RESUMO
SUMO (Small Ubiquitin-like Modifiers) proteins are involved in a crucial post-translational modification commonly termed as SUMOylation. In this work, we have investigated the native-state conformational flexibility of human SUMO2 and its interaction with Cu2+ and Zn2+ ions using 15N-1H based 2D NMR spectroscopy. After SUMO1, SUMO2 is the most studied SUMO isoform in humans which shares 45 % and ~80 % similarity with SUMO1 in terms of sequence and structure, respectively. In this manuscript, we demonstrate that compared to SUMO1, several amino acids around the α1-helix region of SUMO2 access energetically similar near-native conformations. These conformations could play a crucial role in SUMO2's non-covalent interactions with SUMO interaction motifs (SIMs) on other proteins. The C-terminal of SUMO2 was found to bind strongly with Cu2+ ions resulting in a trimeric structure as observed by gel electrophoresis. This interaction seems to interfere in its non-covalent interaction with a V/I-x-V/I-V/I based SIM in Daxx protein.
Assuntos
Cobre , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Zinco , Humanos , Cobre/química , Cobre/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Zinco/química , Zinco/metabolismo , Conformação Proteica , Ressonância Magnética Nuclear Biomolecular , Ligação ProteicaRESUMO
While ionic liquids (ILs) are considered as prospective ingredients of new antimicrobial agents, it is important to understand the adverse effects of these molecules on human cells. Since cholesterol is the essential component of a human cell membrane, in the present study, the effect of an imidazolium-based IL has been investigated on the model membrane in the presence of cholesterol. The area per sphingomyelin lipid is found to reduce in the presence of the IL, which is quantified by the area-surface pressure isotherm of the lipid monolayer formed at the air-water interface. The effect is considerably diminished in the cholesterol-containing monolayer. Further, the IL is observed to decrease the rigidity of the cholesterol-free monolayer. Interestingly, the presence of cholesterol does not allow any change in this property of the layer at lower surface pressure. However, at a higher surface pressure, the IL increases the elasticity in the cholesterol-induced condensed phase of the lipid layer. The X-ray reflectivity measurement on a stack of cholesterol-free lipid bilayers proved the formation of IL-induced phase-separated domains in the matrix of a pure lipid phase. These domains are found to be formed by interdigitating the chains of the lipids, producing a thinner membrane. Such a phase is less intense in the cholesterol-containing membrane. All of these results indicate that the IL molecules may deform the cholesterol-free membrane of a bacterial cell, but the same may not be harmful to human beings as cholesterol could restrict the insertion in the cellular membrane of a human cell.
Assuntos
Líquidos Iônicos , Humanos , Líquidos Iônicos/farmacologia , Estudos Prospectivos , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , ColesterolRESUMO
Ionic liquids based on doubly charged cations, often termed dicationic ionic liquids (DILs), offer robust physicochemical properties and low toxicity than conventional monocationic ionic liquids. In this design-based study, we used solid-state NMR spectroscopy to provide the interaction mechanism of two DILs, 1,n-bis(3-alkylimidazolium-1-yl) alkane dibromide ([C2n(C7-nIM)2]2+·2Br-, n = 1, 6), with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) phospholipid membranes, to explain the low toxicity of DILs toward HeLa, Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae cell lines. Dications with a short linker and long terminal chains cause substantial perturbation to the bilayer structure, making them more membrane permeabilizing, as shown by fluorescence-based dye leakage assays. The structural perturbation is even higher than [C12(MIM)]+ monocations, which carry a single 12-carbon long chain and exhibit a much higher membrane affinity, permeability, and cytotoxicity. These structural details are a crucial contribution to the design strategies aimed at harnessing the biological activity of ionic liquids.
Assuntos
Líquidos Iônicos , Fosfolipídeos , Bicamadas Lipídicas/química , Líquidos Iônicos/toxicidade , Líquidos Iônicos/química , Alcanos/toxicidade , Escherichia coli/metabolismoRESUMO
We have investigated the impact of increasing concentration of imidazolium-based ionic liquids ([CnMIM]+[Br]-) on the structural integrity of large unilamellar vesicles (LUVs) made of pure phosphatidylcholine (PC) and phosphatidylglycerol (PG) lipids. Calcein based dye leakage assays were used to monitor the permeability of LUVs in the presence of ionic liquids. As the ionic liquid concentration approaches the critical micelle value, vesicle fusion occurs resulting in unexpected quenching which is accompanied by rapid dye leakage due to the formation of transiently lived fusion-holes. Vesicle fusion is confirmed using dynamic light scattering based size measurements and fluorescence based lipid mixing assays. 1H-1H NOESY measurements using solid-state NMR spectroscopy were performed to obtain insights into the fusion mechanism. While POPC LUVs are more prone to membrane fusion, the overall extent of fusion is higher in POPG LUVs. Ionic liquid induced splaying of phospholipid chains is crucial for overcoming the hydration barrier between the merging bilayers.
Assuntos
Líquidos Iônicos/química , Lipossomos/química , Lipossomos/metabolismo , Fosfolipídeos/químicaRESUMO
Metal catalyzed post-Ugi cyclization of bis-amides is reported in this study. Exposure of bis-amides to Pd(II) catalyst triggered the formation of seven-membered benzoxazepinones. This investigation established that changing the catalyst to a Echavarren's gold(I) turned off cyclization to seven member ring and turned on 6-exo-dig annulations to afford family of six-membered benzoxazinones. To support the proposed mechanisms, quantum chemical based density functional theory calculations have been performed and validated. This novel method obtained molecular complexity up to four modular inputs and divergence of two different skeletons. 2D NMR spectroscopic techniques and single crystal X-ray diffraction established the proposed structures.
RESUMO
There are three specific regions in the Amyloid beta (Aß) peptide sequence where variations cause enhanced toxicity in Alzheimer's disease: the N-terminus, the central salt bridge, and the C-terminus. Here, we investigate if there is a close conformational connection between these three regions, which may suggest a concerted mechanism of toxicity. We measure the effects of Zn2+ and curcumin on Aß40, and compare these with their previously reported effects on Aß42. Aß42 and Aß40 differ only near the C-terminus, where curcumin interacts, while Zn2+ interacts near the N-terminus. Therefore, this comparison should help us differentiate the effect of modulating the C- and the N-termini. We find that curcumin allows fibril-like structures containing the salt bridge to emerge in the mature Aß40 aggregates, but not in Aß42. In contrast, we find no difference in the effects of Zn+2 on Aß40 and Aß42. In the presence of Zn+2, both of these fail to form proper fibrils, and the salt bridge remains disrupted. These results indicate that modulations of the Aß termini can determine the fate of a salt bridge far away in the sequence, and this has significant consequences for Aß toxicity. We also infer that small molecules can alter oligomer-induced toxicity by modulating the aggregation pathway, without substantially changing the final product of aggregation.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Curcumina/farmacologia , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/metabolismo , Zinco/farmacologia , Amiloide/química , Amiloide/efeitos dos fármacos , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/toxicidade , Animais , Cátions Bivalentes/química , Cátions Bivalentes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Curcumina/química , Microscopia Eletrônica de Transmissão , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/toxicidade , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/metabolismo , Conformação Proteica/efeitos dos fármacos , Ratos , Espectrometria de Fluorescência , Zinco/químicaRESUMO
We report here a novel IBX-promoted oxidative coupling of primary amines and its utilization to Ugi reaction. Advantageously, the reaction could be carried out in choline chloride urea as a natural deep eutectic solvent. A range of imines and bisamides from pseudo-four-component oxidative Ugi reaction could be synthesized under mild and metal-free conditions. Advantageously, the oxidant (IBX) and solvent could be recycled up to five times with only a slight loss in activity.
RESUMO
Aß self-assembles into parallel cross-ß fibrillar aggregates, which is associated with Alzheimer's disease pathology. A central hairpin turn around residues 23-29 is a defining characteristic of Aß in its aggregated state. Major biophysical properties of Aß, including this turn, remain unaltered in the central fragment Aß18-35. Here, we synthesize a single deletion mutant, ΔG25, with the aim of sterically hindering the hairpin turn in Aß18-35. We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of Aß18-35, suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe(19) and Leu(34) residues, observed in full-length Aß and Aß18-35, is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel ß-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel ß-sheet. We conclude that the self-assembly of Aß is critically dependent on the hairpin turn and on the contact between the Phe(19) and Leu(34) regions, making them potentially sensitive targets for Alzheimer's therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions.
Assuntos
Peptídeos beta-Amiloides/química , Dobramento de Proteína , Peptídeos beta-Amiloides/genética , Dicroísmo Circular , Cinética , Mutação , Ressonância Magnética Nuclear Biomolecular , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria InfravermelhoRESUMO
Amyloid ß (Aß) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aß fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid ß(1-42) (Aß42) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23-Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aß42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn(2+) ions, another agent known to disrupt the fibrils and alter Aß42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aß42, which is stabilized both by curcumin and by Zn(2+.)
Assuntos
Peptídeos beta-Amiloides/química , Curcumina/química , Inibidores Enzimáticos/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Células Cultivadas , Curcumina/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Estabilidade Proteica , Estrutura Secundária de Proteína , Ratos , Ratos Wistar , Zinco/química , Zinco/metabolismoRESUMO
Morpholinium-based amide-functionalized ionic liquids (ILs) [C(n)AMorph][Br], where n = 8, 12, and 16, have been synthesized and characterized for their micellization behavior in aqueous medium using a variety of state of the art techniques. The adsorption and micellization behavior of [CnAMorph][Br] ILs at the air-solution interface and in the bulk, respectively, has been found to be much better compared to that observed for nonfunctionalized homologous ILs and conventional cationic surfactants, as shown by the comparatively higher adsorption efficiency, lower surface tension at the critical micelle concentraiton (γ(cmc)), and much lower critical micelle concentration (cmc) for [C(n)AMorph][Br] ILs. Conductivity measurements have been performed to obtain the cmc, degree of counterion binding (ß), and standard free energy of micellization (ΔG(m)°). Isothermal titration calorimetry has provided information specifically about the thermodynamics of micellization, whereas steady-state fluorescence has been used to obtain the cmc, micropolarity of the cybotactic region, and aggregation number (N(agg)) of the micelles. Both dynamic light scattering and atomic force microscopy have provided insights into the size and shape of the micelles. 2D (1)H-(1)H nuclear Overhauser effect spectroscopy experiments have provided insights into the structure of the micelle, where [C16AMorph][Br] has shown distinct micellization behavior as compared to [C8AMorph][Br] and [C12AMorph][Br] in corroboration with observations made from other techniques.
RESUMO
Small oligomers of the amyloidâ ß (Aß) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimer's disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two-dimensional solid-state NMR methods. The smallest Aß40 oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10-21 and 30-40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22-29) and the N-terminal tail (residues 1-9) are strikingly different. Notably, ten of eleven known Aßâ mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.
Assuntos
Peptídeos beta-Amiloides/genética , Fragmentos de Peptídeos/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Membrana Celular/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Mutação , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
In this work, we explored how the amount of cholesterol in the lipid membrane composed of phosphatidylcholine (POPC) or phosphatidylglycerol (POPG) affects the interaction with 1-dodecyl-3-methylimidazolium bromide ([C12MIM]+Br-) ionic liquids using various biophysical techniques. On interacting with the membrane, [C12MIM]+Br- leads to enhanced membrane permeability and induces membrane fusion, leading to an increase in vesicle size. The 2H-based solid-state NMR investigations of cholesterol-containing lipid membranes reveal that [C12MIM]+Br- decreases the lipid chain order parameters and counteracts the lipid condensation effect of cholesterol to some extent. Therefore, as the amount of cholesterol in the membrane increases, the membrane effect of [C12MIM]+Br- decreases. The effect of [C12MIM]+Br- on the membrane properties is more pronounced for POPC compared to that of POPG membranes. This suggests a dependence of these effects on the electrostatic interactions, indicating that the influence of [C12MIM]+Br- varies based on the lipid composition. The findings suggest that the presence of cholesterol can modulate the effect of [C12MIM]+Br- on membrane properties, with variations observed between POPC and POPG membranes, highlighting the importance of lipid composition. In short, this study provides insights into the intricate interplay between cholesterol, the lipid membrane, and the ionic liquid [C12MIM]+Br-.
Assuntos
Colesterol , Imidazóis , Líquidos Iônicos , Fosfatidilcolinas , Fosfatidilgliceróis , Líquidos Iônicos/química , Colesterol/química , Colesterol/metabolismo , Fosfatidilgliceróis/química , Fosfatidilcolinas/química , Imidazóis/química , Permeabilidade , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismoRESUMO
We have studied the impact of the lipid ratio on the membrane permeability of mixed phosphatidylcholine (POPC)/phosphatidylglycerol (POPG) membranes induced by 1-dodecyl-3-methylimidazolium bromide ([C12MIM]+Br-) ionic liquid by evaluating the role of affinity and architecture of the phospholipid bilayer. Nine different model membranes composed of negatively charged POPG and zwitterionic POPC lipids mixed in molar ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9 have been studied. The membrane permeability of each composition has been evaluated using fluorescence-based dye leakage assays. Despite having the highest membrane affinity, POPG-rich membranes doped with 10 and 20 mol % POPC are found to be the least permeable. 31P- and 2H-based solid-state NMR investigations reveal that the minor POPC component is homogeneously dispersed in the PG/PC (8:2) membrane. In contrast, the lipids seem to be segregated into POPG- and POPC-rich domains in the complementary PG/PC (2:8) composition. Although [C12MIM]+ cations have a stronger interaction with the POPG component in the mixed membranes, their insertion has a limited impact on the overall structure and dynamics of the PG/PC (8:2) composition.
Assuntos
Líquidos Iônicos , Fosfatidilgliceróis , Brometos , Imidazóis , Bicamadas Lipídicas , Permeabilidade , FosfatidilcolinasRESUMO
In continuous efforts to develop anti-Alzheimer's agents, we rationally designed and synthesized a series of multitargeting molecules by incorporating the essential molecular features of the standard drug donepezil. Among the series, compound 4b showed multitargeting properties to act as an anti-Alzheimer's agent, which is better tolerable in vivo than donepezil. Acetylcholinesterase (AChE) inhibition data showed that compound 4b inhibits the enzyme with a half-maximal inhibitory concentration (IC50) value of 0.78 µM and also showed DNA protection, which was confirmed through the DNA nicking assay, suggesting the protective effect of 4b against oxidative DNA damage. Compound 4b also showed 53.04% inhibition against Aß1-42 aggregations, which was found comparable to that of the standard compound curcumin. Molecular dynamics simulations were performed to check the stability of compound 4b with the enzyme AChE, which showed that the enzyme-ligand complex is stable enough to block the hydrolysis of acetylcholine in the brain. Its higher LD50 cutoff value (50 mg/kg) in comparison to donepezil (LD50: 25 mg/kg) made it safer, suggesting that it can be used in further clinical experiments. To evaluate its anti-Alzheimer property, a mice model with melamine-induced cognitive dysfunction was used, and Morris water maze and Rotarod tests were performed. A significant improvement in memory was observed after the treatment with compound 4b and donepezil. The study postulated that the introduction of important structural features of donepezil (dimethoxyindanone moiety as ring-A) embarked with terminal aromatic ether (ring-B and ring-C) made 4b a multitargeting molecule that offers a way for developing alternative therapeutics in the future against Alzheimer's disease (AD).
Assuntos
Acetilcolinesterase , Doença de Alzheimer , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Animais , Inibidores da Colinesterase/uso terapêutico , DNA , Donepezila/uso terapêutico , Indanos , Camundongos , Relação Estrutura-AtividadeRESUMO
Observations like high Zn(2+) concentrations in senile plaques found in the brains of Alzheimer's patients and evidences emphasizing the role of Zn(2+) in amyloid-ß (Aß)-induced toxicity have triggered wide interest in understanding the nature of Zn(2+)-Aß interaction. In vivo and in vitro studies have shown that aggregation kinetics, toxicity, and morphology of Aß aggregates are perturbed in the presence of Zn(2+). Structural studies have revealed that Zn(2+) has a binding site in the N-terminal region of monomeric Aß, but not much is precisely known about the nature of binding of Zn(2+) with aggregated forms of Aß or its effect on the molecular structure of these aggregates. Here, we explore this aspect of the Zn(2+)-Aß interaction using one- and two-dimensional (13)C and (15)N solid-state NMR. We find that Zn(2+) causes major structural changes in the N-terminal and the loop region connecting the two ß-sheets. It breaks the salt bridge between the side chains of Asp(23) and Lys(28) by driving these residues into nonsalt-bridge-forming conformations. However, the cross-ß structure of Aß(42) aggregates remains unperturbed though the fibrillar morphology changes distinctly. We conclude that the salt bridge is not important for defining the characteristic molecular architecture of Aß(42) but is significant for determining its fibrillar morphology and toxicity.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico/metabolismo , Lisina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Zinco/metabolismo , Amiloide/química , Amiloide/ultraestrutura , Espectroscopia de Ressonância Magnética , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
We have evaluated ionic liquids based on double-chained 1-alkyl-3-octylimidazolium cations ([CnC8IM]+, n = 2, 4, 6, 8, 10, 12) for their cytotoxicity toward various cell lines. The toxicity of ionic liquids was correlated to their ability to partition into and permeabilize phosphocholine (POPC)- or phosphoglycerol (POPG)-based large unilamellar vesicles. Membrane partitioning of ionic liquids was assessed using the ζ-potential measurements, and membrane permeability was determined using fluorescence-based dye leakage assays. Both cytotoxicity and membrane permeability of these ILs were found to increase in a sigmoidal fashion with increasing chain length on the N1 atom (n in [CnC8IM]+) cations. These results were compared with those for ionic liquids based on single-chained 1-alkyl-3-methylimidazolium cations ([Cn+8C1IM]+), carrying a similar number of carbon atoms but as a single alkyl chain. Our studies show that ionic liquids containing double-chained cations are relatively less cytotoxic and membrane-permeabilizing than the cations bearing a single long alkyl chain.
Assuntos
Líquidos Iônicos , Cátions , Permeabilidade da Membrana Celular , Líquidos Iônicos/toxicidade , Permeabilidade , Lipossomas UnilamelaresRESUMO
The effect of cationic head-group of ionic liquid on the structure and dynamics of phospholipid bilayer was studied to provide insights into the mechanism of ionic liquid-membrane interaction. The effect was observed using six ionic liquids containing benzimidazolium, imidazolium, pyrrolidinium, piperidinium, ammonium, and morpholinium based amphiphilic cations carrying a dodecyl alkyl chain. Unilamellar and multilamellar vesicles composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were used. Permeability of POPC bilayer was found to have a strong dependence on ionic liquid head-group structure. To probe the structural details of interaction, 31P and 2H based solid-state NMR measurements were performed. The cations differed in terms of their effect on the orientation and disorder in the phosphocholine moiety in lipid head-group as revealed by chemical shift anisotropy of 31P. Cations carrying an unshielded charge like benzimidazolium, imidazolium, and ammonium result in strong reorientation of phosphocholine moiety in lipid head-group. Large sized cations like benzimidazolium and piperidinium result in enhanced lipid chain dynamics as revealed by order parameter calculations of deuterated lipid chains. Relatively polar head-group of morpholinium cation neither impacts the phospholipid head-group nor chain packing. Our results suggest that there exists a direct correlation between ionic liquid head-group induced structural changes in bilayer and their ability to permeabilize/disrupt the membrane and be cytotoxic.
RESUMO
Metal ions like Cu2+ and Zn2+ have been shown to impact protein misfolding pathways in neurodegenerative proteinopathies like Alzheimer's and Parkinson's. Also, due to their strong interaction with Ubiquitin, they interfere in degradation of misfolded proteins by impairing the ubiquitin-proteasome system (UPS). In this work, we have studied the interaction of these metal ions with a small Ubiquitin like post-translation modifier SUMO1, which is known to work co-operatively with Ubiquitin to regulate UPS system. Between Cu2+ and Zn2+, the former binds more strongly with SUMO1 as determined using fluorescence spectroscopy. SUMO1 aggregates, forming trimer and higher oligomers in presence of Cu2+ ions which were characterized using gel electrophoresis, Bradford assay, and transmission electron microscopy. Chemical shift analysis using 15N/1H based NMR spectroscopy revealed that SUMO1 retains its structural fold in its trimeric state. Cu2+ induced paramagnetic quenching and Zn2+ induced chemical shift perturbation of 15N-1H cross-peaks were used to identify their respective binding sites in SUMO1. Binding sites so obtained were further validated with molecular dynamics studies. Our findings provide structural insights into the SUMO1-Cu2+/Zn2+ interaction, and its impact on aggregation of SUMO1 which might affect its ability to modify functions of target proteins.
Assuntos
Sítios de Ligação , Cobre/química , Íons , Proteína SUMO-1/química , Zinco/química , Sequência de Aminoácidos , Simulação de Dinâmica Molecular , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes , Proteína SUMO-1/metabolismo , Análise Espectral , Relação Estrutura-AtividadeRESUMO
The proteins secreted by bacteria contribute to immune mediated gastric inflammation and epithelial damage; thus aid bacterial invasion in host tissue, and may also interact with host proteins, conspirating a mechanism against host-immune system. The Histone-like DNA binding protein is one of the most abundant nucleoid-associated proteins in Helicobacter pylori (H. pylori). The protein -referred here as Hup- is also secreted in vitro by H. pylori, thus it may have its role in disease pathogenesis. This is possible only if Hup interact with some human proteins including Small-Ubiquitin-like-Modifier (SUMO) proteins. Studies have established that SUMO-proteins participate in various innate-immune pathways and thus promote an efficient immune response to combat pathogenic infections. Sequence analysis revealed the presence of two SUMO interacting motifs (SIMs) and several positively charged lysine residues on the protein surface of Hup. Additionally, SUMO-proteins epitomize negatively charged surface which confers them the ability to bind to DNA/RNA binding proteins. Based on the presence of SIMs as well as charge complementarity between the proteins, it is legitimate to consider that Hup protein would bind to SUMO-proteins. The present study has been undertaken to establish this interaction for the first time using NMR in combination with ITC and other biophysical techniques.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Helicobacter pylori/química , Ressonância Magnética Nuclear Biomolecular , Proteína SUMO-1/química , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Helicobacter pylori/metabolismo , Humanos , Ligação Proteica , Proteína SUMO-1/metabolismoRESUMO
We present here a simple refocused modification, r TPPM, of the Two-Pulse Phase-Modulation (TPPM) heteronuclear decoupling method, which improves decoupling and makes the sequence much more robust with respect to essential experimental parameters. The modified sequence is compared with the established TPPM sequence and a variety of other decoupling sequences at low to moderate magic-angle spinning frequencies. Simulations are shown to compare TPPM and r TPPM with respect to various experimental parameters. The observations from simulations are corroborated with experimental findings at two spinning frequencies on U-(13)C-glycine and U-(13)C-L-histidine.HCl.H2O.