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1.
J Phys Chem B ; 125(42): 11687-11696, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34652160

RESUMO

Potential energy parameters for α-methyl amino acids were generated with ab initio calculations on α-methyl-N-acetylalanyl-N'-methylamide (the α-methyl "alanine dipeptide") which served as an input to a grid-based correction to the backbone torsional potential (known as CMAP) consistent with the CHARMM36m additive protein force field. The new parameters were validated by comparison with experimentally determined helicities of the 22 residue C-terminal peptide (H10) from apolipoprotein A1 and five α-methylated variants in water and 0.3:0.7 trifluoroethanol (TFE)/water. Conventional molecular dynamics simulation totaling 30 µs for each peptide is in overall good agreement with the experiment, including the increased helicity in 30% TFE. An additional 500 ns of simulation using two-dimensional dihedral biasing (bpCMAP) replica exchange reduced left-handed conformations, increased right-handed helices, and thereby mostly decreased agreement with the experiment. Analysis of side chain-side chain salt bridges suggests that the overestimation of the helical content may be, in part, due to such interactions. The increased helicity of the peptides in 30% TFE arises from decreased hydrogen bonding of the backbone atoms to water and a concomitant increase in intramolecular backbone hydrogen bonds.


Assuntos
Simulação de Dinâmica Molecular , Proteínas , Aminoácidos , Ligação de Hidrogênio , Trifluoretanol
2.
Phys Chem Chem Phys ; 23(32): 17536-17544, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34369530

RESUMO

Water, being an active participant in most of the biophysical processes, is important to trace how protein solvation changes as its conformation evolves in the presence of solutes or co-solvents. In this study, we investigate how the secondary structures of two diverse proteins - lysozyme and ß-lactoglobulin - change in the aqueous mixtures of two alcohols - ethanol and 2,2,2-trifluoroethanol (TFE) using circular dichroism measurements. We observe that these alcohols change the secondary structures of these proteins and the changes are protein-specific. Subsequently, we measure the collective solvation dynamics of these two proteins both in the absence and in the presence of alcohols by measuring the frequency-dependent absorption coefficient (α(ν)) in the THz (0.1-1.2 THz) frequency domain. The alcohol-water mixtures exhibit a non-ideal behaviour with the highest absorption difference (Δα) obtained at Xalcohol = 0.2. The protein solvation in the presence of the alcohols shows an oscillating behaviour in which Δαprotein changes with Xalcohol. Such an oscillatory behaviour of protein solvation results from a delicate interplay between the protein-water, protein-alcohol and water-alcohol associations. We attempt to correlate the various structural conformations of the proteins with the associated solvation.


Assuntos
Etanol/química , Lactoglobulinas/química , Muramidase/química , Trifluoretanol/química , Água/química , Animais , Bovinos , Galinhas , Conformação Proteica , Estrutura Secundária de Proteína , Solubilidade , Espectroscopia Terahertz
3.
Molecules ; 26(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071240

RESUMO

The synthesis of naproxen-containing diaryliodonium salts has been realized from naproxen methyl ester and ArI(OH)OTs activated by trimethylsilyl trifluoromethanesulfonate (TMSOTf) in a solvent mixture comprising dichloromethane and 2,2,2-trifluoroethanol (TFE). Those iodonium salts have been successfully used in the functionalization of an aromatic ring of naproxen methyl ester, including fluorination, iodination, alkynylation, arylation, thiophenolation, and amination and esterification reactions. Moreover, further hydrolysis of the obtained 5-iodo-naproxen methyl ester afforded 5-iodo-naproxen.


Assuntos
Ésteres/química , Naproxeno/síntese química , Sais/química , Catálise , Química Farmacêutica/métodos , Esterificação , Flúor/química , Halogenação , Hidrólise , Iodo/química , Lipase/metabolismo , Espectroscopia de Ressonância Magnética , Fenóis/química , Solventes , Estereoisomerismo , Trifluoretanol/química , Difração de Raios X
4.
J Dairy Res ; 88(2): 221-225, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33985600

RESUMO

Alpha-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) are contained in bovine milk whey. Chemical and physical treatments are known to alter the conformation of these proteins and we have previously reported that α-LA denatured with trifluoroethanol (TFE) and isolated from sterilized market milk inhibited the growth of rat crypt IEC-6 cells. In the present study, we aimed to evaluate the effects of TFE-treated α-LA and ß-LG on cell growth using cultured intestinal cells and on their safety using a suckling mouse model. First, we investigated the effect of the TFE-treated whey proteins on human colonic Caco-2 cells at various differentiation stages. In the undifferentiated stage, we assessed cell growth by a water-soluble tetrazolium-1 method. The native whey proteins enhanced cell proliferation, whereas the TFE-treated whey proteins strongly inhibited cell growth. We investigated cell barrier function in the post-differentiated stage by measuring transepithelial electrical resistance (TER). Not only native but also the TFE-treated whey proteins increased TER. Next, we evaluated whether the TFE-treated α-LA and ß-LG have adverse effects on healthy suckling mice. No mice given by the TFE-treated samples showed any adverse symptoms. We also performed a safety test using a human rotavirus infected gastrointestinal disease suckling mice model. Even the TFE-treated whey proteins appeared to prevent the development of diarrheal symptoms without any adverse effects. Although we cannot know the effect of long-term ingestion of denatured whey proteins, these results suggest that they have no adverse effects on differentiated intestinal cells and digestive tract, at least in short-term ingestion.


Assuntos
Trato Gastrointestinal/efeitos dos fármacos , Desnaturação Proteica , Proteínas do Soro do Leite/química , Proteínas do Soro do Leite/farmacologia , Animais , Animais Lactentes , Células CACO-2 , Bovinos , Diferenciação Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Diarreia/tratamento farmacológico , Humanos , Lactalbumina/química , Lactalbumina/farmacologia , Lactoglobulinas/química , Lactoglobulinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Trifluoretanol/química
5.
Food Funct ; 12(13): 5967-5974, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34032239

RESUMO

To illustrate the relationship between environment hydrophobicity and soybean peptide and its calcium complexes when they are absorbed transmembrane, different solution environments (HBS buffer, TFE hydrophobic solution and cell suspension) were used to simulate hydrophilic and hydrophobic environments. In this study, soybean peptides (10-30 kDa) with a high calcium binding capacity were prepared by enzymatic hydrolysis and ultrafiltration. The results of cell experiments showed that the peptide could transport calcium into cells for absorption. Secondary structure changes of the peptide and its calcium complexes in different solution environments showed that the secondary structure of the peptide changed during the transmembrane absorption, and the contents of α-helix and ß-sheet structures increased. Besides, the ß-sheet structures in the peptide-calcium complexes were further converted to an α-helix structure. This conversion may be induced by the hydrophobicity of peptide solutions. In addition, when the conformation changes, the positively charged peptides in the sample will be exposed and then interact with cells, which is beneficial for the transmembrane of peptide-calcium complexes.


Assuntos
Cálcio/química , Peptídeos/química , Soja/química , Tampões (Química) , Dicroísmo Circular , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Fosfatos/química , Conformação Proteica , Conformação Proteica em alfa-Hélice , Estrutura Secundária de Proteína , Trifluoretanol/química
6.
Phys Chem Chem Phys ; 23(10): 5760-5772, 2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33481971

RESUMO

Inhomogeneous distribution of constituent molecules in a mixed solvent has been known to give remarkable effects on the solute, e.g., conformational changes of biomolecules in an alcohol-water mixture. We investigated the general effects of 2,2,2-trifluoroethanol (TFE) on proteins/peptides in a mixture of water and TFE using melittin as a model protein. Fluctuations and Kirkwood-Buff integrals (KBIs) in the TFE-H2O mixture, quantitative descriptions of inhomogeneity, were determined by small-angle X-ray scattering investigation and compared with those in the aqueous solutions of other alcohols. The concentration fluctuation for the mixtures ranks as methanol < ethanol ≪ TFE < tert-butanol < 1-propanol, indicating that the inhomogeneity of molecular distribution in the TFE-H2O mixture is unexpectedly comparable to those in the series of mono-ols. On the basis of the concentration dependence of KBIs between the TFE molecules, it was found that a strong attraction between the TFE molecules is not necessarily important to induce helix conformation, which is inconsistent with the previously proposed mechanism. To address this issue, by combining the KBIs and the helix contents reported by the experimental spectroscopic studies, we quantitatively evaluated the change in the preferential binding parameter of TFE to melittin attributed to the coil-helix transition. As a result, we found two different regimes on TFE-induced helix formation. In the dilute concentration region of TFE below ∼2 M, where the TFE molecules are not aggregated among themselves, the excess preferential binding of TFE to the helix occurs due to the direct interaction between them, namely independent of the solvent fluctuation. In the higher concentration region above ∼2 M, in addition to the former effect, the excess preferential binding is significantly enhanced by the solvent fluctuation. This scheme should be held as general cosolvent effects of TFE on proteins/peptides.


Assuntos
Álcoois/química , Meliteno/química , Peptídeos/química , Solventes/química , Trifluoretanol/química , Sequência de Aminoácidos , Conformação Molecular , Transição de Fase , Termodinâmica , Água
7.
Molecules ; 25(24)2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322563

RESUMO

The synthesis of a molecularly diverse library of tetrasubstituted alkenes containing a barbiturate motif is described. Base-induced condensation of N1-substituted pyrimidine-2,4,6(1H,3H,5H)-triones with 5-(bis(methylthio)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione gave 3-substituted 5-(methylthio)-2H-pyrano[2,3-d]pyrimidine-2,4,7(1H,3H)-triones ('pyranopyrimidinones'), regioselectively. A sequence of reactions involving ring-opening of the pyran moiety, displacement of the methylthio group with an amine, re-formation of the pyran ring, and after its final cleavage with an amine, gave tetrasubstituted alkenes (3-amino-3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)propanamides) with a diversity of substituents. Cleavage of the pyranopyrimidinones with an aniline was facilitated in 2,2,2-trifluoroethanol under microwave irradiation. Compounds were tested against Escherichia coli, Staphylococcus aureus, the yeast Schizosaccharomyces pombe, and the pathogenic fungus Candida albicans. No compounds exhibited activity against E. coli, whilst one compound was weakly active against S. aureus. Three compounds were strongly active against S. pombe, but none was active against C. albicans.


Assuntos
Alcenos/química , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Bioensaio/métodos , Testes de Sensibilidade Microbiana , Barbitúricos/síntese química , Candida albicans/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Concentração Inibidora 50 , Conformação Molecular , Piranos , Schizosaccharomyces/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Trifluoretanol/química
8.
Biochemistry ; 59(39): 3650-3659, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32924445

RESUMO

Misfolding of Cu, Zn superoxide dismutase (SOD1) variants may lead to protein aggregation and ultimately amyotrophic lateral sclerosis (ALS). The mechanism and protein conformational changes during this process are complex and remain unclear. To study SOD1 variant aggregation at the molecular level and in solution, we chemically induced aggregation of a mutant variant (G93A SOD1) with trifluoroethanol (TFE) and used both native mass spectrometry (MS) to analyze the intact protein and fast photochemical oxidation of proteins (FPOP) to characterize the structural changes induced by TFE. We found partially unfolded G93A SOD1 monomers prior to oligomerization and identified regions of the N-terminus, C-terminus, and strands ß5, ß6 accountable for the partial unfolding. We propose that exposure of hydrophobic interfaces of these unstructured regions serves as a precursor to aggregation. Our results provide a possible mechanism and molecular basis for ALS-linked SOD1 misfolding and aggregation.


Assuntos
Agregados Proteicos/efeitos dos fármacos , Desdobramento de Proteína/efeitos dos fármacos , Superóxido Dismutase/química , Trifluoretanol/farmacologia , Humanos , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Pegadas de Proteínas , Espectrometria de Massas por Ionização por Electrospray
9.
Int J Biol Macromol ; 163: 1697-1706, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32961181

RESUMO

We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity.


Assuntos
Membrana Celular/efeitos dos fármacos , Citotoxinas/metabolismo , Citotoxinas/farmacologia , Lactalbumina/metabolismo , Lactalbumina/farmacologia , Animais , Bovinos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dicroísmo Circular , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Trifluoretanol/metabolismo , Trifluoretanol/farmacologia , Células U937
10.
Molecules ; 25(15)2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32708018

RESUMO

This work reports a straightforward regioselective synthetic methodology to prepare α-aminophosphine oxides and phosphonates through the addition of oxygen and sulfur nucleophiles to the C-N double bond of 2H-azirine derivatives. Determined by the nature of the nucleophile, different α-aminophosphorus compounds may be obtained. For instance, aliphatic alcohols such as methanol or ethanol afford α-aminophosphine oxide and phosphonate acetals after N-C3 ring opening of the intermediate aziridine. However, addition of 2,2,2-trifluoroethanol, phenols, substituted benzenthiols or ethanethiol to 2H-azirine phosphine oxides or phosphonates yields allylic α-aminophosphine oxides and phosphonates in good to high general yields. In some cases, the intermediate aziridine attained by the nucleophilic addition of O- or S-nucleophiles to the starting 2H-azirine may be isolated and characterized before ring opening. Additionally, the cytotoxic effect on cell lines derived from human lung adenocarcinoma (A549) and non-malignant cells (MCR-5) was also screened. Some α-aminophosphorus derivatives exhibited very good activity against the A549 cell line in vitro. Furthermore, selectivity towards cancer cell (A549) over non-malignant cells (MCR-5) has been detected in almost all compounds tested.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos/síntese química , Azirinas/química , Ácidos Fosforosos/síntese química , Antineoplásicos/farmacologia , Aziridinas/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Organofosfonatos/química , Oxigênio/química , Fenóis/química , Fosfinas/química , Ácidos Fosforosos/farmacologia , Estereoisomerismo , Compostos de Sulfidrila/química , Enxofre/química , Trifluoretanol/química
11.
J Phys Chem B ; 124(28): 5993-6003, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32573229

RESUMO

2,2,2-Trifluoroethanol (TFE) is one of the fluoroalcohols that have been known to induce and stabilize an open helical structure in many proteins and peptides. The current study has benchmarked low-field 19F NMR relaxation and 19F Overhauser dynamic nuclear polarization (ODNP) by providing a brief account of TFE solvent dynamics in a model melittin (MLT, an antimicrobial peptide) solution with a TFE-D2O cosolvent mixture at pH 7.4. Further, this approach has been employed to reveal the solvation of MLT by TFE in a nonbuffered solution with pH 2.8 for the first time. The structural transition of MLT has been elucidated via solvent dynamics by measuring the 19F TFE relaxation rates at 0.34 T for various TFE-D2O compositions in the absence (bulk TFE) and in the presence of MLT at both the pH values. A complementary initial record of circular dichroism experiments on these aqueous MLT solutions with TFE as the cosolvent at two different pH conditions demonstrated the structural transition from a random coil to a helical or from a folded helical to an open helical structure. The molecular correlation time derived from the corresponding relaxation rates shows that TFE resides on the MLT surface in both pH conditions. However, the trends in the variation of molecular correlation time ratio as a function of TFE concentration represent that the mechanism and the extent to which TFE affects the MLT structural integrity are different at different pH values. The extraction of the DNP coupling parameter from steady-state 19F ODNP experiments performed in the presence of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl at 0.34 T revealed changes in the solvation dynamics of TFE concomitant with the MLT structural transition. In summary, 19F relaxation and ODNP measurements made at a low field have allowed direct monitoring of TFE dynamics during the MLT structural transition in terms of preferential solvation. The choice of experiments performed at a moderately low field (0.34 T) enabled us to exploit on the one hand almost 1200-fold mitigation of the strong contribution of 19F chemical shift anisotropy at 11.76 T, whereas on the other hand, the ODNP experiment offered a window for probing molecular dynamics on timescales of the order of 10-1000 ps.


Assuntos
Meliteno , Trifluoretanol , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Conformação Molecular , Solventes
12.
Nat Commun ; 11(1): 2756, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488003

RESUMO

Trifluoroethanol and difluoroethanol units are important motifs in bioactive molecules, but the methods to direct incorporate these units are limited. Herein, we report two organosilicon reagents for the transfer of trifluoroethanol and difluoroethanol units into molecules. Through intramolecular C-Si bond activation by alkoxyl radicals, these reagents were applied in allylation, alkylation and alkenylation reactions, enabling efficient synthesis of various tri(di)fluoromethyl group substituted alcohols. The broad applicability and general utility of the approach are highlighted by late-stage introduction of these fluoroalkyl groups to complex molecules, and the synthesis of antitumor agent Z and its difluoromethyl analog Z'.


Assuntos
Etanol/análogos & derivados , Etanol/química , Compostos de Organossilício/química , Trifluoretanol/química , Álcoois/química , Alquilação , Técnicas de Química Sintética , Indicadores e Reagentes/química , Estrutura Molecular
13.
Arch Biochem Biophys ; 684: 108342, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32184088

RESUMO

Proteins of the p53 family are best known for their role in the regulation of cell cycle. The p53 protein, as a model system, has been extensively explored in numerous cancer-related studies. The C-terminal domain (CTD) of p53 is an intrinsically disordered region that gains multiple different conformations at interaction with different binding partners. However, the impact of the surrounding environment on the structural preference of p53-CTD is not known. We investigated the impact of the surrounding environment on the conformational behavior and folding of p53-CTD. Although the entire CTD is predicted as a highly disordered region by several commonly used disorder predictors, based on the secondary structure prediction, we find that a part of the CTD sequence (residues 380-388) is "confused", being predicted to shuffle between the irregular, α-helical and ß-strand structures. First time, we are observing the effect of folding-induced organic solvents, trifluoroethanol and methanol, on the conformation of CTD. Water-miscible organic solvents exert hydrophobic interactions, which are major driving force to trigger structural changes in CTD. By lowering the solution dielectric constant, organic solvents can also strengthen electrostatic interactions. We have also performed Replica Exchange Molecular Dynamic (REMD) simulations for enhanced conformation sampling of the peptide. These simulation studies have also provided detailed insight into the peculiarities of this peptide, explaining its folding behavior in the presence of methanol. We consider that these hydrophobic interactions may have important roles for function-related structural changes of this disordered region.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metanol/química , Simulação de Dinâmica Molecular , Domínios Proteicos , Dobramento de Proteína , Estrutura Terciária de Proteína , Temperatura , Trifluoretanol/química
14.
Chemistry ; 26(27): 5970-5981, 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32073728

RESUMO

To investigate the structural impact of phosphorylation on the human histone H1.0 C-terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T118 -H1.0 (T118 PKK motif) and T140 -H1.0 (T140 PVK motif). Both model peptides are mainly disordered in aqueous solution in their non-phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T118 -H1.0 and pT118 -H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104-115 and a short α/310 helix (residues 119-123), that are almost perpendicular in T118 -H1.0 but have a poorly defined orientation in pT118 -H1.0. Peptides T140 -H1.0 and pT140 -H1.0 form very similar α helices between residues 141-147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side-chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT118 -H1.0 and pT140 -H1.0 has pKa values below the intrinsic values, which can be explained by non-specific charge-charge interactions with nearby Lys. The non-polar Val in the TPVK motif accounts for the pT140 pKa being closer to the intrinsic pKa value than the pT118 pKa . Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short-lived intrinsically disordered proteins and domains.


Assuntos
Histonas/química , Proteínas Intrinsicamente Desordenadas/química , Peptídeos/química , Trifluoretanol/química , Histonas/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Fosforilação
15.
Nat Commun ; 11(1): 488, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980618

RESUMO

Metallic lithium anodes are highly promising for revolutionizing current rechargeable batteries because of their ultrahigh energy density. However, the application of lithium metal batteries is considerably impeded by lithium dendrite growth. Here, a biomacromolecule matrix obtained from the natural membrane of eggshell is introduced to control lithium growth and the mechanism is motivated by how living organisms regulate the orientation of inorganic crystals in biomineralization. Specifically, cryo-electron microscopy is utilized to probe the structure of lithium at the atomic level. The dendrites growing along the preferred < 111 > crystallographic orientation are greatly suppressed in the presence of the biomacromolecule. Furthermore, the naturally soluble chemical species in the biomacromolecules can participate in the formation of solid electrolyte interphase upon cycling, thus effectively homogenizing the lithium deposition. The lithium anodes employing bioinspired design exhibit enhanced cycling capability. This work sheds light on identifying substantial challenges in lithium anodes for developing advanced batteries.


Assuntos
Fontes de Energia Elétrica , Lítio , Animais , Biomineralização , Engenharia Química , Microscopia Crioeletrônica , Cristalização , Casca de Ovo/química , Técnicas Eletroquímicas , Eletrodos , Lítio/química , Substâncias Macromoleculares/química , Trifluoretanol/química
16.
J Phys Chem B ; 123(48): 10171-10180, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31692350

RESUMO

The most common obstacles to the development of therapeutic polypeptides are peptide stability and aggregation. Human calcitonin (hCT) is a 32-residue hormone polypeptide secreted from the C-cells of the thyroid gland and is responsible for calcium and phosphate regulation in the blood. hCT reduces calcium levels by inhibiting the activity of osteoclasts, which are bone cells that are mainly responsible for breaking down the bone tissue or decreasing the resorption of calcium from the kidneys. Thus, calcitonin injection has been used to treat osteoporosis and Paget's disease of bone. hCT is an aggregation-prone peptide with a high tendency to form amyloid fibrils. As a result, salmon calcitonin (sCT), which is different from hCT at 16-residue positions and has a lower propensity to aggregate, has been chosen as a clinical substitute for hCT. However, significant side effects, including immune reactions, have been shown with the use of sCT injection. In this study, we found that two residues, Tyr-12 and Asn-17, play key roles in inducing the fibrillization of hCT. Double mutation of hCT at these two crucial sites could greatly enhance its resistance to aggregation and provide a peptide-based inhibitor to prevent amyloid formation by hCT. Double-mutated hCT retains its ability to interact with its receptor in vivo. These findings suggest that this variant of hCT would serve as a valuable therapeutic alternative to sCT.


Assuntos
Amiloide/química , Calcitonina/química , Cálcio/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Agregados Proteicos/genética , Sequência de Aminoácidos , Amiloide/antagonistas & inibidores , Amiloide/genética , Amiloide/metabolismo , Animais , Calcitonina/genética , Calcitonina/metabolismo , Cálcio/metabolismo , AMP Cíclico/química , AMP Cíclico/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Células MCF-7 , Mutação , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica em alfa-Hélice , Salmão , Alinhamento de Sequência , Trifluoretanol/química , Trifluoretanol/metabolismo
18.
PLoS One ; 14(5): e0216946, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31083701

RESUMO

Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading microorganisms. Although AMPs are known to act on bacterial membranes and increase membrane permeability, the action mechanism of most AMPs still remains unclear. In this report, we found that the TP4 peptides from Nile tilapia anchored on E. coli cells and enabled them permeable to SYTOX Green in few minutes after TP4 addition. TP4 peptides existed in small dots either on live or glutaraldehyde-fixed cells. TP4 peptides were driven into oligomers either in soluble or insoluble form by a membrane-mimicking anionic surfactant, sarkosyl, depending on the concentrations employed. The binding forces among TP4 components were mediated through hydrophobic interaction. The soluble oligomers were negatively charged on surface, while the insoluble oligomers could be fused with each other or piled on existing particles to form larger particles with diameters 0.1 to 20 µm by hydrophobic interactions. Interestingly, the morphology and solubility of TP4 particles changed with the concentration of exogenous sarkosyl or trifluoroethanol. The TP4 peptides were assembled into oligomers on or in bacterial membrane. This study provides direct evidence and a model for the oligomerization and insertion of AMPs into bacterial membrane before entering into cytosol.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Peixes/química , Tensoativos/farmacologia , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Transporte Biológico , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Ciclídeos/fisiologia , Escherichia coli/metabolismo , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/farmacologia , Corantes Fluorescentes/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Compostos Orgânicos/metabolismo , Ligação Proteica , Multimerização Proteica , Sarcosina/análogos & derivados , Sarcosina/farmacologia , Eletricidade Estática , Trifluoretanol/farmacologia
19.
J Phys Chem B ; 123(15): 3248-3258, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30916962

RESUMO

Molecular dynamics simulations of the protein model Trp-cage in 42% trifluoroethanol (TFE)-water at 298 K have been carried out with the goal of exploring peptide hydrogen-solvent fluorine nuclear spin cross-relaxation. The TFE5 model of TFE developed in a previous work was used with the TIP5P-Ew model of water. System densities and component translational diffusion coefficients predicted by the simulations were within 20% of the experimental values. Consideration of the calculated relative amounts of TFE and water surrounding the hydrogens of Trp-cage indicated that the composition of the solvent mixture beyond ∼1.5 nm from the van der Waals surface of the peptide is close to the composition of the bulk solvent, but as observed by others, TFE accumulates preferentially near the peptide surface. In the simulations, both TFE and water molecules make contacts with the peptide surface; water molecules predominate in contacts with the peptide backbone atoms and TFE molecules generally preferentially interact with side chains. Translational diffusion of solvent molecules appears to be slowed near the surface of the peptide. Depending on the location in the structure, TFE molecules form complexes with the peptide that may persist for up to ∼7 ns. Many of the peptide spin-solvent fluorine cross-relaxation parameters (ΣHF) for which experimental values are available are reasonably well-predicted from the simulations. However, the calculated ΣHF values were too small for some hydrogens of the 6Trp indole ring and the amino acid hydrogens near this residue in the native structure, whereas ΣHF values for hydrogens on the side chains of 1Asn, 4Ile, and 7Leu are too large. In 42% TFE-water, persistent conformations of Trp-cage are found, which differ from the conformation found in water by the orientation of the 3Tyr ring.


Assuntos
Simulação de Dinâmica Molecular , Temperatura , Trifluoretanol/química , Triptofano/química , Água/química , Difusão , Hidrogéis/química , Conformação Molecular , Solventes/química
20.
Sci Rep ; 9(1): 3720, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842512

RESUMO

Late Embryogenesis Abundant (LEA) proteins are mostly predicted to be intrinsically disordered proteins (IDPs) that are induced under conditions of cellular dehydration. Their functions, however, are largely unexplored and also their structure and interactions with potential target molecules have only recently been investigated in a small number of proteins. Here, we have characterized the wheat LEA protein TdLEA3, which has sequence homology with the group of LEA_4 proteins that are characterized by the 11-mer repeat motif TAQAAKEKAXE. TdLEA3 has five repeats of this imperfectly conserved 11-mer amino acid motif. To investigate the structure of the protein, we used circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy. The data show that TdLEA3 was largely disordered under fully hydrated conditions and acquired α-helical structure upon drying and in the presence of trifluoroethanol (TFE). Moreover, the addition of increasing glycerol concentrations to the protein solution induced a progressive gain in α-helix content. Activity assays indicated that TdLEA3 was able to prevent the inactivation of lactate dehydrogenase (LDH) under heat, dehydration-rehydration and freeze-thaw treatments. In addition, TdLEA3 reduced aggregate formation in the enzyme during these treatments.


Assuntos
Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Triticum/enzimologia , Motivos de Aminoácidos , Dicroísmo Circular , Estabilidade Enzimática , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Espectroscopia de Infravermelho com Transformada de Fourier , Trifluoretanol/metabolismo
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