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1.
Science ; 374(6564): 156, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34618557

RESUMO

[Figure: see text].


Assuntos
Cátions Bivalentes
2.
Biomacromolecules ; 22(11): 4642-4658, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34670087

RESUMO

Fibrinogen nanofibers are very attractive biomaterials to mimic the native blood clot architecture. Previously, we reported the self-assembly of fibrinogen nanofibers in the presence of monovalent salts and have now studied how divalent salts influence fibrinogen precipitation. Although the secondary fibrinogen structure was significantly altered with divalent metal ions, morphological analysis revealed exclusively smooth fibrinogen precipitates. In situ monitoring of the surface roughness facilitated predicting the tendency of various salts to form fibrinogen fibers or smooth films. Analysis of the chemical composition revealed that divalent salts were removed from smooth fibrinogen films upon rinsing while monovalent Na+ species were still present in fibrinogen fibers. Therefore, we assume that the decisive factor controlling the morphology of fibrinogen precipitates is direct ion-protein contact, which requires disruption of the ion-surrounding hydration shells. We conclude that in fibrinogen aggregates, this mechanism is effective only for monovalent ions, whereas divalent ions are limited to indirect fibrinogen adsorption.


Assuntos
Fibrinogênio , Nanofibras , Adsorção , Cátions Bivalentes , Íons
3.
Biochemistry ; 60(37): 2781-2794, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34472844

RESUMO

RNA is highly negatively charged and often acquires complex structures that require the presence of divalent cations. Subtle changes in conformation resulting from changes in sequence can affect the way ions associate with RNA. Riboswitches are RNA molecules that are involved in the control of gene expression in bacteria and are excellent systems for testing the effects of sequence variations on the conformation of RNA because they contain a highly conserved binding pocket but present sequence variability among different organisms. In this work, we have compared the aptamer domain of a proposed M-box riboswitch from Mycobacterium tuberculosis with the aptamer domain of a validated M-box riboswitch from Bacillus subtilis. We have in vitro transcribed and purified wild-type (WT) M-box riboswitches from M. tuberculosis and B. subtilis as well as a variety of mutated aptamers in which regions from one riboswitch have been replaced with regions from the other riboswitch. We have used ultraviolet unfolding experiments and circular dichroism to characterize the interactions of WT and related M-box riboswitches with divalent cations. Our results show that M-box from M. tuberculosis associates with Mg2+ and Sr2+ in a similar fashion while M-box from B. subtilis discriminates between these two ions and appears to associate better with Mg2+. Our overall results show that M-box from M. tuberculosis interacts differently with cations than M-box from B. subtilis and suggest conformational differences between these two riboswitches.


Assuntos
Cátions Bivalentes/metabolismo , Conformação de Ácido Nucleico/efeitos dos fármacos , Riboswitch/genética , Aptâmeros de Nucleotídeos/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sítios de Ligação/genética , Cátions Bivalentes/química , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/genética , Ligantes , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , RNA Bacteriano/química , Riboswitch/fisiologia , Transcrição Genética/genética
4.
J Phys Chem B ; 125(37): 10419-10431, 2021 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-34515482

RESUMO

Divalent calcium ion (Ca2+) plays an indispensable role as a second messenger in a myriad of signal transduction processes. Of utmost importance for the faultless functioning of calcium-modulated signaling proteins is their binding selectivity of the native metal cation over rival biogenic/abiogenic metal ion contenders in the intra/extracellular fluids. In this Perspective, we summarize recent findings on the competition between the cognate Ca2+ and other biogenic or abiogenic divalent cations for binding to Ca2+-signaling proteins or organic cofactors. We describe the competition between the two most abundant intracellular biogenic metal ions (Mg2+ and Ca2+) for Ca2+-binding sites in signaling proteins, followed by the rivalry between native Ca2+ and "therapeutic" Li+ as well as "toxic" Pb2+. We delineate the key factors governing the rivalry between the native and non-native cations in proteins and highlight key implications for the biological performance of the respective proteins/organic cofactors.


Assuntos
Cálcio , Transdução de Sinais , Sítios de Ligação , Cátions , Cátions Bivalentes
5.
Molecules ; 26(18)2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34577161

RESUMO

Removing toxic heavy metal species from aqueous solutions is a point of concern in our society. In this paper, a promising biomass adsorbent, the modified waste shrimp shell (MS), for Cu (II) removal was successfully prepared using a facile and simple one-step modification, making it possible to achieve high-efficiency recycling of the waste NaOH solution as the modification agent. The outcome shows that with the continuous increase in pH, temperature and ion concentration, the adsorption effect of MS on Cu (II) can also be continuously improved. Adsorption isotherm and adsorption kinetics were fitted with the Langmuir isotherm model and the pseudo-second-order model, respectively, and the maximum adsorption capacity of Cu (II) as obtained from the Langmuir isotherm model fitting reached 1.04 mmol/g. The systematic desorption results indicated that the desorption rate of Cu (II) in the MS could reach 100% within 6 min, where HNO3 is used as the desorption agent. Moreover, experiments have proven that after five successive recycles of NaOH as a modifier, the adsorption capacity of MS on Cu (II) was efficient and stable, maintaining tendency in 0.83-0.85 mmol/g, which shows that waste NaOH solution can be used as a modification agent in the preparation of waste shrimp shell adsorbent, such as waste NaOH solution produced in industrial production, thereby making it possible to turn waste into renewable resources and providing a new way to recycle resources.


Assuntos
Cobre/química , Penaeidae/química , Eliminação de Resíduos Líquidos/métodos , Poluentes Químicos da Água/química , Purificação da Água/métodos , Adsorção , Animais , Cátions Bivalentes , Cobre/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Microscopia Eletrônica de Varredura , Concentração Osmolar , Penaeidae/anatomia & histologia , Frutos do Mar , Hidróxido de Sódio/química , Temperatura , Termodinâmica , Poluentes Químicos da Água/isolamento & purificação
6.
Exp Brain Res ; 239(10): 3045-3057, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34363514

RESUMO

Previously, we reported that distal Schaffer collaterals undergo biphasic changes in excitability during high-frequency stimulation (HFS), with an early hyper-excitability period followed by an excitability depression period. The extracellular divalent cations calcium and magnesium can regulate membrane excitability in neuronal tissue. Therefore, we hypothesized that altering the concentrations of extracellular calcium and magnesium would alter the biphasic excitability changes. We tested this hypothesis by recording distal Schaffer collateral fiber volleys in stratum radiatum of hippocampal area CA1 during 100 Hz HFS in artificial cerebral spinal fluid (ACSF) containing normal and altered concentrations of extracellular divalent cations. Our normal ACSF contained 2.0 mM calcium and 2.0 mM magnesium. We examined four solutions with altered divalent cation concentrations: (1) high-calcium/low-magnesium (3.8 mM/0.2 mM), (2) low-calcium/high-magnesium (0.2 mM/3.8 mM), (3) high-calcium/normal-magnesium (3.8 mM/2.0 mM), or (4) normal-calcium/high-magnesium (2.0 mM/10.0 mM), and assessed the effects on Schaffer collateral responses. Increasing or decreasing extracellular calcium enhanced or reduced (respectively) the early hyper-excitable period whereas increasing extracellular magnesium reduced the later excitability depression. Because these results might be explained by altered calcium influx through voltage-gated calcium (CaV) channels, we tested CaV blockers (ω-agatoxin IVA, ω-conotoxin-GVIA, cadmium), but observed no effects on responses during HFS. Some of the effects of altered divalent cation concentration may be explained by altered membrane surface charge. Although this mechanism does not completely explain our findings, calcium influx through CaV channels is not required.


Assuntos
Hipocampo , Neurônios , Axônios , Cálcio , Cátions Bivalentes , Humanos , ômega-Conotoxina GVIA
7.
Biomolecules ; 11(8)2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34439824

RESUMO

Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr2+ interference with Ca2+ binding to proteins of the EF-hand family, we studied Sr2+/Ca2+ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca2+ binding sites of recombinant human α-PA ('CD' and 'EF' sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 109 M-1 and 4 × 109 M-1 for Ca2+, and 2 × 107 M-1 and 2 × 106 M-1 for Sr2+. Inactivation of the EF site by homologous substitution of the Ca2+-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca2+/Sr2+ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca2+/Sr2+ affinity. These results suggest that Sr2+ and Ca2+ bind to CD/EF sites of α-PA and the Ca2+/Sr2+ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr2+ titration of the Ca2+-loaded α-PA revealed presence of secondary Sr2+ binding site(s) with an apparent equilibrium association constant of 4 × 105 M-1. Fourier-transform infrared spectroscopy data evidence that Ca2+/Sr2+-loaded forms of α-PA exhibit similar states of their COO- groups. Near-UV circular dichroism (CD) data show that Ca2+/Sr2+ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca2+/Sr2+ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr2+-induced increase in stability of tertiary structure of α-PA, compared to the Ca2+-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca2+-binding sites of α-PA are well protected against exchange of Ca2+ for Sr2+ regardless of coordination number of Sr2+, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca2+/Sr2+ selectivity. Overall, despite lowered affinity of α-PA to Sr2+, the latter competes with Ca2+ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr2+ binding site(s) could be a factor contributing to Sr2+ impact on the functional activity of proteins.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Parvalbuminas/metabolismo , Estrôncio/metabolismo , Sítios de Ligação , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Cátions Bivalentes , Clonagem Molecular , Teoria da Densidade Funcional , Motivos EF Hand , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Parvalbuminas/química , Parvalbuminas/genética , Ligação Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Soluções
8.
Int J Mol Sci ; 22(16)2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34445449

RESUMO

The cardiac Mg2+-sensitive, TRPM6, and TRPM7-like channels remain undefined, especially with the uncertainty regarding TRPM6 expression in cardiomyocytes. Additionally, their contribution to the cardiac action potential (AP) profile is unclear. Immunofluorescence assays showed the expression of the TRPM6 and TRPM7 proteins in isolated pig atrial and ventricular cardiomyocytes, of which the expression was modulated by incubation in extracellular divalent cation-free conditions. In patch clamp studies of cells dialyzed with solutions containing zero intracellular Mg2+ concentration ([Mg2+]i) to activate the Mg2+-sensitive channels, raising extracellular [Mg2+] ([Mg2+]o) from the 0.9-mM baseline to 7.2 mM prolonged the AP duration (APD). In contrast, no such effect was observed in cells dialyzed with physiological [Mg2+]i. Under voltage clamp, in cells dialyzed with zero [Mg2+]i, depolarizing ramps induced an outward-rectifying current, which was suppressed by raising [Mg2+]o and was absent in cells dialyzed with physiological [Mg2+]i. In cells dialyzed with physiological [Mg2+]i, raising [Mg2+]o decreased the L-type Ca2+ current and the total delayed-rectifier current but had no effect on the APD. These results suggest a co-expression of the TRPM6 and TRPM7 proteins in cardiomyocytes, which are therefore the molecular candidates for the native cardiac Mg2+-sensitive channels, and also suggest that the cardiac Mg2+-sensitive current shortens the APD, with potential implications in arrhythmogenesis.


Assuntos
Potenciais de Ação , Magnésio/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Cátions Bivalentes , Miócitos Cardíacos/fisiologia , Sus scrofa/metabolismo , Sus scrofa/fisiologia , Canais de Cátion TRPM/fisiologia
9.
J Chromatogr A ; 1652: 462127, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34214833

RESUMO

In this work, the molecular mechanism of Lactobacillus paracasei bio-colloid clumping under divalent metal ions treatment such as zinc, copper and magnesium at constant concentrations was studied. The work involved experimental (electrophoretic - capillary electrophoresis in pseudo-isotachophoresis mode, spectroscopic and spectrometric - FT-IR and MALDI-TOF-MS, microscopic - fluorescent microscopy, and flow cytometry) and theoretical (DFT calculations of model complex systems) characterization. Electrophoretic results have pointed out the formation of aggregates under the Zn2+ and Cu2+ modification, whereas the use of the Mg2+ allowed focusing the zone of L. paracasei biocolloid. According to the FT-IR analysis, the major functional groups involved in the aggregation are deprotonated carboxyl and amide groups derived from the bacterial surface structure. Nature of the divalent metal ions was shown to be one of the key factors influencing the bacterial aggregation process. Proteomic analysis showed that surface modification had a considerable impact on bacteria molecular profiles and protein expression, mainly linked to the activation of carbohydrate and nucleotides metabolism as well with the transcription regulation and membrane transport. Density-functional theory (DFT) calculations of modeled Cu2+, Mg2+ and Zn2+ coordination complexes support the interaction between the divalent metal ions and bacterial proteins. Consequently, the possible mechanism of the aggregation phenomenon was proposed. Therefore, this comprehensive study could be further applied in evaluation of biocolloid aggregation under different types of metal ions.


Assuntos
Cátions Bivalentes , Eletroforese , Íons , Lactobacillus paracasei , Metais , Cátions Bivalentes/química , Íons/química , Lactobacillus paracasei/metabolismo , Metais/química , Proteômica , Espectroscopia de Infravermelho com Transformada de Fourier
10.
Sci Rep ; 11(1): 15445, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326388

RESUMO

The expression of the channels-enzymes TRPM6 and TRPM7 in the human heart remains poorly defined, and TRPM6 is generally considered not to be expressed in cardiomyocytes. We examined their expression at protein and mRNA levels using right atrial samples resected from patients (n = 72) with or without ischemic heart disease (IHD) and samples from all chamber walls of explanted human hearts (n = 9). TRPM6 and TRPM7 proteins were detected using immunofluorescence on isolated cardiomyocytes, ELISA on tissue homogenates, and immunostaining of cardiac tissue, whereas their mRNAs were detected by RT-qPCR. Both TRPM6 and TRPM7 were present in all chamber walls, with TRPM7 being more abundant. TRPM6 was co-expressed with TRPM7. The expression levels were dependent on cell incubation conditions (presence or absence of divalent cations, pH of the extracellular milieu, presence of TRP channel inhibitors 2-aminoethoxydiphenyl-borate and carvacrol). These drugs reduced TRPM7 immunofluorescence but increased that of TRPM6. TRPM6 and TRPM7 expression was increased in tissues from IHD patients. This is the first demonstration of the presence and co-expression of TRPM6 and TRPM7 in cardiomyocytes from all chamber walls of the human heart. The increased TRPM6 and TRPM7 expression in IHD suggests that the chanzymes are involved in the pathophysiology of the disease.


Assuntos
Expressão Gênica , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , /metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Cátions Bivalentes/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Imunofluorescência/métodos , Humanos , Magnésio/metabolismo , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos
11.
Biochemistry ; 60(31): 2374-2386, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34319696

RESUMO

RNA regulates myriad cellular events such as transcription, translation, and splicing. To perform these essential functions, RNA often folds into complex tertiary structures in which its negatively charged ribose-phosphate backbone interacts with metal ions. Magnesium, the most abundant divalent metal ion in cells, neutralizes the backbone, thereby playing essential roles in RNA folding and function. This has been known for more than 50 years, and there are now thousands of in vitro studies, most of which have used ≥10 mM free Mg2+ ions to achieve optimal RNA folding and function. In the cell, however, concentrations of free Mg2+ ions are much lower, with most Mg2+ ions chelated by metabolites. In this Perspective, we curate data from a number of sources to provide extensive summaries of cellular concentrations of metabolites that bind Mg2+ and to estimate cellular concentrations of metabolite-chelated Mg2+ species, in the representative prokaryotic and eukaryotic systems Escherichia coli, Saccharomyces cerevisiae, and iBMK cells. Recent research from our lab and others has uncovered the fact that such weakly chelated Mg2+ ions can enhance RNA function, including its thermodynamic stability, chemical stability, and catalysis. We also discuss how metabolite-chelated Mg2+ complexes may have played roles in the origins of life. It is clear from this analysis that bound Mg2+ should not be simply considered non-RNA-interacting and that future RNA research, as well as protein research, could benefit from considering chelated magnesium.


Assuntos
Magnésio/metabolismo , Dobramento de RNA , RNA/metabolismo , RNA/fisiologia , Animais , Biocatálise , Cátions Bivalentes/química , Cátions Bivalentes/metabolismo , Linhagem Celular , Escherichia coli/metabolismo , Magnésio/química , Metaboloma/fisiologia , Camundongos , RNA/química , Saccharomyces cerevisiae/metabolismo
12.
Molecules ; 26(14)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34299529

RESUMO

Aroma deterioration is one of the biggest problems in processing tea beverages. The aroma of tea infusion deteriorates fast during heat sterilization and the presence of ferrous ion (Fe2+) aggravates it. The underlying mechanism remains unveiled. In this study, Fe2+ was verified to deteriorate the aroma quality of green tea infusion with heat treatment. Catechins were necessary for Fe2+-mediated aroma deterioration. By enhancing the degradation of catechins, Fe2+ dramatically increased the production of hydrogen peroxide (H2O2). Fe2+ and H2O2 together exacerbated the aroma of green tea infusion with heat treatment. GC-MS analysis revealed that the presence of Fe2+ enhanced the loss of green/grassy volatiles and promoted the formation of new volatiles with diversified aroma characteristics, resulting in a dull scent of green tea infusion. Our results revealed how Fe2+ induced aroma deterioration of green tea infusion with heat treatment and could help guide tea producers in attenuating the aroma deterioration of tea infusion during processing.


Assuntos
Compostos Ferrosos/análise , Odorantes/análise , Chá/química , Catequina/química , Cátions Bivalentes/análise , Temperatura Alta , Ferro/análise , Esterilização
13.
Chem Commun (Camb) ; 57(67): 8340-8343, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34328150

RESUMO

Complexing with adenosine-5'-monophosphate (AMP) was proven to be a facile way to enhance the oxidase-mimicking activity of Ce4+, and enabled nanoenzyme recovery and reuse. Additionally, the oxidase-mimicking activity of AMP-Ce4+ infinite coordination polymers (ICPs) could be specifically inhibited by Fe2+. Based on this finding, we developed a simple and highly selective colorimetric assay to detect Fe2+.


Assuntos
Monofosfato de Adenosina/química , Materiais Biomiméticos/química , Cério/química , Complexos de Coordenação/química , Ferro/análise , Nanopartículas Metálicas/química , Oxirredutases/química , Catálise , Cátions Bivalentes/análise , Colorimetria , Corantes Fluorescentes/química , Limite de Detecção , Polímeros/química
14.
Nucleic Acids Res ; 49(12): 7139-7153, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34125892

RESUMO

Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson-Crick base pair to junction J2. Xanthine inserts between a G-U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg2+ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg2+ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.


Assuntos
Magnésio/química , Riboswitch , Xantina/química , Sítios de Ligação , Cátions Bivalentes , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Dobramento de RNA
15.
Langmuir ; 37(24): 7573-7581, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34101478

RESUMO

Self-assembled monolayers are attractive for surface modification due to their ease of synthesis and the range of chemical functionality that can be applied. Metal-phosphonate monolayer properties can be controlled through the metal ions that can be used in their formation. The organization and fluid properties of these monolayers can be understood in the context of their thermodynamic properties and the association and dissociation kinetics that proceed at the metal-phosphonate complex. In this work, four different M(II)-phosphonate monolayers were synthesized and the diffusional behavior of free and tethered chromophores was evaluated using fluorescence recovery after photobleaching measurements. The ω-terminal group identity of the metal-phosphonate monolayer was varied to determine its effect on monolayer dynamics.


Assuntos
Organofosfonatos , Cátions Bivalentes , Difusão , Fotodegradação , Propriedades de Superfície
16.
Nucleic Acids Res ; 49(12): 6863-6879, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34139017

RESUMO

Helicobacter pylori is a gram-negative, microaerophilic, pathogenic bacterium and a widespread colonizer of humans. H. pylori has developed mechanisms that enable it to overcome the harsh environment of the human stomach, including reactive oxygen species (ROS). Interestingly, up to now no typical regulator dedicated to the oxidative-stress response has been discovered. In this work, we reveal that the inhibitor of replication initiation HP1021 functions as a redox switch protein in H. pylori and plays an important role in response to oxidative stress of the gastric pathogen. Each of the two predicted HP1021 domains contains three cysteine residues. We show that the cysteine residues of HP1021 are sensitive to oxidation both in vitro and in vivo, and we demonstrate that HP1021 DNA-binding activity to oriC depends on the redox state of the protein. Moreover, Zn2+ modulates HP1021 affinity towards oriC template DNA. Transcription analysis of selected H. pylori genes by RT-qPCR indicated that HP1021 is directly involved in the oxygen-dependent control of H. pylori fecA3 and gluP genes, which are implicated in response to oxidative stress. In conclusion, HP1021 is a redox switch protein and could be a target for H. pylori control strategies.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Helicobacter pylori/genética , Estresse Oxidativo , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Cátions Bivalentes/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica , Domínios Proteicos , Transcrição Genética
17.
Biochemistry ; 60(24): 1909-1918, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34106684

RESUMO

The RNA-cleaving 17E DNAzyme exhibits different levels of cleavage activity in the presence of various divalent metal ions, with Pb2+ giving the fastest cleavage. In this study, the metal-phosphate interaction is probed to understand the trend of activity with different metal ions. For the first-row transition metals, the lowest activity shown by Ni2+ correlates with the inhibition by the inorganic phosphate and its water ligand exchange rate, suggesting inner-sphere metal coordination. Cleavage activity with the two stereoisomers of the phosphorothioate-modified substrates, Rp and Sp, indicated that Mg2+, Mn2+, Fe2+, and Co2+ had the highest Sp:Rp activity ratio of >900. Comparatively, the activity was much less affected using the thiophilic metals, including Pb2+, suggesting inner-sphere coordination. The pH-rate profiles showed that Pb2+ was different than the rest of the metal ions in having a smaller slope and a similar fitted apparent pKa and the pKa of metal-bound water. Combining previous reports and our current results, we propose that Pb2+ most likely plays the role of a general acid while the other metal ions are Lewis acid catalysts interacting with the scissile phosphate.


Assuntos
Cátions Bivalentes/metabolismo , DNA Catalítico/metabolismo , Fosfatos/metabolismo , Catálise , DNA/química , DNA Catalítico/genética , Hidrólise , Íons , Metais/metabolismo , RNA/metabolismo
18.
Langmuir ; 37(25): 7780-7788, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34129342

RESUMO

Divalent cations, especially Ca2+ and Mg2+, play a vital role in the function of biomolecules and making them important to be constituents in samples for in vitro biophysical and biochemical characterizations. Although lipid nanodiscs are becoming valuable tools for structural biology studies on membrane proteins and for drug delivery, most types of nanodiscs used in these studies are unstable in the presence of divalent metal ions. To avoid the interaction of divalent metal ions with the belt of the nanodiscs, synthetic polymers have been designed and demonstrated to form stable lipid nanodiscs under such unstable conditions. Such polymer-based nanodiscs have been shown to provide an ideal platform for structural studies using both solid-state and solution NMR spectroscopies because of the near-native cell-membrane environment they provide and the unique magnetic-alignment behavior of large-size nanodiscs. In this study, we report an investigation probing the effects of Ca2+ and Mg2+ ions on the formation of polymer-based lipid nanodiscs and the magnetic-alignment properties using a synthetic polymer, styrene maleimide quaternary ammonium (SMA-QA), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids. Phosphorus-31 NMR experiments were used to evaluate the stability of the magnetic-alignment behavior of the nanodiscs for varying concentrations of Ca2+ or Mg2+ at different temperatures. It is remarkable that the interaction of divalent cations with lipid headgroups promotes the stacking up of nanodiscs that results in the enhanced magnetic alignment of nanodiscs. Interestingly, the reported results show that both the temperature and the concentration of divalent metal ions can be optimized to achieve the optimal alignment of nanodiscs in the presence of an applied magnetic field. We expect the reported results to be useful in the design of nanodisc-based nanoparticles for various applications in addition to atomic-resolution structural and dynamics studies using NMR and other biophysical techniques.


Assuntos
Nanoestruturas , Polímeros , Cátions Bivalentes , Íons , Bicamadas Lipídicas , Fenômenos Magnéticos , Espectroscopia de Ressonância Magnética
19.
Sci Rep ; 11(1): 10961, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34040092

RESUMO

Trichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme's biochemical and biophysical properties were assessed. GH54 members show wide functional diversity and specifically remove plant cell substitutions including arabinose and galactose in the presence of a metallic cofactor. Plant cell wall substitution has a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity of the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.


Assuntos
Cátions Bivalentes/química , Proteínas Fúngicas/isolamento & purificação , Glicosídeo Hidrolases/isolamento & purificação , Hypocreales/enzimologia , Família Multigênica , Sequência de Aminoácidos , Sequência de Bases , Biodegradação Ambiental , Simulação por Computador , Sequência Consenso , Mineração de Dados , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/classificação , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hypocreales/genética , Modelos Moleculares , Filogenia , Polissacarídeos/metabolismo , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Açúcares/metabolismo , Temperatura
20.
ACS Chem Neurosci ; 12(11): 2027-2035, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-33973758

RESUMO

In several neurodegenerative diseases, cell toxicity can emerge from damage produced by amyloid aggregates to lipid membranes. The details accounting for this damage are poorly understood including how individual amyloid peptides interact with phospholipid membranes before aggregation. Here, we use all-atom molecular dynamics simulations to investigate the molecular mechanisms accounting for amyloid-membrane interactions and the role played by calcium ions in this interaction. Model peptides known to self-assemble into amyloid fibrils and bilayer made from zwitterionic and anionic lipids are used in this study. We find that both electrostatic and hydrophobic interactions contribute to peptide-bilayer binding. In particular, the attraction of peptides to lipid bilayers is dominated by electrostatic interactions between positive residues and negative phosphate moieties of lipid head groups. This attraction is stronger for anionic bilayers than for zwitterionic ones. Hydrophobicity drives the burial of nonpolar residues into the interior of the bilayer producing strong binding in our simulations. Moreover, we observe that the attraction of peptides to the bilayer is significantly reduced in the presence of calcium ions. This is due to the binding of calcium ions to negative phosphate moieties of lipid head groups, which leaves phospholipid bilayers with a net positive charge. Strong binding of the peptide to the membrane occurs less frequently in the presence of calcium ions and involves the formation of a "Ca2+ bridge".


Assuntos
Peptídeos beta-Amiloides , Bicamadas Lipídicas , Amiloide , Cátions Bivalentes , Simulação de Dinâmica Molecular
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