Ruthenium(II) Complex Enantiomers as Cellular Probes for Diastereomeric Interactions in Confocal and Fluorescence Lifetime Imaging Microscopy

Ruthenium dipyridophenazine (dppz) complexes are sensitive luminescent probes for hydrophobic environments. Here, we apply multiple-frequency fluorescence lifetime imaging microscopy (FLIM) to Δ and Λ enantiomers of lipophilic ruthenium dppz complexes in live and fixed cells, and their different lifetime staining patterns are related to conventional intensity-based microscopy. Excited state lifetimes of the enantiomers determined from FLIM measurements correspond well with spectroscopically measured emission decay curves in pure microenvironments of DNA, phospholipid membrane or a model protein. We show that FLIM can be applied to monitor the long-lived excited states of ruthenium complex enantiomers and, combined with confocal microscopy, give new insight into their biomolecular binding and reveal differences in the microenvironment probed by the complexes.     

Enhanced hydrophobicity of fluorinated lipid bilayer: a molecular dynamics study

A molecular dynamics simulation of a partially fluorinated phospholipid bilayer has been carried out to understand the effects of fluorination of the hydrophobic chains on the structure and water permeability across the membrane. Fluorocarbon chains typically have an all-trans conformation, showing a highly ordered structure in the membrane core compared to ordinary hydrocarbon chains. The free energy profiles of water across the bilayers were successfully estimated by a revised cavity insertion Widom method. The fluorinated bilayer showed a higher free energy barrier than an ordinary nonfluorinated lipid bilayer by about 1.2 kcal/mol, suggesting a lower water permeability of the fluorinated bilayer membrane. A cavity distribution analysis elucidated the reduced free volume in the fluorinated membrane due to the neatly packed chains, which should account for the higher free energy barrier.     

Membrane localization and flexibility of a lipidated ras peptide studied by molecular dynamics simulations

Lipid-modified membrane-binding proteins are essential in signal transduction events of the cell, a typical example being the GTPase ras. Recently, membrane binding of a doubly lipid-modified heptapeptide from the C-terminus of the human N-ras protein was studied by spectroscopic techniques. It was found that membrane binding is mainly due to lipid chain insertion, but it is also favored by interactions between apolar side chains and the hydrophobic region of the membrane. Here, 10 explicit solvent molecular dynamics simulations for a total time of about 150 ns are used to investigate the atomic details of the peptide-membrane association. The 16:0 peptide lipid chains are more mobile than the 14:0 phospholipid chains, which is in agreement with (2)H NMR experiments. Peptide-lipid and peptide-solvent interactions, backbone and side-chain distributions, as well as the effects of lipidated peptide insertion onto the structure, and dynamics of a 1,2-dimyristoylglycero-3-phosphocholine bilayer are described. The simulation results validate the structural model proposed by the analysis of spectroscopic data and highlight the main aspects of the insertion mechanism. The peptide in the membrane is rather rigid over the simulation time scale of about 10 ns, but different partially extended conformations devoid of backbone hydrogen bonds are observed in different trajectories.     

Membrane insertion of a lipidated ras peptide studied by FTIR,solid-state NMR,and neutron diffraction spectroscopy

Membrane binding of a doubly lipid modified heptapeptide from the C-terminus of the human N-ras protein was studied by Fourier transform infrared, solid-state NMR, and neutron diffraction spectroscopy. The 16:0 peptide chains insert well into the 1,2-dimyristoyl-sn-glycero-3-phosphocholine phospholipid matrix. This is indicated by a common main phase transition temperature of 21.5 degrees C for both the lipid and peptide chains as revealed by FTIR measurements. Further, (2)H NMR reveals that peptide and lipid chains have approximately the same chain length in the liquid crystalline state. This is achieved by a much lower order parameter of the 16:0 peptide chains compared to the 14:0 phospholipid chains. Finally, proton/deuterium contrast variation of neutron diffraction experiments indicates that peptide chains are localized in the membrane interior analogous to the phospholipid chains. In agreement with this model of peptide chain insertion, the peptide part is localized at the lipid-water interface of the membrane. This is revealed by (1)H nuclear Overhauser enhancement spectra recorded under magic angle spinning conditions. Quantitative cross-peak analysis allows the examination of the average location of the peptide backbone and side chains with respect to the membrane. While the backbone shows the strongest cross-relaxation rates with the phospholipid glycerol, the hydrophobic side chains of the peptide insert deeper into the membrane interior. This is supported by neutron diffraction experiments that reveal a peptide distribution in the lipid-water interface of the membrane. Concurring with these experimental findings, the amide protons of the peptide show strong water exchange as seen in NMR and FTIR measurements. No indications for a hydrogen-bonded secondary structure of the peptide backbone are found. Therefore, membrane binding of the C-terminus of the N-ras protein is mainly due to lipid chain insertion but also supported by interactions between hydrophobic side chains and the lipid membrane. The peptide assumes a mobile and disordered conformation in the membrane. Since the C-terminus of the soluble part of the ras protein is also disordered, we hypothesize that our model for membrane binding of the ras peptide realistically describes the membrane binding of the lipidated C-terminus of the active ras protein.     

Retinoid chromophores as probes of membrane lipid order

There is a great need for development of independent methods to study the structure and function of membrane-associated proteins and peptides. Polarized light spectroscopy (linear dichroism, LD) using shear-aligned lipid vesicles as model membranes has emerged as a promising tool for the characterization of the binding geometry of membrane-bound biomolecules. Here we explore the potential of retinoic acid, retinol, and retinal to function as probes of the macroscopic alignment of shear-deformed 100 nm liposomes. The retinoids display negative LD, proving their preferred alignment perpendicular to the membrane surface. The magnitude of the LD indicates the order retinoic acid > retinol > retinal regarding the degree of orientation in all tested lipid vesicle types. It is concluded that mainly nonspecific electrostatic interactions govern the apparent orientation of the retinoids within the bilayer. We propose a simple model for how the effective orientation may be related to the polarity of the end groups of the retinoid probes, their insertion depths, and their angular distribution of configurations around the membrane normal. Further, we provide evidence that the retinoids can sense subtle structural differences due to variations in membrane composition and we explore the pH sensitivity of retinoic acid, which manifests in variations in absorption maximum wavelength in membranes of varying surface charge. Based on LD measurements on cholesterol-containing liposomes, the influence of membrane constituents on bending rigidity and vesicle deformation is considered in relation to the macroscopic alignment, as well as to lipid chain order on the microscopic scale.     

Effects of lipid chain length on molecular interactions between paclitaxel and phospholipid within model biomembranes

Molecular interactions between an anticancer drug, paclitaxel, and phosphatidylcholine (PC) of various chain lengths were investigated in the present work by the Langmuir film balance technique and differential scanning calorimetry (DSC). Both the lipid monolayer at the air-water interface and lipid bilayer vesicles (liposomes) were employed as model biological cell membranes. Measurement and analysis of the surface pressure versus molecular area curves of the mixed monolayers of phospholipids and paclitaxel under various molar ratio showed that phospholipids and paclitaxel formed a nonideal miscible system at the interface. Paclitaxel exerted an area-condensing effect on the lipid monolayer at small molecular surface areas and an area-expanding effect at large molecular areas, which could be explained by the intermolecular forces and geometric accommodation between the two components. Paclitaxel and phospholipids could form thermodynamically stable monolayer systems: the stability increased with the chain length in the order DMPC (C14:0)>DPPC (C16:0)>DSPC (C18:0). Investigation of paclitaxel penetration into the pure lipid monolayer showed that DMPC had a higher ability to incorporate paclitaxel and the critical surface pressure for paclitaxel penetration also increased with the chain length in the order DMPC>DPPC>DSPC. A similar trend was testified by DSC studies on vesicles of the mixed paclitaxel/phospholipids bilayer. Paclitaxel showed the greatest interaction with DMPC while little interaction could be measured in the paclitaxel/DSPC liposomes. Paclitaxel caused broadening of the main phase transition without significant change at the peak melting temperature of the phospholipid bilayers, which demonstrated that paclitaxel was localized in the outer hydrophobic cooperative zone of the bilayer. The interaction between paclitaxel and phospholipid was nonspecific and the dominant factor in this interaction was the van der Waals force or hydrophobic force. As the result of the lower net van der Waals interaction between hydrocarbon chains for the shorter acyl chains, paclitaxel interacted more readily with phospholipids of shorter chain length, which also increased the bilayer intermolecular spacing.     

Exploiting Benzophenone Photoreactivity To Probe the Phospholipid Environment and Insertion Depth of the Cell-Penetrating Peptide Penetratin in Model Membranes

Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, thus implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles, depending on their phospholipid content. To evaluate the phospholipid preference of penetratin, as the first step of translocation, we exploited the benzophenone triplet kinetics of hydrogen abstraction, which is slower for secondary than for allylic hydrogen atoms. By using multilamellar vesicles of varying phospholipid content, we identified and characterized the cross-linked products by MALDI-TOF mass spectrometry. Penetratin showed a preference for negatively charged (vs. zwitterionic) polar heads, and for unsaturated (vs. saturated) and short (vs. long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.     

Exploiting Benzophenone Photoreactivity To Probe the Phospholipid Environment and Insertion Depth of the Cell‐Penetrating Peptide Penetratin in Model Membranes

Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, thus implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles, depending on their phospholipid content. To evaluate the phospholipid preference of penetratin, as the first step of translocation, we exploited the benzophenone triplet kinetics of hydrogen abstraction, which is slower for secondary than for allylic hydrogen atoms. By using multilamellar vesicles of varying phospholipid content, we identified and characterized the cross‐linked products by MALDI‐TOF mass spectrometry. Penetratin showed a preference for negatively charged (vs. zwitterionic) polar heads, and for unsaturated (vs. saturated) and short (vs. long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.     

Effect of chain length and asymmetry on material properties of bilayer membranes

Dissipative particle dynamics is used to extract the material parameters (bending and area stretch moduli) of a bilayer membrane patch. Some experiments indicate that the area stretch modulus of lipid vesicles varies little as the chain length of the lipids composing the bilayer increases. Here we show that making the interactions between the hydrophilic head groups of the model amphiphiles proportional to the hydrophobic tail length reproduces the above result for the area stretch modulus. We also show that the area stretch modulus of bilayers composed of amphiphiles with the same number of tail beads but with asymmetric chains is less than that of bilayers with symmetric chains. The effects on the bilayer density and lateral stress profiles of changes to the amphiphile architecture are also presented.     

A Fluorinated Detergent for Membrane‐Protein Applications

Surfactants carrying fluorocarbon chains hold great promise as gentle alternatives to conventional hydrocarbon‐based detergents for the solubilization and handling of integral membrane proteins. However, their inertness towards lipid bilayer membranes has limited the usefulness of fluorinated surfactants in situations where detergent‐like activity is required. We demonstrate that fluorination does not necessarily preclude detergency, as exemplified by a fluorinated octyl maltoside derivative termed F₆OM. This nonionic compound readily interacts with and completely solubilizes phospholipid vesicles in a manner reminiscent of conventional detergents without, however, compromising membrane order at subsolubilizing concentrations. Owing to this mild and unusual mode of detergency, F₆OM outperforms a lipophobic fluorinated surfactant in chaperoning the functional refolding of an integral membrane enzyme by promoting bilayer insertion in the absence of micelles.     

Structural Studies Reveal Enantiospecific Recognition of a DNA G‐Quadruplex by a Ruthenium Polypyridyl Complex

By using X‐ray crystallography, we show that the complexes Λ/Δ‐[Ru(TAP)₂(11‐CN‐dppz)]²⁺ (TAP=1,4,5,8‐tetraazaphenanthrene, dppz=dipyridophenazine) bind DNA G‐quadruplex in an enantiospecific manner that parallels the specificity of these complexes with duplex DNA. The Λ complex crystallises with the normally parallel stranded d(TAGGGTTA) tetraplex to give the first such antiparallel strand assembly in which syn‐guanosine is adjacent to the complex at the 5′ end of the quadruplex core. SRCD measurements confirm that the same conformational switch occurs in solution. The Δ enantiomer, by contrast, is present in the structure but stacked at the ends of the assembly. In addition, we report the structure of Λ‐[Ru(phen)₂(11‐CN‐dppz)]²⁺ bound to d(TCGGCGCCGA), a duplex‐forming sequence, and use both structural models to provide insight into the motif‐specific luminescence response of the isostructural phen analogue enantiomers.     

Sodium ion internalized within phospholipid membranes

Seven phospholipids, modified with ester groups in their hydrophobic chains, were synthesized and examined for their ability to promote sodium ion flux across vesicular membranes. It was found by 23Na NMR that only the phospholipids having short chain segments beyond their terminal ester groups catalyze sodium ion transfer by up to 2 orders of magnitude relative to a conventional phospholipid, POPC. The rates increase with the concentration of the ester-phospholipid admixed with POPC in the bilayer. More surprisingly, the rates increase with the time allowed for the vesicles to age. This was attributed to ester-phospholipid migrating in the bilayers to form domains that solubilize the sodium ion within the hydrocarbon interior of the membrane. Such membrane domains explain why shift reagent-modified NMR spectra display three 23Na signals representing sodium outside the vesicles, sodium within the vesicular water pools, and sodium within the membranes themselves.     

H-ras protein in a bilayer: interaction and structure perturbation

Ras GTPases become functionally active when anchored to membranes by inserting their lipid modified side chains. Their role in cell division, development, and cancer has made them targets of extensive research efforts, yet the mechanism of membrane insertion and the structure of the resulting complex remain elusive. Recently, the structure of the full-length H-ras protein in a DMPC bilayer has been computationally characterized. Here, the atomic interactions between the H-ras membrane anchor and the DMPC bilayer are investigated in detail. We find that the palmitoylated cysteines and Met182 have dual contributions to membrane affinity: hydrogen bonding by their amides and van der Waals interaction by their hydrophobic side chains. The polar side chains help maintain the orientation of the anchor. Although the overall structure of the bilayer is similar to that of a pure DMPC, there are localized perturbations. These perturbations depend on the insertion depth and backbone localization of the anchor, which in turn is modulated by the catalytic domain and the linker. The pattern of anchor amide-DMPC phosphate/carbonyl hydrogen bonds and the flexibility of Palm184 are important in discriminating between different modes of ras-DMPC interactions. The results provide structural arguments in support of the proposed participation of ras in the organization of membrane nanoclusters.     

Comparative molecular dynamics study of ether- and ester-linked phospholipid bilayers

The lipid membranes found in archaea have high bilayer stability and low permeability. The molecular structure of their constituent lipids is characterized by ether-linked, branched hydrophobic chains, whereas the conventional lipids obtained from eukaryotic or eubacterial sources have ester linked straight chains. In order to elucidate the influence of the ether linkage, instead of an ester one, on the physical properties of the lipid bilayers, we have carried out comparative 10 ns molecular dynamics simulations of diphytanyl phosphatidylcholine (ether-DPhPC) and diphytanoyl phosphatidylcholine (ester-DPhPC) bilayers in water, respectively. We analyze bilayer structures, hydration of the lipids, membrane dipole potentials, and free energy profiles of water and oxygen across the bilayers. We observe that the membrane dipole potential for the ether-DPhPC bilayer, which arises mainly from the ether linkage, is about half of that of the ester-DPhPC. The calculated free energy barrier for a water molecule in the ether-DPhPC bilayer system is slightly higher than that in the ester-DPhPC counterpart, which is in accord with experimental data.     

Lateral organization of a membrane protein in a supported binary lipid domain: direct observation of the organization of bacterial light-harvesting complex 2 by total internal reflection fluorescence microscopy

A unique method is described for directly observing the lateral organization of a membrane protein (bacterial light-harvesting complex LH2) in a supported lipid bilayer using total internal reflection fluorescence (TIRF) microscopy. The supported lipid bilayer consisted of anionic 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)] (DOPG) and 1,2-distearoly-sn-3-[phospho-rac-(1'-glycerol)] (DSPG) and was formed through the rupture of a giant vesicle on a positively charged coverslip. TIRF microscopy revealed that the bilayer was composed of phase-separated domains. When a suspension of cationic phospholipid (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine: EDOPC) vesicles (approximately 400 nm in diameter), containing LH2 complexes (EDOPC/LH2 = 1000/1), was put into contact with the supported lipid bilayer, the cationic vesicles immediately began to fuse and did so specifically with the fluid phase (DOPG-rich domain) of the supported bilayer. Fluorescence from the incorporated LH2 complexes gradually (over approximately 20 min) spread from the domain boundary into the gel domain (DSPG-rich domain). Similar diffusion into the domain-structured supported lipid membrane was observed when the fluorescent lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl: N-Rh-DOPE) was incorporated into the vesicles instead of LH2. These results indicate that vesicles containing LH2 and lipids preferentially fuse with the fluid domain, after which they laterally diffuse into the gel domain. This report describes for first time the lateral organization of a membrane protein, LH2, via vesicle fusion and subsequent lateral diffusion of the LH2 from the fluid to the gel domains in the supported lipid bilayer. The biological implications and applications of the present study are briefly discussed.     

Effect of polymer characteristics on structure of polymer-liposome complexes

In the current study, we examined the effect of polymer characteristics on the structure of complexes formed between poly(methacrylic acid-co-n-alkyl methacrylate) and with phosphatidylcholine/cholesterol liposomes. We varied the polymer concentration in the vesicles, the preparation concentration of lipid and polymer components during preparation, the molecular weight of the polymer chain, the molecular weight of the polymer's hydrophobic side groups and their mole fraction. The vesicle behavior indicated polymer-free bilayers and bilayers complexed with polymer coexisted at low polymer concentrations. As the polymer concentration exceeds a critical level, however, the system became homogeneous, indicating bilayer uniformity of the bilayer. As the polymer content was raised, the vesicle size and fluidity increased, and the transition temperature decreased. We found that the vesicle size mostly affects the membrane fluidity. We also found that the thermal properties (transition temperature and the magnitude of heat capacity of the peak, DeltaCp) are governed by the effects of the polymer on the structure of bilayer. The length of the alkyl chain of the polymer is shown to significantly affect the structure of polymer-liposome complexes, as did the chain molecular weight and mole concentration of hydrophobic group in the polymer.     

A COMPARISON OF THERMALLY INDUCED CHANGES IN SONICATED PHOSPHOLIPID AND SURFACTANT VESICLES BY MEANS OF INTRA-MOLECULAR EXCIMER-FORMING PROBE

Abstract The molecule (1,l'-dipyrenyl)-methyl ether (dipyme) was used for monitoring the bilayer fluidity of surfactant and sonicated phospholipid vesicles. In the latter systems, the observed transition temperatures ( Tc ) are identical with those found by different methods. Surfactant vesicles prepared from dioctadecyldimethylammonium bromide (DODAB) and dihexadecylphosphate (DHP) molecules manifest a similar fluidity of their bilayers as those of sonicated phospholipid vesicles below their Tc. However, unlike in phospholipid vesicles, there was no significant change of the bilayer structure above Tc observed in surfactant vesicles. DHP vesicles formed in pure water provide a different solubilization site for dipyme than those prepared in a buffer solution. Such sites are characterized by a relatively high local concentration of the probe and the appearance of the blue shifted spectrum of the excimer.     

Oxidative damage by ruthenium complexes containing the dipyridophenazine ligand or its derivatives: a focus on intercalation

Interactions with DNA by a family of ruthenium(II) complexes bearing the dppz (dppz = dipyridophenazine) ligand or its derivatives have been examined. The complexes include Ru(bpy)(2)(dppx)(2+) (dppx = 7,8-dimethyldipyridophenazine), Ru(bpy)(2)(dpq)(2+) (dpq = dipyridoquinoxaline), and Ru(bpy)(2)(dpqC)(2+) (dpqC = dipyrido-6,7,8,9-tetrahydrophenazine). Their ground and excited state oxidation/reduction potentials have been determined using cyclic voltammetry and fluorescence spectroscopy. An intercalative binding mode has been established on the basis of luminescence enhancements in the presence of DNA, excited state quenching, fluorescence polarization values, and enantioselectivity. Oxidative damage to DNA by these complexes using the flash/quench method has been examined. A direct correlation between the amount of guanine oxidation obtained via DNA charge transport and the strength of intercalative binding was observed. Oxidative damage to DNA through DNA-mediated charge transport was also compared directly for two DNA-tethered ruthenium complexes. One contains the dppz ligand that binds avidly by intercalation, and the other contains only bpy ligands, that, while bound covalently, can only associate with the base pairs through groove binding. Long range oxidative damage was observed only with the tethered, intercalating complex. These results, taken together, all support the importance of close association and intercalation for DNA-mediated charge transport. Electronic access to the DNA base pairs, provided by intercalation of the oxidant, is a prerequisite for efficient charge transport through the DNA pi-stack.     

Molecular dynamics simulations of hydrophobic and amphiphatic proteins interacting with a lipid bilayer membrane

Molecular dynamics simulations of polypeptides at high dilution near a fully hydrated bilayer membrane have been performed. In contrast to previous theoretical predictions, Monte Carlo simulations and conclusions from experiments a spontaneous insertion of amphiphatic or hydrophobic proteins into a membrane is not observed. Rather it is found that an amphiphatic chain has the tendency to remain in proximity to the membrane surface, whereas the location of a hydrophobic chain is more unbound. This is shown using two proteins, melittin and polyleucine. The conformation of the proteins and their orientation with respect to the membrane surface are discussed.     

Binding of a truncated form of lecithin:retinol acyltransferase and its N- and C-terminal peptides to lipid monolayers

Lecithin:retinol acyltransferase (LRAT) is a 230 amino acid membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. A truncated form of LRAT (tLRAT), which contains the residues required for catalysis but which is lacking the N- and C-terminal hydrophobic segments, was produced to study its membrane binding properties. Measurements of the maximum insertion pressure of tLRAT, which is higher than the estimated lateral pressure of membranes, and the positive synergy factor a argue in favor of a strong binding of tLRAT to phospholipid monolayers. Moreover, the binding, secondary structure and orientation of the peptides corresponding to its N- and C-terminal hydrophobic segments of LRAT have been studied by circular dichroism and polarization-modulation infrared reflection absorption spectroscopy in monolayers. The results show that these peptides spontaneously bind to lipid monolayers and adopt an α-helical secondary structure. On the basis of these data, a new membrane topology model of LRAT is proposed where its N- and C-terminal segments allow to anchor this protein to the lipid bilayer.