Free pyrene probes in gel and fluid membranes: perspective through atomistic simulations

We consider the properties of free pyrene probes inside gel- and fluidlike phospholipid membranes and unravel their influence on membrane properties. For this purpose, we employ atomic-scale molecular dynamics simulations at several temperatures for varying pyrene concentrations. Molecular dynamics simulations show that free pyrene molecules prefer to be located in the hydrophobic acyl chain region close to the glycerol group of lipid molecules. Their orientation is shown to depend on the phase of the membrane. In the fluid phase, pyrenes favor orientations where they are standing upright in parallel to the membrane normal, while, in the gel phase, the orientation is affected by the tilt of lipid acyl chains. Pyrenes are found to locally perturb membrane structure, while the nature of perturbations in the gel and fluid phases is completely different. In the gel phase, pyrenes break the local packing of lipids and decrease the ordering of lipid acyl chains around them, while, in the fluid phase, pyrenes increase the ordering of nearby acyl chains, thus having an opposite effect. Interestingly, this proposes a similarity to effects induced by cholesterol on structural membrane properties above and below the gel-fluid transition temperature. Further studies express a view that the orientational ordering of pyrene is not a particularly good measure of the acyl chain ordering of lipids. While pyrene ordering provides the correct qualitative behavior of acyl chain ordering in the fluid phase, its capability to predict the correct temperature dependence is limited.     

Practical Labeling Methodology for Choline‐Derived Lipids and Applications in Live Cell Fluorescence Imaging

Lipids of the plasma membrane participate in a variety of biological processes, and methods to probe their function and cellular location are essential to understanding biochemical mechanisms. Previous reports have established that phosphocholine‐containing lipids can be labeled by alkyne groups through metabolic incorporation. Herein, we have tested alkyne, azide and ketone‐containing derivatives of choline as metabolic labels of choline‐containing lipids in cells. We also show that 17‐octadecynoic acid can be used as a complementary metabolic label for lipid acyl chains. We provide methods for the synthesis of cyanine‐based dyes that are reactive with alkyne, azide and ketone metabolic labels. Using an improved method for fluorophore conjugation to azide or alkyne‐modified lipids by Cu(I)‐catalyzed azide‐alkyne cycloaddition (CuAAC), we apply this methodology in cells. Lipid‐labeled cell membranes were then interrogated using flow cytometry and fluorescence microscopy. Furthermore, we explored the utility of this labeling strategy for use in live cell experiments. We demonstrate measurements of lipid dynamics (lateral mobility) by fluorescence photobleaching recovery (FPR). In addition, we show that adhesion of cells to specific surfaces can be accomplished by chemically linking membrane lipids to a functionalized surface. The strategies described provide robust methods for introducing bioorthogonal labels into native lipids.     

Interleaflet interaction and asymmetry in phase separated lipid bilayers: molecular dynamics simulations

In order to investigate experimentally inaccessible, molecular-level detail regarding interleaflet interaction in membranes, we have run an extensive series of coarse-grained molecular dynamics simulations of phase separated lipid bilayers. The simulations are motivated by differences in lipid and cholesterol composition in the inner and outer leaflets of biological membranes. Over the past several years, this phenomenon has inspired a series of experiments in model membrane systems which have explored the effects of lipid compositional asymmetry in the two leaflets. The simulations are directed at understanding one potential consequence of compositional asymmetry, that being regions of bilayers where liquid-ordered (L(o)) domains in one leaflet are opposite liquid-disordered (L(d)) domains in the other leaflet (phase asymmetry). The simulated bilayers are of two sorts: 1) Compositionally symmetric leaflets where each of the two leaflets contains an identical, phase separated (L(o)/L(d)) mixture of cholesterol, saturated and unsaturated phospholipid; and 2) Compositionally asymmetric leaflets, where one leaflet contains a phase separated (L(o)/L(d)) mixture while the other contains only unsaturated lipid, which on its own would be in the L(d) phase. In addition, we have run simulations where the lengths of the saturated lipid chains as well as the mole ratios of the three lipid components are varied. Collectively, we report on three types of interleaflet coupling within a bilayer. First, we show the effects of compositional asymmetry on acyl chain tilt and order, lipid rotational dynamics, and lateral diffusion in regions of leaflets that are opposite L(o) domains. Second, we show substantial effects of compositional asymmetry on local bilayer curvature, with the conclusion that phase separated leaflets resist curvature, while inducing large degrees of curvature in an opposing L(d) leaflet. Finally, in compositionally symmetric, phase separated bilayers, we find phase asymmetry (domain antiregistration) between the two leaflets occurs as a consequence of mismatched acyl chain-lengths in the saturated and unsaturated lipids.     

Label-free detection of polynucleotide single-base mismatch via pyrene probe excimer emission

The pyrene probe and pyrene-labeled oligonucleotides (ODNs) probe are expected to be candidates as fluorescent probe for DNA assay. In particular, label-free detection is a very hot because of its simpleness, speediness and cheapness. Herein, we have investigated the use of a pyrenylakylammonium salt, a novel fluorescent probe for the detection of one single nucleotide polymorphism (SNP) in double stranded DNA. After S1 nuclease digestion, the pyrene probes bind electrostatically to the perfect complement DNA and emit a strong excimer emission. However, treatment of the non-complementary DNA with S1 nuclease caused nucleotide fragments of less than 5 bases, which could not induce excimer emission. By comparing ratio of excimer to monomer fluorescence between normal and mutant DNA after S1 nuclease digestion, One-base mutation in DNA was detected easily. This new method may be applied to the detection of SNP.     

Fluorescent biomembrane probe for ratiometric detection of apoptosis

Herein, we developed the first ratiometric fluorescent probe for apoptosis detection. This probe incorporates selectively into the outer leaflet of the cell plasma membrane and senses the loss of the plasma membrane asymmetry occurring during the early steps of apoptosis. The high specificity to the plasma membranes was achieved by introduction into the probe of a membrane anchor, composed of a zwitterionic group and a long (dodecyl) hydrophobic tail. The fluorescence reporter of this probe is 4'-(diethylamino)-3-hydroxyflavone, which exhibits excited-state intramolecular proton transfer (ESIPT), resulting in two-band emission highly sensitive to the lipid composition of the biomembranes. Fluorescence spectroscopy, flow cytometry, and microscopy measurements show that the ratio of the two emission bands of the probe changes dramatically in response to apoptosis. This response reflects the changes in the lipid composition of the outer leaflet of the cell plasma membrane because of the exposure of the anionic phospholipids from the inner leaflet at the early steps of apoptosis. Being ratiometric, the response of the new probe can be easily quantified on an absolute scale. This allows monitoring by laser scanning confocal microscopy the degree and spatial distribution of the apoptotic changes at the cell plasma membranes, a feature that can be hardly achieved with the commonly used fluorescently labeled annexin V assay.     

Effect ofcholinergic ligands on the lipids of acetylcholine receptor-rich membrane preparations from Torpedo californica.

Ion permeation, triggered by ligand-receptor interaction, is associated with the primary events of membrane depolarization at the neuromuscular junction and synaptic connections. To explore the possible sites of ion permeation, the long-lived fluorescent probe pyrene (fluorescence lifetime approximately 400 nsec) has been inserted into the lipid phase of acetylcholine receptor-rich membrane (AcChR-M) preparations from Torpedo californica. The pyrene probe is susceptible to both fluidity and permeability changes in the lipid bilayer. These changes are detected by variations in the rate of decay of the excited singlet state of pyrene after pulsation with a 10-nsec ruby laser flash. Variations of these lifetimes in the membrane preparations alone or in the presence of quenchers show that binding of cholinergic agonists and antagonists, neurotoxins, and local anesthetics to AcChR-M produces varying effects on the properties of the pyrene probe in the lipid phase. It is concluded that binding of cholinergic ligands to the receptor does not significantly alter the fluidity of permeability of the lipids in the bilayer contact with pyrene. On the other hand, local anesthetics do affect these properties.     

Layer-by-layer PMIRRAS characterization of DMPC bilayers deposited on a Au111 electrode surface

A combination of Langmuir-Blodgett and Langmuir-Schaefer techniques was employed to deposit 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at a gold electrode surface. One leaflet consisted of hydrogen-substituted acyl chains, and the second leaflet was composed of molecules with deuterium-substituted acyl chains. This architecture allowed for layer-by-layer analysis of the structure of the bilayer. Photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was used to determine the conformation and orientation of the acyl chains of DMPC molecules in the individual leaflets as a function of the potential applied to the gold electrode. The bilayer is adsorbed onto the metal surface when the field applied to the membrane does not exceed approximately 108 V/m. When adsorbed, the bottom leaflet is in contact with a hydrophobic metal surface, and the top leaflet is interacting with the aqueous solution. The asymmetry of the environment has an effect on the orientation of the DMPC molecules in each leaflet. The tilt angle of the acyl chains of the DMPC molecules in the bottom leaflet that is in contact with the gold is approximately 10 degrees smaller than that observed for the top leaflet that is exposed to the solution. These studies provide direct evidence that the structure of a phospholipid bilayer deposited at an electrode surface is affected by interaction with the metal.     

Asymmetric distribution of anionic phospholipids in supported lipid bilayers

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.     

Interrogating interfacial organization in planar bilayer structures

The field of biomimetic planar lipid membranes is finding increased importance as the need to devise sensing systems for biologically important species increases. We approach this area with an eye toward understanding how to interrogate local organization in these complex media. Our primary tools for this purpose are spectroscopy and electrochemistry, where imbedded reporter molecules serve as the information transducers. We use Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) methods to construct planar lipid membranes on hydrophilic solid substrates (Au for electrochemistry, SiO x for spectroscopy). Pyrene tethered to the substrate acts as our probe and AC voltammetry was used to evaluate its redox kinetics, showing slow, distance independent electron transfer between the pyrene moieties and the electrode for both monolayer and bilayer systems. Time-resolved fluorescence data indicate that tethered pyrene resides in a highly rigid environment and that the addition of the top lipid leaflet improves the organization of the bottom lipid leaflet. These data point to the cooperative effect of the bilayer leaflets in creating a system that is comparatively rigid on short length scales, and capable of mediating motion and accessibility of imbedded species.     

Molar absorption coefficient of pyrene aggregates in water

The molar absorption coefficient of pyrene aggregates, epsilon(E0), was determined for a series of pyrene-labeled poly(N,N-dimethylacrylamide)s (Py-PDMA) having different pyrene contents. Aqueous solutions of Py-PDMA having pyrene contents ranging from 263 to 645 mumol of pyrene per gram of polymer were studied by UV-vis absorbance and time-resolved fluorescence spectroscopy. The global analysis of the monomer and excimer fluorescence decays with the fluorescence blob model yielded the fractions of the overall absorption contributed by all the pyrene species present in solution. The combined knowledge of the fractions obtained from the global analysis of the time-resolved fluorescence decays, the total absorption of the Py-PDMA solution obtained from UV-vis spectroscopy, and the total pyrene concentration in the solution obtained from the known pyrene content of each Py-PDMA sample led to the determination of the molar absorption coefficient of pyrene aggregates. Regardless of the pyrene content of the Py-PDMA samples and hence the level of association of the pyrene pendants in solution, all Py-PDMA samples yielded similar epsilon(E0) values over the range of wavelengths studied, namely, from 325 to 350 nm. The averaged epsilon(E0) was found to be red-shifted relative to unassociated pyrenes by 3 nm as well as having a value at the 0-0 peak of 21 000 M(-1).cm(-1) reduced from 34 700 M(-1).cm(-1) for unassociated pyrenes. The determination of epsilon(E0) enabled the first determination of the absolute fraction of associated pyrenes for aqueous solutions of a series of pyrene-labeled water-soluble polymers. The procedure outlined in this study is applicable to any pyrene-labeled water-soluble polymer and provides a new means to study quantitatively the effect of the hydrophilic-to-lipophilic balance on the hydrophobic associations generated by hydrophobically modified water-soluble polymers. As an application, the average number of pyrenes involved in a pyrene aggregate generated by Py-PDMA in water is determined.     

Single molecule probes of membrane structure: orientation of BODIPY probes in DPPC as a function of probe structure

Single molecule fluorescence measurements have recently been used to probe the orientation of fluorescent lipid analogs doped into lipid films at trace levels. Using defocused polarized total internal reflection fluorescence microscopy (PTIRF-M), these studies have shown that fluorophore orientation responds to changes in membrane surface pressure and composition, providing a molecular level marker of membrane structure. Here we extend those studies by characterizing the single molecule orientations of six related BODIPY probes doped into monolayers of DPPC. Langmuir-Blodgett monolayers transferred at various surface pressures are used to compare the response from fluorescent lipid analogs in which the location of the BODIPY probe is varied along the length of the acyl chain. For each BODIPY probe location along the chain, comparisons are made between analogs containing phosphocholine and smaller fatty acid headgroups. Together these studies show a general propensity of the BODIPY analogs to insert into membranes with the BODIPY probe aligned along the acyl chains or looped back to interact with the headgroups. For all BODIPY probes studied, a bimodal orientation distribution is observed which is sensitive to surface pressure, with the population of BODIPY probes aligned along the acyl chains increasing with elevated surface pressure. Trends in the single molecule orientations for the six analogs reveal a configuration where optimal placement of the BODIPY probe within the acyl chain maximizes its sensitivity to the surrounding membrane structure. These results are discussed in terms of balancing the effects of headgroup association with acyl chain length in designing the optimal placement of the BODIPY probe.     

Simultaneous In Situ Quantification of Two Cellular Lipid Pools Using Orthogonal Fluorescent Sensors

Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid‐binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems‐level investigation of lipid‐mediated cell signaling and regulation.     

Lipid lateral mobility and membrane phase structure modulation by protein binding

Using a combination of fluorescence correlation and infrared absorption spectroscopies, we characterize lipid lateral diffusion and membrane phase structure as a function of protein binding to the membrane surface. In a supported membrane configuration, cholera toxin binding to the pentasaccharaide headgroup of membrane-incorporated GM1 lipid alters the long-range lateral diffusion of fluorescently labeled probe lipids, which are not involved in the binding interaction. This effect is prominently amplified near the gel-fluid transition temperature, Tm, of the majority lipid component. At temperatures near Tm, large changes in probe lipid diffusion are measured at average protein coverage densities as low as 0.02 area fraction. Spectral shifts of the methylene symmetric and asymmetric stretching modes in the lipid acyl chain confirm that protein binding alters the fraction of lipid in the gel phase.     

Sensing isothermal changes in the lateral pressure in model membranes using di-pyrenyl phosphatidylcholine

In this work we present data from a homologous series of di-pyrenyl phosphatidylcholine (dipyPC) probes which can sense lateral pressure variations in the chain region of the amphiphilic membrane (lateral pressures are tangential to the interface). The dipyPC has pyrene moieties attached to the ends of equal length acyl chains on a phosphatidylcholine molecule. Ultraviolet stimulation produces both monomer and excimer fluorescence from pyrene. At low dilutions of dipyPC in model membranes the excimer signal is entirely intra-molecular and since it depends on the frequency with which the pyrene moieties are brought into close proximity, the relative intensity of the excimer to monomer signal, eta, is a measure of the pressure. We synthesised or purchased dipyPC probes with the pyrene moieties attached to acyl chains having 4, 6, 8 and 10 carbon atoms and then measured eta in fully hydrated bilayers composed of dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine (DOPC and DOPE respectively). Although the resolution of our measurements of lateral pressure as a function of distance into the monolayer was limited, we did observe a dip in the excimer signal in the region of the DOPC/DOPE cis double bond. As we isothermally increased the DOPE composition, and hence the desire for interfacial curvature, we observed, as expected, that the net excimer signal increased. However this net increase was apparently brought about by a transfer of pressure from the region around the glycerol backbone to the region near the chain ends, with the lateral pressure dropping above the cis double bond but increasing at a greater rate beyond the double bond.     

Dual pyrene-labeled pyrrolidinyl peptide nucleic acid as an excimer-to-monomer switching probe for DNA sequence detection

The unique ability of pyrene to form excimers with distinct emission characteristic from monomer offers an attractive means to signal the interactions between biomolecules. In this work, dual pyrene-labeled pyrrolidinyl peptide nucleic acid probe with a d-prolyl-2-aminocyclopetanecarboxylic acid α,β-dipeptide backbone (acpcPNA) was designed as an excimer-to-monomer switching probe for DNA sequence detection. In single stranded state, the excimer emission at 470 nm was mainly observed in the fluorescence spectrum. In the presence of DNA target, the hybridization resulted in separation of the two pyrene units, therefore the spectrum displayed increased monomer emission at 380 nm with concomitant decreased excimer emission. Switching ratio, which is defined as the ratio of the monomer to excimer in the double stranded form [F₃₈₀/F₄₇₀(ds)] divided by the same value obtained from the single stranded form [F₃₈₀/F₄₇₀(ss)], was used to describe the performance of the probes. Switching ratios in the range of 5–30 were observed with various dual pyrene-labeled acpcPNA probes bearing pyrenebutyryl label attached five-base apart. Practically no excimer-to-monomer switching behavior was observed with DNA targets carrying a single mismatched base as shown by the small switching ratios of ∼1.     

Fluorescence probing of the temperature-induced phase transition in a glycolipid self-assembly: hexagonal ↔ micellar and cubic ↔ lamellar

Water-driven self-assembly of lipids displays a variety of liquid crystalline phases that are crucial for membrane functions. Herein, we characterize the temperature-induced phase transitions in two compositions of an aqueous self-assembly system of the octyl β-D-glucoside (βGlcOC(8)) system, using steady-state and time-resolved fluorescence measurements. The phase transitions hexagonal ? micellar and cubic ? lamellar were investigated using tryptophan (Trp) and two of its ester derivatives (Trp-C(4) and Trp-C(8)) to probe the polar headgroup region and pyrene to probe the hydrophobic tail region. The polarity of the headgroup region was estimated to be close to that of simple alcohols (methanol and ethanol) for all phases. The pyrene fluorescence indicates that the pyrene molecules are dispersed among the tails of the hydrophobic region, yet remain in close proximity to the polar head groups. Comparing the present results with our previously reported one for βMaltoOC(12), increasing the tail length of the hexagonal phase from C(8) to C(12) leads to less interaction with pyrene, which is attributed to the more random and wobbling motion of the longer alkyl tail. We measured a reduction (more hydrophobic) in the ratio of the vibronic peak intensities of pyrene (I(1)/I(3)) for the lamellar phase compared to that of the cubic phase. The higher polarity in the cubic phase can be correlated to the nature of its interface, which curves toward the bulk water. This geometry also explains the slight reduction in polarity of the headgroup region compared to the other phases. Upon the addition of Trp-C(8), the fluorescence lifetime of pyrene is reduced by 28% in the lamellar and cubic phases, whereas the I(1)/I(3) value is only slightly reduced. The results reflect the dominant role of dynamic interaction mechanism between the C(8) chain of Trp-C(8) and pyrene. This mechanism may be important for these two phases since they participate in the process of membrane fusion. Both lipid compositions show completely reversible temperature-induced phase transitions, reflecting the thermodynamic equilibrium structures of their mesophases. Probing both regions of the different lipid phases reveals a large degree of heterogeneity and flexibility of the lipid self-assembly. These properties are crucial for carrying out different biological functions such as the ability to accommodate various molecular sizes.     

Dynamics of short as compared with long poly(acrylic acid) chains hydrophobically modified with pyrene, as followed by fluorescence techniques

New low and high molecular weight poly(acrylic acid), PAA, 2000 g mol(-1) and 450,000 g mol(-1), respectively, were tagged with pyrene (low and high contents of probe) and its behaviour in solution was investigated using absorption and fluorescence (steady-state and time-resolved) techniques. Fluorescence data shows that the degree and level of intramolecular association strongly depends on the molecular weight. With the short pyrene-labeled PAA chains in aqueous solution, the excimer-to-monomer fluorescence ratio I(E)/I(M) decreases with the increase of pH, oppositely to the increase in the I(E)/I(M) ratio with the increase in pH previously observed with the long chain PAA. Time-resolved data suggest that excimer formation with the short pyrene-labeled PAA polymers (ca. 28 acrylic acid monomers per chain) in water is largely due to excitation of Ground State Dimers, GSD. The increment of pH, and the consequent gradual ionization of the carboxylic groups in the chain, initially increases the fraction of GSD, possibly due to the occurrence of special micelle-like chain conformations, inside which the pyrene units are accommodated. A further increase of the pH above the pK(a) values, resulting in the full ionization of carboxylic groups, apparently destabilizes such chain conformations, which leads to a pH effect on the photophysical properties identical to that of the long chain polymers. In water, the dynamic data shows the existence of two excimers coexisting with two monomer classes. In methanol and dioxane (good solvents for the pyrene probe) at room temperature, where one excimer and two monomers are present, all rate constants could be obtained, as well as the fractions of ground-state species. It is thus shown that different types of interactions are produced with small- and long-sized PAA polymers, i.e., the size of the polymer matters.     

Fluorescent oligonucleotide incorporating 5-(1-ethynylpyrenyl)-2-deoxyuridine: sequence-specific fluorescence changes upon duplex formation

We describe the design and properties of a pyrene-labeled deoxyuridine that can be inserted efficiently into oligodeoxynucleotides using phosphoramidite chemistry. An oligonucleotide incorporating the pyrene-labeled deoxyuridine is a sensitive fluorescence probe that can discriminate between perfect and single-base-mismatched pairing by changes in its fluorescence intensity.     

Utilization of heavy-atom effect quenching of pyrene fluorescence to determine the intramembrane distribution of halothane

The interaction of halothane (CF₃CHBrCl) with dipalmitoylphosphatidylcholine (DPPC) membranes containing varying amounts of dipalmitoylphosphatidylglycerol (DPPG) was examined via heavy atom effect quenching of pyrene fluorescence by halothane. The effect of halothane on pyrene fluorescence is consistent with a kinetic model based upon the assumption of the existence of two populations of pyrene in the membrane: one accessible to interactions with halothane; the second inaccessible to halothane on the time scale of the pyrene fluorescence excited state. Both populations of pyrene are affected by the presence of halothane in the membrane. The rate of halothane quenching of pyrene fluorescence is increased significantly for all DPPG/DPPC membranes compared to pure DPPC membranes indicating that any DPPG in the membrane facilitates interaction between halothane and pyrene even though the measured partition coefficients indicate that little change in total halothane concentration in the membrane as a whole occurs as a function of percent DPPG in DPPG/DPPC mixtures.
It is speculated that phase boundaries play an important role in determining the behavior of this model system by determining the location of the pyrene probe. The heavy atom effect quenching of pyrene by halothane provides a useful probe of phase boundaries in membranes.