dimyristoyl-L-α-phosphatidylcholine Research Articles

Competitive binding of nile red between lipids and β-cyclodextrin

Fluorometric measurements are exploited to explore the binding interactions of nile red (NR) with anionic lipid dimyristoyl-L-α-phosphatidylglycerol (DMPG), zwitterionic lipid dimyristoyl-L-α-phosphatidylcholine (DMPC) as well as neutral cyclic oligosaccharide β-cyclodextrin (β-CD) solutions. The binding constants are found to be quite high and comparable (within a factor of five). Series of spectral techniques like steady state fluorescence and fluorescence anisotropy study, micropolarity study, quenching study and time resolved experiments reveal that the addition of β-CD to the probe–lipid complexes results in weakening of the lipid–probe interaction and formation of probe–β-CD inclusion complexes leading to the removal of some of the probe (NR) molecules from the lipid environments. The extent of removal of NR is, however, more from DMPG than DMPC lipid. The phenomena are explained from the concept of competitive binding of the probe between the lipids and β-CD. Since lipids are the principal constituents of the cell walls, the work might make a foundation for the possible removal of excess of molecules like nile red adsorbed on the cell walls.

2,2′-Bipyridyl]-3,3′-diol in lipid vesicles: slowed down dynamics of proton transfer

(2,2′-Bipyridyl)-3-3′-diol [BP(OH)2] is a well-known compound that undergoes double proton transfer (DPT) that has been previously described as an intramolecular and excited state event in neat solvents and certain constrained environments. Herein, we have explored the interaction of BP(OH)2 with small unilamellar vesicles synthesized using two kinds of lipids, dimyristoyl-L-α-phosphatidylcholine (DMPC) and dimyristoyl-L-α-phosphatidylglycerol (DMPG), which are zwitterionic and anionic, respectively. BP(OH)2 is observed to undergo the formation of a monoketo and a diketo species in sequence along with the creation of dianions aided by the lipid membranes. Interestingly, in the present work, we could witness the formation of the dianionic species under neutral conditions in the presence of lipid vesicles. Moreover, the dynamics of the monoketo species could also be observed as the lipid vesicles supposedly slowed down the overall rate of proton transfer in BP(OH)2. In spite of monitoring in different emitting regions of the spectrum of BP(OH)2 in liposomes, no growth component was observed in the time-resolved fluorescence data. This indicates that in the presence of lipid vesicles all the subsequent species from the parent neutral probe develop in the ground electronic state.

Interaction between Soluble and Membrane-Embedded Potassium Channel Peptides Monitored by Fourier Transform Infrared Spectroscopy

Recent studies have explored the utility of Fourier transform infrared spectroscopy (FTIR) in dynamic monitoring of soluble protein-protein interactions. Here, we investigated the applicability of FTIR to detect interaction between synthetic soluble and phospholipid-embedded peptides corresponding to, respectively, a voltage-gated potassium (Kv) channel inactivation domain (ID) and S4–S6 of the Shaker Kv channel (KV1; including the S4–S5 linker “pre-inactivation” ID binding site). KV1 was predominantly α-helical at 30°C when incorporated into dimyristoyl-l-α-phosphatidylcholine (DMPC) bilayers. Cooling to induce a shift in DMPC from liquid crystalline to gel phase reversibly decreased KV1 helicity, and was previously shown to partially extrude a synthetic S4 peptide. While no interaction was detected in liquid crystalline DMPC, upon cooling to induce the DMPC gel phase a reversible amide I peak (1633 cm−1) consistent with novel hydrogen bond formation was detected. This spectral shift was not observed for KV1 in the absence of ID (or vice versa), nor when the non-inactivating mutant V7E ID was applied to KV1 under similar conditions. Alteration of salt or redox conditions affected KV1-ID hydrogen bonding in a manner suggesting electrostatic KV1-ID interaction favored by a hairpin conformation for the ID and requiring extrusion of one or more KV1 domains from DMPC, consistent with ID binding to S4–S5. These findings support the utility of FTIR in detecting reversible interactions between soluble and membrane-embedded proteins, with lipid state-sensitivity of the conformation of the latter facilitating control of the interaction.

Differential interactions of a biological photosensitizer with liposome membranes having varying surface charges

The present work demonstrates the interaction of promising cancer cell photosensitizer, harmane (HM), with liposome membranes of varying surface charges, dimyristoyl-l-α-phosphatidylcholine (DMPC) and dimyristoyl-l-α-phosphatidylglycerol (DMPG). Electrostatic interaction of the cationic probe (HM) with the surface charges of the lipids is responsible for differential modulation of the spectral properties of the drug in different lipid environments. Estimation of partition coefficient (K(p) (±10%) = 5.58 × 10(4) in DMPC and 3.28 × 10(5) in DMPG) of HM between aqueous buffer and lipid phases reflect strong binding interaction of the drug with both the lipids. Evidence for greater degree of partitioning of HM into DMPG membrane compared to DMPC membrane has been deduced and further substantiated from experimental studies such as steady-state fluorescence anisotropy, micropolarity determination. The molecular modeling investigation by docking simulation coupled with fluorescence quenching experiment has been exploited to substantiate the location of drug at the lipid head-group region. Modulation of the dynamical properties of the drug within the lipid environments has also been addressed. Rotational relaxation dynamics studies unravel the impartation of a significant degree of motional restriction on the probe molecule within the lipids and reinforce the differential interactions of HM with the two lipid systems along the lines of other findings. Fluorescence kinetics studies reveal a faster association (in terms of apparent rate constants describing the process of interaction) of the drug with DMPG membrane compared to DMPC. This result is argued in connection with the electrostatic interaction between the drug and the liposome surface charges.

Spectroscopic probing of location and dynamics of an environment-sensitive intramolecular charge transfer probe within liposome membranes

The present work demonstrates the interaction of an intramolecular charge transfer (ICT) probe 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid methyl ester (DPDAME) with liposome membranes of dimyristoyl-l-α-phosphatidylcholine (DMPC) and dimyristoyl-l-α-phosphatidylglycerol (DMPG) studied by steady-state absorption, emission and time-resolved emission techniques. A huge hypsochromic shift together with remarkable enhancement of fluorescence quantum yield of the polarity sensitive ICT emission of DPDAME upon interaction with the lipids has been rationalized in terms of incorporation of the probe into hydrophobic interior of the lipids. Compelling evidences for penetration of the probe into the hydrocarbon interior of the lipids have been deduced from intertwining different experimental results e.g., micropolarity in the immediate vicinity of the probe in lipid environments, steady-state anisotropy, red-edge excitation shift (REES), fluorescence quenching experiments and time-resolved measurements. The rotational relaxation dynamics study of the membrane-bound probe unveils the impartation of high degree of motional rigidity. Wavelength-selective emission behaviour paves way for monitoring of solvent-relaxation in the membranes. Overall, the ICT probe DPDAME displays its commendable sensitivity in deciphering the microheterogeneous environments of liposomal membranes of DMPC and DMPG and promises a new membrane-polarity sensitizing probe.

Modulated Photophysics of an ESIPT Probe 1-Hydroxy-2-naphthaldehyde within Motionally Restricted Environments of Liposome Membranes Having Varying Surface Charges

The present work demonstrates the modulation of excited state intramolecular proton transfer (ESIPT) emission of 1-hydroxy-2-naphthaldehyde (HN12) upon its interaction with the liposomal vesicles/bilayer of dimyristoyl-l-α-phosphatidylcholine (DMPC) and dimyristoyl-l-α-phosphatidylglycerol (DMPG) and its subsequent implementation as an efficient molecular reporter for probing of microheterogeneous environments of lipid-bilayer system. Modifications on the emission profile of HN12 in terms of remarkable emission intensity enhancement coupled with a hypsochromic shift induced by the presence of DMPC and DMPG membranes have been interpreted to be vivid manifestations of the interactions between the two partners. Steady-state anisotropy, red-edge excitation shift (REES), and time-resolved fluorescence measurements have been fruitfully exploited to complement other experimental findings. Probable binding site of HN12 in the lipid-bilayers has been assessed on the basis of intertwining the results of fluorescence quenching with other experimental results and is further substantiated from docking studies.

Quantification of Randomly-methylated-β-cyclodextrin effect on liposome: An ESR study

In the present work, the effect of Randomly-methylated-β-cyclodextrin (Rameb) on the microviscosity of dimyristoyl- l-α phosphatidylcholine (DMPC) bilayer was investigated using the electron spin resonance (ESR) technique. The ability of Rameb to extract membrane cholesterol was demonstrated. For the first time, the percentage of cholesterol extracted by Rameb from cholesterol doped DMPC bilayer was monitored and quantified throughout a wide Rameb concentration range. The effect of cholesterol on the inner part of the membrane was also investigated using 16-doxyl stearic acid spin label (16-DSA). 16-DSA seems to explore two different membrane domains and report their respective microviscosities. ESR experiments also establish that the presence of 30% of cholesterol in DMPC liposomes suppresses the jump in membrane fluidity at lipids phase-transition temperature (23.9 °C).

In vitro characterization of vinculin's lipid membrane-interacting domain, helix 3

The focal adhesion (FA) protein vinculin with its 95 kDa head domain (residues 1-858) and 30 kDa tail domain (residues 858-1066) plays an important role in cell migration and adhesion. Binding of vinculin to lipid membranes ensures these processes. Three potential membrane interaction sites (residues 940-970, 1020-1040 and 1052-1066) have been reported on the tail domain. In pull-down assays using artificial lipid membranes it was demonstrated that, when helix 3 (residues 944-972) was mutated on position K952, K956, R963, R966 to Q, its interaction with acidic phospholipid vesicles was impaired. To date, no data exist on the nature of the interaction.Using differential scanning calorimetry on helix 3 we could show that it inserts into lipid vesicles consisting of dimyristoyl-L-α-phosphatidylcholine (DMPC) and negatively-charged dimyristoyl-L-α-phosphatidylserine (DMPS). However, when mutating the four basic residues (K952, K956, R963, R966 to Q) on helix 3, the insertion into lipid vesicles was reduced. Examining the secondary structure of the non-mutated helix 3 in the presence and absence of DMPC/DMPS lipid vesicles by CD-spectroscopy showed a conformational shift. These observations indicate that the electrostatic interactions of the basic residues on helix 3 induce the insertion into the hydrophobic core and promote the localization of the vinculin molecule to lipid membranes.

Photophysics of a β-carboline based non-ionic probe in anionic and zwitterionic liposome membranes

Interaction of a biologically active β-carboline based non-ionic probe, 3-acetyl-4-oxo-6,7-dihydro-12 H indolo-[2,3- a] quinolizine (AODIQ), with the liposomal vesicles of dimyristoyl- l-α-phosphatidylcholine (DMPC) and dimyristoyl- l-α-phosphatidylglycerol (DMPG) has been demonstrated using steady-state and time-resolved fluorescence and fluorescence anisotropy techniques. Polarity sensitive intramolecular charge transfer of AODIQ shows a large hypsochromic shift along with an enhancement in the fluorescence quantum yield and fluorescence lifetime in the bilayer membranes compared to those in aqueous buffer solution. Polarity of the immediate vicinity of the probe in the lipid environments has been determined. The fluorometric, quenching and micropolarity determination studies reveal that the fluorophore penetrates deeper in the zwitterionic DMPC membrane compared to the anionic DMPG vesicle. Enhancement in the rotational relaxation time of AODIQ in liposomal membranes suggests that the fluorophore exists in motionally restricted environments.

Secondary Structure of the MiRP1 (KCNE2) Potassium Channel Ancillary Subunit

MiRP1 (encoded by the KCNE2 gene) is one of a family of five single transmembrane domain voltage-gated potassium (Kv) channel ancillary subunits currently under intense scrutiny to establish their position in channel complexes and elucidate alpha subunit contact points, but its structure is unknown. MiRP1 mutations are associated with inherited and acquired cardiac arrhythmia. Here, synthetic peptides corresponding to human MiRP1 (full-length and separate domains) were structurally analyzed using FTIR and CD spectroscopy. The N-terminal (extracellular) domain was soluble and predominantly non-ordered in aqueous media, but predominantly alpha-helical in L-alpha-lysophosphatidylcholine (LPC) micelles. The MiRP1 transmembrane domain was predominantly a mixture of alpha-helix and non-ordered structure in LPC micelles, with a minor contribution from non-aggregated beta-strand. The intracellular C-terminal domain was insoluble in aqueous solution; reconstitution into non-aqueous environments resulted in solubility and adoption of increasing amounts of alpha-helix, with the solvent order sodium dodecyl sulphate < dimyristoyl L-alpha-phosphatidylcholine (DMPC) < LPC < trifluoroethanol. Correlation of secondary structure changes with lipid transition temperature during heating suggested that the MiRP1 C-terminus incorporates into DMPC bilayers. Full-length MiRP1 was soluble in SDS micelles and calculated to contain 34% alpha-helix, 23% beta-strand and 43% non-ordered structure in this environment, as determined by CD spectroscopy. Thus, MiRP1 is highly dependent upon hydrophobic interaction via lipid and/or protein contacts for adoption of ordered structure without nonspecific aggregation, consistent with a role as a membrane-spanning subunit within Kv channel complexes. These data will provide a structural framework for ongoing mutagenesis-based in situ structure-function studies of MiRP1 and its relatives.

Does propofol alter membrane fluidity at clinically relevant concentrations? An ESR spin label study

General anesthetics have been shown to perturb the membrane properties of excitable tissues. Due to their lipid solubility, anesthetics dissolve in every membrane, penetrate into organelles and interact with numerous cellular structures in multiple ways. Several studies indicate that anesthetics alter membrane fluidity and decrease the phase-transition temperature. However, the required concentrations to induce such effects on the properties of membrane lipids are by far higher than clinically relevant concentrations. In the present study, the fluidizing effect of the anesthetic agent propofol (2,6-diisopropyl phenol: PPF), a general anesthetic extensively used in clinical practice, has been investigated on liposome dimyristoyl- l-α phosphatidylcholine (DMPC) and cell (erythrocyte, Neuro-2a) membranes using electron spin resonance spectroscopy (ESR) of nitroxide labeled fatty acid probes (5-, 16-doxyl stearic acid). A clear effect of PPF at concentrations higher than the clinically relevant ones was quantified both in liposome and cell membranes, while no evident fluidity effect was measured at the clinical PPF doses. However, absorption spectroscopy of merocyanine 540 (MC540) clearly indicates a PPF fluidizing capacity in liposome membrane even at these clinical concentrations. PPF may locally influence the structure and dynamics of membrane domains, through the formation of small-scale lipid domains, which would explain the lack of ESR information at low PPF concentrations.

Quantification of lipid bilayer effective microviscosity and fluidity effect induced by propofol

Electron spin resonance (ESR) spectroscopy with nitroxide spin probes was used as a method to probe the liposome microenvironments. The effective microviscosities have been determined from the calibration of the ESR spectra of the probes in solvent mixtures of known viscosities. In the first time, by measuring ESR order parameter ( S) and correlation time ( τ c) of stearic spin probes, we have been able to quantify the value of effective microviscosity at different depths inside the liposome membrane. At room temperature, local microviscosities measured in dimyristoyl- l-α phosphatidylcholine (DMPC) liposome membrane at the different depths of 7.8, 16.95, and 27.7 Å were 222.53, 64.09, and 62.56 cP, respectively. In the gel state (10 °C), those microviscosity values increased to 472.56, 370.61, and 243.37 cP. In a second time, we have applied this technique to determine the modifications in membrane microviscosity induced by 2,6-diisopropyl phenol (propofol; PPF), an anaesthetic agent extensively used in clinical practice. Propofol is characterized by a unique phenolic structure, absent in the other conventional anaesthetics. Indeed, given its lipophilic property, propofol is presumed to penetrate into and interact with membrane lipids and hence to induce changes in membrane fluidity. Incorporation of propofol into dimyristoyl- l-α phosphatidylcholine liposomes above the phase-transition temperature (23.9 °C) did not change microviscosity. At 10 °C, an increase of propofol concentration from 0 to 1.0×10 −2 M for a constant lipid concentration mainly induced a decrease in microviscosity. This fluidity effect of propofol has been qualitatively confirmed using merocyanine 540 (MC540) as lipid packing probe. Above 10 −2 M propofol, no further decrease in microviscosity was observed, and the microviscosity at the studied depths (7.8, 16.95, and 27.7 Å) amounted 260.21, 123.87, and 102.27 cP, respectively. The concentration 10 −2 M was identified as the saturation limit of propofol in dimyristoyl- l-α phosphatidylcholine liposomes.

Interaction of rifampicin and isoniazid with large unilamellar liposomes: spectroscopic location studies

The location of isoniazid and rifampicin, two tuberculostatics commonly used for the treatment of Mycobacterium tuberculosis and Mycobacterium avium complex infectious diseases, in bilayers of dimyristoyl- l-α-phosphatidylcholine (DMPC) and dimyristoyl- l-a-phosphatidylglycerol (DMPG) have been studied by 1H NMR and fluorimetric methods. Steady-state fluorescence intensity and fluorescence energy transfer studies between rifampicin and a set of functionalized probes { n-(9-anthroyloxy)stearic acids, n=2, 12} reveal that, in both systems, isoniazid is located at the membrane surface whereas rifampicin is deeply buried inside the lipid bilayers. Steady-state fluorescence anisotropy studies performed with the probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenylhexa-triene (TMA-DPH), not only corroborate the above results, but also show that no changes in membrane fluidity were detected in either liposome. The 1H NMR results, in DMPC liposomes, confirm the location of rifampicin near the methylene group of the acyl chains of the lipid bilayers.

Interaction of an odorant lactone with model phospholipid bilayers and its strong fluidizing action in yeast membrane

Some odorant lactones are naturally present in fruits or in fermented products; they can also be used as food additives and can be produced by microorganisms at the industrial scale by biotechnological processes. γ-Decalactone was previously shown to have antimicrobial properties. We determined by infrared spectroscopy measurements that this compound rapidly diffused into model phospholipid bilayers (within 2 min), modifying the general physical state of a dimyristoyl- l-α-phosphatidylcholine (DMPC) film. In vivo, the lactone strongly increased membrane fluidity in the model yeast Yarrowia lipolytica, as evaluated by fluorescence anisotropy measurements. This effect was more important than that of benzyl alcohol, which is known as a fluidizing agent in living cells, and may explain the toxic action of γ-decalactone in microorganisms.

Spectrophotometric determination of drug partition coefficients in dimyristoyl- l-α-phosphatidylcholine/water: a comparative study using phase separation and liposome suspensions

The partition coefficients ( K p) between lipid bilayers of dimyristoyl- l-α-phosphatidylcholine (DMPC) unilamellar liposomes and water were calculated for four drugs, chlordiazepoxide (a benzodiazepine), isoniazid and rifampicin (tuberculostatic drugs) and griseofulvin (an antifungal antibiotic), using spectrophotometric data and two different approaches. In one, spectrophotometric data were obtained in liposome suspensions and the values of K p were determined from second derivative intensities. In the other, the aqueous and lipid phases were separated by filtering through a Centricon filter unit prior to spectrophotometric determination of drug concentration in each phase. Both methods provide precise and accurate values for the partition coefficient of the drugs in water (Hepes)/DMPC media. None of them presents a clear advantage in the total time needed for the determinations, but for drugs which absorb near or below 275 nm, the need to eliminate liposome scattering may favour the phase separation method.

Carotenoids in Liposomes: Photodegradation, Excited State Lifetimes, and Energy Transfer

DMPC (dimyristoyl-l-α-phosphatidylcholine) liposomes are used as artificial photosynthetic media to study the behavior of carotenoids. 8‘-Apo-β-caroten-8‘-al (I) and β-carotene (II) degrade faster under irradiation in DMPC liposomes than in organic solvents, which is possibly because vibrational deactivation of carotenoid excited states is less efficient in rigid lipid membranes. The lifetime of the first excited singlet state (S1) of I in DMPC liposomes is 27.2 ps, very close to that in 3-methylpentane (26.4 ps), but longer than its lifetime in EtOH (17.1 ps) or CH2Cl2 (14.1 ps). The lifetime of the S1 state of I in DMPC liposomes is as expected for an alkane environment. The lifetime of the S1 state of II in DMPC liposomes is 10.3 ps, very close to its lifetimes in 3-methylpentane (8.1 ps), EtOH (9.2 ps), and CH2Cl2 (8.5 ps). This independence of the S1 state lifetime of II from the matrix agrees with earlier conclusions. Carotenoid I can suppress the photodegradation of chlorophyll a (Chl a) in liposom...

Interaction of DMPC liposomes with a DMPA monolayer: related study of Langmuir-Blodgett films

The formation of a phospholipidic layer was achieved in two steps: (1) a dimyristoyl- l-α-phosphatidic acid (DMPA) Langmuir monolayer was formed by spreading a chloroform/methanol DMPA solution onto an aqueous subphase; after a 10 min period, the monolayer was compressed at 5mNm −1; and (2) keeping the area of the DMPA monolayer constant, a dimyristoyl- l-α-phosphatidylcholine (DMPC) liposomal suspension was added. The progressive incorporation of DMPC molecules into the DMPA monolayer was studied by monitoring the variation of surface pressure with time at constant film area. Three parameters involved in the formation of the interfacial layer DMPA/liposomal DMPC (DMPA/ lip-DMPC) were studied: liposome addition, aqueous subphase composition and initial surface pressure of the DMPA monolayer. The transfer of this mixed layer was controlled through a traceable fluorescent probe incorporated in the liposomes. The thickness and homogeneity of the Langmuir-Blodgett films thus obtained were assessed through Fourier transform infra-red spectroscopy and Nomarski microscopy, respectively. This study shows that the DMPA/ lip-DMPC monolayer could be transferred without dragging of aggregates or mesophases.

Temperature dependent FTIR spectroscopic study of the interaction of α-tocopherol and α-tocopheryl acetate with phospholipid bilayers

FTIR spectroscopy has been employed in order to investigate α-tocopherol (α-T) and α-tocopheryl acetate (α-TA) induced effects on the molecular organization of dimyristoyl- l-α-phosphatidylcholine (DMPC) bilayers, at various temperatures and concentrations. It was concluded that α-T interacts much more strongly than α-TA, indicating that α-T has a more polar location in the membrane than α-TA. The observed changes in the carbonyl and phosphate group vibrational modes of DMPC on addition of α-T or α-TA are discussed

A fluorescence quenching study on protoporphyrin IX in a model membrane system

The interaction of protoporphyrin IX (3,7,12,15-tetramethyl-8,13-divinyl -2,18-porphyrine-dipropionic acid) (PPIX) with unilamellar dimyristoyl-L-α-phosphatidylcholine (DMPC) phospholipid vesicles has been studied by means of steady-state fluorescence quenching spectroscopy. The method of fluorescence-quenching-resolved spectroscopy has been applied in order to resolve the complex emission spectrum of a membrane-bound PPIX into two component spectra, attributed to distinct fluorophore species with different accessibilities to the iodide quencher. It is shown that PPIX associated with liposomes exists in two different microenvironments. One part of the fluorophore is embedded inside the lipid bilayer and is inaccessible to iodide. Its fluorescence spectrum exhibits the maximum characteristic of protoporphyrin found in the apolar medium. The other fraction of PPIX is located near the membrane surface, close to the polar phospholipid heads. Its emission is blue-shifted, resembling that of PPIX in a polar environment. It is quenched by iodide, although it reveals significant shielding from the quencher as compared to a buffer PPIX solution. Fluorescence quenching using l-oxyl-4-oxo-2,2,6,6-tetramethyl-piperidine (TEMPONE) does not discriminate between the two protoporphyrin species. However, the accessibility of protoporphyrin IX to this quencher is much lower in a liposome system than in water.

Macrodipole Generated by Symmetry Breaking due to Phase Separation in Mixed Lipid Vesicles

A simple model calculation demonstrates that phase separation of components with different net charges in spherical mixed lipid vesicles leads to huge dipole moments. Experiments on mixed lipid vesicles composed of 50% dimyristoyl-l-α-phosphatidylcholine (DMPC) and 50% dimyristoylphophatidic acid (DMPA) demonstrate a large permanent dipole moment of about 8.1 × 10-24 C m (≡2.4 × 106 D), which appears below a phase transition at about 30 °C. This phenomenon is characteristic of the mixed lipid system; it has not been observed for vesicles prepared from either DMPC or DMPA alone.