Thermal effects and drugs competition on the palmitate binding capacity of human serum albumin.

Human serum albumin (HSA) is the most abundant plasma protein of the circulatory system. It is a multidomain, multifunctional protein that, combining diverse affinities and wide specificity, binds, stores, and transports a variety of biological compounds, pharmacores, and fatty acids. HSA is finding increasing uses in drug-delivery due to its ability to carry functionalized ligands and prodrugs. All this raises the question of competition for binding sites occupancy in case of multiple ligands, which in turn influences the protein structure/dynamic/function relationship and also has an impact on the biomedical applications. In this work, the effects of interactive binding of palmitic acid (PA), warfarin (War) and ibuprofen (Ibu) on the thermal stability of HSA were studied using DSC, ATR-FTIR, and EPR. PA is a high-affinity physiological ligand, while the two drugs are widely used for their anticoagulant (War) and anti-inflammatory (Ibu) efficacy, and are exogenous compounds that accommodate in the deputed drug site DS1 and DS2, respectively overlapping with some of the fatty acid binding sites. The results indicate that HSA acquires the highest thermal stability when it is fully saturated with PA. The binding of this physiological ligand does not hamper the binding of War or Ibu to the native state of the protein. In addition, the three ligands bind simultaneously, suggesting a synergic cooperative influence due to allosteric effects. The increased thermal stability subsequent to binary and multiple ligands binding moderates protein aggregation propensity and restricts protein dynamics. The biophysics findings provide interesting features about protein stability, aggregation, and dynamics in interaction with multiple ligands and are relevant in drug-delivery.

Cholesterol drives enantiospecific effects of ibuprofen in biomimetic membranes.

The interaction between chiral drugs and biomimetic membranes is of interest in biophysical research and biotechnological applications. There is a belief that the membrane composition, particularly the presence of cholesterol, could play a pivotal role in determining enantiospecific effects of pharmaceuticals. Our study explores this topic focusing on the interaction of ibuprofen enantiomers (S- and R-IBP) with cholesterol-containing model membranes. The effects of S- and R-IBP at 20 mol% on bilayer mixtures of dipalmitoylphosphatidylcholine (DPPC) with 0, 10, 20 and 50 mol% cholesterol were investigated using circular dichroism and spin-label electron spin resonance. Morphological changes due to IBP enantiomers were studied with atomic force microscopy on supported cholesterol-containing DPPC monolayers. The results reveal that IBP isoforms significantly and equally interact with pure DPPC lipid assemblies. Cholesterol content, besides modifying the structure and the morphology of the membranes, triggers the drug enantioselectivity at 10 and 20 mol%, with the enantiomers differently adsorbing on membranes and perturbing them. The spectroscopic and the microscopic data indicate that IBP stereospecificity is markedly reduced at equimolar content of Chol mixed with DPPC. This study provides new insights into the role of cholesterol in modulating enantiospecific effects of IBP in lipid membranes.

Effects of curcumin in the interaction with cardiolipin-containg lipid monolayers and bilayers.

Curcumin, a plant polyphenol extracted from the Chinese herb turmeric, has gained widespread attention in recent years because of its multifunctional properties as antioxidant, antinflammatory, antimicrobial, and anticancer agent. Effects of the molecule on mitochondrial membranes properties have also been evidenced. In this work, the interaction of curcumin with models of mitochondrial membranes composed of dimyristoylphosphatidylcholine (DMPC) or mixtures of DMPC and 4 mol% tetramyristoylcardiolipin (TMCL) has been investigated by using biophysical techniques. Spectrophotometry and fluorescence allowed to determine the association constant and the binding energy of curcumin with pure DMPC and mixed DMPC/TMCL aqueous bilayers. The molecular organization of pure DMPC and cardiolipin-containing Langmuir monolayers at the air-water interface were investigated and the morphology of the monolayers transferred into mica substrates were characterized through atomic force microscopy (AFM). It is found that curcumin associates at the polar/apolar interface of the lipid bilayers and the binding is favored in the presence of cardiolipin. At 2 mol%, curcumin is well miscible with lipid monolayers, particularly with mixed DMPC/TMCL ones, where compact terraces formation characterized by a reduction of the surface roughness is observed in the AFM topographic images. At 10 mol%, curcumin perturbs the stability of DMPC monolayers and morphologically are evident terraces surrounded by cur aggregates. In the presence of TMCL, very few curcumin aggregates and larger compact terraces are observed. The overall results indicate that cardiolipin augments the incorporation of curcumin in model membranes highlighting the mutual interplay cardiolipin-curcumin in mitochondrial membranes.

The interaction of tryptophan enantiomers with model membranes is modulated by polar head type and physical state of phospholipids.

The mutual influence of chiral bioactive molecules and supramolecular assemblies is currently being studied in many research fields, including medical-pharmaceutical applications. Model membranes of phospholipids, such as the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the anionic dipalmitoylphosphatidylglycerol (DPPG), interact with a variety of chiral compounds that include amino acids. In this work, the interaction of tryptophan enantiomers, L-Trp and D-Trp, on DPPC and DPPG bilayers was investigated by using differential scanning calorimetry, attenuated total reflectance-Fourier transform infrared and spin-label electron spin resonance spectroscopies as well as molecular docking simulations. The results show that Trp enantiomers slightly perturb the bilayer thermotropic phase transitions. For both membranes, O atoms in the carbonyl groups have a propensity to act as acceptors of a (weak) hydrogen bond. The Trp chiral forms also promote formation of hydrogen bonds and/or hydration in the PO2- moiety of the phosphate group, especially for the DPPC bilayer. In contrast, they interact more closely with the glycerol group of DPPG polar head. Only for DPPC bilayers, both enantiomers increase the packing of the first hydrocarbon chain segments for temperatures through the gel state, whereas they do not affect the lipid chain order and mobility in the fluid state. The results are consistent with a Trp association in the upper region of the bilayers without permeation in the innermost hydrophobic region. The findings suggest that neutral and anionic lipid bilayers are differently sensitive to amino acid chirality.

Librational Dynamics of Spin-Labeled Membranes at Cryogenic Temperatures From Echo-Detected ED-EPR Spectra.

Methods of electron spin echo of pulse electron paramagnetic resonance (EPR) spectroscopy are increasingly employed to investigate biophysical properties of nitroxide-labeled biosystems at cryogenic temperatures. Two-pulse echo-detected ED-spectra have proven to be valuable tools to describe the librational dynamics in the low-temperature phases of both lipids and proteins in membranes. The motional parameter, α 2 τ C , given by the product of the mean-square angular amplitude, α 2 , and the rotational correlation time, τ C , of the motion, is readily determined from the nitroxide ED-spectra as well as from the W-relaxation rate curves. An independent evaluation of α 2 is obtained from the motionally averaged 14N-hyperfine splitting separation in the continuous wave cw-EPR spectra. Finally, the rotational correlation time τ C can be estimated by combining ED- and cw-EPR data. In this mini-review, results on the librational dynamics in model and natural membranes are illustrated.

Low-temperature librations and dynamical transition in proteins at differing hydration levels.

Hydration of water affects the dynamics and in turn the activity of biomacromolecules. We investigated the dependence of the librational oscillations and the dynamical transition on the hydrating conditions of two globular proteins with different structure and size, namely ß-lactoglobulin (ßLG) and human serum albumin (HSA), by spin-label electron paramagnetic resonance (EPR) in the temperature range of 120-270 K. The proteins were spin-labeled with 5-maleimide spin-label on free cysteins and prepared in the lyophilized state, at low (h = 0.12) and full (h = 2) hydration levels in buffer. The angular amplitudes of librations are small and almost temperature independent for both lyophilized proteins. Therefore, in these samples, the librational dynamics is restricted and the dynamical transition is absent. In the small and compact beta-structured ßLG, the angular librational amplitudes increase with temperature and hydrating conditions, whereas hydration-independent librational oscillations whose amplitudes rise with temperature are recorded in the large and flexible alpha-structured HSA. Both ßLG and HSA at low and fully hydration levels undergo the dynamical transition at about 230 K. The overall results indicate that protein librational dynamics is activated at the low hydration level h = 0.12 and highlight biophysical properties that are common to other biosamples at cryogenic temperatures.

Interactive multiple binding of oleic acid, warfarin and ibuprofen with human serum albumin revealed by thermal and fluorescence studies.

Human serum albumin binds a wide variety of drugs with different structure and affinity to two main binding sites, drug site 1 (DS1) and drug site 2 (DS2), which partially or totally overlap with fatty acid (FA) sites. Although multiple binding sites are available for endogenous compounds, FAs are the primary physiological ligands of albumin and their competition in the occupancy of DS1 and DS2 affects the binding of exogenous molecules, with a possible impact on drug delivery. In this work, we have investigated the simultaneous binding of oleic acid, warfarin and ibuprofen to albumin using differential scanning calorimetry and fluorescence to evaluate the impact on the conformational stability of the protein. The two drugs are widely used for their anticoagulant (warfarin) and anti-inflammatory (ibuprofen) properties, and can be also considered as site markers to probe DS1 and DS2, respectively. Oleic acid is one of the most important fatty acids from a physiological point of view for its role as a source of energy for cells, and also it binds albumin with the highest association constant. When complexed with oleic acid the calorimetric profile of albumin shows a biphasic trend whose line shape depends on the ligand concentration. The binding capacity of either warfarin or ibuprofen to albumin is modulated by oleate molecules in a concentration-dependent mode being synergic cooperative (warfarin) or competitive-like (ibuprofen). The overall results provide insights on the dynamics of albumin/ligands complex, which in turn may have important pharmacokinetic and pharmacodynamic implications.

Influence of hydration on segmental chain librations and dynamical transition in lipid bilayers.

Continuous wave electron paramagnetic resonance spectroscopy of chain-labeled phospholipids is used to investigate the effects of hydration on the librational oscillations and the dynamical transition of phospholipid membranes in the low-temperature range 120-270 K. Bilayers of dipalmitoylphostatidiycholine (DPPC) spin-labeled at the first acyl chain segments and at the methyl ends and prepared at full, low, and very low hydration are considered. The segmental mean-square angular amplitudes of librations, 〈α2〉, are larger in the bilayer interior than at the polar/apolar interface and larger in the fully and low hydrated than in the very low hydrated membranes. For chain segments at the beginning of the hydrocarbon region, 〈α2〉-values are markedly restricted and temperature independent in DPPC with the lowest water content, whereas they increase with temperature in the low and fully hydrated bilayers, particularly at the highest temperatures. For chain segments at the chain termini, the librational amplitudes increase progressively, first slowly and then more rapidly with temperature in bilayers at any level of hydration. From the temperature dependence of the mean-square librational amplitude, the dynamical transition is detected around 240 K at the polar/apolar interface in fully and low hydrated DPPC and at around 225 K at the inner hydrocarbon region for bilayers at any hydration condition. At the dynamical transition the bilayers cross low energy barriers of activation energy in the range 10-20 kJ/mol. The results highlight biophysical properties of DPPC bilayers at low-temperature and provide evidence of the effects of the hydration on the dynamical transition in bilayers.

Geometry and water accessibility of the inhibitor binding site of Na+-pump: Pulse- and CW-EPR study.

Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D2O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.

Cryogenically frozen PEGylated liposomes and micelles: Water penetration and polarity profiles.

Poly(ethylene glycol) (PEG)-grafted lipid dispersions are widely investigated in fundamental and biotechnological research for their successful use in drug-delivery. Here, we consider mixtures of the bilayer-forming lipid dipalmitoylphosphatidylcholine (DPPC) with the micelle-forming lipid PEG:2000-phosphatidilethanolamine (PEG:2000-DPPE) fully hydrated in D2O and measured at 77 K. Electron Spin Echo Envelope Modulation and continuous wave Electron Paramagnetic Resonance of chain-labelled lipids are employed to detect the extent of solvent permeation and the environmental polarity, respectively, across the hydrocarbon regions of the lipid assemblies. Sigmoidal water penetration and polarity profiles are described in sterically stabilized liposomes (SSL) formed at submicellar content of PEG:2000-DPPE incorporated in DPPC. Compared to DPPC bilayers, SSL show increased hydrophobicity at both the polar/apolar interface and the chain termini, and a broader transition that is shifted toward the interface. Solvent exposure and polarity decrease on going down the chain in PEG:2000-DPPE micelles. However, compared to SSL, polymer-lipid micelles show higher solvent permeation at any chain segment and the chain termini are accessible to water. In any sample, heterogeneity is found in H-bond formation between the spin-label nitroxide groups and the solvent molecules. The results at cryogenic temperature add new insights into the biophysico-chemical characterization of PEGylated lipid dispersions.

Binding of warfarin differently affects the thermal behavior and chain packing of anionic, zwitterionic and cationic lipid membranes.

Warfarin is a coumarin derivative drug widely used for its anticoagulant properties. The interaction of warfarin with fully hydrated lipid bilayers has been studied by combining differential scanning calorimetry, spectrophotometry, electron spin resonance of chain-labelled lipids and molecular docking. Bilayers formed by lipids with different chemico-physical properties were considered, namely dimyristoyl-phosphatidylcholine (DMPC), dimyristoyl-phosphatidylglycerol (DMPG), and dioleoyltrimethyl-ammoniumpropane (DOTAP). We observed in all cases the binding of warfarin in proximity of the surface of the bilayers, leading to a variety of distinct effects on key molecular properties of the membranes. The drug associates with the lipid bilayers in the deprotonated open chain form, with an association constant similar for DMPC and DMPG (1.27·104 and 2.82·104 M-1, respectively) and lower for DOTAP (0.46·104 M-1). In DMPC bilayers, which are zwitterionic and with saturated symmetrical chains, warfarin at 10 mol% suppresses the pre-transition, slightly stabilizes the fluid state and reduces the cooperativity of the main transition. Moreover, it alters the lateral packing density of the chain segments close to the polar/apolar interface at any temperature through the gel phase. In anionic DMPG bilayers, the drug slightly perturbs the thermotropic phase behavior, and at 10 mol% markedly loosens the compact gel phase packing of the first chain segments. In cationic DOTAP bilayers, possessing unsaturated acyl chains, the drug induces a slightly higher degree of order and motional restriction in the outer hydrocarbon region in the frozen state. In all cases, as a surface adsorbed molecule, warfarin does not affect the segmental chain order and dynamics for temperatures in the fluid phase. The overall results provide an outline of the action of warfarin on membranes formed by lipids of different types.

Effects of Polar Head Nature and Tail Length of Single-Chain Lipids on the Conformational Stability of ß-Lactoglobulin.

Interaction between ß-lactoglobulin and single-chain lipids, differing for either the length of the aliphatic chain or the molecular properties of the headgroup, was investigated at neutral and acidic pH to determine the impact on the thermal stability of the protein. Differential scanning calorimetry results with different fatty acids (from C10:0 to C18:0) show a correlation of both melting temperature and unfolding enthalpy of the protein with the ligand binding affinity, and the maximum effect was found for palmitic acid (PLM). The influence of the lipid polar head was investigated by comparing PLM with lyso-palmitoylphosphatidylcholine (LPC), which possesses the same aliphatic chain. At neutral pH, the stabilizing effect of LPC is less favorable compared to PLM. However, fluorescence results revealed that LPC can bind into the protein calyx even at acidic pH, at variance with fatty acids. Molecular dynamics simulations indicated that this difference is due to the ability of the polar head of LPC to interact with the protein loop that regulates the shift (Tanford transition) between open and closed state of the binding site of ß-lactoglobulin. The results provide a rationale for how a ligand has the ability to access the protein active site at acidic conditions by overcoming the Tanford transition, and they demonstrate that ß-lactoglobulin can deliver ligands with tailored properties of the polar head in a wide pH range.

Warfarin increases thermal resistance of albumin through stabilization of the protein lobe that includes its binding site.

Protein-drug interaction is of prominent interest in determining the pharmacokinetic and pharmacodynamic consequences on drug delivery. Warfarin is a widely used anticoagulant drug in the treatment of venous thrombosis and pulmonary embolism and is carried in the blood almost exclusively by human serum albumin. The effects of the binding of warfarin to the native state of albumin were characterized by UV-vis absorption, conventional and synchronous fluorescence, isothermal titration calorimetry, differential scanning calorimetry and molecular dynamics simulation. The overall results indicate that, under physiological condition, the binding of warfarin in site DS1 of albumin promotes local stabilization with resulting effects on the global protein dynamics. The increase of the protein stability has both an enthalpic and entropic character. Under denaturing condition, the stabilizing effect of warfarin is evidenced by an increase of both the melting temperature and unfolding enthalpy of albumin with the drug/protein molar ratio. More importantly, thermal resistance is increased due to selective effect on the specific protein lobe that includes the main drug binding site. The comparison of the thermal behavior of the protein-warfarin complex with that in the presence of a typical ligand of the other main protein binding site, i.e. drug site DS2, provides key insight on domain-specific stabilization effects on albumin.

Unsaturated lipid bilayers at cryogenic temperature: librational dynamics of chain-labeled lipids from pulsed and CW-EPR.

Fully hydrated bilayers of monounsaturated palmitoyloleoylphosphatidylcholine (POPC) and diunsaturated dioleoylphosphatidylcholine (DOPC) lipids have low main phase transition temperatures (271 K for POPC and 253 K for DOPC). Two-pulse echo detected spectra, combined with continuous wave electron paramagnetic resonance spectroscopy, are employed to study the low-temperature lamellar phases of the POPC and DOPC unsaturated bilayers that are usually studied in the fluid state. Phosphatidylcholine spin-labeled at C-5 and C-16 carbon atom positions along the acyl chain were used and the temperature varied over the range 77-270 K. Segmental chain librational oscillations of small amplitude and with correlation time in the subnanosecond to nanosecond range are found in both membranes. The mean-square angular amplitude, α2, of librations increases with temperature, is larger close to the bilayer midplane than close to the first acyl chain segments, and is larger in diunsaturated than in monounsaturated bilayers. In the inner hydrocarbon region of both lipid matrices, α2 increases first slowly and linearly with temperature and then more rapidly, and a dynamical transition is detected in the range 190-210 K. Compared to dipalmitoylphosphatidylcholine bilayers of fully saturated symmetric chain lipids, the presence of double bonds in the acyl chain enhances the intensity of librational motion which is characterized by larger angular variations at the terminal methyl ends. These findings highlight biophysical properties of unsaturated bilayers in the frozen state, including a detailed characterization of segmental chain dynamics and the evidence of a dynamical transition that appears to be a generic feature in hydrated macromolecular systems. These results can also be relevant in regulating membrane physical properties and function at higher physiological temperatures.

Interdigitated lamellar phases in the frozen state: Spin-label CW- and FT-EPR.

Interdigitated lamellar phases composed of dipalmitoylphosphatidylcholine (DPPC) and equimolar content of lyso-palmitoylphosphatidylcholine (Lyso-PPC) or DPPC hydrated in ethanol containing water (60% v/v) have been studied in the frozen state. Electron paramagnetic resonance spectra of labeled lipids at C5 or C16 carbon atom positions along the chain are indicative of segmental librational motion over the temperature range 120-260 K. For any dispersion, the mean-square-angular amplitudes of the librations are comparable for both label positions but are larger in DPPC/etOH than in DPPC/Lyso-PPC interdigitated sample. The temperature dependences of the librational amplitudes of the labels in the lipid matrices show a rapid increase at the dynamical transition at Td ≈ 220 K with an activation energy of 20-30 kJ/mol. Three-pulse electron spin echo envelope modulation by D2O revealed comparable solvent accessibility and fractions of singly and doubly hydrogen-bonded nitroxides to deuterons for both positional isomers in the interdigitated lamellae at 77 K. The overall EPR results indicate that the interdigitated DPPC/etOH sample is more loosened packed compared to DPPC/Lyso-PPC sample. The findings of the present work obtained at cryogenic temperatures point out dynamic and molecular properties of interdigitated lamellae that contribute to the biophysical characterization of membrane model systems.

Solvent accessibility in interdigitated and micellar phases formed by DPPC/Lyso-PPC mixtures: D2O-ESEEM of chain labeled lipids.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to investigate binary mixtures of single-chain micelle-forming lipids and diacyl bilayer-forming lipids dispersed in D2O at 77 K. Mixtures of dipalmitoylphosphatidylcholine (DPPC) and lyso-palmitoylphosphatidylcholine (Lyso-PPC) over the entire composition range (0-100 mol%) and phosphatidylcholine spin-labeled at selected carbon atom position along the sn-2 chain (n-PCSL) were considered. On increasing the content of the lysolipids incorporated in DPPC, the lipid bilayers are first transformed in interdigitated lamellae and then converted in micelles of Lyso-PPC. In the interdigitated phase, the profile of translamellae water accessibility is rather uniform as all the hydrocarbon segments are equally exposed to the solvent. In Lyso-PPC micelle, water penetrates at any depth of the hydrocarbon region with a tendency to increase toward the chain termini. The extent of water penetration is higher in the interdigitated DPPC/Lyso-PPC dispersions than in Lyso-PPC micelles. The profiles of water permeation revealed directly by D2O-ESEEM are also confirmed by more indirect evaluation of the polarity profiles based on the 14N-hyperfine splitting in the conventional electron paramagnetic resonance spectra of n-PCSL in frozen DPPC/Lyso-PPC mixtures at 77 K. The ESEEM data reveal that H-bonding formation between the -NO group of the spin-label and the D2O molecules is favored in the intergitated phase with respect to the micellar phase and, in any lipid dispersion, the fraction of nitroxides that are singly H-bonded to deuterons is higher than the fraction that are doubly H-bonded. The overall results highlight the differences in the accessibility and properties of the solvent in the hydrocarbon region of lipid bilayers, interdigitated bilayers and micelles.

Association of ibuprofen at the polar/apolar interface of lipid membranes.

Ibuprofen is a non-steroidal anti-inflammatory drug widely used to treat inflammatory diseases, and for its analgesic and antipyretic activity. Although operating as a protein inhibitor, it is also known to interact with lipid membranes. We combined calorimetry, electron spin resonance, attenuated total reflectance-Fourier transform infrared and molecular docking to characterize the interaction of ibuprofen with dimyristyolphosphatidylcholine (DMPC) bilayers, as a function of temperature and drug concentration. At increasing concentration, ibuprofen first perturbs and then suppresses the DMPC pre-transition, stabilizes the fluid state, and favours gel-fluid phase coexistence. The drug decreases the molecular packing of the polar heads and of the first methylene segments of lipid membranes in the gel phase, whereas it leaves unperturbed the chain flexibility in the liquid-crystalline phase. The action of ibuprofen also leads to a higher degree of hydration of the bilayer polar heads and favours hydrogen bond formation with solvent molecules. The overall results reveal that ibuprofen affects a number of key molecular properties of DMPC bilayers by binding through non-specific interactions at the polar/apolar interface.

Ether-linked lipids: Spin-label EPR and spin echoes.

Electron spin echo envelope modulation (ESEEM) and conventional electron paramagnetic resonance (EPR) of site-specifically spin-labelled phospholipids are used to investigate the effect of ether-linked chains on the water-penetration and polarity profiles, as well as the phase behaviour and chain flexibility profiles, of phospholipid membranes. D2O-ESEEM reveals that water exposure of the terminal methyl groups in the interdigitated phase of dihexadecyl phosphatidylcholine (DHPC) is comparable to that of the methylene groups at the polar head-group end of the chains. Similarly, an uniform transmembrane polarity profile is obtained from the dependence of the outer 14N-hyperfine splitting on the spin-label position along the chain in frozen interdigitated DHPC dispersions. Two-component conventional EPR spectra of spin labels at the terminal methyl end of the chain reveal that the intermediate gel phase above the pretransition of DHPC contains components in which the lipid chains are interdigitated. The polarity and chain-flexibility profiles in the fluid Lα-phase of DHPC with ether-linked chains are shifted outwards, towards the polar-apolar interface, as compared with that of dihexadecanoyl phosphatidylcholine (DPPC) with ester-linked chains. Also, the polarity profile of DHPC is shifted upwards, to higher polarities. These differences reflect those in hydrocarbon thickness and area/lipid molecule reported by x-ray diffraction for the Lα-phases of the two lipids.

Low-Temperature Dynamics of Chain-Labeled Lipids in Ester- and Ether-Linked Phosphatidylcholine Membranes.

Continuous wave electron paramagnetic resonance spectroscopy and two-pulse echo detected spectra of chain-labeled lipids are used to study the dynamics of frozen lipid membranes over the temperature range 77-260 K. Bilayers of ester-linked dihexadecanoylphosphatidylcholine (DPPC) with noninterdigitated chains and ether-linked dihexadecyl phosphatidylcholine (DHPC) with interdigitated chains are considered. Rapid stochastic librations of small angular amplitude are found in both lipid matrices. In noninterdigitated DPPC bilayers, the mean-square angular amplitude, [Formula: see text], of the motion increases with temperature and it is larger close to the chain termini than close to the polar/apolar interface. In contrast, in interdigitated DHPC lamellae, [Formula: see text] is small and temperature and label-position independent at low temperature and increases steeply at high temperature. The rotational correlation time, τc, of librations lies in the subnanosecond range for DPPC and in the nanosecond range for DHPC. In all membrane samples, the temperature dependence of [Formula: see text] resembles that of the mean-square atomic displacement revealed by neutron scattering and a dynamical transition is detected in the range 210-240 K. The results highlight the librational oscillations and the glass-like behavior in bilayer and interdigitated lipid membranes.

Resveratrol induces chain interdigitation in DPPC cell membrane model systems.

Resveratrol is a natural polyphenol found in various plants with potential therapeutic activity as anti-oxidant, anti-inflammatory, cardioprotective and anti-tumoral. Lipid membranes are among cellular components that are targets of its action. In this work ESR of chain labeled lipids, calorimetry, X-ray diffraction and molecular docking are used to study the interaction of resveratrol with membrane model systems of dipalmitoylphosphatidylcholine (DPPC) as a function of resveratrol concentration (0-30 mol% of the lipid) and temperature (10-50°C). Resveratrol incorporated in DPPC bilayers induces considerable motional restriction at the lipid tail termini, removing the gradient of increasing mobility along the chain found in DPPC bilayers in the gel phase. In contrast, it leaves unperturbed the DPPC chain flexibility profile in the liquid-crystalline phase. At low concentration, resveratrol progressively reduces the pre-transition temperature and eliminates the pre-transition for content ≥5mol%. A reduced cooperativity and a downshift of the main transition temperature are observed, especially at high content. The typical diffraction pattern of DPPC multibilayers in the Lß' phase is converted to a lamellar pattern with reduced d-spacing of untilted lipid chain in a hexagonal packing at 30 mol% of resveratrol. Molecular docking indicates that the energetically favoured anchoring site is the polar headgroup region, where resveratrol acts as a spacer. The overall results are consistent with the formation in DPPC of an interdigitated Lßi gel phase induced by 30 mol% resveratrol.