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Expert Review of Proteomics Aug 2006Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding... (Review)
Review
Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.
Topics: Azides; Benzophenones; Diazomethane; Ligands; Mass Spectrometry; Models, Molecular; Photoaffinity Labels; Protein Interaction Mapping; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Ultraviolet Rays
PubMed: 16901199
DOI: 10.1586/14789450.3.4.399 -
PloS One 2014A new oriented method using a diazonium salt reaction was developed for linking β2-adrenoceptor (β2-AR) on the surface of macroporous silica gel. Stationary phase...
Immobilised histidine tagged β2-adrenoceptor oriented by a diazonium salt reaction and its application in exploring drug-protein interaction using ephedrine and pseudoephedrine as probes.
A new oriented method using a diazonium salt reaction was developed for linking β2-adrenoceptor (β2-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β2-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β2-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×103/M for ephedrine and (3.80±0.02) ×103/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10-4 M. Thermodynamic studies showed that the binding of the two compounds to β2-AR was a spontaneous reaction with exothermal processes. The ΔGθ, ΔHθ and ΔSθ for the interaction between ephedrine and β2-AR were -(22.33±0.04) kJ/mol, -(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were -(21.17±0.02) kJ/mol, -(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β2-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.
Topics: Animals; Binding, Competitive; Diazonium Compounds; Ephedrine; Histidine; Immobilized Proteins; Molecular Docking Simulation; Protein Binding; Protein Conformation; Protein Stability; Pseudoephedrine; Receptors, Adrenergic, beta-2; Salts; Silicon Dioxide; Thermodynamics
PubMed: 24747442
DOI: 10.1371/journal.pone.0094955 -
Journal of Clinical Pathology.... 1970
Review
Topics: Alkaline Phosphatase; Aspartate Aminotransferases; Autoanalysis; Computers; Diazonium Compounds; Enzymes; Indicators and Reagents; L-Lactate Dehydrogenase; Malate Dehydrogenase; Methods; Spectrophotometry
PubMed: 4949769
DOI: 10.1136/jcp.s1-4.1.31 -
Biochemistry Jan 2014Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of...
Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus . In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) Nature 469, 428-431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50 values of 20 and 50 μM, respectively. When [(3)H]AziPm (7 μM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziPm bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket.
Topics: Affinity Labels; Amino Acid Sequence; Bacterial Proteins; Binding Sites; Diazomethane; Ion Channels; Ligand-Gated Ion Channels; Models, Molecular; Propofol; Protein Structure, Tertiary; Receptors, GABA-A
PubMed: 24341978
DOI: 10.1021/bi401492k -
ACS Chemical Biology Mar 2020Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles...
Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Advances in our understanding of its function have been hindered by a lack of available tools that can specifically measure the proteolytic activity of cathepsin X. We present a series of activity-based probes that incorporate a sulfoxonium ylide warhead, which exhibit improved specificity for cathepsin X compared to previously reported probes. We apply these probes to detect cathepsin X activity in cell and tissue lysates, in live cells and in vivo, and to localize active cathepsin X in mouse tissues by microscopy. Finally, we utilize an improved method to generate chloromethylketones, necessary intermediates for synthesis of acyloxymethylketones probes, by way of sulfoxonium ylide intermediates. In conclusion, the probes presented in this study will be valuable for investigating cathepsin X pathophysiology.
Topics: Amino Acids; Animals; Cathepsins; Cell Culture Techniques; Cell Line; Diazomethane; Enzyme Inhibitors; Fluorescent Dyes; Humans; Hydrocarbons, Fluorinated; Ketones; Kidney; Kinetics; Male; Mice; Mice, Inbred C57BL; Optical Imaging; Protein Domains; Small Molecule Libraries; Structure-Activity Relationship; Substrate Specificity
PubMed: 32022538
DOI: 10.1021/acschembio.9b00961 -
The Journal of General Physiology Feb 2012Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the...
Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.
Topics: Acinar Cells; Animals; Calcium; Cell Membrane; Chloride Channels; Diazonium Compounds; Indoles; Membrane Potentials; Mice; Mice, Inbred C57BL; Parotid Gland; Phenoxyacetates; Photolysis; Potassium; Potassium Channels, Calcium-Activated; Pyrazoles
PubMed: 22291145
DOI: 10.1085/jgp.201110718 -
The Journal of Organic Chemistry Mar 2014Photoaffinity labeling is a useful technique employed to identify protein-ligand and protein-protein noncovalent interactions. Photolabeling experiments have been...
Photoaffinity labeling is a useful technique employed to identify protein-ligand and protein-protein noncovalent interactions. Photolabeling experiments have been particularly informative for probing membrane-bound proteins where structural information is difficult to obtain. The most widely used classes of photoactive functionalities include aryl azides, diazocarbonyls, diazirines, and benzophenones. Diazirines are intrinsically smaller than benzophenones and generate carbenes upon photolysis that react with a broader range of amino acid side chains compared with the benzophenone-derived diradical; this makes diazirines potentially more general photoaffinity-labeling agents. In this article, we describe the development and application of a new isoprenoid analogue containing a diazirine moiety that was prepared in six steps and incorporated into an a-factor-derived peptide produced via solid-phase synthesis. In addition to the diazirine moiety, fluorescein and biotin groups were also incorporated into the peptide to aid in the detection and enrichment of photo-cross-linked products. This multifuctional diazirine-containing peptide was a substrate for Ste14p, the yeast homologue of the potential anticancer target Icmt, with K(m) (6.6 μM) and V(max) (947 pmol min(-1) mg(-1)) values comparable or better than a-factor peptides functionalized with benzophenone-based isoprenoids. Photo-cross-linking experiments demonstrated that the diazirine probe photo-cross-linked to Ste14p with observably higher efficiency than benzophenone-containing a-factor peptides.
Topics: Benzophenones; Cross-Linking Reagents; Diazomethane; Ligands; Photoaffinity Labels; Photochemistry; Protein Methyltransferases; Solid-Phase Synthesis Techniques; Terpenes
PubMed: 24502619
DOI: 10.1021/jo402600b -
Journal of the American Chemical Society Feb 2019Vinyl cations derived from diazo ketones participate in transition-metal-free C-H insertion reactions, but the corresponding amide and ester analog exhibit divergent...
Vinyl cations derived from diazo ketones participate in transition-metal-free C-H insertion reactions, but the corresponding amide and ester analog exhibit divergent reactivity profiles. Whereas cations formed from diazo ketones undergo a rearrangement and C-H insertion sequence, those from diazo amides do so less efficiently and tend to be competitively trapped before the insertion step occurs. Diazo esters undergo several rearrangement steps and fail to insert. DFT calculations reveal that this disparity stems from two factors: differing levels of electrostatic stabilization of the initially formed vinyl cation by the adjacent carbonyl oxygen and predistortion of the ketone and amide systems toward C-H insertion. The computational data is in strong agreement with experimental results, and this study explains how structural and electronic factors determine the outcome of reactions of diazo carbonyl-derived vinyl cations.
Topics: Amides; Density Functional Theory; Diazonium Compounds; Esters; Ketones; Molecular Structure; Transition Elements
PubMed: 30758200
DOI: 10.1021/jacs.8b12420 -
Molecules (Basel, Switzerland) Jun 2017Diazocoupling reaction of curcumin with different diazonium salts of -toluidine, 2-aminopyridine, and 4-aminoantipyrine in pyridine yielded the arylhydrazones -....
Diazocoupling reaction of curcumin with different diazonium salts of -toluidine, 2-aminopyridine, and 4-aminoantipyrine in pyridine yielded the arylhydrazones -. Arylhydrazone of -toluidine reacted with urea, thiourea, and guanidine nitrate to produce 5,6-dihydropyrimidines. Further reaction of with 2,3-diaminopyrdine in sodium ethoxide solution yielded 1-pyrido[2,3-][1,4]diazepine derivative. (2,5-dihydroisoxazole) is obtained from the reaction of with hydroxylamine hydrochloride, while its reactions with hydrazines afforded the respective 4,5-dihydro-1-pyrazoles. The target compounds were evaluated as antioxidant and antibacterial agents. The tested compounds showed good to moderate activities compared to ascorbic acid and chloramphenicol, respectively.
Topics: Aminopyridines; Ampyrone; Anti-Bacterial Agents; Antioxidants; Ascorbic Acid; Chloramphenicol; Curcumin; Diazonium Compounds; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Microbial Sensitivity Tests; Molecular Structure; Structure-Activity Relationship; Toluidines
PubMed: 28604614
DOI: 10.3390/molecules22060971 -
Molecules (Basel, Switzerland) Dec 2020The mechanism of the carbonylation of diazomethane in the presence of iron-carbonyl-phosphine catalysts has been investigated by means of DFT calculations at the...
The mechanism of the carbonylation of diazomethane in the presence of iron-carbonyl-phosphine catalysts has been investigated by means of DFT calculations at the M06/def-TZVP//B97D3/def2-TZVP level of theory, in combination with the SMD solvation method. The reaction rate is determined by the formation of the coordinatively unsaturated doublet-state Fe(CO)(P) precursor followed by the diazoalkane coordination and the N extrusion. The free energy of activation is predicted to be 18.5 and 28.2 kcal/mol for the PF and PPh containing systems, respectively. Thus, in the presence of less basic P-donor ligands with stronger π-acceptor properties, a significant increase in the reaction rate can be expected. According to energy decomposition analysis combined with natural orbitals of chemical valence (EDA-NOCV) calculations, diazomethane in the Fe(CO)(phosphine)(-CHN) adduct reveals a π-donor-π-acceptor type of coordination.
Topics: Catalysis; Computer Simulation; Diazomethane; Electrons; Hydrogenase; Iron; Iron Compounds; Iron-Sulfur Proteins; Ligands; Methane; Models, Molecular; Molecular Structure; Nickel; Palladium; Phosphines; Phosphorus; Quantum Theory
PubMed: 33322410
DOI: 10.3390/molecules25245860