID:9442102

Sulfur mustard-induced epigenetic modifications over time - a pilot study.

PubMed

Simons, Thilo; Steinritz, Dirk; Bölck, Birgit; Schmidt, Annette; Popp, Tanja; Thiermann, Horst; Gudermann, Thomas; Bloch, Wilhelm; Kehe, Kai

2018-09-01

The chemical warfare agent sulfur mustard (SM) can cause long-term health effects that may occur even years after a single exposure. The underlying pathophysiology is unknown, but epigenetic mechanisms are discussed as feasible explanation. "Epigenetics" depicts regulation of gene function without affecting the DNA sequence itself. DNA-methylation and covalent histone modifications (methylation or acetylation) are regarded as important processes. In the present in vitro study using early endothelial cells (EEC), we analyzed SM-induced DNA methylation over time and compared results to an in vivo skin sample that was obtained approx. one year after an accidental SM exposure. EEC were exposed to low SM concentrations (0.5 and 1.0μM). DNA methylation and histone acetylation (H3-K9, H3-K27, H4-K8) or histone di-methylation (H3-K9, H3-K27, H3-K36) were investigated 24h after exposure, and after 2 or 4 additional cell passages. The human skin sample was assessed in parallel. SM had only some minor effects on histone modifications. However, a significant and pronounced increase of DNA methylation was detected in the late cell passages as well as in the skin sample. Our results indicate that SM does indeed cause epigenetic modifications that appear to persist over time. Copyright © 2017 Elsevier B.V. All rights reserved.

Variation of the chemical reactivity of Thermus thermophilus HB8 ribosomal proteins as a function of pH.

PubMed

Running, William E; Reilly, James P

2010-10-01

Ribosomes occupy a central position in cellular metabolism, converting stored genetic information into active cellular machinery. Ribosomal proteins modulate both the intrinsic function of the ribosome and its interaction with other cellular complexes, such as chaperonins or the signal recognition particle. Chemical modification of proteins combined with mass spectrometric detection of the extent and position of covalent modifications is a rapid, sensitive method for the study of protein structure and flexibility. By altering the pH of the solution, we have induced non-denaturing changes in the structure of bacterial ribosomal proteins and detected these conformational changes by covalent labeling. Changes in ribosomal protein modification across a pH range from 6.6 to 8.3 are unique to each protein, and correlate with their structural environment in the ribosome. Lysine residues whose extent of modification increases as a function of increasing pH are on the surface of proteins, but in close proximity either to glutamate and aspartate residues, or to rRNA backbone phosphates. Increasing pH disrupts tertiary and quaternary interactions mediated by hydrogen bonding or ionic interactions, and regions of protein structure whose conformations are sensitive to these changes are of potential importance in modulating the flexibility of the ribosome or its interaction with other cellular complexes.

Cell Signalling Through Covalent Modification and Allostery

NASA Astrophysics Data System (ADS)

Johnson, Louise N.

Phosphorylation plays essential roles in nearly every aspect of cell life. Protein kinases catalyze the transfer of the γ-phosphate of ATP to a serine, threonine or tyrosine residue in protein substrates. This covalent modification allows activation or inhibition of enzyme activity, creates recognition sites for other proteins and promotes order/disorder or disorder/order transitions. These properties regulate ­signalling pathways and cellular processes that mediate metabolism, transcription, cell cycle progression, differentiation, cytoskeleton arrangement and cell movement, apoptosis, intercellular communication, and neuronal and immunological functions. In this lecture I shall review the structural consequences of protein phosphorylation using our work on glycogen phosphorylase and the cell cycle cyclin dependent protein kinases as illustrations. Regulation of protein phosphorylation may be disrupted in the diseased state and protein kinases have become high profile targets for drug development. To date there are 11 compounds that have been approved for clinical use in the treatment of cancer.

Endowing carbon nanotubes with biological and biomedical properties by chemical modifications.

PubMed

Battigelli, Alessia; Ménard-Moyon, Cécilia; Da Ros, Tatiana; Prato, Maurizio; Bianco, Alberto

2013-12-01

The scope of nanotechnology is gaining importance in biology and medicine. Carbon nanotubes (CNTs) have emerged as a promising tool due to their unique properties, high specific surface area, and capacity to cross biological barriers. These properties offer a variety of opportunities for applications in nanomedicine, such as diagnosis, disease treatment, imaging, and tissue engineering. Nevertheless, pristine CNTs are insoluble in water and in most organic solvents; thereby functionalization of their surface is necessary to increase biocompatibility. Derivatization of CNTs also gives the possibility to conjugate different biological and bioactive molecules including drugs, proteins, and targeting ligands. This review focuses on the chemical modifications of CNTs that have been developed to impart specific properties for biological and medical purposes. Biomolecules can be covalently grafted or non-covalently adsorbed on the nanotube surface. In addition, the inner core of CNTs can be exploited to encapsulate drugs, nanoparticles, or radioactive elements. © 2013.

Advances in covalent organic frameworks in separation science.

PubMed

Qian, Hai-Long; Yang, Cheng-Xiong; Wang, Wen-Long; Yang, Cheng; Yan, Xiu-Ping

2018-03-23

Covalent organic frameworks (COFs) are a new class of multifunctional crystalline organic polymer constructed with organic monomers via robust covalent bonds. The unique properties such as convenient modification, low densities, large specific surface areas, good stability and permanent porosity make COFs great potential in separation science. This review shows the state-of-the art for the application of COFs and their composites in analytical separation science. COFs and their composites have been explored as promising sorbents for solid phase extraction, potential coatings for solid phase microextraction, and novel stationary phases for gas chromatography, high-performance liquid chromatography and capillary electrochromatography. The prospects of COFs for separation science are also presented, which can offer an outlook and reference for further study on the applications of COFs. Copyright © 2018 Elsevier B.V. All rights reserved.

Modification of human serum albumin by the nerve agent VX: microbore liquid chromatography/electrospray ionization high-resolution time-of-flight tandem mass spectrometry method for detection of phosphonylated tyrosine and novel cysteine containing disulfide adducts.

PubMed

Kranawetvogl, Andreas; Worek, Franz; Thiermann, Horst; John, Harald

2016-10-15

Organophosphorus nerve agents still constitute a considerable threat to the health of military personnel and the civilian population. Long-term biomarkers are crucial for reliable verification of exposure to banned substances. Therefore, current research focuses on identification of endogenous protein targets showing covalent modifications by organophosphorus nerve agents (adducts). Purified human serum albumin and human plasma were incubated with the nerve agent VX followed by enzymatic proteolysis with pronase. Resulting peptide cleavage products were separated by microbore liquid chromatography (μLC) online coupled to positive electrospray ionization (ESI) with subsequent high-resolution time-of-flight tandem mass spectrometry (HR MS/MS) allowing identification of known and novel adducts. In addition to known phosphonylation of various tyrosine residues, albumin was found to be modified at diverse cysteine residues by covalent attachment of the leaving group of VX. These novel disulfide adducts were cleaved from at least two regions of the intact protein as dipeptides containing cysteine and proline either as CP or PC. A rapid and sensitive method was developed for simultaneous detection of the diverse covalent modifications of human albumin by VX. Identification of the novel leaving group adducts with human albumin expands the basic knowledge on molecular toxicology of the nerve agent VX. Furthermore, the presented μLC/ESI HR MS/MS method might be of relevance for verification of VX poisoning. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Covalent immobilization of metal organic frameworks onto chemical resistant poly(ether ether ketone) jacket for stir bar extraction.

PubMed

Wang, Chenlu; Zhou, Wei; Liao, Xiaoyan; Wang, Xuemei; Chen, Zilin

2018-09-26

Preparation of stir bar extraction (SBSE) device with high physical and chemical stability is important and challenging by date. A novel poly (ether ether ketone) (PEEK) tube with excellent mechanical property and chemical stability was firstly used as jacket of metal bar for preparation of stir bar. By employing covalent modification method, the inherent chemical resistant problem of PEEK which restricts the modification of sorbents was well solved. After functionalization, plenty of benzoic acid groups were formed onto the PEEK jacket. Metal organic frameworks of aluminium-based Materials of Institute Lavoisier-68 (MIL-68) was in situ immobilized onto the PEEK surface (MIL-68@PEEK) by the bonding with benzoic acid groups. Afterwards, a facile dumbbell-shaped structure was designed for reducing the friction between sorbents and bottom of container. Due to superior property of the PEEK jacket and the covalent modification method, the MIL-68 modified PEEK jacket SBSE device showed good robustness. After coupling with HPLC-MS/MS, the MIL-68@PEEK-based SBSE device was used to analyse of three parabens including methyl paraben, ethyl paraben and propyl paraben. The method had low limit detection up to 1 pg mL -1 with good linearity (R 2  ≥ 0.9978) and good reproducibility (relative standard deviation ≤ 9.74%). The method has been applied to the detection of parabens in cosmetics and rabbit plasma after painted with cosmetics with recoveries between 73.25% and 104.23%. Copyright © 2018 Elsevier B.V. All rights reserved.

Antimicrobial and antioxidant surface modification toward a new silk-fibroin (SF)-L-Cysteine material for skin disease management

NASA Astrophysics Data System (ADS)

Nogueira, Frederico; Granadeiro, Luíza; Mouro, Claudia; Gouveia, Isabel C.

2016-02-01

A novel dressing material - silk fibroin fabric (SF)-L-Cysteine (L-Cys) - is here developed to be used as standard treatment for atopic dermatitis (AD), which combines comfort, thermic, and tensile strength properties of silk materials with antioxidant and antimicrobial effects of L-Cys. A careful understanding about the linking strategies is needed in order not to compromise the bioavailability of L-Cys and deplenish its bioactivity. Durability was also addressed through washing cycles and compared with hospital requirements, according to international Standard EN ISO 105-C06:2010. The present research also analyze the interactions between Staphylococcus aureus and SF-L-Cys under simulating conditions of AD and demonstrated the effectiveness of a double covalent grafting, with the importance of SF tyrosine (Tyr) covalent linkage with L-Cys (SF-g-L-Cys/Tyr-g-L-Cys) even after several washing cycles, twenty five, whereas for a disposable application a single covalent mechanism of grafting L-Cys proved to be sufficient (SF-g-L-Cys). Results showed effective antimicrobial activities exhibiting higher inhibition ratios of 98.65% for SF-g-L-Cys after 5 washing cycles, whereas 97.55% for SF-g-L-Cys/Tyr-g-L-Cys after 25 washing cycles, both at pH 9.5 grafting strategy. Furthermore, it is also reported a non-protumoral effect of L-Cys. A new advance is herein achieved at the world of medical antimicrobial textiles tailored to address wound moisture environment and exudate self-cleaning, which may open novel applications as complementary therapy for AD disease.

Modeling of Interfacial Modification Effects on Thermal Conductivity of Carbon Nanotube Composites

NASA Technical Reports Server (NTRS)

Clancy, Thomas C.; Gates, Thomas S.

2006-01-01

The effect of functionalization of carbon nanotubes on the thermal conductivity of nanocomposites has been studied using a multi-scale modeling approach. These results predict that grafting linear hydrocarbon chains to the surface of a single wall carbon nanotube with covalent chemical bonds should result in a significant increase in the thermal conductivity of these nanocomposites. This is due to the decrease in the interfacial thermal (Kapitza) resistance between the single wall carbon nanotube and the surrounding polymer matrix upon chemical functionalization. The nanocomposites studied here consist of single wall carbon nanotubes in a bulk poly(ethylene vinyl acetate) matrix. The nanotubes are functionalized by end-grafting linear hydrocarbon chains of varying length to the surface of the nanotube. The effect which this functionalization has on the interfacial thermal resistance is studied by molecular dynamics simulation. Interfacial thermal resistance values are calculated for a range of chemical grafting densities and with several chain lengths. These results are subsequently used in an analytical model to predict the resulting effect on the bulk thermal conductivity of the nanocomposite.

Thermally induced alkylation of diamond.

PubMed

Hoeb, Marco; Auernhammer, Marianne; Schoell, Sebastian J; Brandt, Martin S; Garrido, Jose A; Stutzmann, Martin; Sharp, Ian D

2010-12-21

We present an approach for the thermally activated formation of alkene-derived self-assembled monolayers on oxygen-terminated single and polycrystalline diamond surfaces. Chemical modification of the oxygen and hydrogen plasma-treated samples was achieved by heating in 1-octadecene. The resulting layers were characterized using X-ray photoelectron spectroscopy, thermal desorption spectroscopy, atomic force microscopy, Fourier transform infrared spectroscopy, and water contact angle measurements. This investigation reveals that alkenes selectively attach to the oxygen-terminated sites via covalent C-O-C bonds. The hydrophilic oxygen-terminated diamond is rendered strongly hydrophobic following this reaction. The nature of the process limits the organic layer growth to a single monolayer, and FTIR measurements reveal that such monolayers are dense and well ordered. In contrast, hydrogen-terminated diamond sites remain unaffected by this process. This method is thus complementary to the UV-initiated reaction of alkenes with diamond, which exhibits the opposite reactivity contrast. Thermal alkylation increases the range of available diamond functionalization strategies and provides a means of straightforwardly forming single organic layers in order to engineer the surface properties of diamond.

DNA-binding and oxidative properties of cationic phthalocyanines and their dimeric complexes with anionic phthalocyanines covalently linked to oligonucleotides.

PubMed

Kuznetsova, A A; Lukyanets, E A; Solovyeva, L I; Knorre, D G; Fedorova, O S

2008-12-01

Design of chemically modified oligonucleotides for regulation of gene expression has attracted considerable attention over the past decades. One actively pursued approach involves antisense or antigene oligonucleotide constructs carrying reactive groups, many of these based on transition metal complexes. The complexes of Fe(II) and Co(II) with phthalocyanines are extremely good catalysts of oxidation of organic compounds with molecular oxygen and hydrogen peroxide. The binding of positively charged Fe(II) and Co(II) phthalocyanines with single- and double-stranded DNA was investigated. It was shown that these phthalocyanines interact with nucleic acids through an outside binding mode. The site-directed modification of single-stranded DNA by O2 and H2O2 in the presence of dimeric complexes of negatively and positively charged Fe(II) and Co(II) phthalocyanines was investigated. These complexes were formed directly on single-stranded DNA through interaction between negatively charged phthalocyanine in conjugate and positively charged phthalocyanine in solution. The resulting oppositely charged phthalocyanine complexes showed significant increase of catalytic activity compared with monomeric forms of phthalocyanines Fe(II) and Co(II). These complexes catalyzed the DNA oxidation with high efficacy and led to direct DNA strand cleavage. It was determined that oxidation of DNA by molecular oxygen catalyzed by complex of Fe(II)-phthalocyanines proceeds with higher rate than in the case of Co(II)-phthalocyanines but the latter led to a greater extent of target DNA modification.

Single step signal group-imidazole labeling of organic phosphate groups under aqueous conditions

DOEpatents

Giese, R.W.; Wang, P.

1996-04-30

Compounds and methods for single step, covalent labeling of the phosphate group of an organic substance under aqueous conditions are described. The labeling compound includes any kind of detectable signal group covalently bound to an imidazole moiety, which can be imidazole or a substituted imidazole. A preferred labeling compound has the formula shown in the accompanying diagram. 4 figs.

Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I.

PubMed

Peisley, Alys; Wu, Bin; Xu, Hui; Chen, Zhijian J; Hur, Sun

2014-05-01

Ubiquitin (Ub) has important roles in a wide range of intracellular signalling pathways. In the conventional view, ubiquitin alters the signalling activity of the target protein through covalent modification, but accumulating evidence points to the emerging role of non-covalent interaction between ubiquitin and the target. In the innate immune signalling pathway of a viral RNA sensor, RIG-I, both covalent and non-covalent interactions with K63-linked ubiquitin chains (K63-Ubn) were shown to occur in its signalling domain, a tandem caspase activation and recruitment domain (hereafter referred to as 2CARD). Non-covalent binding of K63-Ubn to 2CARD induces its tetramer formation, a requirement for downstream signal activation. Here we report the crystal structure of the tetramer of human RIG-I 2CARD bound by three chains of K63-Ub2. 2CARD assembles into a helical tetramer resembling a 'lock-washer', in which the tetrameric surface serves as a signalling platform for recruitment and activation of the downstream signalling molecule, MAVS. Ubiquitin chains are bound along the outer rim of the helical trajectory, bridging adjacent subunits of 2CARD and stabilizing the 2CARD tetramer. The combination of structural and functional analyses reveals that binding avidity dictates the K63-linkage and chain-length specificity of 2CARD, and that covalent ubiquitin conjugation of 2CARD further stabilizes the Ub-2CARD interaction and thus the 2CARD tetramer. Our work provides unique insights into the novel types of ubiquitin-mediated signal-activation mechanism, and previously unexpected synergism between the covalent and non-covalent ubiquitin interaction modes.

Oriented and covalent immobilization of target molecules to solid supports: synthesis and application of a light-activatable and thiol-reactive cross-linking reagent.

PubMed

Collioud, A; Clémence, J F; Sänger, M; Sigrist, H

1993-01-01

Light-dependent oriented and covalent immobilization of target molecules has been achieved by combining two modification procedures: light-dependent coupling of target molecules to inert surfaces and thiol-selective reactions occurring at macromolecule or substrate surfaces. For immobilization purposes the heterobifunctional reagent N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide was synthesized and chemically characterized. The photosensitivity of the carbene-generating reagent and its reactivity toward thiols were ascertained. Light-induced cross-linking properties of the reagent were documented (i) by reacting first the maleimide function with a thiolated surface, followed by carbene insertion into applied target molecules, (ii) by photochemical coupling of the reagent to an inert support followed by thermochemical reactions with thiol functions, and (iii) by thermochemical modification of target molecules prior to carbene-mediated insertion into surface materials. Procedures mentioned led to light-dependent covalent immobilization of target molecules including amino acids, a synthetic peptide, and antibody-derived F(ab') fragments. Topically selective, light-dependent immobilization was attained with the bifunctional reagent by irradiation of coated surfaces through patterned masks. Glass and polystyrene served as substrates. Molecular orientation is asserted by inherently available or selectively introduced terminal thiol functions in F(ab') fragments and synthetic polypeptides, respectively.

Site-specific covalent modifications of human insulin by catechol estrogens: Reactivity and induced structural and functional changes

NASA Astrophysics Data System (ADS)

Ku, Ming-Chun; Fang, Chieh-Ming; Cheng, Juei-Tang; Liang, Huei-Chen; Wang, Tzu-Fan; Wu, Chih-Hsing; Chen, Chiao-Chen; Tai, Jung-Hsiang; Chen, Shu-Hui

2016-06-01

Proteins, covalently modified by catechol estrogens (CEs), were identified recently from the blood serum of diabetic patients and referred to as estrogenized proteins. Estrogenization of circulating insulin may occur and affect its molecular functioning. Here, the chemical reactivity of CEs towards specific amino acid residues of proteins and the structural and functional changes induced by the estrogenization of insulin were studied using cyclic voltammetry, liquid chromatography-mass spectrometry, circular dichroism spectroscopy, molecular modeling, and bioassays. Our results indicate that CEs, namely, 2- and 4-hydroxyl estrogens, were thermodynamically and kinetically more reactive than the catechol moiety. Upon co-incubation, intact insulin formed a substantial number of adducts with one or multiple CEs via covalent conjugation at its Cys 7 in the A or B chain, as well as at His10 or Lys29 in the B chain. Such conjugation was coupled with the cleavage of inter-chain disulfide linkages. Estrogenization on these sites may block the receptor-binding pockets of insulin. Insulin signaling and glucose uptake levels were lower in MCF-7 cells treated with modified insulin than in cells treated with native insulin. Taken together, our findings demonstrate that insulin molecules are susceptible to active estrogenization, and that such modification may alter the action of insulin.

Detection of Dichlorvos Adducts in a Hepatocyte Cell Line

DTIC Science & Technology

2014-06-30

form adducts to DDVP.16,20−22 Two distinct DDVP adducts are formed by covalent bonding to the hydroxyl group of tyrosine, serine, and threonine ...variable modifications on methionine (oxidation) and on serine, tyrosine, or threonine (phosphorylation, O-methylphosphate, or O-dimethylphosphate). Only...modifications on tyrosine, serine, or threonine residues with a mass of 94 or 108 amu corresponding to O-methyl- or O-dimethyl-phosphate groups. We found 53

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells.

PubMed

Malina, Halina Z

2011-01-19

The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases.

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells

PubMed Central

2011-01-01

Background The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Results Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. Conclusions The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases. PMID:21247434

Cell behavior on gallium nitride surfaces: peptide affinity attachment versus covalent functionalization.

PubMed

Foster, Corey M; Collazo, Ramon; Sitar, Zlatko; Ivanisevic, Albena

2013-07-02

Gallium nitride is a wide band gap semiconductor that demonstrates a unique set of optical and electrical properties as well as aqueous stability and biocompatibility. This combination of properties makes gallium nitride a strong candidate for use in chemical and biological applications such as sensors and neural interfaces. Molecular modification can be used to enhance the functionality and properties of the gallium nitride surface. Here, gallium nitride surfaces were functionalized with a PC12 cell adhesion promoting peptide using covalent and affinity driven attachment methods. The covalent scheme proceeded by Grignard reaction and olefin metathesis while the affinity driven scheme utilized the recognition peptide isolated through phage display. This study shows that the method of attaching the adhesion peptide influences PC12 cell adhesion and differentiation as measured by cell density and morphological analysis. Covalent attachment promoted monolayer and dispersed cell adhesion while affinity driven attachment promoted multilayer cell agglomeration. Higher cell density was observed on surfaces modified using the recognition peptide. The results suggest that the covalent and affinity driven attachment methods are both suitable for promoting PC12 cell adhesion to the gallium nitride surface, though each method may be preferentially suited for distinct applications.

Fabrication of biomolecules self-assembled on Au nanodot array for bioelectronic device.

PubMed

Lee, Taek; Kumar, Ajay Yagati; Yoo, Si-Youl; Jung, Mi; Min, Junhong; Choi, Jeong-Woo

2013-09-01

In the present study, an nano-platform composed of Au nanodot arrays on which biomolecules could be self-assembled was developed and investigated for a stable bioelectronic device platform. Au nanodot pattern was fabricated using a nanoporous alumina template. Two different biomolecules, a cytochrome c and a single strand DNA (ssDNA), were immobilized on the Au nanodot arrays. Cytochorme c and single stranded DNA could be immobilized on the Au nanodot using the chemical linker 11-MUA and thiol-modification by covalent bonding, respectively. The atomic structure of the fabricated nano-platform device was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The electrical conductivity of biomolecules immobilized on the Au nanodot arrays was confirmed by scanning tunneling spectroscopy (STS). To investigate the activity of biomolecule-immobilized Au-nano dot array, the cyclic voltammetry was carried out. This proposed nano-platform device, which is composed of biomolecules, can be used for the construction of a novel bioelectronic device.

S-protected thiolated chitosan for oral delivery of hydrophilic macromolecules: evaluation of permeation enhancing and efflux pump inhibitory properties.

PubMed

Dünnhaupt, Sarah; Barthelmes, Jan; Rahmat, Deni; Leithner, Katharina; Thurner, Clemens C; Friedl, Heike; Bernkop-Schnürch, Andreas

2012-05-07

The objective of this study was the investigation of permeation enhancing and P-glycoprotein (P-gp) inhibition effects of a novel thiolated chitosan, the so-named S-protected thiolated chitosan. Mediated by a carbodiimide, increasing amounts of thioglycolic acid (TGA) were covalently bound to chitosan (CS) in the first step of modification. In the second step, these thiol groups of thiolated chitosan were protected by disulfide bond formation with the thiolated aromatic residue 6-mercaptonicotinamide (6-MNA). Mucoadhesive properties of all conjugates were evaluated in vitro on porcine intestinal mucosa based on tensile strength investigations. Permeation enhancing effects were evaluated ex vivo using rat intestinal mucosa and in vitro via Caco-2 cells using the hydrophilic macromolecule FD(4) as the model drug. Caco-2 cells were further used to show P-gp inhibition effects by using Rho-123 as P-gp substrate. Apparent permeability coefficients (P(app)) were calculated and compared to values obtained from each buffer control. Three different thiolated chitosans were generated in the first step of modification, which displayed increasing amounts of covalently attached free thiol groups on the polymer backbone. In the second modification step, more than 50% of these free thiol groups were covalently linked with 6-MNA. Within 3 h of permeation studies on excised rat intestine, P(app) values of all S-protected chitosans were at least 1.3-fold higher compared to those of corresponding thiomers and more than twice as high as that of unmodified chitosan. Additional permeation studies on Caco-2 cells confirmed these results. Because of the chemical modification and higher amount of reactive thiol groups, all S-protected thiolated chitosans exhibit at least 1.4-fold pronounced P-gp inhibition effects in contrast to their corresponding thiomers. These features approve S-protected thiolated chitosan as a promising excipient for various drug delivery systems providing improved permeation enhancing and efflux inhibition effects.

Formation of Covalently Modified Folding Intermediates of Simian Virus 40 Vp1 in Large T Antigen-Expressing Cells

PubMed Central

Watanabe, Marika; Phamduong, Ellen; Huang, Chu-Han; Itoh, Noriko; Bernal, Janie; Nakanishi, Akira; Rundell, Kathleen; Gjoerup, Ole

2013-01-01

The folding and pentamer assembly of the simian virus 40 (SV40) major capsid protein Vp1, which take place in the infected cytoplasm, have been shown to progress through disulfide-bonded Vp1 folding intermediates. In this report, we further demonstrate the existence of another category of Vp1 folding or assembly intermediates: the nonreducible, covalently modified mdVp1s. These species were present in COS-7 cells that expressed a recombinant SV40 Vp1, Vp1ΔC, through plasmid transfection. The mdVp1s persisted under cell and lysate treatment and SDS-PAGE conditions that are expected to have suppressed the formation of artifactual disulfide cross-links. As shown through a pulse-chase analysis, the mdVp1s were derived from the newly synthesized Vp1ΔC in the same time frame as Vp1's folding and oligomerization. The apparent covalent modifications occurred in the cytoplasm within the core region of Vp1 and depended on the coexpression of the SV40 large T antigen (LT) in the cells. Analogous covalently modified species were found with the expression of recombinant polyomavirus Vp1s and human papillomavirus L1s in COS-7 cells. Furthermore, the mdVp1s formed multiprotein complexes with LT, Hsp70, and Hsp40, and a fraction of the largest mdVp1, md4, was disulfide linked to the unmodified Vp1ΔC. Both mdVp1 formation and most of the multiprotein complex formation were blocked by a Vp1 folding mutation, C87A-C254A. Our observations are consistent with a role for LT in facilitating the folding process of SV40 Vp1 by stimulating certain covalent modifications of Vp1 or by recruiting certain cellular proteins. PMID:23427157

Microwave plasma induced surface modification of diamond-like carbon films

NASA Astrophysics Data System (ADS)

Rao Polaki, Shyamala; Kumar, Niranjan; Gopala Krishna, Nanda; Madapu, Kishore; Kamruddin, Mohamed; Dash, Sitaram; Tyagi, Ashok Kumar

2017-12-01

Tailoring the surface of diamond-like carbon (DLC) film is technically relevant for altering the physical and chemical properties, desirable for useful applications. A physically smooth and sp3 dominated DLC film with tetrahedral coordination was prepared by plasma-enhanced chemical vapor deposition technique. The surface of the DLC film was exposed to hydrogen, oxygen and nitrogen plasma for physical and chemical modifications. The surface modification was based on the concept of adsorption-desorption of plasma species and surface entities of films. Energetic chemical species of microwave plasma are adsorbed, leading to desorbtion of the surface carbon atoms due to energy and momentum exchange. The interaction of such reactive species with DLC films enhanced the roughness, surface defects and dangling bonds of carbon atoms. Adsorbed hydrogen, oxygen and nitrogen formed a covalent network while saturating the dangling carbon bonds around the tetrahedral sp3 valency. The modified surface chemical affinity depends upon the charge carriers and electron covalency of the adsorbed atoms. The contact angle of chemically reconstructed surface increases when a water droplet interacts either through hydrogen or van dear Waals bonding. These weak interactions influenced the wetting property of the DLC surface to a great extent.

Fabrication of biomembrane-like films on carbon electrodes using alkanethiol and diazonium salt and their application for direct electrochemistry of myoglobin.

PubMed

Anjum, Saima; Qi, Wenjing; Gao, Wenyue; Zhao, Jianming; Hanif, Saima; Aziz-Ur-Rehman; Xu, Guobao

2015-03-15

Alkanethiols generally form self-assembled monolayers on gold electrodes and the electrochemical reduction of aromatic diazonium salts is a popular method for the covalent modification of carbon. Based on the reaction of alkanethiol with aldehyde groups covalently bound on carbon surface by the electrochemical reduction of aromatic diazonium salts, a new strategy for the modification of carbon electrodes with alkanethiols has been developed. The modification of carbon surface with aldehyde groups is achieved by the electrochemical reduction of aromatic diazonium salts in situ electrogenerated from a nitro precursor, p-nitrophenylaldehyde, in the presence of nitrous acid. By this way, in situ electrogenerated p-aminophenyl aldehyde from p-nitrophenylaldehyde immediately reacts with nitrous acid, effectively minimizing the side reaction of amine groups and aldehyde groups. The as-prepared alkanethiol-modified glassy carbon electrode was further used to make biomembrane-like films by casting didodecyldimethylammonium bromide on its surface. The biomembrane-like films enable the direct electrochemistry of immobilized myoglobin for the detection of hydrogen peroxide. The response is linear over the range of 1-600μM with a detection limit of 0.3μM. Copyright © 2014 Elsevier B.V. All rights reserved.

Global regulation of post-translational modifications on core histones.

PubMed

Galasinski, Scott C; Louie, Donna F; Gloor, Kristen K; Resing, Katheryn A; Ahn, Natalie G

2002-01-25

Full-length masses of histones were analyzed by mass spectrometry to characterize post-translational modifications of bulk histones and their changes induced by cell stimulation. By matching masses of unique peptides with full-length masses, H4 and the variants H2A.1, H2B.1, and H3.1 were identified as the main histone forms in K562 cells. Mass changes caused by covalent modifications were measured in a dose- and time-dependent manner following inhibition of phosphatases by okadaic acid. Histones H2A, H3, and H4 underwent changes in mass consistent with altered acetylation and phosphorylation, whereas H2B mass was largely unchanged. Unexpectedly, histone H4 became almost completely deacetylated in a dose-dependent manner that occurred independently of phosphorylation. Okadaic acid also partially blocked H4 hyperacetylation induced by trichostatin-A, suggesting that the mechanism of deacetylation involves inhibition of H4 acetyltransferase activity, following perturbation of cellular phosphatases. In addition, mass changes in H3 in response to okadaic acid were consistent with phosphorylation of methylated, acetylated, and phosphorylated forms. Finally, kinetic differences were observed with respect to the rate of phosphorylation of H2A versus H4, suggesting differential regulation of phosphorylation at sites on these proteins, which are highly related by sequence. These results provide novel evidence that global covalent modifications of chromatin-bound histones are regulated through phosphorylation-dependent mechanisms.

Covalent modification of proteins by ligands of steroid hormone receptors.

PubMed Central

Takahashi, N; Breitman, T R

1992-01-01

Retinoylation, acylation with retinoic acid (RA), is a covalent modification of proteins occurring in a variety of eukaryotic cell lines. In this study, we found that proteins in HL-60 cells were labeled by 17 beta-[3H]estradiol (E2), [3H]progesterone (Pg), 1 alpha,25-dihydroxy[3H]vitamin D3 [1,25(OH)2D3], [125I]triiodothyronine (T3), [125I]thyroxine (T4), and [3H]prostaglandin E2 (PGE2). All of these hormones, except PGE2, are ligands of the steroid hormone receptor family. Addition to the growth medium of 5 microM ketoconazole, an inhibitor of cytochrome P450-dependent enzymes, increased about 2-fold the labeling of proteins by T3, T4, 1,25(OH)2D3, and PGE2. In contrast, ketoconazole did not change markedly the extent of labeling by RA, E2, or Pg. Alkaline methanolysis, which cleaves ester bonds, released variable percentages of the radioactive ligands bound to protein. These values were about 80% for RA and PGE2; 50% for T3, T4, and Pg; and 20% for E2 and 1,25(OH)2D3. Treatment with thioether-cleavage reagents, iodomethane or Raney nickel catalyst, released < 2% of the covalently bound ligands. Two-dimensional polyacrylamide gel electrophoresis patterns of labeled proteins were unique for each ligand. Proteins of M(r) 47,000 and 51,000 were labeled by RA, E2, T3, and T4. These proteins had the same mobilities as RI and RII, the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases. 1,25(OH)2D3 also bound to proteins of M(r) 47,000 and 51,000. However, these proteins had pI values different from those of RI or RII. These results suggest that some activities of ligands of the steroid hormone receptor family and of PGE2 may be mediated by their covalent modification of proteins. Images PMID:1438281

Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pletnev, Vladimir Z., E-mail: vzpletnev@gmail.com; Pletneva, Nadya V.; Lukyanov, Konstantin A.

The crystal structure of the novel red emitting fluorescent protein from lancelet Branchiostoma lanceolatum (Chordata) revealed an unusual five residues cyclic unit comprising Gly58-Tyr59-Gly60 chromophore, the following Phe61 and Tyr62 covalently bound to chromophore Tyr59. A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the ‘core’ structure, which can be extended in red-shifted proteins owing to modifications of the protein backbonemore » at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λ{sub ex}/λ{sub em} = 502/511 nm) and red laRFP (λ{sub ex}/λ{sub em} ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C{sup β} atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis.« less

Communication: Low-energy free-electron driven molecular engineering: In situ preparation of intrinsically short-lived carbon-carbon covalent dimer of CO

NASA Astrophysics Data System (ADS)

Davis, Daly; Sajeev, Y.

2017-02-01

Molecular modification induced through the resonant attachment of a low energy electron (LEE) is a novel approach for molecular engineering. In this communication, we explore the possibility to use the LEE as a quantum tool for the in situ preparation of short lived molecules. Using ab initio quantum chemical methods, this possibility is best illustrated for the in situ preparation of the intrinsically short-lived carbon-carbon covalent dimer of CO from a glyoxal molecule. The chemical conversion of glyoxal to the covalent dimer of CO is initiated and driven by the resonant capture of a near 11 eV electron by the glyoxal molecule. The resulting two-particle one-hole (2p-1h) negative ion resonant state (NIRS) of the glyoxal molecule undergoes a barrierless radical dehydrogenation reaction and produces the covalent dimer of CO. The autoionization electron spectra from the 2p-1h NIRS at the dissociation limit of the dehydrogenation reaction provides access to the electronic states of the CO dimer. The overall process is an example of a catalytic electron reaction channel.

Photophysics of covalently functionalized single wall carbon nanotubes with verteporfin

NASA Astrophysics Data System (ADS)

Staicu, Angela; Smarandache, Adriana; Pascu, Alexandru; Pascu, Mihail Lucian

2017-09-01

Covalently functionalized single wall carbon nanotubes (SWCNT) with the photosensitizer verteporfin (VP) were synthesized and studied. Photophysical properties of the obtained compounds like optical absorption, laser-induced fluorescence and generated singlet oxygen were investigated. In order to highlight the features of the conjugated compound, its photophysical characteristics were compared with those of the mixtures of the initial components. The optical absorption data evidenced a compound that combines features of the primary SWCNTs and VP. This is the also the case of the laser induced fluorescence of the synthesized product. Moreover, fluorescence quantum yield (Φf) of the compound (Φf = 2.4%) is smaller than for the mixture of SWCNT and VP in (Φf = 3.2%). The behavior is expected, because linked VP (carrying the fluorescent moiety) transfers easier a part of its excitation energy to the SWCNT in the covalent structure. Relative to the quantum yield of singlet oxygen generation (ΦΔ) by Methylene Blue, it was found that the ΦΔ for the conjugated VP-SWCNT is 51% while for the mixture ΦΔ is 23%. The results indicate covalently functionalized single walled carbon nanotubes with verteporfin as potential compounds of interest in targeted drug delivery and photodynamic therapy.

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics

PubMed Central

Smirnov, Ivan; Carletti, Eugénie; Kurkova, Inna; Nachon, Florian; Nicolet, Yvain; Mitkevich, Vladimir A.; Débat, Hélène; Avalle, Bérangère; Belogurov, Alexey A.; Kuznetsov, Nikita; Reshetnyak, Andrey; Masson, Patrick; Tonevitsky, Alexander G.; Ponomarenko, Natalia; Makarov, Alexander A.; Friboulet, Alain; Tramontano, Alfonso; Gabibov, Alexander

2011-01-01

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (VL) and variable heavy (VH) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes. PMID:21896761

Strategies for generating peptide radical cations via ion/ion reactions.

PubMed

Gilbert, Joshua D; Fisher, Christine M; Bu, Jiexun; Prentice, Boone M; Redwine, James G; McLuckey, Scott A

2015-02-01

Several approaches for the generation of peptide radical cations using ion/ion reactions coupled with either collision induced dissociation (CID) or ultraviolet photo dissociation (UVPD) are described here. Ion/ion reactions are used to generate electrostatic or covalent complexes comprised of a peptide and a radical reagent. The radical site of the reagent can be generated multiple ways. Reagents containing a carbon-iodine (C-I) bond are subjected to UVPD with 266-nm photons, which selectively cleaves the C-I bond homolytically. Alternatively, reagents containing azo functionalities are collisionally activated to yield radical sites on either side of the azo group. Both of these methods generate an initial radical site on the reagent, which then abstracts a hydrogen from the peptide while the peptide and reagent are held together by either electrostatic interactions or a covalent linkage. These methods are demonstrated via ion/ion reactions between the model peptide RARARAA (doubly protonated) and various distonic anionic radical reagents. The radical site abstracts a hydrogen atom from the peptide, while the charge site abstracts a proton. The net result is the conversion of a doubly protonated peptide to a peptide radical cation. The peptide radical cations have been fragmented via CID and the resulting product ion mass spectra are compared to the control CID spectrum of the singly protonated, even-electron species. This work is then extended to bradykinin, a more broadly studied peptide, for comparison with other radical peptide generation methods. The work presented here provides novel methods for generating peptide radical cations in the gas phase through ion/ion reaction complexes that do not require modification of the peptide in solution or generation of non-covalent complexes in the electrospray process. Copyright © 2015 John Wiley & Sons, Ltd.

The Redox Proteome*

PubMed Central

Go, Young-Mi; Jones, Dean P.

2013-01-01

The redox proteome consists of reversible and irreversible covalent modifications that link redox metabolism to biologic structure and function. These modifications, especially of Cys, function at the molecular level in protein folding and maturation, catalytic activity, signaling, and macromolecular interactions and at the macroscopic level in control of secretion and cell shape. Interaction of the redox proteome with redox-active chemicals is central to macromolecular structure, regulation, and signaling during the life cycle and has a central role in the tolerance and adaptability to diet and environmental challenges. PMID:23861437

Mixed non-covalent assemblies of ethynyl nile red and ethynyl pyrene along oligonucleotide templates.

PubMed

Ensslen, Philipp; Fritz, Yannic; Wagenknecht, Hans-Achim

2015-01-14

Ethynyl pyrene and ethynyl nile red as modifications at the 5-position of 2'-deoxyuridines self-assemble non-covalently and specifically along oligo-2'-deoxyadenosines as templates. Oligo-2'-deoxyadenosines of the lengths (dA)10-(dA)20 are able to retain nearly exactly as many ethynyl nile red units in solution as binding sites are available on these templates. In contrast, in the presence of oligo-2'-thymidines the ethynyl nile red moieties are similarly insoluble to those in the absence of any oligonucleotide and yield an aggregate. The mixed assemblies of both chromophores are highly ordered, show left-handed chirality and yield dual fluorescence. The strong excitonic coupling indicates assemblies with a high degree of order. These results show that DNA represents an important supramolecular scaffold for the templated, helical and non-covalent arrangement not only for one type of chromophore but also for mixtures of two different chromophores.

Photogeneration of singlet oxygen by the phenothiazine derivatives covalently bound to the surface-modified glassy carbon

NASA Astrophysics Data System (ADS)

Blacha-Grzechnik, Agata; Piwowar, Katarzyna; Krukiewicz, Katarzyna; Koscielniak, Piotr; Szuber, Jacek; Zak, Jerzy K.

2016-05-01

The selected group of four amine-derivatives of phenothiazine was covalently grafted to the glassy carbon surface in the four-step procedure consisting of the electrochemical reduction of the diazonium salt followed by the electrochemical and chemical post-modification steps. The proposed strategy involves the bonding of linker molecule to which the photosensitizer is attached. The synthesized organic layers were characterized by means of cyclic voltammetry, XPS and Raman Spectroscopy. It was shown that the phenothiazines immobilized via proposed strategy retain their photochemical properties and are able to generate 1O2 when activated by the laser radiation. The effectiveness of in situ singlet oxygen generation by those new solid photoactive materials was determined by means of UVVis spectroscopy. The reported, covalently modified solid surfaces may find their application as the singlet oxygen photogenerators in the fine chemicals' synthesis or in the wastewater treatment.

Covalent Bonding of Chlorogenic Acid Induces Structural Modifications on Sunflower Proteins.

PubMed

Karefyllakis, Dimitris; Salakou, Stavroula; Bitter, J Harry; van der Goot, Atze J; Nikiforidis, Constantinos V

2018-02-19

Proteins and phenols coexist in the confined space of plant cells leading to reactions between them, which result in new covalently bonded complex molecules. This kind of reactions has been widely observed during storage and processing of plant materials. However, the nature of the new complex molecules and their physicochemical properties are largely unknown. Therefore, we investigated the structural characteristics of covalently bonded complexes between sunflower protein isolate (SFPI, protein content 85 wt %) and the dominant phenol in the confined space of a sunflower seed cell (chlorogenic acid, CGA). It was shown that the efficiency of bond formation goes through a maximum as a function of the SFPI:CGA ratio. Moreover, the bonding of CGA with proteins resulted in changes in the secondary and tertiary structure of the protein. It was also shown that the phenol bound strongly to the protein, which resulted in new crosslinks between the polypeptide chains. As a result, secondary structures like α-helices and β-sheets diminished, which in turn resulted in more disordered domains and a subsequent modification of the tertiary structure of the proteins. These findings are relevant for establishing future protocols for extraction of high-quality proteins and phenols when utilizing plant material and offer insight into the impact of processing that these ingredients endure. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tailored functionalization of iron oxide nanoparticles for MRI, drug delivery, magnetic separation and immobilization of biosubstances.

PubMed

Hola, Katerina; Markova, Zdenka; Zoppellaro, Giorgio; Tucek, Jiri; Zboril, Radek

2015-11-01

In this critical review, we outline various covalent and non-covalent approaches for the functionalization of iron oxide nanoparticles (IONPs). Tuning the surface chemistry and design of magnetic nanoparticles are described in relation to their applicability in advanced medical technologies and biotechnologies including magnetic resonance imaging (MRI) contrast agents, targeted drug delivery, magnetic separations and immobilizations of proteins, enzymes, antibodies, targeting agents and other biosubstances. We review synthetic strategies for the controlled preparation of IONPs modified with frequently used functional groups including amine, carboxyl and hydroxyl groups as well as the preparation of IONPs functionalized with other species, e.g., epoxy, thiol, alkane, azide, and alkyne groups. Three main coupling strategies for linking IONPs with active agents are presented: (i) chemical modification of amine groups on the surface of IONPs, (ii) chemical modification of bioactive substances (e.g. with fluorescent dyes), and (iii) the activation of carboxyl groups mainly for enzyme immobilization. Applications for drug delivery using click chemistry linking or biodegradable bonds are compared to non-covalent methods based on polymer modified condensed magnetic nanoclusters. Among many challenges, we highlight the specific surface engineering allowing both therapeutic and diagnostic applications (theranostics) of IONPs and magnetic/metallic hybrid nanostructures possessing a huge potential in biocatalysis, green chemistry, magnetic bioseparations and bioimaging. Copyright © 2015 Elsevier Inc. All rights reserved.

Oxidized methionine is not a prion-specific covalent modification

USDA-ARS?s Scientific Manuscript database

The oxidation of methionine residues in the '-helical region of PrPC has been proposed to be important for prion formation. This proposal has been supported by structural studies, model systems and antibody-based experimental evidence. We developed a sensitive mass spectrometry-based method to stu...

Microcontact Printing Patterning of an HOPG Surface by an Inverse Electron Demand Diels-Alder Reaction.

PubMed

Zhu, Jun; Hiltz, Jonathan; Tefashe, Ushula M; Mauzeroll, Janine; Lennox, R Bruce

2018-06-21

The chemical modification of an sp 2 hybridized carbon surface in a controllable manner is very challenging but also crucial for many applications. An inverse electron demand Diels-Alder (IEDDA) reaction using microcontact printing technique is introduced to spatially control the modification of a highly ordered pyrolytic graphite (HOPG) surface under ambient conditions. The covalent modification was characterized by Raman spectroscopy, XPS, and SECM. Tetrazine derivatives can effectively react with an HOPG surface and with microcontact printing methods resulting in spatially patterned surfaces being produced with micrometer-scale resolution. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein Arginine Methylation in Mammals: Who, What, and Why

PubMed Central

Bedford, Mark T.; Clarke, Steven G.

2012-01-01

The covalent marking of proteins by methyl group addition to arginine residues can promote their recognition by binding partners or can modulate their biological activity. A small family of gene products that catalyze such methylation reactions in eukaryotes (PRMTs) work in conjunction with a changing cast of associated subunits to recognize distinct cellular substrates. These reactions display many of the attributes of reversible covalent modifications such as protein phosphorylation or protein lysine methylation; however, it is unclear to what extent protein arginine demethylation occurs. Physiological roles for protein arginine methylation have been established in signal transduction, mRNA splicing, transcriptional control, DNA repair, and protein translocation. PMID:19150423

Protein mass analysis of histones.

PubMed

Galasinski, Scott C; Resing, Katheryn A; Ahn, Natalie G

2003-09-01

Posttranslational modification of chromatin-associated proteins, including histones and high-mobility-group (HMG) proteins, provides an important mechanism to control gene expression, genome integrity, and epigenetic inheritance. Protein mass analysis provides a rapid and unbiased approach to monitor multiple chemical modifications on individual molecules. This review describes methods for acid extraction of histones and HMG proteins, followed by separation by reverse-phase chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS). Posttranslational modifications are detected by analysis of full-length protein masses. Confirmation of protein identity and modification state is obtained through enzymatic digestion and peptide sequencing by MS/MS. For differentially modified forms of each protein, the measured intensities are semiquantitative and allow determination of relative abundance and stoichiometry. The method simultaneously detects covalent modifications on multiple proteins and provides a facile assay for comparing chromatin modification states between different cell types and/or cellular responses.

Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol).

PubMed

Murad, K L; Mahany, K L; Brugnara, C; Kuypers, F A; Eaton, J W; Scott, M D

1999-03-15

We previously showed that the covalent modification of the red blood cell (RBC) surface with methoxypoly(ethylene glycol) [mPEG; MW approximately 5 kD] could significantly attenuate the immunologic recognition of surface antigens. However, to make these antigenically silent RBC a clinically viable option, the mPEG-modified RBC must maintain normal cellular structure and functions. To this end, mPEG-derivatization was found to have no significant detrimental effects on RBC structure or function at concentrations that effectively blocked antigenic recognition of a variety of RBC antigens. Importantly, RBC lysis, morphology, and hemoglobin oxidation state were unaffected by mPEG-modification. Furthermore, as shown by functional studies of Band 3, a major site of modification, PEG-binding does not affect protein function, as evidenced by normal SO4- flux. Similarly, Na+ and K+ homeostasis were unaffected. The functional aspects of the mPEG-modified RBC were also maintained, as evidenced by normal oxygen binding and cellular deformability. Perhaps most importantly, mPEG-derivatized mouse RBC showed normal in vivo survival ( approximately 50 days) with no sensitization after repeated transfusions. These data further support the hypothesis that the covalent attachment of nonimmunogenic materials (eg, mPEG) to intact RBC may have significant application in transfusion medicine, especially for the chronically transfused and/or allosensitized patient.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick

Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugationmore » to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.« less

The PA influenza virus polymerase subunit is a phosphorylated protein.

PubMed

Sanz-Ezquerro, J J; Fernández Santarén, J; Sierra, T; Aragón, T; Ortega, J; Ortín, J; Smith, G L; Nieto, A

1998-03-01

The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by two-dimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [32P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic alpha subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PA subunit of the influenza virus RNA polymerase is a phosphoprotein.

A regenerating ultrasensitive electrochemical impedance immunosensor for the detection of adenovirus.

PubMed

Lin, Donghai; Tang, Thompson; Jed Harrison, D; Lee, William E; Jemere, Abebaw B

2015-06-15

We report on the development of a regenerable sensitive immunosensor based on electrochemical impedance spectroscopy for the detection of type 5 adenovirus. The multi-layered immunosensor fabrication involved successive modification steps on gold electrodes: (i) modification with self-assembled layer of 1,6-hexanedithiol to which gold nanoparticles were attached via the distal thiol groups, (ii) formation of self-assembled monolayer of 11-mercaptoundecanoic acid onto the gold nanoparticles, (iii) covalent immobilization of monoclonal anti-adenovirus 5 antibody, with EDC/NHS coupling reaction on the nanoparticles, completing the immunosensor. The immunosensor displayed a very good detection limit of 30 virus particles/ml and a wide linear dynamic range of 10(5). An electrochemical reductive desorption technique was employed to completely desorb the components of the immunosensor surface, then re-assemble the sensing layer and reuse the sensor. On a single electrode, the multi-layered immunosensor could be assembled and disassembled at least 30 times with 87% of the original signal intact. The changes of electrode behavior after each assembly and desorption processes were investigated by cyclic voltammetry, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy techniques. Copyright © 2014 Elsevier B.V. All rights reserved.

Single cell and single molecule techniques for the analysis of the epigenome

NASA Astrophysics Data System (ADS)

Wallin, Christopher Benjamin

Epigenetic regulation is a critical biological process for the health and development of a cell. Epigenetic regulation is facilitated by covalent modifications to the underlying DNA and chromatin proteins. A fundamental understanding of these epigenetic modifications and their associated interactions at the molecular scale is necessary to explain phenomena including cellular identity, stem cell plasticity, and neoplastic transformation. It is widely known that abnormal epigenetic profiles have been linked to many diseases, most notably cancer. While the field of epigenetics has progressed rapidly with conventional techniques, significant advances remain to be made with respect to combinatoric analysis of epigenetic marks and single cell epigenetics. Therefore, in this dissertation, I will discuss our development of devices and methodologies to address these pertinent issues. First, we designed a preparatory polydimethylsiloxane (PDMS) microdevice for the extraction, purification, and stretching of human chromosomal DNA and chromatin from small cell populations down to a single cell. The valveless device captures cells by size exclusion within the micropillars, entraps the DNA or chromatin in the micropillars after cell lysis, purifies away the cellular debris, and fluorescently labels the DNA and/or chromatin all within a single reaction chamber. With the device, we achieve nearly 100% extraction efficiency of the DNA. The device is also used for in-channel immunostaining of chromatin followed by downstream single molecule chromatin analysis in nanochannels (SCAN). Second, using multi-color, time-correlated single molecule measurements in nanochannels, simultaneous coincidence detection of 2 epigenetic marks is demonstrated. Coincidence detection of 3 epigenetic marks is also established using a pulsed interleaved excitation scheme. With these two promising results, genome-wide quantification of epigenetic marks was pursued. Unfortunately, quantitative SCAN never materialized. Reasons for this, including poor signal to background, are explained in detail. Third, development of mobility-SCAN, an analytical technique for measuring and analyzing single molecules based on their fluorescent signature and their electrophoretic mobility in nanochannels is described. We use the technique to differentiate biomolecules from complex mixtures and derive parameters such as diffusion coefficients and effective charges. Finally, the device is used to detect binding interactions of various complexes similar to affinity capillary electrophoresis, but on a single molecule level. Fourth, we conclude by briefly discussing SCAN-sort, a technique to sort individual chromatin molecules based on their fluorescent emissions for further downstream analysis such as DNA sequencing. We demonstrate a 2-fold enrichment of chromatin from sorting and discuss possible system modifications for better performance in the future.

The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

PubMed Central

Cross, Janet V; Foss, Frank W; Rady, Joshua M; Macdonald, Timothy L; Templeton, Dennis J

2007-01-01

Background Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. Methods The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. Results ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. Conclusion These results demonstrate that MEKK1 is directly modified and inhibited by ITCs, and that this correlates with inhibition of downstream activation of SAPK. These results support the conclusion that ITCs may carry out many of their actions by directly targeting important cell regulatory proteins. PMID:17894894

Use of Functionalized Carbon Nanotubes for Covalent Attachment of Nanotubes to Silicon

NASA Technical Reports Server (NTRS)

Tour, James M.; Dyke, Christopher A.; Maya, Francisco; Stewart, Michael P.; Chen, Bo; Flatt, Austen K.

2012-01-01

The purpose of the invention is to covalently attach functionalized carbon nanotubes to silicon. This step allows for the introduction of carbon nanotubes onto all manner of silicon surfaces, and thereby introduction of carbon nano - tubes covalently into silicon-based devices, onto silicon particles, and onto silicon surfaces. Single-walled carbon nanotubes (SWNTs) dispersed as individuals in surfactant were functionalized. The nano - tube was first treated with 4-t-butylbenzenediazonium tetrafluoroborate to give increased solubility to the carbon nanotube; the second group attached to the sidewall of the nanotube has a silyl-protected terminal alkyne that is de-protected in situ. This gives a soluble carbon nanotube that has functional groups appended to the sidewall that can be attached covalently to silicon. This reaction was monitored by UV/vis/NJR to assure direct covalent functionalization.

Removal of Covalent Heterogeneity Reveals Simple Folding Behavior for P4-P6 RNA*

PubMed Central

Greenfeld, Max; Solomatin, Sergey V.; Herschlag, Daniel

2011-01-01

RNA folding landscapes have been described alternately as simple and as complex. The limited diversity of RNA residues and the ability of RNA to form stable secondary structures prior to adoption of a tertiary structure would appear to simplify folding relative to proteins. Nevertheless, there is considerable evidence for long-lived misfolded RNA states, and these observations have suggested rugged energy landscapes. Recently, single molecule fluorescence resonance energy transfer (smFRET) studies have exposed heterogeneity in many RNAs, consistent with deeply furrowed rugged landscapes. We turned to an RNA of intermediate complexity, the P4-P6 domain from the Tetrahymena group I intron, to address basic questions in RNA folding. P4-P6 exhibited long-lived heterogeneity in smFRET experiments, but the inability to observe exchange in the behavior of individual molecules led us to probe whether there was a non-conformational origin to this heterogeneity. We determined that routine protocols in RNA preparation and purification, including UV shadowing and heat annealing, cause covalent modifications that alter folding behavior. By taking measures to avoid these treatments and by purifying away damaged P4-P6 molecules, we obtained a population of P4-P6 that gave near-uniform behavior in single molecule studies. Thus, the folding landscape of P4-P6 lacks multiple deep furrows that would trap different P4-P6 molecules in different conformations and contrasts with the molecular heterogeneity that has been seen in many smFRET studies of structured RNAs. The simplicity of P4-P6 allowed us to reliably determine the thermodynamic and kinetic effects of metal ions on folding and to now begin to build more detailed models for RNA folding behavior. PMID:21478155

Effect of Carboxymethylation on the Rheological Properties of Hyaluronan

PubMed Central

Wendling, Rian J.; Christensen, Amanda M.; Quast, Arthur D.; Atzet, Sarah K.; Mann, Brenda K.

2016-01-01

Chemical modifications made to hyaluronan to enable covalent crosslinking to form a hydrogel or to attach other molecules may alter the physical properties as well, which have physiological importance. Here we created carboxymethyl hyaluronan (CMHA) with varied degree of modification and investigated the effect on the viscosity of CMHA solutions. Viscosity decreased initially as modification increased, with a minimum viscosity for about 30–40% modification. This was followed by an increase in viscosity around 45–50% modification. The pH of the solution had a variable effect on viscosity, depending on the degree of carboxymethyl modification and buffer. The presence of phosphates in the buffer led to decreased viscosity. We also compared large-scale production lots of CMHA to lab-scale and found that large-scale required extended reaction times to achieve the same degree of modification. Finally, thiolated CMHA was disulfide crosslinked to create hydrogels with increased viscosity and shear-thinning aspects compared to CMHA solutions. PMID:27611817

Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

NASA Astrophysics Data System (ADS)

El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

2016-02-01

We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

PubMed Central

El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

2016-01-01

We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification. PMID:26899743

Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance.

PubMed

Kang, Yoo Kyung; Kwon, Kyu; Ryu, Jea Sung; Lee, Ha Neul; Park, Chankyu; Chung, Hyun Jung

2017-04-19

The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA-the major gene involved in methicillin resistance-can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.

Covalent modification of pericardial patches for sustained rapamycin delivery inhibits venous neointimal hyperplasia

PubMed Central

Bai, Hualong; Lee, Jung Seok; Chen, Elizabeth; Wang, Mo; Xing, Ying; Fahmy, Tarek M.; Dardik, Alan

2017-01-01

Prosthetic grafts and patches are commonly used in cardiovascular surgery, however neointimal hyperplasia remains a significant concern, especially under low flow conditions. We hypothesized that delivery of rapamycin from nanoparticles (NP) covalently attached to patches allows sustained site-specific delivery of therapeutic agents targeted to inhibit localized neointimal hyperplasia. NP were covalently linked to pericardial patches using EDC/NHS chemistry and could deliver at least 360 ng rapamycin per patch without detectable rapamycin in serum; nanoparticles were detectable in the liver, kidney and spleen but no other sites within 24 hours. In a rat venous patch angioplasty model, control patches developed robust neointimal hyperplasia on the patch luminal surface characterized by Eph-B4-positive endothelium and underlying SMC and infiltrating cells such as macrophages and leukocytes. Patches delivering rapamycin developed less neointimal hyperplasia, less smooth muscle cell proliferation, and had fewer infiltrating cells but retained endothelialization. NP covalently linked to pericardial patches are a novel composite delivery system that allows sustained site-specific delivery of therapeutics; NP delivering rapamycin inhibit patch neointimal hyperplasia. NP linked to patches may represent a next generation of tissue engineered cardiovascular implants. PMID:28071663

Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging

PubMed Central

Liu, Wenhao; Howarth, Mark; Greytak, Andrew B.; Zheng, Yi; Nocera, Daniel G.; Ting, Alice Y.; Bawendi, Moungi G.

2009-01-01

We present a family of water-soluble quantum dots (QDs) that exhibit low nonspecific binding to cells, small hydrodynamic diameter, tunable surface charge, high quantum yield, and good solution stability across a wide pH range. These QDs are amenable to covalent modification via simple carbodiimide coupling chemistry, which is achieved by functionalizing the surface of QDs with a new class of heterobifunctional ligands incorporating dihydrolipoic acid, a short poly(ethylene glycol) (PEG) spacer, and an amine or carboxylate terminus. The covalent attachment of molecules is demonstrated by appending a rhodamine dye to form a QD-dye conjugate exhibiting fluorescence resonance energy transfer (FRET). High-affinity labeling is demonstrated by covalent attachment of streptavidin, thus enabling the tracking of biotinylated epidermal growth factor (EGF) bound to EGF receptor on live cells. In addition, QDs solubilized with the heterobifunctional ligands retain their metal-affinity driven conjugation chemistry with polyhistidine-tagged proteins. This dual functionality is demonstrated by simultaneous covalent attachment of a rhodamine FRET acceptor and binding of polyhistidine-tagged streptavidin on the same nanocrystal to create a targeted QD, which exhibits dual-wavelength emission. Such emission properties could serve as the basis for ratiometric sensing of the cellular receptor’s local chemical environment. PMID:18177042

Covalent modification of pericardial patches for sustained rapamycin delivery inhibits venous neointimal hyperplasia

NASA Astrophysics Data System (ADS)

Bai, Hualong; Lee, Jung Seok; Chen, Elizabeth; Wang, Mo; Xing, Ying; Fahmy, Tarek M.; Dardik, Alan

2017-01-01

Prosthetic grafts and patches are commonly used in cardiovascular surgery, however neointimal hyperplasia remains a significant concern, especially under low flow conditions. We hypothesized that delivery of rapamycin from nanoparticles (NP) covalently attached to patches allows sustained site-specific delivery of therapeutic agents targeted to inhibit localized neointimal hyperplasia. NP were covalently linked to pericardial patches using EDC/NHS chemistry and could deliver at least 360 ng rapamycin per patch without detectable rapamycin in serum; nanoparticles were detectable in the liver, kidney and spleen but no other sites within 24 hours. In a rat venous patch angioplasty model, control patches developed robust neointimal hyperplasia on the patch luminal surface characterized by Eph-B4-positive endothelium and underlying SMC and infiltrating cells such as macrophages and leukocytes. Patches delivering rapamycin developed less neointimal hyperplasia, less smooth muscle cell proliferation, and had fewer infiltrating cells but retained endothelialization. NP covalently linked to pericardial patches are a novel composite delivery system that allows sustained site-specific delivery of therapeutics; NP delivering rapamycin inhibit patch neointimal hyperplasia. NP linked to patches may represent a next generation of tissue engineered cardiovascular implants.

Acetaminophen structure-toxicity studies: In vivo covalent binding of a nonhepatotoxic analog, 3-hydroxyacetanilide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roberts, S.A.; Price, V.F.; Jollow, D.J.

1990-09-01

High doses of 3-hydroxyacetanilide (3HAA), a structural isomer of acetaminophen, do not produce hepatocellular necrosis in normal male hamsters or in those sensitized to acetaminophen-induced liver damage by pretreatment with a combination of 3-methylcholanthrene, borneol, and diethyl maleate. Although 3HAA was not hepatotoxic, the administration of acetyl-labeled (3H or 14C)3HAA (400 mg/kg, ip) produced levels of covalently bound radiolabel that were similar to those observed after an equimolar, hepatotoxic dose of (G-3H)acetaminophen. The covalent nature of 3HAA binding was demonstrated by retention of the binding after repetitive organic solvent extraction following protease digestion. Hepatic and renal covalent binding after 3HAAmore » was approximately linear with both dose and time. In addition, 3HAA produced only a modest depletion of hepatic glutathione, suggesting the lack of a glutathione threshold. 3-Methylcholanthrene pretreatment increased and pretreatment with cobalt chloride and piperonyl butoxide decreased the hepatic covalent binding of 3HAA, indicating the involvement of cytochrome P450 in the formation of the 3HAA reactive metabolite. The administration of multiple doses or a single dose of (ring-3H)3HAA to hamsters pretreated with a combination of 3-methylcholanthrene, borneol, and diethyl maleate produced hepatic levels of 3HAA covalent binding that were in excess of those observed after a single, hepatotoxic acetaminophen dose. These data suggest that the nature and/or the intracellular processing of the reactive metabolites of acetaminophen and 3HAA are different. These data also demonstrate that absolute levels of covalently bound xenobiotic metabolites cannot be utilized as absolute predictors of cytotoxic potential.« less

Regulating the Regulator: Post-Translational Modification of Ras

PubMed Central

Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.

2013-01-01

Ras proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on Ras is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which regulate the activation state of Ras without covalently modifying it. In contrast, post-translational modifications (PTMs) of Ras proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important Ras PTMs include the constitutive and irreversible remodelling of its C-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications including phosphorylation, peptidyl-proly isomerisation, mono- and di-ubiquitination, nitrosylation, ADP ribosylation and glucosylation. PMID:22189424

Plasma immersion ion implantation of polyurethane shape memory polymer: Surface properties and protein immobilization

NASA Astrophysics Data System (ADS)

Cheng, Xinying; Kondyurin, Alexey; Bao, Shisan; Bilek, Marcela M. M.; Ye, Lin

2017-09-01

Polyurethane-type shape memory polymers (SMPU) are promising biomedical implant materials due to their ability to recover to a predetermined shape from a temporary shape induced by thermal activation close to human body temperature and their advantageous mechanical properties including large recovery strains and low recovery stresses. Plasma Immersion Ion Implantation (PIII) is a surface modification process using energetic ions that generates radicals in polymer surfaces leading to carbonisation and oxidation and the ability to covalently immobilise proteins without the need for wet chemistry. Here we show that PIII treatment of SMPU significantly enhances its bioactivity making SMPU suitable for applications in permanent implantable biomedical devices. Scanning Electron Microscopy (SEM), contact angle measurements, surface energy measurements, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to characterise the PIII modified surface, including its after treatment aging kinetics and its capability to covalently immobilise protein directly from solution. The results show a substantial improvement in wettability and dramatic changes of surface chemical composition dependent on treatment duration, due to the generation of radicals and subsequent oxidation. The SMPU surface, PIII treated for 200s, achieved a saturated level of covalently immobilized protein indicating that a full monolayer coverage was achieved. We conclude that PIII is a promising and efficient surface modification method to enhance the biocompatibility of SMPU for use in medical applications that demand bioactivity for tissue integration and stability in vivo.

Covalent modification of a ten-residue cationic antimicrobial peptide with levofloxacin

NASA Astrophysics Data System (ADS)

Rodriguez, Carlos; Papanastasiou, Emilios; Juba, Melanie; Bishop, Barney

2014-09-01

The rampant spread of antibiotic resistant bacteria has spurred interest in alternative strategies for developing next-generation antibacterial therapies. As such, there has been growing interest in cationic antimicrobial peptides (CAMPs) and their therapeutic applications. Modification of CAMPs via conjugation to auxiliary compounds, including small molecule drugs, is a new approach to developing effective, broad-spectrum antibacterial agents with novel physicochemical properties and versatile antibacterial mechanisms. Here, we’ve explored design parameters for engineering CAMPs conjugated to small molecules with favorable physicochemical and antibacterial properties by covalently affixing a fluoroquinolone antibiotic, levofloxacin, to the ten-residue CAMP Pep-4. Relative to the unmodified Pep-4, the conjugate was found to demonstrate substantially increased antibacterial potency under high salt concentrations. Historically, it has been observed that most CAMPs lose antibacterial effectiveness in such high ionic strength environments, a fact that has presented a challenge to their development as therapeutics. Physicochemical studies revealed that P4LC was more hydrophobic than Pep-4, while mechanistic findings indicated that the conjugate was more effective at disrupting bacterial membrane integrity. Although the inherent antibacterial effect of the incorporated levofloxacin molecules did not appear to be substantially realized in this conjugate, these findings nevertheless suggest that covalent attachment of small molecule antibiotics with favorable physicochemical properties to CAMPs could be a promising strategy for enhancing peptide performance and overall therapeutic potential. These results have broader applicability to the development of future CAMP-antibiotic conjugates for potential therapeutic applications.

Small ubiquitin-related modifier is secreted and shows cytokine-like activity.

PubMed

Hosono, Hidetaka; Yokosawa, Hideyoshi

2008-05-01

Small ubiquitin-related modifier (SUMO) is a type I ubiquitin-like protein family member and is covalently attached to various target proteins. Through this post-translational modification, SUMO plays important roles in various cellular events. Here, we show that SUMO is secreted from cultured cells in an endoplasmic reticulum (ER)/Golgi-independent manner and that this secretion occurs without covalent binding to target proteins or chain formation. Overexpression experiments using C-terminally truncated mutants of SUMO revealed that the secretion requires the C-terminal sequence. Recombinant SUMO-3 protein was capable of binding to and promoting the proliferation of cultured cells. Thus, we propose that SUMO functions as a cytokine-like molecule extracellularly.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vega-Arroyo, M.; LeBreton, P. R.; Zapol, P.

Photoinduced charge separation in triads of DNA covalently linked to an anatase nanoparticle via a dopamine bridge was studied by ab initio calculations of the oxidation potentials of carboxyl-DNA trimers and the TiO2/dopamine complex. Conjugation of dopamine to the TiO2 surface results in a lower oxidation potential of the complex relative to the surface and in localization of photogenerated holes on dopamine, while photogenerated electrons are excited into the conduction band of TiO2. Linking dopamine to the DNA trimers at the 5? end of the oligonucleotide may lead to further hole migration to the DNA. Calculations show that for severalmore » different sequences hole migration is favorable in double stranded DNA and unfavorable in single-stranded DNA. This extended charge separation was shown to follow from the redox properties of DNA sequence rather than from the modification of DNA's electron donating properties by the dopamine linker, which explains experimental observations.« less

Whole genome DNA methylation: beyond genes silencing.

PubMed

Tirado-Magallanes, Roberto; Rebbani, Khadija; Lim, Ricky; Pradhan, Sriharsa; Benoukraf, Touati

2017-01-17

The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation at near base pair level resolution, far beyond that of the kilobase-long canonical CpG islands that initially revealed the biological relevance of this covalent DNA modification. The latest high-resolution studies have revealed a role for very punctual DNA methylation in chromatin plasticity, gene regulation and splicing. Here, we aim to outline the major biological consequences of DNA methylation recently discovered. We also discuss the necessity of tuning DNA methylation resolution into an adequate scale to ease the integration of the methylome information with other chromatin features and transcription events such as gene expression, nucleosome positioning, transcription factors binding dynamic, gene splicing and genomic imprinting. Finally, our review sheds light on DNA methylation heterogeneity in cell population and the different approaches used for its assessment, including the contribution of single cell DNA analysis technology.

Whole genome DNA methylation: beyond genes silencing

PubMed Central

Tirado-Magallanes, Roberto; Rebbani, Khadija; Lim, Ricky; Pradhan, Sriharsa; Benoukraf, Touati

2017-01-01

The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation at near base pair level resolution, far beyond that of the kilobase-long canonical CpG islands that initially revealed the biological relevance of this covalent DNA modification. The latest high-resolution studies have revealed a role for very punctual DNA methylation in chromatin plasticity, gene regulation and splicing. Here, we aim to outline the major biological consequences of DNA methylation recently discovered. We also discuss the necessity of tuning DNA methylation resolution into an adequate scale to ease the integration of the methylome information with other chromatin features and transcription events such as gene expression, nucleosome positioning, transcription factors binding dynamic, gene splicing and genomic imprinting. Finally, our review sheds light on DNA methylation heterogeneity in cell population and the different approaches used for its assessment, including the contribution of single cell DNA analysis technology. PMID:27895318

Integrated Affinity Biosensing Platforms on Screen-Printed Electrodes Electrografted with Diazonium Salts

PubMed Central

Yáñez-Sedeño, Paloma

2018-01-01

Adequate selection of the electrode surface and the strategies for its modification to enable subsequent immobilization of biomolecules and/or nanomaterials integration play a major role in the performance of electrochemical affinity biosensors. Because of the simplicity, rapidity and versatility, electrografting using diazonium salt reduction is among the most currently used functionalization methods to provide the attachment of an organic layer to a conductive substrate. This particular chemistry has demonstrated to be a powerful tool to covalently immobilize in a stable and reproducible way a wide range of biomolecules or nanomaterials onto different electrode surfaces. Considering the great progress and interesting features arisen in the last years, this paper outlines the potential of diazonium chemistry to prepare single or multianalyte electrochemical affinity biosensors on screen-printed electrodes (SPEs) and points out the existing challenges and future directions in this field. PMID:29495294

Integrated Affinity Biosensing Platforms on Screen-Printed Electrodes Electrografted with Diazonium Salts.

PubMed

Yáñez-Sedeño, Paloma; Campuzano, Susana; Pingarrón, José M

2018-02-24

Adequate selection of the electrode surface and the strategies for its modification to enable subsequent immobilization of biomolecules and/or nanomaterials integration play a major role in the performance of electrochemical affinity biosensors. Because of the simplicity, rapidity and versatility, electrografting using diazonium salt reduction is among the most currently used functionalization methods to provide the attachment of an organic layer to a conductive substrate. This particular chemistry has demonstrated to be a powerful tool to covalently immobilize in a stable and reproducible way a wide range of biomolecules or nanomaterials onto different electrode surfaces. Considering the great progress and interesting features arisen in the last years, this paper outlines the potential of diazonium chemistry to prepare single or multianalyte electrochemical affinity biosensors on screen-printed electrodes (SPEs) and points out the existing challenges and future directions in this field.

Absolute quantitation of NAPQI-modified rat serum albumin by LC-MS/MS: monitoring acetaminophen covalent binding in vivo.

PubMed

LeBlanc, André; Shiao, Tze Chieh; Roy, René; Sleno, Lekha

2014-09-15

Acetaminophen is known to cause hepatoxicity via the formation of a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liver proteins. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream and is therefore a potential biomarker to assess the levels of protein modification by NAPQI. A newly developed method for the absolute quantitation of serum albumin containing NAPQI covalently bound to its active site cysteine (Cys34) is described. This optimized assay represents the first absolute quantitation of a modified protein, with very low stoichiometric abundance, using a protein-level standard combined with isotope dilution. The LC-MS/MS assay is based on a protein standard modified with a custom-designed reagent, yielding a surrogate peptide (following digestion) that is a positional isomer to the target peptide modified by NAPQI. To illustrate the potential of this approach, the method was applied to quantify NAPQI-modified SA in plasma from rats dosed with acetaminophen. The resulting method is highly sensitive (capable of quantifying down to 0.0006% of total RSA in its NAPQI-modified form) and yields excellent precision and accuracy statistics. A time-course pharmacokinetic study was performed to test the usefulness of this method for following acetaminophen-induced covalent binding at four dosing levels (75-600 mg/kg IP), showing the viability of this approach to directly monitor in vivo samples. This approach can reliably quantify NAPQI-modified albumin, allowing direct monitoring of acetaminophen-related covalent binding.

Preparation of sumoylated substrates for biochemical analysis.

PubMed

Knipscheer, Puck; Klug, Helene; Sixma, Titia K; Pichler, Andrea

2009-01-01

Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via "loss of modification" analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a "gain in modification" analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.

Post-Training Intrahippocampal Inhibition of Class I Histone Deacetylases Enhances Long-Term Object-Location Memory

ERIC Educational Resources Information Center

Hawk, Joshua D.; Florian, Cedrick; Abel, Ted

2011-01-01

Long-term memory formation involves covalent modification of the histone proteins that package DNA. Reducing histone acetylation by mutating histone acetyltransferases impairs long-term memory, and enhancing histone acetylation by inhibiting histone deacetylases (HDACs) improves long-term memory. Previous studies using HDAC inhibitors to enhance…

Exploring Covalent Allosteric Inhibition of Antigen 85C from Mycobacterium tuberculosis by Ebselen Derivatives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goins, Christopher M.; Dajnowicz, Steven; Thanna, Sandeep

Previous studies identified ebselen as a potent in vitro and in vivo inhibitor of the Mycobacterium tuberculosis ( Mtb) antigen 85 (Ag85) complex, comprising three homologous enzymes required for the biosynthesis of the mycobacterial cell wall. In this study, the Mtb Ag85C enzyme was cocrystallized with azido and adamantyl ebselen derivatives, resulting in two crystallographic structures of 2.01 and 1.30 Å resolution, respectively. Both structures displayed the anticipated covalent modification of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide bond. Continuous difference density for both thiol modifiers allowed for the assessment of interactions that influence ebselen binding and inhibitormore » orientation that were unobserved in previous Ag85C ebselen structures. The k inact/ K I values for ebselen, adamantyl ebselen, and azido ebselen support the importance of observed constructive chemical interactions with Arg239 for increased in vitro efficacy toward Ag85C. To better understand the in vitro kinetic properties of these ebselen derivatives, the energetics of specific protein–inhibitor interactions and relative reaction free energies were calculated for ebselen and both derivatives using density functional theory. These studies further support the different in vitro properties of ebselen and two select ebselen derivatives from our previously published ebselen library with respect to kinetics and protein–inhibitor interactions. In both structures, the α9 helix was displaced farther from the enzyme active site than the previous Ag85C ebselen structure, resulting in the restructuring of a connecting loop and imparting a conformational change to residues believed to play a role in substrate binding specific to Ag85C. These notable structural changes directly affect protein stability, reducing the overall melting temperature by up to 14.5 °C, resulting in the unfolding of protein at physiological temperatures. Additionally, this structural rearrangement due to covalent allosteric modification creates a sizable solvent network that encompasses the active site and extends to the modified Cys209 residue. In all, this study outlines factors that influence enzyme inhibition by ebselen and its derivatives while further highlighting the effects of the covalent modification of Cys209 by said inhibitors on the structure and stability of Ag85C. Moreover, the results suggest a strategy for developing new classes of Ag85 inhibitors with increased specificity and potency.« less

Exploring Covalent Allosteric Inhibition of Antigen 85C from Mycobacterium tuberculosis by Ebselen Derivatives.

PubMed

Goins, Christopher M; Dajnowicz, Steven; Thanna, Sandeep; Sucheck, Steven J; Parks, Jerry M; Ronning, Donald R

2017-05-12

Previous studies identified ebselen as a potent in vitro and in vivo inhibitor of the Mycobacterium tuberculosis (Mtb) antigen 85 (Ag85) complex, comprising three homologous enzymes required for the biosynthesis of the mycobacterial cell wall. In this study, the Mtb Ag85C enzyme was cocrystallized with azido and adamantyl ebselen derivatives, resulting in two crystallographic structures of 2.01 and 1.30 Å resolution, respectively. Both structures displayed the anticipated covalent modification of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide bond. Continuous difference density for both thiol modifiers allowed for the assessment of interactions that influence ebselen binding and inhibitor orientation that were unobserved in previous Ag85C ebselen structures. The k inact /K I values for ebselen, adamantyl ebselen, and azido ebselen support the importance of observed constructive chemical interactions with Arg239 for increased in vitro efficacy toward Ag85C. To better understand the in vitro kinetic properties of these ebselen derivatives, the energetics of specific protein-inhibitor interactions and relative reaction free energies were calculated for ebselen and both derivatives using density functional theory. These studies further support the different in vitro properties of ebselen and two select ebselen derivatives from our previously published ebselen library with respect to kinetics and protein-inhibitor interactions. In both structures, the α9 helix was displaced farther from the enzyme active site than the previous Ag85C ebselen structure, resulting in the restructuring of a connecting loop and imparting a conformational change to residues believed to play a role in substrate binding specific to Ag85C. These notable structural changes directly affect protein stability, reducing the overall melting temperature by up to 14.5 °C, resulting in the unfolding of protein at physiological temperatures. Additionally, this structural rearrangement due to covalent allosteric modification creates a sizable solvent network that encompasses the active site and extends to the modified Cys209 residue. In all, this study outlines factors that influence enzyme inhibition by ebselen and its derivatives while further highlighting the effects of the covalent modification of Cys209 by said inhibitors on the structure and stability of Ag85C. Furthermore, the results suggest a strategy for developing new classes of Ag85 inhibitors with increased specificity and potency.

Exploring Covalent Allosteric Inhibition of Antigen 85C from Mycobacterium tuberculosis by Ebselen Derivatives

DOE PAGES

Goins, Christopher M.; Dajnowicz, Steven; Thanna, Sandeep; ...

2017-03-13

Previous studies identified ebselen as a potent in vitro and in vivo inhibitor of the Mycobacterium tuberculosis ( Mtb) antigen 85 (Ag85) complex, comprising three homologous enzymes required for the biosynthesis of the mycobacterial cell wall. In this study, the Mtb Ag85C enzyme was cocrystallized with azido and adamantyl ebselen derivatives, resulting in two crystallographic structures of 2.01 and 1.30 Å resolution, respectively. Both structures displayed the anticipated covalent modification of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide bond. Continuous difference density for both thiol modifiers allowed for the assessment of interactions that influence ebselen binding and inhibitormore » orientation that were unobserved in previous Ag85C ebselen structures. The k inact/ K I values for ebselen, adamantyl ebselen, and azido ebselen support the importance of observed constructive chemical interactions with Arg239 for increased in vitro efficacy toward Ag85C. To better understand the in vitro kinetic properties of these ebselen derivatives, the energetics of specific protein–inhibitor interactions and relative reaction free energies were calculated for ebselen and both derivatives using density functional theory. These studies further support the different in vitro properties of ebselen and two select ebselen derivatives from our previously published ebselen library with respect to kinetics and protein–inhibitor interactions. In both structures, the α9 helix was displaced farther from the enzyme active site than the previous Ag85C ebselen structure, resulting in the restructuring of a connecting loop and imparting a conformational change to residues believed to play a role in substrate binding specific to Ag85C. These notable structural changes directly affect protein stability, reducing the overall melting temperature by up to 14.5 °C, resulting in the unfolding of protein at physiological temperatures. Additionally, this structural rearrangement due to covalent allosteric modification creates a sizable solvent network that encompasses the active site and extends to the modified Cys209 residue. In all, this study outlines factors that influence enzyme inhibition by ebselen and its derivatives while further highlighting the effects of the covalent modification of Cys209 by said inhibitors on the structure and stability of Ag85C. Moreover, the results suggest a strategy for developing new classes of Ag85 inhibitors with increased specificity and potency.« less

Epigenetic control of plant immunity.

PubMed

Alvarez, María E; Nota, Florencia; Cambiagno, Damián A

2010-07-01

In eukaryotic genomes, gene expression and DNA recombination are affected by structural chromatin traits. Chromatin structure is shaped by the activity of enzymes that either introduce covalent modifications in DNA and histone proteins or use energy from ATP to disrupt histone-DNA interactions. The genomic 'marks' that are generated by covalent modifications of histones and DNA, or by the deposition of histone variants, are susceptible to being altered in response to stress. Recent evidence has suggested that proteins generating these epigenetic marks play crucial roles in the defence against pathogens. Histone deacetylases are involved in the activation of jasmonic acid- and ethylene-sensitive defence mechanisms. ATP-dependent chromatin remodellers mediate the constitutive repression of the salicylic acid-dependent pathway, whereas histone methylation at the WRKY70 gene promoter affects the activation of this pathway. Interestingly, bacterial-infected tissues show a net reduction in DNA methylation, which may affect the disease resistance genes responsible for the surveillance against pathogens. As some epigenetic marks can be erased or maintained and transmitted to offspring, epigenetic mechanisms may provide plasticity for the dynamic control of emerging pathogens without the generation of genomic lesions.

Virus-based surface patterning of biological molecules, probes, and inorganic materials.

PubMed

Ahn, Suji; Jeon, Seongho; Kwak, Eun-A; Kim, Jong-Man; Jaworski, Justyn

2014-10-01

An essential requirement for continued technological advancement in many areas of biology, physics, chemistry, and materials science is the growing need to generate custom patterned materials. Building from recent achievements in the site-specific modification of virus for covalent surface tethering, we show in this work that stable 2D virus patterns can be generated in custom geometries over large area glass surfaces to yield templates of biological, biochemical, and inorganic materials in high density. As a nanomaterial building block, filamentous viruses have been extensively used in recent years to produce materials with interesting properties, owing to their ease of genetic and chemical modification. By utilizing un-natural amino acids generated at specific locations on the filamentous fd bacteriophage protein coat, surface immobilization is carried out on APTES patterned glass resulting in precise geometries of covalently linked virus material. This technique facilitated the surface display of a high density of virus that were labeled with biomolecules, fluorescent probes, and gold nanoparticles, thereby opening the possibility of integrating virus as functional components for surface engineering. Copyright © 2014 Elsevier B.V. All rights reserved.

Paradoxical enhancement of chemoreceptor detection sensitivity by a sensory adaptation enzyme

PubMed Central

Han, Xue-Sheng; Dahlquist, Frederick W.; Parkinson, John S.

2017-01-01

A sensory adaptation system that tunes chemoreceptor sensitivity enables motile Escherichia coli cells to track chemical gradients with high sensitivity over a wide dynamic range. Sensory adaptation involves feedback control of covalent receptor modifications by two enzymes: CheR, a methyltransferase, and CheB, a methylesterase. This study describes a CheR function that opposes the signaling consequences of its catalytic activity. In the presence of CheR, a variety of mutant serine chemoreceptors displayed up to 40-fold enhanced detection sensitivity to chemoeffector stimuli. This response enhancement effect did not require the known catalytic activity of CheR, but did involve a binding interaction between CheR and receptor molecules. Response enhancement was maximal at low CheR:receptor stoichiometry and quantitative analyses argued against a reversible binding interaction that simply shifts the ON–OFF equilibrium of receptor signaling complexes. Rather, a short-lived CheR binding interaction appears to promote a long-lasting change in receptor molecules, either a covalent modification or conformation that enhances their response to attractant ligands. PMID:28827352

Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

1989-01-01

Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

One-Dimensional Multichromophor Arrays Based on DNA: From Self-Assembly to Light-Harvesting.

PubMed

Ensslen, Philipp; Wagenknecht, Hans-Achim

2015-10-20

Light-harvesting complexes collect light energy and deliver it by a cascade of energy and electron transfer processes to the reaction center where charge separation leads to storage as chemical energy. The design of artificial light-harvesting assemblies faces enormous challenges because several antenna chromophores need to be kept in close proximity but self-quenching needs to be avoided. Double stranded DNA as a supramolecular scaffold plays a promising role due to its characteristic structural properties. Automated DNA synthesis allows incorporation of artificial chromophore-modified building blocks, and sequence design allows precise control of the distances and orientations between the chromophores. The helical twist between the chromophores, which is induced by the DNA framework, controls energy and electron transfer and thereby reduces the self-quenching that is typically observed in chromophore aggregates. This Account summarizes covalently multichromophore-modified DNA and describes how such multichromophore arrays were achieved by Watson-Crick-specific and DNA-templated self-assembly. The covalent DNA systems were prepared by incorporation of chromophores as DNA base substitutions (either as C-nucleosides or with acyclic linkers as substitutes for the 2'-deoxyribofuranoside) and as DNA base modifications. Studies with DNA base substitutions revealed that distances but more importantly relative orientations of the chromophores govern the energy transfer efficiencies and thereby the light-harvesting properties. With DNA base substitutions, duplex stabilization was faced and could be overcome, for instance, by zipper-like placement of the chromophores in both strands. For both principal structural approaches, DNA-based light-harvesting antenna could be realized. The major disadvantages, however, for covalent multichromophore DNA conjugates are the poor yields of synthesis and the solubility issues for oligonucleotides with more than 5-10 chromophore modifications in a row. A logical alternative approach is to leave out the phosphodiester bridges between the chromophores and let chromophore-nucleoside conjugates self-assemble specifically along single stranded DNA as template. The self-organization of chromophores along the DNA template based on canonical base pairing would be advantageous because sequence selective base pairing could provide a structural basis for programmed complexity within the chromophore assembly. The self-assembly is governed by two interactions. The chromophore-nucleoside conjugates as guest molecules are recognized via hydrogen bonds to the corresponding counter bases in the single stranded DNA template. Moreover, the π-π interactions between the stacked chromophores stabilize these self-assembled constructs with increasing length. Longer DNA templates are more attractive for self-assembled antenna. The helicity in the stack of porphyrins as guest molecules assembled on the DNA template can be switched by environmental changes, such as pH variations. DNA-templated stacks of ethynyl pyrene and nile red exhibit left-handed chirality, which stands in contrast to similar covalent multichromophore-DNA conjugates with enforced right-handed helicity. With ethynyl nile red, it is possible to occupy every available binding site on the templates. Mixed assemblies of ethynyl pyrene and nile red show energy transfer and thereby provide a proof-of-principle that simple light-harvesting antennae can be obtained in a noncovalent and self-assembled fashion. With respect to the next important step, chemical storage of the absorbed light energy, future research has to focus on the coupling of sophisticated DNA-based light-harvesting antenna to reaction centers.

Covalent and non-covalent curcumin loading in acid-responsive polymeric micellar nanocarriers

NASA Astrophysics Data System (ADS)

Gao, Min; Chen, Chao; Fan, Aiping; Zhang, Ju; Kong, Deling; Wang, Zheng; Zhao, Yanjun

2015-07-01

Poor aqueous solubility, potential degradation, rapid metabolism and elimination lead to low bioavailability of pleiotropic impotent curcumin. Herein, we report two types of acid-responsive polymeric micelles where curcumin was encapsulated via both covalent and non-covalent modes for enhanced loading capacity and on-demand release. Biodegradable methoxy poly(ethylene glycol)-poly(lactic acid) copolymer (mPEG-PLA) was conjugated with curcumin via a hydrazone linker, generating two conjugates differing in architecture (single-tail versus double-tail) and free curcumin was encapsulated therein. The two micelles exhibited similar hydrodynamic size at 95 ± 3 nm (single-tail) and 96 ± 3 nm (double-tail), but their loading capacities differed significantly at 15.0 ± 0.5% (w/w) (single-tail) and 4.8 ± 0.5% (w/w) (double-tail). Under acidic sink conditions (pH 5.0 and 6.0), curcumin displayed a faster release from the single-tail nanocarrier, which was correlated to a low IC50 of 14.7 ± 1.6 (μg mL-1) compared to the value of double-tail micelle (24.9 ± 1.3 μg mL-1) in HeLa cells. The confocal imaging and flow cytometry analysis demonstrated a superior capability of single-tail micelle for intracellular curcumin delivery, which was a consequence of the higher loading capacity and lower degree of mPEG surface coverage. In conclusion, the dual loading mode is an effective means to increase the drug content in the micellar nanocarriers whose delivery efficiency is highly dependent on its polymer-drug conjugate architecture. This strategy offers an alternative nanoplatform for intracellularly delivering impotent hydrophobic agents (i.e. curcumin) in an efficient stimuli-triggered way, which is valuable for the enhancement of curcumin’s efficacy in managing a diverse range of disorders.

From 1D Polymers to 2D Polymers: Preparation of Free-Standing Single-Monomer-Thick Two-Dimensional Conjugated Polymers in Water.

PubMed

Zhang, Na; Wang, Taisheng; Wu, Xing; Jiang, Chen; Zhang, Taiming; Jin, Bangkun; Ji, Hengxing; Bai, Wei; Bai, Ruke

2017-07-25

Recently, investigation on two-dimensional (2D) organic polymers has made great progress, and conjugated 2D polymers already play a dynamic role in both academic and practical applications. However, a convenient, noninterfacial approach to obtain single-layer 2D polymers in solution, especially in aqueous media, remains challenging. Herein, we present a facile, highly efficient, and versatile "1D to 2D" strategy for preparation of free-standing single-monomer-thick conjugated 2D polymers in water without any aid. The 2D structure was achieved by taking advantage of the side-by-side self-assembly of a rigid amphiphilic 1D polymer and following topochemical photopolymerization in water. The spontaneous formation of single-layer polymer sheets was driven by synergetic association of the hydrophobic interactions, π-π stacking interactions, and electrostatic repulsion. Both the supramolecular sheets and the covalent sheets were confirmed by spectroscopic analyses and electron microscope techniques. Moreover, in comparison of the supramolecular 2D polymer, the covalent 2D polymer sheets exhibited not only higher mechanical strength but also higher conductivity, which can be ascribed to the conjugated network within the covalent 2D polymer sheets.

Characterization of the covalent binding of N-phenyl-N'-(2-chloroethyl)ureas to {beta}-tubulin: importance of Glu198 in microtubule stability.

PubMed

Fortin, Sébastien; Bouchon, Bernadette; Chambon, Christophe; Lacroix, Jacques; Moreau, Emmanuel; Chezal, Jean-Michel; Degoul, Françoise; C-Gaudreault, René

2011-02-01

N-Phenyl-N'-(2-chloroethyl)ureas (CEUs) are antimicrotubule agents interacting covalently with β-tubulin near the colchicine-binding site (C-BS). Glutamyl 198 residue in β-tubulin (Glu198), which is adjacent to the C-BS behind the two potent nucleophilic residues, Cys239 and Cys354, has been shown to covalently react with 1-(2-chloroethyl)-3-(4-iodophenyl)urea (ICEU). By use of mass spectrometry, we have now identified residues in β-tubulin that have become modified irreversibly by 1-(2-chloroethyl)-3-[3-(5-hydroxypentyl)phenyl]urea (HPCEU), 1-[4-(3-hydroxy-4-methoxystyryl)phenyl]-3-(2-chloroethyl)urea (4ZCombCEU), and N,N'-ethylenebis(iodoacetamide) (EBI). The binding of HPCEU and 4ZCombCEU to β-tubulin resulted in the acylation of Glu198, a protein modification of uncommon occurrence in living cells. Prototypical CEUs then were used as molecular probes to assess, in mouse B16F0 and human MDA-MB-231 cells, the role of Glu198 in microtubule stability. For that purpose, we studied the effect of Glu198 modification by ICEU, HPCEU, and 4ZCombCEU on the acetylation of Lys40 on α-tubulin, a key indicator of microtubule stability. We show that modification of Glu198 by prototypical CEUs correlates with a decrease in Lys40 acetylation, as observed also with other microtubule depolymerizing agents. Therefore, CEU affects the stability and the dynamics of microtubule, likewise a E198G mutation, which is unusual for xenobiotics. We demonstrate for the first time that EBI forms an intramolecular cross-link between Cys239 and Cys354 of β-tubulin in living cells. This work establishes a novel basis for the development of future chemotherapeutic agents and provides a framework for the design of molecules useful for studying the role of Asp and Glu residues in the structure/function and the biological activity of several cellular proteins under physiological conditions.

Characterizing Covalently Sidewall-Functionalized SWCNTs by using 1H NMR Spectroscopy

PubMed Central

Nelson, Donna J.; Kumar, Ravi

2013-01-01

Unambiguous evidence for covalent sidewall functionalization of single-walled carbon nanotubes (SWCNTs) has been a difficult task, especially for nanomaterials in which slight differences in functionality structure produce significant changes in molecular characteristics. Nuclear magnetic resonance (NMR) spectroscopy provides clear information about the structural skeleton of molecules attached to SWCNTs. In order to establish the generality of proton NMR as an analytical technique for characterizing covalently functionalized SWCNTs, we have obtained and analyzed proton NMR data of SWCNT-substituted benzenes across a variety of para substituents. Trends obtained for differences in proton NMR chemical shifts and the impact of o-, p-, and m-directing effects of electrophilic aromatic substituents on phenyl groups covalently bonded to SWCNTs are discussed. PMID:24009779

The PsB glycoprotein complex is secreted as a preassembled precursor of the spore coat in Dictyostelium discoideum.

PubMed

Watson, N; McGuire, V; Alexander, S

1994-09-01

The PsB glycoprotein in Dictyostelium discoideum is one of a diverse group of developmentally regulated, prespore-cell-specific proteins, that contain a common O-linked oligosaccharide. This post-translational modification is dependent on the wild-type modB allele. The PsB protein exists as part of a multiprotein complex of six different proteins, which have different post-translational modifications and are held together by both covalent and non-covalent interactions (Watson et al. (1993). J. Biol. Chem. 268, 22634-22641). In this study we have used microscopic and biochemical analyses to examine the cellular localization and function of the PsB complex during development. We found that the PsB complex first accumulates in prespore vesicles in slug cells and is secreted later during culmination and becomes localized to both the extracellular matrix of the apical spore mass of mature fruiting bodies and to the inner layer of the spore coat. The PsB associated with the spore coat is covalently bound by disulfide bridges. The PsB protein always exists in a multiprotein complex, but the composition of the PsB complex changes during secretion and spore maturation. Some of the PsB complex proteins have been identified as spore coat proteins. These data demonstrate that some of the proteins that form the spore coat exist as a preassembled precursor complex. The PsB complex is secreted in a developmentally regulated manner during the process of spore differentiation, at which time proteins of the complex, as well as additional spore coat proteins, become covalently associated in at least two forms of extracellular matrix: the interspore matrix and the spore coat. These and other studies show that proteins with modB dependent O-linked oligosaccharides are involved in a wide variety of processes underlying morphogenesis in this organism. These developmental processes are the direct result of cellular mechanisms regulating protein targeting, assembly and secretion, and the assembly of specific extracellular matrices.

Direct covalent attachment of small peptide antigens to enzyme-linked immunosorbent assay plates using radiation and carbodiimide activation.

PubMed

Dagenais, P; Desprez, B; Albert, J; Escher, E

1994-10-01

Direct adsorption of small peptide antigens to unaltered, commercially available polystyrene surfaces may be too weak to permit suitable assay by ELISA. We therefore developed a simple method for the covalent attachment of small, potentially single epitope antigens to polystyrene surfaces. Chemical activation of polystyrene plates with carbodiimide considerably improves the total and covalent attachment of radioactive octapeptides. The covalent attachment was demonstrated by washing with hot detergent. A 3.5 Mrad gamma-irradiation of plates also increases total binding, particularly in combination with chemical activation. The covalent attachment presumably occurs through formation and chemical activation of carboxylate functions on the polystyrene surface which form amide bonds with peptides. ELISA test was performed with CGRP and successive smaller CGRP fragments. Covalent attachment of C-terminal peptide fragments as detection antigens allows optimal recognition and sensitivity even for hexapeptides, while decapeptide antigens were already poorly recognized using a conventional antigen plating technique. Repetitive detergent washes and/or prolonged storage of plates with covalently bound antigens did not reduce their ELISA sensitivity. The method with storage and reutilization capacities that we present here will be useful for the development of preplated antibody screening test.

Mechanism of histone survival during transcription by RNA polymerase II

PubMed Central

Kulaeva, Olga I

2010-01-01

Transcription of eukaryotic genes by RNA polymerase II is typically accompanied by minimal exchange of histones H3/H4 carrying various covalent modifications. In vitro studies suggest that histone survival is accompanied by the formation of a small transient DNA loop on the surface of the histone octamer including a molecule of transcribing enzyme. PMID:21326897

Whereas Short-Term Facilitation Is Presynaptic, Intermediate-Term Facilitation Involves Both Presynaptic and Postsynaptic Protein Kinases and Protein Synthesis

ERIC Educational Resources Information Center

Jin, Iksung; Kandel, Eric R.; Hawkins, Robert D.

2011-01-01

Whereas short-term plasticity involves covalent modifications that are generally restricted to either presynaptic or postsynaptic structures, long-term plasticity involves the growth of new synapses, which by its nature involves both pre- and postsynaptic alterations. In addition, an intermediate-term stage of plasticity has been identified that…

Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone.

PubMed

Hurd, P J; Whitmarsh, A J; Baldwin, G S; Kelly, S M; Waltho, J P; Price, N C; Connolly, B A; Hornby, D P

1999-02-19

DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine. Copyright 1999 Academic Press.

Covalently immobilized platelet-derived growth factor-BB promotes angiogenesis in biomimetic poly(ethylene glycol) hydrogels

PubMed Central

Saik, Jennifer E.; Gould, Daniel J.; Watkins, Emily M.; Dickinson, Mary E.; West, Jennifer L.

2011-01-01

The field of tissue engineering is severely limited by a lack of microvascularization in tissue engineered constructs. Biomimetic poly(ethylene glycol) hydrogels containing covalently immobilized platelet-derived growth factor BB (PDGF-BB) were developed to promote angiogenesis. Poly(ethylene glycol) hydrogels resist protein absorption and subsequent non-specific cell adhesion, thus providing a “blank slate”, which can be modified through the incorporation of cell adhesive ligands and growth factors. PDGF-BB is a key angiogenic protein able to support neovessel stabilization by inducing functional anastomoses and recruiting pericytes. Due to the widespread effects of PDGF in the body and a half-life of only 30 min in circulating blood, immobilization of PDGF-BB may be necessary. In this work bioactive, covalently immobilized PDGF-BB was shown to induce tubulogenesis on two-dimensional modified surfaces, migration in three-dimensional (3D) degradable hydrogels and angiogenesis in a mouse cornea micro-pocket angiogenesis assay. Covalently immobilized PDGF-BB was also used in combination with covalently immobilized fibroblast growth factor-2, which led to significantly increased endothelial cell migration in 3D degradable hydrogels compared with the presentation of each factor alone. When a co-culture of endothelial cells and mouse pericyte precursor 10T1/2 cells was seeded onto modified surfaces tubule formation was independent of surface modifications with covalently immobilized growth factors. Furthermore, the combination of soluble PDGF-BB and immobilized PDGF-BB induced a more robust vascular response compared with soluble PDGF-BB alone when implanted into an in vivo mouse cornea micropocket angiogenesis assay. Based on these results, we believe bioactive hydrogels can be tailored to improve the formation of functional microvasculature for tissue engineering. PMID:20801242

Structural and Functional Coordination of DNA and Histone Methylation

PubMed Central

Cheng, Xiaodong

2014-01-01

One of the most fundamental questions in the control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and recognized. This central process in controlling gene expression includes coordinated covalent modifications of DNA and its associated histones. This article focuses on structural aspects of enzymatic activities of histone (arginine and lysine) methylation and demethylation and functional links between the methylation status of the DNA and histones. An interconnected network of methyltransferases, demethylases, and accessory proteins is responsible for changing or maintaining the modification status of specific regions of chromatin. PMID:25085914

Sex pheromone receptor proteins. Visualization using a radiolabeled photoaffinity analog

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vogt, R.G.; Prestwich, G.D.; Riddiford, L.M.

1988-03-15

A tritium-labeled photoaffinity analog of a moth pheromone was used to covalently modify pheromone-selective binding proteins in the antennal sensillum lymph and sensory dendritic membranes of the male silk moth, Antheraea polyphemus. This analog, (E,Z)-6,11-(/sup 3/H)hexadecadienyl diazoacetate, allowed visualization of a 15-kilodalton soluble protein and a 69-kilodalton membrane protein in fluorescence autoradiograms of electrophoretically separated antennal proteins. Covalent modification of these proteins was specifically reduced when incubation and UV irradiation were conducted in the presence of excess unlabeled pheromone, (E,Z)-6,11-hexadecadienyl acetate. These experiments constitute the first direct evidence for a membrane protein of a chemosensory neuron interacting in a specificmore » fashion with a biologically relevant odorant.« less

Resonance of an unshared electron pair between two atoms connected by a single bond

PubMed Central

Pauling, Linus

1983-01-01

The reported structure of the dimer of a compound of bicovalent tin indicates that the tin-tin bond is of a new type. It can be described as involving resonance between two structures in which there is transfer of an electron pair from one tin atom to the other. The tin atoms are connected by a single covalent bond (each also forms two covalent bonds with carbon atoms), and an unshared electron pair resonates between the fourth sp3 orbitals of the two atoms. Similar structures probably occur in digermene and distannene. PMID:16593329

Covalent surface modification of gallium arsenide photocathodes for water splitting in highly acidic electrolyte

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garner, Logan E.; Steirer, K. Xerxes; Young, James L.

Efficient water splitting using light as the only energy input requires stable semiconductor electrodes with favorable energetics for the water-oxidation and proton-reduction reactions. Strategies to tune electrode potentials using molecular dipoles adsorbed to the semiconductor surface have been pursued for decades but are often based on weak interactions and quickly react to desorb the molecule under conditions relevant to sustained photoelectrolysis. Here, we show that covalent attachment of fluorinated, aromatic molecules to p-GaAs(1 0 0) surfaces can be employed to tune the photocurrent onset potentials of p-GaAs(1 0 0) photocathodes and reduce the external energy required for water splitting. Resultsmore » indicate that initial photocurrent onset potentials can be shifted by nearly 150 mV in pH -0.5 electrolyte under 1 Sun (1000 W m -2) illumination resulting from the covalently bound surface dipole. Furthermore, X-ray photoelectron spectroscopy analysis reveals that the covalent molecular dipole attachment is not robust under extended 50 h photoelectrolysis, the modified surface delays arsenic oxide formation that results in a p-GaAs(1 0 0) photoelectrode operating at a sustained photocurrent density of -20.5 mA cm -2 within -0.5 V of the reversible hydrogen electrode.« less

Covalent surface modification of gallium arsenide photocathodes for water splitting in highly acidic electrolyte

DOE PAGES

Garner, Logan E.; Steirer, K. Xerxes; Young, James L.; ...

2016-12-12

Efficient water splitting using light as the only energy input requires stable semiconductor electrodes with favorable energetics for the water-oxidation and proton-reduction reactions. Strategies to tune electrode potentials using molecular dipoles adsorbed to the semiconductor surface have been pursued for decades but are often based on weak interactions and quickly react to desorb the molecule under conditions relevant to sustained photoelectrolysis. Here, we show that covalent attachment of fluorinated, aromatic molecules to p-GaAs(1 0 0) surfaces can be employed to tune the photocurrent onset potentials of p-GaAs(1 0 0) photocathodes and reduce the external energy required for water splitting. Resultsmore » indicate that initial photocurrent onset potentials can be shifted by nearly 150 mV in pH -0.5 electrolyte under 1 Sun (1000 W m -2) illumination resulting from the covalently bound surface dipole. Furthermore, X-ray photoelectron spectroscopy analysis reveals that the covalent molecular dipole attachment is not robust under extended 50 h photoelectrolysis, the modified surface delays arsenic oxide formation that results in a p-GaAs(1 0 0) photoelectrode operating at a sustained photocurrent density of -20.5 mA cm -2 within -0.5 V of the reversible hydrogen electrode.« less

X-ray crystallographic analysis of adipocyte fatty acid binding protein (aP2) modified with 4-hydroxy-2-nonenal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hellberg, Kristina; Grimsrud, Paul A.; Kruse, Andrew C.

2012-07-11

Fatty acid binding proteins (FABP) have been characterized as facilitating the intracellular solubilization and transport of long-chain fatty acyl carboxylates via noncovalent interactions. More recent work has shown that the adipocyte FABP is also covalently modified in vivo on Cys117 with 4-hydroxy-2-nonenal (4-HNE), a bioactive aldehyde linked to oxidative stress and inflammation. To evaluate 4-HNE binding and modification, the crystal structures of adipocyte FABP covalently and noncovalently bound to 4-HNE have been solved to 1.9 {angstrom} and 2.3 {angstrom} resolution, respectively. While the 4-HNE in the noncovalently modified protein is coordinated similarly to a carboxylate of a fatty acid, themore » covalent form show a novel coordination through a water molecule at the polar end of the lipid. Other defining features between the two structures with 4-HNE and previously solved structures of the protein include a peptide flip between residues Ala36 and Lys37 and the rotation of the side chain of Phe57 into its closed conformation. Representing the first structure of an endogenous target protein covalently modified by 4-HNE, these results define a new class of in vivo ligands for FABPs and extend their physiological substrates to include bioactive aldehydes.« less

Detection of histone modifications in plant leaves.

PubMed

Jaskiewicz, Michal; Peterhansel, Christoph; Conrath, Uwe

2011-09-23

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles(1-2). H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues(1-2). These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)(3-7). Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde(8,9), extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies(9,10), de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C(4;) photosynthesis in maize(5,11) and systemic immunity in Arabidopsis(3).

Metal–organic and covalent organic frameworks as single-site catalysts

PubMed Central

Rogge, S. M. J.; Bavykina, A.; Hajek, J.; Garcia, H.; Olivos-Suarez, A. I.; Sepúlveda-Escribano, A.; Vimont, A.; Clet, G.; Bazin, P.; Kapteijn, F.

2017-01-01

Heterogeneous single-site catalysts consist of isolated, well-defined, active sites that are spatially separated in a given solid and, ideally, structurally identical. In this review, the potential of metal–organic frameworks (MOFs) and covalent organic frameworks (COFs) as platforms for the development of heterogeneous single-site catalysts is reviewed thoroughly. In the first part of this article, synthetic strategies and progress in the implementation of such sites in these two classes of materials are discussed. Because these solids are excellent playgrounds to allow a better understanding of catalytic functions, we highlight the most important recent advances in the modelling and spectroscopic characterization of single-site catalysts based on these materials. Finally, we discuss the potential of MOFs as materials in which several single-site catalytic functions can be combined within one framework along with their potential as powerful enzyme-mimicking materials. The review is wrapped up with our personal vision on future research directions. PMID:28338128

Nanoparticle decoration with surfactants: Molecular interactions, assembly, and applications

NASA Astrophysics Data System (ADS)

Heinz, Hendrik; Pramanik, Chandrani; Heinz, Ozge; Ding, Yifu; Mishra, Ratan K.; Marchon, Delphine; Flatt, Robert J.; Estrela-Lopis, Irina; Llop, Jordi; Moya, Sergio; Ziolo, Ronald F.

2017-02-01

Nanostructures of diverse chemical nature are used as biomarkers, therapeutics, catalysts, and structural reinforcements. The decoration with surfactants has a long history and is essential to introduce specific functions. The definition of surfactants in this review is very broad, following its lexical meaning ;surface active agents;, and therefore includes traditional alkyl modifiers, biological ligands, polymers, and other surface active molecules. The review systematically covers covalent and non-covalent interactions of such surfactants with various types of nanomaterials, including metals, oxides, layered materials, and polymers as well as their applications. The major themes are (i) molecular recognition and noncovalent assembly mechanisms of surfactants on the nanoparticle and nanocrystal surfaces, (ii) covalent grafting techniques and multi-step surface modification, (iii) dispersion properties and surface reactions, (iv) the use of surfactants to influence crystal growth, as well as (v) the incorporation of biorecognition and other material-targeting functionality. For the diverse materials classes, similarities and differences in surfactant assembly, function, as well as materials performance in specific applications are described in a comparative way. Major factors that lead to differentiation are the surface energy, surface chemistry and pH sensitivity, as well as the degree of surface regularity and defects in the nanoparticle cores and in the surfactant shell. The review covers a broad range of surface modifications and applications in biological recognition and therapeutics, sensors, nanomaterials for catalysis, energy conversion and storage, the dispersion properties of nanoparticles in structural composites and cement, as well as purification systems and classical detergents. Design principles for surfactants to optimize the performance of specific nanostructures are discussed. The review concludes with challenges and opportunities.

Fabrication of antibody microarrays by light-induced covalent and oriented immobilization.

PubMed

Adak, Avijit K; Li, Ben-Yuan; Huang, Li-De; Lin, Ting-Wei; Chang, Tsung-Che; Hwang, Kuo Chu; Lin, Chun-Cheng

2014-07-09

Antibody microarrays have important applications for the sensitive detection of biologically important target molecules and as biosensors for clinical applications. Microarrays produced by oriented immobilization of antibodies generally have higher antigen-binding capacities than those in which antibodies are immobilized with random orientations. Here, we present a UV photo-cross-linking approach that utilizes boronic acid to achieve oriented immobilization of an antibody on a surface while retaining the antigen-binding activity of the immobilized antibody. A photoactive boronic acid probe was designed and synthesized in which boronic acid provided good affinity and specificity for the recognition of glycan chains on the Fc region of the antibody, enabling covalent tethering to the antibody upon exposure to UV light. Once irradiated with optimal UV exposure (16 mW/cm(2)), significant antibody immobilization on a boronic acid-presenting surface with maximal antigen detection sensitivity in a single step was achieved, thus obviating the necessity of prior antibody modifications. The developed approach is highly modular, as demonstrated by its implementation in sensitive sandwich immunoassays for the protein analytes Ricinus communis agglutinin 120, human prostate-specific antigen, and interleukin-6 with limits of detection of 7.4, 29, and 16 pM, respectively. Furthermore, the present system enabled the detection of multiple analytes in samples without any noticeable cross-reactivities. Antibody coupling via the use of boronic acid and UV light represents a practical, oriented immobilization method with significant implications for the construction of a large array of immunosensors for diagnostic applications.

Palmitoylation as a Functional Regulator of Neurotransmitter Receptors

PubMed Central

Naumenko, Vladimir S.

2018-01-01

The majority of neuronal proteins involved in cellular signaling undergo different posttranslational modifications significantly affecting their functions. One of these modifications is a covalent attachment of a 16-C palmitic acid to one or more cysteine residues (S-palmitoylation) within the target protein. Palmitoylation is a reversible modification, and repeated cycles of palmitoylation/depalmitoylation might be critically involved in the regulation of multiple signaling processes. Palmitoylation also represents a common posttranslational modification of the neurotransmitter receptors, including G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LICs). From the functional point of view, palmitoylation affects a wide span of neurotransmitter receptors activities including their trafficking, sorting, stability, residence lifetime at the cell surface, endocytosis, recycling, and synaptic clustering. This review summarizes the current knowledge on the palmitoylation of neurotransmitter receptors and its role in the regulation of receptors functions as well as in the control of different kinds of physiological and pathological behavior. PMID:29849559

Structure and Modification of Electrode Materials for Protein Electrochemistry.

PubMed

Jeuken, Lars J C

The interactions between proteins and electrode surfaces are of fundamental importance in bioelectrochemistry, including photobioelectrochemistry. In order to optimise the interaction between electrode and redox protein, either the electrode or the protein can be engineered, with the former being the most adopted approach. This tutorial review provides a basic description of the most commonly used electrode materials in bioelectrochemistry and discusses approaches to modify these surfaces. Carbon, gold and transparent electrodes (e.g. indium tin oxide) are covered, while approaches to form meso- and macroporous structured electrodes are also described. Electrode modifications include the chemical modification with (self-assembled) monolayers and the use of conducting polymers in which the protein is imbedded. The proteins themselves can either be in solution, electrostatically adsorbed on the surface or covalently bound to the electrode. Drawbacks and benefits of each material and its modifications are discussed. Where examples exist of applications in photobioelectrochemistry, these are highlighted.

Chromatin immunoprecipitation (ChIP) method for non-model fruit flies (Diptera: Tephritidae) and evidence of histone modifications.

PubMed

Nagalingam, Kumaran; Lorenc, Michał T; Manoli, Sahana; Cameron, Stephen L; Clarke, Anthony R; Dudley, Kevin J

2018-01-01

Interactions between DNA and proteins located in the cell nucleus play an important role in controlling physiological processes by specifying, augmenting and regulating context-specific transcription events. Chromatin immunoprecipitation (ChIP) is a widely used methodology to study DNA-protein interactions and has been successfully used in various cell types for over three decades. More recently, by combining ChIP with genomic screening technologies and Next Generation Sequencing (e.g. ChIP-seq), it has become possible to profile DNA-protein interactions (including covalent histone modifications) across entire genomes. However, the applicability of ChIP-chip and ChIP-seq has rarely been extended to non-model species because of a number of technical challenges. Here we report a method that can be used to identify genome wide covalent histone modifications in a group of non-model fruit fly species (Diptera: Tephritidae). The method was developed by testing and refining protocols that have been used in model organisms, including Drosophila melanogaster. We demonstrate that this method is suitable for a group of economically important pest fruit fly species, viz., Bactrocera dorsalis, Ceratitis capitata, Zeugodacus cucurbitae and Bactrocera tryoni. We also report an example ChIP-seq dataset for B. tryoni, providing evidence for histone modifications in the genome of a tephritid fruit fly for the first time. Since tephritids are major agricultural pests globally, this methodology will be a valuable resource to study taxa-specific evolutionary questions and to assist with pest management. It also provides a basis for researchers working with other non-model species to undertake genome wide DNA-protein interaction studies.

Site-selective protein-modification chemistry for basic biology and drug development

NASA Astrophysics Data System (ADS)

Krall, Nikolaus; da Cruz, Filipa P.; Boutureira, Omar; Bernardes, Gonçalo J. L.

2016-02-01

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.

Modification of silicon nitride surfaces with GOPES and APTES for antibody immobilization: computational and experimental studies

NASA Astrophysics Data System (ADS)

Dien To, Thien; Nguyen, Anh Tuan; Nhat Thanh Phan, Khoa; Thu Thi Truong, An; Doan, Tin Chanh Duc; Mau Dang, Chien

2015-12-01

Chemical modification of silicon nitride (SiN) surfaces by silanization has been widely studied especially with 3-(aminopropyl)triethoxysilane (APTES) and 3-(glycidyloxypropyl) dimethylethoxysilane (GOPES). However few reports performed the experimental and computational studies together. In this study, surface modification of SiN surfaces with GOPES and APTES covalently bound with glutaraldehyde (GTA) was investigated for antibody immobilization. The monoclonal anti-cytokeratin-FITC (MACF) antibody was immobilized on the modified SiN surfaces. The modified surfaces were characterized by water contact angle measurements, atomic force microscopy and fluorescence microscopy. The FITC-fluorescent label indicated the existence of MACF antibody on the SiN surfaces and the efficiency of the silanization reaction. Absorption of APTES and GOPES on the oxidized SiN surfaces was computationally modeled and calculated by Materials Studio software. The computational and experimental results showed that modification of the SiN surfaces with APTES and GTA was more effective than the modification with GOPES.

Site-selective protein-modification chemistry for basic biology and drug development.

PubMed

Krall, Nikolaus; da Cruz, Filipa P; Boutureira, Omar; Bernardes, Gonçalo J L

2016-02-01

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.

Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1

PubMed Central

Chang, Chuang-Rung; Blackstone, Craig

2017-01-01

Mitochondria in cells comprise a tubulovesicular network shaped continuously by complementary fission and fusion events. The mammalian Drp1 protein plays a key role in fission, while Mfn1, Mfn2, and OPA1 are required for fusion. Shifts in the balance between these opposing processes can occur rapidly, indicating that modifications to these proteins may regulate mitochondrial membrane dynamics. We highlight posttranslational modifications of the mitochondrial fission protein Drp1, for which these regulatory mechanisms are best characterized. This dynamin-related GTPase undergoes a number of steps to mediate mitochondrial fission, including translocation from cytoplasm to the mitochondrial outer membrane, higher-order assembly into spirals, GTP hydrolysis associated with a conformational change and membrane deformation, and ultimately disassembly. Many of these steps may be influenced by covalent modification of Drp1. We discuss the dynamic nature of Drp1 modifications and how they contribute not only to the normal regulation of mitochondrial division, but also to neuropathologic processes. PMID:20649536

Human Metabolism and Interactions of Deployment-Related Chemicals

DTIC Science & Technology

2003-08-01

with individual test compounds (final concentration, 100 PM), agent pyridostigmine bromide to protect against possible nerve gas NADPH-generating system...an insect repellent (N,N-diethyl-m- toluamide) a nerve gas prophyllactic (pyridostigmine bromide) did not cause the inhibition of trans-permethrin...mechanism of organophosphorus anticholinesterase agents , namely; covalent modification of the active site of the esterases in question. Carbaryl, another

Covalent surface modification of prions: a mass spectrometry-based means of detecting distinctive structural features of prion strains

USDA-ARS?s Scientific Manuscript database

Prions (PrPSc) are molecular pathogens that are able to convert the isosequential normal cellular prion protein (PrPC) into a prion. The only demonstrated differences between PrPC and PrPSc is conformational, they are isoforms. A given host can be infected by more than one kind or strain of prion. F...

Heat shock protein 70 inhibitors. 2. 2,5'-thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70.

PubMed

Taldone, Tony; Kang, Yanlong; Patel, Hardik J; Patel, Maulik R; Patel, Pallav D; Rodina, Anna; Patel, Yogita; Gozman, Alexander; Maharaj, Ronnie; Clement, Cristina C; Lu, Alvin; Young, Jason C; Chiosis, Gabriela

2014-02-27

The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.

Nacre-mimetic clay/xyloglucan bionanocomposites: a chemical modification route for hygromechanical performance at high humidity.

PubMed

Kochumalayil, Joby J; Morimune, Seira; Nishino, Takashi; Ikkala, Olli; Walther, Andreas; Berglund, Lars A

2013-11-11

Nacre-mimetic bionanocomposites of high montmorillonite (MTM) clay content, prepared from hydrocolloidal suspensions, suffer from reduced strength and stiffness at high relative humidity. We address this problem by chemical modification of xyloglucan in (XG)/MTM nacre-mimetic nanocomposites, by subjecting the XG to regioselective periodate oxidation of side chains to enable it to form covalent cross-links to hydroxyl groups in neighboring XG chains or to the MTM surface. The resulting materials are analyzed by FTIR spectroscopy, thermogravimetric analysis, carbohydrate analysis, calorimetry, X-ray diffraction, scanning electron microscopy, tensile tests, and oxygen barrier properties. We compare the resulting mechanical properties at low and high relative humidity. The periodate oxidation leads to a strong increase in modulus and strength of the materials. A modulus of 30 GPa for cross-linked composite at 50% relative humidity compared with 13.7 GPa for neat XG/MTM demonstrates that periodate oxidation of the XG side chains leads to crucially improved stress transfer at the XG/MTM interface, possibly through covalent bond formation. This enhanced interfacial adhesion and internal cross-linking of the matrix moreover preserves the mechanical properties at high humidity condition and leads to a Young's modulus of 21 GPa at 90%RH.

A novel covalent approach to bio-conjugate silver coated single walled carbon nanotubes with antimicrobial peptide.

PubMed

Chaudhari, Atul A; Ashmore, D'andrea; Nath, Subrata Deb; Kate, Kunal; Dennis, Vida; Singh, Shree R; Owen, Don R; Palazzo, Chris; Arnold, Robert D; Miller, Michael E; Pillai, Shreekumar R

2016-07-13

Due to increasing antibiotic resistance, the use of silver coated single walled carbon nanotubes (SWCNTs-Ag) and antimicrobial peptides (APs) is becoming popular due to their antimicrobial properties against a wide range of pathogens. However, stability against various conditions and toxicity in human cells are some of the major drawbacks of APs and SWCNTs-Ag, respectively. Therefore, we hypothesized that APs-functionalized SWCNTs-Ag could act synergistically. Various covalent functionalization protocols described previously involve harsh treatment of carbon nanotubes for carboxylation (first step in covalent functionalization) and the non-covalently functionalized SWCNTs are not satisfactory. The present study is the first report wherein SWCNTs-Ag were first carboxylated using Tri sodium citrate (TSC) at 37 °C and then subsequently functionalized covalently with an effective antimicrobial peptide from Therapeutic Inc., TP359 (FSWCNTs-Ag). SWCNTs-Ag were also non covalently functionalized with TP359 by simple mixing (SWCNTs-Ag-M) and both, the FSWCNTs-Ag (covalent) and SWCNTs-Ag-M (non-covalent), were characterized by Fourier transform infrared spectroscopy (FT-IR), Ultraviolet visualization (UV-VIS) and transmission electron microscopy (TEM). Further the antibacterial activity of both and TP359 were investigated against two gram positive (Staphylococcus aureus and Streptococcus pyogenes) and two gram negative (Salmonella enterica serovar Typhimurium and Escherichia coli) pathogens and the cellular toxicity of TP359 and FSWCNTs-Ag was compared with plain SWCNTs-Ag using murine macrophages and lung carcinoma cells. FT-IR analysis revealed that treatment with TSC successfully resulted in carboxylation of SWCNTs-Ag and the peptide was indeed attached to the SWCNTs-Ag evidenced by TEM images. More importantly, the present study results further showed that the minimum inhibitory concentration (MIC) of FSWCNTs-Ag were much lower (~7.8-3.9 µg/ml with IC50: ~4-5 µg/ml) compared to SWCNTs-Ag-M and plain SWCNTs-Ag (both 62.6 µg/ml, IC50: ~31-35 µg/ml), suggesting that the covalent conjugation of TP359 with SWCNTs-Ag was very effective on their counterparts. Additionally, FSWCNTs-Ag are non-toxic to the eukaryotic cells at their MIC concentrations (5-2.5 µg/ml) compared to SWCNTs-Ag (62.5 µg/ml). In conclusion, we demonstrated that covalent functionalization of SWCNTs-Ag and TP359 exhibited an additive antibacterial activity. This study described a novel approach to prepare SWCNT-Ag bio-conjugates without loss of antimicrobial activity and reduced toxicity, and this strategy will aid in the development of novel and biologically important nanomaterials.

Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica.

PubMed

Sultan, Nabil Ali Mohammed; Kenoth, Roopa; Swamy, Musti J

2004-12-15

A new galactose-specific lectin has been purified from the extracts of Trichosanthes dioica seeds by affinity chromatography on cross-linked guar gum. The purified lectin (T. dioica seed lectin, TDSL) moved as a single symmetrical peak on gel filtration on Superose-12 in the presence of 0.1 M lactose with an M(r) of 55 kDa. In the absence of ligand, the movement was retarded, indicating a possible interaction of the lectin with the column matrix. In SDS-PAGE, in the presence of beta-mercaptoethanol, two non-identical bands of M(r) 24 and 37 kDa were observed, whereas in the absence of beta-mercaptoethanol, the lectin yielded a single band corresponding to approximately 55,000 Da, indicating that the two subunits of TDSL are connected by one or more disulfide bridges. TDSL is a glycoprotein with about 4.9% covalently bound neutral sugar. Analysis of near-UV CD spectrum by three different methods (CDSSTR, CONTINLL, and SELCON3) shows that TDSL contains 13.3% alpha-helix, 36.7% beta-sheet, 19.4% beta-turns, and 31.6% unordered structure. Among a battery of sugars investigated, TDSL was inhibited strongly by beta-d-galactopyranosides, with 4-methylumbelliferyl-beta-d-galactopyranoside being the best ligand. Chemical modification studies indicate that tyrosine residues are important for the carbohydrate-binding and hemagglutinating activities of the lectin. A partial protection was observed when the tyrosine modification was performed in the presence of 0.2 M lactose. The tryptophan residues of TDSL appear to be buried in the protein interior as they could not be modified under native conditions, whereas upon denaturation with 8 M urea two Trp residues could be selectively modified by N-bromosuccinimide. The subunit composition and size, secondary structure, and sugar specificity of this lectin are similar to those of type-2 ribosome inactivating proteins, suggesting that TDSL may belong to this protein family.

Photoactivable antibody binding protein: site-selective and covalent coupling of antibody.

PubMed

Jung, Yongwon; Lee, Jeong Min; Kim, Jung-won; Yoon, Jeongwon; Cho, Hyunmin; Chung, Bong Hyun

2009-02-01

Here we report new photoactivable antibody binding proteins, which site-selectively capture antibodies and form covalent conjugates with captured antibodies upon irradiation. The proteins allow the site-selective tagging and/or immobilization of antibodies with a highly preferred orientation and omit the need for prior antibody modifications. The minimal Fc-binding domain of protein G, a widely used antibody binding protein, was genetically and chemically engineered to contain a site-specific photo cross-linker, benzophenone. In addition, the domain was further mutated to have an enhanced Fc-targeting ability. This small engineered protein was successfully cross-linked only to the Fc region of the antibody without any nonspecific reactivity. SPR analysis indicated that antibodies can be site-selectively biotinylated through the present photoactivable protein. Furthermore, the system enabled light-induced covalent immobilization of antibodies directly on various solid surfaces, such as those of glass slides, gold chips, and small particles. Antibody coupling via photoactivable antibody binding proteins overcomes several limitations of conventional approaches, such as random chemical reactions or reversible protein binding, and offers a versatile tool for the field of immunosensors.

Oxadiazole-isopropylamides as Potent and Non-covalent Proteasome Inhibitors

PubMed Central

Ozcan, Sevil; Kazi, Aslamuzzaman; Marsilio, Frank; Fang, Bin; Guida, Wayne C.; Koomen, John; Lawrence, Harshani R.; Sebti, Saïd M.

2013-01-01

Screening of the 50,000 ChemBridge compound library led to the identification of the oxadiazole-isopropylamide 1 (PI-1833) which inhibited CT-L activity (IC50 0.60 μM) with little effects on the other 2 major proteasome proteolytic activities, T-L and PGPH-L. LC/MS-MS and dialysis show that 1 is a non-covalent and rapidly reversible CT-L inhibitor. Focused library synthesis provided 11ad (PI-1840) with CT-L activity (IC50 27 nM). Detailed SAR studies indicate that the amide moiety and the 2 phenyl rings are sensitive toward modifications. Hydrophobic residues, such as propyl or butyl, in the para-position (not ortho or meta) of the A-ring and a meta-pyridyl group as B-ring significantly improve activity. Compound 11ad (IC50 0.37 μM) is more potent than 1 (IC50 3.5 μM) at inhibiting CT-L activity in intact MDA-MB-468 human breast cancer cells and inhibiting their survival. The activity of 11ad warrants further pre-clinical investigation of this class as non-covalent proteasome inhibitors. PMID:23547706

Spontaneous modification of carbon surface with neutral red from its diazonium salts for bioelectrochemical systems.

PubMed

Guo, Kun; Chen, Xin; Freguia, Stefano; Donose, Bogdan C

2013-09-15

This study introduces a novel and simple method to covalently graft neutral red (NR) onto carbon surfaces based on spontaneous reduction of in situ generated NR diazonium salts. Immobilization of neutral red on carbon surface was achieved by immersing carbon electrodes in NR-NaNO2-HCl solution. The functionalized electrodes were characterized by cyclic voltammetry (CV), atomic force microscope (AFM), and X-ray photoelectron spectroscopy (XPS). Results demonstrated that NR attached in this way retains high electrochemical activity and proved that NR was covalently bound to the carbon surface via the pathway of reduction of aryl diazonium salts. The NR-modified electrodes showed a good stability when stored in PBS solution in the dark. The current output of an acetate-oxidising microbial bioanode made of NR-modified graphite felts were 3.63±0.36 times higher than the unmodified electrodes, which indicates that covalently bound NR can act as electron transfer mediator to facilitate electron transfer from bacteria to electrodes. Copyright © 2013 Elsevier B.V. All rights reserved.

A simple and widely applicable hit validation strategy for protein-protein interaction inhibitors based on a quantitative ligand displacement assay.

PubMed

Sameshima, Tomoya; Miyahisa, Ikuo; Homma, Misaki; Aikawa, Katsuji; Hixon, Mark S; Matsui, Junji

2014-12-15

Identification of inhibitors for protein-protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

Single-Crystal Structure of a Covalent Organic Framework

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, YB; Su, J; Furukawa, H

2013-11-06

The crystal structure of a new covalent organic framework, termed COF-320, is determined by single-crystal 3D electron diffraction using the rotation electron diffraction (RED) method for data collection. The COF crystals are prepared by an imine condensation of tetra-(4-anilyl)methane and 4,4'-biphenyldialdehyde in 1,4-dioxane at 120 degrees C to produce a highly porous 9-fold interwoven diamond net. COF-320 exhibits permanent porosity with a Langmuir surface area of 2400 m(2)/g and a methane total uptake of 15.0 wt % (176 cm(3)/cm(3)) at 25 degrees C and 80 bar. The successful determination of the structure of COF-320 directly from single-crystal samples is anmore » important advance in the development of COF chemistry.« less

Systematic Tuning and Multifunctionalization of Covalent Organic Polymers for Enhanced Carbon Capture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, Zhonghua; Mercado, Rocio; Huck, Johanna M.

Porous covalent polymers are attracting increasing interest in the fields of gas adsorption, gas separation, and catalysis due to their fertile synthetic polymer chemistry, large internal surface areas, and ultrahigh hydrothermal stabilities. While precisely manipulating the porosities of porous organic materials for targeted applications remains challenging, we show how a large degree of diversity can be achieved in covalent organic polymers by incorporating multiple functionalities into a single framework, as is done for crystalline porous materials. Here, we synthesized 17 novel porous covalent organic polymers (COPs) with finely tuned porosities, a wide range of Brunauer–Emmett–Teller (BET) specific surface areas ofmore » 430–3624 m2 g–1, and a broad range of pore volumes of 0.24–3.50 cm3 g–1, all achieved by tailoring the length and geometry of building blocks. Furthermore, we are the first to successfully incorporate more than three distinct functional groups into one phase for porous organic materials, which has been previously demonstrated in crystalline metal–organic frameworks (MOFs). COPs decorated with multiple functional groups in one phase can lead to enhanced properties that are not simply linear combinations of the pure component properties. For instance, in the dibromobenzene-lined frameworks, the bi- and multifunctionalized COPs exhibit selectivities for carbon dioxide over nitrogen twice as large as any of the singly functionalized COPs. These multifunctionalized frameworks also exhibit a lower parasitic energy cost for carbon capture at typical flue gas conditions than any of the singly functionalized frameworks. Despite the significant improvement, these frameworks do not yet outperform the current state-of-art technology for carbon capture. Nonetheless, the tuning strategy presented here opens up avenues for the design of novel catalysts, the synthesis of functional sensors from these materials, and the improvement in the performance of existing covalent organic polymers by multifunctionalization.« less

Ruthenium based metallopolymer grafted reduced graphene oxide as a new hybrid solar light harvester in polymer solar cells

PubMed Central

Vinoth, R.; Babu, S. Ganesh; Bharti, Vishal; Gupta, V.; Navaneethan, M.; Bhat, S. Venkataprasad; Muthamizhchelvan, C.; Ramamurthy, Praveen C.; Sharma, Chhavi; Aswal, Dinesh K.; Hayakawa, Yasuhiro; Neppolian, B.

2017-01-01

A new class of pyridyl benzimdazole based Ru complex decorated polyaniline assembly (PANI-Ru) was covalently grafted onto reduced graphene oxide sheets (rGO) via covalent functionalization approach. The covalent attachment of PANI-Ru with rGO was confirmed from XPS analysis and Raman spectroscopy. The chemical bonding between PANI-Ru and rGO induced the electron transfer from Ru complex to rGO via backbone of the conjugated PANI chain. The resultant hybrid metallopolymer assembly was successfully demonstrated as an electron donor in bulk heterojunction polymer solar cells (PSCs). A PSC device fabricated with rGO/PANI-Ru showed an utmost ~6 fold and 2 fold enhancement in open circuit potential (Voc) and short circuit current density (Jsc) with respect to the standard device made with PANI-Ru (i.e., without rGO) under the illumination of AM 1.5 G. The excellent electronic properties of rGO significantly improved the electron injection from PANI-Ru to PCBM and in turn the overall performance of the PSC device was enhanced. The ultrafast excited state charge separation and electron transfer role of rGO sheet in hybrid metallopolymer was confirmed from ultrafast spectroscopy measurements. This covalent modification of rGO with metallopolymer assembly may open a new strategy for the development of new hybrid nanomaterials for light harvesting applications. PMID:28225039

Covalent Surface Modification of Gallium Arsenide Photocathodes for Water Splitting in Highly Acidic Electrolyte.

PubMed

Garner, Logan E; Steirer, K Xerxes; Young, James L; Anderson, Nicholas C; Miller, Elisa M; Tinkham, Jonathan S; Deutsch, Todd G; Sellinger, Alan; Turner, John A; Neale, Nathan R

2017-02-22

Efficient water splitting using light as the only energy input requires stable semiconductor electrodes with favorable energetics for the water-oxidation and proton-reduction reactions. Strategies to tune electrode potentials using molecular dipoles adsorbed to the semiconductor surface have been pursued for decades but are often based on weak interactions and quickly react to desorb the molecule under conditions relevant to sustained photoelectrolysis. Here, we show that covalent attachment of fluorinated, aromatic molecules to p-GaAs(1 0 0) surfaces can be employed to tune the photocurrent onset potentials of p-GaAs(1 0 0) photocathodes and reduce the external energy required for water splitting. Results indicate that initial photocurrent onset potentials can be shifted by nearly 150 mV in pH -0.5 electrolyte under 1 Sun (1000 W m -2 ) illumination resulting from the covalently bound surface dipole. Though X-ray photoelectron spectroscopy analysis reveals that the covalent molecular dipole attachment is not robust under extended 50 h photoelectrolysis, the modified surface delays arsenic oxide formation that results in a p-GaAs(1 0 0) photoelectrode operating at a sustained photocurrent density of -20.5 mA cm -2 within -0.5 V of the reversible hydrogen electrode. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Covalent and non-covalent chemical engineering of actin for biotechnological applications.

PubMed

Kumar, Saroj; Mansson, Alf

2017-11-15

The cytoskeletal filaments are self-assembled protein polymers with 8-25nm diameters and up to several tens of micrometres length. They have a range of pivotal roles in eukaryotic cells, including transportation of intracellular cargoes (primarily microtubules with dynein and kinesin motors) and cell motility (primarily actin and myosin) where muscle contraction is one example. For two decades, the cytoskeletal filaments and their associated motor systems have been explored for nanotechnological applications including miniaturized sensor systems and lab-on-a-chip devices. Several developments have also revolved around possible exploitation of the filaments alone without their motor partners. Efforts to use the cytoskeletal filaments for applications often require chemical or genetic engineering of the filaments such as specific conjugation with fluorophores, antibodies, oligonucleotides or various macromolecular complexes e.g. nanoparticles. Similar conjugation methods are also instrumental for a range of fundamental biophysical studies. Here we review methods for non-covalent and covalent chemical modifications of actin filaments with focus on critical advantages and challenges of different methods as well as critical steps in the conjugation procedures. We also review potential uses of the engineered actin filaments in nanotechnological applications and in some key fundamental studies of actin and myosin function. Finally, we consider possible future lines of investigation that may be addressed by applying chemical conjugation of actin in new ways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Noncovalently-functionalized reduced graphene oxide sheets by water-soluble methyl green for supercapacitor application

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Xiaoying; Hu, Zhongai, E-mail: zhongai@nwnu.edu.cn; Hu, Haixiong

2015-10-15

Graphical abstract: Electroactive methyl green (MG) is selected to functionalize reduced graphene oxide (RGO) through non-covalent modification and the composite achieves high specific capacitance, good rate capability and excellent long life cycle. - Highlights: • MG–RGO composites were firstly prepared through non-covalent modification. • The mass ratio in composites is a key for achieving high specific capacitance. • MG–RGO 5:4 exhibits the highest specific capacitance of 341 F g{sup −1}. • MG–RGO 5:4 shows excellent rate capability and long life cycle. - Abstract: In the present work, water-soluble electroactive methyl green (MG) has been used to non-covalently functionalize reduced graphenemore » oxide (RGO) for enhancing supercapacitive performance. The microstructure, composition and morphology of MG–RGO composites are systematically characterized by UV–vis absorption, field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD). The electrochemical performances are investigated by cyclic voltammetry (CV), galvanostatic charge/discharge and electrochemical impedance spectroscopy (EIS). The fast redox reactions from MG could generate additional pseudocapacitance, which endows RGO higher capacitances. As a result, the MG–RGO composite (with the 5:4 mass ratio of MG:RGO) achieve a maximum value of 341 F g{sup −1} at 1 A g{sup −1} within the potential range from −0.25 to 0.75 V and provide a 180% enhancement in specific capacitance in comparison with pure RGO. Furthermore, excellent rate capability (72% capacitance retention from 1 A g{sup −1} to 20 A g{sup −1}) and long life cycle (12% capacitance decay after 5000 cycles) are achieved for the MG–RGO composite electrode.« less

Dimethyl Fumarate Inhibits the Nuclear Factor κB Pathway in Breast Cancer Cells by Covalent Modification of p65 Protein.

PubMed

Kastrati, Irida; Siklos, Marton I; Calderon-Gierszal, Esther L; El-Shennawy, Lamiaa; Georgieva, Gergana; Thayer, Emily N; Thatcher, Gregory R J; Frasor, Jonna

2016-02-12

In breast tumors, activation of the nuclear factor κB (NFκB) pathway promotes survival, migration, invasion, angiogenesis, stem cell-like properties, and resistance to therapy--all phenotypes of aggressive disease where therapy options remain limited. Adding an anti-inflammatory/anti-NFκB agent to breast cancer treatment would be beneficial, but no such drug is approved as either a monotherapy or adjuvant therapy. To address this need, we examined whether dimethyl fumarate (DMF), an anti-inflammatory drug already in clinical use for multiple sclerosis, can inhibit the NFκB pathway. We found that DMF effectively blocks NFκB activity in multiple breast cancer cell lines and abrogates NFκB-dependent mammosphere formation, indicating that DMF has anti-cancer stem cell properties. In addition, DMF inhibits cell proliferation and significantly impairs xenograft tumor growth. Mechanistically, DMF prevents p65 nuclear translocation and attenuates its DNA binding activity but has no effect on upstream proteins in the NFκB pathway. Dimethyl succinate, the inactive analog of DMF that lacks the electrophilic double bond of fumarate, is unable to inhibit NFκB activity. Also, the cell-permeable thiol N-acetyl l-cysteine, reverses DMF inhibition of the NFκB pathway, supporting the notion that the electrophile, DMF, acts via covalent modification. To determine whether DMF interacts directly with p65, we synthesized and used a novel chemical probe of DMF by incorporating an alkyne functionality and found that DMF covalently modifies p65, with cysteine 38 being essential for the activity of DMF. These results establish DMF as an NFκB inhibitor with anti-tumor activity that may add therapeutic value in the treatment of aggressive breast cancers. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Elevated levels of protein-bound p-hydroxyphenylacetaldehyde, an amino-acid-derived aldehyde generated by myeloperoxidase, are present in human fatty streaks, intermediate lesions and advanced atherosclerotic lesions.

PubMed Central

Hazen, S L; Gaut, J P; Crowley, J R; Hsu, F F; Heinecke, J W

2000-01-01

Reactive aldehydes might have a pivotal role in the pathogenesis of atherosclerosis by covalently modifying low-density lipoprotein (LDL). However, the identities of the aldehyde adducts that form on LDL in vivo are not yet clearly established. We previously demonstrated that the haem protein myeloperoxidase oxidizes proteins in the human artery wall. We also have shown that p-hydroxyphenylacetaldehyde (pHA), the aldehyde that forms when myeloperoxidase oxidizes L-tyrosine, covalently modifies the N(epsilon)-lysine residues of proteins. The resulting Schiff base can be quantified as N(epsilon)-[2-(p-hydroxyphenyl)ethyl]lysine (pHA-lysine) after reduction with NaCNBH(3). Here we demonstrate that pHA-lysine is a marker for LDL that has been modified by myeloperoxidase, and that water-soluble, but not lipid-soluble, antioxidants inhibit the modification of LDL protein. To determine whether myeloperoxidase-generated aldehydes might modify LDL in vivo, we used a combination of isotope-dilution GC-MS to quantify pHA-lysine in aortic tissues at various stages of lesion evolution. We also analysed LDL isolated from atherosclerotic aortic tissue. Comparison of normal and atherosclerotic aortic tissue demonstrated a significant elevation (more than 10-fold) of the reduced Schiff base adduct in fatty streaks, intermediate lesions and advanced lesions compared with normal aortic tissue. Moreover, the level of pHA-lysine in LDL recovered from atherosclerotic aortic intima was 200-fold that in plasma LDL of healthy donors. These results indicate that pHA-lysine, a specific covalent modification of LDL, is generated in human atherosclerotic vascular tissue. They also raise the possibility that reactive aldehydes generated by myeloperoxidase have a role in converting LDL into an atherogenic lipoprotein. PMID:11104675

In-situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens.

PubMed

Liang, Huei-Chen; Liu, Yi-Chen; Chen, Hsin; Ku, Ming Chun; Do, Quynh-Trang; Wang, Chih-Yen; Tzeng, Shun-Fen; Chen, Shu-Hui

2018-06-13

Catechol estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in-situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography-mass spectrometry (LC-MS). A total of 334 liver proteins were repeatedly identified (n  2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 non-specific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9) which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems.

Post-Translational Modification Biology of Glutamate Receptors and Drug Addiction

PubMed Central

Mao, Li-Min; Guo, Ming-Lei; Jin, Dao-Zhong; Fibuch, Eugene E.; Choe, Eun Sang; Wang, John Q.

2011-01-01

Post-translational covalent modifications of glutamate receptors remain a hot topic. Early studies have established that this family of receptors, including almost all ionotropic and metabotropic glutamate receptor subtypes, undergoes active phosphorylation at serine, threonine, or tyrosine residues in their intracellular domains. Recent evidence identifies several glutamate receptor subtypes to be direct substrates for palmitoylation at cysteine residues. Other modifications such as ubiquitination and sumoylation at lysine residues also occur to certain glutamate receptors. These modifications are dynamic and reversible in nature and are regulatable by changing synaptic inputs. The regulated modifications significantly impact the receptor in many ways, including interrelated changes in biochemistry (synthesis, subunit assembling, and protein–protein interactions), subcellular redistribution (trafficking, endocytosis, synaptic delivery, and clustering), and physiology, usually associated with changes in synaptic plasticity. Glutamate receptors are enriched in the striatum and cooperate closely with dopamine to regulate striatal signaling. Emerging evidence shows that modification processes of striatal glutamate receptors are sensitive to addictive drugs, such as psychostimulants (cocaine and amphetamine). Altered modifications are believed to be directly linked to enduring receptor/synaptic plasticity and drug-seeking. This review summarizes several major types of modifications of glutamate receptors and analyzes the role of these modifications in striatal signaling and in the pathogenesis of psychostimulant addiction. PMID:21441996

Structure of the TRPA1 ion channel suggests regulatory mechanisms.

PubMed

Paulsen, Candice E; Armache, Jean-Paul; Gao, Yuan; Cheng, Yifan; Julius, David

2015-04-23

The TRPA1 ion channel (also known as the wasabi receptor) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here we use single-particle electron cryo- microscopy to determine the structure of full-length human TRPA1 to ∼4 Å resolution in the presence of pharmacophores, including a potent antagonist. Several unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted transient receptor potential (TRP)-like allosteric domain. These findings provide new insights into the mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents.

Effective, homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine

PubMed Central

Baubec, Tuncay; Pecinka, Ales; Rozhon, Wilfried; Mittelsten Scheid, Ortrun

2009-01-01

Covalent modification by methylation of cytosine residues represents an important epigenetic hallmark. While sequence analysis after bisulphite conversion allows correlative analyses with single-base resolution, functional analysis by interference with DNA methylation is less precise, due to the complexity of methylation enzymes and their targets. A cytidine analogue, 5-azacytidine, is frequently used as an inhibitor of DNA methyltransferases, but its rapid degradation in aqueous solution is problematic for culture periods of longer than a few hours. Application of zebularine, a more stable cytidine analogue with a similar mode of action that is successfully used as a methylation inhibitor in Neurospora and mammalian tumour cell lines, can significantly reduce DNA methylation in plants in a dose-dependent and transient manner independent of sequence context. Demethylation is connected with transcriptional reactivation and partial decondensation of heterochromatin. Zebularine represents a promising new and versatile tool for investigating the role of DNA methylation in plants with regard to transcriptional control, maintenance and formation of (hetero-) chromatin. PMID:18826433

AGR2, an Endoplasmic Reticulum Protein, Is Secreted into the Gastrointestinal Mucus

PubMed Central

Bergström, Joakim H.; Berg, Katarina A.; Rodríguez-Piñeiro, Ana M.; Stecher, Bärbel; Johansson, Malin E. V.; Hansson, Gunnar C.

2014-01-01

The MUC2 mucin is the major constituent of the two mucus layers in colon. Mice lacking the disulfide isomerase-like protein Agr2 have been shown to be more susceptible to colon inflammation. The Agr2−/− mice have less filled goblet cells and were now shown to have a poorly developed inner colon mucus layer. We could not show AGR2 covalently bound to recombinant MUC2 N- and C-termini as have previously been suggested. We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles. In tissue culture (CHO-K1) cells, AGR2 is normally not secreted. Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal. In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine. PMID:25111734

Structure of the TRPA1 ion channel suggests regulatory mechanisms

PubMed Central

Paulsen, Candice E.; Armache, Jean-Paul; Gao, Yuan; Cheng, Yifan; Julius, David

2015-01-01

The TRPA1 ion channel (a.k.a the ‘wasabi receptor’) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here, we use single-particle electron cryo-microscopy to determine the structure of full-length human TRPA1 to ~4Å resolution in the presence of pharmacophores, including a potent antagonist. A number of unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted TRP-like allosteric domain. These findings provide novel insights into mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents. PMID:25855297

Understanding and controlling the substrate effect on graphene electron-transfer chemistry via reactivity imprint lithography

NASA Astrophysics Data System (ADS)

Wang, Qing Hua; Jin, Zhong; Kim, Ki Kang; Hilmer, Andrew J.; Paulus, Geraldine L. C.; Shih, Chih-Jen; Ham, Moon-Ho; Sanchez-Yamagishi, Javier D.; Watanabe, Kenji; Taniguchi, Takashi; Kong, Jing; Jarillo-Herrero, Pablo; Strano, Michael S.

2012-09-01

Graphene has exceptional electronic, optical, mechanical and thermal properties, which provide it with great potential for use in electronic, optoelectronic and sensing applications. The chemical functionalization of graphene has been investigated with a view to controlling its electronic properties and interactions with other materials. Covalent modification of graphene by organic diazonium salts has been used to achieve these goals, but because graphene comprises only a single atomic layer, it is strongly influenced by the underlying substrate. Here, we show a stark difference in the rate of electron-transfer reactions with organic diazonium salts for monolayer graphene supported on a variety of substrates. Reactions proceed rapidly for graphene supported on SiO2 and Al2O3 (sapphire), but negligibly on alkyl-terminated and hexagonal boron nitride (hBN) surfaces, as shown by Raman spectroscopy. We also develop a model of reactivity based on substrate-induced electron-hole puddles in graphene, and achieve spatial patterning of chemical reactions in graphene by patterning the substrate.

2D Covalent Metals: A New Materials Domain of Electrochemical CO2 Conversion with Broken Scaling Relationship.

PubMed

Shin, Hyeyoung; Ha, Yoonhoo; Kim, Hyungjun

2016-10-04

Toward a sustainable carbon cycle, electrochemical conversion of CO 2 into valuable fuels has drawn much attention. However, sluggish kinetics and a substantial overpotential, originating from the strong correlation between the adsorption energies of intermediates and products, are key obstacles of electrochemical CO 2 conversion. Here we show that 2D covalent metals with a zero band gap can overcome the intrinsic limitation of conventional metals and metal alloys and thereby substantially decrease the overpotential for CO 2 reduction because of their covalent characteristics. From first-principles-based high-throughput screening results on 61 2D covalent metals, we find that the strong correlation between the adsorption energies of COOH and CO can be entirely broken. This leads to the computational design of CO 2 -to-CO and CO 2 -to-CH 4 conversion catalysts in addition to hydrogen-evolution-reaction catalysts. Toward efficient electrochemical catalysts for CO 2 reduction, this work suggests a new materials domain having two contradictory properties in a single material: covalent nature and electrical conductance.

Dynamic Testing of Signal Transduction Deregulation During Breast Cancer Initiation

DTIC Science & Technology

2011-07-01

1 at a chamber pressure of ~3 × 10-6 Torr for the electron beam evaporated films. A Hitachi FB2100 Focused Ion Beam milling machine with a gallium ...immobilization. These include physical absorption, layer-by-layer (LBL) assembly, and covalent attachment, and eventually chose the covalent attachment...testing real-time signaling in live breast cancer cells, it is important to evaluate the nanosensors to monitor fluorescent compounds in single

Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications

PubMed Central

McClure, Michelle; DeLucas, Lawrence J.; Wilson, Landon; Ray, Marjorie; Rowe, Steven M.; Wu, Xiaoyun; Dai, Qun; Hong, Jeong S.; Sorscher, Eric J.; Kappes, John C.; Barnes, Stephen

2012-01-01

Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease. PMID:22119790

Optical excitation of carbon nanotubes drives stoichiometric reaction with diazonium salts

NASA Astrophysics Data System (ADS)

Powell, Lyndsey; Piao, Yanmei; Wang, Yuhuang; YuHuang Wang Research Group Team

Covalent chemistry is known to lack the precision required to tailor the physical properties of carbon nanostructures. Here we show that, for the first time, light can be used to drive a typically inefficient reaction with single-walled carbon nanotubes in a more stoichiometric fashion. Specifically, our experimental results suggest that light can enhance the reaction rate of diazonium salt with carbon nanotubes by as much as 35-fold, making possible stoichiometric control of the covalent bonding of a functional group to the sp2 carbon lattice. This light-controlled reaction paves the way for the possibility of highly selective and precise chemistry on single-walled carbon nanotubes and other graphitic nanostructures.

A revised set of values of single-bond radii derived from the observed interatomic distances in metals by correction for bond number and resonance energy

PubMed Central

Pauling, Linus; Kamb, Barclay

1986-01-01

An earlier discussion [Pauling, L. (1947) J. Am. Chem. Soc. 69, 542] of observed bond lengths in elemental metals with correction for bond number and resonance energy led to a set of single-bond metallic radii with values usually somewhat less than the corresponding values obtained from molecules and complex ions. A theory of resonating covalent bonds has now been developed that permits calculation of the number of resonance structures per atom and of the effective resonance energy per bond. With this refined method of correcting the observed bond lengths for the effect of resonance energy, a new set of single-bond covalent radii, in better agreement with values from molecules and complex ions, has been constructed. PMID:16593698

Non-covalent binding of nucleic acids with gold nanoparticles provides their stability and effective desorption in environment mimicking biological media.

PubMed

Epanchintseva, Anna; Dolodoev, Anton; Grigor'eva, Alina; Chelobanov, Boris; Pyshnyi, Dmitrii; Ryabchikova, Elena; Pyshnaya, Inna

2018-08-31

The ability of gold nanoparticles to bind different substances has resulted in the high interest of researchers determining their usage as a promising carrier of various biological substances including nucleic acids (NAs) for therapeutic applications. Most publications report covalent binding (conjugation) of an NA to spherical AuNPs via the Au-S bond. In this work, we obtained non-covalent associates of different ssDNA, ssRNA and siRNAs with spherical gold nanoparticles (AuNPs) and examined their physico-chemical properties and stability in media mimicking intracellular space (bacterial 'cytosol') and cell culture media (10% FBS in DMEM). The 'cytosol' was obtained from E. coli and possessed nuclease activity. For the first time, we used the phosphoryl guanidine (dimethylimidazolidin-2-imine, Dmi) group for modification of 3'-ends to enhance the stability of ssRNAs and siRNAs against nuclease destruction. Trying to evaluate the material balance, we analyzed the whole nucleotide species obtained after incubation of NA-AuNPs associates in 'cytosol' and FBS and evaluated the degree of NAs destruction, a share of full-size NAs remained on the surface of the AuNPs and in the solution. Native ss- and siRNAs, both free and in composition of non-covalent associates with AuNPs, were less resistant to degrading factors than ssDNA. The introduction of two Dmi-groups into the ssDNA increased its stability in 'cytosol' three times within 2.5 h. Dmi-modified siRNAs in non-covalent associates with AuNPs were two times more stable than unmodified siRNA within 4 h. We showed that non-covalent siRNA-AuNPs associates serve as a kind of storage for full-size NAs and thereby prolong their presence in nuclease-active media. Our study showed that non-covalent binding of siRNAs with a surface of AuNPs provides desorption of both strands, which is necessary for siRNA functioning in living cells, and could be considered as an important way to construct siRNA and ssDNA delivery systems based on AuNPs.

Biofunctional Paper via Covalent Modification of Cellulose

PubMed Central

Yu, Arthur; Shang, Jing; Cheng, Fang; Paik, Bradford A.; Kaplan, Justin M.; Andrade, Rodrigo B.; Ratner, Daniel M.

2012-01-01

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to covalently immobilize small molecules, proteins and DNA onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane’s bioactivity was specific, dose-dependent, and stable over a long period of time. Use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices. PMID:22708701

Biofunctional paper via the covalent modification of cellulose.

PubMed

Yu, Arthur; Shang, Jing; Cheng, Fang; Paik, Bradford A; Kaplan, Justin M; Andrade, Rodrigo B; Ratner, Daniel M

2012-07-31

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies, and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to immobilize small molecules, proteins, and DNA covalently onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane's bioactivity was specific, dose-dependent, and stable over a long period of time. The use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices.

Covalent functionalization and passivation of exfoliated black phosphorus via aryl diazonium chemistry.

PubMed

Ryder, Christopher R; Wood, Joshua D; Wells, Spencer A; Yang, Yang; Jariwala, Deep; Marks, Tobin J; Schatz, George C; Hersam, Mark C

2016-06-01

Functionalization of atomically thin nanomaterials enables the tailoring of their chemical, optical and electronic properties. Exfoliated black phosphorus (BP)-a layered two-dimensional semiconductor-exhibits favourable charge-carrier mobility, tunable bandgap and highly anisotropic properties, but it is chemically reactive and degrades rapidly in ambient conditions. Here we show that covalent aryl diazonium functionalization suppresses the chemical degradation of exfoliated BP even after three weeks of ambient exposure. This chemical modification scheme spontaneously forms phosphorus-carbon bonds, has a reaction rate sensitive to the aryl diazonium substituent and alters the electronic properties of exfoliated BP, ultimately yielding a strong, tunable p-type doping that simultaneously improves the field-effect transistor mobility and on/off current ratio. This chemical functionalization pathway controllably modifies the properties of exfoliated BP, and thus improves its prospects for nanoelectronic applications.

Covalent functionalization and passivation of exfoliated black phosphorus via aryl diazonium chemistry

NASA Astrophysics Data System (ADS)

Ryder, Christopher R.; Wood, Joshua D.; Wells, Spencer A.; Yang, Yang; Jariwala, Deep; Marks, Tobin J.; Schatz, George C.; Hersam, Mark C.

2016-06-01

Functionalization of atomically thin nanomaterials enables the tailoring of their chemical, optical and electronic properties. Exfoliated black phosphorus (BP)—a layered two-dimensional semiconductor—exhibits favourable charge-carrier mobility, tunable bandgap and highly anisotropic properties, but it is chemically reactive and degrades rapidly in ambient conditions. Here we show that covalent aryl diazonium functionalization suppresses the chemical degradation of exfoliated BP even after three weeks of ambient exposure. This chemical modification scheme spontaneously forms phosphorus-carbon bonds, has a reaction rate sensitive to the aryl diazonium substituent and alters the electronic properties of exfoliated BP, ultimately yielding a strong, tunable p-type doping that simultaneously improves the field-effect transistor mobility and on/off current ratio. This chemical functionalization pathway controllably modifies the properties of exfoliated BP, and thus improves its prospects for nanoelectronic applications.

Covalent Functionalization of NiTi Surfaces with Bioactive Peptide Amphiphile Nanofibers

PubMed Central

Sargeant, Timothy D.; Rao, Mukti S.; Koh, Chung-Yan

2009-01-01

Surface modification enables the creation of bioactive implants using traditional material substrates without altering the mechanical properties of the bulk material. For applications such as bone plates and stents, it is desirable to modify the surface of metal alloy substrates to facilitate cellular attachment, proliferation, and possibly differentiation. In this work we present a general strategy for altering the surface chemistry of nickel-titanium shape memory alloy (NiTi) in order to covalently attach self-assembled peptide amphiphile (PA) nanofibers with bioactive functions. Bioactivity in the systems studied here includes biological adhesion and proliferation of osteoblast and endothelial cell types. The optimized surface treatment creates a uniform TiO2 layer with low levels of Ni on the NiTi surface, which is subsequently covered with an aminopropylsilane coating using a novel, lower temperature vapor deposition method. This method produces an aminated surface suitable for covalent attachment of PA molecules containing terminal carboxylic acid groups. The functionalized NiTi surfaces have been characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and atomic force microscopy (AFM). These techniques offer evidence that the treated metal surfaces consist primarily of TiO2 with very little Ni, and also confirm the presence of the aminopropylsilane overlayer. Self-assembled PA nanofibers presenting the biological peptide adhesion sequence Arg-Gly-Asp-Ser are capable of covalently anchoring to the treated substrate, as demonstrated by spectrofluorimetry and AFM. Cell culture and scanning electron microscopy (SEM) demonstrate cellular adhesion, spreading, and proliferation on these functionalized metal surfaces. Furthermore, these experiments demonstrate that covalent attachment is crucial for creating robust PA nanofiber coatings, leading to confluent cell monolayers. PMID:18083225

[Preparation and performance characterization of gold nanoparticles modified chiral capillary electrochromatography stationary phase].

PubMed

Xiong, Lele; Li, Ruijun; Ji, Yibing

2017-07-08

Gold nanoparticles (GNPs, 15 nm) were prepared and introduced to amino groups derived silica monolithic column. Bovine serum albumin (BSA) was immobilized via covalent modification method onto the carboxylic functionalized GNPs to afford chiral stationary phase (CSP) for enantioseparation. GNPs were well dispersed and successfully incorporated onto the columns with the contents as high as 17.18% by characterization method such as transmission electron microscopy (TEM), ultraviolet (UV)-visible absorption spectra and scanning electron microscopy (SEM). The preparation conditions of the BSA modified CSP were optimized and 10% (v/v) 3-aminopropyltriethoxysilane (APTES) and 15 g/L BSA were selected as appropriate reaction conditions. The enantioseparation performance of the BSA modified CSP has been investigated by capillary electrochromatography (CEC). Enantiomers of tryptophan, ephedrine and atenolol were resolved, and the baseline separation of tryptophan was achieved. Meanwhile, the influences of pH value, buffer concentrations and applied voltages used on the chiral separation were studied, and the optimal separation conditions were 10 mmol/L phosphate buffer at pH 7.4 and 15 kV applied voltages. In comparison with the BSA modified CSP prepared by physical adsorption, the CSP prepared by covalent modification method had better separation results, and the analytes could be separated directly without pre-column derivatization. In addition, the prepared BSA modified CSP exhibited good run to run repeatability with relative standard deviations (RSDs) of the migration times and selectivity factors not more than 2.3% and 0.96%, respectively. This work offers a good thinking for modification with other proteins or other types of chiral selectors.

Sequential changes in chromatin structure during transcriptional activation in the beta globin LCR and its target gene.

PubMed

Kim, Kihoon; Kim, AeRi

2010-09-01

Chromatin structure is modulated during transcriptional activation. The changes include the association of transcriptional activators, formation of hypersensitive sites and covalent modifications of histones. To understand the order of the various changes accompanying transcriptional activation, we analyzed the mouse beta globin gene, which is transcriptionally inducible in erythroid MEL cells over a time course of HMBA treatment. Transcription of the globin genes requires the locus control region (LCR) consisting of several hypersensitive sites (HSs). Erythroid specific transcriptional activators such as NF-E2, GATA-1, TAL1 and EKLF were associated with the LCR in the uninduced state before transcriptional activation. The HSs of the LCR were formed in this state as revealed by high sensitivity to DNase I and MNase attack. However the binding of transcriptional activators and the depletion of histones were observed in the promoter of the beta globin gene only after transcriptional activation. In addition, various covalent histone modifications were sequentially detected in lysine residues of histone H3 during the activation. Acetylation of K9, K36 and K27 was notable in both LCR HSs and gene after induction but before transcriptional initiation. Inactive histone marks such as K9me2, K36me2 and K27me2 were removed coincident with transcriptional initiation in the gene region. Taken together, these results indicate that LCR has a substantially active structure in the uninduced state while transcriptional activation serially adds active marks, including histone modifications, and removes inactive marks in the target gene of the LCR. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

DTIC Science & Technology

2015-10-01

activation in melanoma cells using chemical biology and functional genomic approaches. In the first year of the study, we have developed more potent...post-translational modification by adding a 16-carbon palmitate) is required for N-RAS proper membrane localization and its oncogenic activities ...RAS regulation could be a novel strategy to treat N-RAS mutant melanoma. We have developed chemical probes that covalently label the active sites of

Activity-based probes for the ubiquitin conjugation-deconjugation machinery: new chemistries, new tools, and new insights.

PubMed

Hewings, David S; Flygare, John A; Bogyo, Matthew; Wertz, Ingrid E

2017-05-01

The reversible post-translational modification of proteins by ubiquitin and ubiquitin-like proteins regulates almost all cellular processes, by affecting protein degradation, localization, and complex formation. Deubiquitinases (DUBs) are proteases that remove ubiquitin modifications or cleave ubiquitin chains. Most DUBs are cysteine proteases, which makes them well suited for study by activity-based probes. These DUB probes report on deubiquitinase activity by reacting covalently with the active site in an enzyme-catalyzed manner. They have proven to be important tools to study DUB selectivity and proteolytic activity in different settings, to identify novel DUBs, and to characterize deubiquitinase inhibitors. Inspired by the efficacy of activity-based probes for DUBs, several groups have recently reported probes for the ubiquitin conjugation machinery (E1, E2, and E3 enzymes). Many of these enzymes, while not proteases, also posses active site cysteine residues and can be targeted by covalent probes. In this review, we will discuss how features of the probe (cysteine-reactive group, recognition element, and reporter tag) affect reactivity and suitability for certain experimental applications. We will also review the diverse applications of the current probes, and discuss the need for new probe types to study emerging aspects of ubiquitin biology. © 2017 Federation of European Biochemical Societies.

Improved estimation of ligand macromolecule binding affinities by linear response approach using a combination of multi-mode MD simulation and QM/MM methods

NASA Astrophysics Data System (ADS)

Khandelwal, Akash; Balaz, Stefan

2007-01-01

Structure-based predictions of binding affinities of ligands binding to proteins by coordination bonds with transition metals, covalent bonds, and bonds involving charge re-distributions are hindered by the absence of proper force fields. This shortcoming affects all methods which use force-field-based molecular simulation data on complex formation for affinity predictions. One of the most frequently used methods in this category is the Linear Response (LR) approach of Åquist, correlating binding affinities with van der Waals and electrostatic energies, as extended by Jorgensen's inclusion of solvent-accessible surface areas. All these terms represent the differences, upon binding, in the ensemble averages of pertinent quantities, obtained from molecular dynamics (MD) or Monte Carlo simulations of the complex and of single components. Here we report a modification of the LR approach by: (1) the replacement of the two energy terms through the single-point QM/MM energy of the time-averaged complex structure from an MD simulation; and (2) a rigorous consideration of multiple modes (mm) of binding. The first extension alleviates the force-field related problems, while the second extension deals with the ligands exhibiting large-scale motions in the course of an MD simulation. The second modification results in the correlation equation that is nonlinear in optimized coefficients, but does not lead to an increase in the number of optimized coefficients. The application of the resulting mm QM/MM LR approach to the inhibition of zinc-dependent gelatinase B (matrix metalloproteinase 9) by 28 hydroxamate ligands indicates a significant improvement of descriptive and predictive abilities.

Role of adsorbed surfactant in the reaction of aryl diazonium salts with single-walled carbon nanotubes.

PubMed

Hilmer, Andrew J; McNicholas, Thomas P; Lin, Shangchao; Zhang, Jingqing; Wang, Qing Hua; Mendenhall, Jonathan D; Song, Changsik; Heller, Daniel A; Barone, Paul W; Blankschtein, Daniel; Strano, Michael S

2012-01-17

Because covalent chemistry can diminish the optical and electronic properties of single-walled carbon nanotubes (SWCNTs), there is significant interest in developing methods of controllably functionalizing the nanotube sidewall. To date, most attempts at obtaining such control have focused on reaction stoichiometry or strength of oxidative treatment. Here, we examine the role of surfactants in the chemical modification of single-walled carbon nanotubes with aryl diazonium salts. The adsorbed surfactant layer is shown to affect the diazonium derivatization of carbon nanotubes in several ways, including electrostatic attraction or repulsion, steric exclusion, and direct chemical modification of the diazonium reactant. Electrostatic effects are most pronounced in the cases of anionic sodium dodecyl sulfate and cationic cetyltrimethylammonium bromide, where differences in surfactant charge can significantly affect the ability of the diazonium ion to access the SWCNT surface. For bile salt surfactants, with the exception of sodium cholate, we find that the surfactant wraps tightly enough such that exclusion effects are dominant. Here, sodium taurocholate exhibits almost no reactivity under the explored reaction conditions, while for sodium deoxycholate and sodium taurodeoxycholate, we show that the greatest extent of reaction is observed among a small population of nanotube species, with diameters between 0.88 and 0.92 nm. The anomalous reaction of nanotubes in this diameter range seems to imply that the surfactant is less effective at coating these species, resulting in a reduced surface coverage on the nanotube. Contrary to the other bile salts studied, sodium cholate enables high selectivity toward metallic species and small band gap semiconductors, which is attributed to surfactant-diazonium coupling to form highly reactive diazoesters. Further, it is found that the rigidity of anionic surfactants can significantly influence the ability of the surfactant layer to stabilize the diazonium ion near the nanotube surface. Such Coulombic and surfactant packing effects offer promise toward employing surfactants to controllably functionalize carbon nanotubes. © 2011 American Chemical Society

Fine Tuning Gene Expression: The Epigenome

PubMed Central

Mohtat, Davoud; Susztak, Katalin

2011-01-01

An epigenetic trait is a stably inherited phenotype resulting from changes in a chromosome without alterations in the DNA sequence. Epigenetic modifications, such as; DNA methylation, together with covalent modification of histones, are thought to alter chromatin density and accessibility of the DNA to cellular machinery, thereby modulating the transcriptional potential of the underlying DNA sequence. As epigenetic marks under environmental influence, epigenetics provides an added layer of variation that might mediate the relationship between genotype and internal and external environmental factors. Integration of our knowledge in genetics, epigenomics and genomics with the use of systems biology tools may present investigators with new powerful tools to study many complex human diseases such as kidney disease. PMID:21044758

Reprogramming cellular events by poly(ADP-ribose)-binding proteins

PubMed Central

Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

2013-01-01

Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

Installation of a reactive site for covalent wiring onto an intrinsically conductive poly(ionic liquid)

DOE PAGES

Brombosz, Scott M.; Lee, Sungwon; Firestone, Millicent A.

2014-11-04

We describe post-polymerization radical bromination of a nanostructured poly(ionic liquid) that selectively introduces a reactive bromo-group onto the polyalkylthiophene backbone. Raman and FT-IR spectroscopy proves that the bromine is successfully introduced at the 3-methyl position of the thiophene and that the molecular structure of the polymer remains largely intact with only minimal chain scission detected. FT-IR and Vis-NIR spectroscopy indicates that incorporation of the bromine induces twisting (loss of co-planarity) of the polythiophene backbone. WAXS confirms retention of an ordered lamellar structure with minor lattice spacing contraction. Cyclic voltammetry confirms spectroscopic findings that the bromination reaction yields a stable p-dopedmore » polymer. The installed bromine is susceptible to nucleophilic displacement permitting the covalent attachment of other functional molecules, such as a dialkylphosphonate. Elemental analysis of such a transformation established that 100 % functionalization can be achieved. These results collectively demonstrate that post-modification of a π-conjugated polymer can be used to both tune electronic and photonic properties, as well as install a chemoselective attachment point for the covalent wiring of other molecules.« less

Molecular Biodynamers: Dynamic Covalent Analogues of Biopolymers

PubMed Central

2017-01-01

Conspectus Constitutional dynamic chemistry (CDC) features the use of reversible linkages at both molecular and supramolecular levels, including reversible covalent bonds (dynamic covalent chemistry, DCC) and noncovalent interactions (dynamic noncovalent chemistry, DNCC). Due to its inherent reversibility and stimuli-responsiveness, CDC has been widely utilized as a powerful tool for the screening of bioactive compounds, the exploitation of receptors or substrates driven by molecular recognition, and the fabrication of constitutionally dynamic materials. Implementation of CDC in biopolymer science leads to the generation of constitutionally dynamic analogues of biopolymers, biodynamers, at the molecular level (molecular biodynamers) through DCC or at the supramolecular level (supramolecular biodynamers) via DNCC. Therefore, biodynamers are prepared by reversible covalent polymerization or noncovalent polyassociation of biorelevant monomers. In particular, molecular biodynamers, biodynamers of the covalent type whose monomeric units are connected by reversible covalent bonds, are generated by reversible polymerization of bio-based monomers and can be seen as a combination of biopolymers with DCC. Owing to the reversible covalent bonds used in DCC, molecular biodynamers can undergo continuous and spontaneous constitutional modifications via incorporation/decorporation and exchange of biorelevant monomers in response to internal or external stimuli. As a result, they behave as adaptive materials with novel properties, such as self-healing, stimuli-responsiveness, and tunable mechanical and optical character. More specifically, molecular biodynamers combine the biorelevant characters (e.g., biocompatibility, biodegradability, biofunctionality) of bioactive monomers with the dynamic features of reversible covalent bonds (e.g., changeable, tunable, controllable, self-healing, and stimuli-responsive capacities), to realize synergistic properties in one system. In addition, molecular biodynamers are commonly produced in aqueous media under mild or even physiological conditions to suit their biorelated applications. In contrast to static biopolymers emphasizing structural stability and unity by using irreversible covalent bonds, molecular biodynamers are seeking relative structural adaptability and diversity through the formation of reversible covalent bonds. Based on these considerations, molecular biodynamers are capable of reorganizing their monomers, generating, identifying, and amplifying the fittest structures in response to environmental factors. Hence, molecular biodynamers have received considerable research attention over the past decades. Accordingly, the construction of molecular biodynamers through equilibrium polymerization of nucleobase-, carbohydrate- or amino-acid-based monomers can lead to the fabrication of dynamic analogues of nucleic acids (DyNAs), polysaccharides (glycodynamers), or proteins (dynamic proteoids), respectively. In this Account, we summarize recent advances in developing different types of molecular biodynamers as structural or functional biomimetics of biopolymers, including DyNAs, glycodynamers, and dynamic proteoids. We introduce how chemists utilize various reversible reactions to generate molecular biodynamers with specific sequences and well-ordered structures in aqueous medium. We also discuss and list their potential applications in various research fields, such as drug delivery, drug discovery, gene sensing, cancer diagnosis, and treatment. PMID:28169527

Covalent Binding Antibodies Suppress Advanced Glycation: On the Innate Tier of Adaptive Immunity

PubMed Central

Shcheglova, T.; Makker, S. P.

2009-01-01

Non-enzymatic protein glycation is a source of metabolic stress that contributes to cytotoxicity and tissue damage. Hyperglycemia has been linked to elevation of advanced glycation endproducts, which mediate much of the vascular pathology leading to diabetic complications. Enhanced glycation of immunoglobulins and their accelerated vascular clearance is proposed as a natural mechanism to intercept alternative advanced glycation endproducts, thereby mitigating microvascular disease. We reported that antibodies against the glycoprotein KLH have elevated reactivity for glycopeptides from diabetic serum. These reactions are mediated by covalent binding between antibody light chains and carbonyl groups of glycated peptides. Diabetic animals that were immunized to induce reactive antibodies had attenuated diabetic nephropathy, which correlated with reduced levels of circulating and kidney-bound glycation products. Molecular analysis of antibody glycation revealed the preferential modification of light chains bearing germline-encoded lambda V regions. We previously noted that antibody fragments carrying V regions in the germline configuration are selected from a human Fv library by covalent binding to a reactive organophosphorus ester. These Fv fragments were specifically modified at light chain V region residues, which map to the combining site at the interface between light and heavy chains. These findings suggest that covalent binding is an innate property of antibodies, which may be encoded in the genome for specific physiological purposes. This hypothesis is discussed in context with current knowledge of the natural antibodies that recognize altered self molecules and the catalytic autoantibodies found in autoimmune disease. PMID:22649604

Facile one-step construction of covalently networked, self-healable, and transparent superhydrophobic composite films

NASA Astrophysics Data System (ADS)

Lee, Yujin; You, Eun-Ah; Ha, Young-Geun

2018-07-01

Despite the considerable demand for bioinspired superhydrophobic surfaces with highly transparent, self-cleaning, and self-healable properties, a facile and scalable fabrication method for multifunctional superhydrophobic films with strong chemical networks has rarely been established. Here, we report a rationally designed facile one-step construction of covalently networked, transparent, self-cleaning, and self-healable superhydrophobic films via a one-step preparation and single-reaction process of multi-components. As coating materials for achieving the one-step fabrication of multifunctional superhydrophobic films, we included two different sizes of Al2O3 nanoparticles for hierarchical micro/nano dual-scale structures and transparent films, fluoroalkylsilane for both low surface energy and covalent binding functions, and aluminum nitrate for aluminum oxide networked films. On the basis of stability tests for the robust film composition, the optimized, covalently linked superhydrophobic composite films with a high water contact angle (>160°) and low sliding angle (<1°) showed excellent thermal stability (up to 400 °C), transparency (≈80%), self-healing, self-cleaning, and waterproof abilities. Therefore, the rationally designed, covalently networked superhydrophobic composite films, fabricated via a one-step solution-based process, can be further utilized for various optical and optoelectronic applications.

Real Time Sensing and Discrimination of Single Chemicals Using Channel of Phi29 DNA Packaging Nanomotor

PubMed Central

Haque, Farzin; Lunn, Jennifer; Fang, Huaming; Smithrud, David; Guo, Peixuan

2012-01-01

A highly sensitive and reliable method to sense and identify a single chemical at extremely low concentrations and high contamination is important for environmental surveillance, homeland security, athlete drug monitoring, toxin/drug screening, and earlier disease diagnosis. This manuscript reports a method for precise detection of single chemicals. The hub of the bacteriophage phi29 DNA packaging motor is a connector consisting of twelve protein subunits encircled into a 3.6-nm channel as a path for dsDNA to enter during packaging and to exit during infection. The connector has previously been inserted into a lipid bilayer to serve as a membrane-embedded channel. Herein we report the modification of the phi29 channel to develop a class of sensors to detect single chemicals. The Lysine-234 of each protein subunit was mutated to cysteine, generating 12-SH ring lining the channel wall. Chemicals passing through this robust channel and interactions with the SH-group generated extremely reliable, precise, and sensitive current signatures as revealed by single channel conductance assays. Ethane (57 Daltons), thymine (167 Daltons), and benzene (105 Daltons) with reactive thioester moieties were clearly discriminated upon interaction with the available set of cysteine residues. The covalent attachment of each analyte induced discrete step-wise blockage in current signature with a corresponding decrease in conductance due to the physical blocking of the channel. Transient binding of the chemicals also produced characteristic fingerprints that were deduced from the unique blockage amplitude and pattern of the signals. This study shows that the phi29 connector can be used to sense chemicals with reactive thioesters or maleimide using single channel conduction assays based on their distinct fingerprints. The results demonstrated that this channel system could be further developed into very sensitive sensing devices. PMID:22458779

Real-time sensing and discrimination of single chemicals using the channel of phi29 DNA packaging nanomotor.

PubMed

Haque, Farzin; Lunn, Jennifer; Fang, Huaming; Smithrud, David; Guo, Peixuan

2012-04-24

A highly sensitive and reliable method to sense and identify a single chemical at extremely low concentrations and high contamination is important for environmental surveillance, homeland security, athlete drug monitoring, toxin/drug screening, and earlier disease diagnosis. This article reports a method for precise detection of single chemicals. The hub of the bacteriophage phi29 DNA packaging motor is a connector consisting of 12 protein subunits encircled into a 3.6 nm channel as a path for dsDNA to enter during packaging and to exit during infection. The connector has previously been inserted into a lipid bilayer to serve as a membrane-embedded channel. Herein we report the modification of the phi29 channel to develop a class of sensors to detect single chemicals. The lysine-234 of each protein subunit was mutated to cysteine, generating 12-SH ring lining the channel wall. Chemicals passing through this robust channel and interactions with the SH group generated extremely reliable, precise, and sensitive current signatures as revealed by single channel conductance assays. Ethane (57 Da), thymine (167 Da), and benzene (105 Da) with reactive thioester moieties were clearly discriminated upon interaction with the available set of cysteine residues. The covalent attachment of each analyte induced discrete stepwise blockage in current signature with a corresponding decrease in conductance due to the physical blocking of the channel. Transient binding of the chemicals also produced characteristic fingerprints that were deduced from the unique blockage amplitude and pattern of the signals. This study shows that the phi29 connector can be used to sense chemicals with reactive thioesters or maleimide using single channel conduction assays based on their distinct fingerprints. The results demonstrated that this channel system could be further developed into very sensitive sensing devices.

Covalent antibody display—an in vitro antibody-DNA library selection system

PubMed Central

Reiersen, Herald; Løbersli, Inger; Løset, Geir Å.; Hvattum, Else; Simonsen, Bjørg; Stacy, John E.; McGregor, Duncan; FitzGerald, Kevin; Welschof, Martin; Brekke, Ole H.; Marvik, Ole J.

2005-01-01

The endonuclease P2A initiates the DNA replication of the bacteriophage P2 by making a covalent bond with its own phosphate backbone. This enzyme has now been exploited as a new in vitro display tool for antibody fragments. We have constructed genetic fusions of P2A with single-chain antibodies (scFvs). Linear DNA of these fusion proteins were processed in an in vitro coupled transcription–translation mixture of Escherichia coli S30 lysate. Complexes of scFv–P2A fusion proteins covalently bound to their own DNA were isolated after panning on immobilized antigen, and the enriched DNAs were recovered by PCR and prepared for the subsequent cycles of panning. We have demonstrated the enrichment of scFvs from spiked libraries and the specific selection of different anti-tetanus toxoid scFvs from a V-gene library with 50 million different members prepared from human lymphocytes. This covalent antibody display technology offers a complete in vitro selection system based exclusively on DNA–protein complexes. PMID:15653626

Cell signaling, post-translational protein modifications and NMR spectroscopy

PubMed Central

Theillet, Francois-Xavier; Smet-Nocca, Caroline; Liokatis, Stamatios; Thongwichian, Rossukon; Kosten, Jonas; Yoon, Mi-Kyung; Kriwacki, Richard W.; Landrieu, Isabelle; Lippens, Guy

2016-01-01

Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy. PMID:23011410

Single-Molecule Reaction Chemistry in Patterned Nanowells

PubMed Central

2016-01-01

A new approach to synthetic chemistry is performed in ultraminiaturized, nanofabricated reaction chambers. Using lithographically defined nanowells, we achieve single-point covalent chemistry on hundreds of individual carbon nanotube transistors, providing robust statistics and unprecedented spatial resolution in adduct position. Each device acts as a sensor to detect, in real-time and through quantized changes in conductance, single-point functionalization of the nanotube as well as consecutive chemical reactions, molecular interactions, and molecular conformational changes occurring on the resulting single-molecule probe. In particular, we use a set of sequential bioconjugation reactions to tether a single-strand of DNA to the device and record its repeated, reversible folding into a G-quadruplex structure. The stable covalent tether allows us to measure the same molecule in different solutions, revealing the characteristic increased stability of the G-quadruplex structure in the presence of potassium ions (K+) versus sodium ions (Na+). Nanowell-confined reaction chemistry on carbon nanotube devices offers a versatile method to isolate and monitor individual molecules during successive chemical reactions over an extended period of time. PMID:27270004

Structural Biology of Non-Ribosomal Peptide Synthetases

PubMed Central

Miller, Bradley R.; Gulick, Andrew M.

2016-01-01

Summary The non-ribosomal peptide synthetases are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain that catalyzes release of the peptide product. This multi-domain modular architecture raises questions about the structural features that enable this assembly line synthesis in an efficient manner. The structures of the core component domains have been determined and demonstrate insights into the catalytic activity. More recently, multi-domain structures have been determined and are providing clues to the features of these enzyme systems that govern the functional interaction between multiple domains. This chapter describes the structures of NRPS proteins and the strategies that are being used to assist structural studies of these dynamic proteins, including careful consideration of domain boundaries for generation of truncated proteins and the use of mechanism-based inhibitors that trap interactions between the catalytic and carrier protein domains. PMID:26831698

Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Persson, Jenna; Ekwall, Karl, E-mail: karl.ekwall@ki.se; School of Life Sciences, University College Sodertorn, NOVUM, Huddinge

Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that canmore » arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.« less

Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-{Delta}{sup 12,14}-prostaglandin J2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mishra, Nandita; Kar, Rekha; Singha, Prajjal K.

2010-04-23

Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 {mu}M concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexesmore » is evident when Drp1 is incubated with a non-cleavable GTP analog, GTP{gamma}S or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.« less

Thermally stable electrolytes for rechargeable lithium batteries, phase 2

NASA Technical Reports Server (NTRS)

Dominey, L. A.; Goldman, J. L.; Koch, V. R.

1989-01-01

During the second year of research under NASA SBIR Contract NAS7-967, Covalent Associates and NASA contract monitors at the Jet Propulsion Laboratory agreed to perform an evaluation of the three best electrolytes developed during Phase 2. Due to the extensive period of time required to collect meaningful cycling data, we realized the study would extend well beyond the original formal end of the Phase 2 program (August 31, 1988). The substitution of this effort in lieu of an earlier proposed 20-cell final deliverable is formally documented in Modification No. 1 of Contract NAS7-967 as task 7. This Addendum contains the results of the cycling studies performed at Covalent Associates. In addition, sealed ampoules of each of these three electrolytes were delivered to the Jet Propulsion Laboratory Electrochemical Power Group. Their concurrent evaluation in a different test vehicle has also been recently concluded and their results are also summarized herein.

Nanoporous Gold for Enzyme Immobilization.

PubMed

Stine, Keith J; Jefferson, Kenise; Shulga, Olga V

2017-01-01

Nanoporous gold (NPG) is a material of emerging interest for immobilization of biomolecules, especially enzymes. The material provides a high surface area form of gold that is suitable for physisorption or for covalent modification by self-assembled monolayers. The material can be used as a high surface area electrode and with immobilized enzymes can be used for amperometric detection schemes. NPG can be prepared in a variety of formats from alloys containing between 20 and 50 % atomic composition of gold and less noble element(s) by dealloying procedures. Materials resembling NPG can be prepared by hydrothermal and electrodeposition methods. Related high surface area gold structures have been prepared using templating approaches. Covalent enzyme immobilization can be achieved by first forming a self-assembled monolayer on NPG bearing a terminal reactive functional group followed by conjugation to the enzyme through amide linkages to lysine residues. Enzymes can also be entrapped by physisorption or immobilized by electrostatic interactions.

Mechanisms of Drug Toxicity and Relevance to Pharmaceutical Development

PubMed Central

Guengerich, F. Peter

2016-01-01

Toxicity has been estimated to be responsible for the attrition of ~ 1/3 of drug candidates and is a major contributor to the high cost of drug development, particularly when not recognized until late in the clinical trials or post-marketing. The causes of drug toxicity can be organized in several ways and include mechanism-based (on-target) toxicity, immune hypersensitivity, off-target toxicity, and bioactivation/covalent modification. In addition, idiosyncratic responses are rare but one of the most problematic issues; several hypotheses for these have been advanced. Although covalent binding of drugs to proteins was described almost 40 years ago, the significance to toxicity has been difficult to establish; recent literature in this field is considered. The development of more useful biomarkers and short-term assays for rapid screening of drug toxicity early in the drug discovery/development process is a major goal, and some progress has been made using “omics” approaches. PMID:20978361

The anticancer natural product ophiobolin A induces cytotoxicity by covalent modification of phosphatidylethanolamine.

PubMed

Chidley, Christopher; Trauger, Sunia A; Birsoy, Kıvanç; O'Shea, Erin K

2016-07-12

Phenotypic screens allow the identification of small molecules with promising anticancer activity, but the difficulty in characterizing the mechanism of action of these compounds in human cells often undermines their value as drug leads. Here, we used a loss-of-function genetic screen in human haploid KBM7 cells to discover the mechanism of action of the anticancer natural product ophiobolin A (OPA). We found that genetic inactivation of de novo synthesis of phosphatidylethanolamine (PE) mitigates OPA cytotoxicity by reducing cellular PE levels. OPA reacts with the ethanolamine head group of PE in human cells to form pyrrole-containing covalent cytotoxic adducts and these adducts lead to lipid bilayer destabilization. Our characterization of this unusual cytotoxicity mechanism, made possible by unbiased genetic screening in human cells, suggests that the selective antitumor activity displayed by OPA may be due to altered membrane PE levels in cancer cells.

Thermally stable electrolytes for rechargeable lithium batteries, phase 2

NASA Astrophysics Data System (ADS)

Dominey, L. A.; Goldman, J. L.; Koch, V. R.

1989-09-01

During the second year of research under NASA SBIR Contract NAS7-967, Covalent Associates and NASA contract monitors at the Jet Propulsion Laboratory agreed to perform an evaluation of the three best electrolytes developed during Phase 2. Due to the extensive period of time required to collect meaningful cycling data, we realized the study would extend well beyond the original formal end of the Phase 2 program (August 31, 1988). The substitution of this effort in lieu of an earlier proposed 20-cell final deliverable is formally documented in Modification No. 1 of Contract NAS7-967 as task 7. This Addendum contains the results of the cycling studies performed at Covalent Associates. In addition, sealed ampoules of each of these three electrolytes were delivered to the Jet Propulsion Laboratory Electrochemical Power Group. Their concurrent evaluation in a different test vehicle has also been recently concluded and their results are also summarized herein.

Exogenous and endogenous DNA modifications as monitored by 32P-postlabeling: relationships to cancer and aging.

PubMed

Randerath, K; Li, D; Nath, R; Randerath, E

1992-01-01

32P-postlabeling analysis, a highly sensitive method for the detection and measurement of covalent carcinogen-DNA adducts and other DNA modifications, does not require radioactive test substances and, therefore, can be applied to DNA of mammals, including humans exposed to low doses of environmental or occupational genotoxicants. The basic procedure entails the enzymatic incorporation of 32P-label into hydrolysis products of DNA, followed by chromatographic mapping and autoradiography of the 32P-labeled digestion products and quantitative scintillation spectrometry. Microgram amounts of DNA are analyzed: Thus the assay is suited for limited amounts of cells or tissues. Various versions of the assay afford different sensitivities of adduct detection. A single aromatic or bulky/hydrophobic adduct in 10(8)-10(10) nucleotides can be detected and measured (corresponding to 0.3-30 amol adduct/micrograms DNA or 0.1-10 nmol adduct/mol DNA-P). In animal models, the assay has been successfully applied to a variety of mutagenic (genotoxic) as well as nonmutagenic carcinogens. In humans, DNA specimens from cigarette smokers, iron foundry workers, and coke oven workers whose total aromatic adduct levels ranged from 1 adduct in 10(6)-10(8) DNA nucleotides have been examined by 32P-postlabeling. The assay also detects DNA modifications--Indigenous (I)-compounds--that increase with age in untreated animals. I-compound profiles and levels are highly species-, strain-, sex-, and tissue-specific, and also depend on diet composition. Caloric restriction, a highly efficient method for improving resistance to carcinogenesis and extending life span, increased rather than decreased I-compound levels in various tissues of male rats. Nonmutagenic hepatocarcinogens reduced levels of I-compounds in the target organ. Because of the specificity of this effect, reduction of I-compound levels appears to represent a novel biomarker for the action of nonmutagenic carcinogens. DNA from various hepatomas was found largely devoid of I-compounds. The results support a possible antineoplastic and antiaging role of these DNA modifications.

Covalent Organic Frameworks as a Platform for Multidimensional Polymerization.

PubMed

Bisbey, Ryan P; Dichtel, William R

2017-06-28

The simultaneous polymerization and crystallization of monomers featuring directional bonding designs provides covalent organic frameworks (COFs), which are periodic polymer networks with robust covalent bonds arranged in two- or three-dimensional topologies. The range of properties characterized in COFs has rapidly expanded to include those of interest for heterogeneous catalysis, energy storage and photovoltaic devices, and proton-conducting membranes. Yet many of these applications will require materials quality, morphological control, and synthetic efficiency exceeding the capabilities of contemporary synthetic methods. This level of control will emerge from an improved fundamental understanding of COF nucleation and growth processes. More powerful characterization of structure and defects, improved syntheses guided by mechanistic understanding, and accessing diverse isolated forms, ranging from single crystals to thin films to colloidal suspensions, remain important frontier problems.

Covalent Organic Frameworks as a Platform for Multidimensional Polymerization

PubMed Central

2017-01-01

The simultaneous polymerization and crystallization of monomers featuring directional bonding designs provides covalent organic frameworks (COFs), which are periodic polymer networks with robust covalent bonds arranged in two- or three-dimensional topologies. The range of properties characterized in COFs has rapidly expanded to include those of interest for heterogeneous catalysis, energy storage and photovoltaic devices, and proton-conducting membranes. Yet many of these applications will require materials quality, morphological control, and synthetic efficiency exceeding the capabilities of contemporary synthetic methods. This level of control will emerge from an improved fundamental understanding of COF nucleation and growth processes. More powerful characterization of structure and defects, improved syntheses guided by mechanistic understanding, and accessing diverse isolated forms, ranging from single crystals to thin films to colloidal suspensions, remain important frontier problems. PMID:28691064

Preserving π-conjugation in covalently functionalized carbon nanotubes for optoelectronic applications.

PubMed

Setaro, Antonio; Adeli, Mohsen; Glaeske, Mareen; Przyrembel, Daniel; Bisswanger, Timo; Gordeev, Georgy; Maschietto, Federica; Faghani, Abbas; Paulus, Beate; Weinelt, Martin; Arenal, Raul; Haag, Rainer; Reich, Stephanie

2017-01-30

Covalent functionalization tailors carbon nanotubes for a wide range of applications in varying environments. Its strength and stability of attachment come at the price of degrading the carbon nanotubes sp 2 network and destroying the tubes electronic and optoelectronic features. Here we present a non-destructive, covalent, gram-scale functionalization of single-walled carbon nanotubes by a new [2+1] cycloaddition. The reaction rebuilds the extended π-network, thereby retaining the outstanding quantum optoelectronic properties of carbon nanotubes, including bright light emission at high degree of functionalization (1 group per 25 carbon atoms). The conjugation method described here opens the way for advanced tailoring nanotubes as demonstrated for light-triggered reversible doping through photochromic molecular switches and nanoplasmonic gold-nanotube hybrids with enhanced infrared light emission.

Indirect photopatterning of functionalized organic monolayers via copper-catalyzed "click chemistry"

NASA Astrophysics Data System (ADS)

Williams, Mackenzie G.; Teplyakov, Andrew V.

2018-07-01

Solution-based lithographic surface modification of an organic monolayer on a solid substrate is attained based on selective area photo-reduction of copper (II) to copper (I) to catalyze the azide-alkyne dipolar cycloaddition "click" reaction. X-ray photoelectron spectroscopy is used to confirm patterning, and spectroscopic results are analyzed and supplemented with computational models to confirm the surface chemistry. It is determined that this surface modification approach requires irradiation of the solid substrate with all necessary components present in solution. This method requires only minutes of irradiation to result in spatial and temporal control of the covalent surface functionalization of a monolayer and offers the potential for wavelength tunability that may be desirable in many applications utilizing organic monolayers.

Epigenetics primer: why the clinician should care about epigenetics.

PubMed

Duarte, Julio D

2013-12-01

Epigenetics describes heritable alterations of gene expression that do not involve DNA sequence variation and are changeable throughout an organism's lifetime. Not only can epigenetic status influence drug response, but it can also be modulated by drugs. In this review, the three major epigenetic mechanisms are described: covalent DNA modification, histone protein modification, and regulation by noncoding RNA. Further, this review describes how drug therapy can influence, and be influenced by, these mechanisms. Drugs with epigenetic mechanisms are already in use, with many more likely to be approved within the next few years. As the understanding of epigenetic processes improves, so will the ability to use these data in the clinic to improve patient care. © 2013 Pharmacotherapy Publications, Inc.

Reductive nanocomplex encapsulation of cRGD-siRNA conjugates for enhanced targeting to cancer cells

PubMed Central

Zhang, Yanfen; Yang, Xiantao; Ma, Yuan; Guan, Zhu; Wu, Yun; Zhang, Lihe; Yang, Zhenjun

2017-01-01

In this study, through covalent conjugation and lipid material entrapment, a combined modification strategy was established for effective delivery of small interfering RNA (siRNA). Single strands of siRNA targeting to BRAFV600E gene (siMB3) conjugated with cRGD peptide at 3′-terminus or 5′-terminus via cleavable disulfide bond was synthesized and then annealed with corresponding strands to obtain single and bis-cRGD-siRNA conjugates. A cationic lipid material (CLD) developed by our laboratory was mixed with the conjugates to generate nanocomplexes; their uniformity and electrical property were revealed by particle size and zeta potential measurement. Compared with CLD/siBraf, CLD/cRGD-siBraf achieved higher cell uptake and more excellent tumor-targeting ability, especially 21 (sense-5′/antisense-3″-cRGD-congjugate) nanocomplex. Moreover, they can regulate multiple pathways to varying degree and reduce acidification of endosome. Compared with the gene silencing of different conjugates, single or bis-cRGD-conjugated siRNA showed little differences except 22 (5/5) which cRGD was conjugated at 5′-terminus of antisense strand and sense strand. However bis-cRGD conjugate 21 nanocomplex exhibited better specific target gene silencing at multiple time points. Furthermore, the serum stabilities of the bis-cRGD conjugates were higher than those of the single-cRGD conjugates. In conclusion, all these data suggested that CLD/bis-conjugates, especially CLD/21, can be an effective system for delivery of siRNA to target BRAFV600E gene for therapy of melanoma. PMID:29042774

49 CFR 232.307 - Modification of the single car air brake test procedures.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 4 2014-10-01 2014-10-01 false Modification of the single car air brake test... Requirements § 232.307 Modification of the single car air brake test procedures. (a) Request. The AAR or other authorized representative of the railroad industry may seek modification of the single car air brake test...

49 CFR 232.307 - Modification of the single car air brake test procedures.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 4 2013-10-01 2013-10-01 false Modification of the single car air brake test... Requirements § 232.307 Modification of the single car air brake test procedures. (a) Request. The AAR or other authorized representative of the railroad industry may seek modification of the single car air brake test...

49 CFR 232.307 - Modification of the single car air brake test procedures.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Requirements § 232.307 Modification of the single car air brake test procedures. (a) Request. The AAR or other authorized representative of the railroad industry may seek modification of the single car air brake test... 49 Transportation 4 2010-10-01 2010-10-01 false Modification of the single car air brake test...

49 CFR 232.307 - Modification of the single car air brake test procedures.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Requirements § 232.307 Modification of the single car air brake test procedures. (a) Request. The AAR or other authorized representative of the railroad industry may seek modification of the single car air brake test... 49 Transportation 4 2011-10-01 2011-10-01 false Modification of the single car air brake test...

49 CFR 232.307 - Modification of the single car air brake test procedures.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Requirements § 232.307 Modification of the single car air brake test procedures. (a) Request. The AAR or other authorized representative of the railroad industry may seek modification of the single car air brake test... 49 Transportation 4 2012-10-01 2012-10-01 false Modification of the single car air brake test...

Yeast One-Hybrid Gγ Recruitment System for Identification of Protein Lipidation Motifs

PubMed Central

Fukuda, Nobuo; Doi, Motomichi; Honda, Shinya

2013-01-01

Fatty acids and isoprenoids can be covalently attached to a variety of proteins. These lipid modifications regulate protein structure, localization and function. Here, we describe a yeast one-hybrid approach based on the Gγ recruitment system that is useful for identifying sequence motifs those influence lipid modification to recruit proteins to the plasma membrane. Our approach facilitates the isolation of yeast cells expressing lipid-modified proteins via a simple and easy growth selection assay utilizing G-protein signaling that induces diploid formation. In the current study, we selected the N-terminal sequence of Gα subunits as a model case to investigate dual lipid modification, i.e., myristoylation and palmitoylation, a modification that is widely conserved from yeast to higher eukaryotes. Our results suggest that both lipid modifications are required for restoration of G-protein signaling. Although we could not differentiate between myristoylation and palmitoylation, N-terminal position 7 and 8 play some critical role. Moreover, we tested the preference for specific amino-acid residues at position 7 and 8 using library-based screening. This new approach will be useful to explore protein-lipid associations and to determine the corresponding sequence motifs. PMID:23922919

Suppression of LPS-induced transcription and cytokine secretion by the dietary isothiocyanate sulforaphane.

PubMed

Folkard, Danielle L; Melchini, Antonietta; Traka, Maria H; Al-Bakheit, Ala'a; Saha, Shikha; Mulholland, Francis; Watson, Andrew; Mithen, Richard F

2014-12-01

Diets rich in cruciferous vegetables are associated with lower levels of pro-inflammatory cytokines, which may contribute to potential health-promoting properties of these vegetables. We investigate whether sulforaphane (SF), an isothiocyanate (ITC) obtained from broccoli, could suppress LPS-induced transcription and subsequent pro-inflammatory cytokine secretion at a physiologically relevant concentration using in vitro models of chronic inflammation. We find that exposure of the LPS receptor Toll-like receptor-4 (TLR4) to physiologically appropriate concentrations of SF under non-reducing conditions results in covalent modification of cysteine residues 246 and 609. We further demonstrate that the changes in expression of 1210 genes (p ≤ 0.01) in THP-1 monocytes and the secretion of pro-inflammatory cytokines in both human peripheral blood mononuclear cells (PBMCs) and THP-1 monocytes induced by LPS exposure can be completely suppressed through exposure with physiologically appropriate concentrations of SF. Finally, we show that in vivo exposure of human PBMCs to ITCs within human circulation reduces secretion of pro-inflammatory cytokines following subsequent ex vivo LPS challenge (p < 0.001). Covalent modification of TLR4 by ITCs and resultant suppression of LPS-induced cell signalling could lead to reductions in levels of pro-inflammatory cytokines in people with chronic diseases who consume diets rich in cruciferous vegetables. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Potent Mechanism-Based Inactivation Of Cytochrome P450 2B4 By 9-Ethynylphenanthrene: Implications For Allosteric Modulation Of Cytochrome P450 Catalysis1

PubMed Central

Zhang, Haoming; Gay, Sean C.; Shah, Manish; Foroozesh, Maryam; Liu, Jiawang; Osawa, Yoichi; Zhang, Qinghai; Stout, C. David; Halpert, James R.; Hollenberg, Paul F.

2013-01-01

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and kinact are 0.2 and 0.25 min−1, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and S50 of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by ESI-LC/MS, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to the Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206 leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low affinity site being the catalytic site and the high affinity site on the protein periphery. Computer-aided docking and MD simulations with one or two ligands bound revealed that the high affinity site is situated at the entrance of a substrate access channel surrounded by the F’ helix, β1/β2 loop and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302. PMID:23276288

Inactivation of Cg10062, a cis-3-chloroacrylic acid dehalogenase homologue in Corynebacterium glutamicum, by (R)- and (S)-oxirane-2-carboxylate: analysis and implications.

PubMed

Robertson, Brooklyn A; Johnson, William H; Lo, Herng-Hsiang; Whitman, Christian P

2008-08-19

( R)- and ( S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the ( R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the ( R) and ( S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by ( R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the ( R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.

Postassembly chemical modification of a highly ordered organosilane multilayer: new insights into the structure, bonding, and dynamics of self-assembling silane monolayers.

PubMed

Wen, Ke; Maoz, Rivka; Cohen, Hagai; Sagiv, Jacob; Gibaud, Alain; Desert, Anne; Ocko, Benjamin M

2008-03-01

Experimental evidence derived from a comprehensive study of a self-assembled organosilane multilayer film system undergoing a process of postassembly chemical modification that affects interlayer-located polar groups of the constituent molecules while preserving its overall molecular architecture allows a quantitative evaluation of both the degree of intralayer polymerization and that of interlayer covalent bonding of the silane headgroups in a highly ordered layer assembly of this type. The investigated system consists of a layer-by-layer assembled multilayer of a bifunctional n-alkyl silane with terminal alcohol group that is in situ converted, via a wet chemical oxidation process conducted on the entire multilayer, to the corresponding carboxylic acid function. A combined chemical-structural analysis of data furnished by four different techniques, Fourier transform infrared spectroscopy (FTIR), synchrotron X-ray scattering, X-ray photoelectron spectroscopy (XPS), and contact angle measurements, demonstrates that the highly ordered 3D molecular arrangement of the initial alcohol-silane multilayer stack is well preserved upon virtually quantitative conversion of the alcohol to carboxylic acid and the concomitant irreversible cleavage of interlayer covalent bonds. Thus, the correlation of quantitative chemical and structural data obtained from such unreacted and fully reacted film samples offers an unprecedented experimental framework within which it becomes possible to differentiate between intralayer and interlayer covalent bonding. In addition, the use of a sufficiently thick multilayer effectively eliminates the interfering contributions of the underlying silicon oxide substrate to both the X-ray scattering and XPS data. The present findings contribute a firm experimental basis to the elucidation of the self-assembly mechanism, the molecular organization, and the modes and dynamics of intra- and interlayer bonding prevailing in highly ordered organosilane films; with further implications for the rational exploitation of some of the unique options such supramolecular surface entities can offer in the advancement of a chemical nanofabrication methodology.

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

PubMed Central

Russell, Susan P.; Limbach, Patrick A.

2013-01-01

Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. The functional dynamics, synergistic nature and regulatory roles of these posttranscriptional nucleoside modifications within the cell are not well characterized. These modifications are present at very low levels and isolation of individual nucleosides for analysis requires a complex multi-step approach. The focus of this study is to characterize the reproducibility of a liquid chromatography method used to isolate and quantitatively characterize modified nucleosides in tRNA and rRNA when nucleoside detection is performed using ultraviolet and mass spectrometric detection (UV and MS, respectively). Despite the analytical challenges of sample isolation and dynamic range, quantitative profiling of modified nucleosides obtained from bacterial tRNAs and rRNAs is feasible at relative standard deviations of 5% RSD or less. PMID:23500350

Inactivation of prion proteins via covalent grafting with methoxypoly(ethylene glycol).

PubMed

Scott, Mark D

2006-01-01

Transmissible spongiform encephalopathies (TSE) such as bovine spongiform encephalitis (BSE), Creutzfeld-Jakob disease (CJD) as well as other proteinaceous infectious particles (prions) mediated diseases have emerged as a significant concern in transfusion medicine. This concern is derived from both the disease causing potential of prion contaminated blood products but also due to tremendous impact of the active deferral of current and potential blood donors due to their extended stays in BSE prevalent countries (e.g., the United Kingdom). To date, there are no effective means by which infectious prion proteins can be inactivated in cellular and acellular blood products. Based on current work on the covalent grafting of methoxypoly(ethylene glycol) [mPEG] to proteins, viruses, and anuclear, and nucleated cells, it is hypothesized that the conversion of the normal PrP protein to its mutant conformation can be prevented by the covalent grafting of mPEG to the mutant protein. Inactivation of infective protein particles (prions) in both cellular blood products as well as cell free solutions (e.g., clotting factors) could be of medical/commercial value. It is hypothesized that consequent to the covalent modification of donor-derived prions with mPEG the requisite nucleation of the normal and mutant PrP proteins is inhibited due to the increased solubility of the modified mutant PrP and that the conformational conversion arising from the mutant PrP is prevented due to obscuration of protein charge by the heavily hydrated and neutral mPEG polymers, as well as by direct steric hindrance of the interaction due to the highly mobile polymer graft.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gifford, Brendan Joel; Kilina, Svetlana; Htoon, Han

Recent spectroscopic studies have revealed the appearance of multiple low-energy peaks in the fluorescence of single-walled carbon nanotubes (SWCNTs) upon their covalent functionalization by aryl groups. The photophysical nature of these low energy optical bands is of significant interest in the quest to understand their appearance and to achieve their precise control via chemical modification of SWCNTs. This theoretical study explains the specific energy dependence of emission features introduced in chemically functionalized (6,5) SWCNTs with aryl bromides at different conformations and in various dielectric media. Calculations using density functional theory (DFT) and time dependent DFT (TD-DFT) show that the specificmore » isomer geometry—the relative position of functional groups on the carbon-ring of the nanotube—is critical for controlling the energies and intensities of optical transitions introduced by functionalization, while the dielectric environment and the chemical composition of functional groups play less significant roles. Furthermore, the predominant effects on optical properties as a result of functionalization conformation are rationalized by exciton localization on the surface of the SWCNT near the dopant sp3-defect but not onto the functional group itself.« less

Nonviral RNA transfection to transiently modify T cells with chimeric antigen receptors for adoptive therapy.

PubMed

Riet, Tobias; Holzinger, Astrid; Dörrie, Jan; Schaft, Niels; Schuler, Gerold; Abken, Hinrich

2013-01-01

Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy.

Note: The performance of new density functionals for a recent blind test of non-covalent interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mardirossian, Narbe; Head-Gordon, Martin

Benchmark datasets of non-covalent interactions are essential for assessing the performance of density functionals and other quantum chemistry approaches. In a recent blind test, Taylor et al. benchmarked 14 methods on a new dataset consisting of 10 dimer potential energy curves calculated using coupled cluster with singles, doubles, and perturbative triples (CCSD(T)) at the complete basis set (CBS) limit (80 data points in total). Finally, the dataset is particularly interesting because compressed, near-equilibrium, and stretched regions of the potential energy surface are extensively sampled.

Note: The performance of new density functionals for a recent blind test of non-covalent interactions

DOE PAGES

Mardirossian, Narbe; Head-Gordon, Martin

2016-11-09

Benchmark datasets of non-covalent interactions are essential for assessing the performance of density functionals and other quantum chemistry approaches. In a recent blind test, Taylor et al. benchmarked 14 methods on a new dataset consisting of 10 dimer potential energy curves calculated using coupled cluster with singles, doubles, and perturbative triples (CCSD(T)) at the complete basis set (CBS) limit (80 data points in total). Finally, the dataset is particularly interesting because compressed, near-equilibrium, and stretched regions of the potential energy surface are extensively sampled.

Discovery of host-targeted covalent inhibitors of dengue virus

PubMed Central

de Wispelaere, Mélissanne; Carocci, Margot; Liang, Yanke; Liu, Qingsong; Sun, Eileen; Vetter, Michael L.; Wang, Jinhua; Gray, Nathanael S.; Yang, Priscilla L.

2017-01-01

We report here on an approach targeting the host reactive cysteinome to identify inhibitors of host factors required for the infectious cycle of Flaviviruses and other viruses. We used two parallel cellular phenotypic screens to identify a series of covalent inhibitors, exemplified by QL-XII-47, that are active against dengue virus. We show that the compounds effectively block viral protein expression and that this inhibition is associated with repression of downstream processes of the infectious cycle, and thus significantly contributes to the potent antiviral activity of these compounds. We demonstrate that QL-XII-47’s antiviral activity requires selective, covalent modification of a host target by showing that the compound's antiviral activity is recapitulated when cells are preincubated with QL-XII-47 and then washed prior to viral infection and by showing that QL-XII-47R, a non-reactive analog, lacks antiviral activity at concentrations more than 20-fold higher than QL-XII-47's IC90. QL-XII-47’s inhibition of Zika virus, West Nile virus, hepatitis C virus, and poliovirus further suggests that it acts via a target mediating inhibition of these other medically relevant viruses. These results demonstrate the utility of screens targeting the host reactive cysteinome for rapid identification of compounds with potent antiviral activity. PMID:28034743

Elastomeric nanoparticle composites covalently bound to Al2O3/GaAs surfaces.

PubMed

Song, Hyon Min; Ye, Peide D; Ivanisevic, Albena

2007-08-28

This article reports the modification of Al2O3/GaAs surfaces with multifunctional soft materials. Siloxane elastomers were covalently bound to dopamine-modified Al2O3/GaAs semiconductor surfaces using MPt (M = Fe, Ni) nanoparticles. The sizes of the monodisperse FePt and NiPt nanoparticles were less than 5 nm. The surfaces of the nanoparticles as well as the Al2O3/GaAs substrates were modified with allyl-functionalized dopamine that utilized a dihydroxy group as a strong ligand. The immobilization of the elastomers was performed via a hydrosilation reaction of the allyl-functionalized dopamines with the siloxane backbones. X-ray photoelectron spectroscopy (XPS) experiments confirmed the covalent bonding of the siloxane elastomers to the oxide layer on the semiconductor surface. Fourier transform-infrared reflection absorption spectroscopy (FT-IRRAS) measurements revealed that the allyl functional groups are bonded to the siloxane backbones. The FT-IRRAS data also showed that the density of the allyl groups on the surface was lower than that of the siloxane backbones. The mechanical properties of the surface-bound nanocomposites were tested using nanoindentation experiments. The nanoindentation data showed that the soft matrix composed of the elastomeric coating on the surfaces behaves differently from the inner, hard Al2O3/GaAs substrate.

Structural and elastic properties and stability characteristics of oxygenated carbon nanotubes under physical adsorption of polymers

NASA Astrophysics Data System (ADS)

Ansari, R.; Ajori, S.; Rouhi, S.

2015-03-01

The importance of covalent and non-covalent functionalization approaches for modification the properties of carbon nanotubes is being more widely recognized. To this end, elastic properties and buckling behavior of oxygenated CNT with atomic oxygen and hydroxyl under physical adsorption of PE (Polyethylene) and PEO (Poly (ethylene oxide)) are determined through employing the molecular dynamics (MD) simulations. The results demonstrate that non-covalent bonding of polymer on the surface of oxygenated CNT causes reductions in the variations of critical buckling load and critical strain compared to oxygenated CNTs. Critical buckling load and critical strain of oxygenated CNT/polymer are higher than those of oxygenated CNT. Also, it is demonstrated that critical buckling load and critical strain values in the case of oxygenated CNT/polymer are independent of polymer type unlike the value of Young's modulus. It is shown that variations of Young's modulus decrease as PE adsorbed on the surface of oxygenated CNT. Moreover, the presence of oxygen atom on PEO chain leads to bigger variations of Young's modulus with weight percentage of chemisorbed component, i.e. atomic oxygen and hydroxyl. It is also demonstrated that Young's modulus reduces more considerably in the presence of PEO chain compared to PE one.

Azidobupramine, an Antidepressant-Derived Bifunctional Neurotransmitter Transporter Ligand Allowing Covalent Labeling and Attachment of Fluorophores

PubMed Central

Werner, Anna M.; Cuboni, Serena; Rudolf, Georg C.; Höfner, Georg; Wanner, Klaus T.; Sieber, Stephan A.; Schmidt, Ulrike; Holsboer, Florian; Rein, Theo; Hausch, Felix

2016-01-01

The aim of this study was to design, synthesize and validate a multifunctional antidepressant probe that is modified at two distinct positions. The purpose of these modifications was to allow covalent linkage of the probe to interaction partners, and decoration of probe-target complexes with fluorescent reporter molecules. The strategy for the design of such a probe (i.e., azidobupramine) was guided by the need for the introduction of additional functional groups, conveying the required properties while keeping the additional moieties as small as possible. This should minimize the risk of changing antidepressant-like properties of the new probe azidobupramine. To control for this, we evaluated the binding parameters of azidobupramine to known target sites such as the transporters for serotonin (SERT), norepinephrine (NET), and dopamine (DAT). The binding affinities of azidobupramine to SERT, NET, and DAT were in the range of structurally related and clinically active antidepressants. Furthermore, we successfully visualized azidobupramine-SERT complexes not only in SERT-enriched protein material but also in living cells stably overexpressing SERT. To our knowledge, azidobupramine is the first structural analogue of a tricyclic antidepressant that can be covalently linked to target structures and further attached to reporter molecules while preserving antidepressant-like properties and avoiding radioactive isotopes. PMID:26863431

Semisynthetic protein nanoreactor for single-molecule chemistry

PubMed Central

Lee, Joongoo; Bayley, Hagan

2015-01-01

The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach. PMID:26504203

Merging constitutional and motional covalent dynamics in reversible imine formation and exchange processes.

PubMed

Kovaříček, Petr; Lehn, Jean-Marie

2012-06-06

The formation and exchange processes of imines of salicylaldehyde, pyridine-2-carboxaldehyde, and benzaldehyde have been studied, showing that the former has features of particular interest for dynamic covalent chemistry, displaying high efficiency and fast rates. The monoimines formed with aliphatic α,ω-diamines display an internal exchange process of self-transimination type, inducing a local motion of either "stepping-in-place" or "single-step" type by bond interchange, whose rate decreases rapidly with the distance of the terminal amino groups. Control of the speed of the process over a wide range may be achieved by substituents, solvent composition, and temperature. These monoimines also undergo intermolecular exchange, thus merging motional and constitutional covalent behavior within the same molecule. With polyamines, the monoimines formed execute internal motions that have been characterized by extensive one-dimensional, two-dimensional, and EXSY proton NMR studies. In particular, with linear polyamines, nondirectional displacement occurs by shifting of the aldehyde residue along the polyamine chain serving as molecular track. Imines thus behave as simple prototypes of systems displaying relative motions of molecular moieties, a subject of high current interest in the investigation of synthetic and biological molecular motors. The motional processes described are of dynamic covalent nature and take place without change in molecular constitution. They thus represent a category of dynamic covalent motions, resulting from reversible covalent bond formation and dissociation. They extend dynamic covalent chemistry into the area of molecular motions. A major further step will be to achieve control of directionality. The results reported here for imines open wide perspectives, together with other chemical groups, for the implementation of such features in multifunctional molecules toward the design of molecular devices presenting a complex combination of motional and constitutional dynamic behaviors.

DNA Detection by Flow Cytometry using PNA-Modified Metal-Organic Framework Particles.

PubMed

Mejia-Ariza, Raquel; Rosselli, Jessica; Breukers, Christian; Manicardi, Alex; Terstappen, Leon W M M; Corradini, Roberto; Huskens, Jurriaan

2017-03-23

A DNA-sensing platform is developed by exploiting the easy surface functionalization of metal-organic framework (MOF) particles and their highly parallelized fluorescence detection by flow cytometry. Two strategies were employed to functionalize the surface of MIL-88A, using either covalent or non-covalent interactions, resulting in alkyne-modified and biotin-modified MIL-88A, respectively. Covalent surface coupling of an azide-dye and the alkyne-MIL-88A was achieved by means of a click reaction. Non-covalent streptavidin-biotin interactions were employed to link biotin-PNA to biotin-MIL-88A particles mediated by streptavidin. Characterization by confocal imaging and flow cytometry demonstrated that DNA can be bound selectively to the MOF surface. Flow cytometry provided quantitative data of the interaction with DNA. Making use of the large numbers of particles that can be simultaneously processed by flow cytometry, this MOF platform was able to discriminate between fully complementary, single-base mismatched, and randomized DNA targets. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

A modified spectroscopic method for the determination of the transbilayer distribution of phosphatidylethanolamine in soya-bean asolectin small unilamellar vesicles.

PubMed Central

Sarti, P; Molinari, A; Arancia, G; Meloni, A; Citro, G

1995-01-01

A spectroscopic kinetic approach for determining the relative concentrations of phosphatidylethanolamine (PE) exposed on the external and internal layers of small unilamellar vesicles (SUVs) used as a model system and prepared by sonication of purified soya-bean asolectin is proposed, based on the use of 2,4,6-trinitrobenzenesulphonic acid (TNBS) and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The known reactions between PE and TNBS and/or SDPD were used, separately or in combination, to derivatize PE in preformed vesicles. We have observed that mixing SUVs with excess TNBS results in a biphasic time course. Kinetic analysis of the data supports the conclusion that external PE is rapidly derivatized (fast phase) with a half-time of 2 min. In the next (slow) phase (half-time 70 min), TNBS permeates the vesicle membrane and also reacts with PE molecules facing the internal liposomal compartment. Under the experimental conditions chosen, SPDP reacted with only the external PE molecules. The reaction of SUVs first derivatized with SPDP and then with TNBS further demonstrates that the two phases, observed with TNBS, are due to modification of external and internal PE. Approx. 30% of PE was found to be facing the external bulk phase, thus confirming the asymmetric distribution of the molecules in SUVs. The maximum number of thiol arms covalently linked by means of SPDP modification of PE on the surface of a single liposome was estimated at about 10(2). Images Figure 1 PMID:8526881

Covalent intermolecular interaction of the nitric oxide dimer (NO)2

NASA Astrophysics Data System (ADS)

Zhang, Hui; Zheng, Gui-Li; Lv, Gang; Geng, Yi-Zhao; Ji, Qing

2015-09-01

Covalent bonds arise from the overlap of the electronic clouds in the internucleus region, which is a pure quantum effect and cannot be obtained in any classical way. If the intermolecular interaction is of covalent character, the result from direct applications of classical simulation methods to the molecular system would be questionable. Here, we analyze the special intermolecular interaction between two NO molecules based on quantum chemical calculation. This weak intermolecular interaction, which is of covalent character, is responsible for the formation of the NO dimer, (NO)2, in its most stable conformation, a cis conformation. The natural bond orbital (NBO) analysis gives an intuitive illustration of the formation of the dimer bonding and antibonding orbitals concomitant with the breaking of the π bonds with bond order 0.5 of the monomers. The dimer bonding is counteracted by partially filling the antibonding dimer orbital and the repulsion between those fully or nearly fully occupied nonbonding dimer orbitals that make the dimer binding rather weak. The direct molecular mechanics (MM) calculation with the UFF force fields predicts a trans conformation as the most stable state, which contradicts the result of quantum mechanics (QM). The lesson from the investigation of this special system is that for the case where intermolecular interaction is of covalent character, a specific modification of the force fields of the molecular simulation method is necessary. Project supported by the National Natural Science Foundation of China (Grant Nos. 90403007 and 10975044), the Key Subject Construction Project of Hebei Provincial Universities, China, the Research Project of Hebei Education Department, China (Grant Nos. Z2012067 and Z2011133), the National Natural Science Foundation of China (Grant No. 11147103), and the Open Project Program of State Key Laboratory of Theoretical Physics, Institute of Theoretical Physics, Chinese Academy of Sciences, China (Grant No. Y5KF211CJ1).

Cell separation in immunoaffinity partition in aqueous polymer two-phase systems

NASA Technical Reports Server (NTRS)

Karr, Laurel J.; Van Alstine, James M.; Snyder, Robert S.; Shafer, Steven G.; Harris, J. Milton

1989-01-01

Two methods for immunoaffinity partitioning are described. One technique involves the covalent coupling of poly (ethylene glycol) (PEG) to immunoglobulin G antibody preparations. In the second method PEG-modified Protein A is used to complex with cells and unmodified antibody. The effects of PEG molecular weight, the degree of modification, and varying phase system composition on antibody activity and its affinity for the upper phase are studied. It is observed that both methods resulted in effective cell separation.

DELIVERY OF THERAPEUTIC PROTEINS

PubMed Central

Pisal, Dipak S.; Kosloski, Matthew P.; Balu-Iyer, Sathy V.

2009-01-01

The safety and efficacy of protein therapeutics are limited by three interrelated pharmaceutical issues, in vitro and in vivo instability, immunogenicity and shorter half-lives. Novel drug modifications for overcoming these issues are under investigation and include covalent attachment of poly(ethylene glycol) (PEG), polysialic acid, or glycolic acid, as well as developing new formulations containing nanoparticulate or colloidal systems (e.g. liposomes, polymeric microspheres, polymeric nanoparticles). Such strategies have the potential to develop as next generation protein therapeutics. This review includes a general discussion on these delivery approaches. PMID:20049941

Triply Periodic Multiply Continuous Lyotropic Liquid Crystals Derived from Gemini Surfactants

NASA Astrophysics Data System (ADS)

Sorenson, Gregory P.

A subtle balance of non-covalent interactions directs the self-assembly of small molecule amphiphiles in aqueous media into supramolecular assemblies known as aqueous lyotropic liquid crystals (LLCs). Aqueous LLCs form many intricate, ordered nanoscale morphologies comprising distinct and structurally periodic hydrophobic and hydrophilic domains. Triply periodic multiply continuous (TPMC) LLC morphologies, which exhibit continuous hydrophobic and aqueous domains that percolate in three-dimensions, are of particular interest by virtue of their potentially wide ranging technological applications including advanced membranes for electrical energy storage and utilization, therapeutic delivery, and templates for new organic and inorganic mesoporous materials. However, robust molecular design criteria for amphiphiles that readily form TMPC morphologies are notably lacking in the literature. Recent reports have described the increased propensity for quaternary ammonium and phosphonium gemini surfactants, derived from dimerization of traditional single-tail surfactants at or near the hydrophilic headgroups through a hydrophobic linker, to stabilize TMPC mesophases. The generality of this surfactant design strategy remains untested in other amphiphiles classes bearing different headgroup chemistries. In this thesis, we describe the unusual aqueous LLC phase behavior of series of gemini dicarboxylate amphiphiles as a function of the alkyl tail length, hydrophobic linker length, and the charge-compensating counterion. These dicarboxylate surfactants unexpectedly exhibit a strong propensity to form TPMC LLCs over amphiphile concentration windows as wide as 20 wt% over a temperature range T = 25--100 °C. Through systematic modifications of the length of the hydrophobic linker and alkyl tails, we use small-angle X-ray scattering to demonstrate that these surfactants adopt new LLC mesophases including the first report of a single-gyroid phase (I4132 symmetry) and a new, tetracontinuous hexagonal network phase ( P63/mcm symmetry). Additionally, we probe the role of the linker position in the surfactant architecture. These data taken together indicate the sensitive dependence of the LLC phase behavior on counterion-headgroup correlations. Based on these molecular design criteria, we demonstrate the synthesis of a polymerizable gemini surfactant that may be self-assembled into a TPMC LLC phase and covalently fixed by a crosslinking photopolymerization. Comprised of aqueous nanochannels lined with metal carboxylates, the resulting LLC membranes exhibit high ionic conductivities.

Epigenome analysis of pluripotent stem cells

PubMed Central

Ricupero, Christopher L.; Swerdel, Mavis R.; Hart, Ronald P.

2015-01-01

Summary Mis-regulation of gene expression due to epigenetic abnormalities has been linked with complex genetic disorders, psychiatric illness and cancer. In addition, the dynamic epigenetic changes that occur in pluripotent stem cells are believed to impact regulatory networks essential for proper lineage development. Chromatin immunoprecipitation (ChIP) is a technique used to isolate and enrich chromatin fragments using antibodies against specific chromatin modifications, such as DNA binding proteins or covalent histone modifications. Until recently, many ChIP protocols required millions of cells for each immunoprecipitation. This severely limited analysis of rare cell populations or post-mitotic, differentiated cell lines. Here, we describe a low cell number ChIP protocol with next generation sequencing and analysis, that has the potential to uncover novel epigenetic regulatory pathways that were previously difficult or impossible to obtain. PMID:23546758

The Discovery of Carboxyethylpyrroles (CEPs): Critical Insights into AMD, Autism, Cancer, and Wound Healing from Basic Research on the Chemistry of Oxidized Phospholipids

PubMed Central

Salomon, Robert G.; Hong, Li; Hollyfield, Joe G.

2011-01-01

Basic research, exploring the hypothesis that 2-(ω-carboxyethyl)pyrrole (CEP) modifications of proteins are generated nonenzymatically in vivo is delivering a bonanza of molecular mechanistic insights into age-related macular degeneration, autism, cancer, and wound healing. CEPs are produced through covalent modification of protein lysyl ε-amino groups by γ-hydroxyalkenal phospholipids that are formed by oxidative cleavage of docosahexaenate-containing phospholipids. Chemical synthesis of CEP-modified proteins and the production of highly specific antibodies that recognize them preceded and facilitated their detection in vivo and enabled exploration of their biological occurrence and activities. This investigational approach –from the chemistry of biomolecules to disease phenotype – is proving to be remarkably productive. PMID:21875030

Noncovalent Ubiquitin Interactions Regulate the Catalytic Activity of Ubiquitin Writers.

PubMed

Wright, Joshua D; Mace, Peter D; Day, Catherine L

2016-11-01

Covalent modification of substrate proteins with ubiquitin is the end result of an intricate network of protein-protein interactions. The inherent ability of the E1, E2, and E3 proteins of the ubiquitylation cascade (the ubiquitin writers) to interact with ubiquitin facilitates this process. Importantly, contact between ubiquitin and the E2/E3 writers is required for catalysis and the assembly of chains of a given linkage. However, ubiquitin is also an activator of ubiquitin-writing enzymes, with many recent studies highlighting the ability of ubiquitin to regulate activity and substrate modification. Here, we review the interactions between ubiquitin-writing enzymes and regulatory ubiquitin molecules that promote activity, and highlight the potential of these interactions to promote processive ubiquitin transfer. Copyright © 2016 Elsevier Ltd. All rights reserved.

Epigenomics.

PubMed

Gomase, Virendra S; Tagore, Somnath

2008-03-01

'Epigenomics' can be termed as the study of the effects of chromatin structure, including the higher order of chromatin folding and attachment to the nuclear matrix, packaging of DNA around nucleosomes, covalent modifications of histone tails and DNA methylation. This has evolved to include any process that alters gene activity without changing the DNA sequence, and leads to modifications that can be transmitted to daughter cells. It also leads to a better knowledge of the changes in the regulation of genes and genomes that occur in major psychosis. It may also aid in understanding why the same gene sequence may predispose an individual to schizophrenia or bipolar disorder and in other cases does not, and elucidate the molecular mechanisms of how harmful; environmental factors interact with the genome. Results from the work may further lead to new diagnostics and effective therapies.

[The effect of hydrophobic surface properties of protein on its resistance to denaturation by organic solvents (using modified alpha-chymotrypsin as an example].

PubMed

Kudriashova, E V; Belova, A B; Vinogradov, A A; Mozhaev, V V

1994-03-01

Catalytic activity of covalently modified alpha-chymotrypsin in water/cosolvent solutions was investigated. The stability of chymotrypsin increases upon modification with hydrophilic reagents, such as glyceraldehyde, pyrometallic and succinic anhydrides, and glucosamine. Correlation was observed between the protein's stability in organic solvents and the degree of hydrophilization of the protein's surface. The protein is the more stable, the higher are the modification degree and the hydrophilicity of the modifying residue. At a certain critical hydrophilization degree of chymotrypsin a limit of stability is achieved. The stabilization effect can be accounted for by the fact that the interaction between water molecules on the surface and protein's functional groups become stronger in the hydrophilized protein.

Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells

PubMed Central

Tsuchiya, Yugo; Peak-Chew, Sew Yeu; Newell, Clare; Miller-Aidoo, Sheritta; Mangal, Sriyash; Zhyvoloup, Alexander; Bakovic´, Jovana; Malanchuk, Oksana; Pereira, Gonçalo C.; Kotiadis, Vassilios; Szabadkai, Gyorgy; Duchen, Michael R.; Campbell, Mark; Cuenca, Sergio Rodriguez; Vidal-Puig, Antonio; James, Andrew M.; Murphy, Michael P.; Filonenko, Valeriy; Skehel, Mark

2017-01-01

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions. PMID:28341808

Lignin Modification for Biopolymer/Conjugated Polymer Hybrids as Renewable Energy Storage Materials.

PubMed

Nilsson, Ting Yang; Wagner, Michal; Inganäs, Olle

2015-12-07

Lignin derivatives, which arise as waste products from the pulp and paper industry and are mainly used for heating, can be used as charge storage materials. The charge storage function is a result of the quinone groups formed in the lignin derivative. Herein, we modified lignins to enhance the density of such quinone groups by covalently linking monolignols and quinones through phenolation. The extra guaiacyl, syringyl, and hydroquinone groups introduced by phenolation of kraft lignin derivatives were monitored by (31) P nuclear magnetic resonance and size exclusion chromatography. Electropolymerization in ethylene glycol/tetraethylammonium tosylate electrolyte was used to synthesize the kraft lignin/polypyrrole hybrid films. These modifications changed the phenolic content of the kraft lignin with attachment of hydroquinone units yielding the highest specific capacity (around 70 mA h g(-1) ). The modification of softwood and hardwood lignin derivatives yielded 50 % and 23 % higher charge capacity than the original lignin, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modification and simulation of Rhizomucor miehei lipase: the influence of surficial electrostatic interaction on enantioselectivity.

PubMed

Xu, Gang; Meng, Xiao; Xu, Lin-Jie; Guo, Li; Wu, Jian-Ping; Yang, Li-Rong

2015-04-01

Surface residues have a significant impact on the enantioselectivity of lipases. But the molecular basis of this has never been explained. In this work, transition state complexes of Rhizomucor miehei lipase (RmL) and (R)- or (S)-n-butyl 2-phenxypropinate were studied using molecular dynamics. According to comparison between B-factor of the two simulated complexes, the β 1-β 2 loop and α 2 helix were considered the enantioselectivity-determining domains of RmL. Interaction analysis of these domains suggested an Asp(61)-Arg(86) electrostatic interaction linking the loop and helix strongly impacting enantioselectivity of RmL. Modification of Arg(86) by 1, 2-cyclohexanedione weakening this interaction decreased the E ratio from 6 to 1, modification by 1-iodo-2, 3-butanedione covalently bonding Asp(61) and Arg(86) strengthening the interaction increased the E ratio to 45. Dynamics simulation and energy calculation of the modified lipases also displayed corresponding decreases or increases of enantioselectivity.

Silane surface modification for improved bioadhesion of esophageal stents

NASA Astrophysics Data System (ADS)

Karakoy, Mert; Gultepe, Evin; Pandey, Shivendra; Khashab, Mouen A.; Gracias, David H.

2014-08-01

Stent migration occurs in 10-40% of patients who undergo placement of esophageal stents, with higher migration rates seen in those treated for benign esophageal disorders. This remains a major drawback of esophageal stent therapy. In this paper, we propose a new surface modification method to increase the adhesion between self-expandable metallic stents (SEMS) and tissue while preserving their removability. Taking advantage of the well-known affinity between epoxide and amine terminated silane coupling agents with amine and carboxyl groups that are abundant in proteins and related molecules in the human body; we modified the surfaces of silicone coated esophageal SEMS with these adhesive self-assembled monolayers (SAMs). We utilized vapor phase silanization to modify the surfaces of different substrates including PDMS strips and SEMS, and measured the force required to slide these substrates on a tissue piece. Our results suggest that surface modification of esophageal SEMS via covalent attachment of protein-binding coupling agents improves adhesion to tissue and could offer a solution to reduce SEMS migration while preserving their removability.

Cisplatin enhances the formation of DNA single- and double-strand breaks by hydrated electrons and hydroxyl radicals.

PubMed

Rezaee, Mohammad; Sanche, Léon; Hunting, Darel J

2013-03-01

The synergistic interaction of cisplatin with ionizing radiation is the clinical rationale for the treatment of several cancers including head and neck, cervical and lung cancer. The underlying molecular mechanism of the synergy has not yet been identified, although both DNA damage and repair processes are likely involved. Here, we investigate the indirect effect of γ rays on strand break formation in a supercoiled plasmid DNA (pGEM-3Zf-) covalently modified by cisplatin. The yields of single- and double-strand breaks were determined by irradiation of DNA and cisplatin/DNA samples with (60)Co γ rays under four different scavenging conditions to examine the involvement of hydrated electrons and hydroxyl radicals in inducing the DNA damage. At 5 mM tris in an N2 atmosphere, the presence of an average of two cisplatins per plasmid increased the yields of single- and double-strand breaks by factors of 1.9 and 2.2, respectively, relative to the irradiated unmodified DNA samples. Given that each plasmid of 3,200 base pairs contained an average of two cisplatins, this represents an increase in radiosensitivity of 3,200-fold on a per base pair basis. When hydrated electrons were scavenged by saturating the samples with N2O, these enhancement factors decreased to 1.5 and 1.2, respectively, for single- and double-strand breaks. When hydroxyl radicals were scavenged using 200 mM tris, the respective enhancement factors were 1.2 and 1.6 for single- and double-strand breaks, respectively. Furthermore, no enhancement in DNA damage by cisplatin was observed after scavenging both hydroxyl radicals and hydrated electrons. These findings show that hydrated electrons can induce both single- and double-strand breaks in the platinated DNA, but not in unmodified DNA. In addition, cisplatin modification is clearly an extremely efficient means of increasing the formation of both single- and double-strand breaks by the hydrated electrons and hydroxyl radicals created by ionizing radiation.

Dynamic Covalent Chemistry within Biphenyl Scaffolds: Reversible Covalent Bonding, Control of Selectivity, and Chirality Sensing with a Single System.

PubMed

Ni, Cailing; Zha, Daijun; Ye, Hebo; Hai, Yu; Zhou, Yuntao; Anslyn, Eric V; You, Lei

2018-01-26

Axial chirality is a prevalent and important phenomenon in chemistry. Herein we report a combination of dynamic covalent chemistry and axial chirality for the development of a versatile platform for the binding and chirality sensing of multiple classes of mononucleophiles. An equilibrium between an open aldehyde and its cyclic hemiaminal within biphenyl derivatives enabled the dynamic incorporation of a broad range of alcohols, thiols, primary amines, and secondary amines with high efficiency. Selectivity toward different classes of nucleophiles was also achieved by regulating the distinct reactivity of the system with external stimuli. Through induced helicity as a result of central-to-axial chirality transfer, the handedness and ee values of chiral monoalcohol and monoamine analytes were reported by circular dichroism. The strategies introduced herein should find application in many contexts, including assembly, sensing, and labeling. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly selective covalent organic functionalization of epitaxial graphene

NASA Astrophysics Data System (ADS)

Bueno, Rebeca A.; Martínez, José I.; Luccas, Roberto F.; Del Árbol, Nerea Ruiz; Munuera, Carmen; Palacio, Irene; Palomares, Francisco J.; Lauwaet, Koen; Thakur, Sangeeta; Baranowski, Jacek M.; Strupinski, Wlodek; López, María F.; Mompean, Federico; García-Hernández, Mar; Martín-Gago, José A.

2017-05-01

Graphene functionalization with organics is expected to be an important step for the development of graphene-based materials with tailored electronic properties. However, its high chemical inertness makes difficult a controlled and selective covalent functionalization, and most of the works performed up to the date report electrostatic molecular adsorption or unruly functionalization. We show hereafter a mechanism for promoting highly specific covalent bonding of any amino-terminated molecule and a description of the operating processes. We show, by different experimental techniques and theoretical methods, that the excess of charge at carbon dangling-bonds formed on single-atomic vacancies at the graphene surface induces enhanced reactivity towards a selective oxidation of the amino group and subsequent integration of the nitrogen within the graphene network. Remarkably, functionalized surfaces retain the electronic properties of pristine graphene. This study opens the door for development of graphene-based interfaces, as nano-bio-hybrid composites, fabrication of dielectrics, plasmonics or spintronics.

Probing the Nanodomain Origin and Phase Transition Mechanisms in (Un)Poled PMN-PT Single Crystals and Textured Ceramics

PubMed Central

Slodczyk, Aneta; Colomban, Philippe

2010-01-01

Outstanding electrical properties of solids are often due to the composition heterogeneity and/or the competition between two or more sublattices. This is true for superionic and superprotonic conductors and supraconductors, as well as for many ferroelectric materials. As in PLZT ferroelectric materials, the exceptional ferro- and piezoelectric properties of the PMN-PT ((1−x)PbMg1/3Nb2/3O3−xPbTiO3) solid solutions arise from the coexistence of different symmetries with long and short scales in the morphotropic phase boundary (MPB) region. This complex physical behavior requires the use of experimental techniques able to probe the local structure at the nanoregion scale. Since both Raman signature and thermal expansion behavior depend on the chemical bond anharmonicity, these techniques are very efficient to detect and then to analyze the subtitle structural modifications with an efficiency comparable to neutron scattering. Using the example of poled (field cooling or room temperature) and unpoled PMN-PT single crystal and textured ceramic, we show how the competition between the different sublattices with competing degrees of freedom, namely the Pb-Pb dominated by the Coulombian interactions and those built of covalent bonded entities (NbO6 and TiO6), determine the short range arrangement and the outstanding ferro- and piezoelectric properties. PMID:28883367

The small molecule 2-phenylethynesulfonamide induces covalent modification of p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jamil, Sarwat; Hojabrpour, Payman; Duronio, Vincent

p53 is a tumor suppressor protein which is either lost or inactivated in a large majority of tumors. The small molecule 2-phenylethynesulfonamide (PES) was originally identified as the inhibitor of p53 effects on the mitochondrial death pathway. In this report we demonstrate that p53 protein from PES-treated cells was detected in reduced mobility bands between molecular weights 95–220 kDa. Resolution of p53 aggregates on urea gel was unable to reduce the high molecular weight p53 aggregates, which were shown to be primarily located in the nucleus. Therefore, our data suggest that PES exerts its effects through covalent cross-linking and nuclear retentionmore » of p53. - Highlights: • p53 protein is in high molecular weight complexes in the nucleus of PES-treated cells. • PES is a drug that inhibits pro-apoptotic p53 action at the mitochondria. • We propose that PES action involves cross-linking and nuclear retention of p53.« less

Immobilization of Chloroperoxidase on Aminopropyl-Glass

PubMed Central

Kadima, Tenshuk A.; Pickard, Michael A.

1990-01-01

Chloroperoxidase (CPO) purified from Caldariomyces fumago CMI 89362 was covalently bound to aminopropyl-glass by using a modification of an established method. Acid-washed glass was derivatized by using aminopropyltriethoxysilane, and the enzyme was ionically bound at low ionic strength. Further treatment with glutaraldehyde covalently linked the enzyme to the glass beads in an active form. No elution of bound activity from glass beads could be detected with a variety of washings. The loading of enzyme protein to the glass beads was highest, 100 mg of CPO per g of glass, at high reaction ratios of CPO to glass, but the specific activity of the immobilized enzyme was highest, 36% of theoretical, at low enzyme-to-carrier ratios. No differences in the properties of the soluble and immobilized enzymes could be detected by a number of criteria: their pH-activity and pH-stability profiles were similar, as were their thermal stabilities. After five uses, the immobilized enzyme retained full activity between pH 6.0 and 6.7. PMID:16348352

Intracellular integration of synthetic nanostructures with viable cells for controlled biochemical manipulation

NASA Astrophysics Data System (ADS)

McKnight, Timothy E.; Melechko, Anatoli V.; Griffin, Guy D.; Guillorn, Michael A.; Merkulov, Vladimir I.; Serna, Francisco; Hensley, Dale K.; Doktycz, Mitchel J.; Lowndes, Douglas H.; Simpson, Michael L.

2003-05-01

We demonstrate the integration of vertically aligned carbon nanofibre (VACNF) elements with the intracellular domains of viable cells for controlled biochemical manipulation. Deterministically synthesized VACNFs were modified with either adsorbed or covalently-linked plasmid DNA and were subsequently inserted into cells. Post insertion viability of the cells was demonstrated by continued proliferation of the interfaced cells and long-term (> 22 day) expression of the introduced plasmid. Adsorbed plasmids were typically desorbed in the intracellular domain and segregated to progeny cells. Covalently bound plasmids remained tethered to nanofibres and were expressed in interfaced cells but were not partitioned into progeny, and gene expression ceased when the nanofibre was no longer retained. This provides a method for achieving a genetic modification that is non-inheritable and whose extent in time can be directly and precisely controlled. These results demonstrate the potential of VACNF arrays as an intracellular interface for monitoring and controlling subcellular and molecular phenomena within viable cells for applications including biosensors, in vivo diagnostics, and in vivo logic devices.

Two-dimensional honeycomb network through sequence-controlled self-assembly of oligopeptides.

PubMed

Abb, Sabine; Harnau, Ludger; Gutzler, Rico; Rauschenbach, Stephan; Kern, Klaus

2016-01-12

The sequence of a peptide programs its self-assembly and hence the expression of specific properties through non-covalent interactions. A large variety of peptide nanostructures has been designed employing different aspects of these non-covalent interactions, such as dispersive interactions, hydrogen bonding or ionic interactions. Here we demonstrate the sequence-controlled fabrication of molecular nanostructures using peptides as bio-organic building blocks for two-dimensional (2D) self-assembly. Scanning tunnelling microscopy reveals changes from compact or linear assemblies (angiotensin I) to long-range ordered, chiral honeycomb networks (angiotensin II) as a result of removal of steric hindrance by sequence modification. Guided by our observations, molecular dynamic simulations yield atomistic models for the elucidation of interpeptide-binding motifs. This new approach to 2D self-assembly on surfaces grants insight at the atomic level that will enable the use of oligo- and polypeptides as large, multi-functional bio-organic building blocks, and opens a new route towards rationally designed, bio-inspired surfaces.

Analysis of the bond-valence method for calculating (29) Si and (31) P magnetic shielding in covalent network solids.

PubMed

Holmes, Sean T; Alkan, Fahri; Iuliucci, Robbie J; Mueller, Karl T; Dybowski, Cecil

2016-07-05

(29) Si and (31) P magnetic-shielding tensors in covalent network solids have been evaluated using periodic and cluster-based calculations. The cluster-based computational methodology employs pseudoatoms to reduce the net charge (resulting from missing co-ordination on the terminal atoms) through valence modification of terminal atoms using bond-valence theory (VMTA/BV). The magnetic-shielding tensors computed with the VMTA/BV method are compared to magnetic-shielding tensors determined with the periodic GIPAW approach. The cluster-based all-electron calculations agree with experiment better than the GIPAW calculations, particularly for predicting absolute magnetic shielding and for predicting chemical shifts. The performance of the DFT functionals CA-PZ, PW91, PBE, rPBE, PBEsol, WC, and PBE0 are assessed for the prediction of (29) Si and (31) P magnetic-shielding constants. Calculations using the hybrid functional PBE0, in combination with the VMTA/BV approach, result in excellent agreement with experiment. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Identification of a Novel Class of Covalent Modifiers of Hemoglobin as Potential Antisickling Agents

PubMed Central

Omar, A. M.; Mahran, M. A.; Ghatge, M. S.; Chowdhury, N.; Bamane, F. H. A.; El-Araby, M. E.; Abdulmalik, O.; Safo, M. K.

2015-01-01

Aromatic aldehydes and ethacrynic acid (ECA) exhibit antipolymerization properties that are beneficial for sickle cell disease therapy. Based on ECA pharmacophore and its atomic interaction with hemoglobin, we designed and synthesized several compounds--designated as KAUS (imidazolylacryloyl derivatives)--that we hypothesized would bind covalently to βCys93 of hemoglobin and inhibit sickling. The compounds surprisingly showed weak allosteric and antisickling properties. X-ray studies of hemoglobin in complex with representative KAUS compounds revealed an unanticipated mode of Michael addition reaction between the β-unsaturated carbon and the N-terminal αVal1 nitrogen at the α-cleft of hemoglobin, with no observable interaction with βCys93. Interestingly, the compounds exhibited almost no reactivity with the free amino acids, L-Val, L-His and L-Lys, however showed some reactivity with both glutathione and L-Cys. Our findings provide a molecular level explanation to the compounds biological activities and an important framework for targeted modifications that would yield novel potent antisickling agents. PMID:25974708

Modified carbon nanotubes: from nanomedicine to nanotoxicology

NASA Astrophysics Data System (ADS)

Bottini, Massimo; Bottini, Nunzio

2012-09-01

Nanomedicine is the science of fabricating smart devices able to diagnose and treat diseases more efficiently than conventional medicine while minimizing costs, complexity and adverse effects. Carbon nanotubes (CNTs) are receiving considerable attention for biomedical applications due to their extraordinary properties. In particular, their chemical nature and high aspect ratio (ratio between the length and the diameter) make them ideal carriers to achieve delivery of high doses of therapeutic and imaging cargo to a specific site of interest. A major obstacle to the use of pristine (unmodified) CNTs in biological systems is their complete aqueous insolubility and low biocompatibility and toxicity profiles. To endow CNTs with solubility in a biological milieu, several non-covalent and covalent modification methods have been explored. Suitably modified CNTs have shown increased solubility under physiological conditions, improved biocompatibility profiles and lack of toxicity after injection in living animals. Additionally, after being loaded with cargo (small molecules, proteins, peptides or nucleic acids) they have been successfully evaluated as pharmaceutical, therapeutic and diagnostic tools.

Surface functionalization of cyclic olefin copolymer with aryldiazonium salts: A covalent grafting method

NASA Astrophysics Data System (ADS)

Brisset, Florian; Vieillard, Julien; Berton, Benjamin; Morin-Grognet, Sandrine; Duclairoir-Poc, Cécile; Le Derf, Franck

2015-02-01

Covalent immobilization of biomolecules on the surface of cyclic olefin copolymer (COC) is still a tough challenge. We developed a robust method for COC surface grafting through reaction with aryldiazonium. Chemical diazonium reduction generated an aryl radical and the formation of a grafted film layer on the organic surface. We also demonstrated that the chemical reduction of diazonium salt was not sufficient to form a film on the COC surface. UV illumination had to be combined with chemical reduction to graft an aryl layer onto the COC surface. We optimized organic film deposition by using different chemical reducers, different reaction times and reagent proportions. We characterized surface modifications by fluorescence microscopy and contact angle measurements, infrared spectroscopy, X-ray photoemission spectroscopy and Raman spectroscopy, and assessed the topography of the aryl film by atomic force microscopy. This original strategy allowed us to evidence various organic functions to graft biomolecules onto COC surfaces with a fast and efficient technique.

Spin Uncoupling in Chemisorbed OCCO and CO 2: Two High-Energy Intermediates in Catalytic CO 2 Reduction

DOE PAGES

Hedstrom, Svante; dos Santos, Egon Campos; Liu, Chang; ...

2018-05-08

Here, the production of useful compounds via the electrochemical carbon dioxide reduction reaction (CO2RR) is a matter of intense research. Although the thermodynamics and kinetic barriers of CO2RR are reported in previous computational studies, the electronic structure details are often overlooked. We study two important CO2RR intermediates: ethylenedione (OCCO) and CO 2 covalently bound to cluster and slab models of the Cu(100) surface. Both molecules exhibit a near-unity negative charge as chemisorbed, but otherwise they behave quite differently, as explained by a spin-uncoupling perspective. OCCO adopts a high-spin, quartetlike geometry, allowing two covalent bonds to the surface with an averagemore » gross interaction energy of –1.82 eV/bond. The energy cost for electronically exciting OCCO– to the quartet state is 1.5 eV which is readily repaid via the formation of its two surface bonds. CO 2, conversely, retains a low-spin, doubletlike structure upon chemisorption, and its single unpaired electron forms a single covalent surface bond of –2.07 eV. The 5.0 eV excitation energy to the CO 2 – quartet state is prohibitively costly and cannot be compensated for by an additional surface bond.« less

Controlling Interfacial Dynamics: Covalent Bonding versus Physical Adsorption in Polymer Nanocomposites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holt, Adam P.; Bocharova, Vera; Cheng, Shiwang

It is generally believed that the strength of the polymer nanoparticle interaction controls the modification of near-interface segmental mobility in polymer nanocomposites (PNCs). However, little is known about the effect of covalent bonding on the segmental dynamics and glass transition of matrix-free polymer-grafted nanoparticles (PGNs), especially when compared to PNCs. In this article, we directly compare the static and dynamic properties of poly(2-vinylpyridine)/silica-based nanocomposites with polymer chains either physically adsorbed (PNCs) or covalently bonded (PGNs) to identical silica nanoparticles (RNP = 12.5 nm) for three different molecular weight (MW) systems. Interestingly, when the MW of the matrix is as lowmore » as 6 kg/mol (RNP/Rg = 5.4) or as high as 140 kg/mol (RNP/Rg= 1.13), both small-angle X-ray scattering and broadband dielectric spectroscopy show similar static and dynamic properties for PNCs and PGNs. However, for the intermediate MW of 18 kg/mol (RNP/Rg = 3.16), the difference between physical adsorption and covalent bonding can be clearly identified in the static and dynamic properties of the interfacial layer. We ascribe the differences in the interfacial properties of PNCs and PGNs to changes in chain stretching, as quantified by self-consistent field theory calculations. These results demonstrate that the dynamic suppression at the interface is affected by the chain stretching; that is, it depends on the anisotropy of the segmental conformations, more so than the strength of the interaction, which suggests that the interfacial dynamics can be effectively tuned by the degree of stretching a parameter accessible from the MW or grafting density.« less

Controlling Interfacial Dynamics: Covalent Bonding versus Physical Adsorption in Polymer Nanocomposites

DOE PAGES

Holt, Adam P.; Bocharova, Vera; Cheng, Shiwang; ...

2016-06-23

It is generally believed that the strength of the polymer nanoparticle interaction controls the modification of near-interface segmental mobility in polymer nanocomposites (PNCs). However, little is known about the effect of covalent bonding on the segmental dynamics and glass transition of matrix-free polymer-grafted nanoparticles (PGNs), especially when compared to PNCs. In this article, we directly compare the static and dynamic properties of poly(2-vinylpyridine)/silica-based nanocomposites with polymer chains either physically adsorbed (PNCs) or covalently bonded (PGNs) to identical silica nanoparticles (RNP = 12.5 nm) for three different molecular weight (MW) systems. Interestingly, when the MW of the matrix is as lowmore » as 6 kg/mol (RNP/Rg = 5.4) or as high as 140 kg/mol (RNP/Rg= 1.13), both small-angle X-ray scattering and broadband dielectric spectroscopy show similar static and dynamic properties for PNCs and PGNs. However, for the intermediate MW of 18 kg/mol (RNP/Rg = 3.16), the difference between physical adsorption and covalent bonding can be clearly identified in the static and dynamic properties of the interfacial layer. We ascribe the differences in the interfacial properties of PNCs and PGNs to changes in chain stretching, as quantified by self-consistent field theory calculations. These results demonstrate that the dynamic suppression at the interface is affected by the chain stretching; that is, it depends on the anisotropy of the segmental conformations, more so than the strength of the interaction, which suggests that the interfacial dynamics can be effectively tuned by the degree of stretching a parameter accessible from the MW or grafting density.« less

Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody

PubMed Central

2015-01-01

Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called “acidic species” that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity. PMID:25136741

Chemical camouflage of antigenic determinants: stealth erythrocytes.

PubMed

Scott, M D; Murad, K L; Koumpouras, F; Talbot, M; Eaton, J W

1997-07-08

In a number of clinical circumstances it would be desirable to artificially conceal cellular antigenic determinants to permit survival of heterologous donor cells. A case in point is the problem encountered in transfusions of patients with rare blood types or chronically transfused patients who become allosensitized to minor blood group determinants. We have tested the possibility that chemical modification of the red blood cell (RBC) membrane might serve to occlude antigenic determinants, thereby minimizing transfusion reactions. To this end, we have covalently bound methoxy(polyethylene glycol) (mPEG) to the surface of mammalian RBC via cyanuric chloride coupling. Human RBC treated with this technique lose ABO blood group reactivity as assessed by solution-phase antisera agglutination. In accord with this, we also find a profound decrease in anti-blood group antibody binding. Furthermore, whereas human monocytes avidly phagocytose untreated sheep RBC, mPEG-derivatized sheep RBC are ineffectively phagocytosed. Surprisingly, human and mouse RBC appear unaffected by this covalent modification of the cell membrane. Thus, mPEG-treated RBC are morphologically normal, have normal osmotic fragility, and mPEG-derivatized murine RBC have normal in vivo survival, even following repeated infusions. Finally, in preliminary experiments, mPEG-modified sheep RBC intraperitoneally transfused into mice show significantly improved (up to 360-fold) survival when compared with untreated sheep RBC. We speculate that similar chemical camouflage of intact cells may have significant clinical applications in both transfusion (e.g., allosensitization and autoimmune hemolytic disease) and transplantation (e.g., endothelial cells and pancreatic beta cells) medicine.

Modification and Functional Inhibition of Regulator of G-Protein Signaling 4 (RGS4) by 4-Hydroxy-2-nonenal

PubMed Central

Monroy, C. Aaron; Doorn, Jonathan A.; Roman, David L.

2015-01-01

Oxidative stress has been implicated as a component of various pathologies including ischemia/reperfusion injury (IRI) and neurodegenerative diseases such as Parkinson's disease (PD) and schizophrenia. Similarly, regulator of G-protein signaling 4 (RGS4) has been implicated as an important player in each of these pathologies. RGS4, like other RGS proteins, is responsible for temporally regulating G-protein coupled receptor signaling by increasing the intrinsic GTPase activity of Gα subunit of the heterotrimeric signaling complex. In this study we evaluated whether modification by 4-hydroxy-2-nonenal (4HNE), a common lipid peroxidation product, inhibits RGS4. Using immunoprecipitation, we first determined RGS4 modification was occurring in cells at concentrations of 4HNE within reported physiological conditions. Following this determination, we evaluated modification of RGS4 by 4HNE by both Western blot and mass spectrometry (MS). Once it was established that covalent modification occurred only on cysteine containing constructs, tryptic digest followed by mass spectrometry analysis revealed modification occurs at cysteine residues 71, 148, and 183. In order to determine the effect 4HNE had on RGS4 activity, a steady-state colorimetric assay was used to analyze the GAP activity of Δ51-RGS4 as well as the cysteine null mutant. From the data, we determined that RGS4 activity can be modulated by 4HNE through modification at cysteine residues similar to previously reported small molecule inhibition of RGS4. PMID:24229325

Reversibly constraining spliceosome-substrate complexes by engineering disulfide crosslinks.

PubMed

McCarthy, Patrick; Garside, Erin; Meschede-Krasa, Yonatan; MacMillan, Andrew; Pomeranz Krummel, Daniel

2017-08-01

The spliceosome is a highly dynamic mega-Dalton enzyme, formed in part by assembly of U snRNPs onto its pre-mRNA substrate transcripts. Early steps in spliceosome assembly are challenging to study biochemically and structurally due to compositional and conformational dynamics. We detail an approach to covalently and reversibly constrain or trap non-covalent pre-mRNA/protein spliceosome complexes. This approach involves engineering a single disulfide bond between a thiol-bearing cysteine sidechain and a proximal backbone phosphate of the pre-mRNA, site-specifically modified with an N-thioalkyl moiety. When distance and angle between reactants is optimal, the sidechain will react with the single N-thioalkyl to form a crosslink upon oxidation. We provide protocols detailing how this has been applied successfully to trap an 11-subunit RNA-protein assembly, the human U1 snRNP, in complex with a pre-mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Single-crystal X-ray diffraction study of SrGeO3 high-pressure perovskite phase at 100 K

NASA Astrophysics Data System (ADS)

Nakatsuka, Akihiko; Arima, Hiroshi; Ohtaka, Osamu; Fujiwara, Keiko; Yoshiasa, Akira

2017-10-01

Single-crystal X-ray diffraction study of SrGeO3 perovskite (cubic; space group Pmɜ¯m) synthesized at 6 GPa and 1223 K was conducted at a low temperature of 100 K. The residual electron density revealed the presence of the bonding electron at the center of the Ge-O bond, in accordance with our previous conclusion that the Ge-O bond is strongly covalent. From comparison with our previous structure-refinement result at 296 K, the mean square displacement (MSD) of the O atom in the direction of the Ge-O bond is suggested to exhibit no significant temperature dependence, in contrast to that in the direction perpendicular to the bond. Thus, the strong covalency of the Ge-O bond can have a large influence on the temperature dependence of thermal vibration of the O atom.

Intramolecular symmetry-adapted perturbation theory with a single-determinant wavefunction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pastorczak, Ewa; Prlj, Antonio; Corminboeuf, Clémence, E-mail: clemence.corminboeuf@epfl.ch

2015-12-14

We introduce an intramolecular energy decomposition scheme for analyzing non-covalent interactions within molecules in the spirit of symmetry-adapted perturbation theory (SAPT). The proposed intra-SAPT approach is based upon the Chemical Hamiltonian of Mayer [Int. J. Quantum Chem. 23(2), 341–363 (1983)] and the recently introduced zeroth-order wavefunction [J. F. Gonthier and C. Corminboeuf, J. Chem. Phys. 140(15), 154107 (2014)]. The scheme decomposes the interaction energy between weakly bound fragments located within the same molecule into physically meaningful components, i.e., electrostatic-exchange, induction, and dispersion. Here, we discuss the key steps of the approach and demonstrate that a single-determinant wavefunction can already delivermore » a detailed and insightful description of a wide range of intramolecular non-covalent phenomena such as hydrogen bonds, dihydrogen contacts, and π − π stacking interactions. Intra-SAPT is also used to shed the light on competing intra- and intermolecular interactions.« less

High-energy anomalies in covalent high-Tc cuprates with large Hubbard Ud on copper

NASA Astrophysics Data System (ADS)

Barišić, O. S.; Barišić, S.

2015-03-01

A large Ud theory is constructed for the metallic state of high-Tc cuprates. The Emery three-band model, extended with Ox-Oy hopping tpp, and with Ud → ∞, is mapped on slave fermions. The Dyson time-dependent diagrammatic theory in terms of the Cu-O hopping tpd, starting from the nondegenerate unperturbed ground state, is translationally and asymptotically locally gauge invariant. The small parameter of the theory is the average hole occupation of Cu sites nd. The lowest order of the theory generates the single particle propagators of the hybridized pdp- and dpd-fermions with the exact covalent three band structure. The leading many-body effect is band narrowing, accompanied by Landau-like damping of the single particle propagation, due to incoherent local charge Cu-O fluctuations. The corresponding continuum is found below and above the Fermi level.

Crystal structure of lactose permease in complex with an affinity inactivator yields unique insight into sugar recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaptal, Vincent; Kwon, Seunghyug; Sawaya, Michael R.

Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl ofmore » the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.« less

Covalent functionalization of single-walled carbon nanotubes with polytyrosine: Characterization and analytical applications for the sensitive quantification of polyphenols.

PubMed

Eguílaz, Marcos; Gutiérrez, Alejandro; Gutierrez, Fabiana; González-Domínguez, Jose Miguel; Ansón-Casaos, Alejandro; Hernández-Ferrer, Javier; Ferreyra, Nancy F; Martínez, María T; Rivas, Gustavo

2016-02-25

This work reports the synthesis and characterization of single-walled carbon nanotubes (SWCNT) covalently functionalized with polytyrosine (Polytyr); the critical analysis of the experimental conditions to obtain the efficient dispersion of the modified carbon nanotubes; and the analytical performance of glassy carbon electrodes (GCE) modified with the dispersion (GCE/SWCNT-Polytyr) for the highly sensitive quantification of polyphenols. Under the optimal conditions, the calibration plot for the amperometric response of gallic acid (GA) shows a linear range between 5.0 × 10(-7) and 1.7 × 10(-4) M, with a sensitivity of (518 ± 5) m AM(-1) cm(-2), and a detection limit of 8.8 nM. The proposed sensor was successfully used for the determination of total polyphenolic content in tea extracts. Copyright © 2016 Elsevier B.V. All rights reserved.

Light-induced covalent immobilization of monolayers of magnetic nanoparticles on hydrogen-terminated silicon.

PubMed

Leem, Gyu; Zhang, Shishan; Jamison, Andrew C; Galstyan, Eduard; Rusakova, Irene; Lorenz, Bernd; Litvinov, Dmitri; Lee, T Randall

2010-10-01

Specifically tailored ω-alkenyl-1-carboxylic acids were synthesized for use as surfactants in the single-step preparation of manganese ferrite (MnFe2O4) nanoparticles (NPs). Monodisperse manganese ferrite NPs terminated with ω-alkenyl moieties were prepared via a one-pot reaction at high temperature without the need of ligand exchange. Using this approach, simple adjustment of the rate of heating allowed precise tuning of the size of the nanoparticles, which were characterized in bulk form by transmission electron microscopy (TEM), Fourier-transform infrared (FT-IR) spectroscopy, and X-ray diffraction (XRD). These surfactant-coated magnetic nanoparticles were then deposited onto hydrogen-terminated silicon(111) wafers and covalently anchored to the surface by UV-initiated covalent bonding. Analysis by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) confirmed that the UV treatment led to covalent immobilization of the NPs on the silicon surface with a consistent packing density across the surface. The magnetic properties of the stable, surface-bound nanoparticle arrays were characterized using a superconducting quantum interference device (SQUID) magnetometer. The materials and methods described here are being developed for use in bit-patterned ultrahigh density magnetic recording media and nanoscale biomagnetic sensing.

Facile fabrication of highly controllable gating systems based on the combination of inverse opal structure and dynamic covalent chemistry.

PubMed

Wang, Chen; Yang, Haowei; Tian, Li; Wang, Shiqiang; Gao, Ning; Zhang, Wanlin; Wang, Peng; Yin, Xianpeng; Li, Guangtao

2017-06-01

A three-dimensional (3D) inverse opal with periodic and porous structures has shown great potential for applications not only in optics and optoelectronics, but also in functional membranes. In this work, the benzaldehyde group was initially introduced into a 3D nanoporous inverse opal, serving as a platform for fabricating functional membranes. By employing the dynamic covalent approach, a highly controllable gating system was facilely fabricated to achieve modulable and reversible transport features. It was found that the physical/chemical properties and pore size of the gating system could easily be regulated through post-modification with amines. As a demonstration, the gated nanopores were modified with three kinds of amines to control the wettability, surface charge and nanopore size which in turn was exploited to achieve selective mass transport, including hydrophobic molecules, cations and anions, and the transport with respect to the physical steric hindrance. In particular, the gating system showed extraordinary reversibility and could recover to its pristine state by simply changing pH values. Due to the unlimited variety provided by the Schiff base reaction, the inverse opal described here exhibits a significant extendibility and could be easily post-modified with stimuli-responsive molecules for special purposes. Furthermore, this work can be extended to employ other dynamic covalent routes, for example Diels-Alder, ester exchange and disulfide exchange-based routes.

A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum

PubMed Central

Wolfram, Stefanie; Wielsch, Natalie; Hupfer, Yvonne; Mönch, Bettina; Lu-Walther, Hui-Wen; Heintzmann, Rainer; Werz, Oliver; Svatoš, Aleš; Pohnert, Georg

2015-01-01

Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways. PMID:26496085

Photochemical properties and sensor applications of modified yellow fluorescent protein (YFP) covalently attached to the surfaces of etched optical fibers (EOFs).

PubMed

Veselov, Alexey A; Abraham, Bobin George; Lemmetyinen, Helge; Karp, Matti T; Tkachenko, Nikolai V

2012-01-01

Fluorescent proteins have the inherent ability to act as sensing components which function both in vitro and inside living cells. We describe here a novel study on a covalent site-specific bonding of fluorescent proteins to form self-assembled monolayers (SAMs) on the surface of etched optical fibers (EOFs). Deposition of fluorescent proteins on EOFs gives the opportunity to increase the interaction of guided light with deposited molecules relative to plane glass surfaces. The EOF modification is carried out by surface activation using 3-aminopropylthrimethoxysilane (APTMS) and bifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) which exposes sulfhydryl-reactive maleimide groups followed by covalent site-specific coupling of modified yellow fluorescent protein (YFP). Steady-state and fluorescence lifetime measurements confirm the formation of SAM. The sensor applications of YPF SAMs on EOF are demonstrated by the gradual increase of emission intensity upon addition of Ca(2+) ions in the concentration range from a few tens of micromolars up to a few tens of millimolars. The studies on the effect of pH, divalent cations, denaturing agents, and proteases reveal the stability of YFP on EOFs at normal physiological conditions. However, treatments with 0.5% SDS at pH 8.5 and protease trypsin are found to denaturate or cleave the YFP from fiber surfaces.

Novel roles for biogenic monoamines: from monoamines in transglutaminase-mediated post-translational protein modification to monoaminylation deregulation diseases.

PubMed

Walther, Diego J; Stahlberg, Silke; Vowinckel, Jakob

2011-12-01

Functional protein serotonylation is a newly recognized post-translational modification with the primary biogenic monoamine (PBMA) serotonin (5-HT). This covalent protein modification is catalyzed by transglutaminases (TGs) and, for example, acts in the constitutive activation of small GTPases. Multiple physiological roles have been identified since its description in 2003 and, importantly, deregulated serotonylation was shown in the etiology of bleeding disorders, primary pulmonary hypertension and diabetes. The PBMAs 5-HT, histamine, dopamine, and norepinephrine all act as neurotransmitters in the nervous system and as hormones in non-neuronal tissues, which points out their physiological importance. In analogy to serotonylation we have found that also the other PBMAs act through the TG-catalyzed mechanisms of 'histaminylation', 'dopaminylation' and 'norepinephrinylation'. Therefore, PBMAs deploy a considerable portion of their effects via protein monoaminylation in addition to their canonical receptor-mediated signaling. Here, the implications of these newly identified post-translational modifications are presented and discussed. Furthermore, the potential regulatory roles of protein monoaminylation in small GTPase, heterotrimeric G-protein and lipid signaling, as well as in modulating metabolic enzymes and nuclear processes, are critically assessed. © 2011 The Authors Journal compilation © 2011 FEBS.

Rapid Characterization of Insulin Modifications and Sequence Variations by Proteinase K Digestion and UHPLC-ESI-MS

NASA Astrophysics Data System (ADS)

Yang, Rong-Sheng; Tang, Weijuan; Sheng, Huaming; Meng, Fanyu

2018-01-01

Discovery of novel insulin analogs as therapeutics has remained an active area of research. Compared with native human insulin, insulin analog molecules normally incorporate either covalent modifications or amino acid sequence variations. From the drug discovery and development perspective, methods for efficient and detailed characterization of these primary structural changes are very important. In this report, we demonstrate that proteinase K digestion coupled with UPLC-ESI-MS analysis provides a simple and rapid approach to characterize the modifications and sequence variations of insulin molecules. A commercially available proteinase K digestion kit was used to process recombinant human insulin (RHI), insulin glargine, and fluorescein isothiocynate-labeled recombinant human insulin (FITC-RHI) samples. The LC-MS data clearly showed that RHI and insulin glargine samples can be differentiated, and the FITC modifications in all three amine sites of the RHI molecule are well characterized. The end-to-end experiment and data interpretation was achieved within 60 min. This approach is fast and simple, and can be easily implemented in early drug discovery laboratories to facilitate research on more advanced insulin therapeutics. [Figure not available: see fulltext.

Rapid Characterization of Insulin Modifications and Sequence Variations by Proteinase K Digestion and UHPLC-ESI-MS

NASA Astrophysics Data System (ADS)

Yang, Rong-Sheng; Tang, Weijuan; Sheng, Huaming; Meng, Fanyu

2018-05-01

Discovery of novel insulin analogs as therapeutics has remained an active area of research. Compared with native human insulin, insulin analog molecules normally incorporate either covalent modifications or amino acid sequence variations. From the drug discovery and development perspective, methods for efficient and detailed characterization of these primary structural changes are very important. In this report, we demonstrate that proteinase K digestion coupled with UPLC-ESI-MS analysis provides a simple and rapid approach to characterize the modifications and sequence variations of insulin molecules. A commercially available proteinase K digestion kit was used to process recombinant human insulin (RHI), insulin glargine, and fluorescein isothiocynate-labeled recombinant human insulin (FITC-RHI) samples. The LC-MS data clearly showed that RHI and insulin glargine samples can be differentiated, and the FITC modifications in all three amine sites of the RHI molecule are well characterized. The end-to-end experiment and data interpretation was achieved within 60 min. This approach is fast and simple, and can be easily implemented in early drug discovery laboratories to facilitate research on more advanced insulin therapeutics. [Figure not available: see fulltext.

Fundamental Studies on Donor-acceptor Conjugated Polymers Containing 'Heavy' Group 14 and Group 16 Elements

NASA Astrophysics Data System (ADS)

Gibson, Gregory Laird

One advantage of conjugated polymers as organic materials is that their properties may be readily tuned through covalent modifications. This thesis presents studies on the structure-property relationships resulting from single- and double-atom substitutions on an alternating donor-acceptor conjugated polymer. Specifically, single selenium and tellurium atoms have been incorporated into the acceptor monomer in place of sulfur; silicon and germanium atoms have been substituted in place of carbon at the donor monomer bridge position. The carbon-donor/ tellurium-acceptor polymer was synthesized by a post-polymerization reaction sequence and demonstrated the utility of heavy group 16 atoms to red shift a polymer absorption spectrum. Density functional theory calculations point to a new explanation for this result invoking the lower heavy atom ionization energy and reduced aromaticity of acceptor monomers containing selenium and tellurium compared to sulfur. Absorption and emission experiments demonstrate that both silicon and germanium substitutions in the donor slightly blue shift the polymer absorption spectrum. Polymers containing sulfur in the acceptor are the strongest light absorbers of all polymers studied here. Molecular weight and phenyl end capping studies show that molecular weight appears to affect polymer absorption to the greatest degree in a medium molecular weight regime and that these effects have a significant aggregation component. Solar cell devices containing either the silicon- or germanium-donor/selenium-acceptor polymer display improved red light harvesting or hole mobility relative to their structural analogues. Overall, these results clarify the effects of single atom substitution on donor-acceptor polymers and aid in the future design of polymers containing heavy atoms.

From genes to protein mechanics on a chip.

PubMed

Otten, Marcus; Ott, Wolfgang; Jobst, Markus A; Milles, Lukas F; Verdorfer, Tobias; Pippig, Diana A; Nash, Michael A; Gaub, Hermann E

2014-11-01

Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

Epigenetic regulation of immune checkpoints: another target for cancer immunotherapy?

PubMed

Ali, Mahmoud A; Matboli, Marwa; Tarek, Marwa; Reda, Maged; Kamal, Kamal M; Nouh, Mahmoud; Ashry, Ahmed M; El-Bab, Ahmed Fath; Mesalam, Hend A; Shafei, Ayman El-Sayed; Abdel-Rahman, Omar

2017-01-01

Epigenetic changes in oncogenes and tumor-suppressor genes contribute to carcinogenesis. Understanding the epigenetic and genetic components of tumor immune evasion is crucial. Few cancer genetic mutations have been linked to direct correlations with immune evasion. Studies on the epigenetic modulation of the immune checkpoints have revealed a critical interaction between epigenetic and immune modulation. Epigenetic modifiers can activate many silenced genes. Some of them are immune checkpoints regulators that turn on immune responses and others turn them off resulting in immune evasion. Many forms of epigenetic inheritance mechanisms may play a role in regulation of immune checkpoints including: covalent modifications, noncoding RNA and histone modifications. In this review, we will show how the potential interaction between epigenetic and immune modulation may lead to new approaches for specific epigenome/immunome-targeted therapies for cancer.

Recent findings and technological advances in phosphoproteomics for cells and tissues.

PubMed

von Stechow, Louise; Francavilla, Chiara; Olsen, Jesper V

2015-01-01

Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins - termed phosphoproteomics - strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.

In the loop: how chromatin topology links genome structure to function in mechanisms underlying learning and memory.

PubMed

Watson, L Ashley; Tsai, Li-Huei

2017-04-01

Different aspects of learning, memory, and cognition are regulated by epigenetic mechanisms such as covalent DNA modifications and histone post-translational modifications. More recently, the modulation of chromatin architecture and nuclear organization is emerging as a key factor in dynamic transcriptional regulation of the post-mitotic neuron. For instance, neuronal activity induces relocalization of gene loci to 'transcription factories', and specific enhancer-promoter looping contacts allow for precise transcriptional regulation. Moreover, neuronal activity-dependent DNA double-strand break formation in the promoter of immediate early genes appears to overcome topological constraints on transcription. Together, these findings point to a critical role for genome topology in integrating dynamic environmental signals to define precise spatiotemporal gene expression programs supporting cognitive processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mass Spectrometric Evidence of Malonaldehyde and 4-Hydroxynonenal Adductions to Radical-Scavenging Soy Peptides

PubMed Central

Zhao, Jing; Chen, Jing; Zhu, Haining; Xiong, Youling L.

2012-01-01

Antioxidative peptides in food systems are potential targets of lipid oxidation-generated reactive aldehydes, such as malonaldehyde (MDA) and 4-hydroxynonenal (HNE). In this study, covalent modifications on radical-scavenging peptides prepared from soy protein hydrolysate by MDA and HNE were characterized by liquid chromatography–electrospray ionization-mass spectrometry (LC-ESI-MS/MS). MS/MS analyses detected the formation of Schiff base type adducts of MDA on the side chain groups of lysine, histidine, arginine, glutamine, and asparagine residues as well as the N-termini of peptides. MDA also formed a fluorescent product with lysine residues. HNE adducted on lysine residues through Schiff base formation and on histidine, arginine, glutamine, and asparagine residues mainly through Michael addition. In spite of the extensive MDA modification, peptide cross-linking by this potential mechanism was undetectable. PMID:22946674

All-in-One Cellulose Nanocrystals for 3D Printing of Nanocomposite Hydrogels.

PubMed

Wang, Jieping; Chiappone, Annalisa; Roppolo, Ignazio; Shao, Feng; Fantino, Erika; Lorusso, Massimo; Rentsch, Daniel; Dietliker, Kurt; Pirri, Candido Fabrizio; Grützmacher, Hansjörg

2018-02-23

Cellulose nanocrystals (CNCs) with >2000 photoactive groups on each can act as highly efficient initiators for radical polymerizations, cross-linkers, as well as covalently embedded nanofillers for nanocomposite hydrogels. This is achieved by a simple and reliable method for surface modification of CNCs with a photoactive bis(acyl)phosphane oxide derivative. Shape-persistent and free-standing 3D structured objects were printed with a mono-functional methacrylate, showing a superior swelling capacity and improved mechanical properties. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Modification of L-asparaginase with colominic acid and the new characteristics of the modified enzyme].

PubMed

Wang, Y D; Guo, L; Qian, S J; Meng, G Z; Zhang, S Z

2000-07-01

The colominic acid was covalently coupled to L-asparaginase molecule by reductive amination. Depending on the molar ratios of colominic acid-asparaginase (30:1, 50:1 and 100:1), a modified enzyme molecule contained 4.7, 7.2 and 12 colominic acid molecule, they retained 58%, 56% and 33.2% of the initial asparaginase activity, respectively. In comparison with the native enzyme, modified enzyme had lower immunogenicity and antigenicity, longer half-life time (in vitro), more resistance ability to trypsin proteolysis, and similar Km value for L-asparagine.

The Ubiquitin–Proteasome System of Saccharomyces cerevisiae

PubMed Central

Finley, Daniel; Ulrich, Helle D.; Sommer, Thomas; Kaiser, Peter

2012-01-01

Protein modifications provide cells with exquisite temporal and spatial control of protein function. Ubiquitin is among the most important modifiers, serving both to target hundreds of proteins for rapid degradation by the proteasome, and as a dynamic signaling agent that regulates the function of covalently bound proteins. The diverse effects of ubiquitylation reflect the assembly of structurally distinct ubiquitin chains on target proteins. The resulting ubiquitin code is interpreted by an extensive family of ubiquitin receptors. Here we review the components of this regulatory network and its effects throughout the cell. PMID:23028185

S-Nitrosylation: NO-Related Redox Signaling to Protect Against Oxidative Stress

PubMed Central

STEENBERGEN, CHARLES; MURPHY, ELIZABETH

2007-01-01

Nitric oxide (NO) plays an important role in the regulation of cardiovascular function. S-nitrosylation, the covalent attachment of an NO moiety to sulfhydryl residues of proteins, resulting in the formation of S-nitrosothiols (SNOs), is a prevalent posttranslational protein modification involved in redox-based cellular signaling. Under physiologic conditions, protein S>-nitrosylation and SNOs provide protection preventing further cellular oxidative and nitrosative stress. However, oxidative stress and the resultant dysfunction of NO signaling have been implicated in the pathogenesis of cardiovascular diseases. PMID:16987022

Characterization of a PEGylated protein therapeutic by ion exchange chromatography with on-line detection by native ESI MS and MS/MS.

PubMed

Muneeruddin, K; Bobst, C E; Frenkel, R; Houde, D; Turyan, I; Sosic, Z; Kaltashov, I A

2017-01-16

Detailed profiling of both enzymatic (e.g., glycosylation) and non-enzymatic (e.g., oxidation and deamidation) post-translational modifications (PTMs) is frequently required for the quality assessment of protein-based drugs. Challenging as it is, this task is further complicated for the so-called second-generation biopharmaceuticals, which also contain "designer PTMs" introduced to either enhance their pharmacokinetic profiles (e.g., PEGylated proteins) or endow them with therapeutic activity (e.g., protein-drug conjugates). Such modifications of protein covalent structure can dramatically increase structural heterogeneity, making the very notion of "molecular mass" meaningless, as ions representing different glycoforms of a PEGylated protein may have nearly identical distributions of ionic current as a function of m/z, making their contributions to the mass spectrum impossible to distinguish. In this work we demonstrate that a combination of ion exchange chromatography (IXC) with on-line detection by electrospray ionization mass spectrometry (ESI MS) and methods of ion manipulation in the gas phase (limited charge reduction and collision-induced dissociation) allows meaningful structural information to be obtained on a structurally heterogeneous sample of PEGylated interferon β-1a. IXC profiling of the protein sample gives rise to a convoluted chromatogram with several partially resolved peaks which can represent both deamidation and different glycosylation patterns within the protein, as well as varying extent of PEGylation. Thus, profiling the protein with on-line IXC/ESI/MS/MS allows it to be characterized by providing information on three different types of PTMs (designer, enzymatic and non-enzymatic) within a single protein therapeutic.

Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region*

PubMed Central

Yasui, Norihisa; Kitago, Yu; Beppu, Ayako; Kohno, Takao; Morishita, Shunsuke; Gomi, Hiroki; Nagae, Masamichi; Hattori, Mitsuharu; Takagi, Junichi

2011-01-01

Reelin is a 3461-residue secreted glycoprotein that plays a critical role in brain development through its action on target neurons. Although it is known that functional reelin protein exists as multimer formed by interchain disulfide bond(s) as well as through non-covalent interactions, the chemical nature of the multimer assembly has been elusive. In the present study, we identified, among 122 cysteines present in full-length reelin, the single critical cysteine residue (Cys2101) responsible for the covalent multimerization. C2101A mutant reelin failed to assemble into disulfide-bonded multimers, whereas it still exhibited non-covalently associated high molecular weight oligomeric states in solution. Detailed analysis of tryptic fragments produced from the purified reelin proteins revealed that the minimum unit of the multimer is a homodimeric reelin linked via Cys2101 present in the central region and that this cysteine does not connect to the N-terminal region of reelin, which had been postulated as the primary oligomerization domain. A surface plasmon resonance binding assay confirmed that C2101A mutant reelin retained binding capability toward two neuronal receptors apolipoprotein E receptor 2 and very low density lipoprotein receptor. However, it failed to show signaling activity in the assay using the cultured neurons. These results indicate that an intact higher order architecture of reelin multimer maintained by both Cys2101-mediated homodimerization and other non-covalent association present elsewhere in the reelin primary structure are essential for exerting its full biological activity. PMID:21844191

Efficient Covalent Modification of Multiwalled Carbon Nanotubes with Diazotized Dyes in Water at Room Temperature.

PubMed

Bensghaïer, Asma; Lau Truong, Stéphanie; Seydou, Mahamadou; Lamouri, Aazdine; Leroy, Eric; Mičušik, Matej; Forro, Klaudia; Beji, Mohamed; Pinson, Jean; Omastová, Mária; Chehimi, Mohamed M

2017-07-11

Tetrafluoroborate salts of diazotized Azure A (AA-N 2 + ), Neutral Red (NR-N 2 + ) and Congo Red (CR-N 2 + ) dyes were prepared and reacted with multiwalled carbon nanotubes (MWCNTs) at room temperature, in water without any reducing agent. The as-modified MWCNTs were examined by IRATR, Raman spectroscopy, XPS, TGA, TEM, and cyclic voltammetry. The diazonium band located at ∼2350 cm -1 in the diazotized dye IR spectra vanished after attachment to the nanotubes whereas the Raman D/G peak ratio slightly increased after dye covalent attachment at a high initial diazonium/CNT mass ratio. XPS measurements show the loss of F 1s from the BF 4 - anion together with a clear change in the high-resolution C 1s region from the modified nanotubes. Thermogravimetric analyses proved substantial mass loadings of the organic grafts leveling off at 40.5, 34.3, and 50.7 wt % for AA, NR, and CR, respectively. High-resolution TEM pictures confirmed the presence of 1.5-7-nm-thick continuous amorphous layers on the nanotubes assigned to the aryl layers from the dyes. Cyclic voltammetry studies in acetonitrile (ACN) confirmed the grafting of the dyes; the latter retain their electrochemical behavior in the grafted state. The experimental results correlate remarkably well with quantum chemical calculations that indicate high binding energies between the dyes and the CNTs accounting for true covalent bonding (140-185 kJ/mol with the CNT-aryl distance <1.6 nm), though attachment by π stacking also contributes to obtaining stable hybrids. Finally, the pH-responsive character of the robust hybrids was demonstrated by a higher degree of protonation of Neutral Red-grafted CNTs at pH 2 compared to that of the neutral aqueous medium. This work demonstrates that diazotized dyes can be employed for the surface modification of MWCNTs in a very simple and efficient manner in water and at room temperature. The hybrids could be employed for many purposes such as optically pH-responsive materials, biosensors, and optothermal composite actuators to name a few.

Protein S-Nitrosylation Regulates Xylem Vessel Cell Differentiation in Arabidopsis.

PubMed

Kawabe, Harunori; Ohtani, Misato; Kurata, Tetsuya; Sakamoto, Tomoaki; Demura, Taku

2018-01-01

Post-translational modifications of proteins have important roles in the regulation of protein activity. One such modification, S-nitrosylation, involves the covalent binding of nitric oxide (NO)-related species to a cysteine residue. Recent work showed that protein S-nitrosylation has crucial functions in plant development and environmental responses. In the present study, we investigated the importance of protein S-nitrosylation for xylem vessel cell differentiation using a forward genetics approach. We performed ethyl methanesulfonate mutagenesis of a transgenic Arabidopsis 35S::VND7-VP16-GR line in which the activity of VASCULAR-RELATED NAC-DOMAIN7 (VND7), a key transcription factor involved in xylem vessel cell differentiation, can be induced post-translationally by glucocorticoid treatment, with the goal of obtaining suppressor mutants that failed to differentiate ectopic xylem vessel cells; we named these mutants suppressor of ectopic vessel cell differentiation induced by VND7 (seiv) mutants. We found the seiv1 mutant to be a recessive mutant in which ectopic xylem cell differentiation was inhibited, especially in aboveground organs. In seiv1 mutants, a single nucleic acid substitution (G to A) leading to an amino acid substitution (E36K) was present in the gene encoding S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which regulates the turnover of the natural NO donor, S-nitrosoglutathione. An in vitro S-nitrosylation assay revealed that VND7 can be S-nitrosylated at Cys264 and Cys320 located near the transactivation activity-related domains, which were shown to be important for transactivation activity of VND7 by transient reporter assay. Our results suggest crucial roles for GSNOR1-regulated protein S-nitrosylation in xylem vessel cell differentiation, partly through the post-translational modification of VND7. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

76 FR 34801 - Petition for Modification of Single Car Air Brake Test Procedures

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-14

...] Petition for Modification of Single Car Air Brake Test Procedures In accordance with Part 232 of Title 49... Railroad Administration (FRA) grant a modification of the single car air brake test procedures as... of 25 flat cars in consist with revenue cars utilized as locomotives in ``work'' trains, where the...

Extensive sequence-influenced DNA methylation polymorphism in the human genome

PubMed Central

2010-01-01

Background Epigenetic polymorphisms are a potential source of human diversity, but their frequency and relationship to genetic polymorphisms are unclear. DNA methylation, an epigenetic mark that is a covalent modification of the DNA itself, plays an important role in the regulation of gene expression. Most studies of DNA methylation in mammalian cells have focused on CpG methylation present in CpG islands (areas of concentrated CpGs often found near promoters), but there are also interesting patterns of CpG methylation found outside of CpG islands. Results We compared DNA methylation patterns on both alleles between many pairs (and larger groups) of related and unrelated individuals. Direct observation and simulation experiments revealed that around 10% of common single nucleotide polymorphisms (SNPs) reside in regions with differences in the propensity for local DNA methylation between the two alleles. We further showed that for the most common form of SNP, a polymorphism at a CpG dinucleotide, the presence of the CpG at the SNP positively affected local DNA methylation in cis. Conclusions Taken together with the known effect of DNA methylation on mutation rate, our results suggest an interesting interdependence between genetics and epigenetics underlying diversity in the human genome. PMID:20497546

Solid phase monofunctionalization of gold nanoparticles using ionic exchange resin as polymer support.

PubMed

Zou, Jianhua; Dai, Qiu; Wang, Jinhai; Liu, Xiong; Huo, Qun

2007-07-01

A solid phase modification method using anionic exchange resin as polymer support was developed for the synthesis of monofunctional gold nanoparticles. Based on a "catch and release" mechanism to control the number of functional groups attached to the nanoparticle surface, bifunctional thiol ligands with a carboxylic acid end group were first immobilized at a controlled density on anionic exchange resin through electrostatic interactions. Gold nanoparticles were then immobilized to the anionic exchange resin by a one-to-one place exchange reaction between resin-bound thiol ligands and butanethiol-protected gold nanoparticles in solution. After cleaving off from the resin under mild conditions, gold nanoparticles with a single carboxyl group attached to the surface were obtained as the major product. Experimental conditions such as the solvents used for ligand loading and solid phase place exchange reaction, and the loading density of the ligands, were found to play a critical role towards the successful synthesis of monofunctional nanoparticles. Overall, the noncovalent bond-based ligand immobilization technique reported here greatly simplified the process of solid phase monofunctionalization of nanoparticles compared to a previously reported covalent bond-based ligand immobilization technique.

Exciton Localization and Optical Emission in Aryl-Functionalized Carbon Nanotubes

DOE PAGES

Gifford, Brendan Joel; Kilina, Svetlana; Htoon, Han; ...

2017-10-26

Recent spectroscopic studies have revealed the appearance of multiple low-energy peaks in the fluorescence of single-walled carbon nanotubes (SWCNTs) upon their covalent functionalization by aryl groups. The photophysical nature of these low energy optical bands is of significant interest in the quest to understand their appearance and to achieve their precise control via chemical modification of SWCNTs. This theoretical study explains the specific energy dependence of emission features introduced in chemically functionalized (6,5) SWCNTs with aryl bromides at different conformations and in various dielectric media. Calculations using density functional theory (DFT) and time dependent DFT (TD-DFT) show that the specificmore » isomer geometry—the relative position of functional groups on the carbon-ring of the nanotube—is critical for controlling the energies and intensities of optical transitions introduced by functionalization, while the dielectric environment and the chemical composition of functional groups play less significant roles. Furthermore, the predominant effects on optical properties as a result of functionalization conformation are rationalized by exciton localization on the surface of the SWCNT near the dopant sp3-defect but not onto the functional group itself.« less

Carbon-Fiber Microelectrodes for In Vivo Applications

PubMed Central

Huffman, Megan L.; Venton, B. Jill

2009-01-01

Carbon-fiber microelectrodes (CFMEs) have been a useful tool for measuring rapid changes in neurotransmitters because of their small size, sensitivity, and good electrochemical properties. In this article, we highlight recent advances using CFMEs for measuring neurotransmitters in vivo. Dopamine has been a primary neurotransmitter of interest but direct electrochemical detection of other neurochemicals including nitric oxide and adenosine has also been investigated. Surface treatments have been studied to enhance electrode sensitivity, such as covalent modification or the addition of a layer of carbon nanotubes. Enzyme-modified microelectrodes that detect non-electroactive compounds further extend the usefulness of CFMEs beyond the traditional monoamines. CFMEs continue to be used in vivo to understand basic neurobiological mechanisms and the actions of pharmacological agents, including drugs of abuse. Advances in sensitivity and instrumentation now allow CFMEs to be used for measurements of natural dopamine release that occur during behavioral experiments. A new technique combining electrochemistry with electrophysiology at a single microelectrode facilitates a better understanding of neurotransmitter concentrations and their effects on cell firing. Future research in this field will likely concentrate on fabricating smaller electrodes and electrode arrays, as well as expanding the use of CFMEs in neuroscience beyond dopamine. PMID:19082168

Non-surgical sterilisation methods may offer a sustainable solution to feral horse (Equus caballus) overpopulation.

PubMed

Hall, Sally Elizabeth; Nixon, Brett; Aitken, R John

2017-09-01

Feral horses are a significant pest species in many parts of the world, contributing to land erosion, weed dispersal and the loss of native flora and fauna. There is an urgent need to modify feral horse management strategies to achieve public acceptance and long-term population control. One way to achieve this is by using non-surgical methods of sterilisation, which are suitable in the context of this mobile and long-lived species. In this review we consider the benefits of implementing novel mechanisms designed to elicit a state of permanent sterility (including redox cycling to generate oxidative stress in the gonad, random peptide phage display to target non-renewable germ cells and the generation of autoantibodies against proteins essential for conception via covalent modification) compared with that of traditional immunocontraceptive approaches. The need for a better understanding of mare folliculogenesis and conception factors, including maternal recognition of pregnancy, is also reviewed because they hold considerable potential in providing a non-surgical mechanism for sterilisation. In conclusion, the authors contend that non-surgical measures that are single shot and irreversible may provide a sustainable and effective strategy for feral horse control.

Agonist trapped in ATP-binding sites of the P2X2 receptor.

PubMed

Jiang, Ruotian; Lemoine, Damien; Martz, Adeline; Taly, Antoine; Gonin, Sophie; Prado de Carvalho, Lia; Specht, Alexandre; Grutter, Thomas

2011-05-31

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent "tethering" reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.

Mre11 and Exo1 contribute to the initiation and processivity of resection at meiotic double-strand breaks made independently of Spo11.

PubMed

Hodgson, Adam; Terentyev, Yaroslav; Johnson, Rebecca A; Bishop-Bailey, Anna; Angevin, Thibaut; Croucher, Adam; Goldman, Alastair S H

2011-02-07

During meiosis DNA double-strand breaks (DSBs) are induced and repaired by homologous recombination to create gene conversion and crossover products. Mostly these DSBs are made by Spo11, which covalently binds to the DSB ends. More rarely in Saccharomyces cerevisiae, other meiotic DSBs are formed by self-homing endonucleases such as VDE, which is site specific and does not covalently bind to the DSB ends. We have used experimentally located VDE-DSB sites to analyse an intermediate step in homologous recombination, resection of the single-strand ending 5' at the DSB site. Analysis of strains with different mutant alleles of MRE11 (mre11-58S and mre11-H125N) and deleted for EXO1 indicated that these two nucleases make significant contributions to repair of VDE-DSBs. Physical analysis of single-stranded repair intermediates indicates that efficient initiation and processivity of resection at VDE-DSBs require both Mre11 and Exo1, with loss of function for either protein causing severe delay in resection. We propose that these experiments model what happens at Spo11-DSBs after removal of the covalently bound protein, and that Mre11 and Exo1 are the major nucleases involved in creating resection tracts of widely varying lengths typical of meiotic recombination. Copyright © 2010 Elsevier B.V. All rights reserved.

Post-translational modifications of linker histone H1 variants in mammals

NASA Astrophysics Data System (ADS)

Starkova, T. Yu; Polyanichko, A. M.; Artamonova, T. O.; Khodorkovskii, M. A.; Kostyleva, E. I.; Chikhirzhina, E. V.; Tomilin, A. N.

2017-02-01

The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2-H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

Close encounters for the first time: Helicase interactions with DNA damage.

PubMed

Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

2015-09-01

DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. Published by Elsevier B.V.

Enhanced enzymatic saccharification of corn stover by in situ modification of lignin with poly (ethylene glycol) ether during low temperature alkali pretreatment.

PubMed

Lai, Chenhuan; Tang, Shuo; Yang, Bo; Gao, Ziqi; Li, Xin; Yong, Qiang

2017-11-01

A novel pretreatment process of corn stover was established in this study by in situ modification of lignin with poly (ethylene glycol) diglycidyl ether (PEGDE) during low temperature alkali pretreatment. The addition of PEGDE obviously improved the enzymatic hydrolysis by covalently modifying the residual lignins in substrates. Under the optimized conditions (pretreated with 10% (w/w) NaOH and 10% (w/w) PEGDE at 70°C for 2.5h), the total fermentable sugar yield was increased by 46.4%, from 23.7g to 34.7g per 100g raw materials. Additionally, the remaining activities of exo-glucanase and β-glucosidase in supernatant were increased by 58.6% and 40.6% respectively, demonstrating that the enhancement of enzymatic hydrolysis was mainly due to the alleviation of enzyme non-productive binding. Although the isolated lignin modified with PEGDE enhanced the enzymatic hydrolysis of substrates as well, this in situ lignin modification provided an efficient but simple way to improve enzymatic saccharification. Copyright © 2017. Published by Elsevier Ltd.

Polyester modification of the mammalian TRPM8 channel protein: Implications for structure and function

PubMed Central

Bikard, Yann; Chen, Wei; Liu, Tong; Li, Hong; Jendrossek, Dieter; Cohen, Alejandro; Pavlov, Evgeny; Rohacs, Tibor; Zakharian, Eleonora

2013-01-01

SUMMARY The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmental cues such as cold temperatures and chemical compounds, including menthol and icilin. The channel functional activity is regulated by various physical and chemical factors, and is likely to be pre-conditioned by its molecular composition. Our studies indicate that TRPM8 channel forms a structural-functional complex with the polyester, poly-(R)-3hydroxybutyrate (PHB). We identified by mass spectrometry a number of PHB-modified peptides in the N-terminus of the TRPM8 protein and in its extracellular S3–S4 linker. Removal of PHB by enzymatic hydrolysis, and site-directed mutagenesis of both the serine residues that serve as covalent anchors for PHB and adjacent hydrophobic residues that interact with the methyl groups of the polymer, resulted in significant inhibition of TRPM8 channel activity. We conclude that the TRPM8 channel undergoes post-translational modification by PHB and that this modification is required for its normal function. PMID:23850286

Formation of oligonucleotide adducts in pharmaceutical formulations.

PubMed

Krotz, Achim H; Gaus, Hans; Hardee, Gregory E

2005-01-01

During preformulation studies, we observed that oligonucleotide extracted from topical formulations contained considerable amounts of covalently modified oligonucleotide adducts. In this report, we describe the identification and characterization of reaction products that form when PS-oligodeoxyribonucleotide ISIS 2302 (1) is brought into contact with aqueous solutions of glycerol-derived excipients. Compatibility tests showed that the presence of certain glycerides in the formulation lead to adduct formation (1+58x amu, 1+72x amu, 1+58x+72y amu, x, and y are the number of modifications on one oligonucleotide strand). No adduct formation was observed in the presence of triglycerides or propylene glycol-derived excipients used in the study. Using nucleosides as model compounds, two modifications of deoxyguanosine were isolated by preparative reversed phase (RP)-high pressure liquid chromatography (HPLC) and characterized by nuclear magnetic resonance (NMR) and HPLC-mass spectrometry (MS). Modifications were identified as N2-(1-carboxymethyl)- and N2-(1-carboxyethyl) derivatives of 2'-deoxyguanosine. The mechanism of formation of these adducts may involve advanced glycation reactions possibly caused by excipient impurities or degradation products such as glyceraldehyde or glyceraldehyde derivatives.

Sequence and Structure Dependent DNA-DNA Interactions

NASA Astrophysics Data System (ADS)

Kopchick, Benjamin; Qiu, Xiangyun

Molecular forces between dsDNA strands are largely dominated by electrostatics and have been extensively studied. Quantitative knowledge has been accumulated on how DNA-DNA interactions are modulated by varied biological constituents such as ions, cationic ligands, and proteins. Despite its central role in biology, the sequence of DNA has not received substantial attention and ``random'' DNA sequences are typically used in biophysical studies. However, ~50% of human genome is composed of non-random-sequence DNAs, particularly repetitive sequences. Furthermore, covalent modifications of DNA such as methylation play key roles in gene functions. Such DNAs with specific sequences or modifications often take on structures other than the canonical B-form. Here we present series of quantitative measurements of the DNA-DNA forces with the osmotic stress method on different DNA sequences, from short repeats to the most frequent sequences in genome, and to modifications such as bromination and methylation. We observe peculiar behaviors that appear to be strongly correlated with the incurred structural changes. We speculate the causalities in terms of the differences in hydration shell and DNA surface structures.

Chemical modification of poly(ethylene terephthalate) and immobilization of the selected enzymes on the modified film

NASA Astrophysics Data System (ADS)

Irena, Gancarz; Jolanta, Bryjak; Karolina, Zynek

2009-07-01

Poly(ethylene terephthalate) (PET) film was modified by reaction with hydrazine (HD), ethylenediamine (EDA), 1,2-diaminopropane (1,2-DAP) and 1,3-diaminopropane (1,3-DAP). The maximal amount of amine functionalities introduced in the chosen conditions on the surface was found as 0.07, 3.35, 0.76 and 1.99 nmol cm -2 for HD, EDA, 1,2-DAP and 1,3-DAP respectively. During the modification process etching of the sample and an increase of stiffness takes place. FTIR-ATR spectra prove that the surface chemistry after modification in amine solution is very complex. The lack of clear correlation between the surface tension and surface concentration of amine functionalities seems to confirm that. For immobilization purpose invertase, laccase and tyrosinase were used. The amount of covalently attached proteins at first increases with the increase of surface concentration of amine groups but after reaching a certain level of amine groups, decrease of the immobilization level was observed. All enzymes tested showed highest activity for a moderate level of aminolysis and this activity had the highest values for EDA-modified PET.

Crosslinking Protein Glutathionylation Mediated by O2-Arylated Bis-Diazeniumdiolate “Double JS-K”

PubMed Central

Holland, Ryan J.; Maciag, Anna E.; Kumar, Varun; Shi, Lei; Saavedra, Joseph E.; Prud’homme, Robert K.; Chakrapani, Harinath; Keefer, Larry K.

2012-01-01

Attachment of glutathione (GSH) to cysteine residues in proteins (S-glutathionylation) is a reversible post-translational modification that can profoundly alter protein structure and function. Often serving in a protective role, e.g., by temporarily saving protein thiols from irreversible oxidation and inactivation, glutathionylation can be identified and semi-quantitatively assessed using anti-GSH antibodies, thought to be specific for recognition of the S-glutathionylation modification. Here we describe an alternate mechanism of protein glutathionylation in which the sulfur atoms of the GSH and the protein’s thiol group are covalently bound via a crosslinking agent, rather than through a disulfide bond. This form of thiol crosslinking has been shown to occur and confirmed by mass spectrometry at the solution chemistry level, as well as in experiments documenting the potent antiproliferative activity of the bis-diazeniumdiolate Double JS-K in H1703 cells in vitro and in vivo. The modification is recognized by the anti-GSH antibody as if it were authentic S-glutathionylation, requiring mass spectrometry to distinguish between them. PMID:23106594

Silane surface modification for improved bioadhesion of esophageal stents

PubMed Central

Karakoy, Mert; Gultepe, Evin; Pandey, Shivendra; Khashab, Mouen A.; Gracias, David H.

2014-01-01

Stent migration occurs in 10-40% of patients who undergo placement of esophageal stents, with higher migration rates seen in those treated for benign esophageal disorders. This remains a major drawback of esophageal stent therapy. In this paper, we propose a new surface modification method to increase the adhesion between self-expandable metallic stents (SEMS) and tissue while preserving their removability. Taking advantage of the well-known affinity between epoxide and amine terminated silane coupling agents with amine and carboxyl groups that are abundant in proteins and related molecules in the human body; we modified the surfaces of silicone coated esophageal SEMS with these adhesive self-assembled monolayers (SAMs). We utilized vapor phase silanization to modify the surfaces of different substrates including PDMS strips and SEMS, and measured the force required to slide these substrates on a tissue piece. Our results suggest that surface modification of esophageal SEMS via covalent attachment of protein-binding coupling agents improves adhesion to tissue and could offer a solution to reduce SEMS migration while preserving their removability. PMID:25663731

Germline mutations affecting the histone H4 core cause a developmental syndrome by altering DNA damage response and cell cycle control.

PubMed

Tessadori, Federico; Giltay, Jacques C; Hurst, Jane A; Massink, Maarten P; Duran, Karen; Vos, Harmjan R; van Es, Robert M; Scott, Richard H; van Gassen, Koen L I; Bakkers, Jeroen; van Haaften, Gijs

2017-11-01

Covalent modifications of histones have an established role as chromatin effectors, as they control processes such as DNA replication and transcription, and repair or regulate nucleosomal structure. Loss of modifications on histone N tails, whether due to mutations in genes belonging to histone-modifying complexes or mutations directly affecting the histone tails, causes developmental disorders or has a role in tumorigenesis. More recently, modifications affecting the globular histone core have been uncovered as being crucial for DNA repair, pluripotency and oncogenesis. Here we report monoallelic missense mutations affecting lysine 91 in the histone H4 core (H4K91) in three individuals with a syndrome of growth delay, microcephaly and intellectual disability. Expression of the histone H4 mutants in zebrafish embryos recapitulates the developmental anomalies seen in the patients. We show that the histone H4 alterations cause genomic instability, resulting in increased apoptosis and cell cycle progression anomalies during early development. Mechanistically, our findings indicate an important role for the ubiquitination of H4K91 in genomic stability during embryonic development.

Hardening Mechanisms of Silicon Nanospheres: A Molecular Dynamics Study

DTIC Science & Technology

2011-05-01

in single oxide system 111 Figure 5.9 Dislocation motion in double oxide systems 112 x Figure 5.10 Dislocation response to incremental...addressed as no single dislocation loops were ever separated and no diffraction peaks indicative of the -Sn phase were observed. The load vs. displacement...as the diamond cubic structure has angle dependent covalent bonds. Therefore, other potentials have been 20 developed that model the

Computational prediction of methylation types of covalently modified lysine and arginine residues in proteins.

PubMed

Deng, Wankun; Wang, Yongbo; Ma, Lili; Zhang, Ying; Ullah, Shahid; Xue, Yu

2017-07-01

Protein methylation is an essential posttranslational modification (PTM) mostly occurs at lysine and arginine residues, and regulates a variety of cellular processes. Owing to the rapid progresses in the large-scale identification of methylation sites, the available data set was dramatically expanded, and more attention has been paid on the identification of specific methylation types of modification residues. Here, we briefly summarized the current progresses in computational prediction of methylation sites, which provided an accurate, rapid and efficient approach in contrast with labor-intensive experiments. We collected 5421 methyllysines and methylarginines in 2592 proteins from the literature, and classified most of the sites into different types. Data analyses demonstrated that different types of methylated proteins were preferentially involved in different biological processes and pathways, whereas a unique sequence preference was observed for each type of methylation sites. Thus, we developed a predictor of GPS-MSP, which can predict mono-, di- and tri-methylation types for specific lysines, and mono-, symmetric di- and asymmetrical di-methylation types for specific arginines. We critically evaluated the performance of GPS-MSP, and compared it with other existing tools. The satisfying results exhibited that the classification of methylation sites into different types for training can considerably improve the prediction accuracy. Taken together, we anticipate that our study provides a new lead for future computational analysis of protein methylation, and the prediction of methylation types of covalently modified lysine and arginine residues can generate more useful information for further experimental manipulation. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Polymer-based protein engineering grown ferrocene-containing redox polymers improve current generation in an enzymatic biofuel cell.

PubMed

Campbell, Alan S; Murata, Hironobu; Carmali, Sheiliza; Matyjaszewski, Krzysztof; Islam, Mohammad F; Russell, Alan J

2016-12-15

Enzymatic biofuel cells (EBFCs) are capable of generating electricity from physiologically present fuels making them promising power sources for the future of implantable devices. The potential application of such systems is limited, however, by inefficient current generation. Polymer-based protein engineering (PBPE) offers a unique method to tailor enzyme function through tunable modification of the enzyme surface with functional polymers. In this study, we report on the modification of glucose oxidase (GOX) with ferrocene-containing redox polymers to increase current generation efficiency in an enzyme-modified anode. Poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide) (pFcAc) was grown from covalently attached, water-soluble initiator molecules on the surface of GOX in a "grafting-from" approach using atom transfer radical polymerization (ATRP). The covalently-coupled ferrocene-containing polymers on the enzyme surface promoted the effective "wiring" of the GOX active site to an external electrode. The resulting GOX-pFcAc conjugates generated over an order of magnitude increase in current generation efficiency and a 4-fold increase in maximum EBFC power density (≈1.7µWcm(-2)) with similar open circuit voltage (0.27V) compared to native GOX when physically adsorbed onto paddle-shaped electrodes made up of electrospun polyacrylonitrile fibers coated with gold nanoparticles and multi-wall carbon nanotubes. The formation of electroactive enzyme-redox polymer conjugates using PBPE represents a powerful new tool for the improvement of mediated enzyme-based bioelectronics without the need for free redox mediators or anode/cathode compartmentalization. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Effect of malondialdehyde modification on the binding of aroma compounds to soy protein isolates.

PubMed

Wang, Juan; Zhao, Mouming; Qiu, Chaoying; Sun, Weizheng

2018-03-01

The interactions of soy protein isolate (SPI) and flavor compounds (hexanal, trans-2-hexenal, 1-octen-3-ol, trans-2-octenal, nonanal, and trans-2-nonenal) were investigated. The influence of SPI structure modified by malondialdehyde (MDA) and flavor compound structure on the interactions were determined by using headspace solid-phase microextraction (SPME) and gas chromatography (GC) combined with mass spectrometry (MS). The binding of native SPI to the flavor compounds decreased in the order trans-2-nonenal>nonanal>trans-2-octenal>trans-2-hexenal>hexanal>1-octen-3-ol. It might be attributed to that aldehydes are more hydrophobic than alcohols. The former is more conducive to hydrophobic binding with the SPI. Furthermore, the aldehydes, in particular trans-s-undecenal, could also react covalently. The effect of MDA modification on protein-flavor interactions depended on the structure of the flavor compound. Upon low concentration of MDA (≤1mM), the binding of all six flavors to SPI increased. However, a further increase in the extent of MDA (≥2.5mM), more soluble and even insoluble aggregates formed, which reduced the binding of hexanal and nonanal to SPI. The other four flavors with double bond revealed little changes in binding (trans-2-octenal, and trans-2-nonenal) or even an increase in binding (trans-2-hexenal, and 1-octen-3-ol). The results suggested that hydrophobic interactions were weakened upon high extent of oxidation, whereas covalent interactions were enhanced. Copyright © 2017 Elsevier Ltd. All rights reserved.

A reusable device for electrochemical applications of hydrogel supported black lipid membranes.

PubMed

Mech-Dorosz, Agnieszka; Heiskanen, Arto; Bäckström, Sania; Perry, Mark; Muhammad, Haseena B; Hélix-Nielsen, Claus; Emnéus, Jenny

2015-02-01

Black lipid membranes (BLMs) are significant in studies of membrane transport, incorporated proteins/ion transporters, and hence in construction of biosensor devices. Although BLMs provide an accepted mimic of cellular membranes, they are inherently fragile. Techniques are developed to stabilize them, such as hydrogel supports. In this paper, we present a reusable device for studies on hydrogel supported (hs) BLMs. These are formed across an ethylene tetrafluoroethylene (ETFE) aperture array supported by the hydrogel, which is during in situ polymerization covalently "sandwiched" between the ETFE substrate and a gold electrode microchip, thus allowing direct electrochemical studies with the integrated working electrodes. Using electrochemical impedance spectroscopy (EIS), X-ray photoelectron spectroscopy and contact angle measurements, we demonstrate the optimized chemical modifications of the gold electrode microchips and plasma modification of the ETFE aperture arrays facilitating covalent "sandwiching" of the hydrogel. Both fluorescence microscopy and EIS were used to demonstrate the induced spontaneous thinning of a deposited lipid solution, leading to formation of stabilized hsBLMs on average in 10 min. The determined specific membrane capacitance and resistance were shown to vary in the range 0.31-0.49 μF/cm(2) and 45-65 kΩ cm(2), respectively, corresponding to partially solvent containing BLMs with an average life time of 60-80 min. The characterized hsBLM formation and devised equivalent circuit models lead to a schematic model to illustrate lipid molecule distribution in hydrogel-supported apertures. The functionality of stabilized hsBLMs and detection sensitivity of the platform were verified by monitoring the effect of the ion transporter valinomycin.

Structural and Functional Modifications of Corneal Crystallin ALDH3A1 by UVB Light

PubMed Central

Estey, Tia; Chen, Ying; Carpenter, John F.; Vasiliou, Vasilis

2010-01-01

As one of the most abundantly expressed proteins in the mammalian corneal epithelium, aldehyde dehydrogenase 3A1 (ALDH3A1) plays critical and multifaceted roles in protecting the cornea from oxidative stress. Recent studies have demonstrated that one protective mechanism of ALDH3A1 is the direct absorption of UV-energy, which reduces damage to other corneal proteins such as glucose-6-phosphate dehydrogenase through a competition mechanism. UV-exposure, however, leads to the inactivation of ALDH3A1 in such cases. In the current study, we demonstrate that UV-light caused soluble, non-native aggregation of ALDH3A1 due to both covalent and non-covalent interactions, and that the formation of the aggregates was responsible for the loss of ALDH3A1 enzymatic activity. Spectroscopic studies revealed that as a result of aggregation, the secondary and tertiary structure of ALDH3A1 were perturbed. LysC peptide mapping using MALDI-TOF mass spectrometry shows that UV-induced damage to ALDH3A1 also includes chemical modifications to Trp, Met, and Cys residues. Surprisingly, the conserved active site Cys of ALDH3A1 does not appear to be affected by UV-exposure; this residue remained intact after exposure to UV-light that rendered the enzyme completely inactive. Collectively, our data suggest that the UV-induced inactivation of ALDH3A1 is a result of non-native aggregation and associated structural changes rather than specific damage to the active site Cys. PMID:21203538

Automated Solid-Phase Protein Modification with Integrated Enzymatic Digest for Reaction Validation: Application of a Compartmented Microfluidic Reactor for Rapid Optimization and Analysis of Protein Biotinylation

PubMed Central

Fraas, Regina; Diehm, Juliane; Franzreb, Matthias

2017-01-01

Protein modification by covalent coupling of small ligands or markers is an important prerequisite for the use of proteins in many applications. Well-known examples are the use of proteins with fluorescent markers in many in vivo experiments or the binding of biotinylated antibodies via biotin–streptavidin coupling in the frame of numerous bioassays. Multiple protocols were established for the coupling of the respective molecules, e.g., via the C and N-terminus, or via cysteines and lysines exposed at the protein surface. Still, in most cases the conditions of these standard protocols are only an initial guess. Optimization of the coupling parameters like reagent concentrations, pH, or temperature may strongly increase coupling yield and the biological activity of the modified protein. In order to facilitate the process of optimizing coupling conditions, a method was developed which uses a compartmented microfluidic reactor for the rapid screening of different coupling conditions. In addition, the system allows for the integration of an enzymatic digest of the modified protein directly after modification. In combination with a subsequent MALDI-TOF analysis of the resulting fragments, this gives a fast and detailed picture not only of the number and extent of the generated modifications but also of their position within the protein sequence. The described process was demonstrated for biotinylation of green fluorescent protein. Different biotin-excesses and different pH-values were tested in order to elucidate the influence on the modification extent and pattern. In addition, the results of solid-phase based modifications within the microfluidic reactor were compared to modification patterns resulting from coupling trials with unbound protein. As expected, modification patterns of immobilized proteins showed clear differences to the ones of dissolved proteins. PMID:29181376

Ubiquitinated Proteome: Ready for Global?*

PubMed Central

Shi, Yi; Xu, Ping; Qin, Jun

2011-01-01

Ubiquitin (Ub) is a small and highly conserved protein that can covalently modify protein substrates. Ubiquitination is one of the major post-translational modifications that regulate a broad spectrum of cellular functions. The advancement of mass spectrometers as well as the development of new affinity purification tools has greatly expedited proteome-wide analysis of several post-translational modifications (e.g. phosphorylation, glycosylation, and acetylation). In contrast, large-scale profiling of lysine ubiquitination remains a challenge. Most recently, new Ub affinity reagents such as Ub remnant antibody and tandem Ub binding domains have been developed, allowing for relatively large-scale detection of several hundreds of lysine ubiquitination events in human cells. Here we review different strategies for the identification of ubiquitination site and discuss several issues associated with data analysis. We suggest that careful interpretation and orthogonal confirmation of MS spectra is necessary to minimize false positive assignments by automatic searching algorithms. PMID:21339389

Monoaminylation of Fibrinogen and Glia-Derived Proteins: Indication for Similar Mechanisms in Posttranslational Protein Modification in Blood and Brain.

PubMed

Hummerich, René; Costina, Victor; Findeisen, Peter; Schloss, Patrick

2015-07-15

Distinct proteins have been demonstrated to be posttranslationally modified by covalent transamidation of serotonin (5-hydropxytryptamin) to glutamine residues of the target proteins. This process is mediated by transglutaminase (TGase) and has been termed "serotonylation." It has also been shown that other biogenic amines, including the neurotransmitters dopamine and norepinephrine, can substitute for serotonin, implying a more general mechanism of "monoaminylation" for this kind of protein modification. Here we transamidated the autofluorescent monoamine monodansylcadaverine (MDC) to purified plasma fibrinogen and to proteins from a primary glia cell culture. Electrophoretic separation of MDC-conjugated proteins followed by mass spectrometry identified three fibrinogen subunits (Aα, Bβ, γ), a homomeric Aα2 dimer, and adducts of >250 kDa molecular weight, as well as several glial proteins. TGase-mediated MDC incorporation was strongly reduced by serotonin, underlining the general mechanism of monoaminylation.

Post-Translational Modification Control of Innate Immunity.

PubMed

Liu, Juan; Qian, Cheng; Cao, Xuetao

2016-07-19

A coordinated balance between the positive and negative regulation of pattern-recognition receptor (PRR)-initiated innate inflammatory responses is required to ensure the most favorable outcome for the host. Post-translational modifications (PTMs) of innate sensors and downstream signaling molecules influence their activity and function by inducing their covalent linkage to new functional groups. PTMs including phosphorylation and polyubiquitination have been shown to potently regulate innate inflammatory responses through the activation, cellular translocation, and interaction of innate receptors, adaptors, and downstream signaling molecules in response to infectious and dangerous signals. Other PTMs such as methylation, acetylation, SUMOylation, and succinylation are increasingly implicated in the regulation of innate immunity and inflammation. In this review, we focus on the roles of PTMs in controlling PRR-triggered innate immunity and inflammatory responses. The emerging roles of PTMs in the pathogenesis and potential treatment of infectious and inflammatory immune diseases are also discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Ionic liquid-modified materials for solid-phase extraction and separation: a review.

PubMed

Vidal, Lorena; Riekkola, Marja-Liisa; Canals, Antonio

2012-02-17

In recent years, materials science has propelled to the research forefront. Ionic liquids with unique and fascinating properties have also left their footprints to the developments of materials science during the last years. In this review we highlight some of their recent advances and provide an overview at the current status of ionic liquid-modified materials applied in solid-phase extraction, liquid and gas chromatography and capillary electrochromatography with reference to recent applications. In addition, the potential of ionic liquids in the modification of capillary inner wall in capillary electrophoresis is demonstrated. The main target material modified with ionic liquids is silica, but polymers and monoliths have recently joined the studies. Although imidazolium is still clearly the most commonly used ionic liquid for the covalently modification of materials, the exploitation of pyridinium and phosphonium will most probably increase in the future. Copyright © 2011 Elsevier B.V. All rights reserved.

Functionalization of multi-walled carbon nanotubes with thermo-responsive azide-terminated poly(N-isopropylacrylamide) via click reactions.

PubMed

Su, Xin; Shuai, Ya; Guo, Zanru; Feng, Yujun

2013-04-18

Covalently functionalized multi-walled carbon nanotubes (MWNTs) were prepared by grafting well-defined thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) via click reactions. First, azide-terminated poly(N-isopropylacrylamide) (N3-PNIPAM) was synthesized by reversible addition fragmentation chain-transfer (RAFT) polymerization, and then the N₃-PNIPAM moiety was connected onto MWNTs by click chemistry. The products were characterized by means of FT-IR, TGA and TEM. The results show that the modification of MWNTs is very successful and MWNTs functionalized by N₃-PNIPAM (MWNTs-PNIPAM) have good solubility and stability in water. TEM images show the functionalized MWNTs are dispersed individually, indicating that the bundles of original MWNTs are separated into individual tubes by surface modification with polymer chains. These MWNTs modified with PNIPAM represent a potential nano-material for preparation of hydrophilic composite materials.

Regulation of parkin and PINK1 by neddylation

PubMed Central

Choo, Yeun Su; Vogler, Georg; Wang, Danling; Kalvakuri, Sreehari; Iliuk, Anton; Tao, W. Andy; Bodmer, Rolf; Zhang, Zhuohua

2012-01-01

Neddylation is a posttranslational modification that plays important roles in regulating protein structure and function by covalently conjugating NEDD8, an ubiquitin-like small molecule, to the substrate. Here, we report that Parkinson's disease (PD)-related parkin and PINK1 are NEDD8 conjugated. Neddylation of parkin and PINK1 results in increased E3 ligase activity of parkin and selective stabilization of the 55 kDa PINK1 fragment. Expression of dAPP-BP1, a NEDD8 activation enzyme subunit, in Drosophila suppresses abnormalities induced by dPINK1 RNAi. PD neurotoxin MPP+ inhibits neddylation of both parkin and PINK1. NEDD8 immunoreactivity is associated with Lewy bodies in midbrain dopaminergic neurons of PD patients. Together, these results suggest that parkin and PINK1 are regulated by neddylation and that impaired NEDD8 modification of these proteins likely contributes to PD pathogenesis. PMID:22388932

Extracellular Matrix and Redox Signaling in Cellular Responses to Stress.

PubMed

Roberts, David D

2017-10-20

Cells in multicellular organisms communicate extensively with neighboring cells and distant organs using a variety of secreted proteins and small molecules. Cells also reside in a structural extracellular matrix (ECM), and changes in its composition, mechanical properties, and post-translational modifications provide additional layers of communication. This Forum addresses emerging mechanisms by which redox signaling controls and is controlled by changes in the ECM, focusing on the roles of matricellular proteins. These proteins engage specific cell surface signaling receptors, integrins, and proteoglycans to regulate the biosynthesis and catabolism of redox signaling molecules and the activation of their signal transducers. These signaling pathways, in turn, regulate the composition of ECM and its function. Covalent post-translational modifications of ECM by redox molecules further regulate its structure and function. Recent studies of acute injuries and chronic disease have identified important pathophysiological roles for this cross-talk and new therapeutic opportunities. Antioxid. Redox Signal. 27, 771-773.

Resistive sensing of gaseous nitrogen dioxide using a dispersion of single-walled carbon nanotubes in an ionic liquid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mishra, Prabhash; Department of Nanoengineering, Samara State Aerospace University, 443086 Samara; Pavelyev, V.S.

2016-06-15

Graphical abstract: Ionic liquid ([C6-mim]PF6) used as dispersant agent for SWCNTs: An investigations were carried out to find the structural quality and surface modification for sensor application. - Highlights: • An effective technique based on Ionic liquids (IL) and their use as a dispersant. • Electron microscopy and spectroscopy for structure characterization. • Covalent linkage of ILs with SWNTs and dispersion of SWCNTs. • The IL-wrapped sensing film, capable for detecting trace levels of gas. - Abstract: Single-walled carbon nanotubes (SWCNTs) were dispersed in an imidazolium-based ionic liquid (IL) and investigated in terms of structural quality, surface functionalization and inter-CNTmore » force. Analysis by field emission electron microscopy and transmission electron microscopy shows the IL layer to coat the SWNTs, and FTIR and Raman spectroscopy confirm strong binding of the ILs to the SWNTs. Two kinds of resistive sensors were fabricated, one by drop casting of IL-wrapped SWCNTs, the other by conventional dispersion of SWCNTs. Good response and recovery to NO{sub 2} is achieved with the IL-wrapped SWCNTs material upon UV-light exposure, which is needed because decrease the desorption energy barrier to increase the gas molecule desorption. NO{sub 2} can be detected in the 1–20 ppm concentration range. The sensor is not interfered by humidity due to the hydrophobic tail of PF6 (ionic liquid) that makes our sensor highly resistant to moisture.« less

Covalent modification and time-dependent inhibition of human CYP2E1 by the meta-isomer of acetaminophen.

PubMed

Harrelson, John P; Stamper, Brendan D; Chapman, John D; Goodlett, David R; Nelson, Sidney D

2012-08-01

The hypothesis that N-acetyl-m-aminophenol (AMAP), the meta isomer of acetaminophen, will covalently bind to and inhibit human CYP2E1 in a time- and NADPH-dependent manner was investigated. Liquid chromatography/electrospray ionization-mass spectrometry analysis indicated that AMAP metabolites (i.e., AMAP*) selectively and covalently modified CYP2E1 apoprotein in a ratio of 1.4:1 (AMAP*/CYP2E1) in a reconstituted system. The deconvoluted spectra of CYP2E1 apoprotein from incubations containing NADPH and AMAP displayed mass shifts of 167.2 ± 7.1 and 334.4 ± 6.5 Da, suggesting the addition of one and two hydroxylated AMAP metabolites to CYP2E1, respectively. Mass shifts in cytochrome P450 reductase, cytochrome b(5), and heme from these samples were not observed. CYP2E1 inhibition by AMAP increased with time in the presence of NADPH; a reversible inhibition component was also observed. The results support a bioactivation process that involves formation of a hydroquinone metabolite that undergoes further oxidation to a quinone, which reacts with CYP2E1 nucleophilic residues. The data are consistent with evidence from previous studies that identified hydroxylated AMAP glutathione conjugates collected from mice and indicate that cysteine residues are the most likely sites for adduct formation. This study reports the first direct evidence of AMAP-derived hydroquinone metabolites bound to human CYP2E1.

Manipulative interplay of two adozelesin molecules with d(ATTAAT)₂achieving ligand-stacked Watson-Crick and Hoogsteen base-paired duplex adducts.

PubMed

Hopton, Suzanne R; Thompson, Andrew S

2011-05-17

Previous structural studies of the cyclopropapyrroloindole (CPI) antitumor antibiotics have shown that these ligands bind covalently edge-on into the minor groove of double-stranded DNA. Reversible covalent modification of the DNA via N3 of adenine occurs in a sequence-specific fashion. Early nuclear magnetic resonance and molecular modeling studies with both mono- and bis-alkylating ligands indicated that the ligands fit tightly within the minor groove, causing little distortion of the helix. In this study, we propose a new binding model for several of the CPI-based analogues, in which the aromatic secondary rings form π-stacked complexes within the minor groove. One of the adducts, formed with adozelesin and the d(ATTAAT)(2) sequence, also demonstrates the ability of these ligands to manipulate the DNA of the binding site, resulting in a Hoogsteen base-paired adduct. Although this type of base pairing has been previously observed with the bisfunctional CPI analogue bizelesin, this is the first time that such an observation has been made with a monoalkylating nondimeric analogue. Together, these results provide a new model for the design of CPI-based antitumor antibiotics, which also has a significant bearing on other structurally related and structurally unrelated minor groove-binding ligands. They indicate the dynamic nature of ligand-DNA interactions, demonstrating both DNA conformational flexibility and the ability of two DNA-bound ligands to interact to form stable covalent modified complexes.

Mapping a protein recognition centre with chiral photoactive ligands. An integrated approach combining photophysics, reactivity, proteomics and molecular dynamics simulation studies.

PubMed

Limones-Herrero, Daniel; Pérez-Ruiz, Raúl; Lence, Emilio; González-Bello, Concepción; Miranda, Miguel A; Jiménez, M Consuelo

2017-04-01

A multidisciplinary strategy to obtain structural information on the intraprotein region is described here. As probe ligands, ( S )- and ( R )- CPFMe (the methyl esters of the chiral drug carprofen) have been selected, while bovine α 1 -acid glycoprotein (BAAG) has been chosen as a biological host. The procedure involves the separate irradiation of the BAAG/( S )- CPFMe and BAAG/( R )- CPFMe complexes, coupled with fluorescence spectroscopy, laser flash photolysis, proteomic analysis, docking and molecular dynamics simulations. Thus, irradiation of the BAAG/ CPFMe complexes at λ = 320 nm was followed by fluorescence spectroscopy. The intensity of the emission band obtained after irradiation indicated photodehalogenation, whereas its structureless shape suggested covalent binding of the resulting radical CBZMe˙ to the biopolymer. After gel filtration chromatography, the spectra still displayed emission, in agreement with covalent attachment of CBZMe˙ to BAAG. Stereodifferentiation was observed in this process. After trypsin digestion and ESI-MS/MS, the incorporation of CBZMe was detected at Phe68. Docking and molecular dynamics simulation studies, which were carried out using a homology model of BAAG, reveal that the closer proximity of the aromatic moiety of the ( S )-enantiomer to the phenyl group of Phe68 would be responsible for the experimentally observed, more effective chemical modification of the protein. The proposed tridimensional structure of BAAG covalently modified by the two enantiomers is also provided. In principle, this approach can be extended to a variety of protein/ligand complexes.

Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.

PubMed

Mosqueira, V C; Legrand, P; Gulik, A; Bourdon, O; Gref, R; Labarre, D; Barratt, G

2001-11-01

The aim of our work was to examine the relationship between modifications of the surface of nanocapsules (NC) by adsorption or covalent grafting of poly(ethylene oxide) (PEG), and changes in their phospholipid (PL) content on complement activation (C3 cleavage) and on uptake by macrophages. The physicochemical characterization of the NC included an investigation of their properties, such as surface charge, size, hydrophilicity, morphology and homogeneity. This is the first time that such properties have been correlated with biological interactions for NC, a novel carrier system with a structure more complex than nanospheres. C3 crossed immunoelectrophoresis revealed the reduced activation for NC with longer PEG chain and higher density, although all formulations induced C3 cleavage to a lesser or greater extent. NC bearing PEG covalently bound to the surface were weaker activators of complement than plain PLA [poly(D,L-lactide)] NC or nanospheres (NS). Furthermore, the fluorescent/confocal microscopy of J774A1 cells in contact with NC reveal a dramatically reduced interaction with PEG-bearing NC. However, the way in which PEG was attached (covalent or adsorbed) seemed to affect the mechanism of uptake. Taken together, these results suggest that the low level of protein binding to NC covered with a high density of 20kDa PEG chains is likely to be due to the steric barriers surrounding these particles, which prevents protein adsorption and reduces their interaction with macrophages.

Role of composition, bond covalency, and short-range order in the disordering of stannate pyrochlores by swift heavy ion irradiation

NASA Astrophysics Data System (ADS)

Tracy, Cameron L.; Shamblin, Jacob; Park, Sulgiye; Zhang, Fuxiang; Trautmann, Christina; Lang, Maik; Ewing, Rodney C.

2016-08-01

A2S n2O7 (A =Nd ,Sm,Gd,Er,Yb,and Y) materials with the pyrochlore structure were irradiated with 2.2 GeV Au ions to systematically investigate disordering of this system in response to dense electronic excitation. Structural modifications were characterized, over multiple length scales, by transmission electron microscopy, x-ray diffraction, and Raman spectroscopy. Transformations to amorphous and disordered phases were observed, with disordering dominating the structural response of materials with small A -site cation ionic radii. Both the disordered and amorphous phases were found to possess weberite-type local ordering, differing only in that the disordered phase exhibits a long-range, modulated arrangement of weberite-type structural units into an average defect-fluorite structure, while the amorphous phase remains fully aperiodic. Comparison with the behavior of titanate and zirconate pyrochlores showed minimal influence of the high covalency of the Sn-O bond on this phase behavior. An analytical model of damage accumulation was developed to account for simultaneous amorphization and recrystallization of the disordered phase during irradiation.

Investigating the Non-Covalent Functionalization and Chemical Transformation of Graphene

NASA Astrophysics Data System (ADS)

Sham, Chun-Hong

Trend in device miniatures demands capabilities to produce rationally designed patterns in ever-shrinking length scale. The research community has examined various techniques to push the current lithography resolution to sub-10nm scale. One of the ideas is to utilize the natural nanoscale patterns of molecular assemblies. In this thesis, the self-assembling phenomenon of a photoactive molecule on epitaxial graphene (EG) grown on SiC was discussed. This molecular assembly enables manipulation of chemical contrast in nanoscale through UV exposure or atomic layer deposition. Future development of nanoelectronics industry will be fueled by innovations in electronics materials, which could be discovered through covalent modification of graphene. In a study reported in this thesis, silicon is deposited onto EG. After annealing, a new surface reconstruction, identified to be (3x3)-SiC, was formed. Raman spectroscopy finds no signature of graphene after annealing, indicating a complete chemical transformation of graphene. DFT calculations reveal a possible conversion mechanism. Overall, these studies provide insights for future device miniaturization; contribute to the search of novel materials and help bridging the gap between graphene and current silicon-based industrial infrastructures.

Comparative investigation of two methods for Acetylcholinesterase enzyme immobilization on modified porous silicon

NASA Astrophysics Data System (ADS)

Khaldi, Khadidja; Sam, Sabrina; Lounas, Amel; Yaddaden, Chafiaa; Gabouze, Noure-Eddine

2017-11-01

In this work, Acetylcholinesterase enzyme (AChE) was immobilized on porous silicon (PSi) surface using two strategies. In the first method, acid chains were covalently grafted on the hydrogenated PSi by hydrosilylation reaction. The obtained acid-terminated surface was activated by a reaction with N-hydroxysuccinimide (NHS) in the presence of a peptide-coupling agent N-ethyl-N‧-(3-dimethylaminopropyl)-carbodiimide (EDC), and then reacted with the amino linker of the lysine residues AChE to anchor the enzyme by a covalent amide bond. In the second procedure, the PSi surface was first hydroxylated in piranha solution, followed by a silanization reaction with 3-aminopropyltriethoxysilane (APTES) to form amine-terminated surface. Finally, AChE was attached to the terminal amine groups by an aminolysis reaction with carboxylic acid groups of AChE in the presence of NHS/EDC mixture. Fourier transform infrared spectroscopy (FTIR) confirmed the efficiency of the surface modifications. The enzymatic activity of immobilized AChE was determined by means of a colorimetric test and was discussed according to the enzyme orientation on the surface which was revealed by contact angle measurements.

Actin filaments – a target for redox regulation

PubMed Central

Wilson, Carlos; Terman, Jonathan R.; González-Billault, Christian; Ahmed, Giasuddin

2016-01-01

Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through non-covalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates – the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL – and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. PMID:27309342

Covalent Surface Modification of Silicon Oxides with Alcohols in Polar Aprotic Solvents.

PubMed

Lee, Austin W H; Gates, Byron D

2017-09-05

Alcohol-based monolayers were successfully formed on the surfaces of silicon oxides through reactions performed in polar aprotic solvents. Monolayers prepared from alcohol-based reagents have been previously introduced as an alternative approach to covalently modify the surfaces of silicon oxides. These reagents are readily available, widely distributed, and are minimally susceptible to side reactions with ambient moisture. A limitation of using alcohol-based compounds is that previous reactions required relatively high temperatures in neat solutions, which can degrade some alcohol compounds or could lead to other unwanted side reactions during the formation of the monolayers. To overcome these challenges, we investigate the condensation reaction of alcohols on silicon oxides carried out in polar aprotic solvents. In particular, propylene carbonate has been identified as a polar aprotic solvent that is relatively nontoxic, readily accessible, and can facilitate the formation of alcohol-based monolayers. We have successfully demonstrated this approach for tuning the surface chemistry of silicon oxide surfaces with a variety of alcohol containing compounds. The strategy introduced in this research can be utilized to create silicon oxide surfaces with hydrophobic, oleophobic, or charged functionalities.

Silica nanoparticles with a substrate switchable luminescence

NASA Astrophysics Data System (ADS)

Bochkova, O. D.; Mustafina, A. R.; Fedorenko, S. V.; Konovalov, A. I.

2011-04-01

Silica nanoparticles with visible (Tb and Ru doped), near IR (Yb doped) and dual visible-near IR luminescence (Ru-Yb doped) were obtained by reverse w/o microemulsion procedure. Plenty of luminescent complexes (from 4900 to 10000) encapsulated into each nanoparticle ensures the intensive luminescence of nanoparticles and their applicability as biomarkers. The silica surface decoration by definite anchor groups is the required step for the gaining to these nanoparticles marking and sensing functions. Thus covalent and non-covalent surface modification of these nanoparticles was developed to provide the binding with biotargets and sensing of anions. The dicationic surfactant coating of negatively charged Tb(III)-TCAS doped silica nanoparticles was chosen as the basis for the anion responsible system. The reversible insertion of the quenching anions (namely phenol red) into the surfactant based layer at the surface of luminescent nanoparticles switches off the Tb-centered luminescence. In turn the reversible reestablishment of the luminescence results from the competitive insertion of the non-quenching anions into the surfactant layer at the silica/water interface. The hydrophobic anions exemplified by dodecylsulfates versus hydrophilic ones (hydrophosphates) are preferable in the competition with phenol red anions.

Derivatizations of Sgc8-c aptamer to prepare metallic radiopharmaceuticals as imaging diagnostic agents: Syntheses, isolations, and physicochemical characterizations.

PubMed

Sicco, Estefanía; Báez, Jessica; Margenat, Jimena; García, Fernanda; Ibarra, Manuel; Cabral, Pablo; Moreno, María; Cerecetto, Hugo; Calzada, Victoria

2018-03-01

Aptamers, oligonucleotides with the capability to bind to a target through non-covalent bonds with high affinity and specificity, have a great number of advantages as scaffold to prepare molecular imaging agents. In this sense, we have performed post-SELEX modifications of a truncated aptamer, Sgc8-c, which bind to protein tyrosine kinase 7 to obtain a specific molecular targeting probe for in vivo diagnosis and in vivo therapy. Herein, we describe the synthetic efforts to prepare conjugates between Sgc8-c and different metallic ions chelator moieties in short times, high purities, and adequate yields. The selected chelator moieties, derived from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 2-benzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid, and 6-hydrazinonicotinic acid, were covalently attached at the 5'-aptamer position yielding the expected products which were stable in aqueous solution up to 75°C and in typical aptamer storage conditions at least for 30 days. © 2017 John Wiley & Sons A/S.

Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry.

PubMed

Sanderson, Patience; Stickney, Morgan; Leach, Franklin E; Xia, Qiangwei; Yu, Yanlei; Zhang, Fuming; Linhardt, Robert J; Amster, I Jonathan

2018-04-13

Reverse polarity capillary zone electrophoresis coupled to negative ion mode mass spectrometry (CZE-MS) is shown to be an effective and sensitive tool for the analysis of glycosaminoglycan mixtures. Covalent modification of the inner wall of the separation capillary with neutral or cationic reagents produces a stable and durable surface that provides reproducible separations. By combining CZE-MS with a cation-coated capillary and a sheath flow interface, a rapid and reliable method has been developed for the analysis of sulfated oligosaccharides from dp4 to dp12. Several different mixtures have been separated and detected by mass spectrometry. The mixtures were selected to test the capability of this approach to resolve subtle differences in structure, such as sulfation position and epimeric variation of the uronic acid. The system was applied to a complex mixture of heparin/heparan sulfate oligosaccharides varying in chain length from dp3 to dp12 and more than 80 molecular compositions were identified by accurate mass measurement. Copyright © 2018 Elsevier B.V. All rights reserved.

Spontaneous modification of graphite anode by anthraquinone-2-sulfonic acid for microbial fuel cells.

PubMed

Tang, Xinhua; Li, Haoran; Du, Zhuwei; Ng, How Yong

2014-07-01

In this study, anthraquinone-2-sulfonic acid (AQS), an electron transfer mediator, was immobilized onto graphite felt surface via spontaneous reduction of the in situ generated AQS diazonium cations. Cyclic voltammetry (CV) and energy dispersive spectrometry (EDS) characterizations of AQS modified graphite demonstrated that AQS was covalently grafted onto the graphite surface. The modified graphite, with a surface AQS concentration of 5.37 ± 1.15 × 10(-9)mol/cm(2), exhibited good electrochemical activity and high stability. The midpoint potential of the modified graphite was about -0.248 V (vs. normal hydrogen electrode, NHE), indicating that electrons could be easily transferred from NADH in bacteria to the electrode. AQS modified anode in MFCs increased the maximum power density from 967 ± 33 mW/m(2) to 1872 ± 42 mW/m(2). These results demonstrated that covalently modified AQS functioned as an electron transfer mediator to facilitate extracellular electron transfer from bacteria to electrode and significantly enhanced the power production in MFCs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Tomato apical stunt viroid - Data Sheet

USDA-ARS?s Scientific Manuscript database

Tomato apical stunt viroid (TASVd), a member of the family Pospiviroidae, genus Pospiviroid, is a small, covalently closed, circular single-stranded, highly base-paired RNA molecule that ranges in size from 362 to 364 nucleotides. Viroids do not encode peptides or proteins, and use host proteins for...

Optimized assembly and covalent coupling of single-molecule DNA origami nanoarrays.

PubMed

Gopinath, Ashwin; Rothemund, Paul W K

2014-12-23

Artificial DNA nanostructures, such as DNA origami, have great potential as templates for the bottom-up fabrication of both biological and nonbiological nanodevices at a resolution unachievable by conventional top-down approaches. However, because origami are synthesized in solution, origami-templated devices cannot easily be studied or integrated into larger on-chip architectures. Electrostatic self-assembly of origami onto lithographically defined binding sites on Si/SiO2 substrates has been achieved, but conditions for optimal assembly have not been characterized, and the method requires high Mg2+ concentrations at which most devices aggregate. We present a quantitative study of parameters affecting origami placement, reproducibly achieving single-origami binding at 94±4% of sites, with 90% of these origami having an orientation within ±10° of their target orientation. Further, we introduce two techniques for converting electrostatic DNA-surface bonds to covalent bonds, allowing origami arrays to be used under a wide variety of Mg2+-free solution conditions.

SPM for functional identification of individual biomolecules

NASA Astrophysics Data System (ADS)

Ros, Robert; Schwesinger, Falk; Padeste, Celestino; Plueckthun, Andreas; Anselmetti, Dario; Guentherodt, Hans-Joachim; Tiefenauer, Louis

1999-06-01

The identification of specific binding molecules is of increasing interest in the context of drug development based on combinatorial libraries. Scanning Probe Microscopy (SPM) is the method of choice to image and probe individual biomolecules on a surface. Functional identification of biomolecules is a first step towards screening on a single molecule level. As a model system we use recombinant single- chain Fv fragment (scFv) antibody molecules directed against the antigen fluorescein. The scFv's are covalently immobilized on a flat gold surface via the C-terminal cysteine, resulting in a high accessibility of the binding site. The antigen is immobilized covalently via a long hydrophilic spacer to the silicon nitride SPM-tip. This arrangement allows a direct measurement of binding forces. Thus, closely related antibody molecules differing in only one amino acid at their binding site could be distinguished. A novel SPM-software has been developed which combines imaging, force spectroscopic modes, and online analysis. This is a major prerequisite for future screening methods.

Non-covalent biofunctionalization of single-walled carbon nanotubes via biotin attachment by π-stacking interactions and pyrrole polymerization.

PubMed

Haddad, R; Cosnier, S; Maaref, A; Holzinger, M

2009-12-01

Single-walled carbon nanotubes were functionalized with biotin using either electropolymerization or formation of pi-stacking interactions for the construction of biosensors. Thanks to the high affinity of the avidin-biotin interactions, a biotinylated glucose oxidase (B-GOX) as a biomolecule model was immobilized on the biotinylated nanotubes. The influence of the biosensor configuration on their amperometric performances was investigated by changing the amount of nanotubes and the numbers of avidin/B-GOX layers. By increasing the amount of nanotube and avidin/B-GOX layers, both sensor setups show a perfect linear increase of immobilized enzymes reflecting a high reproducibility of our systems. The highest sensitivities (up to 5.2 mA M(-1) cm(-2)) and maximum current densities (up to 55 microA cm(-2)) were obtained using nanotube deposits modified by electrochemical coatings. In contrast, non-covalently functionalized biotin-nanotubes show a better permeability for the enzymatically generated hydrogen peroxide.

Methods of making non-covalently bonded carbon-titania nanocomposite thin films and applications of the same

DOEpatents

Liang, Yu Teng; Vijayan, Baiju K.; Gray, Kimberly A.; Hersam, Mark C.

2016-07-19

In one aspect, a method of making non-covalently bonded carbon-titania nanocomposite thin films includes: forming a carbon-based ink; forming a titania (TiO.sub.2) solution; blade-coating a mechanical mixture of the carbon-based ink and the titania solution onto a substrate; and annealing the blade-coated substrate at a first temperature for a first period of time to obtain the carbon-based titania nanocomposite thin films. In certain embodiments, the carbon-based titania nanocomposite thin films may include solvent-exfoliated graphene titania (SEG-TiO.sub.2) nanocomposite thin films, or single walled carbon nanotube titania (SWCNT-TiO.sub.2) nanocomposite thin films.

RUNX family members are covalently modified and regulated by PIAS1-mediated sumoylation

PubMed Central

Kim, J-H; Jang, J-W; Lee, Y-S; Lee, J-W; Chi, X-Z; Li, Y-H; Kim, M-K; Kim, D-M; Choi, B-S; Kim, J; Kim, H-M; van Wijnen, A; Park, IlY; Bae, S-C

2014-01-01

Transcription factors of the RUNX family (RUNXs), which play pivotal roles in normal development and neoplasia, are regulated by various post-translational modifications. To understand the molecular mechanisms underlying the regulation of RUNXs, we performed a large-scale functional genetic screen of a fly mutant library. The screen identified dPias (the fly ortholog of mammalian PIASs), an E3 ligase for the SUMO (small ubiquitin-like modifier) modification, as a novel genetic modifier of lz (the fly ortholog of mammalian RUNX3). Molecular biological analysis revealed that lz/RUNXs are sumoylated by dPias/PIAS1 at an evolutionarily conserved lysine residue (K372 of lz, K144 of RUNX1, K181 of RUNX2 and K148 of RUNX3). PIAS1-mediated sumoylation inhibited RUNX3 transactivation activity, and this modification was promoted by the AKT1 kinase. Importantly, PIAS1 failed to sumoylate some RUNX1 mutants associated with breast cancer. In nude mice, tumorigenicity was promoted by RUNX3 bearing a mutation in the sumoylation site, but suppressed by wild-type RUNX3. Our results suggest that RUNXs are sumoylated by PIAS1, and that this modification could play a critical role in the regulation of the tumor-suppressive activity of these proteins. PMID:24777122

Two supramolecular complexes based on polyoxometalates and Co-EDTA units via covalent connection or non-covalent interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teng, Chunlin; Xiao, Hanxi; Cai, Qing

Two new 3D network organic-inorganic hybrid supramolecular complexes ([Na{sub 6}(CoEDTA){sub 2}(H{sub 2}O){sub 13}]·(H{sub 2}SiW{sub 12}O{sub 40})·xH{sub 2}O)n (1) and [CoH{sub 4}EDTA(H{sub 2}O)]{sub 2}(SiW{sub 12}O{sub 40})·15H{sub 2}O (2) (H{sub 4}EDTA=Ethylenediamine tetraacetic acid) have been successfully synthesized by solution method, and characterized by infrared spectrum (IR), thermogravimetric-differential thermal analysis (TG-DTA), cyclic voltammetry (CV) and single{sup −}crystal X-ray diffraction (XRD). Both of the complexes are the supramolecules, but with different liking mode, they are two representative models of supramolecule. complex (1) is a 3D infinite network supramolecular coordination polymer with a rare multi-metal sturcture of sodium-cobalt-containing, which is mainly linked through coordinate-covalent bonds.more » While complex (2) is normal supramolecule, which linked by non-covalent interactions, such as H-bonding interaction, electrostatic interaction and van der waals force. Both of complex (1) and (2) exhibit good catalytic activities for catalytic oxidation of methanol, when the initial concentration of methanol is 3.0 g m{sup −3}, flow rate is 10 mL min{sup −1}, and the quality of catalyst is 0.2 g, for complex (1) and complex (2) the maximum elimination rates of methanol are 85% (150 °C) and 92% (120 °C), respectively. - Graphical abstract: Two new organic-inorganic hybrid supramolecular complexes based on Co-EDTA, and Keggin polyanions have been successfully synthesized with different pH value by solution method. They are attributed to two representative models of supramolecule. Complex(1) is an infinite coordination polymer with a rare multi-metal sturcture of sodium-cobalt-containing, which is mainly linked through covalent bonds. Complex (2) is a normal supramolecule, which linked by non-covalent interactions of H-bonding interaction, electrostatic interaction and van der waals force. - Highlights: • Two supramolecules are linked by covalent or non-covalent interactions. • They are attributed to two representative models of supramolecule. • A rare multi-metal infinite supramolecular coordination polymer was formed. • They exhibit good catalytic activities for catalytic oxidation of methanol.« less

Final Report: DOE Award Number: DE-SC0006398, University of CA, San Diego

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cha, Jennifer

The focus of the proposed research is to direct the assembly of single or binary nanoparticles into meso- or macroscale three-dimensional crystals of any desired configuration and crystallographic orientation without using prohibitively expensive lithographic processes. The epitaxial nucleation of defect-free, surface-bound bulk single crystals will revolutionize technologies for energy to generate new types of solar cells that yield maximum conversion efficiencies. It has been proposed that having a nanostructured bulk hetero-interface will enable efficient charge-carrier separations, similar to organic based heterojunction cells but with potential improvements, including thermal and long-term stability, tunability of energy levels, large adsorption coefficients and carriermore » multiplication. However, engineering such devices requires nanoscale control and ordering in both 2- and 3-dimensions over macroscopic areas and this has yet to be achieved. In Nature, bulk organic and inorganic materials are arranged into precise and ordered programmed assemblies through the sequestration of raw materials into confined spaces and association through highly specific non-covalent interactions between biomolecules. Using similar strategies, the proposed research will focus on confining metal and semiconductor nanocrystals to pre-determined surface patterns and controlling their arrangement through tunable, orthogonal biomolecular binding. Once a perfect two-dimensional seed layer has been constructed, successive layers of single nanocrystals will be nucleated epitaxially with long-range order and tunable crystallographic orientations. The proposed research exploits the ability of biomolecules to bind specific targets in a tunable, orthogonal, multivalent, and reversible manner to the arrangements of DNA-nanoparticle conjugates on chemically defined surfaces. Through careful balance of the attractive and repulsive forces between the particles, the array, and the outside surface, it is envisioned that single or mixed nanoparticles can be packed to adopt uniform crystal orientation in two and three dimensions from simple mixing and annealing of biomolecule-nanoparticle conjugates with biomolecule-stamped surfaces. To control the crystallographic alignment of each particle with its neighbors, the nanoparticles will be assembled using a mixture of non-covalent biomolecular interactions. To create solar cells in which layers of donor and acceptor nanocrystals that are not only oriented normal to the top and bottom electrodes but are also arranged in a checkerboard pattern, multicomponent nanocrystals (e.g. CdSe, CdTe) will be conjugated with biochemical linkers such that only interactions between the CdTe and CdSe promote particle packing within the array. The proposed research will: (1) elucidate the role of single and binary cooperative particle-DNA interactions in influencing nanoparticle crystallographic orientation in two and three dimensions; (2) understand how confinement of nanoparticles on patterned arrays of biomolecules and modification of the surrounding substrate can nucleate long-range order over macroscopic areas via predefined grain boundaries; and (3) synthesize and characterize DNA conjugated semiconductor nanocrystals and assemble them into 2- and 3-D binary superlattice arrays for photovoltaics.« less

Functionalization of single-walled carbon nanotubes regulates their effect on hemostasis

NASA Astrophysics Data System (ADS)

Sokolov, A. V.; Aseychev, A. V.; Kostevich, V. A.; Gusev, A. A.; Gusev, S. A.; Vlasova, I. I.

2011-04-01

Applications of single-walled carbon nanotubes (SWNTs) in medical field imply the use of drug-coupled carbon nanotubes as well as carbon nanotubes functionalized with different chemical groups that change nanotube surface properties and interactions between nanotubes and cells. Covalent attachment of polyethylene glycol (PEG) to carboxylated single-walled carbon nanotubes (c-SWNT) is known to prevent the nanotubes from interaction with macrophages. Here we characterized nanotube's ability to stimulate coagulation processes in platelet-poor plasma (PPP), and evaluated the effect of SWNTs on platelet aggregation in platelet-rich plasma (PRP). Our study showed that PEG-SWNT did not affect the rate of clotting in PPP, while c-SWNT shortened the clot formation time five times compared to the control PPP. Since c-SWNT failed to accelerate coagulation in plasma lacking coagulation factor XI, it may be suggested that c-SWNT affects the contact activation pathway. In PRP, platelets responded to both SWNT types with irreversible aggregation, as evidenced by changes in the aggregate mean radius. However, the rate of aggregation induced by c-SWNT was two times higher than it was with PEG-SWNT. Cytological analysis also showed that c-SWNT was two times more efficient when compared to PEG-SWNT in aggregating platelets in PRP. Taken together, our results show that functionalization of nanoparticles can diminish their negative influence on blood cells. As seen from our data, modification of c-SWNT with PEG, when only a one percent of carbon atoms is bound to polymer (70 wt %), decreased the nanotube-induced coagulation in PRP and repelled the accelerating effect on the coagulation in PPP. Thus, when functionalized SWNTs are used for administration into bloodstream of laboratory animals, their possible pro-coagulant and pro-aggregating properties must be taken into account.

Permuting the PGF Signature Motif Blocks both Archaeosortase-Dependent C-Terminal Cleavage and Prenyl Lipid Attachment for the Haloferax volcanii S-Layer Glycoprotein.

PubMed

Abdul Halim, Mohd Farid; Karch, Kelly R; Zhou, Yitian; Haft, Daniel H; Garcia, Benjamin A; Pohlschroder, Mechthild

2015-12-28

For years, the S-layer glycoprotein (SLG), the sole component of many archaeal cell walls, was thought to be anchored to the cell surface by a C-terminal transmembrane segment. Recently, however, we demonstrated that the Haloferax volcanii SLG C terminus is removed by an archaeosortase (ArtA), a novel peptidase. SLG, which was previously shown to be lipid modified, contains a C-terminal tripartite structure, including a highly conserved proline-glycine-phenylalanine (PGF) motif. Here, we demonstrate that ArtA does not process an SLG variant where the PGF motif is replaced with a PFG motif (slg(G796F,F797G)). Furthermore, using radiolabeling, we show that SLG lipid modification requires the PGF motif and is ArtA dependent, lending confirmation to the use of a novel C-terminal lipid-mediated protein-anchoring mechanism by prokaryotes. Similar to the case for the ΔartA strain, the growth, cellular morphology, and cell wall of the slg(G796F,F797G) strain, in which modifications of additional H. volcanii ArtA substrates should not be altered, are adversely affected, demonstrating the importance of these posttranslational SLG modifications. Our data suggest that ArtA is either directly or indirectly involved in a novel proteolysis-coupled, covalent lipid-mediated anchoring mechanism. Given that archaeosortase homologs are encoded by a broad range of prokaryotes, it is likely that this anchoring mechanism is widely conserved. Prokaryotic proteins bound to cell surfaces through intercalation, covalent attachment, or protein-protein interactions play critical roles in essential cellular processes. Unfortunately, the molecular mechanisms that anchor proteins to archaeal cell surfaces remain poorly characterized. Here, using the archaeon H. volcanii as a model system, we report the first in vivo studies of a novel protein-anchoring pathway involving lipid modification of a peptidase-processed C terminus. Our findings not only yield important insights into poorly understood aspects of archaeal biology but also have important implications for key bacterial species, including those of the human microbiome. Additionally, insights may facilitate industrial applications, given that photosynthetic cyanobacteria encode uncharacterized homologs of this evolutionarily conserved enzyme, or may spur development of unique drug delivery systems. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Preparation and Characterization of Single Ion Conductors from High Surface Area Fumed Silica

NASA Technical Reports Server (NTRS)

Zhang, H.; Maitra, P.; Liu, B.; Wunder, S. L.; Lin, H.-P.; Salomon, M.; Hagedorn, Norman H. (Technical Monitor)

2002-01-01

Anions that can form dissociative salts with Li(+) have been prepared and covalently attached to high surface area fumed silica. When blended with polyethylene oxide (PEO), the functionalized fumed silica suppresses the crystallization of the PEO, provides dimensional stability, and serves as a single ion conductor. Since functionalized fumed silica is easily dispersed in common polar solvents, it can be incorporated in both the polymer electrolyte and the electrodes.

Impact of acid and oxidative modifications, single or dual, of sorghum starch on biodegradable films.

PubMed

Biduski, Bárbara; Silva, Francine Tavares da; Silva, Wyller Max da; Halal, Shanise Lisie de Mello El; Pinto, Vania Zanella; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa

2017-01-01

The objective of this study was to evaluate the effects of acid and oxidation modifications on sorghum starch, as well as the effect of dual modification of starch on the physical, morphological, mechanical, and barrier properties of biodegradable films. The acid modification was performed with 3% lactic acid and the oxidation was performed with 1.5% active chlorine. For dual modification, the acid modification was performed first, followed by oxidation under the same conditions as above. Both films of the oxidized starches, single and dual, had increased stiffness, providing a higher tensile strength and lower elongation when compared to films based on native and single acid modified starches. However, the dual modification increased the water vapor permeability of the films without changing their solubility. The increase in sorghum starch concentration in the filmogenic solution increased the thickness, water vapor permeability, and elongation of the films. Copyright © 2016. Published by Elsevier Ltd.

Experimental evidence of zone-center optical phonon softening by accumulating holes in thin Ge

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kabuyanagi, Shoichi; Nishimura, Tomonori; Yajima, Takeaki

2016-01-15

We discuss the impact of free carriers on the zone-center optical phonon frequency in germanium (Ge). By taking advantage of the Ge-on-insulator structure, we measured the Raman spectroscopy by applying back-gate bias. Phonon softening by accumulating holes in Ge film was clearly observed. This fact strongly suggests that the phonon softening in heavily-doped Ge is mainly attributed to the free carrier effect rather than the dopant atom counterpart. Furthermore, we propose that the free carrier effect on phonon softening is simply understandable from the viewpoint of covalent bonding modification by free carriers.

Photoactuation behavior of styrene-b-isoprene-b-styrene filled with covalently modified carbon nanotubes

NASA Astrophysics Data System (ADS)

Mosnáček, Jaroslav; Ilčíková, Markéta; Chorvát, Dušan; Czaniková, Klaudia; Krupa, Igor

2012-07-01

Styrene-b-isoprene-b-styrene (Kraton) was used as polymer matrix for preparation of multiwall carbon nanotubes (MWCNT) based nanocomposites. In order to suppress aggregation of the he carbon nanotubes and to improve the interations with the Kraton matrix, the MWCNT were modified with cholesteryl molecules and/or polystyrene chains. The effect of the modification on the composite materials was evaluated by using DMTA. The nanocomposite materials were thermoformed to achieve Braille text elements and their elastic response to light (photoactuation) was tested by atomic force microscopy in a contact mode.

Rapid step-gradient purification of mitochondrial DNA.

PubMed

Welter, C; Meese, E; Blin, N

1988-01-01

A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5'-end labeling, gel retention assays, and various types of hybridization.

[Regulation of thermal stability of enzymes by changing the composition of media. Native and modified alpha-chymotrypsin].

PubMed

Levitskiĭ, V Iu; Melik-Nubarov, N S; Slepnev, V I; Shikshnis, V A; Mozhaev, V V

1990-01-01

Stabilizing effect of denaturing salts on irreversible thermoinactivation of native and modified alpha-chymotrypsin at elevated temperatures is observed. The effect is caused by a shift of conformational equilibrium, at the primary step of reversible unfolding in the course of thermoinactivation, to a more unfolded form which is not able to refold "incorrectly". The stability of alpha-chymotrypsin is regulated within a wide range by medium alteration: the stabilizing effects are similar to those achieved by multipoint attachment of the enzyme to a support or by hydrophilization of protein by covalent modification.

From Genes to Protein Mechanics on a Chip

PubMed Central

Milles, Lukas F.; Verdorfer, Tobias; Pippig, Diana A.; Nash, Michael A.; Gaub, Hermann E.

2014-01-01

Single-molecule force spectroscopy enables mechanical testing of individual proteins, however low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip protein expression and measurement of single-molecule mechanical properties. We constructed microarrays of proteins covalently attached to a chip surface, and found that a single cohesin-modified cantilever that bound to the terminal dockerin-tag of each protein remained stable over thousands of pulling cycles. The ability to synthesize and mechanically probe protein libraries presents new opportunities for high-throughput mechanical phenotyping. PMID:25194847

Agonist trapped in ATP-binding sites of the P2X2 receptor

PubMed Central

Jiang, Ruotian; Lemoine, Damien; Martz, Adeline; Taly, Antoine; Gonin, Sophie; Prado de Carvalho, Lia; Specht, Alexandre; Grutter, Thomas

2011-01-01

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent “tethering” reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel. PMID:21576497

Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

PubMed

Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

2016-04-01

An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay. © The Author(s) 2016.

Synthesis of 1D-glyconanomaterials by a hybrid noncovalent-covalent functionalization of single wall carbon nanotubes: a study of their selective interactions with lectins and with live cells.

PubMed

Pernía Leal, M; Assali, M; Cid, J J; Valdivia, V; Franco, J M; Fernández, I; Pozo, D; Khiar, N

2015-12-07

To take full advantage of the remarkable applications of carbon nanotubes in different fields, there is a need to develop effective methods to improve their water dispersion and biocompatibility while maintaining their physical properties. In this sense, current approaches suffer from serious drawbacks such as loss of electronic structure together with low surface coverage in the case of covalent functionalizations, or instability of the dynamic hybrids obtained by non-covalent functionalizations. In the present work, we examined the molecular basis of an original strategy that combines the advantages of both functionalizations without their main drawbacks. The hierarchical self-assembly of diacetylenic-based neoglycolipids into highly organized and compacted rings around the nanotubes, followed by photopolymerization leads to the formation of nanotubes covered with glyconanorings with a shish kebab-type topology exposing the carbohydrate ligands to the water phase in a multivalent fashion. The glyconanotubes obtained are fully functional, and able to establish specific interactions with their cognate receptors. In fact, by taking advantage of this selective binding, an easy method to sense lectins as a working model of toxin detection was developed based on a simple analysis of TEM images. Remarkably, different experimental settings to assess cell membrane integrity, cell growth kinetics and cell cycle demonstrated the cellular biocompatibility of the sugar-coated carbon nanotubes compared to pristine single-walled carbon nanotubes.

Caenorhabditis elegans syndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva.

PubMed

Minniti, Alicia N; Labarca, Mariana; Hurtado, Claudia; Brandan, Enrique

2004-10-01

In Caenorhabditis elegans, the identification of many enzymes involved in the synthesis and modification of glycosaminoglycans (GAGs), essential components of proteoglycans, has attained special attention in recent years. Mutations in all the genes that encode for GAG biosynthetic enzymes show defects in the development of the vulva, specifically in the invagination of the vulval epithelium. Mutants for certain heparan sulfate modifying enzymes present axonal and cellular guidance defects in specific neuronal classes. Although most of the enzymes involved in the biosynthesis and modification of heparan sulfate have been characterized in C. elegans, little is known regarding the core proteins to which these GAGs covalently bind in proteoglycans. A single syndecan homologue (sdn-1) has been identified in the C. elegans genome through sequence analysis. In the present study, we show that C. elegans synthesizes sulfated proteoglycans, seen as three distinct species in western blot analysis. In the sdn-1 (ok449) deletion mutant allele we observed the lack of one species, which corresponds to a 50 kDa product after heparitinase treatment. The expression of sdn-1 mRNA and sequencing revealed that sdn-1 (ok449) deletion mutants lack two glycosylation sites. Hence, the missing protein in the western blot analysis probably corresponds to SDN-1. In addition, we show that SDN-1 localizes to the C. elegans nerve ring, nerve cords and to the vulva. SDN-1 is found specifically phosphorylated in nerve ring neurons and in the vulva, in both wild-type worms and sdn-1 (ok449) deletion mutants. These mutants show a defective egg-laying phenotype. Our results show for the first time, the identification, localization and some functional aspects of syndecan in the nematode C. elegans.

Surface modification of CoCr alloy using varying concentrations of phosphoric and phosphonoacetic acids: albumin and fibrinogen adsorption, platelet adhesion, activation, and aggregation studies.

PubMed

Thiruppathi, Eagappanath; Larson, Mark K; Mani, Gopinath

2015-01-01

CoCr alloy is commonly used in various cardiovascular medical devices for its excellent physical and mechanical properties. However, the formation of blood clots on the alloy surfaces is a serious concern. This research is focused on the surface modification of CoCr alloy using varying concentrations (1, 25, 50, 75, and 100 mM) of phosphoric acid (PA) and phosphonoacetic acid (PAA) to generate various surfaces with different wettability, chemistry, and roughness. Then, the adsorption of blood plasma proteins such as albumin and fibrinogen and the adhesion, activation, and aggregation of platelets with the various surfaces generated were investigated. Contact angle analysis showed PA and PAA coatings on CoCr provided a gradient of hydrophilic surfaces. FTIR showed PA and PAA were covalently bound to CoCr surface and formed different bonding configurations depending on the concentrations of coating solutions used. AFM showed the formation of homogeneous PA and PAA coatings on CoCr. The single and dual protein adsorption studies showed that the amount of albumin and fibrinogen adsorbed on the alloy surfaces strongly depend on the type of PA and PAA coatings prepared by different concentrations of coating solutions. All PA coated CoCr showed reduced platelet adhesion and activation when compared to control CoCr. Also, 75 and 100 mM PA-CoCr showed reduced platelet aggregation. For PAA coated CoCr, no significant difference in platelet adhesion and activation was observed between PAA coated CoCr and control CoCr. Thus, this study demonstrated that CoCr can be surface modified using PA for potentially reducing the formation of blood clots and improving the blood compatibility of the alloy.

Construction of a Hierarchical Architecture of Covalent Organic Frameworks via a Postsynthetic Approach.

PubMed

Zhang, Gen; Tsujimoto, Masahiko; Packwood, Daniel; Duong, Nghia Tuan; Nishiyama, Yusuke; Kadota, Kentaro; Kitagawa, Susumu; Horike, Satoshi

2018-02-21

Covalent organic frameworks (COFs) represent an emerging class of crystalline porous materials that are constructed by the assembly of organic building blocks linked via covalent bonds. Several strategies have been developed for the construction of new COF structures; however, a facile approach to fabricate hierarchical COF architectures with controlled domain structures remains a significant challenge, and has not yet been achieved. In this study, a dynamic covalent chemistry (DCC)-based postsynthetic approach was employed at the solid-liquid interface to construct such structures. Two-dimensional imine-bonded COFs having different aromatic groups were prepared, and a homogeneously mixed-linker structure and a heterogeneously core-shell hollow structure were fabricated by controlling the reactivity of the postsynthetic reactions. Solid-state nuclear magnetic resonance (NMR) spectroscopy and transmission electron microscopy (TEM) confirmed the structures. COFs prepared by a postsynthetic approach exhibit several functional advantages compared with their parent phases. Their Brunauer-Emmett-Teller (BET) surface areas are 2-fold greater than those of their parent phases because of the higher crystallinity. In addition, the hydrophilicity of the material and the stepwise adsorption isotherms of H 2 O vapor in the hierarchical frameworks were precisely controlled, which was feasible because of the distribution of various domains of the two COFs by controlling the postsynthetic reaction. The approach opens new routes for constructing COF architectures with functionalities that are not possible in a single phase.

Survey of diagnostic tools for detection of viroids and impacts of test results on the seed industry

USDA-ARS?s Scientific Manuscript database

Viroids are unencapsidated, single-stranded, covalently closed circular, highly structured noncoding RNAs of 239 – 401 nucleotides that are replicated by host enzymes and cause disease in several economically important crop plants. Although viroids are primarily and easily transmitted mechanically t...

Potato spindle tuber viroid detection in phloem exudates and guttation fluid of tomato plants (Solanum lycopersicum)

USDA-ARS?s Scientific Manuscript database

Potato spindle tuber viroid (PSTVd) is a single-stranded, non protein-encoding, covalently-closed circular RNA molecule (359nt) that infects many horticultural and agricultural crops. PSTVd is mechanically transmitted, replicates in the nucleus, and moves cell-to-cell through plasmodesmata. Though i...

Effect of Hyaluronic Acid Incorporation Method on the Stability and Biological Properties of Polyurethane-Hyaluronic Acid Biomaterials

PubMed Central

Ruiz, Amaliris; Rathnam, Kashmila R.; Masters, Kristyn S.

2014-01-01

The high failure rate of small diameter vascular grafts continues to drive the development of new materials and modification strategies that address this clinical problem, with biomolecule incorporation typically achieved via surface-based modification of various biomaterials. In this work, we examined whether the method of biomolecule incorporation (i.e., bulk vs. surface modification) into a polyurethane (PU) polymer impacted biomaterial performance in the context of vascular applications. Specifically, hyaluronic acid (HA) was incorporated into a poly(ether urethane) via bulk copolymerization or covalent surface tethering, and the resulting PU-HA materials characterized with respect to both physical and biological properties. Modification of PU with HA by either surface or bulk methods yielded materials that, when tested under static conditions, possessed no significant differences in their ability to resist protein adsorption, platelet adhesion, and bacterial adhesion, while supporting endothelial cell culture. However, only bulk-modified PU-HA materials were able to fully retain these characteristics following material exposure to flow, demonstrating a superior ability to retain the incorporated HA and minimize enzymatic degradation, protein adsorption, platelet adhesion, and bacterial adhesion. Thus, despite bulk methods rarely being implemented in the context of biomolecule attachment, these results demonstrate improved performance of PU-HA upon bulk, rather than surface, incorporation of HA. Although explored only in the context of PU-HA, the findings revealed by these experiments have broader implications for the design and evaluation of vascular graft modification strategies. PMID:24276670

Oriented Covalent Organic Framework Film on Graphene for Robust Ambipolar Vertical Organic Field-Effect Transistor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Bing; Zhu, Chen-Hui; Liu, Yi

Periodically eclipsed π-stacking columns in two-dimensional covalent organic frameworks (2D COFs) could function as direct channel paths for charge carrier transport. Incorporating a welldefined 2D COF into organic electronic devices, however, is still a challenge. Herein, we reported the solvothermal synthesis of a COF TFPy-PPDA film on single layer graphene (SLG), which was constructed via covalent imine-type linkage by employing 1,3,6,8-tetrakis(p-formylphenyl)pyrene (TFPy) and p-phenylenediamine (PPDA) as building blocks. A vertical field-effect transistor (VFET) based on the heterostructure of COF TFPy-PPDA film and SLG shows ambipolar charge carrier behavior under lower modulating voltages. Work-function-tunable contact between SLG and COFTFPy-PPDA film andmore » suitable injection barriers of charge carriers lead to the ambipolar transport with high current density on/off ratio (>10 5) and high on-current density (>4.1 Acm -2). Interfacing 2D COF with graphene for VFET could shed the promising application prospect of 2D COFs in organic electronics and optoelectronics.« less

Biochemical transformation of lignin for deriving valued commodities from lignocellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gall, Daniel L.; Ralph, John; Donohue, Timothy J.

The biochemical properties of lignin present major obstacles to deriving societally beneficial entities from lignocellulosic biomass, an abundant and renewable feedstock. Similar to other biopolymers such as polysaccharides, polypeptides, and ribonucleic acids, lignin polymers are derived from multiple types of monomeric units. However, lignin’s renowned recalcitrance is largely attributable to its racemic nature and the variety of covalent inter-unit linkages through which its aromatic monomers are linked. Indeed, unlike other biopolymers whose monomers are consistently inter-linked by a single type of covalent bond, the monomeric units in lignin are linked via non-enzymatic, combinatorial radical coupling reactions that give rise tomore » a variety of inter-unit covalent bonds in mildly branched racemic polymers. Yet, despite the chemical complexity and stability of lignin, significant strides have been made in recent years to identify routes through which valued commodities can be derived from it. This paper discusses emerging biological and biochemical means through which degradation of lignin to aromatic monomers can lead to the derivation of commercially valuable products.« less

Biochemical transformation of lignin for deriving valued commodities from lignocellulose

DOE PAGES

Gall, Daniel L.; Ralph, John; Donohue, Timothy J.; ...

2017-03-24

The biochemical properties of lignin present major obstacles to deriving societally beneficial entities from lignocellulosic biomass, an abundant and renewable feedstock. Similar to other biopolymers such as polysaccharides, polypeptides, and ribonucleic acids, lignin polymers are derived from multiple types of monomeric units. However, lignin’s renowned recalcitrance is largely attributable to its racemic nature and the variety of covalent inter-unit linkages through which its aromatic monomers are linked. Indeed, unlike other biopolymers whose monomers are consistently inter-linked by a single type of covalent bond, the monomeric units in lignin are linked via non-enzymatic, combinatorial radical coupling reactions that give rise tomore » a variety of inter-unit covalent bonds in mildly branched racemic polymers. Yet, despite the chemical complexity and stability of lignin, significant strides have been made in recent years to identify routes through which valued commodities can be derived from it. This paper discusses emerging biological and biochemical means through which degradation of lignin to aromatic monomers can lead to the derivation of commercially valuable products.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lawler, Keith V.; Childs, Bradley C.; Mast, Daniel S.

The molecular and electronic structures for the Group 7b 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 Re 2O 7 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. The calculations accurately reproduce the experimental crystal structures within a few percent. Analysis of the band structures and density ofmore » states shows similar bonding for all of the solid-state heptoxides, including the presence of the three-center covalent bond. DFT+U simulations show that PBE-D3 underpredicts the band gap by ~0.2 eV due to an under-correlation of the metal d conducting states. As a result, homologue and compression studies show that Re 2O 7 adopts a polymeric structure because the Re-oxide tetrahedra are easily distorted by packing stresses to form additional three-center covalent bonds.« less

Air and moisture stable covalently-bonded tin(ii) coordination polymers.

PubMed

de Lima, G M; Walton, R I; Clarkson, G J; Bitzer, R S; Ardisson, J D

2018-06-05

Four covalently-bonded tin(ii) coordination polymers, (1)-(4), were hydrothermally prepared in aqueous alkaline media by the reactions of SnSO4 with 1,2,4,5-benzenetetracarboxylic acid (1), 1,3,5-benzenetricarboxylic acid (2), 4-hydroxypyridine-2,6-dicarboxylic acid (3), and 1,3,5-cyclohexanetricarboxylic acid (4). All products were structurally authenticated by single-crystal X-ray diffraction, and the number of different tin centres and their oxidation states were confirmed by 119Sn Mössbauer spectroscopy. In addition, the comparison between experimental and simulated X-ray powder diffraction patterns confirmed the authenticity of the samples. Our crystallographic results for (1)-(4) show that the Sn(ii) centres are tetracoordinated and exhibit distorted disphenoidal geometries, corroborating the presence of one stereochemically active lone electron pair at each metal site. Products (1) and (2) display bi-dimensional polymeric structures, (3) exhibits a one-dimensional architecture, whereas (4) shows a remarkable three-dimensional coordination network. Hirshfeld surface and supramolecular analyses for the repeating units of (1)-(4) were also performed in order to identify structurally important non-covalent interactions.

Oriented Covalent Organic Framework Film on Graphene for Robust Ambipolar Vertical Organic Field-Effect Transistor

DOE PAGES

Sun, Bing; Zhu, Chen-Hui; Liu, Yi; ...

2017-04-13

Periodically eclipsed π-stacking columns in two-dimensional covalent organic frameworks (2D COFs) could function as direct channel paths for charge carrier transport. Incorporating a welldefined 2D COF into organic electronic devices, however, is still a challenge. Herein, we reported the solvothermal synthesis of a COF TFPy-PPDA film on single layer graphene (SLG), which was constructed via covalent imine-type linkage by employing 1,3,6,8-tetrakis(p-formylphenyl)pyrene (TFPy) and p-phenylenediamine (PPDA) as building blocks. A vertical field-effect transistor (VFET) based on the heterostructure of COF TFPy-PPDA film and SLG shows ambipolar charge carrier behavior under lower modulating voltages. Work-function-tunable contact between SLG and COFTFPy-PPDA film andmore » suitable injection barriers of charge carriers lead to the ambipolar transport with high current density on/off ratio (>10 5) and high on-current density (>4.1 Acm -2). Interfacing 2D COF with graphene for VFET could shed the promising application prospect of 2D COFs in organic electronics and optoelectronics.« less

Controlled chain polymerisation and chemical soldering for single-molecule electronics.

PubMed

Okawa, Yuji; Akai-Kasaya, Megumi; Kuwahara, Yuji; Mandal, Swapan K; Aono, Masakazu

2012-05-21

Single functional molecules offer great potential for the development of novel nanoelectronic devices with capabilities beyond today's silicon-based devices. To realise single-molecule electronics, the development of a viable method for connecting functional molecules to each other using single conductive polymer chains is required. The method of initiating chain polymerisation using the tip of a scanning tunnelling microscope (STM) is very useful for fabricating single conductive polymer chains at designated positions and thereby wiring single molecules. In this feature article, developments in the controlled chain polymerisation of diacetylene compounds and the properties of polydiacetylene chains are summarised. Recent studies of "chemical soldering", a technique enabling the covalent connection of single polydiacetylene chains to single functional molecules, are also introduced. This represents a key step in advancing the development of single-molecule electronics.

A Comparison of Cocrystal Structure Solutions from Powder and Single Crystal Techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Lapidus; P Stephens; K Arora

We demonstrate the effectiveness and accuracy of high resolution powder diffraction for determination of cocrystal structures through a double-blind study. Structures of 10 cocrystals of varying complexity were determined independently using single crystal and powder techniques. The two methodologies give identical molecular packing and hydrogen bond topology, and an rms difference in covalent bond lengths of 0.035 {angstrom}. Powder techniques are clearly sufficient to establish a complete characterization of cocrystal geometry.

7.87 eV Laser Desorption Postionization Mass Spectrometry of Adsorbed and Covalently Bound Bisphenol A Diglycidyl Methacrylate

PubMed Central

Zhou, Manshui; Wu, Chunping; Akhmetov, Artem; Edirisinghe, Praneeth D.; Drummond, James L.; Hanley, Luke

2007-01-01

Bisphenol A diglycidyl methacrylate (Bis-GMA) was adsorbed onto or covalently bound to a porous silicon oxide surface. Laser desorption 10.5 eV postionization mass spectrometry (LDPI-MS) was previously demonstrated for surface analysis of adsorbed and surface bound Bis-GMA, but signal to noise levels were low and ion fragmentation was extensive. 7.87 eV postionization using the fluorine laser was demonstrated here for Bis-GMA. However, signal levels remained low for LDPI-MS of Bis-GMA as its ionization potential was only ∼7.8 eV, near threshold for single photon ionization by the 7.87 eV fluorine laser. It is known that aromatic tagging of molecular analytes can lower the overall IP of the tagged molecular complex, allowing 7.87 eV single photon ionization. Therefore, Bis-GMA was also derivatized with several tags whose IPs were either below or above 7.87 eV: the tag with an IP below 7.87 eV enhanced single photon ionization while the tags with higher IPs did not. However, signal intensities were enhanced by resonant laser desorption for two of the derivatized Bis-GMAs. Intact ions of Bis-GMA and its derivatives were generally observed by 7.87 eV LDPI-MS, consistent with the formation of ions with relatively little internal energy upon threshold single photon ionization. PMID:17449273

Adapting photosynthesis to the near-infrared: non-covalent binding of phycocyanobilin provides an extreme spectral red-shift to phycobilisome core-membrane linker from Synechococcus sp. PCC7335.

PubMed

Miao, Dan; Ding, Wen-Long; Zhao, Bao-Qing; Lu, Lu; Xu, Qian-Zhao; Scheer, Hugo; Zhao, Kai-Hong

2016-06-01

Phycobiliproteins that bind bilins are organized as light-harvesting complexes, phycobilisomes, in cyanobacteria and red algae. The harvested light energy is funneled to reaction centers via two energy traps, allophycocyanin B and the core-membrane linker, ApcE1 (conventional ApcE). The covalently bound phycocyanobilin (PCB) of ApcE1 absorbs near 660 nm and fluoresces near 675 nm. In cyanobacteria capable of near infrared photoacclimation, such as Synechococcus sp. PCC7335, there exist even further spectrally red shifted components absorbing >700 nm and fluorescing >710 nm. We expressed the chromophore domain of the extra core-membrane linker from Synechococcus sp. PCC7335, ApcE2, in E. coli together with enzymes generating the chromophore, PCB. The resulting chromoproteins, PCB-ApcE2(1-273) and the more truncated PCB-ApcE2(24-245), absorb at 700 nm and fluoresce at 714 nm. The red shift of ~40 nm compared with canonical ApcE1 results from non-covalent binding of the chromophore by which its full conjugation length including the Δ3,3(1) double bond is preserved. The extreme spectral red-shift could not be ascribed to exciton coupling: dimeric PCB-ApcE2(1-273) and monomeric-ApcE2(24-245) absorbed and fluoresced similarly. Chromophorylation of ApcE2 with phycoerythrobilin- or phytochromobilin resulted in similar red shifts (absorption at 615 and 711 nm, fluorescence at 628 or 726 nm, respectively), compared to the covalently bound chromophores. The self-assembled non-covalent chromophorylation demonstrates a novel access to red and near-infrared emitting fluorophores. Brightly fluorescent biomarking was exemplified in E. coli by single-plasmid transformation. Copyright © 2016 Elsevier B.V. All rights reserved.

Reconfigurable and Reprocessable Thermoset Shape Memory Polymer with Synergetic Triple Dynamic Covalent Bonds.

PubMed

Wang, Yongwei; Pan, Yi; Zheng, Zhaohui; Ding, Xiaobin

2018-04-20

Degradable shape memory polymers (SMPs), especially for polyurethane-based SMPs, have shown great potential for biomedical applications. How to reasonably fabricate SMPs with the ideal combination of degradability, shape reconfigurability, and reprocessability is a critical issue and remains a challenge for medical disposable materials. Herein, a shape memory poly(urethane-urea) with synergetic triple dynamic covalent bonds is reported via embedding polycaprolactone unit into poly(urethane-urea) with the hindered urea dynamic bond. The single polymer network is biodegradable, thermadapt, and reprocessable, without sacrificing the outstanding shape memory performance. Such a shape memory network with plasticity and reprocessability is expected to have significant and positive impact on the medical device industry. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry

NASA Astrophysics Data System (ADS)

Hook, Vivian; Lietz, Christopher B.; Podvin, Sonia; Cajka, Tomas; Fiehn, Oliver

2018-05-01

Neuropeptides are short peptides in the range of 3-40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2-4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell-cell signaling. [Figure not available: see fulltext.

Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry

NASA Astrophysics Data System (ADS)

Hook, Vivian; Lietz, Christopher B.; Podvin, Sonia; Cajka, Tomas; Fiehn, Oliver

2018-04-01

Neuropeptides are short peptides in the range of 3-40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2-4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell-cell signaling. [Figure not available: see fulltext.

Applications of nuclear magnetic resonance spectroscopy for the understanding of enantiomer separation mechanisms in capillary electrophoresis.

PubMed

Salgado, Antonio; Chankvetadze, Bezhan

2016-10-07

This review deals with the applications of nuclear magnetic resonance (NMR) spectroscopy to understand the mechanisms of chiral separation in capillary electrophoresis (CE). It is accepted that changes observed in the separation process, including the reversal of enantiomer migration order (EMO), can be caused by subtle modifications in the molecular recognition mechanisms between enantiomer and chiral selector. These modifications may imply minor structural differences in those selector-selectand complexes that arise from the above mentioned interactions. Therefore, it is mandatory to understand the fine intermolecular interactions between analytes and chiral selectors. In other words, it is necessary to know in detail the structures of the complexes formed by the enantiomer (selectand) and the selector. Any differences in the structures of these complexes arising from either enantiomer should be detected, so that enantiomeric bias in the separation process could be explained. As to the nature of these interactions, those have been extensively reviewed, and it is not intended to be discussed here. These interactions contemplate ionic, ion-dipole and dipole-dipole interactions, hydrogen bonding, van der Waals forces, π-π stacking, steric and hydrophobic interactions. The main subject of this review is to describe how NMR spectroscopy helps to gain insight into the non-covalent intermolecular interactions between selector and selectand that lead to enantiomer separation by CE. Examples in which diastereomeric species are created by covalent (irreversible) derivatization will not be considered here. This review is structured upon the different structural classes of chiral selectors employed in CE, in which NMR spectroscopy has made substantial contributions to rationalize the observed enantioseparations. Cases in which other techniques complement NMR spectroscopic data are also mentioned. Copyright © 2016 Elsevier B.V. All rights reserved.

Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of its Active Site Covalent Adduct.

PubMed

González-Bello, Concepción; Tizón, Lorena; Lence, Emilio; Otero, José M; van Raaij, Mark J; Martinez-Guitian, Marta; Beceiro, Alejandro; Thompson, Paul; Hawkins, Alastair R

2015-07-29

The first example of an ammonium derivative that causes a specific modification of the active site of type I dehydroquinase (DHQ1), a dehydratase enzyme that is a promising target for antivirulence drug discovery, is described. The resolution at 1.35 Å of the crystal structure of DHQ1 from Salmonella typhi chemically modified by this ammonium derivative revealed that the ligand is covalently attached to the essential Lys170 through the formation of an amine. The detection by mass spectroscopy of the reaction intermediates, in conjunction with the results of molecular dynamics simulations, allowed us to explain the inhibition mechanism and the experimentally observed differences between S. typhi and Staphylococcus aureus enzymes. The results presented here reveal that the replacement of Phe225 in St-DHQ1 by Tyr214 in Sa-DHQ1 and its hydrogen bonding interaction with the conserved water molecule observed in several crystal structures protects the amino adduct against further dehydration/aromatization reactions. In contrast, for the St-DHQ1 enzyme, the carboxylate group of Asp114, with the assistance of this water molecule, would trigger the formation of a Schiff base that can undergo further dehydration reactions until full aromatization of the cyclohexane ring is achieved. Moreover, in vitro antivirulence studies showed that the reported compound is able to reduce the ability of Salmonella Enteritidis to kill A459 respiratory cells. These studies have identified a good scaffold for the design of irreversible inhibitors that can be used as drugs and has opened up new opportunities for the development of novel antivirulence agents by targeting the DHQ1 enzyme.

Direct detection of OTA by impedimetric aptasensor based on modified polypyrrole-dendrimers.

PubMed

Mejri-Omrani, Nawel; Miodek, Anna; Zribi, Becem; Marrakchi, Mouna; Hamdi, Moktar; Marty, Jean-Louis; Korri-Youssoufi, Hafsa

2016-05-12

Ochratoxin A (OTA) is a carcinogenic mycotoxin that contaminates food such as cereals, wine and beer; therefore it represents a risk for human health. Consequently, the allowed concentration of OTA in food is regulated by governmental organizations and its detection is of major agronomical interest. In the current study we report the development of an electrochemical aptasensor able to directly detect trace OTA without any amplification procedure. This aptasensor was constructed by coating the surface of a gold electrode with a film layer of modified polypyrrole (PPy), which was thereafter covalently bound to polyamidoamine dendrimers of the fourth generation (PAMAM G4). Finally, DNA aptamers that specifically binds OTA were covalently bound to the PAMAM G4 providing the aptasensor, which was characterized by using both Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR) techniques. The study of OTA detection by the constructed electrochemical aptasensor was performed using Electrochemical Impedance Spectroscopy (EIS) and revealed that the presence of OTA led to the modification of the electrical properties of the PPy layer. These modifications could be assigned to conformational changes in the folding of the aptamers upon specific binding of OTA. The aptasensor had a dynamic range of up to 5 μg L(-1) of OTA and a detection limit of 2 ng L(-1) of OTA, which is below the OTA concentration allowed in food by the European regulations. The efficient detection of OTA by this electrochemical aptasensor provides an unforeseen platform that could be used for the detection of various small molecules through specific aptamer association. Copyright © 2016 Elsevier B.V. All rights reserved.

Homogeneous screening assay for human tankyrase.

PubMed

Narwal, Mohit; Fallarero, Adyary; Vuorela, Pia; Lehtiö, Lari

2012-06-01

Tankyrase, a member of human PARP protein superfamily, catalyzes a covalent post-translational modification of substrate proteins. This modification, poly(ADP-ribos)ylation, leads to changes in protein interactions and modifies downstream signaling events. Tankyrase 1 is a potential drug target due to its functions in telomere homeostasis and in Wnt signaling. We describe here optimization and application of an activity-based homogenous assay for tankyrase inhibitors in a high-throughput screening format. The method measures the consumption of substrate by the chemical conversion of the remaining NAD(+) into a stable fluorescent condensation product. Conditions were optimized to measure the enzymatic auto-modification of a recombinant catalytic fragment of tankyrase 1. The fluorescence assay is inexpensive, operationally easy and performs well according to the statistical analysis (Z'= 0.7). A validatory screen with a natural product library confirmed suitability of the assay for finding new tankyrase inhibitors. Flavone was the most potent (IC(50)=325 nM) hit from the natural compounds. A flavone derivative, apigenin, and isopropyl gallate showed potency on the micromolar range, but displayed over 30-fold selectivity for tankyrase over the studied isoenzymes PARP1 and PARP2. The assay is robust and will be useful for screening new tankyrase inhibitors.

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

PubMed

Thinon, Emmanuelle; Fernandez, Joseph P; Molina, Henrik; Hang, Howard C

2018-05-04

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

Synthesis and effect of modification on methacylate - acrylate microspheres for Trametes versicolor laccase enzyme immobilization

NASA Astrophysics Data System (ADS)

Mazlan, Siti Zulaikha; Hanifah, Sharina Abu

2014-09-01

Immobilization of laccase on the modified copolymer methacrylate-acrylate microspheres was studied. A poly (glycidyl methacrylate-co-n-butyl acrylate) microsphere consists of epoxy groups were synthesized using suspension photocuring technique. The epoxy group in poly (GMA-nBA) microspheres were converted into amino groups with aldehyde group. Laccase immobilization is based on having the amino groups on the enzyme surface and aldehyde group on the microspheres via covalent binding. Fourier transform infrared spectroscopy (FT-IR) analysis proved the successful surface modification on microspheres. The FTIR spectrum shows the characteristic peaks at 1646 cm-1 assigned to the conformation of the polymerization that took place between monomer GMA and nBA respectively. In addition, after modification, FTIR peaks that assigned to the epoxy ring (844 cm-1 and 904 cm-1) were decreased. The results obtained from FTIR method signify good agreement with the epoxy content method. Hence, the activity of the laccase-immobilized microspheres increased upon increasing the epoxy content. Furthermore, poly (GMA-nBA) exhibited uniform microspheres with below 2 μm surface. Immobilized enzyme showed a broader pH profile and higher temperature compared native enzyme.

Soft Cysteine Signaling Network: The Functional Significance of Cysteine in Protein Function and the Soft Acids/Bases Thiol Chemistry That Facilitates Cysteine Modification.

PubMed

Wible, Ryan S; Sutter, Thomas R

2017-03-20

The unique biophysical and electronic properties of cysteine make this molecule one of the most biologically critical amino acids in the proteome. The defining sulfur atom in cysteine is much larger than the oxygen and nitrogen atoms more commonly found in the other amino acids. As a result of its size, the valence electrons of sulfur are highly polarizable. Unique protein microenvironments favor the polarization of sulfur, thus increasing the overt reactivity of cysteine. Here, we provide a brief overview of the endogenous generation of reactive oxygen and electrophilic species and specific examples of enzymes and transcription factors in which the oxidation or covalent modification of cysteine in those proteins modulates their function. The perspective concludes with a discussion of cysteine chemistry and biophysics, the hard and soft acids and bases model, and the proposal of the Soft Cysteine Signaling Network: a hypothesis proposing the existence of a complex signaling network governed by layered chemical reactivity and cross-talk in which the chemical modification of reactive cysteine in biological networks triggers the reorganization of intracellular biochemistry to mitigate spikes in endogenous or exogenous oxidative or electrophilic stress.

Ultrathin Polymer Films, Patterned Arrays, and Microwells

NASA Astrophysics Data System (ADS)

Yan, Mingdi

2002-05-01

The ability to control and tailor the surface and interface properties of materials is important in microelectronics, cell growth control, and lab-on-a-chip devices. Modification of material surfaces with ultrathin polymer films is attractive due to the availability of a variety of polymers either commercially or by synthesis. We have developed two approaches to the attachment of ultrathin polymer films on solid substrates. In the first method, a silane-functionalized perfluorophenyl azide (PFPA-silane) was synthesized and used to covalently immobilize polymer thin films on silicon wafers. Silanization of the wafer surface with the PFPA-silane introduced a monolayer of azido groups which in turn covalently attached the polymer film by way of photochemically initiated insertion reactions. The thickness of the film could be adjusted by the type and the molecular weight of the polymer. The method is versatile due to the general C-H and/or N-H insertion reactions of crosslinker; and therefore, no specific reactive functional groups on the polymers are required. Using this method, a new type of microwell array was fabricated from covalently immobilized polymer thin films on flat substrates. The arrays were characterized with AFM, XPS, and TOF-SIMS. The second method describes the attachment of polymer thin films on solid substrates via UV irradiation. The procedure consisted of spin-coating a polymer film and irradiating the film with UV light. Following solvent extraction, a thin film remained. The thickness of the film, from a few to over a hundred nanometers, was controlled by varying solution concentration and the molecular weight of the polymer.

1,4-Naphthoquinone activates the HSP90/HSF1 pathway through the S-arylation of HSP90 in A431 cells: Negative regulation of the redox signal transduction pathway by persulfides/polysulfides.

PubMed

Abiko, Yumi; Sha, Liang; Shinkai, Yasuhiro; Unoki, Takamitsu; Luong, Nho Cong; Tsuchiya, Yukihiro; Watanabe, Yasuo; Hirose, Reiko; Akaike, Takaaki; Kumagai, Yoshito

2017-03-01

The current consensus is that environmental electrophiles activate redox signal transduction pathways through covalent modification of sensor proteins with reactive thiol groups at low concentrations, while they cause cell damage at higher concentrations. We previously exposed human carcinoma A431 cells to the atmospheric electrophile 1,4-naphthoquinone (1,4-NQ) and found that heat shock protein 90 (HSP90), a negative regulator of heat shock factor 1 (HSF1), was a target of 1,4-NQ. In the study presented here, we determined whether 1,4-NQ activates HSF1. We also examined whether such redox signaling could be regulated by nucleophilic sulfur species. Exposure of A431 cells to 1,4-NQ covalently modified cellular HSP90, resulting in repression of the association between HSF1 with HSP90, thereby enhancing HSF1 translocation into the nuclei. Liquid chromatography-tandem mass spectrometry analysis with recombinant HSP90 revealed that the modifications site were Cys412 and Cys564. We found that HSF1 activation mediated by 1,4-NQ upregulated downstream genes, such as HSPA6. HSF1 knockdown accelerated 1,4-NQ-mediated cytotoxicity in the cells. While simultaneous treatment with reactive persulfide and polysulfide, Na 2 S 2 and Na 2 S 4 , blocked 1,4-NQ-dependent protein modification and HSF1 activation in A431 cells, the knockdown of Cys persulfide producing enzymes cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE) enhanced these phenomena. 1,4-NQ-thiol adduct and 1,4-NQ-S-1,4-NQ adduct were produced during the enzymatic reaction of recombinant CSE in the presence of 1,4-NQ. The results suggest that activation of the HSP90-HSF1 signal transduction pathway mediated by 1,4-NQ protects cells against 1,4-NQ and that per/polysulfides can diminish the reactivity of 1,4-NQ by forming sulfur adducts. Copyright © 2017 Elsevier Inc. All rights reserved.

A machine-learning approach for predicting palmitoylation sites from integrated sequence-based features.

PubMed

Li, Liqi; Luo, Qifa; Xiao, Weidong; Li, Jinhui; Zhou, Shiwen; Li, Yongsheng; Zheng, Xiaoqi; Yang, Hua

2017-02-01

Palmitoylation is the covalent attachment of lipids to amino acid residues in proteins. As an important form of protein posttranslational modification, it increases the hydrophobicity of proteins, which contributes to the protein transportation, organelle localization, and functions, therefore plays an important role in a variety of cell biological processes. Identification of palmitoylation sites is necessary for understanding protein-protein interaction, protein stability, and activity. Since conventional experimental techniques to determine palmitoylation sites in proteins are both labor intensive and costly, a fast and accurate computational approach to predict palmitoylation sites from protein sequences is in urgent need. In this study, a support vector machine (SVM)-based method was proposed through integrating PSI-BLAST profile, physicochemical properties, [Formula: see text]-mer amino acid compositions (AACs), and [Formula: see text]-mer pseudo AACs into the principal feature vector. A recursive feature selection scheme was subsequently implemented to single out the most discriminative features. Finally, an SVM method was implemented to predict palmitoylation sites in proteins based on the optimal features. The proposed method achieved an accuracy of 99.41% and Matthews Correlation Coefficient of 0.9773 for a benchmark dataset. The result indicates the efficiency and accuracy of our method in prediction of palmitoylation sites based on protein sequences.

Pharmaceutical cocrystals: along the path to improved medicines.

PubMed

Duggirala, Naga K; Perry, Miranda L; Almarsson, Örn; Zaworotko, Michael J

2016-01-14

Cocrystals, a long known but understudied class of crystalline solids, have attracted interest from crystal engineers and pharmaceutical scientists in the past decade and are now an integral part of the preformulation stage of drug development. This is largely because cocrystals that contain a drug molecule, pharmaceutical cocrystals, can modify physicochemical properties without the need for covalent modification of the drug molecule. This review presents a brief history of cocrystals before addressing recent advances in design, discovery and development of pharmaceutical cocrystals that have occurred since an earlier review published in 2004. We address four aspects of cocrystals: nomenclature; design using hydrogen-bonded supramolecular synthons; methods of discovery and synthesis; development of pharmaceutical cocrystals as drug products. Cocrystals can be classified into molecular cocrystals (MCCs) that contain only neutral components (coformers) and ionic cocrystals (ICCs), which are comprised of at least one ionic coformer that is a salt. That cocrystals, especially ICCs, offer much greater diversity in terms of composition and properties than single component crystal forms and are amenable to design makes them of continuing interest. Seven recent case studies that illustrate how pharmaceutical cocrystals can improve physicochemical properties and clinical performance of drug substances, including a recently approved drug product based upon an ICC, are presented.

Psoralen-induced DNA adducts are substrates for the base excision repair pathway in human cells

PubMed Central

Couvé-Privat, Sophie; Macé, Gaëtane; Saparbaev, Murat K.

2007-01-01

Interstrand cross-link (ICL) is a covalent modification of both strands of DNA, which prevents DNA strand separation during transcription and replication. Upon photoactivation 8-methoxypsoralen (8-MOP+UVA) alkylates both strands of DNA duplex at the 5,6-double bond of thymidines, generating monoadducts (MAs) and ICLs. It was thought that bulky DNA lesions such as MAs are eliminated only in the nucleotide excision repair pathway. Instead, non-bulky DNA lesions are substrates for DNA glycosylases and AP endonucleases which initiate the base excision repair (BER) pathway. Here we examined whether BER might be involved in the removal of psoralen–DNA photoadducts. The results show that in human cells DNA glycosylase NEIL1 excises the MAs in duplex DNA, subsequently the apurinic/apyrimidinic endonuclease 1, APE1, removes the 3′-phosphate residue at single-strand break generated by NEIL1. The apparent kinetic parameters suggest that NEIL1 excises MAs with high efficiency. Consistent with these results HeLa cells lacking APE1 and/or NEIL1 become hypersensitive to 8-MOP+UVA exposure. Furthermore, we demonstrate that bacterial homologues of NEIL1, the Fpg and Nei proteins, also excise MAs. New substrate specificity of the Fpg/Nei protein family provides an alternative repair pathway for ICLs and bulky DNA damage. PMID:17715144

78 FR 44189 - Petition for Modification of Single Car Air Brake Test Procedures

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-23

...] Petition for Modification of Single Car Air Brake Test Procedures In accordance with Part 232 of Title 49... Administration (FRA) per 49 CFR 232.307 to modify the single car air brake test procedures located in AAR Standard S-486, Code of Air Brake System Tests for Freight Equipment-- Single Car Test, and required...

Surface modification of silicon carbide with silane coupling agent and hexadecyl iodiele

NASA Astrophysics Data System (ADS)

Shang, Xujing; Zhu, Yumei; Li, Zhihong

2017-02-01

In this paper, two kinds of silane coupling agents, namely 3-aminopropyl triethoxysilane (KH550) and 3-mercaptopropyl trimethoxysilane (KH590), were adopted as preliminary modifiers to improve the hydrophobic surface properties of silicon carbide (SiC) powder for the first step. The factors that influence the modification effects were investigated by measuring the contact angle. The results showed that KH590 has a better effect than KH550 for the hydrophobic modification of SiC, and the contact angle improved most after SiC powder was reacted with 0.3 g KH590 at 75 °C in aqueous/alcohol solution for 4 h. On account of further enhancement of hydrophobicity, the study was focused on utilizing nucleophilic substitution between KH590 and hexadecyl iodiele to extend the length of alkyl chain. Compared with using KH590 alone, SiC powder modified by KH590 and hexadecyl iodiele showed better water resistance with an increase of contact angle from 106.8° to 127.5°. The Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectra (XPS) as well as X-ray diffraction (XRD) analysis results showed that KH550/KH590 and hexadecyl iodiele can be covalently bonded to the surface of SiC powder without altering its crystal configuration. This methodology may provide a new way of the modification of inorganic materials in further.

Control of peroxisome proliferator-activated receptor gamma2 stability and activity by SUMOylation.

PubMed

Floyd, Z Elizabeth; Stephens, Jacqueline M

2004-06-01

To determine whether small ubiquitin-related modifier (SUMO)ylation of lysine 107 plays a role in regulating the activity of peroxisome proliferator-activated receptor gamma (PPARgamma). Transient expression of wild-type and K107R-PPARgamma2 in the NIH 3T3 fibroblast cell line was carried out in conjunction with half-life studies, luciferase activity assays, and indirect immunofluorescence localization studies. Additional in vitro analysis was carried out using recombinant SUMOylation pathway proteins along with in vitro transcribed and translated wild-type or K107R-PPARgamma2 to examine the SUMO-1 modification state of wild-type and SUMO-deficient K107R-PPARgamma2. While examining PPARgamma2 for potential ubiquitylation sites, we identified a strong consensus site for SUMO modification that contains lysine 107. In vitro, SUMOylation studies showed that lysine 107 of PPARgamma2 is a major SUMOylation site and that at least one other SUMOylation site is present in PPARgamma. In addition, our results demonstrated that SUMO-1 affects PPARgamma stability and transcriptional activity but not the nuclear localization of PPARgamma. These results indicated that SUMOylation plays a role in regulating PPARgamma, both indirectly and directly by modification of lysine 107. Because PPARgamma is regulated in numerous animal models of obesity, understanding the covalent modifications of PPARgamma may enhance our understanding of the metabolic syndrome.

REDOX REGULATION OF SIRT1 IN INFLAMMATION AND CELLULAR SENESCENCE

PubMed Central

Hwang, Jae-woong; Yao, Hongwei; Caito, Samuel; Sundar, Isaac K.; Rahman, Irfan

2013-01-01

Sirtuin1 (SIRT1) regulates inflammation, aging (lifespan and healthspan), calorie restriction/energetics, mitochondrial biogenesis, stress resistance, cellular senescence, endothelial functions, apoptosis/autophagy, and circadian rhythms through deacetylation of transcription factors and histones. SIRT1 level and activity are decreased in chronic inflammatory conditions and aging where oxidative stress occurs. SIRT1 is regulated by a NAD+-dependent DNA repair enzyme poly(ADP-ribose)-polymerase-1 (PARP-1), and subsequent NAD+ depletion by oxidative stresses may have consequent effects on inflammatory and stress responses as well as cellular senescence. SIRT1 has been shown to undergo covalent oxidative modifications by cigarette smoke-derived oxidants/aldehydes, leading to post-translational modifications, inactivation, and protein degradation. Furthermore, oxidant/carbonyl stress-mediated reduction of SIRT1 leads to the loss of its control on acetylation of target proteins including p53, RelA/p65 and FOXO3, thereby enhancing the inflammatory, pro-senescent and apoptotic responses, as well as endothelial dysfunction. In this review, the mechanisms of cigarette smoke/oxidant-mediated redox post-translational modifications of SIRT1 and its role in PARP1, NF-κB activation, FOXO3 and eNOS regulation, as well as chromatin remodeling/histone modifications during inflammaging are discussed. Furthermore, we also discussed various novel ways to activate SIRT1 either directly or indirectly, which may have therapeutic potential in attenuating inflammation and premature senescence involved in chronic lung diseases. PMID:23542362

Four-color single molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes

PubMed Central

DeRocco, Vanessa C.; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A.; Weninger, Keith

2010-01-01

To allow studies of conformational changes within multi-molecular complexes, we present a simultaneous, 4-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera based, wide-field detection. We further demonstrate labeling histidine-tagged proteins non-covalently with tris-Nitrilotriacetic acid (tris-NTA) conjugated dyes to achieve single molecule detection. We combine these methods to co-localize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes. PMID:21091445

Maximum-valence radii of transition metals

PubMed Central

Pauling, Linus

1975-01-01

In many of their compounds the transition metals have covalence 9, forming nine bonds with use of nine hybrid spd bond orbitals. A set of maximum-valence single-bond radii is formulated for use in these compounds. These radii are in reasonably good agreement with observed bond lengths. Quadruple bonds between two transition metal atoms are about 50 pm (iron-group atoms) or 55 pm (palladium and platinum-group atoms) shorter than single bonds. This amount of shortening corresponds to four bent single bonds with the best set of bond angles, 79.24° and 128.8°. PMID:16578730

Semiconducting carbon nanotube and covalent organic polyhedron-C60 nanohybrids for light harvesting.

PubMed

Lohrman, Jessica; Zhang, Chenxi; Zhang, Wei; Ren, Shenqiang

2012-08-28

We demonstrate noncovalent electrostatic and π-π interactions to assemble semiconducting single wall carbon nanotube (SWCNT)-C(60)@COP nanohybrids. The C(60)@COP light harvesting complexes bind strongly to SWCNTs due to significant π-π-stacking between C(60), the aromatic dicarbazolylacetylene moieties and the nanotube surfaces.

Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

PubMed

Hill, A S; Giersch, T M; Loh, C S; Skerritt, J H

1999-10-01

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.

Organophosphonate biofunctionalization of diamond electrodes.

PubMed

Caterino, R; Csiki, R; Wiesinger, M; Sachsenhauser, M; Stutzmann, M; Garrido, J A; Cattani-Scholz, A; Speranza, Giorgio; Janssens, S D; Haenen, K

2014-08-27

The modification of the diamond surface with organic molecules is a crucial aspect to be considered for any bioapplication of this material. There is great interest in broadening the range of linker molecules that can be covalently bound to the diamond surface. In the case of protein immobilization, the hydropathicity of the surface has a major influence on the protein conformation and, thus, on the functionality of proteins immobilized at surfaces. For electrochemical applications, particular attention has to be devoted to avoid that the charge transfer between the electrode and the redox center embedded in the protein is hindered by a thick insulating linker layer. This paper reports on the grafting of 6-phosphonohexanoic acid on OH-terminated diamond surfaces, serving as linkers to tether electroactive proteins onto diamond surfaces. X-ray photoelectron spectroscopy (XPS) confirms the formation of a stable layer on the surface. The charge transfer between electroactive molecules and the substrate is studied by electrochemical characterization of the redox activity of aminomethylferrocene and cytochrome c covalently bound to the substrate through this linker. Our work demonstrates that OH-terminated diamond functionalized with 6-phosphonohexanoic acid is a suitable platform to interface redox-proteins, which are fundamental building blocks for many bioelectronics applications.

Optimization of amino group density on surfaces of titanium dioxide nanoparticles covalently bonded to a silicone substrate for antibacterial and cell adhesion activities.

PubMed

Okada, Masahiro; Yasuda, Shoji; Kimura, Tsuyoshi; Iwasaki, Mitsunobu; Ito, Seishiro; Kishida, Akio; Furuzono, Tsutomu

2006-01-01

A composite consisting of titanium dioxide (TiO2) particle, the surface of which was modified with amino groups, and a silicone substrate through covalent bonding at their interface was developed, and antibacterial and cell adhesion activities of the composite were evaluated. The density of the amino groups on the TiO2 particle surface was controlled by the reaction time of the modification reaction. The degradation rate of CH3CHO in the presence of the TiO2 particles under UV irradiation decreased with an increase in the amino group density on the TiO2 surface. On the other hand, the number of L929 cells adhering on the TiO2/silicone composite increased with an increase in the amino group density. From the above two results, the optimum density of amino groups for both photoreactivity and cell adhesiveness was estimated to be 2.0-4.0 molecules/nm2. The optimum amino group-modified TiO2/silicone composite sheet (amino group density, 3.0 molecules/nm2) showed an effective antibacterial activity for Escherichia coli bacteria under UV irradiation. (c) 2005 Wiley Periodicals, Inc

Products obtained after in vitro reaction of 7,12-dimethylbenz[alpha]anthracene 5,6-oxide with nucleic acids.

PubMed

Blobstein, S H; Weinstein, I B; Grunberger, D; Weisgras, J; Harvey, R G

1975-07-29

Several lines of evidence suggest that oxide derivatives of carcinogenic polycyclic hydrocarbons are the reactive intermediates for in vivo binding to cellular nucleic acids. In the present study the covalent binding of 7,12-dimethylbenz[alpha]anthracene 5,6-oxide to synthetic homopolymers and nucleic acids in aqueous-acetone solutions has been investigated. Poly(G) was found to be the most reactive nucleic acid and underwent approximately 7-10% modification. Alkaline hydrolysis of the poly(G)-dimethylbenzathracene conjugate yielded chromatographically distinct polycyclic hydrocarbon-modified nucleotides which were further characterized by spectral analyses and enzymatic and chemical degradation. When the oxide was allowed to react with GMP or dGMP, at least two products were obtained in about 1% yield. Acid hydrolysis of the dGMP-dimethylbenzanthracene conjugates liberated the corresponding guanine-dimethylbenzathracene products. Mass spectral analysis of the modified bases provided direct evidence that we had obtained covalent binding of the poly-cyclic hydrocarbon to guanine. The mass spectral cleavage pattern suggest that one of these products is a hydroxydihydro derivative of dimethylbenzanthracene bound to guanine and the other is a dimethylbenzanthracene-guanine conjugate. Additional structural aspects of these guanine derivatives are discussed.

Direct Evidence for the Formation of Diastereoisomeric Benzylpenicilloyl Haptens from Benzylpenicillin and Benzylpenicillenic Acid in PatientsS⃞

PubMed Central

Meng, Xiaoli; Jenkins, Rosalind E.; Berry, Neil G.; Maggs, James L.; Farrell, John; Lane, Catherine S.; Stachulski, Andrew V.; French, Neil S.; Naisbitt, Dean J.; Pirmohamed, Munir

2011-01-01

Covalent binding to proteins to form neoantigens is thought to be central to the pathogenesis of penicillin hypersensitivity reactions. We have undertaken detailed mass spectrometric studies to define the mechanism and protein chemistry of hapten formation from benzylpenicillin (BP) and its rearrangement product, benzylpenicillenic acid (PA). Mass spectrometric analysis of human serum albumin exposed to BP and PA in vitro revealed that at low concentrations (drug protein molar ratio 0.001:1) and during short time incubations BP and PA selectively target different residues, Lys199 and Lys525, respectively. Molecular modeling showed that the selectivity was a function of noncovalent interaction before covalent modification. With increased exposure to higher concentrations of BP and PA, multiple epitopes were detected on albumin, demonstrating that the multiplicity of hapten formation is a function of time and concentration. More importantly, we have demonstrated direct evidence that PA is a hapten accounting for the diastereoisomeric BP antigen formation in albumin isolated from the blood of patients receiving penicillin. Furthermore, PA was found to be more potent than BP with respect to stimulation of T cells from patients with penicillin hypersensitivity, illustrating the functional relevance of diastereoisomeric hapten formation. PMID:21680886

A “fullerene-carbon nanotube” structure with tunable mechanical properties

NASA Astrophysics Data System (ADS)

Ji, W. M.; Zhang, L. W.; Liew, K. M.

2018-03-01

Carbon-based nanostructures have drawn tremendous research interest and become promising building blocks for the new generation of smart sensors and devices. Utilizing a bottom-up strategy, the chemical interconnecting sp 3 covalent bond between carbon building blocks is an efficient way to enhance its Young's modulus and ductility. The formation of sp 3 covalent bond, however, inevitably degrades its ultimate tensile strength caused by stress concentration at the junction. By performing a molecular dynamics simulation of tensile deformation for a fullerene-carbon nanotube (FCNT) structure, we propose a tunable strategy in which fullerenes with various angle energy absorption capacities are utilized as building blocks to tune their ductile behavior, while still maintaining a good ultimate tensile strength of the carbon building blocks. A higher ultimate tensile strength is revealed with the reduction of stress concentration at the junction. A brittle-to-ductile transition during the tensile deformation is detected through the structural modification. The development of ductile behavior is attributed to the improvement of energy propagation ability during the fracture initiation, in which the released energy from bonds fracture is mitigated properly, leading to the further development of mechanical properties.

3-Nitropropionic Acid is a Suicide Inhibitor of MitochondrialRespiration that, Upon Oxidation by Complex II, Forms a Covalent AdductWith a Catalytic Base Arginine in the Active Site of the Enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Li-shar; Sun, Gang; Cobessi, David

We report three new structures of mitochondrial respiratory Complex II (succinate ubiquinone oxidoreductase, E.C. 1.3.5.1) at up to 2.1 {angstrom} resolution, with various inhibitors. The structures define the conformation of the bound inhibitors and suggest the residues involved in substrate binding and catalysis at the dicarboxylate site. In particular they support the role of Arg297 as a general base catalyst accepting a proton in the dehydrogenation of succinate. The dicarboxylate ligand in oxaloacetate-containing crystals appears to be the same as that reported for Shewanella flavocytochrome c treated with fumarate. The plant and fungal toxin 3-nitropropionic acid, an irreversible inactivator ofmore » succinate dehydrogenase, forms a covalent adduct with the side chain of Arg297. The modification eliminates a trypsin cleavage site in the flavoprotein, and tandem mass spectroscopic analysis of the new fragment shows the mass of Arg 297 to be increased by 83 Da and to have potential of losing 44 Da, consistent with decarboxylation, during fragmentation.« less

Improved Composites Using Crosslinked, Surface-Modified Carbon Nanotube Materials

NASA Technical Reports Server (NTRS)

Baker, James Stewart

2014-01-01

Individual carbon nanotubes (CNTs) exhibit exceptional tensile strength and stiffness; however, these properties have not translated well to the macroscopic scale. Premature failure of bulk CNT materials under tensile loading occurs due to the relatively weak frictional forces between adjacent CNTs, leading to poor load transfer through the material. When used in polymer matrix composites (PMCs), the weak nanotube-matrix interaction leads to the CNTs providing less than optimal reinforcement.Our group is examining the use of covalent crosslinking and surface modification as a means to improve the tensile properties of PMCs containing carbon nanotubes. Sheet material comprised of unaligned multi-walled carbon nanotubes (MWCNT) was used as a drop-in replacement for carbon fiber in the composites. A variety of post-processing methods have been examined for covalently crosslinking the CNTs to overcome the weak inter-nanotube shear interactions, resulting in improved tensile strength and modulus for the bulk sheet material. Residual functional groups from the crosslinking chemistry may have the added benefit of improving the nanotube-matrix interaction. Composites prepared using these crosslinked, surface-modified nanotube sheet materials exhibit superior tensile properties to composites using the as received CNT sheet material.

Electron mobility enhancement in ZnO thin films via surface modification by carboxylic acids

NASA Astrophysics Data System (ADS)

Spalenka, Josef W.; Gopalan, Padma; Katz, Howard E.; Evans, Paul G.

2013-01-01

Modifying the surface of polycrystalline ZnO films using a monolayer of organic molecules with carboxylic acid attachment groups increases the field-effect electron mobility and zero-bias conductivity, resulting in improved transistors and transparent conductors. The improvement is consistent with the passivation of defects via covalent bonding of the carboxylic acid and is reversible by exposure to a UV-ozone lamp. The properties of the solvent used for the attachment are crucial because solvents with high acid dissociation constants (Ka) for carboxylic acids lead to high proton activities and etching of the nanometers-thick ZnO films, masking the electronic effect.

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, L.I.; Bodwell, J.E.; Mendel, D.B.

1988-05-17

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. The authors have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with (/sup 3/H)dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single (/sup 3/H)dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the (/sup 3/H)dexamethasone 21-mesylate was located at position 5 frommore » the amino terminus. Dual-isotope labeling studies with (/sup 3/H)dexamethasone 21-mesylate and (/sup 35/S)methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of (/sup 3/H)dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, their data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate. They have confirmed this finding by demonstrating that a synthetic peptide representing the amino acid sequence 640-650 of the murine glucocorticoid receptor behaves in an identical manner on reversed-phase HPLC as the trypsin-generated peptide from intact cells.« less

Loss of conformational stability in calmodulin upon methionine oxidation.

PubMed Central

Gao, J; Yin, D H; Yao, Y; Sun, H; Qin, Z; Schöneich, C; Williams, T D; Squier, T C

1998-01-01

We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM. PMID:9512014

Signal transduction in a covalent post-assembly modification cascade

NASA Astrophysics Data System (ADS)

Pilgrim, Ben S.; Roberts, Derrick A.; Lohr, Thorsten G.; Ronson, Tanya K.; Nitschke, Jonathan R.

2017-12-01

Natural reaction cascades control the movement of biomolecules between cellular compartments. Inspired by these systems, we report a synthetic reaction cascade employing post-assembly modification reactions to direct the partitioning of supramolecular complexes between phases. The system is composed of a self-assembled tetrazine-edged FeII8L12 cube and a maleimide-functionalized FeII4L6 tetrahedron. Norbornadiene (NBD) functions as the stimulus that triggers the cascade, beginning with the inverse-electron-demand Diels-Alder reaction of NBD with the tetrazine moieties of the cube. This reaction generates cyclopentadiene as a transient by-product, acting as a relay signal that subsequently undergoes a Diels-Alder reaction with the maleimide-functionalized tetrahedron. Cyclooctyne can selectively inhibit the cascade by outcompeting NBD as the initial trigger. Initiating the cascade with 2-octadecyl NBD leads to selective alkylation of the tetrahedron upon cascade completion. The increased lipophilicity of the C18-tagged tetrahedron drives this complex into a non-polar phase, allowing its isolation from the initially inseparable mixture of complexes.

Photochemical coatings for the prevention of bacterial colonization.

PubMed

Dunkirk, S G; Gregg, S L; Duran, L W; Monfils, J D; Haapala, J E; Marcy, J A; Clapper, D L; Amos, R A; Guire, P E

1991-10-01

Biomaterials are being used with increasing frequency for tissue substitution. Implantable, prosthetic devices are instrumental in the saving of patients' lives and enhancing the quality of life for many others. However, the greatest barrier to expanding the use of biomedical devices is the high probability of bacterial adherence and proliferation, causing very difficult and often untreatable medical-device centered infections. The difficulty in treating such infections results in great danger to the patient, and usually retrieval of the device with considerable pain and suffering. Clearly, development of processes that make biomedical devices resistant to bacterial adherence and colonization would have widespread application in the field of biomedical technology. A photochemical surface modification process is being investigated as a generic means of applying antimicrobial coatings to biomedical devices. The photochemical process results in covalent immobilization of coatings to all classes of medical device polymers. A discussion of the photochemical surface modification process and preliminary results demonstrating the success of photochemical coatings in formulating microbial-resistant surfaces are presented in this paper.

Effects of malondialdehyde modification on the in vitro digestibility of soy protein isolate.

PubMed

Chen, Nannan; Zhao, Qiangzhong; Sun, Weizheng; Zhao, Mouming

2013-12-11

Soy protein isolate (SPI) was modified by lipid peroxidation product malondialdehyde (MDA), and the in vitro digestibility of modified SPI was investigated. Results indicated that incubation with increasing MDA concentration resulted in significant carbonyl group generation and loss of free amino groups of SPI. Fluorescence loss of natural tryptophan and formation of Schiff base were observed. Noncovalent interaction between molecules was enhanced and became the main force that led to the solubility reduction of MDA-modified SPI. Differential scanning calorimetry (DSC) indicated that SPI had higher thermal stability and lower total calorimetric enthalpy after MDA pretreatment. Electrophoresis showed that β-conglycinin was more sensitive to MDA modification. In vitro digestion indicated that MDA could induce non-disulfide covalent polymer of SPI, which could not be digested by pepsin and pancreatin. β subunits of β-conglycinin became more resistant to digestion with increasing MDA concentration. Evaluation of the free amino acid profile in the digests indicated that MDA-modified SPI had deteriorating nutritive quality.

Viral Mimicry to Usurp Ubiquitin and SUMO Host Pathways

PubMed Central

Wimmer, Peter; Schreiner, Sabrina

2015-01-01

Posttranslational modifications (PTMs) of proteins include enzymatic changes by covalent addition of cellular regulatory determinants such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) moieties. These modifications are widely used by eukaryotic cells to control the functional repertoire of proteins. Over the last decade, it became apparent that the repertoire of ubiquitiylation and SUMOylation regulating various biological functions is not restricted to eukaryotic cells, but is also a feature of human virus families, used to extensively exploit complex host-cell networks and homeostasis. Intriguingly, besides binding to host SUMO/Ub control proteins and interfering with the respective enzymatic cascade, many viral proteins mimic key regulatory factors to usurp this host machinery and promote efficient viral outcomes. Advanced detection methods and functional studies of ubiquitiylation and SUMOylation during virus-host interplay have revealed that human viruses have evolved a large arsenal of strategies to exploit these specific PTM processes. In this review, we highlight the known viral analogs orchestrating ubiquitin and SUMO conjugation events to subvert and utilize basic enzymatic pathways. PMID:26343706

Perspectives on Intra- and Intercellular Trafficking of Hedgehog for Tissue Patterning

PubMed Central

Simon, Eléanor; Aguirre-Tamaral, Adrián; Aguilar, Gustavo; Guerrero, Isabel

2016-01-01

Intercellular communication is a fundamental process for correct tissue development. The mechanism of this process involves, among other things, the production and secretion of signaling molecules by specialized cell types and the capability of these signals to reach the target cells in order to trigger specific responses. Hedgehog (Hh) is one of the best-studied signaling pathways because of its importance during morphogenesis in many organisms. The Hh protein acts as a morphogen, activating its targets at a distance in a concentration-dependent manner. Post-translational modifications of Hh lead to a molecule covalently bond to two lipid moieties. These lipid modifications confer Hh high affinity to lipidic membranes, and intense studies have been carried out to explain its release into the extracellular matrix. This work reviews Hh molecule maturation, the intracellular recycling needed for its secretion and the proposed carriers to explain Hh transportation to the receiving cells. Special focus is placed on the role of specialized filopodia, also named cytonemes, in morphogen transport and gradient formation. PMID:29615597

Perspectives on Intra- and Intercellular Trafficking of Hedgehog for Tissue Patterning.

PubMed

Simon, Eléanor; Aguirre-Tamaral, Adrián; Aguilar, Gustavo; Guerrero, Isabel

2016-12-02

Intercellular communication is a fundamental process for correct tissue development. The mechanism of this process involves, among other things, the production and secretion of signaling molecules by specialized cell types and the capability of these signals to reach the target cells in order to trigger specific responses. Hedgehog (Hh) is one of the best-studied signaling pathways because of its importance during morphogenesis in many organisms. The Hh protein acts as a morphogen, activating its targets at a distance in a concentration-dependent manner. Post-translational modifications of Hh lead to a molecule covalently bond to two lipid moieties. These lipid modifications confer Hh high affinity to lipidic membranes, and intense studies have been carried out to explain its release into the extracellular matrix. This work reviews Hh molecule maturation, the intracellular recycling needed for its secretion and the proposed carriers to explain Hh transportation to the receiving cells. Special focus is placed on the role of specialized filopodia, also named cytonemes, in morphogen transport and gradient formation.

Effects of multiple enzyme-substrate interactions in basic units of cellular signal processing

NASA Astrophysics Data System (ADS)

Seaton, D. D.; Krishnan, J.

2012-08-01

Covalent modification cycles are a ubiquitous feature of cellular signalling networks. In these systems, the interaction of an active enzyme with the unmodified form of its substrate is essential for signalling to occur. However, this interaction is not necessarily the only enzyme-substrate interaction possible. In this paper, we analyse the behaviour of a basic model of signalling in which additional, non-essential enzyme-substrate interactions are possible. These interactions include those between the inactive form of an enzyme and its substrate, and between the active form of an enzyme and its product. We find that these additional interactions can result in increased sensitivity and biphasic responses, respectively. The dynamics of the responses are also significantly altered by the presence of additional interactions. Finally, we evaluate the consequences of these interactions in two variations of our basic model, involving double modification of substrate and scaffold-mediated signalling, respectively. We conclude that the molecular details of protein-protein interactions are important in determining the signalling properties of enzymatic signalling pathways.

Drosophila variable nurse cells encodes Arrest defective 1 (ARD1), the catalytic subunit of the major N-terminal acetyltransferase complex

PubMed Central

Wang, Ying; Mijares, Michelle; Gall, Megan D.; Turan, Tolga; Javier, Anna; Bornemann, Douglas J; Manage, Kevin; Warrior, Rahul

2010-01-01

Mutations in the Drosophila variable nurse cells (vnc) gene result in female sterility and oogenesis defects, including egg chambers with too many or too few nurse cells. We show that vnc corresponds to Arrest Defective1 (Ard1) and encodes the catalytic subunit of NatA, the major N-terminal acetyl-transferase complex. While N-terminal acetylation is one of the most prevalent covalent protein modifications in eukaryotes, analysis of its role in development has been challenging since mutants that compromise NatA activity have not been described in any multicellular animal. Our data show that reduced ARD1 levels result in pleiotropic oogenesis defects including abnormal cyst encapsulation, desynchronized cystocyte division, disrupted nurse cell chromosome dispersion and abnormal chorion patterning, consistent with the wide range of predicted NatA substrates. Further we find that loss of Ard1 affects cell survival/proliferation and is lethal for the animal, providing the first demonstration that this modification is essential in higher eukaryotes. PMID:20882681

Replacement of Cetyltrimethylammoniumbromide Bilayer on Gold Nanorod by Alkanethiol Crosslinker for Enhanced Plasmon Resonance Sensitivity

PubMed Central

Casas, Justin; Venkataramasubramani, Meenakshi; Wang, Yanyan; Tang, Liang

2013-01-01

Surface modification of gold nanorods (GNRs) is often problematic due to tightly packed cetyltrimethylammoniumbromide (CTAB) bilayer. Herein, we performed a double phase transfer ligand exchange to achieve displacement of CTAB on nanorods. During the removal, 11-mercaptoundecanoic acid (MUDA) crosslinker is simultaneously assembled on nanorod surfaces to prevent aggregation. The resulting MUDA-GNRs retain the shape and position of plasmon peaks similar to CTAB-capped GNRs. The introduction of carboxyl groups allows covalent conjugation of biological receptors in a facile fashion to construct a robust, label-free biosensor based on localized surface plasmon resonance (LSPR) transduction of biomolecular interaction. More importantly, smaller MUDA layer on the GNRs reduces the distance of target binding to the plasmonic nanostructure interface, leading to a significant enhancement in LSPR assay sensitivity and specificity. Compared to modification using conventional electropolymer adsorption, MUDA-coated gold nanosensor exhibits five times lower detection limit for cardiac troponin I assay with a high selectivity. PMID:23816849

Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius

NASA Astrophysics Data System (ADS)

Anjum, Rana S.; Bray, Sian M.; Blackwood, John K.; Kilkenny, Mairi L.; Coelho, Matthew A.; Foster, Benjamin M.; Li, Shurong; Howard, Julie A.; Pellegrini, Luca; Albers, Sonja-Verena; Deery, Michael J.; Robinson, Nicholas P.

2015-09-01

In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.

A novel surface modification technique for forming porous polymer monoliths in poly(dimethylsiloxane).

PubMed

Burke, Jeffrey M; Smela, Elisabeth

2012-03-01

A new method of surface modification is described for enabling the in situ formation of homogenous porous polymer monoliths (PPMs) within poly(dimethylsiloxane) (PDMS) microfluidic channels that uses 365 nm UV illumination for polymerization. Porous polymer monolith formation in PDMS can be challenging because PDMS readily absorbs the monomers and solvents, changing the final monolith morphology, and because PDMS absorbs oxygen, which inhibits free-radical polymerization. The new approach is based on sequentially absorbing a non-hydrogen-abstracting photoinitiator and the monomers methyl methacrylate and ethylene diacrylate within the walls of the microchannel, and then polymerizing the surface treatment polymer within the PDMS, entangled with it but not covalently bound. Four different monolith compositions were tested, all of which yielded monoliths that were securely anchored and could withstand pressures exceeding the bonding strength of PDMS (40 psi) without dislodging. One was a recipe that was optimized to give a larger average pore size, required for low back pressure. This monolith was used to concentrate and subsequently mechanical lyse B lymphocytes.

Immobilized copper(II) macrocyclic complex on MWCNTs with antibacterial activity

NASA Astrophysics Data System (ADS)

Tarlani, Aliakbar; Narimani, Khashayar; Mohammadipanah, Fatemeh; Hamedi, Javad; Tahermansouri, Hasan; Amini, Mostafa M.

2015-06-01

In a new approach, a copper(II) tetraaza macrocyclic complex (CuTAM) was covalently bonded on modified multi-walled carbon nanotubes (MWCNTs). To achieve this purpose, MWCNTs were converted to MWCNT-COCl and then reacted to NH groups of TAM ligand. The prepared material was characterized by Fourier Transform Infrared (FT-IR), X-ray diffraction (XRD), Raman spectroscopy, thermal gravimetric analysis (TGA), and FESEM (field emission scanning electron microscopy). FT-IR and TGA demonstrated the presence of the organic moieties, and XRD proved that the structure of MWCNTs remained intact during the three modification steps. An increase in the ID/IG ratio in Raman spectra confirmed the surface modifications. Finally, the samples were subjected to an antibacterial assessment to compare their biological activity. The antibacterial test showed that the grafted complex on the surface of the nanotube (MWCNT-CO-CuTAM) has higher antibacterial activity against Bacillus subtilis ATCC 6633 than the MWCNT-COOH and CuTAM with 1000 and 2000 μg/mL.

Covalently bonded networks through surface-confined polymerization

NASA Astrophysics Data System (ADS)

El Garah, Mohamed; MacLeod, Jennifer M.; Rosei, Federico

2013-07-01

The prospect of synthesizing ordered, covalently bonded structures directly on a surface has recently attracted considerable attention due to its fundamental interest and for potential applications in electronics and photonics. This prospective article focuses on efforts to synthesize and characterize epitaxial one- and two-dimensional (1D and 2D, respectively) polymeric networks on single crystal surfaces. Recent studies, mostly performed using scanning tunneling microscopy (STM), demonstrate the ability to induce polymerization based on Ullmann coupling, thermal dehalogenation and dehydration reactions. The 2D polymer networks synthesized to date have exhibited structural limitations and have been shown to form only small domains on the surface. We discuss different approaches to control 1D and 2D polymerization, with particular emphasis on the surface phenomena that are critical to the formation of larger ordered domains.

Method of using a germanium layer transfer to Si for photovoltaic applications and heterostructure made thereby

DOEpatents

Atwater, Jr., Harry A.; Zahler, James M.

2006-11-28

Ge/Si and other nonsilicon film heterostructures are formed by hydrogen-induced exfoliation of the Ge film which is wafer bonded to a cheaper substrate, such as Si. A thin, single-crystal layer of Ge is transferred to Si substrate. The bond at the interface of the Ge/Si heterostructures is covalent to ensure good thermal contact, mechanical strength, and to enable the formation of an ohmic contact between the Si substrate and Ge layers. To accomplish this type of bond, hydrophobic wafer bonding is used, because as the invention demonstrates the hydrogen-surface-terminating species that facilitate van der Waals bonding evolves at temperatures above 600.degree. C. into covalent bonding in hydrophobically bound Ge/Si layer transferred systems.

Fabrication of nanoscale heterostructures comprised of graphene-encapsulated gold nanoparticles and semiconducting quantum dots for photocatalysis.

PubMed

Li, Yuan; Chopra, Nitin

2015-05-21

Patterned growth of multilayer graphene shell encapsulated gold nanoparticles (GNPs) and their covalent linking with inorganic quantum dots are demonstrated. GNPs were grown using a xylene chemical vapor deposition process, where the surface oxidized gold nanoparticles catalyze the multilayer graphene shell growth in a single step process. The graphene shell encapsulating gold nanoparticles could be further functionalized with carboxylic groups, which were covalently linked to amine-terminated quantum dots resulting in GNP-quantum dot heterostructures. The compositions, morphologies, crystallinity, and surface functionalization of GNPs and their heterostructures with quantum dots were evaluated using microscopic, spectroscopic, and analytical methods. Furthermore, optical properties of the derived architectures were studied using both experimental methods and simulations. Finally, GNP-quantum dot heterostructures were studied for photocatalytic degradation of phenol.

Biofunctionalized Zinc Oxide Field Effect Transistors for Selective Sensing of Riboflavin with Current Modulation

PubMed Central

Hagen, Joshua A.; Kim, Sang N.; Bayraktaroglu, Burhan; Leedy, Kevin; Chávez, Jorge L.; Kelley-Loughnane, Nancy; Naik, Rajesh R.; Stone, Morley O.

2011-01-01

Zinc oxide field effect transistors (ZnO-FET), covalently functionalized with single stranded DNA aptamers, provide a highly selective platform for label-free small molecule sensing. The nanostructured surface morphology of ZnO provides high sensitivity and room temperature deposition allows for a wide array of substrate types. Herein we demonstrate the selective detection of riboflavin down to the pM level in aqueous solution using the negative electrical current response of the ZnO-FET by covalently attaching a riboflavin binding aptamer to the surface. The response of the biofunctionalized ZnO-FET was tuned by attaching a redox tag (ferrocene) to the 3′ terminus of the aptamer, resulting in positive current modulation upon exposure to riboflavin down to pM levels. PMID:22163977

Molecular and electronic structures of M 2O 7 (M = Mn, Tc, Re)

DOE PAGES

Lawler, Keith V.; Childs, Bradley C.; Mast, Daniel S.; ...

2017-02-21

The molecular and electronic structures for the Group 7b 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 Re 2O 7 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. The calculations accurately reproduce the experimental crystal structures within a few percent. Analysis of the band structures and density ofmore » states shows similar bonding for all of the solid-state heptoxides, including the presence of the three-center covalent bond. DFT+U simulations show that PBE-D3 underpredicts the band gap by ~0.2 eV due to an under-correlation of the metal d conducting states. As a result, homologue and compression studies show that Re 2O 7 adopts a polymeric structure because the Re-oxide tetrahedra are easily distorted by packing stresses to form additional three-center covalent bonds.« less

Surface modification of copolymerized films from three-armed biodegradable macromers - An analytical platform for modified tissue engineering scaffolds.

PubMed

Müller, Benno M; Loth, Rudi; Hoffmeister, Peter-Georg; Zühl, Friederike; Kalbitzer, Liv; Hacker, Michael C; Schulz-Siegmund, Michaela

2017-03-15

The concept of macromers allows for a broad adjustment of biomaterial properties by macromer chemistry or copolymerization. Copolymerization strategies can also be used to introduce reactive sites for subsequent surface modification. Control over surface features enables adjustment of cellular reactions with regard to site and object of implantation. We designed macromer-derived polymer films which function as non-implantable analytical substrates for the investigation of surface properties of equally composed scaffolds for bone tissue engineering. To this end, a toolbox of nine different biodegradable, three-armed macromers was thermally cross-copolymerized with poly(ethylene glycol)-methacrylate (PEG-MA) to films. Subsequent activation of PEG-hydroxyl groups with succinic anhydride and N-hydroxysuccinimid allowed for covalent surface modification. We quantified the capacity to immobilize analytes of low (amino-functionalized fluorescent dye, Fcad, and RGD-peptides) and high (alkaline phosphatase, ALP) molecular weight. Fcad grafting level was controlled by macromer chemistry, content and molecular weight of PEG-MA, but also the solvent used for film synthesis. Fcad molar amount per surface area was twentyfive times higher on high-swelling compared to low-swelling films, but differences became smaller when large ALP (appr. 2:1) were employed. Similarly, small differences were observed on RGD peptide functionalized films that were investigated by cell adhesion studies. Presentation of PEG-derivatives on surfaces was visualized by atomic force microscopy (AFM) which unraveled composition-dependent domain formation influencing fluorescent dye immobilization. Surface wetting characteristics were investigated via static water contact angle. We conclude that macromer ethoxylation and lactic acid content determined film swelling, PEG domain formation and eventually efficiency of surface decoration. Surfaces of implantable biomaterials are the site of interaction with a host tissue. Accordingly, modifications in the composition of the surface will determine cellular response towards the material which is crucial for the success of innovations and control of tissue regeneration. We employed a macromer approach which is most flexible for the design of biomaterials with a broad spectrum of physicochemical characteristics. For ideal analytical accessibility of the material platform, we cross-copolymerized films on solid supports. Films allowed for the covalent immobilization of fluorescent labels, peptides and enzymes and thorough analytical characterization revealed that macromer hydrophilicity is the most relevant design parameter for surface analyte presentation in these materials. All analytical results were combined in a model describing PEG linker domain formation and ligand presentation. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Protein electrochemistry using graphene-based nano-assembly: an ultrasensitive electrochemical detection of protein molecules via nanoparticle-electrode collisions.

PubMed

Li, Da; Liu, Jingquan; Barrow, Colin J; Yang, Wenrong

2014-08-04

We describe a new electrochemical detection approach towards single protein molecules (microperoxidase-11, MP-11), which are attached to the surface of graphene nanosheets. The non-covalently functionalized graphene nanosheets exhibit enhanced electroactive surface area, where amplified redox current is produced when graphene nanosheets collide with the electrode.

Infrared and Raman Spectroscopy: A Discovery-Based Activity for the General Chemistry Curriculum

ERIC Educational Resources Information Center

Borgsmiller, Karen L.; O'Connell, Dylan J.; Klauenberg, Kathryn M.; Wilson, Peter M.; Stromberg, Christopher J.

2012-01-01

A discovery-based method is described for incorporating the concepts of IR and Raman spectroscopy into the general chemistry curriculum. Students use three sets of springs to model the properties of single, double, and triple covalent bonds. Then, Gaussian 03W molecular modeling software is used to illustrate the relationship between bond…

Chemistry of carbon nanomaterials: Uses of lithium nanotube salts in organic syntheses and functionalization of graphite

NASA Astrophysics Data System (ADS)

Chattopadhyay, Jayanta

The effective utilization of carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs) and graphite, has been hindered due to difficulties (poor solubility, poly-dispersity) in processing. Therefore, a high degree of sidewall functionalization, either covalent or non-covalent, is often required to overcome these difficulties as the functionalized nanomaterials exhibit better solubility (either in organic solvents or in water), dispersity, manipulation, and processibility. This thesis presents a series of convenient and efficient organic synthetic routes to functionalize carbon nanomaterials. Carbon nanotube salts, prepared by treating SWNTs with lithium in liquid ammonia, react readily with aryl halides to yield aryl-functionalized SWNTs. These arylated SWNTs are soluble in methanol and water upon treatment with oleum. Similarly, SWNTs can be covalently functionalized by different heteroatoms (nitrogen, oxygen, and sulfur). Using the reductive alkylation approach, a synthetic scheme is designed to prepare long chain carboxylic acid functionalized SWNTs [SWNTs-(RCOOH)] that can react with (1) amine-terminated polyethylene glycol (PEG) chains to yield water-soluble biocompatible PEGylated SWNTs that are likely to be useful in a variety of biomedical applications; (2) polyethyleneimine (PEI) to prepare a SWNTs-PEI based adsorbent material that shows a four-fold improvement in the adsorption capacity of carbon dioxide over commonly used materials, making it useful for regenerable carbon dioxide removal in spaceflight; (3) chemically modified SWNTs-(RCOOH) to permit covalent bonding to the nylon matrix, thus allowing the formation of nylon 6,10 and nylon 6,10/SWNTs-(RCOOH) nanocomposites. Furthermore, we find that the lithium salts of carbon nanotubes serve as a source of electrons to induce polymerization of simple alkenes and alkynes onto the surface of carbon nanotubes. In the presence of sulfide/disulfide bonds, SWNT salts can initiate the single electron transfer (SET) mechanism to functionalize carbon nanotubes with different alkyl/aryl groups. Using the reductive alkylation approach, we can also functionalize graphites by alkyl/carboxylic acid groups, making graphite soluble in organic solvents and water. Tailoring of graphite layers is also accomplished by using different metals in liquid ammonia. Finally, SWNT-epoxides/graphite epoxides are synthesized using m-CPBA. Quantification of the epoxide substituents on the nanotube/graphite surface is evaluated through the catalytic de-epoxidation reaction using MeReO 3/PPh3 as heterogeneous catalyst. In summary, the proposed covalent functionalization methods yield derivatized nanomaterials that can provide a solid platform for a number of exciting applications, ranging from material science to biomedical devices. Furthermore, the results presented in this thesis provide insight into the molecular chemistry at nano-resolution.

An In-Line Photonic Biosensor for Monitoring of Glucose Concentrations

PubMed Central

Al-Halhouli, Ala'aldeen; Demming, Stefanie; Alahmad, Laila; LIobera, Andreu; Büttgenbach, Stephanus

2014-01-01

This paper presents two PDMS photonic biosensor designs that can be used for continuous monitoring of glucose concentrations. The first design, the internally immobilized sensor, consists of a reactor chamber, micro-lenses and self-alignment structures for fiber optics positioning. This sensor design allows optical detection of glucose concentrations under continuous glucose flow conditions of 33 μL/h based on internal co-immobilization of glucose oxidase (GOX) and horseradish peroxidase (HRP) on the internal PDMS surface of the reactor chamber. For this design, two co-immobilization methods, the simple adsorption and the covalent binding (PEG) methods were tested. Experiments showed successful results when using the covalent binding (PEG) method, where glucose concentrations up to 5 mM with a coefficient of determination (R2) of 0.99 and a limit of detection of 0.26 mM are detectable. The second design is a modified version of the internally immobilized sensor, where a microbead chamber and a beads filling channel are integrated into the sensor. This modification enabled external co-immobilization of enzymes covalently onto functionalized silica microbeads and allows binding a huge amount of HRP and GOX enzymes on the microbeads surfaces which increases the interaction area between immobilized enzymes and the analyte. This has a positive effect on the amount and rate of chemical reactions taking place inside the chamber. The sensor was tested under continuous glucose flow conditions and was found to be able to detect glucose concentrations up to 10 mM with R2 of 0.98 and a limit of detection of 0.7 mM. Such results are very promising for the application in photonic LOC systems used for online analysis. PMID:25157552

Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains.

PubMed

de la Peña, Pilar; Domínguez, Pedro; Barros, Francisco

2018-03-01

Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4-S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4-S5 linker induces an increasing negative shift in activation voltage dependence, a reduced z g value and a more negative ΔG 0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4-S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4-S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.

A perspective on reagent diversity and non-covalent binding of reactive carbonyl species (RCS) and effector reagents in nonenzymatic glycation (NEG): Mechanistic considerations and implications for future research

NASA Astrophysics Data System (ADS)

Rodnick, Kenneth J.; Holman, R. W.; Ropski, Pamela S.; Huang, Mingdong; Swislocki, Arthur L. M.

2017-06-01

This perspective focuses on illustrating the underappreciated connections between reactive carbonyl species (RCS), initial binding in the nonenzymatic glycation (NEG) process, and nonenzymatic covalent protein modification (here termed NECPM). While glucose is the central species involved in NEG, recent studies indicate that the initially-bound glucose species in the NEG of human hemoglobin (HbA) and human serum albumin (HSA) are non-RCS ring-closed isomers. The ring-opened glucose, an RCS structure that reacts in the NEG process, is most likely generated from previously-bound ring-closed isomers undergoing concerted acid/base reactions while bound to protein. The generation of the glucose RCS can involve concomitantly-bound physiological species (e.g., inorganic phosphate, water, etc.); here termed effector reagents. Extant NEG schemes do not account for these recent findings. In addition, effector reagent reactions with glucose in the serum and erythrocyte cytosol can generate RCS (e.g., glyoxal, glyceraldehyde, etc.). Recent research has shown that these RCS covalently modify proteins in vivo via NECPM mechanisms. A general scheme that reflects both the reagent and mechanistic diversity that can lead to NEG and NECPM is presented here. A perspective that accounts for the relationships between RCS, NEG, and NECPM can facilitate the understanding of site selectivity, may help explain overall glycation rates, and may have implications for the clinical assessment/control of diabetes mellitus. In view of this perspective, concentrations of ribose, fructose, Pi, bicarbonate, counter ions, and the resulting RCS generated within intracellular and extracellular compartments may be of importance and of clinical relevance. Future research is also proposed.

Site specific modification of the human plasma proteome by methylglyoxal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimzey, Michael J.; Kinsky, Owen R.; Yassine, Hussein N.

Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-argininemore » modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may represent a useful monitor of effective therapy of T2DM.« less

DehydroalanylGly, a new post translational modification resulting from the breakdown of glutathione.

PubMed

Friedrich, Michael G; Wang, Zhen; Schey, Kevin L; Truscott, Roger J W

2018-04-01

The human body contains numerous long-lived proteins which deteriorate with age, typically by racemisation, deamidation, crosslinking and truncation. Previously we elucidated one reaction responsible for age-related crosslinking, the spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine and cysteine. This resulted in non-disulphide covalent crosslinks. The current paper outlines a novel posttranslational modification (PTM) in human proteins, which involves the addition of dehydroalanylglycine (DHAGly) to Lys residues. Human lens digests were examined by mass spectrometry for the presence of (DHA)Gly (+144.0535 Da) adducts to Lys residues. Peptide model studies were undertaken to elucidate the mechanism of formation. In the lens, this PTM was detected at 18 lysine sites in 7 proteins. Using model peptides, a pathway for its formation was found to involve initial formation of the glutathione degradation product, γ-Glu(DHA)Gly from oxidised glutathione (GSSG). Once the Lys adduct formed, the Glu residue was lost in a hydrolytic mechanism apparently catalysed by the ε-amino group of the Lys. This discovery suggests that within cells, the functional groups of amino acids in proteins may be susceptible to modification by reactive metabolites derived from GSSG. Our finding demonstrates a novel +144.0535 Da PTM arising from the breakdown of oxidised glutathione. Copyright © 2018. Published by Elsevier B.V.

Suppression of protein adsorption on a charged phospholipid polymer interface.

PubMed

Xu, Yan; Takai, Madoka; Ishihara, Kazuhiko

2009-02-09

High capability of a charged interface to suppress adsorption of both anionic and cationic proteins was reported. The interface was covalently constructed on quartz by modifying with an anionic phospholipid copolymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-potassium 3-methacryloyloxypropyl sulfonate (PMPS)-co-3-methacryloxypropyl trimethoxysilane (MPTMSi)) (PMBSSi). The PMBSSi interfaces were very hydrophilic and homogeneous and could function effectively for a long time even under long-term fluidic working conditions. The PMBSSi density on the interface, which was controllable by adjusting the PMBSSi concentration of the modification solution, affected the surface properties, including the surface contact angle, the surface roughness, and the surface zeta-potential. When a PMBSSi modification was applied, the adsorption of various proteins (isoelectric point varying from 1.0 to 11.0) on quartz was reduced to at least 87% in amount, despite the various electrical natures these proteins have. The protein adsorption behavior on the PMBSSi interface depended more on the PMBSSi density than on the surface charge. The PMBSSi modification had a stable impact on the surface, not only at the physiologic ionic strength, but also over a range of the ionic strength, suggesting that electrostatic interactions do not dominate the behavior of protein adsorption to the PMBSSi surface.

Identification of the lipopolysaccharide modifications controlled by the Salmonella PmrA/PmrB system mediating resistance to Fe(III) and Al(III)

PubMed Central

Nishino, Kunihiko; Hsu, Fong-Fu; Turk, John; Cromie, Michael J; Wösten, Marc M S M; Groisman, Eduardo A

2006-01-01

Iron is an essential metal but can be toxic in excess. While several homeostatic mechanisms prevent oxygen-dependent killing promoted by Fe(II), little is known about how cells cope with Fe(III), which kills by oxygen-independent means. Several Gram-negative bacterial species harbour a regulatory system – termed PmrA/PmrB – that is activated by and required for resistance to Fe(III). We now report the identification of the PmrA-regulated determinants mediating resistance to Fe(III) and Al(III) in Salmonella enterica serovar Typhimurium. We establish that these determinants remodel two regions of the lipopolysaccharide, decreasing the negative charge of this major constituent of the outer membrane. Remodelling entails the covalent modification of the two phosphates in the lipid A region with phosphoethanolamine and 4-aminoarabinose, which has been previously implicated in resistance to polymyxin B, as well as dephosphorylation of the Hep(II) phosphate in the core region by the PmrG protein. A mutant lacking the PmrA-regulated Fe(III) resistance genes bound more Fe(III) than the wild-type strain and was defective for survival in soil, suggesting that these PmrA-regulated lipopolysaccharide modifications aid Salmonella's survival and spread in non-host environments. PMID:16803591

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

PubMed Central

Yu, Seok-Ho; Boyce, Michael; Wands, Amberlyn M.; Bond, Michelle R.; Bertozzi, Carolyn R.; Kohler, Jennifer J.

2012-01-01

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation. PMID:22411826

Mechanical gating of a mechanochemical reaction cascade

NASA Astrophysics Data System (ADS)

Wang, Junpeng; Kouznetsova, Tatiana B.; Boulatov, Roman; Craig, Stephen L.

2016-11-01

Covalent polymer mechanochemistry offers promising opportunities for the control and engineering of reactivity. To date, covalent mechanochemistry has largely been limited to individual reactions, but it also presents potential for intricate reaction systems and feedback loops. Here we report a molecular architecture, in which a cyclobutane mechanophore functions as a gate to regulate the activation of a second mechanophore, dichlorocyclopropane, resulting in a mechanochemical cascade reaction. Single-molecule force spectroscopy, pulsed ultrasonication experiments and DFT-level calculations support gating and indicate that extra force of >0.5 nN needs to be applied to a polymer of gated gDCC than of free gDCC for the mechanochemical isomerization gDCC to proceed at equal rate. The gating concept provides a mechanism by which to regulate stress-responsive behaviours, such as load-strengthening and mechanochromism, in future materials designs.

Self-generated covalent cross-links in the cell-surface adhesins of Gram-positive bacteria.

PubMed

Baker, Edward N; Squire, Christopher J; Young, Paul G

2015-10-01

The ability of bacteria to adhere to other cells or to surfaces depends on long, thin adhesive structures that are anchored to their cell walls. These structures include extended protein oligomers known as pili and single, multi-domain polypeptides, mostly based on multiple tandem Ig-like domains. Recent structural studies have revealed the widespread presence of covalent cross-links, not previously seen within proteins, which stabilize these domains. The cross-links discovered so far are either isopeptide bonds that link lysine side chains to the side chains of asparagine or aspartic acid residues or ester bonds between threonine and glutamine side chains. These bonds appear to be formed by spontaneous intramolecular reactions as the proteins fold and are strategically placed so as to impart considerable mechanical strength. © 2015 Authors; published by Portland Press Limited.

Covalently Bound Monomolecular Layers on Si Single Crystals

NASA Astrophysics Data System (ADS)

Chidsey, Christopher E. D.

1996-03-01

Methods and reagents borrowed from the molecular synthetic chemistry of silicon compounds have been used to form covalently bound monomolecular layers on silicon single crystals. Organic monolayers bound covalently to silicon could form the basis for silicon/organic interfaces useful in sensor structures. In a representative reaction, alkyl monolayers with densities approaching that of crystalline polyethylene have been prepared by the radical-initiated insertion of 1-alkenes into the Si-H bonds of hydrogen-terminated Si(111) surfaces footnote M. R. Linford, P. Fenter, P. M. Eisenberger and C. E. D Chidsey, J. Am. Chem. Soc. 117, 3145-3155 (1995). It has recently been found that this insertion reaction can also be initiated by illumination with UV light having sufficient energy to break the Si-H bond. Synchrotron-based high-resolution photoelectron spectroscopy and diffraction have demonstrated the expected Si-C bond in such monolayers footnote J. H. Terry, R. Cao, P. A. Pianetta, M. R. Linford and C. E. D. Chidsey, unpublished results. An alternate approach to similar monolayers has been found to be the chlorination of hydrogen-terminated Si(111) with Cl_2, followed by the nucleophilic displacement of chlorine with alkyl lithium reagents. The well-behaved chemical transformations of the hydrogen-terminated silicon surfaces appear to result from the essentially bulk termination of the silicon lattice with closed-shell silicon hydride "functional groups" on the surface. In addition to the formation of novel organic layers, a full understanding of the reactivity of the hydrogen-terminated silicon surfaces should lead to better control of key technological silicon interfaces such as Si/SiO_2, Si/epi-Si, and Si/metal.

Molecular cloning and biochemical characterization of rabbit factor XI.

PubMed Central

Sinha, Dipali; Marcinkiewicz, Mariola; Gailani, David; Walsh, Peter N

2002-01-01

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160 kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80 kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process. PMID:12084014

Facile and green reduction of covalently PEGylated nanographene oxide via a `water-only' route for high-efficiency photothermal therapy

NASA Astrophysics Data System (ADS)

Chen, Jingqin; Wang, Xiaoping; Chen, Tongsheng

2014-02-01

A facile and green strategy is reported for the fabrication of nanosized and reduced covalently PEGylated graphene oxide (nrGO-PEG) with great biocompatibility and high near-infrared (NIR) absorbance. Covalently PEGylated nGO (nGO-PEG) was synthesized by the reaction of nGO-COOH and methoxypolyethylene glycol amine (mPEG-NH2). The neutral and purified nGO-PEG solution was then directly bathed in water at 90°C for 24 h without any additive to obtain nrGO-PEG. Covalent PEGylation not only prevented the aggregation of nGO but also dramatically promoted the reduction extent of nGO during this reduction process. The resulting single-layered nrGO-PEG sheets were approximately 50 nm in average lateral dimension and exhibited great biocompatibility and approximately 7.6-fold increment in NIR absorption. Moreover, this facile reduction process repaired the aromatic structure of GO. CCK-8 and flow cytometry (FCM) assays showed that exposure of A549 cells to 100 μg/mL of nrGO-PEG for 2 h, exhibiting 71.5% of uptake ratio, did not induce significant cytotoxicity. However, after irradiation with 808 nm laser (0.6 W/cm2) for 5 min, the cells incubated with 6 μg/mL of nrGO-PEG solution showed approximately 90% decrease of cell viability, demonstrating the high-efficiency photothermal therapy of nrGO-PEG to tumor cells in vitro. This work established nrGO-PEG as a promising photothermal agent due to its small size, great biocompatibility, high photothermal efficiency, and low cost.

Activity-based assay for human mono-ADP-ribosyltransferases ARTD7/PARP15 and ARTD10/PARP10 aimed at screening and profiling inhibitors.

PubMed

Venkannagari, Harikanth; Fallarero, Adyary; Feijs, Karla L H; Lüscher, Bernhard; Lehtiö, Lari

2013-05-13

Poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin like ADP-ribosyl transferases (ARTDs) are enzymes that catalyze the covalent modification of proteins by attachment of ADP-ribose units to the target amino acid residues or to the growing chain of ADP-ribose. A subclass of the ARTD superfamily consists of mono-ADP-ribosyl transferases that are thought to modify themselves and other substrate proteins by covalently adding only a single ADP-ribose moiety to the target. Many of the ARTD enzymes are either established or potential drug targets and a functional activity assay for them will be a valuable tool to identify selective inhibitors for each enzyme. Existing assays are not directly applicable for screening of inhibitors due to the different nature of the reaction and different target molecules. We modified and applied a fluorescence-based assay previously described for PARP1/ARTD1 and tankyrase/ARTD5 for screening of PARP10/ARTD10 and PARP15/ARTD7 inhibitors. The assay measures the amount of NAD(+) present after chemically converting it to a fluorescent analog. We demonstrate that by using an excess of a recombinant acceptor protein the performance of the activity-based assay is excellent for screening of compound libraries. The assay is homogenous and cost effective, making it possible to test relatively large compound libraries. This method can be used to screen inhibitors of mono-ARTDs and profile inhibitors of the enzyme class. The assay was optimized for ARTD10 and ARTD7, but it can be directly applied to other mono-ARTDs of the ARTD superfamily. Profiling of known ARTD inhibitors against ARTD10 and ARTD7 in a validatory screening identified the best inhibitors with submicromolar potencies. Only few of the tested ARTD inhibitors were potent, implicating that there is a need to screen new compound scaffolds. This is needed to create small molecules that could serve as biological probes and potential starting points for drug discovery projects against mono-ARTDs. Copyright © 2013 Elsevier B.V. All rights reserved.

Decoding Ca2+ signals in plants

NASA Technical Reports Server (NTRS)

Sathyanarayanan, P. V.; Poovaiah, B. W.

2004-01-01

Different input signals create their own characteristic Ca2+ fingerprints. These fingerprints are distinguished by frequency, amplitude, duration, and number of Ca2+ oscillations. Ca(2+)-binding proteins and protein kinases decode these complex Ca2+ fingerprints through conformational coupling and covalent modifications of proteins. This decoding of signals can lead to a physiological response with or without changes in gene expression. In plants, Ca(2+)-dependent protein kinases and Ca2+/calmodulin-dependent protein kinases are involved in decoding Ca2+ signals into phosphorylation signals. This review summarizes the elements of conformational coupling and molecular mechanisms of regulation of the two groups of protein kinases by Ca2+ and Ca2+/calmodulin in plants.

[Coactivators in energy metabolism: peroxisome proliferator-activated receptor-gamma coactivator 1 family].

PubMed

Wang, Rui; Chang, Yong-sheng; Fang, Fu-de

2009-12-01

Peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1) family is highly expressed in tissues with high energy metabolism. They coactivate transcription factors in regulating genes engaged in processes such as gluconeogenesis, adipose beta-oxydation, lipoprotein synthesis and secretion, mitochondrial biogenesis, and oxidative metabolism. Protein conformation studies demonstrated that they lack DNA binding domains and act as coactivators through physical interaction with transcription factors. PGC1 activity is regulated at transcription level or by multiple covalent chemical modifications such as phosphorylation, methylation and acetylation/deacetylation. Abnormal expression of PGC1 coactivators usually is closely correlated with diseases such as diabetes, obesity, hyperglycemia, hyperlipemia, and arterial and brain neuron necrosis diseases.

Ubiquitin-dependent endocytosis, trafficking and turnover of neuronal membrane proteins

PubMed Central

Schwarz, Lindsay A.; Patrick, Gentry N.

2011-01-01

Extracellular signaling between cells is often transduced via receptors that reside at the cell membrane. In neurons this receptor-mediated signaling can promote a variety of cellular events such as differentiation, axon outgrowth and guidance, synaptic development and function. Endocytic membrane trafficking of receptors can ensure that the strength and duration of an extracellular signal is properly regulated. The covalent modification of membrane proteins by ubiquitin is a key biological mechanism to control receptor internalization and endocytic sorting to recycling and degradative pathways in many cell types. In this review we highlight recent findings regarding the ubiquitin-dependent trafficking and turnover of receptors in neurons and the implications for neuronal development and function. PMID:21884797

Thiol-ene mediated neoglycosylation of collagen patches: a preliminary study.

PubMed

Russo, Laura; Battocchio, Chiara; Secchi, Valeria; Magnano, Elena; Nappini, Silvia; Taraballi, Francesca; Gabrielli, Luca; Comelli, Francesca; Papagni, Antonio; Costa, Barbara; Polzonetti, Giovanni; Nicotra, Francesco; Natalello, Antonino; Doglia, Silvia M; Cipolla, Laura

2014-02-11

Despite the relevance of carbohydrates as cues in eliciting specific biological responses, the covalent surface modification of collagen-based matrices with small carbohydrate epitopes has been scarcely investigated. We report thereby the development of an efficient procedure for the chemoselective neoglycosylation of collagen matrices (patches) via a thiol-ene approach, between alkene-derived monosaccharides and the thiol-functionalized material surface. Synchrotron radiation-induced X-ray photoelectron spectroscopy (SR-XPS), Fourier transform-infrared (FT-IR), and enzyme-linked lectin assay (ELLA) confirmed the effectiveness of the collagen neoglycosylation. Preliminary biological evaluation in osteoarthritic models is reported. The proposed methodology can be extended to any thiolated surface for the development of smart biomaterials for innovative approaches in regenerative medicine.