Assessing the size, stability, and utility of isotropically tumbling bicelle systems for structural biology

Aqueous phospholipid mixtures that form bilayered micelles (bicelles) have gained wide use by molecular biophysicists during the past 20 years for spectroscopic studies of membrane-bound peptides and structural refinement of soluble protein structures. Nonetheless, the utility of bicelle systems may be compromised by considerations of cost, chemical stability, and preservation of the bicelle aggregate organization under a broad range of temperature, concentration, pH, and ionic strength conditions. In the current work, ³¹P nuclear magnetic resonance (NMR) and atomic force microscopy (AFM) have been used to monitor the size and morphology of isotropically tumbling small bicelles formed by mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DIOMPC) with either 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) or 1,2-di-O-hexyl-sn-glycero-3-phosphocholine (DIOHPC), testing their tolerance of variations in commonly used experimental conditions. ¹H-¹⁵N 2D NMR has been used to demonstrate the usefulness of the robust DMPC-DIOHPC system for conformational studies of a fatty acid-binding protein that shuttles small ligands to and from biological membranes.     

2H and 31P NMR study of pentalysine interaction with headgroup deuterated phosphatidylcholine and phosphatidylserine

The interaction of cationic pentalysine with phospholipid membranes was studied by using phosphorus and deuterium Nuclear Magnetic Resonance (NMR) of headgroup deuterated dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS). In the absence of pentalysine, some of the deuterium and phosphorus spectra of DMPC/DMPS 5:1 (m:m) membranes gave lineshapes similar to those of partially-oriented bilayers with the planes of the bilayers being parallel to the magnetic field. The deuterium NMR data show that the quadrupolar splittings of the deuterated methylenes of the DMPC headgroup are not affected by adsorption of pentalysine on the PC/PS membranes. By contrast, the pentalysine produces significant changes in the quadrupolar splittings of the negatively charged DMPS headgroup. The results are discussed in relation to previous ²H NMR investigations of phospholipid headgroup perturbations arising from bilayer interaction with cationic molecules.Abbreviations NMR nuclear magnetic resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPS 1,2-dimyristoyl-sn-glycero-3-phosphoserine - POPC 1-palmitoyl, 2-oleyl-sn-glycero-3-phosphocholine - POPG 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol - PC phosphatidylcholine - PS phosphatidyl serine - PG phosphatidylglycerol - HEPES N-(2-hydroxy-ethyl)piperazine-N-2-ethanesulfonic acid - TRIS tris-(hydroxymethyl)aminoethane - EDTA ethylenediamine-tetra-acetic acid     

A 2H solid-state NMR spectroscopic investigation of biomimetic bicelles containing cholesterol and polyunsaturated phosphatidylcholine

Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.     

Investigating the structural and dynamic properties of n-doxylstearic acid in magnetically-aligned phospholipid bilayers by X-band EPR spectroscopy

X-band electron paramagnetic resonance (EPR) spectroscopy has been employed to investigate the dynamic properties of magnetically-aligned phospholipid bilayers (bicelles) based on the molecular order parameters (S(mol)), the hyperfine splitting values and the line shapes of the EPR spectra. For the first time, a series of EPR spectra of n-doxylstearic acid spin-labels (n = 5, 7, 12, and 16) incorporated into Tm3+-doped parallel-aligned, Dy3+-doped perpendicular-aligned, and randomly dispersed 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DMPC/DHPC) bicelles with respect to the direction of the static magnetic field have been investigated as a function of cholesterol content and temperature variation to characterize the orientational aspects along the hydrocarbon acyl chains. Important general observations are that under conditions for which the bicelle is poised in the liquid crystalline phase, the degree of ordering decreases as the nitroxide moiety is transferred toward the end of the stearic acid acyl chains. The addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC phospholipid bilayers and increases the chain order. However, increasing the temperature of the bicelle system decreases the chain order. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other biological and model membrane systems. The results indicate that magnetically-aligned phospholipid bilayers are an excellent model membrane system.     

Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules

Several complementary physical techniques have been used to characterize the aggregate structures formed in solutions containing dimyristoylphosphatidylcholine (DMPC)/dihexanoylphosphatidylcholine (DHPC) at ratios of < or =0.5 and to establish their morphology and lipid organization as that of bicelles. (31)P NMR studies showed that the DMPC and DHPC components were highly segregated over a wide range of DMPC/DHPC ratios (q = 0.05-0.5) and temperatures (15 degrees C and 37 degrees C). Only at phospholipid concentrations below 130 mM did the bicelles appear to undergo a change in morphology. These results were corroborated by fluorescence data, which demonstrated the inverse dependence of bicelle size on phospholipid concentration as well as a distinctive change in phospholipid arrangement at low concentrations. In addition, dynamic light scattering and electron microscopy studies supported the hypothesis that the bicellar phospholipid aggregates are disk-shaped. The radius of the planar domain of the disk was found to be directly proportional to the ratio of DMPC/DHPC and inversely proportional to the total phospholipid concentration when the DMPC/DHPC ratio was held constant at 0.5. Taken together, these results suggest that bicelles with low q retain the morphology and bilayer organization typical of their liquid-crystalline counterparts, making them useful membrane mimetics.     

Atomistic Simulations of Bicelle Mixtures

Mixtures of long- and short-tail phosphatidylcholine lipids are known to self-assemble into a variety of aggregates combining flat bilayerlike and curved micellelike features, commonly called bicelles. Atomistic simulations of bilayer ribbons and perforated bilayers containing dimyristoylphosphatidylcholine (DMPC, di-C₁₄ tails) and dihexanoylphosphatidylcholine (DHPC, di-C₆ tails) have been carried out to investigate the partitioning of these components between flat and curved microenvironments and the stabilization of the bilayer edge by DHPC. To approach equilibrium partitioning of lipids on an achievable simulation timescale, configuration-bias Monte Carlo mutation moves were used to allow individual lipids to change tail length within a semigrand-canonical ensemble. Since acceptance probabilities for direct transitions between DMPC and DHPC were negligible, a third component with intermediate tail length (didecanoylphosphatidylcholine, di-C₁₀ tails) was included at a low concentration to serve as an intermediate for transitions between DMPC and DHPC. Strong enrichment of DHPC is seen at ribbon and pore edges, with an excess linear density of ∼3 nm⁻¹. The simulation model yields estimates for the onset of edge stability with increasing bilayer DHPC content between 5% and 15% DHPC at 300 K and between 7% and 17% DHPC at 323 K, higher than experimental estimates. Local structure and composition at points of close contact between pores suggest a possible mechanism for effective attractions between pores, providing a rationalization for the tendency of bicelle mixtures to aggregate into perforated vesicles and perforated sheets.     

Interaction of eosinophil granule major basic protein with synthetic lipid bilayers: A mechanism for toxicity

Summary Eosinophil granule major basic protein (MBP) is a potent toxin for mammalian cells and helminths, but the mechaism of its toxicity is not known. Here we tested whether MBP toxicity is exerted through its effect on the lipid bilayer of its targets. Liposomes prepared from synthetic phospholipids were used as targets for MBP and their properties examined by fluorescence and circular dichroism (CD) spectroscopy. MBP caused a change in the temperature transition profiles of acidic liposomes (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl serine or an equimolar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid) and induced their aggregation as shown by fluorescence resonance energy transfer experiments. The CD spectra and fluorescence characteristics of MBP itself were altered by its interaction with acidic lipids. Blue shifts in the emission maxima of the Trp, and of the dimethylaminonaphthyl moiety in acrylodan-labeled MBP, and a reduction in the effectiveness of quenching of Trp fluorescence by acrylamide were observed in the presence of acidic lipids. None of these effects were noted with zwitterionic lipids. This MBP : lipid bilayer interaction resulted in fusion and lysis of liposomes as indicated by the fluorescent indicator calcein. The results demonstrate that MBP associates with acidic lipids and that it disrupts, aggregates, fuses, and lyses liposomes prepared from such lipids. Such interaction might account for its wide range of toxicity.Abbreviations used Acrylodan 6-acryloyl-2-dimethylam-inonaphthalene - CD circular dichroism - DMPA 1,2-dimyrist-oyl-sn-glycero-3-phosphatidic acid - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DPH 1,6-diphenyl-1,3,5-hexatriene - DTT dithiothreitol - FRET fluorescence resonance energy transfer - HEPES N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid - K _sv Stern-Volmer constant - K _q bimolecular quenching coefficient - _em emission wavelength - _ex excitation wavelength - MBP major basic protein - MOPC 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine - NBD-PE N-(7-nitro-2,1,3-benzo-xadiazol-4-yl)-phosphatidylethanolamine - nMBP native major basic protein - PBS phosphate-buffered saline - POPC 1-palmit-oyl-2-oleoyl-sn-glycero-3-phosphocholine - POPS 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl serine - raMBP reduced and alkylated major basic protein - RHO-PE rhodamine-phosphatidylethanolamine - Tes N-tris[hydroxymethyl]-methyl-2-amino-ethane-sulfonic acid - Tris tris[hydroxymethyl]-amino-methane We would like to thank Dr. Predrag J.K. Ilich for assistance with initial data analysis, Dr. Salah S. Sedarous for the lifetime data and for helpful discussions, Dr. S. Yu. Venyaminov for helpful discussions, Mr. Kenneth D. Peters and Mr. Peter J. Callahan for assistance with some of the illustrations, and Ms. Jill Wagner for performing the radioimmunoassays. We would also like to thank Ms. Jill Kappers for excellent secretarial work. This work was supported in part by a Fellowship grant from the American Heart Association, Minnesota Affiliate, and by grants from the National Institutes of Health AI 09728 and from the Mayo Foundation. RIA-G is a Fellow of the American Heart Association.     

Opsin stability and folding: the role of Cys185 and abnormal disulfide bond formation in the intradiscal domain

The structure in the extracellular, intradiscal domain of rhodopsin surrounding the Cys110–Cys187 disulfide bond has been shown to be important for correct folding of this receptor in vivo. Retinitis pigmentosa misfolding mutants of the apoprotein opsin (such as P23H) misfold, as defined by a deficiency in ability to bind 11-cis retinal and form rhodopsin. These mutants also possess an abnormal Cys185–Cys187 disulfide bond in the intradiscal domain. Here, by mutating Cys185 to alanine, we eliminate the possibility of forming this abnormal disulfide bond and investigate the effect of combining the C185A mutation with the retinitis pigmentosa mutation P23H. Both the P23H and P23H/C185A double mutant suffer from low expression and poor 11-cis retinal binding. Our data suggest that misfolding events occur that do not have an absolute requirement for abnormal Cys185–Cys187 disulfide bond formation. In the detergent-solubilised, purified state, the C185A mutation allows formation of rhodopsin at wild-type (WT) levels, but has interesting effects on protein stability. C185A rhodopsin is less thermally stable than WT, whereas C185A opsin shows the same ability to regenerate rhodopsin in detergent as WT. Purified C185A and WT opsins, however, have contrasting 11-cis retinal binding kinetics. A high proportion of C185A opsin binds 11-cis retinal with a slow rate that reflects a denatured state of opsin reverting to a fast-binding, open-pocket conformation. This slower rate is not observed in a stabilising lipid/detergent system, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Chaps, in which C185A exhibits WT (fast) retinal binding. We propose that the C185A mutation destabilises the open-pocket conformation of opsin in detergent resulting in an equilibrium between correctly folded and denatured states of the protein. This equilibrium can be driven towards the correctly folded rhodopsin state by the binding of 11-cis retinal.     

Reinvestigation by phosphorus NMR of lipid distribution in bicelles

Mixtures of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC) in water form disks also called bicelles and different bilayer organizations when the mol ratio of the two lipids and the temperature are varied. The spontaneous alignment in a magnetic field of these bilayers above the transition temperature T(m) of DMPC is an attractive property that was successfully used to investigate protein structure by NMR. In this article, we have attempted to give an overview of all structural transformations of DMPC/DHPC mixtures that can be inferred from broad band (31)P-NMR spectroscopy between 5 and 60 degrees C. We show that above a critical temperature, T(v), perforated vesicles progressively replace alignable structures. The holes in these vesicles disappear above a new temperature threshold, T(h). The driving force for these temperature-dependent transformations that has been overlooked in previous studies is the increase of DHPC miscibility in the bilayer domain above T(m). Accordingly, we propose a new model (the "mixed bicelle" model) that emphasizes the consequence of the mixing. This investigation shows that the various structures of DMPC in the presence of increasing mol ratios of the short-chain DHPC is reminiscent of the observation put forward by several laboratories investigating solubilization and reconstitution of biological membranes.     

Development of magnetically aligned phospholipid bilayers in mixtures of palmitoylstearoylphosphatidylcholine and dihexanoylphosphatidylcholine by solid-state NMR spectroscopy

This study reports the solid-state NMR spectroscopic characterization of a long chain phospholipid bilayer system which spontaneously aligns in a static magnetic field. Magnetically aligned phospholipid bilayers or bicelles are model systems which mimic biological membranes for magnetic resonance studies. The oriented membrane system is composed of a mixture of the bilayer forming phospholipid palmitoylstearoylphosphatidylcholine (PSPC) and the short chain phospholipid dihexanoylphosphatidylcholine (DHPC) that breaks up the extended bilayers into bilayered micelles or bicelles that are highly hydrated (approx. 75% aqueous). Traditionally, the shorter 14 carbon chain phospholipid dimyristoylphosphatidylcholine (DMPC) has been utilized as the bilayer forming phospholipid in bicelle studies. Alignment (perpendicular) was observed with a PSPC/DHPC q ratio between 1.6 and 2.0 slightly above T(m) at 50 degrees C with (2)H and (31)P NMR spectroscopy. Paramagnetic lanthanide ions (Yb(3+)) were added to flip the bilayer discs such that the bilayer normal was parallel with the static magnetic field. The approx. 1.8 (PSPC/DHPC) molar ratio yields a thicker membrane due to the differences in the chain lengths of the DMPC and PSPC phospholipids. The phosphate-to-phosphate thickness of magnetically aligned PSPC/DHPC phospholipid bilayers in the L(alpha) phase may enhance the activity and/or incorporation of different types of integral membrane proteins for solid-state NMR spectroscopic studies.     

Morphology of fast-tumbling bicelles: a small angle neutron scattering and NMR study

Bilayered micelles, or bicelles, which consist of a mixture of long- and short-chain phospholipids, are a popular model membrane system. Depending on composition, concentration, and temperature, bicelle mixtures may adopt an isotropic phase or form an aligned phase in magnetic fields. Well-resolved (1)H NMR spectra are observed in the isotropic or so-called fast-tumbling bicelle phase, over the range of temperatures investigated (10-40 degrees C), for molar ratios of long-chain lipid to short-chain lipid between 0.20 and 1.0. Small angle neutron scattering data of this phase are consistent with the model in which bicelles were proposed to be disk-shaped. The experimentally determined dimensions are roughly consistent with the predictions of R.R. Vold and R.S. Prosser (J. Magn. Reson. B 113 (1996)). Differential paramagnetic shifts of head group resonances of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC), induced by the addition of Eu(3+), are also consistent with the bicelle model in which DHPC is believed to be primarily sequestered to bicelle rims. Selective irradiation of the DHPC aliphatic methyl resonances results in no detectable magnetization transfer to the corresponding DMPC methyl resonances (and vice versa) in bicelles, which also suggests that DHPC and DMPC are largely sequestered in the bicelle. Finally, (1)H spectra of the antibacterial peptide indolicidin (ILPWKWPWWPWRR-NH(2)) are compared, in a DPC micellar phase and the above fast-tumbling bicellar phases for a variety of compositions. The spectra exhibit adequate resolution and improved dispersion of amide and aromatic resonances in certain bicelle mixtures.     

Modulation of Plasma Membrane Ca2+-ATPase by Neutral Phospholipids: EFFECT OF THE MICELLE-VESICLE TRANSITION AND THE BILAYER THICKNESS*

The effects of lipids on membrane proteins are likely to be complex and unique for each membrane protein. Here we studied different detergent/phosphatidylcholine reconstitution media and tested their effects on plasma membrane Ca²⁺ pump (PMCA). We found that Ca²⁺-ATPase activity shows a biphasic behavior with respect to the detergent/phosphatidylcholine ratio. Moreover, the maximal Ca²⁺-ATPase activity largely depends on the length and the unsaturation degree of the hydrocarbon chain. Using static light scattering and fluorescence correlation spectroscopy, we monitored the changes in hydrodynamic radius of detergent/phosphatidylcholine particles during the micelle-vesicle transition. We found that, when PMCA is reconstituted in mixed micelles, neutral phospholipids increase the enzyme turnover. The biophysical changes associated with the transition from mixed micelles to bicelles increase the time of residence of the phosphorylated intermediate (EP), decreasing the enzyme turnover. Molecular dynamics simulations analysis of the interactions between PMCA and the phospholipid bilayer in which it is embedded show that in the 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer, charged residues of the protein are trapped in the hydrophobic core. Conversely, in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer, the overall hydrophobic-hydrophilic requirements of the protein surface are fulfilled the best, reducing the thermodynamic cost of exposing charged residues to the hydrophobic core. The apparent mismatch produced by a 1,2-dioleoyl-sn-glycero-3-phosphocholine thicker bilayer could be a structural foundation to explain its functional effect on PMCA.     

Dynamics of transportan in bicelles is surface charge dependent

In this study we investigated the dynamic behavior of the chimeric cell-penetrating peptide transportan in membrane-like environments using NMR. Backbone amide ¹⁵N spin relaxation was used to investigate the dynamics in two bicelles: neutral DMPC bicelles and partly negatively charged DMPG-containing bicelles. The structure of the peptide as judged from CD and chemical shifts is similar in the two cases. Both the overall motion as well as the local dynamics is, however, different in the two types of bicelles. The overall dynamics of the peptide is significantly slower in the partly negatively charged bicelle environment, as evidenced by longer global correlation times for all measured sites. The local motion, as judged from generalized order parameters, is for all sites in the peptide more restricted when bound to negatively charged bicelles than when bound to neutral bicelles (increase in S ² is on average 0.11 ± 0.07). The slower dynamics of transportan in charged membrane model systems cause significant line broadening in the proton NMR spectrum, which in certain cases limits the observation of ¹H signals for transportan when bound to the membrane. The effect of transportan on DMPC and DHPC motion in zwitterionic bicelles was also investigated, and the motion of both components in the bicelle was found to be affected.Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s10858-006-9008-y and is accessible for authorized users.     

Perturbation of DPPC bilayers by high concentrations of pulmonary surfactant protein SP-B

Deuterium (²H) NMR has been used to observe perturbation of dipalmitoylphosphatidylcholine (DPPC) bilayers by the pulmonary surfactant protein B (SP-B) at concentrations up to 17% (w/w). Previous ²H NMR studies of DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3) bilayers containing up to 11% (w/w) SP-B and DPPC bilayers containing up to 11% (w/w) synthetic SP-B indicated a slight effect on bilayer chain order and a more substantial effect on motions that contribute to decay of quadrupole echoes obtained from bilayers of deuterated DPPC. This is consistent with the perturbation of headgroup-deuterated DPPC reported here for bilayers containing 6 and 9% (w/w) SP-B. For the higher concentrations of SP-B investigated in the present work, ²H NMR spectra of DPPC deuterated in both the headgroup and chain display a prominent narrow component consistent with fast, large amplitude reorientation of some labeled lipid. Similar spectral perturbations have been reported for bilayers in the presence of the antibiotic polypeptide nisin. The observation of large amplitude lipid reorientation at high SP-B concentration could indicate that SP-B can induce regions of high bilayer curvature and thus provides some insight into local interaction of SP-B with DPPC. Such local interactions may be relevant to the formation, in vitro and in vivo, of tubular myelin, a unique structure found in extracellular pulmonary surfactant, and to the delivery of surfactant material to films at the air–water interface.Abbreviations DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol - DPPC-d₆₂ 1,2-perdeuterodipalmitoyl-sn-glycero-3-phosphocholine - DPPC-d₄ 1,2-dipalmitoyl-sn-glycero-3-phospho-(, perdeutero)-choline     

Magnetically oriented dodecylphosphocholine bicelles for solid-state NMR structure analysis

A mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the short-chain detergent n-dodecylphosphocholine (DPC) is introduced here as a new membrane-mimetic bicelle system for solid-state NMR structure analysis of membrane proteins in oriented samples. Magnetically aligned DMPC/DPC bicelles are stable over a range of concentrations, with an optimum lipid ratio of q=3:1, and they can be flipped with lanthanide ions. The advantage of DMPC/DPC over established bicelle systems lies in the possibility to use one and the same detergent for purification and NMR analysis of the membrane protein, without any need for detergent exchange. Furthermore, the same batch of protein can be studied in both micelles and bicelles, using liquid-state and solid-state NMR, respectively. The applicability of the DMPC/DPC bicelles is demonstrated here with the (15)N-labeled transmembrane protein TatA.     

Closely related oxidized phospholipids differentially modulate the physicochemical properties of lipid particles

Oxidation of glycerophospholipids results in the formation of large variety of oxidized phospholipid products that differs significantly in their chemical compositions and molecular structures. Biological activities of these oxidized products also differ considerably. Here we report the comparisons of the physicochemical properties of non-oxidized phospholipid particle containing two closely related tOx-PLs: 1-palmitoyl-2-(5-keto-6-octendioyl)-sn-glycero-3-phosphocholine (KOdiA-PC) and 1-palmitoyl-2-(9-keto-10-dodecendioyl)-sn-glycero-3-phosphocholine (KDdiA-PC). DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) was used as a model membrane non-oxidized phospholipid. Physicochemical properties of the lipid particles were characterized by using fluorescence spectroscopy, native polyacrylamide gel and agarose gel electrophoresis. Our result shows that the presence of closely related tOx-PLs, which differ only in the chemical composition of the oxidized fatty acyl chains at the sn-2 position, exerts considerably different effect on the physicochemical properties of non-oxidized phospholipid particles containing them.     

Lateral diffusion of PEG-Lipid in magnetically aligned bicelles measured using stimulated echo pulsed field gradient 1H NMR

Lateral diffusion measurements of PEG-lipid incorporated into magnetically aligned bicelles are demonstrated using stimulated echo (STE) pulsed field gradient (PFG) proton (1H) nuclear magnetic resonance (NMR) spectroscopy. Bicelles were composed of dimyristoyl phosphatidylcholine (DMPC) plus dihexanoyl phosphatidylcholine (DHPC) (q = DMPC/DHPC molar ratio = 4.5) plus 1 mol % (relative to DMPC) dimyristoyl phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG 2000) at 25 wt % lipid. 1H NMR STE spectra of perpendicular aligned bicelles contained only resonances assigned to residual HDO and to overlapping contributions from a DMPE-PEG 2000 ethoxy headgroup plus DHPC choline methyl protons. Decay of the latter's STE intensity in the STE PFG 1H NMR experiment (g(z) = 244 G cm(-1)) yielded a DMPE-PEG 2000 (1 mol %, 35 degrees C) lateral diffusion coefficient D = 1.35 x 10(-11) m2 s(-1). Hence, below the "mushroom-to-brush" transition, DMPE-PEG 2000 lateral diffusion is dictated by its DMPE hydrophobic anchor. D was independent of the diffusion time, indicating unrestricted lateral diffusion over root mean-square diffusion distances of microns, supporting the "perforated lamellae" model of bicelle structure under these conditions. Overall, the results demonstrate the feasibility of lateral diffusion measurements in magnetically aligned bicelles using the STE PFG NMR technique.     

Label-free,chronological and selective detection of aggregation and fibrillization of amyloid β protein in serum by microcantilever sensor immobilizing cholesterol-incorporated liposome

To facilitate the early diagnosis of Alzheimer's disease and mild cognitive impairment patients, we developed a cantilever-based microsensor that immobilized liposomes of various phospholipids to detect a trace amount of amyloid β (Aβ) protein, and investigated its aggregation and fibrillization on model cell membranes in human serum. Three species of liposomes composed of different phospholipids of 1,2-dipalmtoyl-sn-glycero-3-phosphocholine (DPPC), DPPC/phosphatidyl ethanolamine and 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol having varied hydrophilic groups were applied, which showed different chronological interactions with Aβ(1–40) protein and varied sensitivities of the cantilever sensor, depending on their specific electrostatic charged conditions, hydrophilicity, and membrane fluidity. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) having short hydrophobic carbon chains confirmed to show a large interaction with Aβ(1–40) and a high sensitivity. Furthermore, the incorporation of cholesterol into DMPC was effective to selectively detect Aβ(1–40) in human serum, which effect was also checked by quartz crystal microbalance. Finally, Aβ detection of 100-pM order was expected selectively in the serum by using the developed biosensor.     

Dynamics of formation of lysoforms on enzymatic hydrolysis of phosphatidylalkanols

Hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphomethanol (DOPM), 1,2-dioleoyl-sn-glycero-3-phosphoethanol (DOPEt), 1,2-dioleoyl-sn-glycero-3-phosphoethyleneglycol (DOPEG), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) catalyzed by phospholipase A₂ (PLA₂) from porcine pancreas was studied in single-component and binary model bilayer membranes (liposomes). The presence of anionic phosphatidylalkanols increases the rate of hydrolysis of zwitterionic DOPC in liposomes by the action of PLA₂. The rate of formation of lysoforms of anionic (acidic) lipids at the initial reaction stage in single-component liposomes increased in the following sequence: DOPEG < DOPM < DOPEt (compared with that for the zwitterionic DOPC). In binary liposomes formation of lyso-DOPC increased in the presence of acidic lipids in the following sequence: DOPM < DOPEt < DOPEG. This indicates that the size of polar fragment of the second substrate and the presence of free hydroxy groups in the head of DOPEG may possibly activate DOPC hydrolysis by the action of PLA₂ in the presence of anionic phospholipids including cardiolipin; the studied phospholipids model fragments of the latter.     

Conformational analysis of steroid hormone molecules in the lipid environment--a solid-state NMR approach

Solid-state ¹H/¹³C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy has been applied to two steroid compounds: dehydroepiandrosterone (DHEA) and spironolactone (SPI), to analyze their conformations at the atomic level. In the absence of lipid, the high-resolution ¹³C CP/MAS NMR signals of DHEA and SPI in a powder form reveal multiple patterns, with splittings of 30-160 Hz, indicating the existence of multiple conformations. In the mimic lipid environment formed by mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) in a molar ratio 3:1, the resulting DHEA and SPI spectra revealed mostly singlet patterns, suggesting that these steroids undergo a conformational change leading to a specific conformation in the lipid environment. Evidence from chemical shift isotropy and anisotropy analysis indicates that DHEA might adopt conformations subtly different from that seen in solution and in the powder form. In conclusion, we demonstrate by solid-state NMR that the structures of DHEA and SPI may adopt slightly different conformations in different chemical environments.