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1.
Front Microbiol ; 12: 684591, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335511

RESUMO

Antibiotic-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) has become a serious threat to public health worldwide. Cationic α-helical antimicrobial peptides (CαAMPs) have attracted much attention as promising solutions in post-antibiotic era. However, strong hemolytic activity and in vivo inefficacy have hindered their pharmaceutical development. Here, we attempt to address these obstacles by investigating BmKn2 and BmKn2-7, two scorpion-derived CαAMPs with the same hydrophobic face and a distinct hydrophilic face. Through structural comparison, mutant design and functional analyses, we found that while keeping the hydrophobic face unchanged, increasing the number of alkaline residues (i.e., Lys + Arg residues) on the hydrophilic face of BmKn2 reduces the hemolytic activity and broadens the antimicrobial spectrum. Strikingly, when keeping the total number of alkaline residues constant, increasing the number of Lys residues on the hydrophilic face of BmKn2-7 significantly reduces the hemolytic activity but does not influence the antimicrobial activity. BmKn2-7K, a mutant of BmKn2-7 in which all of the Arg residues on the hydrophilic face were replaced with Lys, showed the lowest hemolytic activity and potent antimicrobial activity against antibiotic-resistant ESKAPE pathogens. Moreover, in vivo experiments indicate that BmKn2-7K displays potent antimicrobial efficacy against both the penicillin-resistant S. aureus and the carbapenem- and multidrug-resistant A. baumannii, and is non-toxic at the antimicrobial dosages. Taken together, our work highlights the significant functional disparity of Lys vs Arg in the scorpion-derived antimicrobial peptide BmKn2-7, and provides a promising lead molecule for drug development against ESKAPE pathogens.

2.
Toxins (Basel) ; 13(5)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064808

RESUMO

Scorpion venoms are rich resources of antimicrobial peptides (AMPs). While the short-chain noncysteine-containing AMPs have attracted much attention as templates for drug development, the antimicrobial potential of long-chain noncysteine-containing AMPs has been largely overlooked. Here, by using the online HeliQuest server, we designed and analyzed a series of 14-residue fragments of Smp43, a 43-residue long-chain noncysteine-containing AMP identified from the venom of Scorpio maurus palmatus. We found that Smp43(1-14) shows high antimicrobial activity against both Gram-positive and Gram-negative bacteria and is nontoxic to mammalian cells at the antimicrobial dosage. Sequence alignments showed that the designed Smp43(1-14) displays a unique primary structure that is different from other natural short-chain noncysteine-containing AMPs from scorpions, such as Uy17, Uy192 and IsCT. Moreover, the peptide Smp43(1-14) caused concentration-dependent fluorescence increases in the bacteria for all of the tested dyes, propidium iodide, SYTOXTM Green and DiSC3-5, suggesting that the peptide may kill the bacteria through the formation of pore structures in the plasma membrane. Taken together, our work sheds light on a new avenue for the design of novel short-chain noncysteine-containing AMPs and provides a good peptide template with a unique sequence for the development of novel drugs for use against bacterial infectious diseases.


Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Venenos de Escorpião/química , Animais , Antibacterianos/isolamento & purificação , Membrana Celular/metabolismo , Proteínas Citotóxicas Formadoras de Poros/isolamento & purificação , Conformação Proteica em alfa-Hélice , Escorpiões
3.
Microb Pathog ; 157: 104960, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34022355

RESUMO

Carbapenem-resistant Acinetobacter baumannii (CRAB) is becoming a troublesome issue worldwide, and anti-CRAB drug research and development is urgently needed. To identify new anti-CRAB drug leads, we investigated seven scorpion venom-derived α-helical peptides that differ in their sequence composition and length. Three peptides, Hp1404, ctriporin and Im5, showed antimicrobial activities against Acinetobacter baumannii. Further antimicrobial assays revealed that Hp1404 exhibited the best cell selectivity with high anti-CRAB and low hemolytic activities. Fluorescence assays demonstrated that Hp1404 can induce dose-dependent disruptions of the bacterial cell membrane, implying a membrane-lytic mode of action. Taken together, our work sheds light on the potential of the scorpion venom-derived peptide Hp1404 for the development of novel antimicrobial agents against CRAB infections.


Assuntos
Acinetobacter baumannii , Anti-Infecciosos , Venenos de Escorpião , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Carbapenêmicos/farmacologia , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros , Venenos de Escorpião/farmacologia
4.
J Phys Chem Lett ; 12(20): 5009-5015, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34018746

RESUMO

We considered a database of tens of thousands of known organic semiconductors and identified those compounds with computed electronic properties (orbital energies, excited state energies, and oscillator strengths) that would make them suitable as nonfullerene electron acceptors in organic solar cells. The range of parameters for the desirable acceptors is determined from a set of experimentally characterized high-efficiency nonfullerene acceptors. This search leads to ∼30 lead compounds never considered before for organic photovoltaic applications. We then proceed to modify these compounds to bring their computed solubility in line with that of the best small-molecule nonfullerene acceptors. A further refinement of the search can be based on additional properties like the reorganization energy for chemical reduction. This simple strategy, which relies on a few easily computable parameters and can be expanded to a larger set of molecules, enables the identification of completely new chemical families to be explored experimentally.

5.
Toxicon ; 196: 63-73, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33836178

RESUMO

Methicillin-resistant staphylococci have become growing threats to human health, and novel antimicrobials are urgently needed. Natural antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics. Here, two novel cationic α-helical antimicrobial peptides, Lausporin-1 and Lausporin-2, were identified from the venom gland of the scorpion L. australasiae through a cDNA library screening strategy. Biochemical analyses demonstrated that Lausporin-1 and Lausporin-2 are cationic α-helical amphipathic molecules. Antimicrobial assays demonstrated that the two peptides possess antibacterial activities against several species of antibiotic-resistant staphylococci. Importantly, they are active against methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus capitis, with the minimum inhibitory concentrations ranging from 2.5 to 10 µg/ml. Moreover, both peptides can induce dose-dependent plasma membrane disruptions of the bacteria. In short, our work expands the knowledge of the scorpion L. australasiae venom-derived AMPs and sheds light on the potential of Lausporin-1 and Lausporin-2 in the development of novel drugs against methicillin-resistant staphylococci.


Assuntos
Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Animais , Antibacterianos/farmacologia , Humanos , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Escorpiões
6.
Chemistry ; 27(37): 9571-9579, 2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-33786898

RESUMO

Quantum mechanical and molecular dynamics simulations have been carried out on a series of anthracene-o-carborane derivatives (ANT-H, ANT-Ph, ANT-Me and ANT-TMS) with rare red-light emission in the solid state. The simulation of the heating process of the crystals and further comparison of the molecular structures and excited-state properties before and after heating help us to disclose the thermochromic behavior, that is, the red-shift emission is caused by elongation of the C1-C2 bond in the carborane moiety after heating. Thus, we believe that the molecular structure in the crystal is severely affected by heating. Transformation of the molecular conformation appears in the ANT-H crystal with increasing temperature. More specifically, the anthracene moiety moves from nearly parallel to the C1-C2 bond to nearly perpendicular, causing the short-wavelength emission to disappear after heating. As for the aggregation-induced emission phenomenon, the structures and photophysical properties were investigated comparatively in both the isolated and crystal states; the results suggested that the energy dissipation in crystal surroundings was greatly reduced through hindering structure relaxation from the excited to the ground state. We expect that discussion of the thermochromic behavior will provide a new analysis perspective for the molecular design of o-carborane derivatives.

7.
J Org Chem ; 86(1): 484-492, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33295780

RESUMO

Recent experimental work reported that visible-light photoredox catalysis coupled with primary sulfonamides and electron-deficient alkenes could efficiently construct C-C bonds at the α-position of primary amine derivatives under mild conditions. Here, a systematic study was conducted to explore the non-negligible excited-state single-electron-transfer (SET) processes and the catalytic cycle. Hydrogen atom transfer (HAT) catalysis containing different site-selective functionalization, involved as a critical process during the reaction, was computationally characterized. The superiorities of iridium-based photoredox catalysts in terms of photoabsorption properties, phosphorescence rates, and electron-transfer rates for SET processes were focused on. In addition, the function of quinuclidine in the entire photocatalytic reaction was also probed. These intrinsic properties and detailed insights into the mechanism are supposed to be helpful to the understanding of the C-C bond functionalization reaction and the future application of the iridium-based photoredox catalyst.

8.
J Bacteriol ; 203(1)2020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-33046561

RESUMO

The invasion and colonization of host plants by the destructive pathogen Ralstonia solanacearum rely on its cell motility, which is controlled by multiple factors. Here, we report that the LysR-type transcriptional regulator CrgA (RS_RS16695) represses cell motility in R. solanacearum GMI1000. CrgA possesses common features of a LysR-type transcriptional regulator and contains an N-terminal helix-turn-helix motif as well as a C-terminal LysR substrate-binding domain. Deletion of crgA results in an enhanced swim ring and increased transcription of flhDC In addition, the ΔcrgA mutant possesses more polar flagella than wild-type GMI1000 and exhibits higher expression of the flagellin gene fliC Despite these alterations, the ΔcrgA mutant did not have a detectable growth defect in culture. Yeast one-hybrid and electrophoretic mobility shift assays revealed that CrgA interacts directly with the flhDC promoter. Expressing the ß-glucuronidase (GUS) reporter under the control of the crgA promoter showed that crgA transcription is dependent on cell density. Soil-soaking inoculation with the crgA mutant caused wilt symptoms on tomato (Solanum lycopersicum L. cv. Hong yangli) plants earlier than inoculation with the wild-type GMI1000 but resulted in lower disease severity. We conclude that the R. solanacearum regulator CrgA represses flhDC expression and consequently affects the expression of fliC to modulate cell motility, thereby conditioning disease development in host plants.IMPORTANCE Ralstonia solanacearum is a widely distributed soilborne plant pathogen that causes bacterial wilt disease on diverse plant species. Motility is a critical virulence attribute of R. solanacearum because it allows this pathogen to efficiently invade and colonize host plants. In R. solanacearum, motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum motility is more complex than previously thought and enhances our understanding of flagellum regulation in R. solanacearum.


Assuntos
Proteínas de Bactérias/fisiologia , Flagelos/fisiologia , Ralstonia solanacearum/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Lycopersicon esculentum/microbiologia , Microscopia Eletrônica de Transmissão , Regiões Promotoras Genéticas/fisiologia , Ralstonia solanacearum/genética , Ralstonia solanacearum/patogenicidade , Ralstonia solanacearum/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real , Elementos Reguladores de Transcrição/fisiologia , Microbiologia do Solo , Técnicas do Sistema de Duplo-Híbrido , Virulência
9.
J Mol Graph Model ; 94: 107488, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31707196

RESUMO

In order to compare the main difference of two diimide derivatives on the modulation of electronic and optical properties of P3HT-based organic solar cell, the density functional theory and molecular dynamics simulations were implemented to achieve elementary data on geometrical structure, molecular orbital, open-circuit voltage, absorption spectra, energetic driving force, and interface parameter of P3HT/D1 and P3HT/D2 systems. According to the investigation, P3HT/D1 system not only exhibits higher open circuit voltage and enough energetic driving force than P3HT/D2 system, but also possesses low-lying LUMO +1 orbital which can favor the exciton separation efficiency. Moreover, on the basis of some typical interface models choose from MD simulation, the estimation of the interface rate manifests that the P3HT/D1 interface possesses the smaller charge recombination rates and larger charge separation rates than those of the P3HT/D2 interface. It is expect that this work can provide certain guidelines for the further develop the performance of organic solar cell. We hope this work can further study on non-fullerene acceptor materials as a certain guides.


Assuntos
Simulação de Dinâmica Molecular , Teoria da Densidade Funcional
10.
Anal Chem ; 91(9): 6097-6102, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30966737

RESUMO

A variety of fluorophores have been designed and created to fabricate organic fluorescent probes. Among these fluorophores, benzopyran-coumarin (BC) based fluorescent platform has attracted increasing attention as it shows multiple appropriate fluorescent and imaging capacities. Nevertheless, the analytical potential of BC is still urgently needed to be further excavated as its detection performance is hindered by the inherent drawbacks of current BC skeleton, that is, limited number of reactive sites. As such, in this work, by simply introducing electron-withdrawing (EW) substituent groups, we reconstructed BC skeleton to afford two fluorescent probes, BCB (-Br substitued) and BCN (-NO2 substitued), both of which featured two highly reactive sites. These two probes were capable of detecting peroxynitrite (ONOO-) and biothiols (hydrogen sulfide, glutathione, cysteine, and homocysteine) through naked eye and UV-vis absorption analysis in buffer solution. In addition, BCB was able to specifically sense biothiols with fluorescent analysis while BCN, with - NO2 instead of -Br, displayed more prominent fluorescent specificity toward ONOO-. This work provided a new strategy for the reactivity regulation of fluorophore through EW group introduction, as well as an alternative approach and method for the construction of fluorescent probes for other important biological species.


Assuntos
Benzopiranos/química , Cumarínicos/química , Elétrons , Corantes Fluorescentes/química , Ácido Peroxinitroso/análise , Compostos de Sulfidrila/análise , Animais , Corantes Fluorescentes/síntese química , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos , Estrutura Molecular , Imagem Óptica , Células PC12 , Ratos
11.
Mol Plant Pathol ; 20(3): 372-381, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30353625

RESUMO

Xanthomonas citri ssp. citri, a polar flagellated bacterium, causes citrus canker disease worldwide. In this study, we found that the X. citri ssp. citri response regulator VemR plays a regulatory role in flagellum-derived cell motility. Deletion of the vemR gene resulted in a reduction in cell motility, as well as reductions in virulence and exopolysaccharide production. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that vemR is transcribed in an operon together with rpoN2 and fleQ. In the vemR mutant, the flagellar distal rod gene flgG was significantly down-regulated. Because flgG is also rpoN2 dependent, we speculated that VemR and RpoN2 physically interact, which was confirmed by yeast two-hybrid and maltose-binding protein (MBP) pull-down assays. This suggested that the transcription of flgG is synergistically controlled by VemR and RpoN2. To confirm this, we constructed a vemR and rpoN2 double mutant. In this mutant, the reductions in cell motility and flgG transcription were unable to be restored by the expression of either vemR or rpoN2 alone. In contrast, the expression of both vemR and rpoN2 together in the double mutant restored the wild-type phenotype. Together, our data demonstrate that the response regulator VemR functions as an RpoN2 cognate activator to positively regulate the transcription of the rod gene flgG in X. citri ssp. citri.


Assuntos
Doenças das Plantas/microbiologia , Xanthomonas/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Virulência
12.
Angew Chem Int Ed Engl ; 58(4): 1148-1152, 2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30411835

RESUMO

A general and efficient protocol was developed for the synthesis of polysubstituted pyridines from propargyl amines and unsaturated carbonyl compounds through a tandem condensation/alkyne isomerization/6π 3-azatriene electrocyclization sequence. This process was found to be applicable to a wide range of readily available substrates (30 examples, up to 95 % yield) and could be readily performed on a preparative (20 g) scale. By taking advantage of this method for late-stage pyridine incorporation, we successfully completed the collective total synthesis of suveoline, norsuveoline, and macrophylline.

13.
Mol Plant Microbe Interact ; 32(6): 697-707, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30540527

RESUMO

Ralstonia solanacearum is the causal agent of bacterial wilt disease. Here, we report that a large FAD-linked oxidase encoded by RSc0454 in GMI1000 is required for pathogenicity. The FAD-linked oxidase encoded by RSc0454 is composed of 1,345 amino acids, including DUF3683, lactate dehydrogenase (LDH), and succinate dehydrogenase (SDH) domains. The RSc0454 protein showed both LDH and SDH activities. To investigate its role in pathogenicity, a deletion mutant of the RSc0454 gene was constructed in GMI1000, which was impaired in its ability to cause bacterial wilt disease in tomato. A single DUF3683, LDH, or SDH domain was insufficient to restore bacterial pathogenicity. Mutagenesis of the RSc0454 gene did not affect growth rate but caused cell aggregation at the bottom of the liquid nutrient medium, which was reversed by exogenous applications of lactate, fumarate, pyruvate, and succinate. qRT-PCR and promoter LacZ fusion experiments demonstrated that RSc0454 gene transcription was induced by lactate and fumarate (both substrates of LDH). Compared with the downregulation of the succinate dehydrogenase gene sdhBADC and the lactate dehydrogenase gene ldh, RSc0454 gene transcription was enhanced in planta. This suggests that the oxidase encoded by RSc0454 was involved in a redox balance, which is in line with the different living conditions of R. solanacearum.


Assuntos
Oxirredutases , Ralstonia solanacearum , Virulência , Flavina-Adenina Dinucleotídeo/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Doenças das Plantas/microbiologia , Ralstonia solanacearum/enzimologia , Ralstonia solanacearum/genética , Ralstonia solanacearum/patogenicidade , Deleção de Sequência , Virulência/genética
14.
Chem Commun (Camb) ; 54(84): 11965-11968, 2018 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-30289138

RESUMO

Utilizing the oxidation/elimination tandem reaction of the α-phenylseleno carbonyl moiety, a two-photon fluorescent probe for ratiometric visualization of hypochlorous acid was developed. Its superior sensing performance and practical applications were well demonstrated.

15.
J Inequal Appl ; 2018(1): 82, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29674836

RESUMO

In this paper, we consider the variable selection problem of the generalized random coefficient autoregressive model (GRCA). Instead of parametric likelihood, we use non-parametric empirical likelihood in the information theoretic approach. We propose an empirical likelihood-based Akaike information criterion (AIC) and a Bayesian information criterion (BIC).

16.
J Mol Graph Model ; 77: 9-16, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28802153

RESUMO

A series of polymer donor materials 1-5 based on diketopyrrolopyrrole and thiophene unit which have been widely used in organic solar cells (OSCs) were investigated based on quantum chemical calculations. The effect of fluorine and cyano substitutions in polymer donor materials was focused on. Based on the investigation on electronic structures and optical properties of the reported molecules 1 and 2 and the analysis on some parameters relevant to charge dissociation ability at donor/acceptor interface constituted by 1 and 2 with PC61BM such as intermolecular charge transfer and recombination, driving force and Coulombic bound energy, we explained why fluorine substitution can improve OPV efficiency through strengthening eletron-withdrawing ability from a theoretical perspective. Then we designed cyano-substituted polymers 3-5 with the aim of obtaining better photovoltaic donor materials. The results reveal that our attempt to design donor materials which can balance large open-circuit voltage (Voc) and high short-circuit current (Jsc) in OSCs has worked out. It is worth noting that the substitutions of fluorine and cyano groups synergistically reduce energy gap and HOMO energy level of polymers 3 and 4. Moreover, 3/PC61BM and 4/PC61BM heterojunctions show over 107 and 104 times higher than 1/PC61BM on the ratios of intermolecular charge transfer and recombination rates (kinter-CT/kinter-CR). Thus, our work here may provide an efficient strategy to design promising donor materials in OPVs and we hope it could be useful in the future experimental synthesis.


Assuntos
Flúor/química , Modelos Teóricos , Polímeros/química , Energia Solar , Elétrons , Tiofenos
17.
Inorg Chem ; 56(8): 4569-4576, 2017 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-28345901

RESUMO

Ionic rare-earth metal complexes 1-4 bearing an imidazolium cation were synthesized, which, as single-component catalysts, showed good activity in catalyzing cyclic carbonate synthesis from epoxides and CO2. In the presence of 0.2 mol % catalyst, monosubstituted epoxides bearing different functional groups were converted into cyclic carbonates in 60-97% yields under atmospheric pressure. In addition, bulky/internal epoxides with low reactivity yielded cyclic carbonates in 40-95% yields. More importantly, the readily available samarium complex 2 was reused for six successive cycles without any significant loss in its catalytic activity. This is the first recyclable rare-earth metal-based catalyst in cyclic carbonate synthesis.

18.
Prep Biochem Biotechnol ; 45(2): 192-204, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-24678584

RESUMO

A novel exopolysaccharide (EPS), namely, B4-EPS, is produced by Arthrobacter sp. B4. Response surface methodology (RSM) was employed to optimize the fermentation medium for increasing B4-EPS production. Based on Plackett-Burman design (PBD), glucose, yeast extract, and KH2PO4 were selected as significant variables, which were further optimized by a central composite design (CCD). According to response surface and canonical analysis, the optimal medium was composed of 16.94 g/L glucose, 2.33 g/L yeast extract, and 5.32 g/L KH2PO4. Under this condition, the maximum yield of B4-EPS reached about 8.54 g/L after 72 hr of batch fermentation, which was pretty close to the predicted value (8.52 g/L). Furthermore, B4-EPS was refined by column chromatography. The main homogeneous fraction (B4-EPS1) was collected and applied to assay of antibiofilm activity. B4-EPS1 exhibited a dose-dependent inhibitory effect on biofilm formation of Pseudomonas aeruginosa PAO1 without antibacterial activity. About 86.1% of biofilm formation of P. aeruginosa PAO1 was inhibited in the presence of 50 µg/mL B4-EPS1, which was more effective than the peer published data. Moreover, B4-EPS1 could prevent biofilm formation of other strains. These data suggest B4-EPS may represent a promising strategy to combat bacterial biofilms in the future.


Assuntos
Arthrobacter , Biofilmes/efeitos dos fármacos , Polissacarídeos Bacterianos , Pseudomonas aeruginosa/fisiologia , Arthrobacter/química , Arthrobacter/crescimento & desenvolvimento , Meios de Cultura/química , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/isolamento & purificação , Polissacarídeos Bacterianos/farmacologia
19.
J Biol Chem ; 289(24): 16914-23, 2014 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-24764303

RESUMO

Large conductance Ca(2+)- and voltage-activated potassium (BK) channels, composed of pore-forming α subunits and auxiliary ß subunits, play important roles in diverse physiological activities. The ß1 is predominately expressed in smooth muscle cells, where it greatly enhances the Ca(2+) sensitivity of BK channels for proper regulation of smooth muscle tone. However, the structural basis underlying dynamic interaction between BK mSlo1 α and ß1 remains elusive. Using macroscopic ionic current recordings in various Ca(2+) and Mg(2+) concentrations, we identified two binding sites on the cytosolic N terminus of ß1, namely the electrostatic enhancing site (mSlo1(K392,R393)-ß1(E13,T14)), increasing the calcium sensitivity of BK channels, and the hydrophobic site (mSlo1(L906,L908)-ß1(L5,V6,M7)), passing the physical force from the Ca(2+) bowl onto the enhancing site and S6 C-linker. Dynamic binding of these sites affects the interaction between the cytosolic domain and voltage-sensing domain, leading to the reduction of Mg(2+) sensitivity. A comprehensive structural model of the BK(mSlo1 α-ß1) complex was reconstructed based on these functional studies, which provides structural and mechanistic insights for understanding BK gating.


Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Magnésio/metabolismo , Potenciais de Ação , Sequência de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Gelo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
20.
PLoS One ; 8(5): e64286, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23696876

RESUMO

Voltage-gated sodium channel Nav1.5 has been linked to the cardiac cell excitability and a variety of arrhythmic syndromes including long QT, Brugada, and conduction abnormalities. Nav1.5 exhibits a slow inactivation, corresponding to a duration-dependent bi-exponential recovery, which is often associated with various arrhythmia syndromes. However, the gating mechanism of Nav1.5 and the physiological role of slow inactivation in cardiac cells remain elusive. Here a 12-state two-step inactivation Markov model was successfully developed to depict the gating kinetics of Nav1.5. This model can simulate the Nav1.5 channel in not only steady state processes, but also various transient processes. Compared with the simpler 8-state model, this 12-state model is well-behaved in simulating and explaining the processes of slow inactivation and slow recovery. This model provides a good framework for further studying the gating mechanism and physiological role of sodium channel in excitable cells.


Assuntos
Modelos Teóricos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Linhagem Celular , Eletrofisiologia , Humanos , Cinética
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