RESUMEN
Investigating how hydrophobic molecules mix with phospholipid bilayers and how they affect membrane properties is commonplace in biophysics. Despite this, a molecular-level empirical description of a membrane model as simple as a phospholipid bilayer with long linear hydrophobic chains incorporated is still missing. Here, we present an unprecedented molecular characterization of the incorporation of two long n-alkanes, n-eicosane (C20) and n-triacontane (C30) with 20 and 30 carbons, respectively, in phosphatidylcholine (PC) bilayers using a combination of experimental techniques (2H NMR, 31P NMR, 1H-13C dipolar recoupling solid-state NMR, X-ray scattering, and cryogenic electron microscopy) and atomistic molecular dynamics (MD) simulations. At low hydration, deuterated C20 and C30 yield 2H NMR spectra evidencing anisotropic-motion, which demonstrates their miscibility in PC membranes up to a critical alkane-to-acyl-chain volume fraction, Ïc. The acquired 2H NMR spectra of C20 and C30 have notably different lineshapes. At low alkane volume fractions below Ïc, CHARMM36 MD simulations predict such 2H NMR spectra qualitatively and thus enable an atomistic-level interpretation of the spectra. Above Ïc, the 2H NMR lineshapes become characteristic of motions in the intermediate-regime that, together with the MD simulation results, suggest the onset of immiscibility between the alkane molecules and the acyl chains. For all the systems investigated, the phospholipid molecular structure is unperturbed by the presence of the alkanes. However, at conditions of excess hydration and at surprisingly low alkane fractions below Ïc, a peak characteristic of isotropic motion is observed in both the 2H spectra of the alkanes and 31P spectra of the phospholipids, strongly indicating that the incorporation of the alkanes induces a reduction on the average radius of the lipid vesicles.
Asunto(s)
Membrana Dobles de Lípidos , Fosfolípidos , Alcanos , Membrana Dobles de Lípidos/química , Espectroscopía de Resonancia Magnética , Fosfatidilcolinas/química , Fosfolípidos/químicaRESUMEN
Phospholipid-coated targeted microbubbles are ultrasound contrast agents that can be used for molecular imaging and enhanced drug delivery. However, a better understanding is needed of their targeting capabilities and how they relate to microstructures in the microbubble coating. Here, we investigated the ligand distribution, lipid phase behavior, and their correlation in targeted microbubbles of clinically relevant sizes, coated with a ternary mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), with PEG40-stearate and DSPE-PEG2000. To investigate the effect of lipid handling prior to microbubble production in DSPC-based microbubbles, the components were either dispersed in aqueous medium (direct method) or first dissolved and mixed in an organic solvent (indirect method). To determine the lipid-phase behavior of all components, experiments were conducted on monolayers at the air/water interface. In comparison to pure DSPC and DPPC, the ternary mixtures had an additional transition plateau around 10-12 mN/m. As confirmed by infrared reflection absorption spectroscopy (IRRAS), this plateau was due to a transition in the conformation of the PEGylated components (mushroom to brush). While the condensed phase domains had a different morphology in the ternary DPPC and DSPC monolayers on the Langmuir trough, the domain morphology was similar in the coating of both ternary DPPC and DSPC microbubbles (1.5-8 µm diameter). The ternary DPPC microbubbles had a homogenous ligand distribution and significantly less liquid condensed (LC) phase area in their coating than the DSPC-based microbubbles. For ternary DSPC microbubbles, the ligand distribution and LC phase area in the coating depended on the lipid handling. The direct method resulted in a heterogeneous ligand distribution, less LC phase area than the indirect method, and the ligand colocalizing with the liquid expanded (LE) phase area. The indirect method resulted in a homogenous ligand distribution with the largest LC phase area. In conclusion, lipid handling prior to microbubble production is of importance for a ternary mixture of DSPC, PEG40-stearate, and DSPE-PEG2000.
RESUMEN
Tween (polysorbate) 20 and 80 are surfactants used for the development of parenteral protein drugs, due to their beneficial safety profile and stabilisation properties. To elucidate the mechanism by which Tween 20 and 80 stabilise proteins in aqueous solutions, either by a "direct" protein to surfactant interaction and/or by an interaction with the protein film at the air-water interface, we used spectroscopic (Infrared Reflection Absorption Spectroscopy, IRRAS) and microscopic techniques (Brewster Angle Microscopy, BAM) in combination with surface pressure measurements. To this end, the impact of both types of Tweens with regard to the displacement of the protein from the air-water interface was studied. As a model protein, human serum albumin (HSA) was used. The results for the displacement of the adsorbed HSA films by Tweens 20 and 80 can partially be understood on the basis of an orogenic displacement mechanism, which depends on the critical surface pressure of the adsorbed protein film. With increasing concentration of Tween in the sub-phase, BAM images showed the formation of different domain morphologies. IRRA-spectra supported the finding that at high protein concentration in the sub-phase, the protein film could not be completely displaced by the surfactants. Comparing the impact of both surfactants, we found that Tween 20 adsorbed faster to the protein film than Tween 80. The adsorption kinetics of both Tweens and the speed of protein displacement increased with rising surfactant concentration. Tween 80 reached significant lower surface pressures than Tween 20, which led to an incomplete displacement of the observed HSA film.
Asunto(s)
Aire , Albúminas/química , Polisorbatos/química , Agua/química , Adsorción , Biofisica , Humanos , Iones , Ensayo de Materiales , Proteínas/química , Albúmina Sérica Humana/química , Espectrofotometría Infrarroja , Propiedades de Superficie , Tensoactivos/químicaRESUMEN
The binding of alkaline earth cations Mg2+, Ca2+, and Sr2+ (M2+) to unilamellar 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA) vesicles was analysed by pH potentiometry, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and FT-IR spectroscopy. The binding of alkaline earth cations induces deprotonation of the DMPA headgroup even at very low concentration of divalent cations (~ 100 µM). The amount of deprotonated DMPA was measured by pH potentiometry as a function of divalent cation concentration. The thermotropic phase behaviour of DMPA:M2+ complexes was studied by DSC and FT-IR as a function of pH of the dispersion (pH 7 and pH 3-5). The formation of metastable phases was observed, especially for Ca2+ and Sr2+ at pH 3-5. In unbuffered solutions, the divalent cations bind to single and/or double negatively charged DMPA, leading to the formation of different complexes and changes in the mixing behaviour of the two complexes. At pH 7, all three equimolar lipid/cation mixtures form a very stable, highly ordered 1:1 DMPA:M2+ complex. At lower divalence, the presence of a mixture of 2:1 and 1:1 complexes was observed. FT-IR spectroscopy experiments indicated an ordering of the acyl chains of DMPA after ion binding even in the liquid-crystalline phase and the induction of the dissociation of the second proton from the headgroup induced by Ca2+ or Sr2+ binding at pH 7. With ITC, the binding enthalpy ΔH of Mg2+, Ca2+, and Sr2+ to DMPA model membranes in the gel and in the liquid-crystalline phase was measured. Evidence for dehydration of hydrophobic surfaces due to cation binding was derived from changes in heat capacity.
Asunto(s)
Compuestos de Anilina/química , Membranas Artificiales , Metales Alcalinotérreos/química , Electricidad Estática , Concentración de Iones de Hidrógeno , Fosfatos/química , VibraciónRESUMEN
In the present work, we describe the synthesis and the temperature-dependent aggregation behavior of a new class of asymmetrical glycerol diether bolalipids. These bolalipids are composed of a membrane-spanning alkyl chain with 32 carbon atoms (C32) in the sn-3 position, a methyl-branched C16 alkyl chain in the sn-2 position, and a zwitterionic phosphocholine headgroup in the sn-1 position of a glycerol moiety. The long C32 alkyl chain is terminated either by a second phosphocholine (PC-Gly(2C16Me)C32-PC) or by a phosphodimethylethanolamine headgroup (PC-Gly(2C16Me)C32-Me2PE). The temperature- and pH-dependent aggregation behavior of both lipids was studied using differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS) experiments. The morphology of the formed aggregates in an aqueous suspension was visualized by transmission electron microscopy (TEM). We show that PC-Gly(2C16Me)C32-PC and PC-Gly(2C16Me)C32-Me2PE at pH 5 self-assemble into large lamellar aggregates and large lipid vesicles. Within these structures, the bolalipid molecules are probably assembled in a monolayer with fully interdigitated chains. The lipid molecules seem to be tilted with respect to the layer normal to ensure a dense packing of the alkyl chains. A temperature increase leads to a transition from a lamellar gel phase to the liquid-crystalline phase at about 28-30 °C for both bolalipids. The lamellar aggregates of PC-Gly(2C16Me)C32-Me2PE started to transform into nanofibers when the pH value of the suspension was increased to above 11. At pH 12, these nanofibers were the dominant aggregates.
RESUMEN
Hsp12 of S. cerevisiae is upregulated several 100-fold in response to stress. Our phenotypic analysis showed that this protein is important for survival of a variety of stress conditions, including high temperature. In the absence of Hsp12, we observed changes in cell morphology under stress conditions. Surprisingly, in the cell, Hsp12 exists both as a soluble cytosolic protein and associated to the plasma membrane. The in vitro analysis revealed that Hsp12, unlike all other Hsps studied so far, is completely unfolded; however, in the presence of certain lipids, it adopts a helical structure. The presence of Hsp12 does not alter the overall lipid composition of the plasma membrane but increases membrane stability.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Choque Térmico/genética , Fluidez de la Membrana , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Membrana Celular/ultraestructura , Citosol/metabolismo , Regulación Fúngica de la Expresión Génica , Genotipo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Lípidos de la Membrana/metabolismo , Presión Osmótica , Estrés Oxidativo , Fenotipo , Pliegue de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia , Estrés Fisiológico , Relación Estructura-ActividadRESUMEN
The binding of cationic peptides of the sequence (KX)4K to lipid vesicles of negatively charged dipalmitoyl-phosphatidylglycerol (DPPG) was investigated by differential scanning calorimetry (DSC) and temperature dependent Fourier-transformed infrared (FT-IR) spectroscopy. The hydrophobicity of the uncharged amino acid X was changed from G (glycine) over A (alanine), Abu (α-aminobutyric acid), V (valine) to L (leucine). The binding of the peptides caused an increase of the phase transition temperature (Tm) of DPPG by up to 20°C. The shift depended on the charge ratio and on the hydrophobicity of the amino acid X. Unexpectedly, the upward shift of Tm increased with increasing hydrophobicity of X. FT-IR spectroscopy showed a shift of the CH2 stretching vibrations of DPPG to lower frequency, particularly for bilayers in the liquid-crystalline phase, indicating an ordering of the hydrocarbon chains when the peptides were bound. Changes in the lipid C=O vibrational band indicated a dehydration of the lipid headgroup region after peptide binding. (KG)4K was bound in an unordered structure at all temperatures. All other peptides formed intermolecular antiparallel ß-sheets, when bound to gel phase DPPG. However, for (KA)4K and (KAbu)4K, the ß-sheets converted into an unordered structure above Tm. In contrast, the ß-sheet structures of (KV)4K and (KL)4K remained stable even at 80°C when bound to the liquid-crystalline phase of DPPG. Strong aggregation of DPPG vesicles occurred after peptide binding. For the aggregates, we suggest a structure, where aggregated single ß-sheets are sandwiched between opposing DPPG bilayers with a dehydrated interfacial region.
Asunto(s)
Rastreo Diferencial de Calorimetría/métodos , Membrana Dobles de Lípidos , Péptidos/metabolismo , Fosfatidilgliceroles/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Unión ProteicaRESUMEN
Differential Scanning Calorimetry (DSC) and Fourier transformed Infrared (FT-IR) spectroscopy were used to test the influence of acyl chain length, acyl chain saturation, and chemical structure of anionic phospholipids on the interaction with cationic model peptides (KX)4K, with amino acid X=A, Abu, and L. The lipids used were phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), and cardiolipin (CL). DSC was used to monitor the phase transition of lipid vesicles before and after peptide binding. The electrostatic attraction is the main driving force for binding. The hydrophobicity of the amino acid X influences the binding strength as well as the secondary structure of the bound peptide. Binding of peptides leads to an upshift of the lipid phase transition. Lipids with smaller headgroups show a larger upshift of the main phase transition temperature. Data from FT-IR spectroscopy show in addition that the stability of the bound ß-sheets of (KX)4K depends on the hydrophobicity of the uncharged amino acid X and on the size of the lipid headgroup. For lipids with large anionic headgroups, such as PS, the antiparallel ß-sheet of (KAbu)4K bound to gel phase bilayers is converted to an unordered structure upon heating through the lipid phase transition. Reducing the size of the headgroup, as in PG, increases the stability of the bound peptide ß-sheets. For the smallest headgroups, present in PA and CL, stably bound ß-sheets are observed even above the lipid phase transition.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Aniones/química , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Rastreo Diferencial de Calorimetría , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Transición de Fase , Fosfatidilgliceroles/química , Unión Proteica , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , Electricidad Estática , Temperatura de TransiciónRESUMEN
Mixtures of anionic phospholipids (PG, PA, PS, and CL) with cationic peptides were cospread from a common organic solvent at the air-water interface. The compression of the mixed film was combined with epifluorescence microscopy or infrared reflection adsorption spectroscopy (IRRAS) to gain information on the interactions of the peptide with the different lipids. To evaluate the influence of the amino acid X of peptides with the sequence (KX)4K on the binding, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) was mixed with different peptides with increasing hydrophobicity of the uncharged amino acid X. The monolayer isotherms of DPPG/(KX)4K mixtures show an increased area for the lift-off due to incorporation of the peptide into the liquid-expanded (LE) state of the lipid. The surface pressure for the transition from LE to the liquid-condensed (LC) state is slightly increased for peptides with amino acids X with moderate hydrophobicity. For the most hydrophobic peptide (KL)4K two plateaus are seen at a charge ratio PG to K of 5:1, and a strongly increased transition pressure is observed for a charge ratio of 1:1. Epifluorescence microscopy images and infrared spectroscopy show that the lower plateau corresponds to the LE-LC phase transition of the lipid. The upper plateau is connected with a squeeze-out of the peptide into the subphase. To test the influence of the lipid headgroup structure on peptide binding (KL)4K was cospread with different anionic phospholipids. The shift of the isotherm to larger areas for lift-off and to higher surface pressure for the LE-LC phase transition was observed for all tested anionic lipids. Epifluorescence microscopy reveals the formation of LC domains with extended filaments indicating a decrease in line tension due to accumulation of the peptides at the LC-domain boundaries. This effect depends on the size of the headgroup of the anionic phospholipid.
Asunto(s)
Péptidos/química , Aniones , Interacciones Hidrofóbicas e Hidrofílicas , Fosfolípidos , Propiedades de Superficie , AguaRESUMEN
In the present work, we describe the synthesis of a single-chain, phenylene-modified bolalipid with two phosphocholine headgroups, PC-C18pPhC18-PC, using a Sonogashira cross-coupling reaction as a key step. The aggregation behaviour was studied as a function of temperature using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small angle neutron scattering (SANS). We show that our new bolalipid self-assembles into nanofibres, which transform into flexible nanofibres at 27 °C and further to small elongated micelles at 45 °C. Furthermore, the miscibility of the bolalipid with bilayer-forming phosphatidylcholines (DMPC, DPPC, and DSPC) was investigated by means of DSC, TEM, FTIR, and small angle X-ray scattering (SAXS). We could show that the PC-C18pPhC18-PC is partially miscible with saturated phosphatidylcholines; however, closed lipid vesicles with an increased thermal stability were not found. Instead, bilayer fragments and disk-like aggregates are formed.
RESUMEN
Various models have been proposed for the sequence of events occurring after binding of specific antimicrobial peptides to lipid membranes. The lipid clustering model arose by the finding that antimicrobial peptides can induce a segregation of certain negatively charged lipids in lipid model membranes. Anionic lipid segregation by cationic peptides is initially an effect of charge interaction where the ratio of peptide and lipid charges is thought to be the decisive parameter in the peptide induced lipid demixing. However, the sequence of events following this initial lipid clustering is more complex and can lead to deactivation of membrane proteins involved in cell division or perturbation of lipid reorganization essential for cell division. In this study we used DSC and ITC techniques to investigate the effect of binding different cyclic hexapeptides with varying antimicrobial efficacy, to phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) lipid membranes and their ability to induce lipid segregation in these mixtures. We found that these cyclic hexapeptides consisting of three charged and three aromatic amino acids showed indeed different abilities to induce lipid demixing depending on their amino acid composition and their sequence. The results clearly showed that the cationic amino acids are essential for electrostatic binding but that the three hydrophobic amino acids in the peptides and their position in the sequence also contribute to binding affinity and to the extent of induction of lipid clustering. The efficacy of these different hexapeptides to induce PG clusters in PG/PE membranes was found to be correlated with their antimicrobial activity.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Lípidos de la Membrana/química , Oligopéptidos/química , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Calorimetría/métodos , Rastreo Diferencial de Calorimetría , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos de la Membrana/metabolismo , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceroles/química , Fosfatidilgliceroles/metabolismo , Unión Proteica , Temperatura , TermodinámicaRESUMEN
The interaction of amphiphilic and triphilic block copolymers with lipid monolayers has been studied. Amphiphilic triblock copolymer PGMA20-PPO34-PGMA20 (GP) is composed of a hydrophobic poly(propylene oxide) (PPO) middle block that is flanked by two hydrophilic poly(glycerol monomethacrylate) (PGMA) side blocks. The attachment of a perfluoro-n-nonyl residue (F9) to either end of GP yields a triphilic polymer with the sequence F9-PGMA20-PPO34-PGMA20-F9 (F-GP). The F9 chains are fluorophilic, i.e., they have a tendency to demix in hydrophilic as well as in lipophilic environments. We investigated (i) the adsorption of both polymers to differently composed lipid monolayers and (ii) the compression behavior of mixed polymer/lipid monolayers. The lipid monolayers are composed of phospholipids with PC or PE headgroups and acyl chains of different length and saturation. Both polymers interact with lipid monolayers by inserting their hydrophobic moieties (PPO, F9). The interaction is markedly enhanced in the presence of F9 chains, which act as membrane anchors. GP inserts into lipid monolayers up to a surface pressure of 30 mN/m, whereas F-GP inserts into monolayers at up to 45 mN/m, suggesting that F-GP also inserts into lipid bilayer membranes. The adsorption of both polymers to lipid monolayers with short acyl chains is favored. Upon compression, a two-step squeeze-out of F-GP occurs, with PPO blocks being released into the aqueous subphase at 28 mN/m and the F9 chains being squeezed out at 48 mN/m. GP is squeezed out in one step at 28 mN/m because of the lack of F9 anchor groups. The liquid expanded (LE) to liquid condensed (LC) phase transition of DPPC and DMPE is maintained in the presence of the polymers, indicating that the polymers can be accommodated in LE- and LC-phase monolayers. These results show how fluorinated moieties can be included in the rational design of membrane-binding polymers.
RESUMEN
A novel class of rigid-rod bolapolyphilic molecules with three philicities (rigid aromatic core, mobile aliphatic side chains, polar end groups) has recently been demonstrated to incorporate into and span lipid membranes, and to exhibit a rich variety of self-organization modes, including macroscopically ordered snowflake structures with 6-fold symmetry. In order to support a structural model and to better understand the self-organization on a molecular scale, we here report on proton and carbon-13 high-resolution magic-angle spinning solid-state NMR investigations of two different bolapolyphiles (BPs) in model membranes of two different phospholipids (DPPC, DOPC). We elucidate the changes in molecular dynamics associated with three new phase transitions detected by calorimetry in composite membranes of different composition, namely, a change in π-π-packing, the melting of lipid tails associated with the superstructure, and the dissolution and onset of free rotation of the BPs. We derive dynamic order parameters associated with different H-H and C-H bond directions of the BPs, demonstrating that the aromatic cores are well packed below the final phase transition, showing only 180° flips of the phenyl ring, and that they perform free rotations with additional oscillations of the long axis when dissolved in the fluid membrane. Our data suggests that BPs not only form ordered superstructures, but also rather homogeneously dispersed π-packed filaments within the lipid gel phase, thus reducing the corrugation of large vesicles.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Membrana Dobles de Lípidos/química , Fosfatidilcolinas/química , Espectroscopía de Resonancia Magnética/métodos , Simulación de Dinámica Molecular , Transición de FaseRESUMEN
A novel class of bolapolyphile (BP) molecules are shown to integrate into phospholipid bilayers and self-assemble into unique sixfold symmetric domains of snowflake-like dendritic shapes. The BPs comprise three philicities: a lipophilic, rigid, π-π stacking core; two flexible lipophilic side chains; and two hydrophilic, hydrogen-bonding head groups. Confocal microscopy, differential scanning calorimetry, XRD, and solid-state NMR spectroscopy confirm BP-rich domains with transmembrane-oriented BPs and three to four lipid molecules per BP. Both species remain well organized even above the main 1,2-dipalmitoyl-sn-glycero-3-phosphocholine transition. The BP molecules only dissolve in the fluid membrane above 70 °C. Structural variations of the BP demonstrate that head-group hydrogen bonding is a prerequisite for domain formation. Independent of the head group, the BPs reduce membrane corrugation. In conclusion, the BPs form nanofilaments by π stacking of aromatic cores, which reduce membrane corrugation and possibly fuse into a hexagonal network in the dendritic domains.
RESUMEN
The influence of the peptide sequence on the binding of short cationic peptides composed of five lysines alternating with uncharged amino acids within the series (KX)4K to negatively charged monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) was investigated by adsorption experiments in combination with epifluorescence microscopy. To evaluate the impact of electrostatic and hydrophobic contributions, different uncharged amino acids X with increasing hydrophobicity, where X = G (glycine), A (alanine), Abu (α-aminobutyric acid), V (valine), or L (leucine) were introduced into the peptide sequence to tune the peptide hydrophobicity. The adsorption kinetics of these peptides to a DPPG monolayer always showed two superimposed processes, one leading to an increase and another to a decrease of the surface pressure Π. Thus, the plots of the change in Π after peptide binding vs initial surface pressure of the monolayer showed an unusual behavior with maxima and negative changes in Π at high initial Π values. Epifluorescence microscopy confirmed that electrostatic binding of the peptides with a concomitant decrease in Π leads to a condensation of the lipid monolayer and the formation of liquid-condensed (LC) domains even at Π values where the monolayer is supposedly in the liquid-expanded (LE) state. An increase in hydrophobicity of the amino acid X was found to counteract the condensation and an increase in Π upon peptide binding is observed at low Π values, also concomitant with the formation of LC-domains. Compression of monolayers after peptide adsorption at low surface pressure for 4 h leads to a change of the isotherms compared to pure DPPG isotherms. The phase transition of DPPG from LE to LC state is smeared out or is shifted to higher surface pressure. Considerable changes in the shapes of LC-domains were observed after peptide binding. Growth of the LC-domains was hindered in most cases and regular domain patterns were formed. Binding of (KL)4K leads to a decrease in line tension and the formation of extended filaments protruding from initially circular domains.
Asunto(s)
Péptidos/química , Fosfatidilgliceroles/química , Adsorción , Cationes , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Fluorescente , Electricidad EstáticaRESUMEN
In the present work, we describe the synthesis and temperature-dependent aggregation behavior of two examples of a new class of highly asymmetrical glycerol diether bolaphospholipids. The bolalipids contain a long alkyl chain (C32) bound to glycerol in the sn-3 position, carrying a hydroxyl group at the ω position. The C16 alkyl chain in the sn-2 position either possesses a racemic methyl branch at the 10 position of the short alkyl chain (lipid II) or does not (lipid I). The sn-1 position of the glycerol is linked to a zwitterionic phosphocholine moiety. The temperature-dependent aggregation behavior of both bolalipids was studied using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and X-ray scattering. Aggregate structures were visualized by transmission electron microscopy (TEM). We show that both bolalipids self-assemble into large lamellar sheetlike aggregates. Closed lipid vesicles or other aggregate structures such as tubes or nanofibers, as usually found for diglycerol tetraether lipids, were not observed. Within the lamellae the bolalipid molecules are arranged in an antiparallel (interdigitated) orientation. Lipid I, without an additional methyl moiety in the short alkyl chain, shows a lamellar phase with high crystallinity up to a temperature of 34 °C, which was not observed before for other phospholipids.
Asunto(s)
Éteres de Glicerilo/química , Fosfolípidos/síntesis química , Rastreo Diferencial de Calorimetría , Microscopía Electrónica de Transmisión , Fosfolípidos/química , TemperaturaRESUMEN
Polyphilic compound B12 is an X-shaped molecule with a stiff aromatic core, flexible aliphatic side chains, and hydrophilic end groups. Forming a thermotropic triangular honeycomb phase in the bulk between 177 and 182 °C but no lyotropic phases, it is designed to fit into DPPC or DMPC lipid bilayers, in which it phase separates at room temperature, as observed in giant unilamellar vesicles (GUVs) by fluorescence microscopy. TEM investigations of bilayer aggregates support the incorporation of B12 into intact membranes. The temperature-dependent behavior of the mixed samples was followed by differential scanning calorimetry (DSC), FT-IR spectroscopy, fluorescence spectroscopy, and X-ray scattering. DSC results support in-membrane phase separation, where a reduced main transition and new B12-related transitions indicate the incorporation of lipids into the B12-rich phase. The phase separation was confirmed by X-ray scattering, where two different lamellar repeat distances are visible over a wide temperature range. Polarized ATR-FTIR and fluorescence anisotropy experiments support the transmembrane orientation of B12, and FT-IR spectra further prove a stepwise "melting" of the lipid chains. The data suggest that in the B12-rich domains the DPPC chains are still rigid and the B12 molecules interact with each other via π-π interactions. All results obtained at temperatures above 75 °C confirm the formation of a single, homogeneously mixed phase with freely mobile B12 molecules.
Asunto(s)
Membrana Dobles de Lípidos/química , Conformación Molecular , Polímeros/química , Temperatura , 1,2-Dipalmitoilfosfatidilcolina/química , Membrana Celular/química , Dimiristoilfosfatidilcolina/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos MolecularesRESUMEN
Cholesterol (Ch) linked to a linear-hyperbranched block copolymer composed of poly(ethylene glycol) (PEG) and poly(glycerol) (hbPG) was investigated for its membrane anchoring properties. Two polyether-based linear-hyperbranched block copolymers with and without a covalently attached rhodamine fluorescence label (Rho) were employed (Ch-PEG30-b-hbPG23 and Ch-PEG30-b-hbPG17-Rho). Compression isotherms of co-spread 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with the respective polymers were measured on the Langmuir trough and the morphology development of the liquid-condensed (LC) domains was studied by epi-fluorescence microscopy. LC domains were strongly deformed due to the localization of the polymers at the domain interface, indicating a line activity for both block copolymers. Simultaneously, it was observed that the presence of the fluorescence label significantly influences the domain morphology, the rhodamine labelled polymer showing higher line activity. Adsorption isotherms of the polymers to the water surface or to monolayers of DPPC and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), respectively, were collected. Again the rhodamine labelled polymer showed higher surface activity and a higher affinity for insertion into lipid monolayers, which was negligibly affected when the sub-phase was changed to aqueous sodium chloride solution or phosphate buffer. Calorimetric investigations in bulk confirmed the results found using tensiometry. Confocal laser scanning microscopy (CLSM) of giant unilamellar vesicles (GUVs) also confirmed the polymers' fast adsorption to and insertion into phospholipid membranes.
Asunto(s)
Colesterol/química , Fosfolípidos/química , Polímeros/química , Liposomas Unilamelares/química , Glicerilfosforilcolina/análogos & derivados , Ácido Láctico/química , Membrana Dobles de Lípidos/química , Microscopía Fluorescente , Fosfatidilcolinas , Poliésteres , Polietilenglicoles/química , Rodaminas/químicaRESUMEN
A major aspect determining the colloidal properties of proteins in solution is the interaction between them and with surrounding molecules. These interactions can be described by the concentration dependency of the protein diffusivity (kD), as derived by dynamic light scattering and was determined for different solutions of monoclonal antibodies varying in pH, ionic strength and presence/absence of co-solute(s). Concerning colloidal stability, protein solutions of different kD values are evaluated, based on their initial solution opalescence, to assess protein association. The current investigation shows that solution conditions with large kD values, indicating high repulsive protein-protein interactions, show lower initial opalescence, compared to solution conditions with low kD values. Upon applying stirring stress, to assess colloidal stability, the trend is such that, the higher kD values are, the more stable the protein solutions are, as long as the thermodynamic and conformational stability is not impaired. Besides, kD allows ranking of solution conditions for highly concentrated immunoglobulin solutions up to concentrations of â¼200 mg mL(-1) with regard to protein self-association and thus opalescent properties. The present study shows that the protein interaction parameter kD can be used as a surrogate parameter for a qualitative prediction of protein association and, thus, colloidal protein stability.
Asunto(s)
Anticuerpos Monoclonales/química , Química Farmacéutica/métodos , Proteínas/química , Coloides , Concentración de Iones de Hidrógeno , Luz , Concentración Osmolar , Soluciones Farmacéuticas , Estabilidad Proteica , Dispersión de Radiación , TermodinámicaRESUMEN
Lipid monolayers at the air-water interface represent half of a lipid bilayer and are therefore suitable model systems for studying the binding of peripheral proteins and polypeptides as well as proteins containing hydrophobic membrane anchors to membrane interfaces. Infrared reflection-absorption spectroscopy (IRRAS) of these monolayer films at the air-water interface provides information on the state of the lipid monolayers as well as on the conformational and orientational order of the film constituents. We will review shortly the experimental set-up and the possibilities for obtaining structural information before several applications of the method to lipid-protein monolayers will be described. We will focus on examples where the analysis of the protein and peptide bands for pure monolayers of these compounds are combined with experiments where the same compounds are bound to lipid monolayers. Combination of these experiments leads to detailed information about the conformational properties and the orientation of the molecules at the air-water interface in contrast to being bound to the lipid-water interface. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.