RESUMO
Dipeptidyl peptidase III (DPP III, EC 3.4.14.4) is a monozinc metalloexopeptidase that hydrolyzes dipeptides from the N-terminus of peptides consisting of three or more amino acids. Recently, DPP III has attracted great interest from scientists, and numerous studies have been conducted showing that it is involved in the regulation of various physiological processes. Since it is the only metalloenzyme among the dipeptidyl peptidases, we considered it important to study the process of binding and exchange of physiologically relevant metal dications in DPP III. Using fluorimetry, we measured the Kd values for the binding of Zn2+, Cu2+, and Co2+ to the catalytic site, and using isothermal titration calorimetry (ITC), we measured the Kd values for the binding of these metals to an additional binding site. The structure of the catalytic metal's binding site is known from previous studies, and in this work, the affinities for this site were calculated for Zn2+, Cu2+, Co2+, and Mn2+ using the QM approach. The structures of the additional binding sites for the Zn2+ and Cu2+ were also identified, and MD simulations showed that two Cu2+ ions bound to the catalytic and inhibitory sites exchanged less frequently than the Zn2+ ions bound to these sites.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases , Humanos , Sítios de Ligação , Domínio Catalítico , Domínios ProteicosRESUMO
Two novel conjugate molecules were designed: pyrene and phenanthridine-amino acid units with a different linker length between the aromatic fragments. Molecular modelling combined with spectrophotometric experiments revealed that in neutral and acidic buffered water solutions conjugates predominantly exist in intramolecularly stacked conformations because of the π-π stacking interaction between pyrene and phenanthridine moieties. The investigated systems exhibited a pH-dependent excimer formation that is significantly red-shifted relative to the pyrene and phenanthridine fluorescence. While the conjugate with a short linker showed negligible spectrophotometric changes due to the polynucleotide addition, the conjugate with a longer and more flexible linker exhibited a micromolar and submicromolar binding affinity for ds-polynucleotides and inactivated a mutant of dipeptidyl peptidase enzyme E451A. Confocal microscopy revealed that the conjugate with the longer linker entered the HeLa cell membranes and blue fluorescence was visualized as the dye accumulated in the cell membrane.
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A novel naphthalene diimde analogue (NDI) equipped at the imide positions with two guanidinio-carbonyl-pyrrole (GCP) pendant arms interacted significantly stronger with ds-DNA at pH 5 than at pH 7, due to reversible protonation of the GCP arms. This was consequence of a pH-switchable threading intercalation into ds-DNAs only at pH 5, while at neutral conditions (pH 7) NDI-GCP2 switched to the DNA minor groove binding. Intriguingly, NDI-GCP2 was at both pH values studied bound to the ds-RNA major groove, still showing a higher affinity and thermal denaturation effect at pH 5 due to GCP protonation. At excess over the DNA/RNA conjugate NDI-GCP2 showed also aggregation along the ds-polynucleotide and AFM and DLS demonstrated that NDI-GCP2 has pronounced ds-DNA condensation ability.
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Brassica rapa auxin amidohydrolase (BrILL2) participates in the homeostasis of the plant hormones auxins by hydrolyzing the amino acid conjugates of auxins, thereby releasing the free active form of hormones. Herein, the potential role of the two conserved Cys residues of BrILL2 (at sequence positions 139 and 320) has been investigated by using interdisciplinary approaches and methods of molecular biology, biochemistry, biophysics and molecular modelling. The obtained results show that both Cys residues participate in the regulation of enzyme activity. Cys320 located in the satellite domain of the enzyme is mainly responsible for protein stability and regulation of enzyme activity through polymer formation, as has been revealed by enzyme kinetics and differential scanning calorimetry analysis of the BrILL2 wild type and mutants C320S and C139S. Cys139 positioned in the active site of the catalytic domain is involved in the coordination of one Mn(2+) ion of the bimetal center and is crucial for the enzymatic activity. Although the point mutation Cys139 to Ser causes the loss of enzyme activity, it does not affect the metal binding to the BrILL2 enzyme, as has been shown by isothermal titration calorimetry, circular dichroism spectropolarimetry and differential scanning calorimetry data. MD simulations (200 ns) revealed a different active site architecture of the BrILL2C139S mutant in comparison to the wild type enzyme. Additional possible reasons for the inactivity of the BrILL2C139S mutant have been discussed based on MD simulations and MM-PBSA free energy calculations of BrILL2 enzyme complexes (wt and C139S mutant) with IPA-Ala as a substrate.
Assuntos
Amidoidrolases/metabolismo , Brassica rapa/enzimologia , Cisteína/química , Ácidos Indolacéticos/metabolismo , Calorimetria , Estabilidade Enzimática , Espectrometria de Massas , Simulação de Dinâmica Molecular , Mutagênese Sítio-DirigidaRESUMO
We present a multidisciplinary study on the hematite (001)-aqueous solution interface, in particular the relationship between surface structure (studied via surface diffraction in a humid atmosphere) and the macroscopic charging (studied via surface- and zeta-potential measurements in electrolyte solutions as a function of pH). Upon aging in water changes in the surface structure are observed, that are accompanied by drastic changes in the zeta-potential. Surprisingly the surface potential is not accordingly affected. We interpret our results by increasing hydration of the surface with time and enhanced reactivity of singly-coordinated hydroxyl groups that cause the isoelectric point of the surface to shift to values that are reminiscent of those typically reported for hematite particles. In its initial stages after preparation the hematite surface is very flat and only weakly hydrated. Our model links the entailing weak water structure with the observed low isoelectric point reminiscent of hydrophobic surfaces. The absence of an aging effect on the surface potential vs. pH curves is interpreted as domination of the surface potential by the doubly coordinated hydroxyls, which are present on both surfaces.
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Kombucha is a traditional beverage obtained by the microbial fermentation of tea using a symbiotic culture of bacteria and yeasts. In addition to several documented functional properties, such as anti-inflammatory activity and antioxidant activity, kombucha is often credited with high levels of vitamins, including riboflavin. To our knowledge, the vitamin B2 content in traditionally prepared kombucha has been determined in only two studies, in which the concentration measured by the HPLC technique ranged from 2.2 × 10-7 to 2.1 × 10-4 mol dm-3. These unexplained differences of three orders of magnitude in the vitamin B2 content prompted us to determine its concentration during the cultivation of kombucha under very similar conditions by spectrofluorimetry. The B2 concentrations during the 10-day fermentation of black tea ranged from 7.6 × 10-8 to 3.3 × 10-7 mol dm-3.
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Dipeptidyl peptidase III (DPP III) is a cytosolic, two-domain zinc-exopeptidase. It is widely distributed in mammalian tissues, where it's involved in the final steps of normal intracellular protein degradation. However, its pronounced affinity for some bioactive peptides (angiotensins, enkephalins, and endomorphins) suggests more specific functions such as blood pressure regulation and involvement in pain regulation. We have investigated several different neuropeptides as potential substrates and inhibitors of human DPP III. The binding affinities and kinetic data determined by isothermal titration calorimetry, in combination with measurements of enzyme inhibition identified the hemorphin-related valorphin, tynorphin, S-tynorphin, and I-tynorphin as the most potent inhibitors of DPP III (actually slow substrates), whereas hemorphin-4 proved to be the best substrate of all neuropeptides examined. In addition, we have shown that the neuropeptides valorphin, Leu-valorphin-Arg, and the opioid peptide ß-casomorphin, are DPP III substrates. The molecular modelling of selected peptides shows uniform binding to the lower domain ß-strand residues of DPP III via peptide backbone atoms, but also previously unrecognized stabilizing interactions with conserved residues of the metal-binding site and catalytic machinery in the upper domain. The computational data helped explain the differences between substrates that are hydrolyzed effectively and those hydrolysed slowly by DPP III.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases , Zinco , Adamantano/análogos & derivados , Angiotensinas , Encefalinas , Humanos , Peptídeos Opioides , Zinco/metabolismoRESUMO
Interactions of an array of nucleic acid structures with a small series of benzothiazole ligands (bis-benzothiazolyl-pyridines-group 1, 2-thienyl/2-benzothienyl-substituted 6-(2-imidazolinyl)benzothiazoles-group 2, and three 2-aryl/heteroaryl-substituted 6-(2-imidazolinyl)benzothiazoles-group 3) were screened by competition dialysis. Due to the involvement of DNA:RNA hybrids and triplex helices in many essential functions in cells, this study's main aim is to detect benzothiazole-based moieties with selective binding or spectroscopic response to these nucleic structures compared to regular (non-hybrid) DNA and RNA duplexes and single-stranded forms. Complexes of nucleic acids and benzothiazoles, selected by this method, were characterized by UV/Vis, fluorescence and circular dichroism (CD) spectroscopy, isothermal titration calorimetry, and molecular modeling. Two compounds (1 and 6) from groups 1 and 2 demonstrated the highest affinities against 13 nucleic acid structures, while another compound (5) from group 2, despite lower affinities, yielded higher selectivity among studied compounds. Compound 1 significantly inhibited RNase H. Compound 6 could differentiate between B- (binding of 6 dimers inside minor groove) and A-type (intercalation) helices by an induced CD signal, while both 5 and 6 selectively stabilized ATT triplex in regard to AT duplex. Compound 3 induced strong condensation-like changes in CD spectra of AT-rich DNA sequences.
Assuntos
Ácidos Nucleicos , RNA , Benzotiazóis , Dicroísmo Circular , DNA/química , Ligantes , Conformação de Ácido Nucleico , RNA/química , Diálise RenalRESUMO
This work is about synergy of theory and experiment in revealing mechanism of binding of dipeptidyl peptidase III (DPP III) and Kelch-like ECH-associated protein 1 (KEAP1), the main cellular sensor of oxidative stress. The NRF2 ̶ KEAP1 signaling pathway is important for cell protection, but it is also impaired in many cancer cells where NRF2 target gene expression leads to resistance to chemotherapeutic drugs. DPP III competitively binds to KEAP1 in the conditions of oxidative stress and induces release of NRF2 and its translocation into nucleus. The binding is established mainly through the ETGE motif of DPP III and the Kelch domain of KEAP1. However, although part of a flexible loop, ETGE itself is firmly attached to the DPP III surface by strong hydrogen bonds. Using combined computational and experimental study, we found that DPP III ̶ Kelch binding is a two-step process comprising the endergonic loop detachment and exergonic DPP III ̶ Kelch interaction. Substitution of arginines, which keep the ETGE motif attached, decreases the work needed for its release and increases DPP III ̶ Kelch binding affinity. Interestingly, mutations of one of these arginine residues have been reported in cBioPortal for cancer genomics, implicating its possible involvement in cancer development. Communicated by Ramaswamy H. Sarma.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases , Fator 2 Relacionado a NF-E2 , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse OxidativoRESUMO
The binding of four phenanthridine-guanidine peptides to DNA/RNA was evaluated via spectrophotometric/microcalorimetric methods and computations. The minor structural modifications-the type of the guanidine group (pyrrole guanidine (GCP) and arginine) and the linker length (presence or absence of glycine)-greatly affected the conformation of compounds and consequently the binding to double- (ds-) and single-stranded (ss-) polynucleotides. GCP peptide with shorter linker was able to distinguish between RNA (A-helix) and DNA (B-helix) by different circular dichroism response at 295â¯nm and thus can be used as a chiral probe. Opposed to the dominant stretched conformation of GCP peptide with shorter linker, the more flexible and longer linker of its analogue enabled the molecule to adopt the intramolecularly stacked form which resulted in weaker yet selective binding to DNA. Beside efficient organization of ss-polynucleotide structures, GCP peptide with shorter linker bound stronger to ss-DNA/RNA compared to arginine peptides which emphasize the importance of GCP unit.
Assuntos
DNA , Glicina , Guanidina , Peptídeos , Fenantridinas , RNA , Pareamento de Bases , Dicroísmo Circular , DNA/química , Glicina/química , Guanidina/química , Conformação Molecular , Conformação de Ácido Nucleico , Peptídeos/química , Fenantridinas/química , RNA/química , Análise Espectral , TermodinâmicaRESUMO
BACKGROUND: The aim of this study was to evaluate high voltage electrical discharges (HVED) as a green technology, in order to establish the effectiveness of phenolic extraction from olive leaves against conventional extraction (CE). HVED parameters included different green solvents (water, ethanol), treatment times (3 and 9 min), gases (nitrogen, argon), and voltages (15, 20, 25 kV). METHODS: Phenolic compounds were characterized by ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS), while antioxidant potency (total phenolic content and antioxidant capacity) were monitored spectrophotometrically. Data for Near infrared spectroscopy (NIR) spectroscopy, colorimetry, zeta potential, particle size, and conductivity were also reported. RESULTS: The highest yield of phenolic compounds was obtained for the sample treated with argon/9 min/20 kV/50% (3.2 times higher as compared to CE). Obtained results suggested the usage of HVED technology in simultaneous extraction and nanoformulation, and production of stable emulsion systems. Antioxidant capacity (AOC) of obtained extracts showed no significant difference upon the HVED treatment. CONCLUSIONS: Ethanol with HVED destroys the linkage between phenolic compounds and components of the plant material to which they are bound. All extracts were compliant with legal requirements regarding content of contaminants, pesticide residues and toxic metals. In conclusion, HVED presents an excellent potential for phenolic compounds extraction for further use in functional food manufacturing.
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Two bis-polyaza pyridinophane derivatives and their monomeric reference compounds revealed strong interactions with ds-DNA and RNA. The bis-derivatives show a specific condensation of GC- and IC-DNA, which is almost two orders of magnitude more efficient than the well-known condensation agent spermine. The type of condensed DNA was identified as ψ-DNA, characterized by the exceptionally strong CD signals. At variance to the almost silent AT(U) polynucleotides, these strong CD signals allow the determination of GC-condensates at nanomolar nucleobase concentrations. Detailed thermodynamic characterisation by ITC reveals significant differences between the DNA binding of the bis-derivative compounds (enthalpy driven) and that of spermine and of their monomeric counterparts (entropy driven). Atomic force microscopy confirmed GC-DNA compaction by the bis-derivatives and the formation of toroid- and rod-like structures responsible for the ψ-type pattern in the CD spectra.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Piridonas/química , Calorimetria , Dicroísmo Circular , Microscopia de Força Atômica , Conformação de Ácido Nucleico/efeitos dos fármacos , Desnaturação de Ácido Nucleico , Piridonas/farmacologia , RNA de Cadeia Dupla/química , TemperaturaRESUMO
The hypothesis that pH dependent charge of interfacial water affects electrokinetic charge and electrokinetic potential of hydrophobic colloids, but not the (inner) surface potential was tested. It was found that isoelectric points of silver chloride, bromide and iodide shift to the higher pAg values in the acidic solutions, but that surface potential did not depend on pH. Isoelectric points of water at inert surfaces lie in the range 2
RESUMO
Dependence of surface potential (electrostatic potential at the inner Helmholtz plane, Ψ(0)) at the silver bromide aqueous electrolyte interface was measured as a function of the activities of Br(-) and Ag(+) by using a single crystal silver bromide electrode (SCr-AgBr). Absolute values of surface potentials were obtained from electrode potentials of SCr-AgBr and isoelectric points. Measurements were performed at different temperatures in the range from 10 to 50°C. Corresponding equilibrium constants of interfacial reactions were obtained using the surface complexation model and interpreted via the van't Hoff equation. As a result of the interpretation for the binding of bromide ions leading to a negative surface charge, the thermodynamic parameters obtained were Δ(n)H(∘)=-33kJmol(-1) and Δ(n)S(∘)=-31Jmol(-1)K(-1); and for the binding of silver ions leading to a positive surface charge, Δ(p)H(∘)=-72kJmol(-1) and Δ(p)S(∘)=-196Jmol(-1)K(-1). Association of counterions (CI) with oppositely charged surface sites partially compensates the surface charge. Assuming approximately the same affinities for anions (NO(3)(-)) and cations (K(+)) thermodynamic parameters for their binding were obtained as Δ(CI)H(∘)≈7kJmol(-1) and Δ(CI)S(∘)≈105Jmol(-1)K(-1).
RESUMO
A single crystal silver bromide electrode (SCr-AgBr) was used to measure the inner surface potential (Ψ(0)) at the silver bromide aqueous electrolyte interface as a function of the activities of Br(-) and Ag(+). Absolute values of the surface potential were calculated from electrode potentials of SCr-AgBr using the value of point of zero charge (pBr(pzc)=6.9 [H.A. Hoyen, R.M. Cole, J. Colloid Interface Sci. 41 (1972) 93.]) as the value of point of zero potential. Measurements were performed in potassium nitrate aqueous solutions. The Ψ(0)(pBr) function was linear and slightly dependent on the ionic strength. The reduction values of the slope with respect to the Nernst equation, expressed by the α coefficient, were 0.880,0.935, and 0.950 at ionic strengths of 10(-4), 10(-3), and 10(-2) mol dm(-3), respectively. The results were successfully interpreted by employing the surface complexation model, developed originally for metal oxides and adapted for silver halides. The thermodynamic ("intrinsic") equilibrium constants for binding of bromide (K(n)(∘)) and silver (K(p)(∘)) ions on the corresponding sites at the silver bromide surface were evaluated as lgK(n)(∘)=3.98; lgK(p)(∘)=2.48. Symmetrical counterion surface association was assumed and equilibrium constants were obtained as lgK(NO(3)(-))(∘)=lgK(K(+))(∘)=4.30.
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A single crystal silver chloride electrode (SCr-AgCl) was used to measure the inner surface potential (Psi(0)) at the silver chloride aqueous electrolyte interface as a function of activity of Cl(-) ions as determined by the Ag/AgCl electrode. Absolute values of the surface potential were calculated from electrode potentials of SCr-AgCl using the value of point of zero charge (pCl(pzc)=5.2) as the value of point of zero potential. Measurements were performed in potassium nitrate aqueous solutions, as well as in the presence of Li, Na, Cs, Mg, and La nitrates. The Psi(0) (pCl) function was found to be linear within the experimental error and practically the same for all the examined electrolytes and almost independent of ionic strength. The reduction of the slope with respect to the Nernst equation, expressed by the alpha coefficient, was (0.88+/-0.01) at I(c)=10(-1) mol dm(-3), (0.87+/-0.01) at I(c)=10(-2) mol dm(-3), and (0.84+/-0.01) at I(c)=10(-3) mol dm(-3). The results were successfully interpreted by employing the surface complexation model developed originally for metal oxides and adapted for silver chloride. The standard ("intrinsic") equilibrium constants for the binding of chloride (K(o)(n)) and silver ions (K(o)(p)) on the corresponding sites at the silver chloride surface were evaluated as lg K(o)(n)=2.67+/-0.05; lg K(o)(p)=2.07+/-0.05. Counterion surface association equilibrium constants were also obtained as lg K(o)(NO3(-))=lg K(o)(K+)=274+/-0.05.
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The surface potential at the silver chloride aqueous interface was measured by means of a single-crystal silver chloride electrode (SCr-AgCl). The measurements were conducted by titration of the KCl solution with AgNO3, and vice versa. The SCr-AgCl electrode potentials were converted to surface potentials psi(0) by setting zero at the point of zero charge at pCl = 5.2. The psi(0)(pCl) function was linear, with a slope 12% lower with respect to the Nernst equation. It was demonstrated that the surface potential at the silver halide aqueous interface could be interpreted by means of the surface complexation model, originally developed for metal oxides.