Depth-dependent investigation of the apolar zone of lipid membranes using a series of fluorescent probes,Me<Subscript>4</Subscript>-BODIPY-8-labeled phosphatidylcholines

A series of lipid probes, phosphatidylcholines labeled with Me₄-BODIPY-8 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl) fluorophore attached to the end of acyl residue at different distances from the polar head, were used as depth-dependent probes for the apolar zone of the model membrane systems, large unilamellar vesicles (LUV). Data on the anisotropy of probe fluorescence demonstrated a different mobility profiles for the fluorophore microenvironment in LUVs of different composition at various temperatures, which indicates a high sensitivity of these probes as tools for studying membrane systems. An interesting anomaly was observed for LUVs from dimiristoylphosphatidylcholine (DMPC) or from a DMPC-cholesterol mixture: the anisotropy of the fluorophore located near the bilayer center is larger than that of the fluorophore located further from the center; i.e., the mobility of the microenvironment is lower in the first case. This anomaly is supposed to result from the penetration of unlabeled long chain of the probes to the opposite bilayer leaflet. Such a possibility should be taken into account when constructing the fluorescent probes and interpreting the results.     

Rotation of fluorescent probes localized within lipid bilayer membranes

Measurements of the steady state polarization of fluorescence from perylene and 9-vinylanthracene embedded in bilayer membranes were performed as a function of temperature. Similar measurements were made when these probes were dissolved in hydrocarbons as model solvents. The effects of cholesterol and n-alkyl alcohol additions to bilayers and head group variation were also examined. Results were expressed in terms of the average rotation rates of the probes.At 25°C, the calculated rotation rate for perylene in egg phosphatidylcholine vesicles was 275 × 10⁶ sec^?1 as compared to 2400 × 10⁶ sec^?1 for perylene in n-hexadecane. However, the activation energies for probe rotation in both environments was about 7 kcal/mole suggesting similar rotational diffusion mechanisms. Membrane microviscosity evaluations were performed according to a recently published scheme and an assessment of this method of viscosity estimation was given. The presence of an approximately equimolar amount of cholesterol impeded probe rotation (90 × 10⁶ sec^?1 at 25°C) and reduced the activation energy (4.9 kcal/mole) for probe rotation. In contrast, addition of n-alkyl alcohols to the vesicle suspension acted to increase probe rotation rates, an indication of fluidization of the membranes. This is in accord with spin label and cation permeability data for similar membranes.It was concluded that this method of probing can adequately report changes in membrane dynamic structure when these changes occur uniformly over the membrane surface. The interpretation is less clear when structural changes occur only in patches or domains of the membrane thereby producing a non-uniform surface distribution of probes.     

Anion binding to lipid bilayers: a study using fluorescent lipid probes

Anion-induced fluorescence quenching of lipid probes incorporated into the liposomal membrane was used to study the binding of anions to the lipid membrane. Lipid derivatives bearing nonpolar fluorophore located either in the proximity of the polar headgroups (anthrylvinyl-labelled phosphatidylcholine, ApPC; methyl 4-pyrenylbutyrate, MPB) or in the polar region (rhodamine 19 oleyl ester, OR19) of the bilayer were used as probes. The binding of iodide to the bilayers of different compositions was studied. Based on the anion-induced quenching of the fluorescence, the isotherm of adsorption of the quencher (iodide) to the membrane was plotted. For anions, which are non-quenchers or weak quenchers (thiocyanate, perchlorate or trichloroacetate), the binding parameters were obtained from the data of the competitive displacement of iodide by these anions. The association constants of the anion binding to the bilayer (Ka) were determined for the stoichiometry of 1 ion/1 lipid and also for the case of independent anion binding. At the physiological concentration of the salt, which does not bind noticeably to the membrane (150 mM NaCl), anion binding could be satisfactorily described by the Langmuir isotherm. The approach applied here offers new possibilities for the studies of ion-membrane interactions using fluorescent probes.     

Effect of cholesterol on lateral diffusion of fluorescent lipid probes in native hippocampal membranes

Cholesterol is an abundant lipid of mammalian membranes and plays a crucial role in membrane organization, dynamics, function and sorting. The role of cholesterol in membrane organization has been a subject of intense investigation that has largely been carried out in model membrane systems. An extension of these studies in natural membranes, more importantly in neuronal membranes, is important to establish a relationship between disease states and changes in membrane physical properties resulting from an alteration in lipid composition. We have monitored the lateral diffusion of lipid probes, DiIC(18)(3) and FAST DiI which are similar in their intrinsic fluorescence properties but differ in their structure, in native and cholesterol-depleted hippocampal membranes using the fluorescence recovery after photobleaching (FRAP) approach. Our results show that the mobility of these probes is in general higher in hippocampal membranes depleted of cholesterol. Interestingly, the increase in mobility of these probes does not linearly correlate with the extent of cholesterol depletion. These results assume significance in the light of recent reports on the requirement of cholesterol to support the function of the G-protein coupled serotonin(1A) receptor present endogenously in hippocampal membranes.     

Study of the effect of tegalide and bithionol on biological membranes using fluorescent probes

Fluorescent probes were used to study structural changes in different membranes affected by gamma-radiation, protonophores and radioprotective agents, tegalide and bithionol. The preparations of the defined concentrations decreased the microviscosity of membranes, lowered the peaks and changed the temperature of phase transitions in liposomes from dipalmitoyl lecithin, and induced the output of Ca2+ from mitochondria. The effects depended on the radiation dose, the structure, concentration and lipophilicity of the preparation; protonophores produced a specific effect.     

Direct measurement of antigen binding properties of CD1 proteins using fluorescent lipid probes

CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses. In the current study, we investigated ligand binding by CD1 proteins by developing a fluorescent probe binding approach using soluble recombinant human CD1 proteins. To increase stability and yield, soluble group 1 CD1 (CD1b and CD1c) and group 2 CD1 (CD1d) proteins were produced as single chain secreted CD1 proteins in which beta2-microglobulin was fused to the N termini of the CD1 heavy chains by a flexible peptide linker sequence. Analysis of ligand binding properties of single chain secreted CD1 proteins by using fluorescent lipid probes indicated significant differences in ligand preference and in pH dependence of binding by group 1 versus group 2 CD1 proteins. Whereas group 1 CD1 isoforms (CD1b and CD1c) show stronger binding of nitrobenzoxadiazole (NBD)-labeled dialkyl-based ligands (phosphatidylcholine, sphingomyelin, and ceramide), group 2 CD1 (CD1d) proteins were stronger binders of small hydrophobic probes such as 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-naphthyl-5,5'-disulfonic acid. Competition studies indicated that binding of fluorescent lipid probes involved association of the probe with the hydrophobic ligand binding groove of CD1 proteins. Analysis of selected alanine substitution mutants of human CD1b known to inhibit antigen presentation showed that NBD-labeled lipid probe binding could be used to distinguish mutations that interfere with ligand binding from those that affect T cell receptor docking. Our findings provide further evidence for the functional specialization of different CD1 isoforms and demonstrate the value of the fluorescent lipid probe binding method for assisting structure-based studies of CD1 function.     

Study of adsorption of Influenza virus matrix protein M1 on lipid membranes by the technique of fluorescent probes

Matrix protein M1 of Influenza virus, which forms its inner scaffold, is the most abundant amongst viral proteins. Functions of M1 protein are highly diverse, as it has to ensure both the entry of the viral genetic material into the cytoplasm of the infected cell and the assembly of new viral particles for multiplication of infection. In all these processes matrix protein interacts with lipid membranes–either viral external lipid envelope or plasma membrane of a virus-infected cell. However, molecular mechanisms of such interactions are still unclear. In this work, we used the method of fluorescent probes on the example of 1-anilinonaphthalene- 8-sulfonate to determine components of the lipid bilayer required for binding of the M1 protein to the membrane, as well as possible orientations of the protein relative to the lipid membrane. We found that for the adsorption of matrix protein M1 lipid bilayer had to contain phosphatidylserines, while neither phosphatidylethanolamine nor cholesterol promoted protein binding to the membrane. Furthermore, our data suggest that M1 protein binds negatively charged lipid bilayer by positively charged amino acids exhibiting outward anionic sites.     

Monitoring the permeability profile of lipid membranes with spin probes

The rate of reaction of the ascorbate ion with the nitroxide group of spin probes intercalated in lipid bilayers has been studied to examine the mechanism of transport of solutes across membranes. The loss of electron spin resonance (ESR) signal follows first-order kinetics. For a given bilayer system, the half-time of the process increases with the distance of the reacting group from the aqueous interface, according to an approximately linear permeation profile. The dependence on phospholipid headgroup is that which would be predicted from the net charge; addition of negatively charged headgroups increases the half-time of reaction, and positively charged headgroups decrease it, compared with bilayers having no net charge. Addition of cholesterol, which is known to decrease the fluidity of the hydrocarbon core of the bilayer, is found to increase the half-time of reaction. The results have been analyzed in terms of a partition-diffusion mechanism. It is suggested that the rate-limiting step for partitioning the solute into the bilayer might be removal of water of hydration. Cholesterol increases the activation energy, most probably by increasing the height of the barriers to diffusion. Quantitation of the changes in reaction rates gives an estimate of the change in bilayer surface potential on changing the headgroup composition. Examination of the permeation profile supports a diffusive mechanism, from which it can be estimated that the diffusion coefficient is approximately halved on adding 35 mol% cholesterol to egg lecithin bilayers.     

Determination of intracellular pH using sensitive, clickable fluorescent probes

We synthesized and evaluated a series of acidic fluorescent pH probes exhibiting robust pH dependence, high sensitivity and photostability, and excellent cell membrane permeability. Titration analyses indicated that probe 3 could increase its fluorescence intensity 800-fold between pH 8.0 and 4.1. Additionally, its pK(a) value is optimal for intracellular probing of acidic organelles. Fluorescent imaging of HepG2 and Hela cells further revealed that probe 3 demonstrates outstanding capacity for monitoring of intracellular [H(+)] levels. The easily accessible terminal alkyne/azido function groups of these probes offer the possibility of rapidly constructing sensor molecule libraries using 'click' chemistry.     

Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers

Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of “impurities” into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2 mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2 mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.     

Studies of the ligand binding reaction of adipocyte lipid binding protein using the fluorescent probe 1, 8-anilinonaphthalene-8-sulfonate.

The fluorescent probe anilinonaphthalene-8-sulfonate binds to adipocyte lipid binding protein at a site that competes with normal physiological ligands, such as fatty acids. Binding to the protein is accompanied by a relatively large increase in fluorescent intensity. To correlate the major change in optical properties and to determine the mechanism of competitive inhibition with fatty acids, the crystal structure of the protein with the bound fluorophore has been determined. In addition, the thermodynamic contributions to the binding reaction have been studied by titration calorimetry. Because the binding site is in a relatively internal position, kinetic studies have also been carried out to determine k(on). The results indicate that binding is not accompanied by any major conformational change. However, the negatively charged sulfonate moiety is not positioned the same as the carboxylate of fatty acid ligands as determined in previous studies. Nonetheless, the binding reaction is still driven by enthalpic effects. As judged by the crystallographic structure, a significant amount of the surface of the fluorophore is no longer exposed to water in the bound state.     

Two-color fluorescent probes for imaging the dipole potential of cell plasma membranes

The dipole potential (Psi(d)) constitutes a large and functionally important part of the electrostatic potential of cell plasma membranes. However, its direct measurement is not possible. Herein, new 3-hydroxyflavone fluorescent probes were developed that respond strongly to Psi(d) changes by a variation of the intensity ratio of their two well-separated fluorescence bands. Using fluorescence spectroscopy with cell suspensions and confocal microscopy with adherent cells, we showed, for the first time, two-color fluorescence ratiometric measurement and visualization of Psi(d) in cell plasma membranes. Using this new tool, a heterogeneous distribution of this potential within the membrane was evidenced.     

Study of effects of cholesterol on the polar groups of the lipid bilayer using a fluorescent probe, 4-dimethylaminochalcone

Fluorescence parameters of a probe 4-dimethylaminochalcone (DMAC) in egg phosphatidylcholine bilayer lipid membranes are extremely sensitive to cholesterol, since the latter influences the solvation shells of DMAC formed by lipid molecules. The membrane population of DMAC is heterogeneous and is represented by two populations of molecules. The first of them contains about 60% of DMAC molecules, which fluoresce with a short (0.3 ns) decay time at 485 nm; their solvation shells are insensitive to 30 mol. % cholesterol. The second DMAC population is responsible for more than 50% of total fluorescence. Reorientation of the probe solvation shell dipoles is accompanied by a noticeable Stokes shift of the DMAC fluorescence spectrum; in the absence of cholesterol the Stokes shift amounts to 50 nm (i.e., from 485 to 535 nm). In the presence of 30 mol. % cholesterol, the effect of relaxation of the solvation shells on the DMAC fluorescence shift is reduced to 22 nm. At the same time, cholesterol reduces fluorescence quenching in the second population of DMAC molecules due to a twofold increase in their fluorescence quantum yield. Reorientation of solvation shell dipoles inducing the Stokes shift is complete within less than 0.1 ns, apparently, at the expense of fast-relaxing dipoles, viz., water molecules penetrating into the lipid bilayer. The data obtained suggest that cholesterol induces only partial modification of the lipid bilayer; some membrane regions are not exposed to its effect even when the cholesterol fraction constitutes up to 30% of the total lipid content.     

Binding of a fluorescent lipid amphiphile to albumin and its transfer to lipid bilayer membranes

Kinetics and thermodynamics of the binding of a fluorescent lipid amphiphile, Rhodamine Green(TM)-tetradecylamide (RG-C(14:0)), to bovine serum albumin were characterized in an equilibrium titration and by stopped-flow fluorimetry. The binding equilibrium of RG-C(14:0) to albumin was then used to reduce its concentration in the aqueous phase to a value below its critical micelle concentration. Under these conditions, the only two species of RG-C(14:0) in the system were the monomer in aqueous solution in equilibrium with the protein-bound species. After previous determination of the kinetic and thermodynamic parameters for association of RG-C(14:0) with albumin, the kinetics of insertion of the amphiphile into and desorption off lipid bilayer membranes in different phases (solid, liquid-ordered, and liquid-disordered phases, presented as large unilamellar vesicles) were studied by stopped-flow fluorimetry at 30 degrees C. Insertion and desorption rate constants for association of the RG-C(14:0) monomer with the lipid bilayers were used to obtain lipid/water equilibrium partition coefficients for this fluorescent amphiphile. The direct measurement of these partition coefficients is shown to provide a new method for the indirect determination of the equilibrium partition coefficient of similar molecules between two defined lipid phases if they coexist in the same membrane.     

Structural investigation of loose connective tissue by using a series of dextran fractions as non-interacting macromolecular probes.

The ability of the uncharged open-coil dextran molecules to penetrate tissue space, without coil-shape change, was utilized to probe (by partitioning experiments) the structural arrangement of the collagen-fibre network and the proteoglycan system. Hyaluronidase digests most of the proteoglycans away and enables the respective contributions to the exclusion volume to be evaluated by using a series of different-molecular-weight dextrans. It appears that the major part of the exclusion volume is due to the collagen-fibril as a rod and the dextran coil as an impenetrable sphere. The additional exclusion due to the proteoglycans could be accounted for by a set of points (regions of high proteoglycan-segment density) over which the dextran coild cannot pass. These points are an average of 50 nm apart and are indicative of local extensive entanglement of high-molecular-weight proteoglycans with each other. Reasons are given why these entanglements could not act as cross-links in long-term elastic loading of the tissue.     

Dipolar relaxation in a lipid bilayer detected by a fluorescent probe, 4'-dimethylaminochalcone

The dynamic behavior of polar molecules in egg phosphatidylcholine (PC) bilayers has been studied using a membrane fluorescent probe, 4'-dimethylaminochalcone (DMAC). Time and spectrally resolved fluorescence spectroscopy of DMAC incorporated in PC liposomes, as compared to studies of the probe in organic solvents, shows the existence of two independent populations, associated with different extent and speed of dipolar solvent relaxation. The first DMAC population represents approximately 69% of the fluorescence-emitting molecules, has a short fluorescence decay time (0.32 ns) and undergoes Stokes shift of 80 nm. The remaining 31% fraction of DMAC molecules has a decay time of 0.74 ns and undergoes a high (106 nm) Stokes shift. A fraction of the shift, ca. 24 nm for the first and 46 nm for the second population, is attributed to the fast (<0.1 ns) rotational relaxation of nearby dipolar molecules, which might be water. This two-state model accounts well for the detailed fluorescence properties of DMAC in egg PC, i.e. its broadened steady-state spectrum, its average fluorescence quantum yield and its complex wavelength-dependent fluorescence decays.     

The rate of lipid transfer during fusion depends on the structure of fluorescent lipid probes: a new chain-labeled lipid transfer probe pair.

A number of fluorescent probes have been used to follow membrane fusion events, particularly intermixing of lipids. None of them is ideal. The most popular pair of probes is NBD-PE and Rh-PE, in which the fluorescent groups are attached to the lipid headgroups, making them sensitive to changes in the surrounding medium. Here we present a new assay for monitoring lipid transfer during membrane fusion using the acyl chain tagged fluorescent probes BODIPY500-PC and BODIPY530-PE. Like the NBD-PE/Rh-PE assay, this assay is based on fluorescence resonance energy transfer (FRET) between the donor, BODIPY500, and the acceptor, BODIPY530. The magnitude of FRET is sensitive to the probe surface concentration, allowing one to detect movement of probes from labeled to unlabeled vesicles during fusion. The high quantum yield of fluorescence, high efficiency of FRET (R(o) is estimated to be approximately 60 A), photostability, and localization in the central hydrophobic region of a bilayer all make this pair of probes quite promising for detecting fusion. We have compared this and two other lipid mixing assays for their abilities to detect the initial events of poly(ethylene glycol) (PEG)-mediated fusion of small unilamellar vesicles (SUVs). We found that the BODIPY500/530 assay showed lipid transfer rates consistent with those obtained using the DPHpPC self-quenching assay, while lipid mixing rates measured with the NBD-PE/Rh-PE RET assay were significantly slower. We speculate that the bulky labeled headgroups of NBD-PE and especially Rh-PE molecules hamper movement of probes through the stalk between fusing vesicles, and thus reduce the apparent rate of lipid mixing.