Amphiphilic dynamic NDI and PDI probes: imaging microdomains in giant unilamellar vesicles

Dynamic amphiphiles provide access to transmembrane ion transport, differential sensing and cellular uptake. In this report, we introduce dynamic amphiphiles with fluorescent tails. Core-substituted naphthalenediimides (cNDIs) and perylenediimides (cPDIs) are tested. Whereas the latter suffer from poor partitioning, dynamic cNDI amphiphiles are found to be purifiable by RP-HPLC, to partition selectively into liquid-disordered (Ld) microdomains of mixed lipid bilayers and to activate DNA as transporters. Importantly, fluorescence properties, partitioning and activity can be modulated by changes in the structure of mixed amphiphiles. These results confirm the potential of dynamic fluorescent amphiphiles to selectively label extra- and intracellular membrane domains and visualize biological function.     

Interaction of tetraphenyl-porphyrin derivatives with DPPC-liposomes: an EPR study

The effect of the symmetry and polarity of the porphyrin molecules on their membrane localization and interaction with membrane lipids were investigated by electron paramagnetic resonance (EPR). For this purpose, two glycoconjugated tetraphenyl porphyrin derivatives were selected, respectively, symmetrically and asymmetrically substituted. Small unilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and spin labeled stearic acids were prepared. The spin probe was located at the 5th or 7th or 12th or 16th position of the hydrocarbon chain in order to monitor various regions of the lipid bilayer. EPR spectra of porphyrin-free and porphyrin-bound liposomes were recorded at various temperatures below and above the phase transition temperature of DPPC. The effect on membrane fluidity proved to be stronger with the asymmetrical porphyrin derivative than with the symmetrical one. The rigidity increased when the spin label was near lipid head groups. The difference observed between control and porphyrin-treated samples when measured below the main lipid transition temperature disappeared at higher temperature. When the spin label was near the end of the hydrophobic tails, the symmetrical porphyrin derivative caused increase in fluidity, while the asymmetrical one slightly decreased it. To explain this phenomenon we propose that the asymmetrical derivative exerts a stronger ordering effect caused by its fluorophenyl group located at the level of the lipid heads, which is attenuated to the hydrophobic tails. The perturbing effect of the symmetric derivative could not lead to similar extent of ordering at the head groups and looses the hydrocarbon chains deeper in the membrane.     

Effect of tetracaine on model and erythrocyte membranes by DSC and EPR

Summary DSC and EPR experiments were performed on human erythrocyte membranes and DPPC vesicles in order to study the effect of the anaesthetic drug tetracaine on structure and dynamics of the lipid region. Experiments using spin label technique showed that tetracaine induced fluidity changes of the lipid region in the environment of the fatty acid probe molecules incorporated into the membranes in the vicinity of the lipid-water interface. Similarly to EPR observations, DSC measurements reported decrease of the main melting and the pretransition temperature in comparison to control DPPC vesicles, which is the sign of destabilisation of the structure in the head group region of the lipids. Similar effect was observed in the case of erythrocytes where the protein conformation was also controlled in the presence of drug. A separated membrane melting with well distinguished membrane protein phase transition was found that was affected significantly by tetracaine. These results suggest that tetracaine is able to modify not only the internal dynamics of erythrocyte membranes and produce destabilisation of the lipid structure, but the protein system as well. These might lead to further damage of the biological functions.     

Selective High‐Resolution Detection of Membrane Protein–Ligand Interaction in Native Membranes Using Trityl–Nitroxide PELDOR

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero‐oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro–electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter‐subunit interaction and the intra‐subunit conformational changes in hetero‐oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein–ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.     

Electron paramagnetic resonance studies of an integral membrane peptide inserted into aligned phospholipid bilayer nanotube arrays

This communication reports for the first time the determination of the helical tilt of an integral membrane peptide inserted into aligned phospholipids bilayer nanotube arrays using spin label EPR spectroscopy. Also, we demonstrate herein how the helical tilt of the peptide can be easily calculated using the hyperfine splitting values gleaned from a perpendicularly aligned bilayer in phospholipid bilayer nanotube arrays. EPR spectral simulations were used to verify the method.     

EPR spin probe investigation of irradiated wheat,rice and sunflower seeds

TEMPO and 4-nitro-TEMPO spin probes were used to monitor dose-dependent changes in the EPR spectra of irradiated wheat and rice embryos and sunflower embryo parts. Rice embryos were studied in the 233–293 K temperature range using 4-nitro-TEMPO. TEMPAMINE, TEMPYO and DTBN spin probes were also studied for their applicability in the determination of irradiated seeds. All the recorded spectra were simulated, and spectral parameters and partition of the probes among various domains were determined. Despite the contribution of the signal from extracellular regions, it was possible to detect the changes in the water/lipid ratios with dose. The hydrophilic character of the probe alone was not sufficient to distinguish the different doses of irradiation. Line widths and rotational correlation times of various domains within embryo also play an important role. Partition after dehydration was another measure in the selection of the suitable probes for irradiation studies. Better results were obtained in dehydrated embryos for the probes preferring lipid bodies.     

Specific binding structures of dendrimers on lipid bilayer membranes

Dissipative particle dynamics simulations are used to study the specific binding structures of polyamidoamine (PAMAM) dendrimers on amphiphilic membranes and the permeation mechanisms. Mutually consistent coarse-grained (CG) models both for PAMAM dendrimers and for dimyristoylphosphatidylcholine (DMPC) lipid molecules are constructed. The PAMAM CG model describes correctly the conformational behavior of the dendrimers, and the DMPC CG model can properly give the surface tension of the amphiphilic membrane. A series of systematic simulations is performed to investigate the binding structures of the dendrimers on membranes with varied length of the hydrophobic tails of amphiphiles. The permeability of dendrimers across membranes is enhanced upon increasing the dendrimer size (generation). The length of the hydrophobic tails of amphiphiles in turn affects the dendrimer conformation, as well as the binding structure of the dendrimer-membrane complexes. The negative curvature of the membrane formed in the dendrimer-membrane complexes is related to dendrimer concentration. Higher dendrimer concentration together with increased dendrimer generation is observed to enhance the permeability of dendrimers across the amphiphilic membranes.     

Membrane dynamics of the amphiphilic siderophore, acinetoferrin

Acinetobacter haemolyticus is an antibiotic resistant, pathogenic bacterium responsible for an increasing number of hospital infections. Acinetoferrin (Af), the amphiphilic siderophore isolated from this organism, contains two unusual trans-2-octenoyl hydrocarbon chains reminiscent of a phospholipid structural motif. Here, we have investigated the membrane affinity of Af and its iron complex, Fe-Af, using small and large unilamellar phospholipid vesicles (SUV and LUV) as model membranes. Af shows a high membrane affinity with a partition coefficient, K(x)= 6.8 x 10(5). Membrane partitioning and trans-membrane flip-flop of Fe-Af have also been studied via fluorescence quenching of specifically labeled vesicle leaflets and (1)H NMR line-broadening techniques. Fe-Af is found to rapidly redistribute between lipid and aqueous phases with dissociation/partitioning rates of k(off) = 29 s(-1) and k(on) = 2.4 x 10(4) M(-1) s(-1), respectively. Upon binding iron, the membrane affinity of Af is reduced 30-fold to K'(x) = 2.2 x 10(4) for Fe-Af. In addition, trans-membrane flip-flop of Fe-Af occurs with a rate constant, k(p) = 1.2 x 10(-3) s(-1), with egg-PC LUV and a half-life time around 10 min with DMPC SUV. These properties are due to the phospholipid-like conformation of Af and the more extended conformation of Fe-Af that is enforced by iron binding. Remarkable similarities and differences between Af and another amphiphilic siderophore, marinobactin E, are discussed. The potential biological implications of Af and Fe-Af are also addressed. Our approaches using inner- and outer-leaflet-labeled fluorescent vesicles and (1)H NMR line-broadening techniques to discern Af-mediated membrane partitioning and trans-membrane diffusion are amenable to similar studies for other paramagnetic amphiphiles.     

LIGHT-INDUCED LEAKAGE OF SPIN LABEL MARKER FROM LIPOSOMES IN THE PRESENCE OF PHOTOTOXIC PHENOTHIAZINES

Abstract— Liposomes prepared from dipalmitoyl lecithin, cholesterol and dicetyl phosphate and containing a trapped spin label marker were exposed to long wavelength UV light in the presence of a series of phenothiazine tranquilizers. EPR spectroscopy was used to detect spin label marker released from liposomes, taking advantage of the disappearance of line broadening from electron spin exchange which occurred on spin label release. The minimum effective phototoxic dose in mice of these phenothiazines was also determined. Kinetic studies of light-induced spin label release from phenothiazine-sensitized liposomes showed that membrane damage was rapidly induced and that the damaging species were short-lived. The damage process was oxygen dependent and could be temporarily prevented by cysteamine or α-tocopherol added immediately before irradiation. Only those phenothiazines which mediated light-dependent liposomal membrane damage had phototoxic activity in mice and the degree of photosensitization was parallel in the two systems. In both photosensitization phenomena, the nature of the substituent at the phenothiazine 2-position was more important than the phenothiazine side chain.     

The use of simulation in the EPR spin probe technique for detection of irradiated seeds

An electron paramagnetic resonance (EPR) spin probe study of irradiated wheat seeds was performed depending on irradiation dose. The structural changes in the membrane integrity were followed using aqueous solutions of 4-hydroxy-TEMPO (TANOL) spin probe and a line broadening material. In the studies dry seed embryos were kept in these solutions for 150 min. The spectra were recorded at various times of air drying process. The simulation of these spectra indicated a decrease in the water content of the embryos depending on the increasing irradiation dose. This indicates the increase in the permeability of the membranes as a result of the radiation damage. From the decay curves it is possible to determine about irradiation dose, however, this approach is not very successful at close irradiation doses.     

A Bifunctional Spin Label for Ligand Recognition on Surfaces

In situ monitoring of biomolecular recognition, especially at surfaces, still presents a significant technical challenge. Electron paramagnetic resonance (EPR) of biomolecules spin‐labeled with nitroxides can offer uniquely sensitive and selective insights into these processes, but new spin‐labeling strategies are needed. The synthesis and study of a bromoacrylaldehyde spin label (BASL), which features two attachment points with orthogonal reactivity is reported. The first examples of mannose and biotin ligands coupled to aqueous carboxy‐functionalized gold nanoparticles through a spin label are presented. EPR spectra were obtained for the spin‐labeled ligands both free in solution and attached to nanoparticles. The labels were recognized by the mannose‐binding lectin, Con A, and the biotin‐binding protein avidin‐peroxidase. Binding gave quantifiable changes in the EPR spectra from which binding profiles could be obtained that reflect the strength of binding in each case.     

Effect of bilayer thickness on membrane bending rigidity

The bending rigidity k(c) of bilayer vesicles self-assembled from amphiphilic diblock copolymers has been measured using single- and dual-micropipet techniques. These copolymers are nearly a factor of 5 greater in hydrophobic membrane thickness d than their lipid counterparts and an order of magnitude larger in molecular weight M(n). The macromolecular structure of these amphiphiles lends insight into and extends relationships for traditional surfactant behavior. We find the scaling of k(c) with thickness to be nearly quadratic, in good agreement with existing theories for bilayer membranes. The results here are key to understanding and designing soft interfaces such as biomembrane mimetics.     

Bis-Thioether-Containing Lipid Chains in Cationic Amphiphiles: Physicochemical Properties and Applications in Gene Delivery

Cationic amphiphiles featuring two thioether functions in each lipid chain of bicatenar cationic amphiphiles are reported here for the first time. The physicochemical properties and transfection abilities of these new amphiphiles were compared with those of already reported analogues featuring either (i) saturated, (ii) unsaturated or (iii) mono-thioether containing lipid chains. The homogeneity of the series of new compounds allowed to clearly underscore the effect of bis-thioether containing lipid chains. This study shows that besides previous strategies based on unsaturation or ramification, the incorporation of two thioether functions per lipid chain constitutes an original complementary alternative to tune the supramolecular properties of amphiphilic compounds. The potential of this strategy was evaluated in the context of gene delivery and report that two cationic amphiphiles (i. e. 4 a and 4 b) can be proposed as new efficient transfection reagents.     

New synthesis of a spin-labeled peptide nucleic acid and its interactions with nucleic acids

A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) is developed. The methodology involved initial preparation of a protected PNA on solid phase, followed by efficient solution phase coupling to a spin label containing a reactive carboxylic group. This strategy allows to maintain the integrity of the nitroxide moiety during the various steps of chemical synthesis assuring in the same time the fidelity of the hybridization assay. This compound can be used as a reporter molecule to investigate the binding of peptide nucleic acids to oligonucleotide sequences (DNA or RNA) by EPR spectroscopy.     

Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties

The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane?polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic bacterial cellulose membranes versus hydrophobic modified poly(ether)sulfone membranes. These results are discussed with reference to analysis and utilization of biofunctional membranes.     

A new glucosamine-containing amphiphilic spin probe

A new, nonionic amphiphilic spin probe for investigating the extracellular matrix close to the cell membrane by EPR spectroscopy has been synthesized and characterized. A pyrrolidine type nitroxide spin-label has been introduced to the third position of a nonionic sugar polar head (glucosamine) bonded to a lipophilic stearic acid acyl chain anchor. The compound is soluble in polar organic solvents such as ethanol and chloroform, but is sparingly soluble in water.     

A Spirocyclohexyl Nitroxide Amino Acid Spin Label for Pulsed EPR Spectroscopy Distance Measurements

Site‐directed spin labeling and EPR spectroscopy offer accurate, sensitive tools for the characterization of structure and function of macromolecules and their assemblies. A new rigid spin label, spirocyclohexyl nitroxide α‐amino acid and its N‐(9‐fluorenylmethoxycarbonyl) derivative, have been synthesized, which exhibit slow enough spin‐echo dephasing to permit accurate distance measurements by pulsed EPR spectroscopy at temperatures up to 125 K in 1:1 water/glycerol and at higher temperatures in matrices with higher glass transition temperatures. Distance measurements in the liquid nitrogen temperature range are less expensive than those that require liquid helium, which will greatly facilitate applications of pulsed EPR spectroscopy to the study of structure and conformation of peptides and proteins.     

In‐Situ Spin Labeling of His‐Tagged Proteins: Distance Measurements under In‐Cell Conditions

New spin labeling strategies have immense potential in studying protein structure and dynamics under physiological conditions with electron paramagnetic resonance (EPR) spectroscopy. Here, a new spin‐labeled chemical recognition unit for switchable and concomitantly high affinity binding to His‐tagged proteins was synthesized. In combination with an orthogonal site‐directed spin label, this novel spin probe, Proxyl‐trisNTA (P‐trisNTA) allows the extraction of structural constraints within proteins and macromolecular complexes by EPR. By using the multisubunit maltose import system of E. coli: 1) the topology of the substrate‐binding protein, 2) its substrate‐dependent conformational change, and 3) the formation of the membrane multiprotein complex can be extracted. Notably, the same distance information was retrieved both in vitro and in situ allowing for site‐specific spin labeling in cell lysates under in‐cell conditions. This approach will open new avenues towards in‐cell EPR.     

Fast stochastic librations and slow rotations of spin labeled stearic acids in a model phospholipid bilayer at cryogenic temperatures

The spin label DOXYL (4,4-dimethyl-oxazolidine-1-oxyl) is a nitroxyl ring that can be attached rigidly at specific C-atom positions in the stearic acid. 5-DOXYL-stearic acid and 16-DOXYL-stearic acid in 1-Palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) lipid bilayers were studied using electron spin echo (ESE) spectroscopy at low temperatures. The anisotropy of ESE decay across the electron paramagnetic resonance (EPR) spectrum evidence that these spin labels participate in orientational motions at temperatures down to approximately 120 K for 5-DOXYL-stearic acid and down to approximately 80 K for 16-DOXYL-stearic acid. Fast stochastic librations, with correlation time at the nanosecond time scale, manifest itself in a two-pulse ESE experiment. Stimulated three-pulse ESE experiment is sensitive to motions of ultrasmall amplitude, approximately 0.1-1 degrees , developing at the microsecond time scale. The stimulated ESE decays were found to depend on the product of the two time delays of the pulse sequence. This fact may be described within a simple model of slow inertial rotations developing within this small range of angles with a rate of approximately 1 kHz. Both types of motion evidence the pronounced motional heterogeneity across the bilayer at cryogenic temperatures, with a remarkable increase of motion in the bilayer interior. The found low-temperature motions imply that hydrophobic parts of amphiphilic biomolecules may possess a noticeable mobility even at temperatures as low as approximately 100 K.     

Conformationally Restricted Isoindoline‐Derived Spin Labels in Duplex DNA: Distances and Rotational Flexibility by Pulsed Electron–Electron Double Resonance Spectroscopy

Three structurally related isoindoline‐derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation‐dependent pulsed electron–electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, ^ExIm U , was synthesized and incorporated into DNA oligonucleotides. ^ExIm U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N?O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline‐based spin label to a uridine base. Continuous‐wave (CW) EPR measurements of ^ExIm U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label ^Im U , which has restricted mobility due to an intramolecular hydrogen bond. The X‐band CW‐EPR spectra of ^ExIm U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels ^Im U , ^Ox U , and ^ExIm U in duplex DNA showed a strong angular dependence for ^Im U , a medium dependence for ^Ox U , and no orientation effect for ^ExIm U . Thus, precise distances can be extracted from ^ExIm U without having to take orientational effects into account.