共查询到20条相似文献,搜索用时 125 毫秒
1.
《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2017,129(10):2788-2792
Biochemical studies of cellular processes involving polyubiquitin have gained increasing attention. More tools are needed to identify ubiquitin (Ub)‐binding proteins. We report diazirine‐based photoaffinity probes that can capture Ub‐binding proteins in cell lysates, and show that diazirines are preferable to aryl azides as the photo‐crosslinking group, since they decrease non‐selective capture. Photoaffinity probes containing at least two Ub units were required to effectively capture Ub‐binding proteins. Different capture selectivity was observed for probes containing diubiquitin moieties with different types of linkages, thus indicating the potential to develop linkage‐dependent probes for selectively profiling Ub‐binding proteins under various cellular conditions. 相似文献
2.
Inorganic arsenic that is ingested through drinking water or inhalation is metabolized by biological methylation pathways into organoarsenical metabolites. It is now becoming understood that this metabolism that was formerly considered to be detoxification may contribute as much or more to increasing the toxicity of arsenic. One proposed mode of the toxic action of arsenic and its organoarsenic metabolites is through its binding to proteins and inactivating their enzymatic activity. The classic case has been considered the affinity of the proximal 1,3 sulfhydryl groups of the lipoic acid cofactor of the pyruvate dehydrogenase complex for arsenic. A 2:1 stoichiometry of sulfhydryl to arsenic groups has been measured in proteins and arsenical complexes can be synthesized using free d,l-lipoic acid. The relative importance of this site for arsenic binding has come in to question through the use of methylating bifunctional arsenic complexes, and the suggestion that arsenic inhibits the pyruvate dehydrogenase complex indirectly by elevating mitochondrial hydrogen peroxide generation. In order to separate the effects of direct trivalent arsenite toxicity from that of hydrogen peroxide and activated oxygen, we studied the inhibition of the PDH complex under conditions that did not generate hydrogen peroxide but did expose the lipoic acid group in its reduced state to arsenicals. We also studied the effects of arsenicals in the inhibition of the α-ketoglutarate dehydrogenase complex. We found that only trivalent arsenical compounds inhibited the activity of both dehydrogenase complexes and only when the lipoic acid was in its reduced form. Arsenite inhibited both enzyme complexes approximately equivalently while monomethylarsenite inhibited the PDH complex to a greater extent than the KGDH complex - although both complexes were very sensitive to inhibition by this complex. Dimethylarsenite inhibition of both complexes was only observed with longer pre-incubation periods. Cumulative inhibition by the reduced arsenical was observed for all complexes indicating a binding mode of inhibition that is dependent upon lipoic acid being in its reduced state. 相似文献
3.
Fluorophore‐Conjugated Holliday Junctions for Generating Super‐Bright Antibodies and Antibody Fragments 下载免费PDF全文
Zeyang Li Christopher S. Theile Guan‐Yu Chen Angelina M. Bilate Joao N. Duarte Ana M. Avalos Tao Fang Roberto Barberena Shuji Sato Hidde L. Ploegh 《Angewandte Chemie (International ed. in English)》2015,54(40):11706-11710
The site‐specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self‐quenching from multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site‐specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooctyne substituent to render it reactive with an azide‐modified fluorescent single‐domain antibody fragment or an intact immunoglobulin produced in a sortase‐catalyzed reaction. These fluorescent Holliday junctions improve fluorescence yields for both single‐domain and full‐sized antibodies without deleterious effects on antigen binding. 相似文献
4.
Mara Colzani Willy V. Bienvenut Eveline Faes Manfredo Quadroni 《Rapid communications in mass spectrometry : RCM》2009,23(22):3570-3578
Stable isotope labels are routinely introduced into proteomes for quantification purposes. Full labeling of cells in varying biological states, followed by sample mixing, fractionation and intensive data acquisition, is used to obtain accurate large‐scale quantification of total protein levels. However, biological processes often affect only a small group of proteins for a short time, resulting in changes that are difficult to detect against the total proteome background. An alternative approach could be the targeted analysis of the proteins synthesized in response to a given biological stimulus. Such proteins can be pulse‐labeled with a stable isotope by metabolic incorporation of ‘heavy’ amino acids. In this study we investigated the specific detection and identification of labeled proteins using acquisition methods based on Precursor Ion Scans (PIS) on a triple‐quadrupole ion trap mass spectrometer. PIS‐based methods were set to detect unique immonium ions originating from labeled peptides. Different labels and methods were tested in standard mixtures to optimize performance. We showed that, in comparison with an untargeted analysis on the same instrument, the approach allowed a several‐fold increase in the specificity of detection of labeled proteins over unlabeled ones. The technique was applied to the identification of proteins secreted by human cells into growth media containing bovine serum proteins, allowing the preferential detection of labeled cellular proteins over unlabeled bovine ones. However, compared with untargeted acquisitions on two different instruments, the PIS‐based strategy showed some limitations in sensitivity. We discuss possible perspectives of the technique. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
5.
This work is part of an ongoing research study towards an understanding of the complete metabolism of arsenosugars in mammalian organisms when ingesting seaweed, using the North Ronaldsay (NR) sheep as a model organism. We focus on the analysis of only those arsenic species bound to the lipids of the feed (Laminaria digitata), faeces and the tissues of the NR sheep using a novel enzymatic hydrolytic method that is simple and reliable. This rare breed of sheep, found in the remote Orkney Islands in the north of Scotland, live the entire year on the beaches and eat seaweed that is washed ashore (up to 3 kg daily). Previous studies on arsenic fractionation in muscle, kidney and liver tissues revealed that most of the arsenic is concentrated in the fat fractions of these tissues (muscle fat: 61%; liver fat: 66%; kidney fat: 25%) rather than in the non‐lipid fractions. Hence, this study was undertaken in order to determine the arsenic species bound to lipids in the muscle, kidney and faeces of NR sheep and to compare these with the arsenic species bound to the lipids of the L. digitata consumed. The enzymatic hydrolytic procedure has been successfully employed for the first time to cleave the arsenic species cleanly from the rest of the lipid structure. This makes the arsenic species water soluble and enables their direct determination by high‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry. Dimethylarsinic acid (DMA(V)) and monomethylarsonic acid (MA(V)) were found to be the major hydrolysed arsenic species bound to the kidney and muscle lipids, whereas arsenosugar‐1 was found to be the major hydrolysed arsenic species in L. digitata lipids. On the other hand, DMA(V) was found to be the major arsenical obtained after the enzymatic hydrolysis of the faeces lipids. These results seem to suggest that both direct absorption and biotransformation of the absorbed organoarsenicals are the likely reasons for their occurrence and accumulation in the NR sheep tissues. Copyright © 2003 John Wiley & Sons, Ltd. 相似文献
6.
Labeling Cell Surface GPIs and GPI‐Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives 下载免费PDF全文
Dr. Lili Lu Dr. Jian Gao Prof. Dr. Zhongwu Guo 《Angewandte Chemie (International ed. in English)》2015,54(33):9679-9682
Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI‐anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell‐surface GPIs and GPI‐anchored proteins by using inositol derivatives carrying an azido group. The azide‐labeled GPIs and GPI‐anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin‐conjugated dyes or other molecules. The strategy can be used to label GPI‐anchored proteins with various tags for biological studies. 相似文献
7.
Arsenic‐Containing Phosphatidylcholines: A New Group of Arsenolipids Discovered in Herring Caviar 下载免费PDF全文
Sandra A. Viczek Dr. Kenneth B. Jensen Prof. Dr. Kevin A. Francesconi 《Angewandte Chemie (International ed. in English)》2016,55(17):5259-5262
A new group of arsenolipids based on cell‐membrane phosphatidylcholines has been discovered in herring caviar (fish roe). A combination of HPLC with elemental and molecular mass spectrometry was used to identify five arsenic‐containing phosphatidylcholines; the same technique applied to salmon caviar identified an arsenic‐containing phosphatidylethanolamine. The arsenic group in these membrane lipids might impart particular properties to the molecules not displayed by their non‐arsenic analogues. Additionally, the new compounds have human health implications according to recent results showing high cytotoxicity for some arsenolipids. 相似文献
8.
Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short‐Chain Dehydrogenases/Reductases 下载免费PDF全文
Antonios Lygidakis Dr. Vijaykumar Karuppiah Dr. Robin Hoeven Dr. Aisling Ní Cheallaigh Prof. David Leys Dr. John M. Gardiner Dr. Helen S. Toogood Prof. Nigel S. Scrutton 《Angewandte Chemie (International ed. in English)》2016,55(33):9596-9600
Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (?)‐menthone:(?)‐menthol reductase and (?)‐menthone:(+)‐neomenthol reductase, and the “ene” reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue‐swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β‐unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases. 相似文献
9.
Stefan Pieper Sebastian Beck Robert Ahrends Christian Scheler Michael W. Linscheid 《Rapid communications in mass spectrometry : RCM》2009,23(13):2045-2052
Quantitative proteomics has become an important method in modern life sciences. Besides protein identification, the aspect of quantification is of rapidly increasing relevance. MeCAT (metal‐coded affinity tagging) is able to provide a tool that enables relative as well as absolute quantification. For structural elucidation, knowledge on the fragmentation behavior of MeCAT‐modified peptides is highly beneficial. Therefore, the fragmentation behavior of MeCAT‐labeled peptides under collision induced dissociation (CID), electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) conditions was studied. Application of CID and ECD allowed a straight‐forward sequence elucidation of MeCAT‐labeled peptides. During IRMPD all MeCAT‐labeled peptides form characteristic fragments resulting from the fragmentational cleavage of the tagging group, thus, providing a screening method for the identification of labeled compounds. Furthermore, occurring side reactions during the labeling process were investigated. By‐products were structurally characterized and reaction conditions were optimized in order to prevent the formation of these. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
10.
It is demonstrated that bifunctionalized polythiophenes involving thiol and azide end‐functional groups can be synthesized by chain‐growth Suzuki‐Miyaura type polymerization. The bifunctionalized polythiophenes are successfully characterized by 1H NMR, gel permeation chromatography (GPC), and matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF). Furthermore, the azide end‐group reacts with DNA via “click chemistry” to form a polythiophene/DNA hybrid structure, which is characterized by ESI‐MS. The described synthetic approaches will lead to the synthesis of novel multi‐block copolymers as well as biomolecule‐based conjugated polymer structures. 相似文献
11.
S. G. Ramkumar K. A. Amala Rose S. Ramakrishnan 《Journal of polymer science. Part A, Polymer chemistry》2010,48(14):3200-3208
1,3‐Dipolar cycloaddition of an organic azide and an acetylenic unit, often referred to as the “click reaction”, has become an important ligation tool both in the context of materials chemistry and biology. Thus, development of simple approaches to directly generate polymers that bear either an azide or an alkyne unit has gained considerable importance. We describe here a straightforward approach to directly prepare linear and hyperbranched polyesters that carry terminal propargyl groups. To achieve the former, we designed an AB‐type monomer that carries a hydroxyl group and a propargyl ester, which upon self‐condensation under standard transesterification conditions yielded a polyester that carries a single propargyl group at one of its chain‐ends. Similarly, an AB2 type monomer that carries one hydroxyl group and two propargyl ester groups, when polymerized under the same conditions yielded a hyperbranched polymer with numerous “clickable” propargyl groups at its molecular periphery. These propargyl groups can be readily clicked with different organic azides, such as benzyl azide, ω‐azido heptaethyleneglycol monomethylether or 9‐azidomethyl anthracene. When an anthracene chromophore is clicked, the molecular weight of the linear polyester could be readily estimated using both UV–visible and fluorescence spectroscopic measurements. Furthermore, the reactive propargyl end group could also provide an opportunity to prepare block copolymers in the case of linear polyesters and to generate nanodimensional scaffolds to anchor a variety of functional units, in the case of the hyperbranched polymer. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3200–3208, 2010 相似文献
12.
Optimized two‐dimensional gel electrophoresis in an alkaline pH range improves the identification of intracellular CFDA‐cisplatin‐protein adducts in ovarian cancer cells 下载免费PDF全文
《Electrophoresis》2018,39(12):1488-1496
Intracellular binding of cisplatin to proteins has been associated with acquired resistance to chemotherapy. In our previous study we established an analytical method for the identification of intracellular cisplatin‐binding proteins. The method used a fluorescent carboxyfluorescein‐diacetate‐labeled cisplatin analogue (CFDA‐cisplatin), two‐dimensional gel electrophoresis (2DE) and mass spectrometry, which allows detecting and identifying intracellular CFDA‐cisplatin‐containing protein adducts in the acidic pH range (pH 4–7). Based on this analytical method we extended the identification of intracellular cisplatin‐protein adducts to the alkaline pH range (pH 6–10) giving chance to discover new important binding partners. 2DE analysis of alkaline proteins is challenging due to the difficult separation of basic proteins during the isoelectric focusing (IEF). The establishment of an optimized IEF protocol for basic proteins enabled us to identify several intracellular CFDA‐cisplatin‐binding proteins including enzymes of the glucose and serine metabolism like alpha enolase and D‐3‐phosphoglycerate 1‐dehydrogenase. 相似文献
13.
Double Strain‐Promoted Macrocyclization for the Rapid Selection of Cell‐Active Stapled Peptides 下载免费PDF全文
Dr. Maxim Rossmann Dr. Ban Xiong Tan Dr. Peterson de Andrade Dr. Yaw Sing Tan Dr. Chandra Verma Dr. Grahame J. McKenzie Prof. Ashok R. Venkitaraman Dr. Marko Hyvönen Prof. David R. Spring 《Angewandte Chemie (International ed. in English)》2015,54(51):15410-15413
Peptide stapling is a method for designing macrocyclic alpha‐helical inhibitors of protein–protein interactions. However, obtaining a cell‐active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain‐promoted azide–alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell‐active stapled peptides. As a proof of concept, MDM2‐binding peptides were stapled in parallel, directly in cell culture medium in 96‐well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α‐Helicity was confirmed by a crystal structure of the MDM2‐peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high‐throughput biological applications. 相似文献
14.
Markus Leimbacher Dr. Yixin Zhang Dr. Luca Mannocci Michael Stravs Dr. Tim Geppert Dr. Jörg Scheuermann Prof. Gisbert Schneider Prof. Dario Neri 《Chemistry (Weinheim an der Bergstrasse, Germany)》2012,18(25):7729-7737
Libraries of chemical compounds individually coupled to encoding DNA tags (DNA‐encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high‐quality DNA‐encoded chemical library comprising 30 000 drug‐like compounds; this was screened in 170 different affinity capture experiments. High‐throughput sequencing allowed the evaluation of 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor‐associated antigen carbonic anhydrase IX (CA IX) and the pro‐inflammatory cytokine interleukin‐2 (IL‐2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL‐2 was confirmed by molecular docking. Our findings suggest that DNA‐encoded chemical libraries allow the facile identification of drug‐like ligands principally to any protein of choice, including molecules capable of disrupting high‐affinity protein–protein interactions. 相似文献
15.
Heal WP Jovanovic B Bessin S Wright MH Magee AI Tate EW 《Chemical communications (Cambridge, England)》2011,47(14):4081-4083
We report the first chemical probe for bioorthogonal chemical tagging of post-translationally cholesterylated proteins with an azide in living cells. This enables rapid multiplexed fluorescence detection and affinity labelling of protein cholesterylation, as exemplified by Sonic hedgehog protein, opening up new approaches for the de novo identification of cholesterylated proteins. 相似文献
16.
Ozcan Altintas Gurkan Hizal Umit Tunca 《Journal of polymer science. Part A, Polymer chemistry》2006,44(19):5699-5707
Hetero‐arm star ABC‐type terpolymers, poly(methyl methacrylate)‐polystyrene‐poly(tert‐butyl acrylate) (PMMA‐PS‐PtBA) and PMMA‐PS‐poly(ethylene glycol) (PEG), were prepared by using “Click” chemistry strategy. For this, first, PMMA‐b‐PS with alkyne functional group at the junction point was obtained from successive atom transfer radical polymerization (ATRP) and nitroxide‐mediated radical polymerization (NMP) routes. Furthermore, PtBA obtained from ATRP of tBA and commercially available monohydroxyl PEG were efficiently converted to the azide end‐functionalized polymers. As a second step, the alkyne and azide functional polymers were reacted to give the hetero‐arm star polymers in the presence of CuBr/N,N,N′,N″,N″‐pentamethyldiethylenetriamine ( PMDETA) in DMF at room temperature for 24 h. The hetero‐arm star polymers were characterized by 1H NMR, GPC, and DSC. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5699–5707, 2006 相似文献
17.
Matthias Schulz Susanne Tanner Haitham Barqawi Wolfgang H. Binder 《Journal of polymer science. Part A, Polymer chemistry》2010,48(3):671-680
The end‐to‐end cyclization of telechelic polyisobutylenes (PIB's) toward cyclic polyisobutylenes is reported, using either ring‐closing metathesis (RCM) or the azide/alkyne‐“click”‐reaction. The first approach uses bisallyl‐telchelic PIB's (Mn = 1650, 3680, 9770 g mol?1) and Grubbs 1st‐, 2nd‐, and 3rd‐generation catalyst leading to cyclic PIB's in 60–80% yield, with narrow polydispersities (Mw/Mn = 1.25). Azide/alkyne‐“click”‐reactions of bisalkyne‐telechelic PIB's (Mn = 3840 and 9820 g mol?1) with excess of 1,11‐diazido‐undecane leads to the formation of mixtures of linear/cyclic PIB's under formation of oligomeric cycles. Subsequent reaction of the residual azide‐moieties in the linear PIB's with excess of alkyne‐telechelic PEO enables the chromatographic removal of the resulting linear PEO‐PIB‐block copolymers by column chromatography. Thus pure cyclic PIB's can be obtained using this double‐“click”‐method, devoid of linear contaminants. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 671–680, 2010 相似文献
18.
《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2018,130(28):8795-8799
We report a simple, low‐symmetry 2‐(1‐(pyridine‐4‐methyl)‐1H‐1,2,3‐triazol‐4‐yl)pyridine ligand that has both monodentate and bidentate binding sites. With platinum(II) and/or palladium(II) ions, two examples of a new nona‐nuclear metallo‐assembly have been accessed. These complexes were characterized by NMR spectroscopy, electrospray mass spectrometry (ESI‐MS), and in key cases, X‐ray crystallography. The cages possess three clefts comprised of planar cationic panels. This structural feature enables the binding of planar aromatic guests such as anthracene. More interestingly, the heterometallic assembly was able to catalyze the light‐induced [4+2] cycloaddition of anthracene with singlet oxygen. 相似文献
19.
Jianfu Zhang Jianzhao Peng Yiran Huang Ling Meng Qingrong Li Feng Xiong Xiaoyu Li 《Angewandte Chemie (International ed. in English)》2020,59(40):17525-17532
Histone deacetylase (HDAC) is a major class of deacetylation enzymes. Many HDACs exist in large protein complexes in cells and their functions strongly depend on the complex composition. The identification of HDAC‐associated proteins is highly important in understanding their molecular mechanisms. Although affinity probes have been developed to study HDACs, they were mostly targeting the direct binder HDAC, while other proteins in the complex remain underexplored. We report a DNA‐based affinity labeling method capable of presenting different probe configurations without the need for preparing multiple probes. Using one binding probe, 9 probe configurations were created to profile HDAC complexes. Notably, this method identified indirect HDAC binders that may be inaccessible to traditional affinity probes, and it also revealed new biological implications for HDAC‐associated proteins. This study provided a simple and broadly applicable method for characterizing protein‐protein interactions. 相似文献
20.
Hongjuan Dong Sau Ha Cheung Yimin Liang Baojiang Wang Rajkumar Ramalingam Ping Wang Hongyan Sun Shuk Han Cheng Yun Wah Lam 《Electrophoresis》2013,34(13):1957-1964
Organelle‐specific cell‐permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR‐labeled proteins by 2DE. The MTR‐specific fluorescent signals on the gel revealed the spots that contained MTR‐conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock‐like responses. The identification of the molecular targets of biological dyes, or “stainomics,” can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes. 相似文献