Doxyl Group Research Articles

Characterization of the immersion properties of the peripheral membrane anchor of the FATC domain of the kinase "target of rapamycin" by NMR, oriented CD spectroscopy, and MD simulations.

The multidomain ser/thr kinase "target of rapamycin" (TOR) centrally controls eukaryotic growth and metabolism. The C-terminal FATC domain is important for TOR regulation and was suggested to directly mediate TOR-membrane interactions. Here, we present a detailed characterization of the membrane immersion properties of the oxidized and reduced yeast TOR1 FATC domain (2438-2470 = y1fatc). The immersion depth was characterized by NMR-monitored interaction studies with DPC micelles containing paramagnetically tagged 5- or 16-doxyl stearic acid (5-/16-SASL) and by analyzing the paramagnetic relaxation enhancement (PRE) from Mn(2+) in the solvent. Complementary MD-simulations of micellar systems in the absence and presence of protein showed that 5-/16-SASL can move in the micelle and that 16-SASL can bend such that the doxyl group is close to the headgroup region and not deep in the interior as commonly assumed. Based on oriented CD (OCD) data, the single α-helix of oxidized/reduced y1fatc has an angle to the membrane normal of ∼30-60°/∼35-65° in neutral and ∼5-35°/∼0-30° in negatively charged bilayers. The presented experimentally well-founded models help to better understand how this redox-sensitive peripheral membrane anchor may be part of a network of protein-protein and protein-membrane interactions regulating TOR localization at different cellular membranes. Moreover, the presented work provides a good methodological reference for the structural characterization of other peripherally membrane associating proteins.

Self-assembled monolayers of radical molecules physisorbed on HOPG(0 0 0 1) substrate studied by scanning tunnelling microscopy and electron paramagnetic resonance techniques

In this paper, we present a combined STM and EPR study on the adsorption and self-organization of monolayers formed from 2-(14-Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy (16DS) and 4′,4′-Dimethylspiro(5α-cholestane-3,2′-oxazolidin)-3′-yloxy (CSL) adsorbed on a highly oriented pyrolitic graphite HOPG(0 0 0 1) substrate. Both 16DS and CSL molecules are persistent free radicals containing a paramagnetic doxyl group. The STM measurements of 16DS on HOPG(0 0 0 1) were performed at the liquid–solid interface while the studies of CSL on HOPG(0 0 0 1) were carried out under ultrahigh vacuum conditions. It was found that the 16DS molecules on the HOPG(0 0 0 1) surface form a highly-ordered monolayer with a domain structure. The high-resolution STM images show structural details of 16DS molecules on HOPG(0 0 0 1) revealing the paramagnetic doxyl group. In contrast, CSL molecules on HOPG(0 0 0 1) form a well-ordered monolayer without domain structure. The EPR results indicate that both compounds deposited on HOPG(0 0 0 1) substrate are not reduced and retain their paramagnetic character. We believe that the molecular systems described can be used in single spin detection experiments using an electron spin noise-scanning tunnelling microscopy (ESN-STM) technique. In particular, the possibility of obtaining contrast spin signals from the paramagnetic and diamagnetic parts of molecules increases the significance of our results.

Biophysical Characterization of a New Phospholipid Analogue with a Spin-Labeled Unsaturated Fatty Acyl Chain

Spin-labeled analogs of phospholipids have been used widely to characterize the biophysical properties of membranes. We describe synthesis and application of a new spin-labeled phospholipid analog, SL-POPC. The advantage of this molecule is that the EPR active doxyl group is linked to an unsaturated fatty acyl chain different to saturated phospholipid analogs used so far. The need for those analogs arises from the fact that biological membranes contain unsaturated phospholipids to a large extent. The biophysical properties of SL-POPC in membranes were characterized using EPR and NMR spectroscopy and compared with those of the saturated spin-labeled phospholipid, SL-PSPC. To this end, POPC membranes were labeled with either analog to assess whether the spin-labeled counterpart SL-POPC mimics the membrane properties better than the often used SL-PSPC. The results show that SL-POPC and SL-PSPC explore different molecular environments of the bilayer, and that the type and degree of perturbation of bilayer caused by the label moiety also differs between both analogs. We found that SL-POPC is more appropriate to assess the versatile dynamics of POPC membranes than SL-PSPC.

Synthesis of novel amphiphilic spin probes with the paramagnetic doxyl group in the polar region

The use of ESR and specially designed spin probes has led to major breakthroughs in understanding the complexity of biological membranes. Research has been focused mainly on molecular events within the lipid bilayer, and few probes have been designed for studying events in the extracellular space near the membrane surface. We have prepared a series of amphiphilic spin probes in which an ethylene glycol type hydrophilic spacer was introduced between a hydrophobic anchor and the doxyl group, placing the latter above the membrane in the extracellular space. Furthermore, the 2pπ orbital, containing the unpaired electron of the nitroxide group, would be orientated perpendicular to the membrane surface, making it more useful for ESR investigations of structural and dynamic properties close to the membrane surface in different situations of the cell life.

Spin-Labeled Alkylphospholipids in a Dipalmitoylphosphatidylcholine Bilayer: Molecular Dynamics Simulations

Molecular dynamics simulation has been performed to investigate the structural properties of perifosine and its synthetic spin-labeled alkylphospholipid analogues. The conformations adopted by these compounds in water and in a dipalmitoylphosphatidylcholine bilayer as a function of the presence and position of the N-oxyl-4',4'-dimethyloxazolidine ring (doxyl group) have been investigated by all-atom molecular dynamics. No predominant conformation was observed in water, but the molecules adopt specific orientations and conformations in the lipid bilayer. As is expected, alkyl chains tend to insert into the hydrophobic core, while charged groups stay at the lipid-water interface. A doxyl group in the middle of the alkyl chain moves up to the interface region, thus preventing adoption of the extended conformation. Compounds with a doxyl group close to the polar head group adopt conformations similar to that of unlabeled perifosine within the first nanoseconds of simulation. When the doxyl group is at the end of alkyl chain, the spin-labeled molecule needs more time to reach equilibrium. These results indicate a considerable effect of the doxyl position within the alkyl chain on its localization in the lipid bilayer and can be extended further to other similar spin probes used in the electron paramagnetic resonance spectroscopy of biological membranes.

The Interaction of Fusarium solani pisi Cutinase with Long Chain Spin Label Esters

We here present a study of the interaction between the Fusarium solani pisi cutinase mutant S120A and spin-labeled 4,4-dimethyloxazoline-N-oxyl-(DOXYL)-stearoyl-glycerol substrates in a micellar system. The interaction is detected by NMR measuring changes in chemical shift for 1H and 15N as well as relaxation parameters for backbone 1H (T1) and 15N (T1, T2) atoms as well as for side chain methyl groups 1H (T1). The detected interaction shows a weak binding of cutinase to the lipid micelles. Structural and mobility changes are located inside and around the active site, its flanking loops, and the oxyanion hole, respectively. Relaxation changes in the amino acid pairs Ser 92, Ala 93 and Thr 173, Gly 174 positioned at the edge of each of the active site flanking loops make these residues prime candidates for hinges, allowing for structural rearrangement during substrate binding. The cutinase mutant S120A used carries a 15 amino acid pro-peptide; the significance of this pro-peptide was so far undetermined. We show here that the pro-peptide is affected by the presence of the micellar substrate. Relaxation enhancements indicative of spatial proximity between the DOXYL group in the lipid chain and some hydrophobic residues surrounding the active site could be found.

Basis for Selectivity of Cationic Antimicrobial Peptides for Bacterial Versus Mammalian Membranes

Novel cationic antimicrobial peptides typified by structures such as KKKKKKAAXAAWAAXAA-NH2, where X = Phe/Trp, and several of their analogues display high activity against a variety of bacteria but exhibit no hemolytic activity even at high dose levels in mammalian erythrocytes. To elucidate their mechanism of action and source of selectivity for bacterial membranes, phospholipid mixtures mimicking the compositions of natural bacterial membranes (containing anionic lipids) and mammalian membranes (containing zwitterionic lipids + cholesterol) were challenged with the peptides. We found that peptides readily inserted into bacterial lipid mixtures, although no insertion was detected in model "mammalian" membranes. The depth of peptide insertion into model bacterial membranes was estimated by Trp fluorescence quenching using doxyl groups variably positioned along the phospholipid acyl chains. Peptide antimicrobial activity generally increased with increasing depth of peptide insertion. The overall results, in conjunction with molecular modeling, support an initial electrostatic interaction step in which bacterial membranes attract and bind peptide dimers onto the bacterial surface, followed by the "sinking" of the hydrophobic core segment to a peptide sequence-dependent depth of approximately 2.5-8 A into the membrane, largely parallel to the membrane surface. Antimicrobial activity was likely enhanced by the fact that the peptide sequences contain AXXXA sequence motifs, which promote their dimerization, and possibly higher oligomerization, as assessed by SDS-polyacrylamide gel analysis and fluorescence resonance energy transfer experiments. The high selectivity of these peptides for nonmammalian membranes, combined with their activity toward a wide spectrum of Gram-negative and Gram-positive bacteria and yeast, while retaining water solubility, represent significant advantages of this class of peptides.

Structure and dynamics of ionic aggregates in ethylene ionomer membranes: recent electron spin resonance (ESR) studies

In this review we present recent advances in the understanding of self-assembling, structure, and dynamics in poly(ethylene- co-methacrylic acid) (EMAA) ionomers as membranes and water dispersions, with focus on the information obtained by electron spin resonance (ESR) studies of amphiphilic and hydrophobic nitroxide spin probes. The probes selected for this study were doxylstearic acids and esters, with the doxyl group attached to carbon atoms at different positions relative to the head group, and a nitroxide group attached to a nonadecane backbone. Application of this approach to the study of EMAA ionomers has demonstrated that, by a judicious choice of the polarity, size, and chemical structure of the probe, it is possible to explore specific regions of the microphase-separated system; an important advantage of the ‘position-selectivity’ method is the ability to bypass the often difficult synthetic step of spin labeling. Information on the local environment of the probes and on chain mobility in, and in the vicinity of, the ionic aggregates in the EMAA ionomers will be discussed, and compared with results obtained by other techniques.

The Potential of Fluorescent and Spin-labeled Steroid Analogs to Mimic Natural Cholesterol

Cholesterol analogs are often used to investigate lipid trafficking and membrane organization of native cholesterol. Here, the potential of various spin (doxyl moiety) and fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group) labeled cholesterol analogs as well as of fluorescent cholestatrienol and the naturally occurring dehydroergosterol to mimic the unique properties of native cholesterol in lipid membranes was studied in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes by electron paramagnetic resonance, nuclear magnetic resonance, and fluorescence spectroscopy. As cholesterol, all analogs undergo fluctuating motions of large amplitude parallel to the bilayer normal. Native cholesterol keeps a strict orientation in the membrane with the long axis parallel to the bilayer normal. Depending on the chemical modification or the position of the label, cholesterol analogs may adopt an "up-side-down" orientation in the membrane or may even fluctuate between "upright" and up-side-down orientation by rotational motions about the short axis not typical for native cholesterol. Those analogs are not able to induce a comparable condensation of phospholipid membranes as known for native cholesterol revealed by 2H nuclear magnetic resonance. However, cholesterol-induced lipid condensation is one of the key properties of native cholesterol, and, therefore, a well suited parameter to assess the potential of steroid analogs to mimic cholesterol. The study points to extreme caution when studying cholesterol behavior by the respective analogs. Among seven analogs investigated, only a spin-labeled cholesterol with the doxyl group at the end of the acyl chain and the fluorophore cholestatrienol mimic cholesterol satisfactorily. Dehydroergosterol has a similar upright orientation as cholesterol and could be used at low concentration (about 1 mol %), at which its lower potential to enhance lipid packing density does not perturb membrane organization.

Copper Excess Reduces the Fluidity of Plasma Membrane Lipids of Wheat Roots: a Spin Probe EPR Study

Electron paramagnetic resonance spectroscopy was applied to investigate changes in the organization and dynamics of plasma membrane (PM) lipids of roots caused by growing wheat (Triticum durum Desf. cv. Creso) seedlings under copper excess (50 μM Cu2+). To this purpose, unilamellar vesicles were made from total lipid extracts of PMs isolated either from copper-stressed or control roots, and probed at different bilayer depth using stearic acids bearing a doxyl group at two different positions of the alkyl chain, namely at the fifth (5-DSA) and 16th (16-DSA) carbon. EPR spectra were recorded as a function of temperature in the range between 273 and 323 K and interpreted in terms of a microscopically ordered and macroscopically disordered model, in which orientational ordering and dynamics of the spin probes were described by means of the order parameter S and the rotational diffusion coefficient R, respectively. It was found that growth in Cu excess resulted in a decreased root PM fluidity. The results were...

The Distribution of Lipid Attached Spin Probes in Bilayers: Application to Membrane Protein Topology

The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by 1H and 13C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, 1H and 13C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both 1H and 13C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that 1H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by 13C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.

Elasticity of vesicles assessed by electron spin resonance, electron microscopy and extrusion measurements

The composition of vesicles determines the physical state and elasticity of their bilayers. Fatty acid spin labels were incorporated into vesicles, composed of the single chain non-ionic surfactant octaoxyethylenelaurate-ester (PEG-8-L), the sucrose laurate-ester l-595 and cholesterol sulfate (CS) to monitor local dynamic properties of lipid molecules in vesicle bilayers and to study the elasticity of vesicle bilayers. Studies with the spin label probes 5-, 12- and 16-doxyl stearic acid (DSA) indicated that both the order parameter and the rotational correlation times increased when the doxyl group was positioned closer to the headgroup region. These findings indicate that the fluidity of membranes decreased near the headgroup region. Comparing 16-DSA incorporated in vesicle formulations with either 30 or 70 mol% showed no difference in alkyl chain mobility as was reflected by the order parameter. The rotational correlation times, however, showed a slowdown from 0.38 to 0.71 and 1.13 ns when the PEG-8-L molar content was decreased from 100 to 70 and 30 mol% for PEG-8-L:L-595:CS vesicles, respectively. Extrusion measurements indicated an increase in elasticity of vesicle bilayers as the molar content of PEG-8-L was increased from 10 to 90 mol%. Incorporation of cholesterol sulfate stabilizes vesicles and thereby, decreases the elasticity. The increased elasticity correlated excellent with a reduction in the rotational correlation times observed. In conclusion, these results demonstrate that when the molar content of the single chain non-ionic surfactant PEG-8-L in vesicles is increased the elasticity is enhanced and the rotational correlation time is reduced. The enhanced elasticity might contribute to an optimal design of vesicles as drug carriers for transdermal application.

Spin-labeling study of membranes in wheat embryo axes. 1. Partitioning of doxyl stearates into the lipid domains

The interaction of lipid soluble spin labels with wheat embryo axes has been investigated to obtain insight into the structural organization of lipid domains in embryo cell membranes, using conventional electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy. Stearic acid spin labels ( n-SASL) and their methylated derivatives ( n-MeSASL), labelled at different positions of their doxyl group ( n=5, 12 and 16), were used to probe the ordering and molecular mobility in different regions of the lipid moiety of axis cell membranes. The ordering and local polarity in relation to the position of the doxyl group along the hydrocarbon chain of SASL, determined over the temperature range from −50 to +20°C, are typical for biological and model lipid membranes, but essentially differ from those in seed oil droplets. Positional profiles for ST-EPR spectra show that the flexibility profile along the lipid hydrocarbon chain does exist even at low temperatures, when most of the membrane lipids are in solid state (gel phase). The ordering of the SASL nitroxide radical in the membrane surface region is essentially higher than that in the depth of the membrane. The doxyl groups of MeSASLs are less ordered (even at low temperatures) than those of the corresponding SASLs, indicating that the MeSASLs are located in the bulk of membrane lipids rather than in the protein boundary lipids. The analysis of the profiles of EPR and ST-EPR spectral parameters allows us to conclude that the vast majority of SASL and MeSASL molecules accumulated in embryo axes is located in the cell membranes rather than in the interior of the oil bodies. The preferential partitioning of the doxyl stearates into membranes demonstrates the potential of the EPR spin-labelling technique for the in situ study of membrane behavior in seeds of different hydration levels.

Electron spin resonance (ESR) spectra of amphiphilic spin probes in the triblock copolymer EO13PO30EO13 (Pluronic L64): hydration, dynamics and order in the polymer aggregates

Aqueous solutions of the triblock copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) EO13PO30EO13 (Pluronic L64) were investigated over a wide concentration range (20–100%, (w/w) polymer) in the micellar, liquid crystalline and reverse micellar phases, using electron spin resonance (ESR) spectroscopy of spin probes. A series of amphiphilic nitroxide spin probes based on n-doxylstearic acid (nDSA) with n, the carbon atom to which the doxyl group is attached, equal to 5 and 10 were used to measure the local polarity, dynamics and degree of order in the self-assembled system. The 14N isotropic hyperfine splitting, aN, was the polarity sensitive parameter. The probe location and the corresponding effective local hydration, Zeff, were deduced by comparing ESR spectra of the probes in L64 solutions with spectra of the probes in aqueous solutions of poly(ethylene oxide) (PEO), poly(propylene oxide) (PPO) and a mixture of PEO and PPO containing the monomer molar ratio 26:30, as in L64. The results indicate that the probes reside in and provide evidence for the presence of hydrophobic and non-hydrated regions consisting of PO blocks and that the order in the aggregates decreases from the PO/EO interface toward the PO domains. Additional support for these conclusions was obtained from ESR spectra of the probes in the lamellar phase as a function of added cholesterol and by simulations of the ESR spectra of the probes in the lamellar phase of L64. This study of the hydrophobic part of the aggregates, together with our previous study based on ESR spectra of cationic probes that reside in the polar and hydrated EO regions, lead to a detailed description of the nature of L64 aggregates in aqueous solutions.

Diffusion Coefficients of Small Molecules as Guests in Various Phases of Pluronic L64 Measured by One-Dimensional Electron Spin Resonance Imaging

The diffusion coefficients of small nitroxide probes as guests in aqueous solutions of the triblock copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) EO13PO30EO13 (Pluronic L64) were measured at 300 K by one-dimensional electron spin resonance imaging (1D ESRI). The method is based on encoding the spatial distribution of the probes as a function of time in ESR spectra recorded in the presence of magnetic field gradients and simulation of these spectra in order to extract the diffusion coefficient, D. The rate of transport of each probe as a function of polymer content in the various phases (micellar, hexagonal, lamellar, and reverse micellar) of aqueous L64 depends on the probe location in the self-assembled system. The probe site was deduced from the analysis of the ESR spectra; the isotropic hyperfine splitting, aN, from the 14N nucleus of the >NO fragment in the probes was the polarity sensitive parameter. D values for the cationic probe 4-(N,N,N-trimethyl)ammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl iodide that is known to reside in the water domains follow the expression D = D0 exp(−aw2), where D0 is the diffusion coefficient in the neat solvent and w2 is the weight fraction of the polymer. For the hydrophobic probe 5DSE, the methyl ester of doxylstearic acid where 5 indicates the carbon atom to which the doxyl group is attached, D is significantly lower and almost constant for w2 in the range 0.20−0.80. For the probe perdeuterio-2,2‘,6,6‘-tetramethyl-piperidone-N-oxide that is located at the interface between water and the EO domains, D decreases with increase in the polymer content, but the decrease is more prominent for w2 in the range 0.10−0.30; at w2 = 0.90, D is similar to that of the polymer chains. The method of 1D ESRI enables the measurement of D values for guests present in small concentrations, typically ≤2 × 10-3 mol/L. This method and the results obtained in this study are relevant for assessing both the rate of transport of drugs in drug delivery systems and the partitioning of various guests in complex systems, for instance in biological membranes.

Structural Modifications of DMPC Vesicles upon Interaction with Poly(amidoamine) Dendrimers Studied by CW-Electron Paramagnetic Resonance and Electron Spin−Echo Techniques

This report describes a computer-aided CW- and pulsed-electron paramagnetic resonance (EPR) investigation on the structural modification of dimyristoylphosphatidylcoline (DMPC) vesicles, which occur upon interaction with starburst dendrimers (SBDs). Probes used for this study included doxyl-functionalized stearic acids, with the doxyl group attached at different positions of the stearic chain (5DXSA, 12DXSA, and 16DXSA). Mainly mobility and polarity parameters were evaluated from the analysis of the CW-EPR spectra, whereas the analysis of the decay and modulation of the electron spin-echo (ESE) signal provided information on the structural environment of the paramagnetic center. Due to the interaction with the SBD surface, the vesicle structure became more rigid and ordered. The enhanced rigidity of the structure also caused the tilting of the chains of about 50° with respect to the surface line. The permeability of water at the chain beginning level increased, thus increasing the rotational mobility of the probe. The perturbing effects lessened toward the end of the chains. A fraction of 16DXSA (15%) was in the bent conformation, with the chain inserted into the lipid layer and the two polar heads at the external surface. The interaction with protonated dendrimers caused the swelling of the vesicle structure. This study indicated that the bilayer structure is modified but only partially perturbed by the addition of the dendrimers, and the integrity of the vesicle, as a model cell membrane, is preserved after the interaction with the dendrimers. This is encouraging for the use of the SBDs as drug and gene carriers.

Topological disposition of Cys 222 in the alpha-subunit of nicotinic acetylcholine receptor analyzed by fluorescence-quenching and electron paramagnetic resonance measurements.

The structure of the nicotinic acetylcholine receptor (AChR) has been studied using a combination of fluorescence quenching and electron paramagnetic resonance (EPR) collision gradient methods. The AChR from Torpedo californica was labeled with a fluorescent probe, N-(1-pyrenyl)maleimide, specific for sulfhydryls in a hydrophobic environment, under conditions of selective labeling of Cys222 in the alpha-subunit. alphaCys222 is located in the postulated M1 transmembrane domain and predicted to be at the center of an alpha-helical secondary structure. The spatial disposition of the acetylcholine receptor-bound pyrene with respect to the membrane bilayer was assessed by fluorescence quenching measurements. Quenching of pyrene fluorescence by spin-labeled fatty acids with the doxyl group at positions C-5 and C-12 revealed that the former was more effective, suggesting that the fluorophore is located closer to the membrane-water interface than to the hydrophobic interior. Power saturation EPR spectroscopy was also used to examine the effect of molecular oxygen and water-soluble paramagnetic reagents on the saturation behavior of a nitroxide spin label, which was specifically attached to the same alphaCys222 residue. Using the gradients of these paramagnetic reagents through the membrane-solution interface, the distance for the nitroxide derivative from the membrane-solution interface was measured to be approximately 7 A from the headgroup region of the phospholipid bilayer, in agreement with fluorescence quenching results. These results suggest that the M1 transmembrane domain of the AChR probably forms an irregular structure, a beta-strand, or an alpha-helical structure that may span the membrane in a way different from a linear alpha-helix.

Interaction of glucagon with dimyristoylphosphatidylcholine in vesicular and discoidal complexes.

Glucagon fragments dimyristoylphosphatidylcholine liposomes into discoidal complex under appropriate conditions. The concentration of glucagon required to fragment the vesicles increases with increasing pH, which appears to be related to the glucagon binding. It was also observed that the fragmentation is facilitated by NaCl, which is also due to increased glucagon binding. From the quenching of Trp fluorescence by doxyl group located at various positions of the acyl chain of the lipid, Trp of glucagon was found to be located close to the bilayer surface in the vesicular complex. However, the Trp fluorescence was quenched by the doxyl group in the discoidal complex to an equal extent regardless of the position of this spin label in the acyl chain. This and the results of second derivative UV spectroscopy of Tyr suggested that segments including Tyr-13 and Trp-25 are involved in the discoidal complex formation and that the orientation of glucagon is not normal to the bilayer surface.

Interaction between Starburst Dendrimers and SDS micelles studied by continuous-wave and pulsed electron spin resonances

A computer aided analysis of both cw-and pulsed-Electron Paramagnetic Resonance (EPR) spectra of 5doxylstearic acid (5DXSA) as a probe was carried out to compare the aggregation process of Sodium Dodecyl Sulfate (SDS) surfactants in the absence and in the presence of Starburst Dendrimers (SBDs), and to provide information on the interactions between SDS and SBDs. Mobility and polarity parameters were extracted from the cw-EPR analysis whereas the analysis of the Electron Spin Echo Envelope Modulation (ESEEM) signal provided details about the doxyl environment in the SDS micelles. In the absence of SBDs, the formation of SDS micelles was revealed by the decrease in mobility of the probes inserted in the micelles. The high packing of SDS chains in the micelles prevented the water permeability at the doxyl site. In the presence of the dendrimers, the analysis of the EPR spectra suggested the formation of SDS aggregates at the dendrimer surface (cooperative interaction). The larger the size of the dendrimers and the protonation of their surface, the stronger the interactions resulted between the SDS surfactants and the SBD surface. The analysis of the ESEEM pattern indicated that the cooperative interaction of the surfactant with the SBD surface led to a less packed structure of the aggregates. A schematic view was proposed to describe the local structure of the doxyl group and its environment in the absence and in the presence of the dendrimers.

Hydration and Dynamics in Reverse Micelles of the Triblock Copolymer EO13PO30EO13 in Water/o-Xylene Mixtures: A Spin Probe Study

The reverse micellar phase L2 of the triblock copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) EO13PO30EO13 (commercial name Pluronic L64), where EO is ethylene oxide and PO is propylene oxide, was studied in the ternary mixture L64/water/o-xylene and in the binary mixture L64/water, using ESR spectroscopy with nitroxide spin probes. The spin probes differed in size, structure, and polarity and belong to two main types: (a) cationic probes 4-(N,N-dimethyl-N-alkyl)ammonio)-2,2‘,6,6‘-tetramethylpiperidine-1-oxyl iodide (CATn) with n, the number of carbon atoms in the alkyl substituent, equal to 1, 4, 8, 11, and 16; (b) amphiphilic probes based on x-doxylstearic acid (xDSA) with x, the carbon atom to which the doxyl group is attached, equal to 5, 7, 10, and 16. X-band ESR spectra reflect the intercalation of the probes in the self-assembled L64 system, but different probes choose different locations in the aggregates and report on the local polarity, hydration, and degree of ord...