Dynamics of lipid domain formation: Fluctuation analysis

Scanning-fluctuation correlation spectroscopy was used to detect subresolution organizational fluctuations in the lipid liquid-crystalline phase for single lipid model systems. We used the fluorescent probe Laurdan which is sensitive to the amount of water in the membrane to show that there is a spatial heterogeneity on the scale of few pixels (the size of the pixel is 50 nm). We calculated the pixel variance of the GP function and we found that the variance has a peak at the phase transition for 3 different samples made of pure lipids. The pixel variance has an abrupt change at the phase transition of the membrane and then it slowly decreases at higher temperature. The relatively large variance of the GP indicates that the liquid phase of the membrane is quite heterogeneous even several degrees higher than the phase transition temperature. We interpreted this result as evidence of an underlying microscale structure of the membrane in which water is not uniformly distributed at the micron scale. Imaging of these microstructures shows that the pixels with different GP tend to concentrate in specific domains in the membrane. In the case of single lipid membrane, the statistical and fluctuation analysis of the GP data shows that even such simple lipid systems are capable of generating and maintaining stable structural and organizational heterogeneities.     

Thermotropic phase behaviour of lipid bilayers containing carotenoid pigment canthaxanthin: a differential scanning calorimetry study

In this study we address the problem of the effect of canthaxanthin on the thermotropic properties of lipid membranes formed with lipids which differ in the thickness of their hydrophobic core, size of polar heads or presence of the ester carbonyl group. For all the lipids a decrease in main transition enthalpy has been observed, indicating that canthaxanthin alters the membrane properties in its gel phase. The strongest influence of canthaxanthin on main phase transition and pretransition has been observed for the lipid having the thinnest hydrophobic region. Component analysis indicates a distinct cooperativity change, which most probably colligates with the formation of new thermotropic phases. The effect of canthaxanthin has been almost negligible in the case of phosphatidylethanolamines. The absence of the ester carbonyl group results in different thermotropic behavior, especially for low canthaxanthin concentrations. The effect of canthaxanthin is explained in terms of its organization within the membrane.     

Projection map of aquaporin-9 at 7 A resolution

Aquaporin-9, an aquaglyceroporin present in diverse tissues, is unique among aquaporins because it is not only permeable to water, urea and glycerol, but also allows passage of larger uncharged solutes. Single particle analysis of negatively stained recombinant rat aquaporin-9 revealed a particle size characteristic of the tetrameric organization of all members of the aquaporin family. Reconstitution of aquaporin-9 into two-dimensional crystals enabled us to calculate a projection map at 7 A resolution. The projection structure indicates a tetrameric structure, similar to GlpF, with each square-like monomer forming a pore. A comparison of the pore-lining residues between the crystal structure of GlpF and a homology model of aquaporin-9 locates substitutions in these residues predominantly to the hydrophobic edge of the tripathic pore of GlpF, providing first insights into the structural basis for the broader substrate specificity of aquaporin-9.     

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.     

Correlation between the hydration of acyl chains and phosphate groups in lipid bilayers: Effect of phase state,head group,chain length,double bonds and carbonyl groups

This paper demonstrates by means of FTIR/ATR analysis that water molecules intercalate at different extents in the acyl chain region of lipid membranes in correlation with the hydration of the phosphate groups.This correlation is sensible to the chain length, the presence of double bonds and the phase state of the lipid membrane.The presence of carbonyl groups CO modifies the profile of hydration of the two regions as observed from the comparison of DMPC and 14:0 Diether PC.The different water populations in lipid interphases would give arrangements with different free energy states that could drive the interaction of biological effectors with membranes.     

Effect of ionic strength on the organization and dynamics of membrane-bound melittin

Melittin, a cationic hemolytic peptide, is intrinsically fluorescent due to the presence of a single functionally important tryptophan residue. We have previously shown that the sole tryptophan of melittin is localized in a motionally restricted environment in the membrane interface. We have monitored the effect of ionic strength on the organization and dynamics of membrane-bound melittin utilizing fluorescence and circular dichroism (CD) spectroscopic approaches. Our results show that red edge excitation shift (REES) of melittin bound to membranes is sensitive to the change in ionic strength of the medium. This could be attributed to a change in the immediate environment around melittin tryptophan with increasing ionic strength due to differential solvation of ions. Interestingly, the rotational mobility of melittin does not appear to be affected with change in ionic strength. In addition, fluorescence parameters such as lifetime and acrylamide quenching of melittin indicate an increase in water penetration in the membrane interface upon increasing ionic strength. Our results suggest that the solvent dynamics and water penetration in the interfacial region of the membranes are significantly affected at physiologically relevant ionic strength. These results assume significance in the overall context of the influence of ionic strength in the organization and dynamics of membrane proteins and membrane-active peptides.     

Effect of hydrophobic surfactant proteins SP-B and SP-C on binary phospholipid monolayers: II. Infrared external reflectance-absorption spectroscopy

In situ external reflection infrared spectroscopy at the air-water interface was used to study the influence on phospholipid structure of an endogenous mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, which are thought to play pivotal roles in the adsorption and function of pulmonary surfactant. Mixtures studied were 1:1, 2:1, and 7:1 (mol:mol) DPPC-d(62):DPPG, and 7:1 DPPC-d(62):DOPG, alone and in the presence of 0.5-10 wt % mixed SP-B/C purified chromatographically from calf lung surfactant extract. Perdeuteration of DPPC produced a shift in vibrational frequencies so that it could be differentiated spectroscopically from the phosphoglycerol component in the surface monolayer. CH(2) antisymmetric and symmetric stretching bands ( approximately 2920 and 2852 cm(-1)) along with the analogous CD(2) stretching bands ( approximately 2194 and 2089 cm(-1)) were analyzed, and band heights and peak wavenumber positions were assessed as a function of monolayer surface pressure. Small, near-physiological contents of 1-2 wt % SP-B/C typically produced the maximum observed spectroscopic effects, which were abolished at high protein contents of 10 wt %. Analysis of CH(2) and CD(2) stretching bands and C-H/C-D band height ratios indicated that SP-B/C affected PC and PG lipids differently within the surface monolayer. SP-B/C had preferential interactions with DPPG in 1:1, 2:1, and 7:1 DPPC-d(62):DPPG films that increased its acyl chain order. SP-B/C also interacted specifically with DOPG in 7:1 DPPC-d(62):DOPG monolayers, but in this case an increase in CH(2) band heights and peak wavenumber positions indicated a further disordering of the already fluid DOPG acyl chains. CD(2) band height and peak wavenumber analysis indicated that SP-B/C had no significant effect on the structure of DPPC-d(62) chains in 7:1 films with DPPG or DOPG, and had only a slight tendency to increase the acyl chain order in 1:1 films of DPPC-d(62):DPPG. SP-B/C had no significant effect on DPPC-d(62) structure in films with DOPG. Infrared results also indicated that interactions involving SP-B/C and lipids led to exclusion of PC and PG lipids from the compressed interfacial monolayer, in agreement with our previous report on the phase morphology of lipid monolayers containing 1 wt % SP-B/C.     

Alzheimer's disease amyloid-β peptide analogue alters the ps-dynamics of phospholipid membranes

We have investigated the influence of the neurotoxic Alzheimer's disease peptide amyloid-β (25-35) on the dynamics of phospholipid membranes by means of quasi-elastic neutron scattering in the picosecond time-scale. Samples of pure phospholipids (DMPC/DMPS) and samples with amyloid-β (25-35) peptide included have been compared. With two different orientations of the samples the directional dependence of the dynamics was probed. The sample temperature was varied between 290 K and 320 K to cover both the gel phase and the liquid-crystalline phase of the lipid membranes. The model for describing the dynamics combines a long-range translational diffusion of the lipid molecules and a spatially restricted diffusive motion. Amyloid-β (25-35) peptide affects significantly the ps-dynamics of oriented lipid membranes in different ways. It accelerates the lateral diffusion especially in the liquid-crystalline phase. This is very important for all kinds of protein-protein interactions which are enabled and strongly influenced by the lateral diffusion such as signal and energy transducing cascades. Amyloid-β (25-35) peptide also increases the local lipid mobility as probed by variations of the vibrational motions with a larger effect in the out-of-plane direction. Thus, the insertion of amyloid-β (25-35) peptide changes not only the structure of phospholipid membranes as previously demonstrated by us employing neutron diffraction (disordering effect on the mosaicity of the lipid bilayer system) but also the dynamics inside the membranes. The amyloid-β (25-35) peptide induced membrane alteration even at only 3 mol% might be involved in the pathology of Alzheimer's disease as well as be a clue in early diagnosis and therapy.     

Multilayer structures in lipid monolayer films containing surfactant protein C: effects of cholesterol and POPE

The influence of cholesterol and POPE on lung surfactant model systems consisting of DPPC/DPPG (80:20) and DPPC/DPPG/surfactant protein C (80:20:0.4) has been investigated. Cholesterol leads to a condensation of the monolayers, whereas the isotherms of model lung surfactant films containing POPE exhibit a slight expansion combined with an increased compressibility at medium surface pressure (10-30 mN/m). An increasing amount of liquid-expanded domains can be visualized by means of fluorescence light microscopy in lung surfactant monolayers after addition of either cholesterol or POPE. At surface pressures of 50 mN/m, protrusions are formed which differ in size and shape as a function of the content of cholesterol or POPE, but only if SP-C is present. Low amounts of cholesterol (10 mol %) lead to an increasing number of protrusions, which also grow in size. This is interpreted as a stabilizing effect of cholesterol on bilayers formed underneath the monolayer. Extreme amounts of cholesterol (30 mol %), however, cause an increased monolayer rigidity, thus preventing reversible multilayer formation. In contrast, POPE, as a nonbilayer lipid thought to stabilize the edges of protrusions, leads to more narrow protrusions. The lateral extension of the protrusions is thereby more influenced than their height.     

Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles

The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu(2-30+), a 36-residue polypeptide containing residues 2-30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional (1)H/(15)N PISEMA spectra of uniformly (15)N-labeled Vpu(2-30+) obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a (1)H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane alpha-helix spanning residues 8-25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent (15)N chemical shift and (1)H-(15)N dipolar coupling frequencies. Tilt angles of 30 degrees and 21 degrees are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers.     

Transbilayer movement of phospholipids at the main phase transition of lipid membranes: implications for rapid flip-flop in biological membranes

The transbilayer movement of fluorescent phospholipid analogs in liposomes was studied at the lipid phase transition of phospholipid membranes. Two NBD-labeled analogs were used, one bearing the fluorescent moiety at a short fatty acid chain in the sn-2 position (C(6)-NBD-PC) and one headgroup-labeled analog having two long fatty acyl chains (N-NBD-PE). The transbilayer redistribution of the analogs was assessed by a dithionite-based assay. We observed a drastic increase of the transbilayer movement of both analogs at the lipid phase transition of DPPC (T(c) = 41 degrees C) and DMPC (T(c) = 23 degrees C). The flip-flop of analogs was fast at the T(c) of DPPC with a half-time (t(1/2)) of ~6-10 min and even faster at the T(c) of DMPC with t(1/2) on the order of <2 min, as shown for C(6)-NBD-PC. Suppressing the phase transition by the addition of cholesterol, the rapid transbilayer movement was abolished. Molecular packing defects at the phase transition are assumed to be responsible for the rapid transbilayer movement. The relevance of those defects for understanding of the activity of flippases is discussed.     

Complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: A review

Plasma membranes are complex entities common to all living cells. The basic principle of their organization appears very simple, but they are actually of high complexity and represent very dynamic structures. The interactions between bioactive molecules and lipids are important for numerous processes, from drug bioavailability to viral fusion. The cell membrane is a carefully balanced environment and any change inflicted upon its structure by a bioactive molecule must be considered in conjunction with the overall effect that this may have on the function and integrity of the membrane. Conceptually, understanding the molecular mechanisms by which bioactive molecules interact with cell membranes is of fundamental importance.     

Conformational changes of chicken liver bile acid-binding protein bound to anionic lipid membrane are coupled to the lipid phase transitions

Chicken liver bile acid-binding protein (L-BABP) binds to anionic lipid membranes by electrostatic interactions and acquires a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B. and Montich, G. G. (2003) Biochim. Biophys. Acta 1611, 98-106]. We studied the infrared amide I′ band of L-BABP bound to dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG) and palmitoyloleoylphosphatidylglycerol (POPG) in the range of 7 to 60 °C. Besides, the thermotrophic behaviour of DPPG and DMPG was studied in the absence and in the presence of bound-protein by differential scanning calorimetry (DSC) and infrared spectra of the stretching vibration of methylene and carbonyl groups. When L-BABP was bound to lipid membranes in the liquid-crystalline state (POPG between 7 and 30 °C) acquired a more unfolded conformation that in membranes in the gel state (DPPG between 7 and 30 °C). Nevertheless, this conformational change of the protein in DMPG did not occur at the temperature of the lipid gel to liquid-crystalline phase transition detected by infrared spectroscopy. Instead, the degree of unfolding in the protein was coincident with a phase transition in DMPG that occurs with heat absorption and without change in the lipid order.     

The two-fold aspect of the interplay of amyloidogenic proteins with lipid membranes

Investigating the pathways leading to the formation of amyloid protein aggregates and the mechanism of their cytotoxicity is fundamental for a deeper understanding of a broad range of human diseases. Increasing evidence indicates that early aggregates are responsible for the cytotoxic effects. This paper addresses the catalytic role of lipid surfaces in promoting aggregation of amyloid proteins and the permeability changes that these aggregates induce on lipid membranes. Effects of amyloid aggregates on model systems such as monolayers, vesicles, liposomes and supported lipid bilayers are reviewed. In particular, the relevance of atomic force microscopy in detecting both kinetics of amyloid formation and amyloid-membrane interactions is emphasized.     

Structure and Dynamics of the Membrane-Bound Form of Pf1 Coat Protein: Implications of Structural Rearrangement for Virus Assembly

The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16-A46) around its long axis changes dramatically (by 160°) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2-G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state ¹H/¹⁵N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly.     

Polyglutamine expansion in huntingtin increases its insertion into lipid bilayers

An expanded polyglutamine (Q) tract (>37Q) in huntingtin (htt) causes Huntington disease. Htt associates with membranes and polyglutamine expansion in htt may alter membrane function in Huntington disease through a mechanism that is not known. Here we used differential scanning calorimetry to examine the effects of polyQ expansion in htt on its insertion into lipid bilayers. We prepared synthetic lipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine and tested interactions of htt amino acids 1-89 with 20Q, 32Q or 53Q with the vesicles. GST-htt1-89 with 53Q inserted into synthetic lipid vesicles significantly more than GST-htt1-89 with 20Q or 32Q. We speculate that by inserting more into cell membranes, mutant huntingtin could increase disorder within the lipid bilayer and thereby disturb cellular membrane function.     

Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba delta-endotoxin complex revealed by electron crystallography: implications for toxin-pore formation

The insecticidal nature of Cry delta-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore.     

Influence of the lipid composition on the kinetics of concerted insertion and folding of melittin in bilayers

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the ¹⁹Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the ¹⁹Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an α-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of ¹⁹Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the α-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.     

Apolipophorin III interaction with model membranes composed of phosphatidylcholine and sphingomyelin using differential scanning calorimetry

Apolipophorin III (apoLp-III) from Locusta migratoria was employed as a model apolipoprotein to gain insight into binding interactions with lipid vesicles. Differential scanning calorimetry (DSC) was used to measure the binding interaction of apoLp-III with liposomes composed of mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SM). Association of apoLp-III with multilamellar liposomes occurred over a temperature range around the liquid crystalline phase transition (L_α). Qualitative and quantitative data were obtained from changes in the lipid phase transition upon addition of apoLp-III. Eleven ratios of DMPC and SM were tested from pure DMPC to pure SM. Broadness of the phase transition (T_1/2), melting temperature of the phase transition (T_m) and enthalpy were used to determine the relative binding affinity to the liposomes. Multilamellar vesicles composed of 40% DMPC and 60% SM showed the greatest interaction with apoLp-III, indicated by large T_1/2 values. Pure DMPC showed the weakest interaction and liposomes with lower percentage of DMPC retained domains of pure DMPC, even upon apoLp-III binding indicating demixing of liposome lipids. Addition of apoLp-III to rehydrated liposomes was compared to codissolved trials, in which lipids were rehydrated in the presence of protein, forcing the protein to interact with the lipid system. Similar trends between the codissolved and non-codissolved trials were observed, indicating a similar binding affinity except for pure DMPC. These results suggested that surface defects due to non-ideal packing that occur at the phase transition temperature of the lipid mixtures are responsible for apolipoprotein-lipid interaction in DMPC/SM liposomes.