11 research outputs found
Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements
The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor
Incorporation of β-Alanine in Cu(II) ATCUN Peptide Complexes Increases ROS Levels, DNA Cleavage and Antiproliferative Activity
Redox-active Cu(II) complexes are able to form reactive oxygen species (ROS) in the presence of oxygen and reducing agents. Recently, Faller et al. reported that ROS generation by Cu(II) ATCUN complexes is not as high as assumed for decades. High complex stability results in silencing of the Cu(II)/Cu(I) redox cycle and therefore leads to low ROS generation. In this work, we demonstrate that an exchange of the α-amino acid Gly with the β-amino acid β-Ala at position 2 (Gly2→β-Ala2) of the ATCUN motif reinstates ROS production (•OH and H2O2). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these β-Ala2-containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for β-Ala2 compared to the Gly2 complexes. Modifications with positively charged Lys residues increased the DNA affinity through electrostatic interactions as determined by UV/VIS, fluorescence, and CD spectroscopy, and consequently led to a further increase in nuclease activity. A similar trend was observed regarding the cytotoxic activity of the complexes against several human cancer cell lines where β-Ala2 peptide complexes had lower IC50 values compared to Gly2. The higher cytotoxicity could be attributed to an increased cellular uptake as determined by ICP-MS measurements
Hydroxylated derivatives of dimethoxy-1,4-benzoquinone as redox switchable earth-alkaline metal ligands and radical scavengers
Benzoquinones (BQ) have important functions in many biological processes. In alkaline environments, BQs
can be hydroxylated at quinoid ring proton positions. Very little is known about the chemical reaction
leading to these structural transformations as well as about the properties of the obtained hydroxyl
benzoquinones. We analyzed the behavior of the naturally occurring 2,6-dimethoxy-1,4-benzoquinone
under alkaline conditions and show that upon substitution of methoxy-groups, poly-hydroxyl-derivatives
(OHBQ) are formed. The emerging compounds with one or several hydroxyl-substituents on single or fused
quinone-rings exist in oxidized or reduced states and are very stable under physiological conditions. In
comparison with the parent BQs, OHBQs are stronger radical scavengers and redox switchable
earth-alkaline metal ligands. Considering that hydroxylated quinones appear as biosynthetic intermediates
or as products of enzymatic reactions, and that BQs present in food or administered as drugs can be
hydroxylated by enzymatic pathways, highlights their potential importance in biological systems
An EPR and DFT study on the primary radical formed in hydroxylation reactions of 2,6-dimethoxy-1,4-benzoquinone
Concentration Effects in the Interaction of Monoclonal Antibodies (mAbs) with their Immediate Environment Characterized by EPR Spectroscopy
Monoclonal antibodies (mAbs) are often needed and applied in high concentration solutions, >100 mg/mL. Due to close intermolecular distances between mAbs at high concentrations (~10–20 nm at 200 mg/mL), intermolecular interactions between mAbs and mAbs and solvent/co-solute molecules become non-negligible. Here, EPR spectroscopy is used to study the high-concentration solutions of mAbs and their effect on co-solvated small molecules, using EPR “spin probing” assay in aqueous and buffered solutions. Such, information regarding the surrounding environments of mAbs at high concentrations were obtained and comparisons between EPR-obtained micro-viscosities (rotational correlation times) and macroscopic viscosities measured by rheology were possible. In comparison with highly viscous systems like glycerol-water mixtures, it was found that up to concentrations of 50 mg/mL, the mAb-spin probe systems have similar trends in their macro- (rheology) and micro-viscosities (EPR), whereas at very high concentrations they deviate strongly. The charged spin probes sense an almost unchanged aqueous solution even at very high concentrations, which in turn indicates the existence of large solvent regions that despite their proximity to large mAbs essentially offer pure water reservoirs for co-solvated charged molecules. In contrast, in buffered solutions, amphiphilic spin probes like TEMPO interact with the mAb network, due to slight charge screening. The application of EPR spectroscopy in the present work has enabled us to observe and discriminate between electrostatic and hydrophobic kinds of interactions and depict the potential underlying mechanisms of network formation at high concentrations of mAbs. These findings could be of importance as well for the development of liquid-liquid phase separations often observed in highly concentrated protein solutions
An EPR and DFT study on the primary radical formed in hydroxylation reactions of 2,6-dimethoxy-1,4-benzoquinone
The quinone compound 2,6-dimethoxy-1,4-benzoquinone is hydroxylated in alkaline aqueous solutionwithpHabove12.Electronparamagneticresonanceexperimentsshowedthattwotransientradicals are formed in this reaction. The radical appearing first is assigned to a one electron reduced 2,6-dimethoxy-1,4-benzoquinone,receivingtheelectronfromanintermediateanionichydroxylated species.Forthisprimaryradical,allprotoncouplingsweredetermined(quinoidringprotons:1.453G, methylprotons:0.795G).Thedensityfunctionaltheorymethodwasappliedtoobtainelectronicand structuralinformationoftheprimaryradicalandasolutionstructureissuggested.Forapproaching the experimental hyperfine couplings in theoretical models, it was necessary to consider effects of external polarisation arising from water molecules near one carbonyl group, and the orientation of methoxygroupstowardsthequinonering.Withthisapproach,thesecondaryradicalformedinthe hydroxylation reaction, and the transient radicals found for other biologically important quinones (includingcoenzymesQ)andtheirhydroxylatedspeciesmaybecomeaccessible
Electronic and Geometric Structures of Paramagnetic Diazadiene Complexes of Lithium and Sodium
The electronic and molecular structures of the lithium and sodium complexes of 1,4‐bis(2,6‐diisopropylphenyl)‐2,3‐dimethyl‐1,4‐diazabutadiene (Me2DADDipp) were fully characterized by using a multi‐frequency electron paramagnetic resonance (EPR) spectroscopy approach and crystallography, together with density functional theory (DFT) calculations. EPR measurements, using T1 relaxation‐time‐filtered pulse EPR spectroscopy, revealed the diagonal elements of the A and g tensors for the metal and ligand sites. It was found that the central metals in the lithium complexes had sizable contributions to the SOMO, whereas this contribution was less strongly observed for the sodium complex. Such strong contributions were attributed to structural specifications (e.g. geometrical data and atomic size) rather than electronic effects
New insights into the chemistry of Coenzyme Q-0: A voltammetric and spectroscopic study
Coenzyme Q-0 (CoQ-0) is the only Coenzyme Q lacking an isoprenoid group on the quinoid ring, a feature important
for its physico-chemical properties. Here, the redox behavior of CoQ-0 in buffered and non-buffered aqueous
media was examined. In buffered aqueous media CoQ-0 redox chemistry can be described by a 2-electron–2-proton
redox scheme, characteristic for all benzoquinones. In non-buffered media the number of electrons involved
in the electrode reaction of CoQ-0 is still 2; however, the number of protons involved varies between 0 and 2. This
results in two additional voltammetric signals, attributed to 2-electrons–1H+ and 2-electrons–0H redox processes,
in which mono- and di-anionic compounds of CoQ-0 are formed. In addition, CoQ-0 exhibits a complex
chemistry in strong alkaline environment. The reaction of CoQ-0 and OH− anions generates several hydroxyl derivatives
as products. Their structures were identified with HPLC/MS. The prevailing radical reaction mechanism
was analyzed by electron paramagnetic resonance spectroscopy. The hydroxyl derivatives of CoQ-0 have a strong antioxidative potential and form stable complexes with Ca2+ions. In summary, our results allowmechanistic insights into the redox properties of CoQ-0 and its hydroxylated derivatives and provide hints on possible
applications.
Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements
The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor
Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements
The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor