RESUMO
Heat shock proteins 90 (Hsp90) are chaperones that promote the proper folding of other proteins under high temperature stress situations. Hsp90s are highly conserved and ubiquitous proteins, and in mammalian cells, they are localized in the cytoplasm, endoplasmic reticulum, and mitochondria. Cytoplasmic Hsp90 are named Hsp90α and Hsp90ß and differ mainly in their expression pattern: Hsp90α is expressed under stress conditions, while Hsp90ß is a constitutive protein. Structurally, both share the same characteristics by presenting three well-conserved domains, one of which, the N-terminal domain, has a binding site for ATP to which various drugs targeting this protein, including radicicol, can bind. The protein is mainly found in dimeric form and adopts different conformations depending on the presence of ligands, co-chaperones and client proteins. In this study, some aspects of structure and thermal unfolding of cytoplasmic human Hsp90 were analysed by infrared spectroscopy. The effect on Hsp90ß of binding with a non-hydrolysable ATP analogue and radicicol was also examined. The results obtained showed that despite the high similarity in secondary structure the two isoforms exhibit substantial differences in their behaviour during thermal unfolding, as Hsp90α exhibits higher thermal stability, slower denaturation process and different event sequence during unfolding. Ligand binding strongly stabilizes Hsp90ß and slightly modifies the secondary structure of the protein as well. Most likely, these structural and thermostability characteristics are closely related to the conformational cycling of the chaperone and its propensity to exist in monomer or dimer form.
Assuntos
Proteínas de Choque Térmico HSP90 , Chaperonas Moleculares , Animais , Humanos , Proteínas de Choque Térmico HSP90/química , Espectroscopia de Infravermelho com Transformada de Fourier , Chaperonas Moleculares/metabolismo , Isoformas de Proteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Mamíferos/metabolismoRESUMO
The C2 domain of novel protein kinases C (nPKC) binds to membranes in a Ca2+-independent way contributing to the activation of these enzymes. We have studied the C2 domain of one of these nPKCs, namely PKCε, and confirmed that it establishes a strong interaction with POPA, which is clearly visible through changes in chemical shifts detected through 31P-MAS-NMR and the protection that it exerts on the domain against thermal denaturation seen through DSC and FT-IR. In this study, using two-dimensional correlation analysis (2D-COS) applied to infrared spectra, we determined the sequence of events that occur during the thermal unfolding of the domain and highlighted some differences when phosphatidic acid or cardiolipin are present. Finally, by means of FRET and DLS experiments, we wanted to determine the effect of membrane curvature on the domain/membrane interaction by using lysophosphatidylcholine to introduce positive curvature as a control and we observed that the effect of these phospholipids on the protein binding is not exerted through the change of membrane curvature.
Assuntos
Membrana Celular/metabolismo , Proteína Quinase C-épsilon/química , Proteína Quinase C-épsilon/metabolismo , Desdobramento de Proteína , Cardiolipinas/metabolismo , Membrana Celular/química , Modelos Moleculares , Ácidos Fosfatídicos/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Vitamin K1 and vitamin K2 play very important biological roles as members of chains of electron transport as antioxidants in membranes and as cofactors for the posttranslational modification of proteins that participate in a number of physiological functions such as coagulation. The interaction of these vitamins with dimyristoylphosphatidylcholine (DMPC) model membranes has been studied by using a biophysical approach. It was observed by using differential scanning calorimetry that both vitamins have a very limited miscibility with DMPC and they form domains rich in the vitamins at high concentrations. Experiments using X-ray diffraction also showed the formation of different phases as a consequence of the inclusion of either vitamin K at temperatures below the phase transition. However, in the fluid state, a homogeneous phase was detected, and a decrease in the thickness of the membrane was accompanied by an increase in the water layer thickness. 2H NMR spectroscopy showed that both vitamins K induced a decrease in the onset of the phase transition, which was bigger for vitamin K1, and both vitamins decreased the order of the membrane as seen through the first moment (M1). 1H NOESY MAS-NMR showed that protons located at the rings or at the beginning of the lateral chain of both vitamins K interacted with a clear preference with protons located in the polar part of DMPC. On the other hand, protons located on the lateral chain have a nearer proximity with the methyl end of the myristoyl chains of DMPC. In agreement with the 2H NMR, ATR-FTIR (attenuated total reflectance Fourier transform infrared spectroscopy) indicated that both vitamins decreased the order parameters of DMPC. It was additionally deduced that the lateral chains of both vitamins were oriented almost in parallel to the myristoyl chains of the phospholipid.
Assuntos
Dimiristoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Vitamina K 1/química , Vitamina K 2/químicaRESUMO
α-Tocopherol is considered to carry on a very important role as an antioxidant for membranes and lipoproteins and other biological roles as membrane stabilizers and bioactive lipids. Given its essential role, it is very important to fully understand its location in the membrane. In this work, the vertical location of vitamin E in saturated membranes has been studied using biophysical techniques. Small- and wide-angle X-ray diffraction experiments show that α-tocopherol alters the water layer between bilayers in both 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), indicating its proximity to this surface. The quenching of the intrinsic fluorescence of α-tocopherol indicates a low quenching efficiency by acrylamide and a higher quenching by 5-doxyl-PC than by 9- and 16-doxyl-PC. These results suggest that in both DMPC and DPPC membranes, the chromanol ring is not far away from the surface of the membrane but within the bilayer. 1H nuclear Overhauser enhancement spectroscopy magic-angle spinning-nuclear magnetic resonance studies showed that α-tocopherol is localized in a similar manner in DMPC and DPPC membranes, with the chromanol ring embedded in the upper part of the hydrophobic bilayer. Using attenuated total reflection-Fourier transform infrared spectroscopy, it was observed that the tail chain of α-tocopherol lies nearly parallel to the acyl chains of DMPC and DPPC. Taking these results together, it was concluded that in both DMPC and DPPC, the hydroxyl group of the chromanol ring will establish hydrogen bonding with water on the membrane surface, and the main axis of the α-tocopherol molecule will be perpendicular to the bilayer plane.
Assuntos
Dimiristoilfosfatidilcolina/química , Lipídeos/química , Fenóis/química , Fosfatidilcolinas/química , Água/química , alfa-Tocoferol/química , Bicamadas Lipídicas/químicaRESUMO
Edelfosine is an anticancer drug with an asymmetric structure because, being a derivative of glycerol, it possesses two hydrophobic substituents of very different lengths. We showed that edelfosine destabilizes liquid-ordered membranes formed by either 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine, sphingomyelin (SM), and cholesterol (1:1:1 molar ratio) or SM and cholesterol (2:1 molar ratio). This was observed by differential scanning calorimetry in which phase transition arises from either of these membrane systems after the addition of edelfosine. The alteration in the liquid-ordered domains was characterized by using a small-angle X-ray diffraction that revealed the formation of gel phases as a consequence of the addition of edelfosine at low temperatures and by a wide-angle X-ray diffraction that confirmed changes in the membranes, indicating the formation of these gel phases. The increase in phase transition derived by the edelfosine addition was further confirmed by Fourier-transform infrared spectroscopy. The effect of edelfosine was compared with that of structurally analogue lipids: platelet-activating factor and 1-palmitoyl-2-acetyl- sn-glycero-3-phosphocholine, which also have the capacity of destabilizing liquid-ordered domains, although they are less potent than edelfosine for this activity, and lysophosphatidylcholine, which lacks this capacity. It was concluded that edelfosine may be associated with cholesterol favorably competing with sphingomyelin, and that this sets sphingomyelin free to undergo a phase transition. Finally, the experimental observations can be described by molecular dynamics calculations in terms of intermolecular interaction energies in phospholipid-cholesterol membranes. Higher interaction energies between asymmetric phospholipids and cholesterol than between sphingomyelin and cholesterol were obtained. These results are interesting because they biophysically characterize one of the main molecular mechanisms to trigger apoptosis of the cancer cells.
Assuntos
Membrana Celular/efeitos dos fármacos , Colesterol/química , Éteres Fosfolipídicos/química , Éteres Fosfolipídicos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Membrana Celular/química , Bicamadas Lipídicas/químicaRESUMO
In this paper, we present WaterSpy, a project developing an innovative, compact, cost-effective photonic device for pervasive water quality sensing, operating in the mid-IR spectral range. The approach combines the use of advanced Quantum Cascade Lasers (QCLs) employing the Vernier effect, used as light source, with novel, fibre-coupled, fast and sensitive Higher Operation Temperature (HOT) photodetectors, used as sensors. These will be complemented by optimised laser driving and detector electronics, laser modulation and signal conditioning technologies. The paper presents the WaterSpy concept, the requirements elicited, the preliminary architecture design of the device, the use cases in which it will be validated, while highlighting the innovative technologies that contribute to the advancement of the current state of the art.
RESUMO
A comparative study of three synthetic peptides, namely neutral Cecropin D-like G. mellonella (WT) and two cationic peptides derived from its sequence, ΔM1 (+5) and ΔM2 (+9) is reported in this work. The influence of charge on the interactions between peptides and membranes and its effect on phase were studied by calorimetric assays. Differential scanning calorimetry (DSC) showed that ΔM2 peptide showed the strongest effect when the membrane contained phosphatidylcholine (PC) and phosphatidylglycerol (PG), increasing membrane fluidization. Fourier transform infrared spectroscopy (FTIR) was used to determine lipid segregation in the presence of peptides. When WT and ΔM1 bound to model membrane containing PG and PC (1:1 molar ratio) a separation of both lipids was observed. Meanwhile, ΔM2 peptide also induced a demixing of PG-peptide rich domains separated from PC. FTIR experiments also suggested that the presence of ΔM1 and ΔM2 peptides increased lipid carbonyl group hydration in DMPG membrane fluid phase, However, hydration at the interface level in fluid phase was notably increased in the presence of WT and ΔM1 peptides in DMPC/DMPG. Overall the increase in positively charged residues favors the interaction of the peptides with the negatively charged membrane and its perturbation.
Assuntos
Bactérias/citologia , Cecropinas/química , Cecropinas/metabolismo , Membrana Celular/metabolismo , Lepidópteros/química , Membranas Artificiais , Sequência de Aminoácidos , Animais , Ligação Proteica , Especificidade por SubstratoRESUMO
The Thermotoga maritima arginine-binding protein (TmArgBP) has been modified to create a reagentless fluorescent protein biosensor. Two design methods for biosensor construction are compared: 1) solvent accessibility of environmentally-sensitive probes and 2) fluorescence deactivation due to photo-induced electron transfer (PET). Nine single cysteine TmArgBP mutants were created and labeled with three different environmentally sensitive fluorescent probes. These mutants demonstrated limited changes in fluorescence emission upon the addition of arginine. In contrast, the PET-based biosensor provides significant enhancements over the traditional approach and provides a fluorescence quenching mechanism that was capable of providing quantitative detection of arginine. Site-directed mutagenesis of TmArgBP was used to create attachment points for the fluorescent probe (K145C) and for an internal aromatic residue (D18X) to serve as the PET quencher. Both tyrosine and tryptophan, but not phenylalanine, were able to quench the emission of the fluorescent probe by more than 80% upon the addition of arginine. The dissociation constant for arginine ranged from 0.87 to 1.5 µM across the different sensors. This PET-based strategy provides a simple and broadly applicable approach for the analytical detection of small molecules that may be applied to any protein that exhibits conformational switching in a ligand dependent manner.
Assuntos
Arginina/análise , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Proteínas Periplásmicas de Ligação/metabolismo , Thermotoga maritima/metabolismo , Arginina/genética , Arginina/metabolismo , Proteínas de Bactérias , Sítios de Ligação , Fluorescência , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Ligação Proteica , Espectrometria de Fluorescência , Thermotoga maritima/genética , Thermotoga maritima/crescimento & desenvolvimento , Triptofano/química , Triptofano/genética , Triptofano/metabolismoRESUMO
α-Tocopherol is a natural preservative that prevents free radical chain oxidations in biomembranes. We have studied the location of α-tocopherol in model membranes formed by different unsaturated phosphatidylcholines, namely 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC). Small angle X-ray diffraction revealed that α-tocopherol was well mixed with all the phospholipids. In all the cases only one lamellar phase was detected. Very modest changes occasioned by α-tocopherol were observed in the electron density profiles. The results obtained from quenching of α-tocopherol intrinsic fluorescence by acrylamide showed that this vitamin was inefficiently quenched in the four types of membranes, indicating that the fluorescent chromanol ring was poorly accessible for this hydrophilic quencher. Compatible with that, quenching by doxyl derivatives of phosphatidylcholines indicated that the chromanol ring was close in the four membranes to the nitroxide probe located at position 5. Quenching by doxyl-phosphatidylcholines also indicated that the efficiency of quenching was higher in POPC than in the other unsaturated phospholipids. 1H-MAS-NMR showed that α-tocopherol induced chemical shifts of protons from the phospholipids, especially of those bonded to carbons 2 and 3 of the acyl chains of the four phospholipids studied. The 1H-MAS-NMR NOESY results suggested that the lower part of the chromanol ring was located between the C3 of the fatty acyl chains and the centre of the hydrophobic monolayer for the four phospholipid membranes studied. Taken together, these results suggest that α-tocopherol is located, in all the membranes studied, with the chromanol ring within the hydrophobic palisade but not far away from the lipid-water interface.
Assuntos
Fosfatidilcolinas/química , alfa-Tocoferol/química , Gorduras Insaturadas , Membranas Artificiais , Fosfolipídeos/química , Difração de Raios XRESUMO
Saporin-S6 is a plant toxin belonging to the type 1 ribosome-inactivating protein (RIP) family. Since it was extracted and isolated from Saponaria officinalis for the first time almost thirty years ago, the protein has been widely studied mainly for its potential applications in anti-tumour and anti-viral infection therapy. Like other RIPs, saporin-S6 is particularly effective in the form of immunotoxin conjugated with monoclonal antibodies and its chemico-physical characteristics made the protein a perfect candidate for the synthesis, development and use of saporin-S6-based chimeric toxins. The high stability of the protein against different denaturing agents has been broadly demonstrated, however, its complete thermal unfolding characterization has not already been performed. In this work we analyse in detail structure, thermostability and unfolding features by means of infrared spectroscopy coupled with two-dimensional correlation spectroscopy. Our data showed that saporin-S6 in solution at neutral pH exhibits a secondary structure analogue to that of the crystal and confirmed its good stability at moderately high temperatures, with a temperature of melting of 58°C. Our results also demonstrated that the thermal unfolding process is non-cooperative and occurs in two steps, and revealed the sequence of the events that take place during the denaturation, showing a higher stability of the N-terminal domain of the protein.
Assuntos
Proteínas Inativadoras de Ribossomos Tipo 1/química , Saponaria/química , Sementes/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Modelos Moleculares , Desnaturação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Desdobramento de Proteína , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saporinas , Soluções , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The C2 domain of PKCε binds to negatively charged phospholipids but little is known so far about the docking orientation of this domain when it is bound. By using a FRET assay we have studied the binding of this domain to model membranes. We have also used ATR-Fourier transform infrared spectroscopy with polarized light (ATR-FTIR) to determine the docking mode by calculating the ß-sandwich orientation when the domain is bound to different types of model membranes. The vesicle lipid compositions were: POPC/POPE/POPA (22:36:42) imitating the inner leaflet of a plasma membrane, POPC/POPA (50:50) in which POPE has been eliminated with respect to the former composition and POPC/POPE/CL (43:36:21) imitating the inner mitochondrial membrane. Results show that the ß-sandwich of the PKCα-C2 domain is inclined at an angle α close to 45° to the membrane normal. Some differences were found with respect to the extent of binding as a function of phospholipid composition and small changes on secondary structure were only evident when the domain was bound to model membranes of POPC/POPA: in this case, the percentage of ß-sheet of the C2 domain increases if compared with the secondary structure of the domain in the absence of vesicles. With respect to the ß-sandwich orientation, when the domain is bound to POPC/POPE/CL membranes it forms an angle with the normal to the surface of the lipid bilayer (39°) smaller than that one observed when the domain interacts with vesicles of POPC/POPA (49°).
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Bicamadas Lipídicas/química , Proteína Quinase C-épsilon/química , Espectrofotometria Infravermelho/métodos , Adenosina/análogos & derivados , Adenosina/química , Cálcio/química , Glicerofosfolipídeos/química , Humanos , Lipídeos/química , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Modelos Estatísticos , Conformação Molecular , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfolipídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP(2) in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP(2). The results best fitted a binding mechanism in which protein bound to the membrane following a two-phase mechanism with a first bimolecular reaction followed by a slow unimolecular reaction. In the absence of PIP(2), the rapid protein binding rate was especially dependent on POPS concentration. Formation of the slow high affinity complex during the second phase seems to involve specific interactions with POPS and DOG since it is only sensitive to changes within relatively low concentration ranges of these lipids. Both the association and dissociation rate constants fell in the presence of PIP(2). We propose a model in which PKCα binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKCα driven by interactions with POPS and/or PIP(2) although interactions with DOG are involved too. PKCα searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands.
Assuntos
Membrana Celular/metabolismo , Diglicerídeos/metabolismo , Lipídeos de Membrana/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Proteína Quinase C-alfa/metabolismo , Animais , Transferência Ressonante de Energia de Fluorescência , Cinética , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Ligação Proteica , Proteína Quinase C-alfa/isolamento & purificação , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , SuínosRESUMO
We have used attenuated total internal reflection infrared spectroscopy (ATR-IR) spectroscopy to study the association of the C2 domain from protein kinase Cα (PKCα) with different phospholipid membranes, so as to characterise the mode of membrane docking and its modulation by the second-messenger lipid PIP2. In parallel, we have also examined the membrane interaction of the C2 domain from cytosolic phospholipase A2. PIP2 did not induce significant changes in secondary structure of the membrane-bound PKCα-C2 domain, nor did binding of the PKCα-C2 domain change the dichroic ratios of the lipid chains, whereas the C2 domain from phospholipase A2 did perturb the lipid chain orientation. Measurements of the dichroic ratios for the amide I and amide II protein bands were combined so as to distinguish the tilt of the ß-sheets from that of the ß-strands within the sheet. When associated with POPC/POPS membranes, the ß-sandwich of the PKCα-C2 domain is inclined at an angle α=35° to the membrane normal, i.e., is oriented more nearly perpendicular than parallel to the membrane. In the process of membrane docking, the tilt angle increases to α=44° in the presence of PIP2, indicating that the ß-sandwich comes closer to the membrane surface, so confirming the importance of this lipid in determining docking of the C2 domain and consequent activation of PKCα.
Assuntos
Membrana Celular/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipases A2/metabolismo , Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/metabolismo , Citosol/metabolismo , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The structural and stability properties of a novel zinc-dependent alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII) were investigated by Fourier transformed infrared spectroscopy (FTIR). This enzyme is a thermostable homo-tetramer belonging to the GroES-fold motif proteins characterized by an irregular ß-barrel conformation. Our results revealed a protein with a secondary structure rich in ß-sheet (32% of the total secondary elements) and it showed a three-step thermal unfolding pathway. The complete enzyme denaturation was preceded by the formation of a relaxed tertiary/quaternary structure and previously by an excited native-like conformation. Two-dimensional correlation spectroscopy analysis (2D-COS) and differential scanning calorimetry (DSC) experiments supported these data and allowed us to determine the protein melting temperature at 96.9 °C as well as to suggest the sequence of the events that occurred during the protein denaturation process.
Assuntos
Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Pyrobaculum/enzimologia , Temperatura , Estabilidade Enzimática , Desnaturação Proteica , Estrutura Secundária de ProteínaRESUMO
Clotrimazole (1-[(2-chlorophenyl)-diphenylmethyl]-imidazole) is an azole antifungal drug belonging to the imidazole subclass that is widely used in pharmacology and that can be incorporated in membranes. We studied its interaction with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) phospholipid vesicles by using differential scanning calorimetry and found that the transition temperature decreases progressively as the concentration of clotrimazole increases. However, the temperature of completion of the transition remained constant despite the increase of clotrimazole concentration, suggesting the formation of fluid immiscibility. 1H-NMR and 1H NOESY MAS-NMR were employed to investigate the location of clotrimazole in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid membranes. In the presence of clotrimazole, all the resonances originating from POPC were shifted upfield, but mainly those corresponding to C2 and C3 of the fatty acyl, chains suggesting that clotrimazole aromatic rings preferentially locate near these carbons. In the same way, 2D-NOESY measurements showed that the highest cross-relaxation rates between protons of clotrimazole and POPC were with those bound to the C2 and C3 carbons of the fatty acyl chains. Molecular dynamics simulations indicated that clotrimazole is located near the top of the hydrocarbon-chain phase, with the nitrogen atoms of the imidazole ring of clotrimazole being closest to the polar group of the carbonyl moiety. These results are in close agreement with the NMR and the conclusion is that clotrimazole is located near the water-lipid interface and in the upper part of the hydrophobic bilayer.
Assuntos
Membrana Celular/química , Clotrimazol/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Fluidez de Membrana , Fosfolipídeos/química , Varredura Diferencial de Calorimetria , Clotrimazol/química , Dimiristoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Fluidez de Membrana/efeitos dos fármacos , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Espectroscopia de Prótons por Ressonância Magnética , Temperatura , Água/químicaRESUMO
The synthetic estrogen diethylstilbestrol (DES) is used to treat metastatic carcinomas and prostate cancer. We studied its interaction with membranes and its localization to understand its mechanism of action and side-effects. We used differential scanning calorimetry (DSC) showing that DES fluidized the membrane and has poor solubility in DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) in the fluid state. Using small-angle X-ray diffraction (SAXD), it was observed that DES increased the thickness of the water layer between phospholipid membranes, indicating effects on the membrane surface. DSC, X-ray diffraction, and 31P-NMR spectroscopy were used to study the effect of DES on the Lα-to-HII phase transition, and it was observed that negative curvature of the membrane is promoted by DES, and this effect may be significant to understand its action on membrane enzymes. Using the 1H-NOESY-NMR-MAS technique, cross-relaxation rates for different protons of DES with POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) protons were calculated, suggesting that the most likely location of DES in the membrane is with the main axis parallel to the surface and close to the first carbons of the fatty acyl chains of POPC. Molecular dynamics simulations were in close agreements with the experimental results regarding the location of DES in phospholipids bilayers.
Assuntos
Varredura Diferencial de Calorimetria/métodos , Carbono/química , Dietilestilbestrol/química , Ácidos Graxos/química , Membranas Artificiais , Difração de Raios X/métodos , Dietilestilbestrol/análise , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Transição de Fase , Fosfatidilcolinas/química , Fosfolipídeos/química , Radioisótopos de Fósforo/química , Prótons , Solubilidade , Temperatura , TermodinâmicaRESUMO
Curcumin and two bivalent compounds, namely 17MD and 21MO, both obtained by conjugation of curcumin with a steroid molecule that acts as a membrane anchor, were comparatively studied. When incorporated into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine the compounds showed a very limited solubility in the model membranes. Curcumin and the two bivalent compounds were also incorporated in membranes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and quenching the fluorescence of pure curcumin or of the curcumin moiety in the bivalent compounds by acrylamide it was seen that curcumin was accessible to this water soluble quencher but the molecule was somehow located in a hydrophobic environment. This was confirmed by quenching with doxyl-phosphatidylcholines, indicating that the curcumin moieties of 17MD and 21MO were in a more polar environment than pure curcumin itself. 1H NOESY MAS-NMR analysis supports this notion by showing that the orientation of curcumin was parallel to the plane of the membrane surface close to C2 and C3 of the fatty acyl chains, while the curcumin moiety of 17MD and 21MO positioned close to the polar part of the membrane with the steroid moiety in the centre of the membrane. Molecular dynamics studies were in close agreement with the experimental results with respect to the likely proximity of the protons studied by NMR and show that 17MD and 21MO have a clear tendency to aggregate in a fluid membrane. The anchorage of the bivalent compounds to the membrane leaving the curcumin moiety near the polar part may be very important to facilitate the bioactivity of the curcumin moiety when used as anti-Alzheimer drugs.
Assuntos
Doença de Alzheimer , Curcumina , Doença de Alzheimer/tratamento farmacológico , Curcumina/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas , Espectroscopia de Ressonância Magnética , Fosfatidilcolinas , ÁguaRESUMO
The C-terminal domain of the pro-apoptotic protein Bax (Bax-C) is supposed to act as a membrane anchor motif when Bax is activated leading to programmed cell death. A synthetic peptide which imitates this domain has been used to study the mechanism of peptide-phospholipid interaction. We have used static and MAS-NMR techniques to show that the interaction of Bax-C with membranes is modulated by the presence of a negatively charged phospholipid like phosphatidylglycerol. Bax-C slightly shifted upfield the (31)P resonances coming from phosphatidylglycerol and phosphatidylcholine. However the width of the resonance peaks was considerably higher when phosphatidylglycerol was present. Bax-C substantially decreased the T(1) relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol, but T(1) values were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. (13)C-MAS-NMR showed that T(1) values were decreased when Bax-C was incorporated into the lipid vesicles and this reduction affected similarly to carbons located in different regions of the membrane when the only phospholipid present was phosphatidylcholine. However, when phosphatidylglycerol was also present, the decrease in T(1) affected considerably more to some carbons in the polar region. These results indicate that Bax-C interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of Bax-C with the membrane determines the location of this domain. Fluorescence spectroscopy showed that the Trp residues of Bax-C were placed in a microenvironment more hydrophobic and less accessible to quenching by acrylamide when phosphatidylglycerol was present.
Assuntos
Fosfolipídeos/metabolismo , Estrutura Terciária de Proteína , Proteína X Associada a bcl-2/metabolismo , Isótopos de Carbono , Ressonância Magnética Nuclear Biomolecular , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Espectrometria de FluorescênciaRESUMO
Gelonin from the Indian plant Gelonium multiflorum belongs to the type I ribosome-inactivating proteins (RIPs). Like other members of RIPs, this toxin glycoprotein inhibits protein synthesis of eukaryotic cells; hence, it is largely used in the construction of immunotoxins composed of cell-targeted antibodies. Lysosomal degradation is one of the main issues in targeted tumor therapies, especially for type I RIP-based toxins, as they lack the translocation domains. The result is an attenuated cytosolic delivery and a decrease of the antitumor efficacy of these plant-derived toxins; therefore, strategies to permit their release from endosomal vesicles or modifications of the toxins to make them resistant to degradation are necessary to improve their efficacy. Using infrared spectroscopy, we thoroughly analyzed both the secondary structure and the thermal unfolding of gelonin. Moreover, by the combination of two-dimensional correlation spectroscopy and phase diagram method, it was possible to deduce the sequence of events during the unfolding, confirming the typical characteristic of the RIP members to denature in two steps, as a sequential loss of tertiary and secondary structure was detected at 58 °C and at 65 °C, respectively. Additionally, some discrepancies in the unfolding process between gelonin and saporin-S6, another type I RIP protein, were detected.
Assuntos
Temperatura Alta , Desdobramento de Proteína , Proteínas Inativadoras de Ribossomos Tipo 1/química , Suregada/química , Toxinas Biológicas/química , Dicroísmo Circular , Estrutura Secundária de Proteína , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Sementes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Toxinas Biológicas/isolamento & purificaçãoRESUMO
Antimicrobial peptides (AMPs) are effector molecules of the innate immune system and have been isolated from multiple organisms. Their antimicrobial properties are due to the fact that they interact mainly with the anionic membrane of the microorganisms, permeabilizing it and releasing the cytoplasmic content. Alyteserin 1c (+2), an AMP isolated from Alytes obstetricans and its more cationic and hydrophilic analogue (+5) were synthesized using the solid phase method, in order to study the interaction with model membranes by calorimetric and spectroscopic assays. Differential scanning calorimetry (DSC) showed that both peptides had a strong effect when the membrane contained phosphatidylcholine (PC) alone or was mixed with phosphatidylglycerol (PG), increasing membrane fluidization. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to study the secondary structure of the peptide. Peptide +2 exhibited a transition from ß-sheet/turns to ß-sheet/α-helix structures after binding with model membranes, whereas peptide +5 had a transition from aggregation/unordered to ß-sheet/α-helix structures after binding with membrane-contained PC. Interestingly, the latter showed a ß-sheet structure predominantly in the presence of PG lipids. Additionally, molecular dynamics (MD) results showed that the carboxy-terminal of the peptide +5 has the ability to insert into the surface of the PC/PG membranes, resulting in the increase of the membrane fluidity.