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2.
Science ; 369(6500): 138-139, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32646984
3.
Chemosphere ; 255: 126948, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32387733

RESUMO

Uranium phosphites have recently emerged as promising materials to remediate radioactive contamination. In this study, the redox mechanisms of uranyl phosphites at mineral surfaces have been addressed by periodic DFT calculations with dispersion corrections. Different from other ligands, the phosphite anions (H2PO3-, HPO32-) are efficient reducing agents for uranyl reduction, and the redox reactions are divided into three steps, as isomerization between two phosphite anion isomers (Step 1), conformational transition (Step 2) and dissociation of the water molecule (Step 3). A second water molecule is critical to lower the activation barriers of Step 1, and all activation barriers are moderate so that the redox reactions occur favorably under normal conditions, which are further dramatically accelerated by the highly exergonic Step 3. Accordingly, formation of uranyl phosphites becomes an effective approach to manage uranium pollution. Moreover, the lower activation barriers for H2PO3- rather than HPO32- rationalize the superior reduction activities of uranyl phosphites and the enhanced stability of U(IV) products at lower pH conditions. Owing to the cooperative proton/electron transfer, the U(VI) reduction to U(IV) and P(III) oxidation to P(V) are completed within one step, with transition states being featured by the U(V) and P(IV) species.


Assuntos
Minerais/química , Fosfitos/química , Urânio/química , Transporte de Elétrons , Elétrons , Oxirredução , Prótons
4.
PLoS One ; 15(4): e0228350, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320391

RESUMO

Dosimetry is an important tool for triage and treatment planning following any radiation exposure accident, and biological dosimetry, which estimates exposure dose using a biological parameter, is a practical means of determining the specific dose an individual receives. The cytokinesis-blocked micronucleus assay (CBMN) is an established biodosimetric tool to measure chromosomal damage in mitogen-stimulated human lymphocytes. The CBMN method is especially valuable for biodosimetry in triage situations thanks to simplicity in scoring and adaptability to high-throughput automated sample processing systems. While this technique produces dose-response data which fit very well to a linear-quadratic model for exposures to low linear energy transfer (LET) radiation and for doses up for 5 Gy, limitations to the accuracy of this method arise at larger doses. Accuracy at higher doses is limited by the number of cells reaching mitosis. Whereas it would be expected that the yield of micronuclei increases with the dose, in many experiments it has been shown to actually decrease when normalized over the total number of cells. This variation from a monotonically increasing dose response poses a limitation for retrospective dose reconstruction. In this study we modified the standard CBMN assay to increase its accuracy following exposures to higher doses of photons or a mixed neutron-photon beam. The assay is modified either through inhibitions of the G2/M and spindle checkpoints with the addition of caffeine and/or ZM447439 (an Aurora kinase inhibitor), respectively to the blood cultures at select times during the assay. Our results showed that caffeine addition improved assay performance for photon up to 10 Gy. This was achieved by extending the assay time from the typical 70 h to just 74 h. Compared to micronuclei yields without inhibitors, addition of caffeine and ZM447439 resulted in improved accuracy in the detection of micronuclei yields up to 10 Gy from photons and 4 Gy of mixed neutrons-photons. When the dose-effect curves were fitted to take into account the turnover phenomenon observed at higher doses, best fitting was achieved when the combination of both inhibitors was used. These techniques permit reliable dose reconstruction after high doses of radiation with a method that can be adapted to high-throughput automated sample processing systems.


Assuntos
Citogenética , Doses de Radiação , Radiometria , Adulto , Benzamidas/farmacologia , Cafeína/farmacologia , Células Cultivadas , Relação Dose-Resposta à Radiação , Feminino , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Masculino , Testes para Micronúcleos , Pessoa de Meia-Idade , Nêutrons , Prótons , Quinazolinas/farmacologia
5.
Chemistry ; 26(35): 7881-7888, 2020 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-32315472

RESUMO

Fast magic-angle spinning (MAS), frequency selective (FS) heteronuclear multiple quantum coherence (HMQC) experiments which function in an analogous manner to solution SOFAST HMQC NMR experiments, are demonstrated. Fast MAS enables efficient FS excitation of 1 H solid-state NMR signals. Selective excitation and observation preserves 1 H magnetization, leading to a significant shortening of the optimal inter-scan delay. Dipolar and scalar 1 H{14 N} FS HMQC solid-state NMR experiments routinely provide 4- to 9-fold reductions in experiment times as compared to conventional 1 H{14 N} HMQC solid-state NMR experiments. 1 H{14 N} FS resonance-echo saturation-pulse double-resonance (RESPDOR) allowed dipolar dephasing curves to be obtained in minutes, enabling the rapid determination of NH dipolar coupling constants and internuclear distances. 1 H{14 N} FS RESPDOR was used to assign multicomponent active pharmaceutical ingredients (APIs) as salts or cocrystals. FS HMQC also provided enhanced sensitivity for 1 H{17 O} and 1 H{35 Cl} HMQC experiments on 17 O-labeled Fmoc-alanine and histidine hydrochloride monohydrate, respectively. FS HMQC and FS RESPDOR experiments will provide access to valuable structural constraints from materials that are challenging to study due to unfavorable relaxation times or dilution of the nuclei of interest.


Assuntos
Histidina/química , Núcleo Celular/química , Espectroscopia de Ressonância Magnética , Prótons
7.
Ecotoxicol Environ Saf ; 197: 110615, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32325328

RESUMO

The respiratory deposition rates are the important analytical parameters for human health risk assessment related to the environmental volatile organic compounds (VOCs). In present study, the deposition rates from the linear regressions of CH2O, CH5N, C2H6O, C2H4O2, C3H8O, C6H6, C7H8, C8H8, and C8H10 of 120 healthy volunteers were obtained with significantly different from the respective calculated deposition rates. The CH2O (formaldehyde) has the highest deposition rate, indicating the highest associated exposure risk of CH2O if the persons are exposed to the same concentrations of these VOCs through inhalation. In order to explore the effects of the breathing models and sampling time on the deposition rates of VOCs, volunteers were first asked to breathe successively with nasal-in-nasal-out, oral-in-nasal-out, and oral-in-oral-out breathing models before and after three meals for three days. Sampling time variation has no effect on the deposition rates of selected VOCs, while the deposition rates of C2H4O2, C3H8O, C6H6, C7H8 and C8H10 by nasal-in-nasal-out were significantly different from oral-in-oral-out and nasal-in-oral-out models. Among all the breathing models, nasal-in-oral-out comprises the entire respiratory system. In order to further validate the results, the deposition rates of the selected VOCs were calculated in 120 healthy volunteers using nasal-in-oral-out breathing model for unlimited time after the conventional lung function examination. Difference in gender and body mass index had no effect on the deposition rates of VOCs, while the age affects the deposition rates of CH2O, CH5N and C2H4O2. Positive correlation analysis between lung function factors and deposition rates revealed that the individuals with larger lung function factors are more susceptible to deposit the VOCs. Overall, the main conclusion can be drawn that the respiratory deposition rates were influenced by the physiological factors. Therefore, the major objective for future research is to accurately calculate the deposition rates of environmental VOCs for health-risk assessment.


Assuntos
Poluentes Atmosféricos/análise , Exposição por Inalação , Compostos Orgânicos Voláteis/análise , Adulto , Poluentes Atmosféricos/toxicidade , Feminino , Humanos , Masculino , Espectrometria de Massas , Prótons , Respiração , Taxa Respiratória , Medição de Risco , Compostos Orgânicos Voláteis/toxicidade
8.
Proc Natl Acad Sci U S A ; 117(15): 8455-8461, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234783

RESUMO

The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is the penultimate step of chlorophyll biosynthesis. In oxygenic photosynthetic bacteria, algae, and plants, this reaction can be catalyzed by the light-dependent Pchlide oxidoreductase (LPOR), a member of the short-chain dehydrogenase superfamily sharing a conserved Rossmann fold for NAD(P)H binding and the catalytic activity. Whereas modeling and simulation approaches have been used to study the catalytic mechanism of this light-driven reaction, key details of the LPOR structure remain unclear. We determined the crystal structures of LPOR from two cyanobacteria, Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus Structural analysis defines the LPOR core fold, outlines the LPOR-NADPH interaction network, identifies the residues forming the substrate cavity and the proton-relay path, and reveals the role of the LPOR-specific loop. These findings provide a basis for understanding the structure-function relationships of the light-driven Pchlide reduction.


Assuntos
Cianobactérias/enzimologia , Luz , NADP/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Protoclorifilida/metabolismo , Synechocystis/enzimologia , Catálise , Clorofila/metabolismo , Cristalografia por Raios X , Modelos Moleculares , NADP/química , Conformação Proteica , Protoclorifilida/química , Prótons
9.
PLoS Comput Biol ; 16(4): e1007405, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32315300

RESUMO

Understanding the gating mechanism of ion channel proteins is key to understanding the regulation of cell signaling through these channels. Channel opening and closing are regulated by diverse environmental factors that include temperature, electrical voltage across the channel, and proton concentration. Low permeability in voltage-gated potassium ion channels (Kv) is intimately correlated with the prolonged action potential duration observed in many acidosis diseases. The Kv channels consist of voltage-sensing domains (S1-S4 helices) and central pore domains (S5-S6 helices) that include a selectivity filter and water-filled cavity. The voltage-sensing domain is responsible for the voltage-gating of Kv channels. While the low permeability of Kv channels to potassium ion is highly correlated with the cellular proton concentration, it is unclear how an intracellular acidic condition drives their closure, which may indicate an additional pH-dependent gating mechanism of the Kv family. Here, we show that two residues E327 and H418 in the proximity of the water cavity of Kv1.2 play crucial roles as a pH switch. In addition, we present a structural and molecular concept of the pH-dependent gating of Kv1.2 in atomic detail, showing that the protonation of E327 and H418 disrupts the electrostatic balance around the S6 helices, which leads to a straightening transition in the shape of their axes and causes dewetting of the water-filled cavity and closure of the channel. Our work offers a conceptual advancement to the regulation of the pH-dependent gating of various voltage-gated ion channels and their related biological functions.


Assuntos
Canal de Potássio Kv1.2/química , Simulação de Dinâmica Molecular , Água/química , Animais , Hidrogênio/química , Concentração de Íons de Hidrogênio , Lipídeos/química , Mutação , Permeabilidade , Domínios Proteicos , Prótons , Ratos , Transdução de Sinais , Software , Eletricidade Estática , Temperatura
10.
Radiology ; 295(2): 397-406, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32154775

RESUMO

Background Amide proton transfer (APT) MRI has the potential to demonstrate antitumor effects by reflecting biologically active tumor portion, providing different information from diffusion-weighted imaging (DWI) or dynamic susceptibility contrast (DSC) imaging. Purpose To evaluate whether a change in APT signal intensity after antiangiogenic treatment is predictive of early treatment response in recurrent glioblastoma. Materials and Methods In this retrospective study, APT MRI, DWI, and DSC imaging were performed in patients with recurrent glioblastoma from July 2015 to April 2019, both before treatment and 4-6 weeks after initiation of bevacizumab (follow-up). Progression was based on pathologic confirmation or clinical-radiologic assessment, and progression patterns were defined as local enhancing or diffuse nonenhancing. Changes in mean and histogram parameters (fifth and 95th percentiles) of APT signal intensity, apparent diffusion coefficient, and normalized cerebral blood volume (CBV) between imaging time points were calculated. Predictors of 12-month progression and progression-free survival (PFS) were determined by using logistic regression and Cox proportional hazard modeling and according to progression type. Results A total of 54 patients were included (median age, 56 years [interquartile range, 49-64 years]; 24 men). Mean APT signal intensity change after bevacizumab treatment indicated a low 12-month progression rate (odds ratio [OR], 0.36; 95% confidence interval [CI]: 0.13, 0.90; P = .04) and longer PFS (hazard ratio: 0.38; 95% CI: 0.20, 0.74; P = .004). High mean normalized CBV at follow-up was associated with a high 12-month progression rate (OR, 20; 95% CI: 2.7, 32; P = .04) and shorter PFS (hazard ratio, 9.4; 95% CI: 2.3, 38; P = .002). Mean APT signal intensity change was a significant predictor of diffuse nonenhancing progression (OR, 0.27; 95% CI: 0.06, 0.85; P = .047), whereas follow-up 95th percentile of the normalized CBV was a predictor of local enhancing progression (OR, 7.1; 95% CI: 2.4, 15; P = .04). Conclusion Early reduction in mean amide proton transfer signal intensity at 4-6 weeks after initiation of antiangiogenic treatment was predictive of a better response at 12 months and longer progression-free survival in patients with recurrent glioblastoma, especially in those with diffuse nonenhancing progression. © RSNA, 2020 Online supplemental material is available for this article.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Bevacizumab/uso terapêutico , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/tratamento farmacológico , Imagem de Difusão por Ressonância Magnética , Glioblastoma/diagnóstico por imagem , Glioblastoma/tratamento farmacológico , Angiografia por Ressonância Magnética , Progressão da Doença , Feminino , Humanos , Interpretação de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Prótons , Reprodutibilidade dos Testes , Estudos Retrospectivos
11.
Artigo em Inglês | MEDLINE | ID: mdl-32186230

RESUMO

In this study, a green adsorbent was synthesized for the removal of nitrate ions from water. The adsorbent consisted of carbonaceous particles with high specific surface area (1,240 m2 g-1) and porosity derived from pyrolysis of cornelian cherry stone and modified by protonated cross-linked chitosan. The adsorbent was characterized using various techniques like SEM, FTIR, BJH and zeta potential measurements. Dynamic behavior of the adsorbent in the nitrate adsorption was studied in a packed bed system at various operating conditions and in the presence of other competing anions (PO43-, HCO3-, SO42-). Based on the error analysis, the optimum operating conditions were considered at flow rate of 3.8 mL min-1, bed depth of 10 cm and nitrate concentration of 75 mg L-1. The kinetics of the adsorption process was studied using Adams-Bohart and Thomas models and the qmax was calculated to be about 12.4 mg g-1 at neutral pH and room temperature. Furthermore, the relationship between the bed height and the breakthrough time was described by bed depth service time (BDST) model. The experimental results suggested that the adsorbent possessed significant ability in nitrate removal from water due to the desired chemistry of the biopolymer and the excellent textural properties of the carbon support.


Assuntos
Quitosana/química , Reagentes para Ligações Cruzadas/química , Modelos Teóricos , Nitratos/análise , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Adsorção , Ânions , Biomassa , Concentração de Íons de Hidrogênio , Cinética , Prótons , Propriedades de Superfície , Águas Residuárias/química
12.
Biochim Biophys Acta Bioenerg ; 1861(7): 148185, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32171794

RESUMO

In the aerobic respiratory chains of many organisms, complex I functions as the first electron input. By reducing ubiquinone (Q) to ubiquinol, it catalyzes the translocation of protons across the membrane as far as ~200 Å from the site of redox reactions. Despite significant amount of structural and biochemical data, the details of redox coupled proton pumping in complex I are poorly understood. In particular, the proton transfer pathways are extremely difficult to characterize with the current structural and biochemical techniques. Here, we applied multiscale computational approaches to identify the proton transfer paths in the terminal antiporter-like subunit of complex I. Data from combined classical and quantum chemical simulations reveal for the first time structural elements that are exclusive to the subunit, and enables the enzyme to achieve coupling between the spatially separated Q redox reactions and proton pumping. By studying long time scale protonation and hydration dependent conformational dynamics of key amino acid residues, we provide novel insights into the proton pumping mechanism of complex I.


Assuntos
Antiporters/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Subunidades Proteicas/metabolismo , Força Próton-Motriz/fisiologia , Sequência de Aminoácidos , Antiporters/química , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Prótons , Sais/química , Thermus thermophilus/metabolismo , Água/química
13.
Proc Natl Acad Sci U S A ; 117(11): 5818-5825, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123084

RESUMO

Gram-negative bacteria expressing class A ß-lactamases pose a serious health threat due to their ability to inactivate all ß-lactam antibiotics. The acyl-enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A ß-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved ß-lactamase inhibitor avibactam to trap the acyl-enzyme complex of class A ß-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pKa) compared with acyl-enzyme complexes with ß-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pKa to be ∼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl-enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.


Assuntos
Compostos Azabicíclicos/química , Compostos Azabicíclicos/farmacologia , Prótons , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , beta-Lactamases/efeitos dos fármacos , Acilação , Compostos Azabicíclicos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos dos fármacos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Inibidores de beta-Lactamases/química , beta-Lactamases/metabolismo
14.
Biochemistry ; 59(12): 1289-1297, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32167292

RESUMO

Cobalt-mimochrome VI*a (CoMC6*a) is a synthetic mini-protein that catalyzes aqueous proton reduction to hydrogen (H2). In buffered water, there are multiple possible proton donors, complicating the elucidation of the mechanism. We have found that the buffer pKa and sterics have significant effects on activity, evaluated via cyclic voltammetry (CV). Protonated buffer is proposed to act as the primary proton donor to the catalyst, specifically through the protonated amine of the buffers that were tested. At a constant pH of 6.5, catalytic H2 evolution in the presence of buffer acids with pKa values ranging from 5.8 to 11.6 was investigated, giving rise to a potential-pKa relationship that can be divided into two regions. For acids with pKa values of ≤8.7, the half-wave catalytic potential (Eh) changes as a function of pKa with a slope of -128 mV/pKa unit, and for acids with pKa of ≥8.7, Eh changes as a function of pKa with a slope of -39 mV/pKa unit. In addition, a series of buffer acids were synthesized to explore the influence of steric bulk around the acidic proton on catalysis. The catalytic current in CV shows a significant decrease in the presence of the sterically hindered buffer acids compared to those of their parent compounds, also consistent with the added buffer acid acting as the primary proton donor to the catalyst and showing that acid structure in addition to pKa impacts activity. These results demonstrate that buffer acidity and structure are important considerations when optimizing and evaluating systems for proton-dependent catalysis in water.


Assuntos
Cobalto/química , Deuteroporfirinas/química , Hidrogênio/química , Metaloproteínas/química , Prótons , Tampões (Química) , Catálise , Concentração de Íons de Hidrogênio , Água/química
15.
Chemosphere ; 252: 126292, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32203779

RESUMO

New particle formation (NPF) involving amines in the atmosphere is considered an aggregation process, during which stable molecular clusters are formed from amines and sulfuric acid via hydrogen bond interaction. In this work, ab initio dynamics simulations of ammonium bisulfate formation from a series of amines, SO3, and H2O molecules were carried out in the gas phase and at the air-water interface. The results show that reactions between amines and hydrated SO3 molecules in the gas phase are barrierless or nearly barrierless processes. The reaction rate is related to the basicity of gas-phase amines-the stronger the basicity, the faster the reaction. Furthermore, SO3 hydrolysis catalyzed by amines occurs simultaneously with H2SO4-amine cluster formation. At the air-water interface, reactions between amines and SO3 involve multiple water molecules. The reaction center's ring structure (amine-SO3-nH2O) promotes the transfer of protons in the water molecules. The formed ammonium cation (-RNH3+) and the bisulfate anion (HSO4-) are present and stable by means of hydrogen bond interaction. The cluster formation mechanism provides new insights into NPF involving amines, which may play an important role in the formation of aerosols in some heavily polluted areas - e.g., those with a high amine concentration.


Assuntos
Aminas/química , Modelos Químicos , Sulfitos/química , Aerossóis , Atmosfera/química , Catálise , Ligação de Hidrogênio , Hidrólise , Prótons , Ácidos Sulfúricos , Água/química
16.
Phys Chem Chem Phys ; 22(14): 7537-7545, 2020 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-32219231

RESUMO

Understanding how electrons and protons move in a coupled manner and affect one another is important to the design of proton-electron conductors and achieving biological transport in synthetic materials. In this study, a new methodology is proposed that allows for the quantification of the degree of coupling between electrons and protons in tyrosine-rich peptides and metal oxide hybrid films at room temperature under a voltage bias. This approach is developed according to the Onsager principle, which has been thoroughly established for the investigation of mixed ion-electron conductors with electron and oxide ion vacancies as carriers at high temperatures. Herein, a new device platform using electron-blocking electrodes provides a new strategy to investigate the coupling of protons and electrons in bulk materials beyond the molecular level investigation of coupled proton and electron transfer. Two Onsager transport parameters, αi* and σe', are obtained from the device, and the results of these transport parameters demonstrate that the coupled transport of electrons and protons inside the hybrid film plays an important role in the macroscopic-scale conduction. The results suggest that an average of one electron is dragged by one proton in the absence of a direct driving force for electron movement ∇ηe.


Assuntos
Técnicas de Química Analítica/instrumentação , Transporte de Elétrons/fisiologia , Elétrons , Compostos de Manganês/química , Óxidos/química , Peptídeos/química , Prótons , Transporte Biológico/fisiologia
17.
Proc Natl Acad Sci U S A ; 117(7): 3583-3591, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015120

RESUMO

The matrix-2 (M2) protein from influenza A virus is a tetrameric, integral transmembrane (TM) protein that plays a vital role in viral replication by proton flux into the virus. The His37 tetrad is a pH sensor in the center of the M2 TM helix that activates the channel in response to the low endosomal pH. M2 consists of different regions that are believed to be involved in membrane targeting, packaging, nucleocapsid binding, and proton transport. Although M2 has been the target of many experimental and theoretical studies that have led to significant insights into its structure and function under differing conditions, the main mechanism of proton transport, its conformational dynamics, and the role of the amphipathic helices (AHs) on proton conductance remain elusive. To this end, we have applied explicit solvent constant pH molecular dynamics using the multisite λ-dynamics approach (CpHMDMSλD) to investigate the buried ionizable residues comprehensively and to elucidate their effect on the conformational transition. Our model recapitulates the pH-dependent conformational transition of M2 from closed to open state when the AH domain is included in the M2 construct, revealing the role of the amphipathic helices on this transition and shedding light on the proton-transport mechanism. This work demonstrates the importance of including the amphipathic helices in future experimental and theoretical studies of ion channels. Finally, our work shows that explicit solvent CpHMDMSλD provides a realistic pH-dependent model for membrane proteins.


Assuntos
Vírus da Influenza A/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Motivos de Aminoácidos , Transporte Biológico , Concentração de Íons de Hidrogênio , Vírus da Influenza A/química , Vírus da Influenza A/genética , Cinética , Estrutura Secundária de Proteína , Prótons , Proteínas da Matriz Viral/genética
18.
Proc Natl Acad Sci U S A ; 117(6): 3144-3149, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32001509

RESUMO

Glycogen plays a central role in glucose homeostasis and is abundant in several types of tissue. We report an MRI method for imaging glycogen noninvasively with enhanced detection sensitivity and high specificity, using the magnetic coupling between glycogen and water protons through the nuclear Overhauser enhancement (NOE). We show in vitro that the glycogen NOE (glycoNOE) signal is correlated linearly with glycogen concentration, while pH and temperature have little effect on its intensity. For validation, we imaged glycoNOE signal changes in mouse liver, both before and after fasting and during glucagon infusion. The glycoNOE signal was reduced by 88 ± 16% (n = 5) after 24 h of fasting and by 76 ± 22% (n = 5) at 1 h after intraperitoneal (i.p.) injection of glucagon, which is known to rapidly deplete hepatic glycogen. The ability to noninvasively image glycogen should allow assessment of diseases in which glucose metabolism or storage is altered, for instance, diabetes, cardiac disease, muscular disorders, cancer, and glycogen storage diseases.


Assuntos
Glicogênio , Imagem por Ressonância Magnética/métodos , Animais , Jejum/fisiologia , Glicogênio/análise , Glicogênio/química , Glicogênio/metabolismo , Fígado/diagnóstico por imagem , Fígado/metabolismo , Camundongos , Prótons , Água/química
19.
Inorg Chem ; 59(4): 2594-2603, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32011880

RESUMO

Glyoxalase I (GlxI) is a member of the glyoxalase system, which is important in cell detoxification and converts hemithioacetals of methylglyoxal (a cytotoxic byproduct of sugar metabolism that may react with DNA or proteins and introduce nucleic acid strand breaks, elevated mutation frequencies, and structural or functional changes of the proteins) and glutathione into d-lactate. GlxI accepts both the S and R enantiomers of hemithioacetal, but converts them to only the S-d enantiomer of lactoylglutathione. Interestingly, the enzyme shows this unusual specificity with a rather symmetric active site (a Zn ion coordinated to two glutamate residues; Glu-99 and Glu-172), making the investigation of its reaction mechanism challenging. Herein, we have performed a series of combined quantum mechanics and molecular mechanics calculations to study the reaction mechanism of GlxI. The substrate can bind to the enzyme in two different modes, depending on the direction of its alcoholic proton (H2; toward Glu-99 or Glu-172). Our results show that the S substrate can react only if H2 is directed toward Glu-99 and the R substrate only if H2 is directed toward Glu-172. In both cases, the reactions lead to the experimentally observed S-d enantiomer of the product. In addition, the results do not show any low-energy paths to the wrong enantiomer of the product from neither the S nor the R substrate. Previous studies have presented several opposing mechanisms for the conversion of R and S enantiomers of the substrate to the correct enantiomer of the product. Our results confirm one of them for the S substrate, but propose a new one for the R substrate.


Assuntos
Glutationa/análogos & derivados , Lactoilglutationa Liase/química , Aldeído Pirúvico/análogos & derivados , Teoria da Densidade Funcional , Humanos , Modelos Químicos , Prótons , Estereoisomerismo
20.
Proc Natl Acad Sci U S A ; 117(9): 4732-4740, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32075917

RESUMO

Multidrug and toxic compound extrusion (MATE) transporters are ubiquitous ion-coupled antiporters that extrude structurally and chemically dissimilar cytotoxic compounds and have been implicated in conferring multidrug resistance. Here, we integrate double electron-electron resonance (DEER) with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of ligand-dependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeon Pyrococcus furiosus Pairs of spin labels monitoring the two sides of the transporter reconstituted into nanodiscs reveal large-amplitude movement of helices that alter the orientation of a putative substrate binding cavity. We found that acidic pH favors formation of an inward-facing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. The lipid-dependent PfMATE isomerization between OF and IF conformation is driven by protonation of a previously unidentified intracellular glutamate residue that is critical for drug resistance. Our results can be framed in a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.


Assuntos
Antiporters/química , Antiporters/metabolismo , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Antiporters/genética , Resistência a Múltiplos Medicamentos , Espectroscopia de Ressonância de Spin Eletrônica , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas de Transporte de Cátions Orgânicos/genética , Conformação Proteica , Prótons , Pyrococcus furiosus/metabolismo
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