RESUMO
Multiple molecular dynamics simulations were performed to investigate the association of stearic acid into the highest affinity binding site of human serum albumin. All binding events ended with a rapid (<10 ps) lock-in of the fatty acid due to formation of a hydrogen bond with Tyr401. The kinetics and energetics of the penetration process both depended linearly on the positional shift of the fatty acid, with an average insertion time and free energy reduction of, respectively, 32 ± 20 ps and 0.70 ± 0.15 kcal/mol per methylene group absorbed. Binding events of longer duration (tbind>1 ns) were characterized by a slow exploration of the pocket entry and, frequently, of a nearby protein crevice corresponding to a metastable state along the route to the binding site. Taken all together, these findings reconstruct the following pathway for the binding process of stearic acid: (i) contact with the protein surface, possibly facilitated by the presence of an intermediate location, (ii) probing of the site entry, (iii) insertion into the protein, and (iv) lock-in at the final position. This general description may also apply to other long-chain fatty acids binding into any of the high-affinity sites of albumin, or to specific sites of other lipid-binding proteins.
Assuntos
Modelos Químicos , Simulação de Dinâmica Molecular , Albumina Sérica/química , Albumina Sérica/ultraestrutura , Ácidos Esteáricos/química , Sítios de Ligação , Humanos , Cinética , Ligação Proteica , Conformação ProteicaRESUMO
Librational motions in the region of the protein "glass" (or dynamic) transition are analysed for spin-labelled haemoglobin, serum albumin and ß-lactoglobulin by EPR spectroscopy. A discontinuity in the temperature dependence of the mean-square librational amplitude, <α(2)>, occurs in the region of 200K as found for the mean-square atomic displacement,
Assuntos
Vidro/química , Hemoglobinas/química , Lactoglobulinas/química , Proteínas/química , Albumina Sérica/química , ATPase Trocadora de Sódio-Potássio/química , Animais , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Marcadores de SpinRESUMO
Denaturant-perturbation and pulsed EPR spectroscopy are combined to probe the folding of the membrane-bound Na,K-ATPase active transport system. The Na,K-ATPase enzymes from shark salt gland and pig kidney are covalently spin labelled on cysteine residues that either do not perturb or are essential to hydrolytic activity (Class I and Class II -SH groups, respectively). Urea increases the accessibility of water to the spin-labelled groups and increases their mutual separations, as recorded by D2O interactions from ESEEM spectroscopy and instantaneous spin diffusion from echo-detected EPR spectra, respectively. The greater effects of urea are experienced by Class I groups, which indicates preferential unfolding of the extramembrane domains. Conformational heterogeneity induced by urea causes dispersion in spin-echo phase-memory times to persist to higher temperatures. Analysis of lineshapes from partially relaxed echo-detected EPR spectra indicates that perturbation by urea enhances the amplitude and rate of fluctuations between conformational substates, in the higher temperature regime, and also depresses the glasslike transition in the protein. These non-native substates that are promoted by urea lie off the enzymatic pathway and contribute to the loss of function.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Desdobramento de Proteína/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/química , Ureia/farmacologia , Algoritmos , Animais , Medição da Troca de Deutério , Óxido de Deutério/química , Relação Dose-Resposta a Droga , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Cinética , Modelos Químicos , Conformação Proteica/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Glândula de Sal/enzimologia , Tubarões , ATPase Trocadora de Sódio-Potássio/metabolismo , Marcadores de Spin , Especificidade por Substrato , Suínos , Temperatura , Água/químicaRESUMO
The copper site and overall structures of azurin (AZ) variants in which the amicyanin (AMI) and plastocyanin (PC) metal binding loops have been introduced, AZAMI and AZPC, respectively, are similar to that of AZ, whereas the loop conformations resemble those in the native proteins. To assess the influence of these loop mutations on stability, the thermal unfolding of AZAMI and AZPC has been investigated by differential scanning calorimetry, absorption and fluorescence spectroscopy. The calorimetric profiles of both variants exhibit a complex shape consisting of two endothermic peaks and an exothermic peak. The temperature of the maximum heat of absorption for the single endothermic peak is 82.7°C for AZ, whereas for AZAMI and AZPC the most intense endothermic peaks are at 74.9 and 68.1°C comparable to values for AMI and PC, respectively. Denaturation investigated using the temperature dependence of the absorbance at â¼600nm and Trp emission, also demonstrates decreased stability for both loop mutants. The thermal transition between the native and the denaturated states is irreversible, scan rate dependent and consistent with the two-state irreversible model. The structure of the active-site loop has a dramatic effect on the kinetic stability and the unfolding pathway of cupredoxins.
Assuntos
Azurina/química , Azurina/genética , Azurina/metabolismo , Varredura Diferencial de Calorimetria , Domínio Catalítico/genética , Cinética , Mutagênese Sítio-Dirigida , Conformação Proteica , Desnaturação Proteica , Estabilidade Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Espectrometria de Fluorescência , Espectrofotometria , Termodinâmica , Resposta a Proteínas não DobradasRESUMO
The interaction between the natural polyphenol resveratrol and human serum albumin (HSA), the most abundant transport protein in plasma, has been studied in the absence and in the presence of up to six molecules of stearic acids (SA) pre-complexed with the protein. The study has been carried out by using the intrinsic fluorescence of both HSA and resveratrol. Protein and polyphenol fluorescence data indicate that resveratrol binds to HSA with an association constant k(a) = (1.10 ± 0.14) × 10(5) M(-1) and (1.09 ± 0.02) × 10(5) M(-1), respectively, whereas Job plot evidences the formation of an equimolar protein/drug complex. Low SA content associated with HSA does not affect significantly the structural conformation of the protein and its interaction with resveratrol, whereas high SA content induces conformational changes in the protein, and reduces resveratrol binding affinity. The photostability of resveratrol in the different samples changes in the order: buffer < (high [SA]/HSA) < HSA < (low [SA]/HSA). The results on (SA/HSA)-resveratrol samples highlight the ability of the protein to bind hydrophobic and amphiphilic ligands and to protect from degradation an important antioxidant molecule under biologically relevant conditions.
Assuntos
Albumina Sérica/química , Ácidos Esteáricos/química , Estilbenos/química , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Resveratrol , Albumina Sérica/metabolismo , Ácidos Esteáricos/farmacologia , Estilbenos/farmacologiaRESUMO
Two-pulse, echo-detected electron paramagnetic resonance (ED-EPR) spectra and continuous-wave EPR (CW-EPR) spectra were used to investigate the solvent effect on the librational motion of human haemoglobin spin-labelled on cysteine ß93 with the nitroxide derivative of maleimide, 6-MSL. Protein samples fully hydrated in phosphate buffer solution (PBS), in a 60% v/v glycerol/water mixture and in the lyophilized form were measured at cryogenic temperature in the frozen state. The protein librational motion was characterized by the amplitude-correlation time product, <α²>τ(c), deduced from the ED-EPR spectra. The librational amplitude, <α²>τ(c), was determined independently, from the motionally averaged hyperfine splitting in the CW-EPR spectra, and the librational correlation time, τ(c), was derived from the combination of the pulsed and conventional EPR data. Rapid librational motion of small amplitude was detected in all samples. In each case, the librational dynamics was restricted up to 180 K, beyond which it increased steeply for the hydrated protein in PBS and in the presence of glycerol. In contrast, in the dehydrated protein, the librational dynamics was hindered and less dependent on temperature up to ~240 K. In all samples, <α²> deviated from small values only for T > 200 K, where a rapid increase of <α²> was evident for the hydrated samples, whereas limited temperature variation was shown in the lyophilized samples. The librational correlation time was in the sub-nanosecond regime and weakly dependent on temperature. The results evidence that solvent favours protein dynamics.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hemoglobinas/química , Modelos Químicos , Solventes/química , Marcadores de Spin/síntese química , Humanos , Movimento (Física) , TemperaturaRESUMO
Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between human serum albumin (HSA) and sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Protein/lipid dispersions are considered in which spin-labelled stearic acids at the 16th carbon atom along the acyl chain (16-SASL) are inserted either in the protein or in the SSL. Two component ESR spectra with different rotational mobility are obtained over a broad range of temperature and membrane composition. Indeed, superimposed to an anisotropic protein-signal, appears a more isotropic lipid-signal. Since in the samples only one matrix (protein or membranes) is spin-labelled, the other component accounts for the transfer of 16-SASL between albumin and membranes. The two components have been resolved and quantified by spectral subtractions, and the fraction, f (p) (16-SASL), of spin labels bound non-covalently to the protein has been used to monitor the transfer. It is found that it depends on the type of donor and acceptor matrix, on the physical state of the membranes and on the grafting density of the polymer-lipids. Indeed, it is favoured from SSL to HSA and the fraction of stearic acids transferred increases with temperature in both directions of transfer. Moreover, in the presence of polymer-lipids, the transfer from HSA to SSL is slightly attenuated, especially in the brush regime of the polymer-chains. Instead, the transfer from SSL to HSA is favoured by the polymer-lipids much more in the mushroom than in the brush regime.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/análogos & derivados , 1,2-Dipalmitoilfosfatidilcolina/química , Doxorrubicina/análogos & derivados , Polietilenoglicóis/química , Ácidos Esteáricos/química , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Humanos , Membranas/química , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacocinéticaRESUMO
The thermal stability of umecyanin, a stellacyanin from horseradish roots, has been investigated by differential scanning calorimetry, optical absorption and fluorescence spectroscopy at neutral and alkaline pH. Above pH 9 the Cu(II) protein experiences a blue shift of the main visible absorption band at approximately 600 nm and changes colour from blue to violet. The thermal transition of the protein is irreversible and occurs between 61.4 and 68.8 degrees C at pH 7.5 and between 50.7 and 57.4 degrees C at pH 9.8. The calorimetric data indicates that at both pH values the thermally induced transition of the protein between the native and denaturated states can be described in terms of the classical Lumry-Eyring unfolding model Native<-->Unfolded-->Final. The analysis of the reversible step in the unfolding pathway demonstrates a significant reduction in conformational stability (DeltaG) of the alkaline form of the protein. Such a reduction is consistent with an enhanced flexibility of UMC at high pH and has mainly entropic character.
Assuntos
Armoracia/química , Metaloproteínas/química , Modelos Moleculares , Proteínas de Plantas/química , Dobramento de Proteína , Varredura Diferencial de Calorimetria/métodos , Entropia , Temperatura Alta , Concentração de Íons de Hidrogênio , Espectrometria de Fluorescência/métodosRESUMO
Molecular dynamics simulation has been carried out for the blue copper protein amicyanin from two different sources, Paracoccus denitrificans and Paraccocus versutus, to investigate the structural and dynamical properties common to the two molecules and to identify prominent features shared with proteins of the same family, the monomeric cupredoxins. The two amicyanins have almost identical secondary and tertiary structure. In the simulation, they differ for the number of hydrogen bonds in the main chain and the conformation of some beta-strands. However, they strictly maintain the arrangement of the portions of the beta-barrel that are conserved in the folding architecture of the blue copper proteins. Paracoccus versutus amicyanin equilibrates more rapidly, shows lower atomic deviation values, and is less rigid with respect to Paracoccus denitrificans amicyanin. Principal component analysis reveals that the conformational subspaces corresponding to eigenvectors with the same index for each of the two molecules are not necessarily equivalent. Nevertheless, a core scaffold with constrained dynamics exist for both amicyanins. In addition, two fairly flexible regions that are located on the opposite side with respect to the interaction sites with the partner molecules in the redox process have been evidenced in the protein structure. This description of amicyanin, with a few mobile regions remote from the active site and a rigid scaffold including most of the protein beta-barrel, has a close similarity with that of azurin and plastocyanin, two other cupredoxins previously investigated in simulation. Furthermore, similarities in the distribution of the atomic fluctuations indicate that amicyanin, azurin, and plastocyanin possess common dynamical features, in spite of differences in their structure. On the basis of these findings, we suggest that topological constraints imposed by the folding in correspondence of protein regions that are the most conserved determine the protein dynamics of the cupredoxin family. The dynamical properties of the cupredoxins might be controlled for functional advantages that include the binding mechanism with the biological partners and the collective inner motions of the protein matrix required for the electron transfer, whereas long-range conformational changes in the redox reaction should be excluded.
Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Sítios de Ligação , Simulação por Computador , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Paracoccus denitrificans/metabolismo , Conformação ProteicaRESUMO
Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2tau. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.
Assuntos
Alameticina/química , Óxidos N-Cíclicos/química , Dimiristoilfosfatidilcolina/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Bicamadas Lipídicas/química , Fluidez de Membrana , Sítios de Ligação , Cinética , Conformação Molecular , Marcadores de SpinRESUMO
Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D(2)O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D(2)O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.
Assuntos
Membranas/química , Modelos Moleculares , Albumina Sérica/química , Ácidos Esteáricos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Adsorção , Espectroscopia de Ressonância de Spin Eletrônica , Ácidos Graxos não Esterificados/química , Humanos , Fosfolipídeos/química , TemperaturaRESUMO
Molecular dynamics was applied to dissect out the internal motions of azurin, a copper protein performing electron transfer. Simulations of 16.5 ns were analyzed in search of coordinated displacements of amino acid residues that are important for the protein function. A region with high conformational instability was found in the 'southern' end of the molecule, far away from the copper site and the binding sites for the redox partners of azurin. By excluding the 'southern' region from the subsequent analysis, correlated motions were identified in the hydrophobic patch that surrounds the protein active site. The simulation results are in excellent agreement with recent NMR data on azurin in solution [A. V. Zhuravleva, D. M. Korzhnev, E. Kupce, A. S. Arseniev, M. Billeter, V. Y. Orekhov, Gated electron transfers and electron pathways in azurin: a NMR dynamic study at multiple fields and temperatures, J. Mol. Biol. 342 (2004) 1599-1611] and suggest a rationale for cooperative displacements of protein residues that are thought to be critical for the electron transfer process. A number of other structural and dynamic features of azurin are discussed in the context of the blue copper protein family and an explanation is proposed to account for the variability/conservation of some regions in the cupredoxins.
Assuntos
Azurina/química , Sítios de Ligação , Transporte de Elétrons , Interações Hidrofóbicas e Hidrofílicas , Metaloproteínas/química , Movimento (Física) , Conformação ProteicaRESUMO
Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T(m) approximately 100 degrees C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry-Eyring model, N<-->U-->F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed.
Assuntos
Alcaligenes faecalis/enzimologia , Nitrito Redutases/química , Varredura Diferencial de Calorimetria , Cobre/análise , Espectroscopia de Ressonância de Spin Eletrônica , Estabilidade Enzimática , Temperatura Alta , Desnaturação Proteica , TermodinâmicaRESUMO
Recently, developments in time-resolved spin-label electron spin resonance (ESR) spectroscopy have contributed considerably to the study of biomembranes. Two different applications of electron spin echo spectroscopy of spin-labelled phospholipids are reviewed here: (1) the use of partially relaxed echo-detected ESR spectra to study the librational lipid-chain motions in the low-temperature phases of phospholipid bilayers; (2) the use of electron spin echo envelope modulation spectroscopy to determine the penetration of water into phospholipid membranes. Results are described for phosphatidylcholine bilayer membranes, with and without equimolar cholesterol, that are obtained with phosphatidylcholine spin probes site-specifically labelled throughout the sn-2 chain.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Bicamadas Lipídicas/química , Lipídeos/química , Fluidez de Membrana , Membranas/química , Colesterol/química , Modelos Moleculares , Fosfatidilcolinas/química , Marcadores de Spin , Fatores de Tempo , Água/químicaRESUMO
In this work, the pro-oxidant behavior of oleuropein (OLP, 1) is characterized in a Fenton-like experiment by means of ESR spectroscopy using the spin trap system DMSO and 4-(pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) in phosphate buffer (PB) solution. Ferrous ions in the absence of hydrogen peroxide cause the formation of the stable nitroxide species 4 and 5 through the intermediate perferryl species. OLP displays its antioxidant activity in vitro blocking the oxidation path that leads to methoxyl radicals hence to the formation of the stable radical species 5. The role of the catechol moiety was proved when the perferryl experiments were repeated in the presence of the dimethylated oleuropein homologue (OLP-Met2, 2). The dual behavior of oleuropein, similar to that ascertained for other catechol and non-catechol natural active species, should provide warnings for its use as nutraceutical or as drug with manifold healing effects.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Oxidantes/farmacologia , Piranos/farmacologia , Antioxidantes , Óxidos N-Cíclicos , Dimetil Sulfóxido , Glucosídeos Iridoides , Iridoides , Olea/química , Folhas de Planta/química , PiridinasRESUMO
Several phenolic compounds bind to proteins and show the ability to interfere with their aggregation process. The impact of the natural polyphenol resveratrol on the stability and heat induced aggregation of human serum albumin (HSA) was investigated by differential scanning calorimetry (DSC), attenuated total reflectance Fourier transform infrared (ATR-FTIR), UV-vis absorbance, ThT fluorescence, atomic force microscopy (AFM) and molecular modeling. The binding of resveratrol to HSA improves the stability of the protein to thermal unfolding, particularly for the energetic domain containing the ligand binding site, as modeled by computational techniques. The thermal unfolding is irreversible and after the melting the protein aggregates, either with or without the ligand. The kinetics of HSA aggregation between 70 and 80°C shows an exponential growth of the absorbance change and it slows down when resveratrol is added. The aggregates have fibril-like morphology and resveratrol attenuates the formation of ß-structured species. The overall results suggest that resveratrol stabilizes the protein structure and modulates the formation of fibrils along the initial stage of the HSA aggregation pathway.
Assuntos
Agregados Proteicos , Albumina Sérica/química , Estilbenos/química , Sítios de Ligação , Temperatura Alta , Humanos , Cinética , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Estrutura Secundária de Proteína , Desdobramento de Proteína , Resveratrol , TermodinâmicaRESUMO
Membranes grafted with water-soluble polymers resist protein adsorption and adhesion to cellular surfaces. Liposomes with surface-grafted polymers therefore find applications in drug delivery. The physicochemical properties of polymer-grafted lipid membranes are reviewed with mean-field and scaling theories from polymer physics. Topics covered are: mushroom-brush transitions, membrane expansion and elasticity, bilayer-micelle transitions, membrane-membrane interactions and protein-membrane interactions.
Assuntos
Membrana Celular/fisiologia , Bicamadas Lipídicas/metabolismo , Polímeros/metabolismo , Animais , Membrana Celular/química , Humanos , Bicamadas Lipídicas/química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polímeros/química , Proteínas/metabolismoRESUMO
The adsorption of human serum albumin (HSA) to dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was studied as a function of content and headgroup size of the polymer lipid. In the absence of protein, conversion from the low-density mushroom regime to the high-density brush regime of polymer-lipid content is detected by the change in ESR outer hyperfine splitting, 2A(max), of chain spin-labelled phosphatidylcholine in gel-phase membranes. The values of 2A(max) remain constant in the mushroom regime, but decrease on entering the brush regime. Conversion between the two regimes occurs at mole fractions X(PEG)(m-->b) approximately 0.04, 0.01-0.02 and 0.005-0.01 for PEG-DPPE with mean PEG molecular masses of 350, 2000 and 5000 Da, respectively, as expected theoretically. Adsorption of HSA to DPPC membranes is detected as a decrease of the spin label 2A(max) hyperfine splitting in the gel phase. Saturation is obtained at a protein/lipid ratio of ca. 1:1 w/w. In the presence of polymer-grafted lipids, HSA adsorbs to DPPC membranes only in the mushroom regime, irrespective of polymer length. In the brush regime, the spin-label values of 2A(max) are unchanged in the presence of protein. Even in the mushroom regime, protein adsorption progressively becomes strongly attenuated as a result of the steric stabilization exerted by the polymer lipid. These results are in agreement with theoretical estimates of the lateral pressure exerted by the grafted polymer in the brush and mushroom regimes, respectively.
Assuntos
Lipídeos de Membrana , Albumina Sérica , 1,2-Dipalmitoilfosfatidilcolina , Adsorção , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Técnicas In Vitro , Bicamadas Lipídicas/química , Fosfatidiletanolaminas , Polietilenoglicóis , Marcadores de SpinRESUMO
The chain-melting transition temperature of dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was determined by optical turbidity measurements. The dependence on content, Xp, of PEG-DPPE lipid was studied for different polar headgroup sizes, np, of the polymer lipid, throughout the lamellar phase of the mixtures with DPPC. Mean-field theory for the polymer brush regime predicts that the downward shift in transition temperature should vary with polymer size and content as npXp(5/3) (approximately npXp(11/6) for scaling theory). Any shift induced by the charge on PEG-lipids is independent of polymer size. These predictions are reasonably borne out for the longer polymer lipids (PEG molecular masses 750, 2000 and 5000 Da). Transition temperature shifts in the lamellar phase, before the onset of micellisation, are in the region of -1 to -2 degrees C (+/-0.1-0.2 degrees C) in reasonable accord with theoretical estimates of the lateral pressure exerted by the polymer brush. Shifts of this size are significant to the design of liposomes for controlled release of contents by mild hyperthermia.
Assuntos
Lipossomos/química , Polímeros/química , Temperatura de Transição , 1,2-Dipalmitoilfosfatidilcolina/química , Sistemas de Liberação de Medicamentos , Transição de Fase , Fosfatidiletanolaminas/química , Polietilenoglicóis/químicaRESUMO
Electron spin-echo envelope modulation (ESEEM) spectroscopy of phospholipids spin-labeled systematically down the sn-2 chain was used to detect the penetration of water (D2O) into bilayer membranes of dipalmitoyl phosphatidylcholine with and without 50 mol % cholesterol. Three-pulse stimulated echoes allow the resolution of two superimposed 2H-ESEEM spectral components of different widths, for spin labels located in the upper part of the lipid chains. Quantum chemical calculations (DFT) and ESEEM simulations assign the broad spectral component to one or two D2O molecules that are directly hydrogen bonded to the N-O group of the spin label. Classical ESEEM simulations establish that the narrow spectral component arises from nonbonded water (D2O) molecules that are free in the hydrocarbon chain region of the bilayer membrane. The amplitudes of the broad 2H-ESEEM spectral component correlate directly with those of the narrow component for spin labels at different positions down the lipid chain, reflecting the local H-bonding equilibria. The D2O-ESEEM amplitudes decrease with position down the chain toward the bilayer center, displaying a sigmoidal dependence on position that is characteristic of transmembrane polarity profiles established by other less direct spin-labeling methods. The midpoint of the sigmoidal profile is shifted toward the membrane center for membranes without cholesterol, relative to those with cholesterol, and the D2O-ESEEM amplitude in the outer regions of the chain is greater in the presence of cholesterol than in its absence. For both membrane types, the D2O amplitude is almost vanishingly small at the bilayer center. The water-penetration profiles reverse correlate with the lipid-chain packing density, as reflected by 1H-ESEEM intensities from protons of the membrane matrix. An analysis of the H-bonding equilibria provides essential information on the binding of water molecules to H-bond acceptors within the hydrophobic interior of membranes. For membranes containing cholesterol, approximately 40% of the nitroxides in the region adjacent to the lipid headgroups are H bonded to water, of which ca. 15% are doubly H bonded. Corresponding H-bonded populations in membranes without cholesterol are ca. 20%, of which ca. 6% are doubly bonded.