Opsin stability and folding: modulation by phospholipid bicelles

Integral membrane proteins do not fare well when extracted from biological membranes and are unstable or lose activity in detergents commonly used for structure and function investigations. We show that phospholipid bicelles provide a valuable means of preserving alpha-helical membrane proteins in vitro by supplying a soluble lipid bilayer fragment. Both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (Chaps) and DMPC/l-α-1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles dramatically increase the stability of the mammalian vision receptor rhodopsin as well as its apoprotein, opsin. Opsin is particularly unstable in detergent solution but can be directly purified into DMPC/Chaps. We show that opsin can also be directly purified in DMPC/DHPC bicelles to give correctly folded functional opsin, as shown by the ability to regenerate rhodopsin to  70% yield. These well-characterised DMPC/DHPC bicelles enable us to probe the influence of bicelle properties on opsin stability. These bicelles are thought to provide DMPC bilayer fragments with most DHPC capping the bilayer edge, giving a soluble bilayer disc. Opsin stability is shown to be modulated by the q value, the ratio of DMPC to DHPC, which reflects changes in the bicelle size and, thus, proportion of DMPC bilayer present. The observed changes in stability also correlate with loss of opsin secondary structure as determined by synchrotron far-UV circular dichroism spectroscopy; the most stable bicelle results in the least helix loss. The inclusion of Chaps rather than DHPC in the DMPC/Chaps bicelles, however, imparts the greatest stability. This suggests that it is not just the DMPC bilayer fragment in the bicelles that stabilises the protein, but that Chaps provides additional stability either through direct interaction with the protein or by altering the DMPC/Chaps bilayer properties within the bicelle. The significant stability enhancements and preservation of secondary structure reported here in bicelles are pertinent to other membrane proteins, notably G-protein-coupled receptors, which are unstable in detergent solution.     

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.     

Influence of the long-chain/short-chain amphiphile ratio on lateral diffusion of PEG-lipid in magnetically aligned lipid bilayers as measured via pulsed-field-gradient NMR

Lateral diffusion measurements of polyethylene glycol(PEG)-lipid incorporated into magnetically aligned lipid bilayers, composed of dimyristoyl phosphatidylcholine (DMPC) plus dihexanoyl phosphatidylcholine (DHPC) plus 1 mol % (relative to DMPC) dimyristoyl phosphatidylethanolamine-n-[methoxy(polyethylene glycol)-2000] (DMPE-PEG 2000), were performed using stimulated-echo pulsed-field-gradient proton (¹H) nuclear magnetic resonance spectroscopy. The DMPE-PEG 2000 (1 mol %, 35°C) lateral diffusion coefficient D varied directly with the mole fraction of DMPC, X_DMPC = q/(1+q) where q = DMPC/DHPC molar ratio, decreasing progressively from D = 1.65 × 10⁻¹¹ m² s⁻¹ at q ≈ 4.7 to D = 0.65 × 10⁻¹¹ m² s⁻¹ at q ≈ 2.5. Possible sources of this dependence, including orientational disorder, obstruction, and PEG-lipid sequestration, were simulated using, respectively, a diffusion-in-a-cone model, percolation theory, and a two-phase PEG distribution model. Orientational disorder alone was not capable of reproducing the observations, but in combination with either obstruction or PEG-lipid two-phase distribution models did so satisfactorily. A combination of all three models yielded the most reasonable fit to the observed dependence of lateral diffusion on q. These same effects would be expected to influence lateral diffusion of any bilayer-associating species in such systems.     

Membrane interactions in small fast-tumbling bicelles as studied by 31P NMR

Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used ³¹P NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and ³¹P T₂ relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T₂ relaxation are observed at higher temperatures. A comparison of ³¹P T₁ values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T₁ relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules  – in particular for the negatively charged DMPG – while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide–membrane interactions.     

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.     

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K channels

The membrane location of two fragments in two different K⁺-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T₁ and ¹³C-¹H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. ²H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.     

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.     

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.     

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.     

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.     

The membrane-induced structure of melittin is correlated with the fluidity of the lipids

The effect of the bee toxin melittin on DMPC dynamics in fast-tumbling bicelles has been investigated. The ¹³C R₁ and ¹³C-¹H NOE relaxation parameters for DMPC were used to monitor the effect of melittin and cholesterol on lipid dynamics. It was found that melittin has the largest effect on the DMPC mobility in DMPC/DHPC bicelles, while less effect was observed in cholesterol-doped bicelles, or in bicelles made with CHAPS, indicating that the rigidity of the membrane affects the melittin-membrane interaction. CD spectra were analysed in terms of cooperativity of the α-helix to random coil transition in melittin, and these results also indicated similar differences between the bicelles. The study shows that bicelles can be used to investigate lipid dynamics by spin relaxation, and in particular of peptide-induced changes in membrane fluidity.     

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.     

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.     

Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules

Weak alignment of solute molecules with the magnetic field can be achieved in a dilute liquid crystalline medium, consisting of an aqueous mixture of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). For a certain range of molar ratios, DMPC and DHPC can form large, disc-shaped particles, commonly referred to as bicelles (Sanders and Schwonek, 1992), which cooperatively align in the magnetic field and induce a small degree of alignment on asymmetrically shaped solute molecules. As a result, dipolar couplings between pairs of ¹H, ¹³C or ¹⁵N nuclei are no longer averaged to zero by rotational diffusion and they can be readily measured, providing valuable structural information. The stability of these liquid crystals and the degree of alignment of the solute molecules depend strongly on experimental variables such as the DMPC:DHPC ratio and concentration, the preparation protocol of the DMPC/DHPC mixtures, as well as salt, temperature, and pH. The lower temperature limit for which the liquid crystalline phase is stable can be reduced to 20 °C by using a ternary mixture of DHPC, DMPC, and 1-myristoyl-2-myristoleoyl-sn-glycero-3-phosphocholine, or a binary mixture of DHPC and ditridecanoyl-phosphatidylcholine. These issues are discussed, with an emphasis on the use of the medium for obtaining weak alignment of biological macromolecules.     

Sign determination of dipolar couplings in field-oriented bicelles by variable angle sample spinning (VASS)

Residual dipolar couplings are being increasingly used as structural constraints for NMR studies of biomolecules. A problem arises when dipolar coupling contributions are larger than scalar contributions for a given spin pair, as is commonly observed in solid state NMR studies, in that signs of dipolar couplings cannot easily be determined. Here the sign ambiguities of dipolar couplings in field-oriented bicelles are resolved by variable angle sample spinning (VASS) techniques. The director behavior of field-oriented bicelles (DMPC/DHPC, DMPC/CHAPSO) in VASS is studied by ³¹P NMR. A stable configuration occurs when the spinning angle is smaller than the magic angle, 54.7°, and the director (or bicelle normal) of the disks is mainly distributed in a plane perpendicular to the rotation axis. Since the dipolar couplings depend on how the bicelles are oriented with respect to the magnetic field, it is shown that the dipolar interaction can be scaled to the same order as the J-coupling by moving the spinning axis from 0° toward 54.7°. Thus the relative sign of dipolar and scalar couplings can be determined.     

Acidic phospholipid bicelles: a versatile model membrane system

With the aim of establishing acidic bicellar solutions as a useful membrane model system, we have used deuterium NMR spectroscopy to investigate the properties of dimyristoyl/dihexanoylphosphatidylcholine (DMPC/DHPC) bicelles containing 25% (w/w in H(2)O) of either dimyristoylphosphatidylserine (DMPS) or dimyristoylphosphatidylglycerol (DMPG). The addition of the acidic lipid component to this lyotropic liquid crystalline system reduces its range of stability because of poor miscibility of the two dimyristoylated phospholipids. Compared to the neutral bicelles, which are stable at pH 4 to pH 7, acidic bicelles are stable only from pH 5.5 to pH 7. Solid-state deuterium NMR analysis of d(54)-DMPC showed similar ordering in neutral and acidic bicelles. Fully deuterated DMPS or DMPG is ordered in a way similar to that of DMPC. Study of the binding of the myristoylated N-terminal 14-residue peptide mu-GSSKSKPKDPSQRR from pp60(nu-src) to both neutral and acidic bicelles shows the utility of these novel membrane mimetics.     

Lipid dynamics in fast-tumbling bicelles with varying bilayer thickness: Effect of model transmembrane peptides

The morphology of q = 0.5 fast-tumbling bicelles prepared with three different acyl chain lengths has been investigated by NMR. It is shown that bicelles prepared with DLPC (12 C) and DHPC are on average larger than those containing DMPC or DPPC (14 and 16 C) and DHPC, which may be due to a higher degree of mixing between DLPC and DHPC. The fast internal mobility of the lipids was determined from natural abundance carbon-13 relaxation. A similar dynamical behaviour of the phospholipids in the three different bicelles was observed, although the DPPC lipid acyl chain displayed a somewhat lower degree of mobility, as evidenced by higher generalized order parameters throughout the acyl chain. Carbon-13 relaxation was also used to determine the effect of different model transmembrane peptides, with flanking Lys residues, on the lipid dynamics in the three different bicelles. All peptides had the effect of increasing the order parameters for the DLPC lipid, while no effect was observed on the longer lipid chains. This effect may be explained by a mismatch between the hydrophobic length of the peptides and the DLPC lipid acyl chain.     

Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label: The effect of cholesterol

X-band EPR spectroscopy has been employed to study the dynamic properties of magnetically aligned phospholipid bilayers (bicelles) utilizing a variety of phosphocholine spin labels (n-PCSL) as a function of cholesterol content. The utilization of both perpendicular and parallel aligned bicelles in EPR spectroscopy provides a more detailed structural and orientational picture of the phospholipid bilayers. The magnetically aligned EPR spectra of the bicelles and the hyperfine splitting values reveal that the addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC bicelles. The corresponding molecular order parameter, S_mol, of the DMPC/DHPC bicelles increased upon addition of cholesterol. Cholesterol also decreased the rotational motion and increased the degree of anisotropy in the interior region of the bicelles. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other model membrane systems and that the magnetically aligned bicelles are an excellent model membrane system.     

Controlled release mechanisms of spontaneously forming unilamellar vesicles

Spontaneously forming small unilamellar vesicles (SULVs) are easy to prepare and show great promise for use in delivering therapeutic payloads. We report of SULVs made up of the ternary phospholipid mixture, dimyristoyl-phosphatidylcholine (DMPC), dihexanoyl-phosphatidylcholine (DHPC) and dimyristoyl-phosphatidylglycerol (DMPG), which have been characterized by small angle neutron scattering (SANS). These low-polydispersity (0.14-0.19) SULVs range in size (i.e., radius) from 110 to 215 Å and are capable of entrapping, and subsequently releasing, hydrophilic molecules (e.g., fluorescent dyes and quenchers) in a controlled fashion over two different temperature ranges. The low-temperature release mechanism involves the SULVs transforming into discoidal micelles, with an onset temperature (T_o) of ~ 32 °C, while the high-temperature release mechanism is more gradual, presumably the result of defects formed through the continuous dissolution of DHPC into solution. Both of these mechanisms differ from other, previously reported thermosensitive liposomes.     

A comparative study of the effect of cholesterol on bicelle model membranes using X-band and Q-band EPR spectroscopy

X-band and Q-band electron paramagnetic resonance (EPR) spectroscopic techniques were used to investigate the structure and dynamics of cholesterol containing phospholipid bicelles based upon molecular order parameters (S_mol), orientational dependent hyperfine splittings and line shape analysis of the corresponding EPR spectra. The nitroxide spin-label 3-β-doxyl-5-α-cholestane (cholestane) was incorporated into DMPC/DHPC bicelles to report the alignment of bicelles in the static magnetic field. The influence of cholesterol on aligned phospholipid bicelles in terms of ordering, the ease of alignment, phase transition temperature have been studied comparatively at X-band and Q-band. At a magnetic field of 1.25 T (Q-band), bicelles with 20 mol% cholesterol aligned at a much lower temperature (313 K), when compared to 318 K at a 0.35 T field strength for X-band, showed better hyperfine splitting values (18.29 G at X-band vs. 18.55 G at Q-band for perpendicular alignment and 8.25 G at X-band vs. 7.83 G at Q-band for the parallel alignment at 318 K) and have greater molecular order parameters (0.76 at X-band vs. 0.86 at Q-band at 318 K). Increasing cholesterol content increased the bicelle ordering, the bicelle-alignment temperature and the gel to liquid crystalline phase transition temperature. We observed that Q-band is more effective than X-band for studying aligned bicelles, because it yielded a higher ordered bicelle system for EPR spectroscopic studies.