Incorporation of synthetic peptide helices in membranes of tetraether lipids from Thermoplasma acidophilum. A calorimetric study

Four analogues of the membrane-modifying, alpha-helical polypeptide antibiotic alamethicin were synthesized. the alpha-helical deca-, undeca-, heptadeca-, and icosapeptides were mixed with the main tetraether lipid of the Archaebacterium Thermoplasma acidophilum (MPL), dipalmitoylphosphatidylcholine (DPPC) and dihexadecylmaltosylglycerol (DHMG) in various ratios and the modification of the lipid phase transition was determined by differential thermal analysis (DTA). The polypeptides form mixed phases with MPL and DPPC, however, not with DHMG. Heptadeca- and icosapeptide exert a much stronger reduction of enthalpy (delta H) than deca- and undecapeptide and bind about 0.5 molecule of MPL (or one molecule of DPPC) per peptide molecule. delta H of the DPPC pretransition is reduced by the deca- and the undecapeptides and completely disappears with heptadeca- and icosapeptides (at 0.2 mole of peptide/mole of lipid). The modulation of the melting point Tm by the incorporation of peptides is more pronounced with MPL than with DPPC, the heptadecapeptide exhibiting the strongest reduction (with MPL) and the strongest broadening of the transition peak (with DPPC). Helix length, amphiphilicity and charge of the polypeptides can be correlated with the observed modifications of the lipid phase transitions.     

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum.

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.     

Effects of a squalene epoxidase inhibitor, terbinafine, on ether lipid biosyntheses in a thermoacidophilic archaeon, Thermoplasma acidophilum

The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-(14)C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum.     

Current-voltage studies on the thylakoid membrane in the presence of ionophores.

The reversibility of the binding of ionophores to the thylakoid membrane is studied. While gramicidin binds practically irreversibly, valinomycin and nonactin bind reversibly, however, only a small fraction (about 1%) of the membrane-bound valinomycin or nonactin is active in ion transport. The current-voltage relationship is evaluated under these circumstances. We have found that it is practically linear. This together with the relationship between current and ion concentration agrees qualitatively with the results reported for bimolecular lipid membranes, which contain a large fraction of negatively charged lipids. For the ionophores, valinomycin and nonactin, the binding equilibria (K approximately equal to 10-4) and the turnover numbers (approximately equal to 3-10-4/s) are evaluated for their action on the thylakoid membrane. Possible reasons for the inactivity of the majority of membrane-bound ionophore molecules are discussed.     

Current-voltage studies on the thylakoid membrane in the presence of ionophores

The reversibility of the binding of ionophores to the thylakoid membrane is studied. While gramicidin binds practically irreversibly, valinomycin and nonactin bind reversibly, however, only a small fraction (about 1 %) of the membrane-bound valinomycin or nonactin is active in ion transport. The current-voltage relationship is evaluated under these circumstances. We have found that it is practically linear. This together with the relationship between current and ion concentration agrees qualitatively with the results reported for bimolecular lipid membranes, which contain a large fraction of negatively charged lipids. For the ionophores, valinomycin and nonactin, the binding equilibria (K ≈ 10⁴) and the turnover numbers (≈ 3 · 10⁴/s) are evaluated for their action on the thylakoid membrane. Possible reasons for the inactivity of the majority of membrane-bound ionophore molecules are discussed.     

Phosphatidic acid regulates the activity of the channel-forming ionophores alamethicin,melittin, and nystatin: A1H-NMR study using phospholipid membranes

The regulation of ion channels by phosphatidic acid (a proposed active metabolite in the phosphatidylinositol effect) was investigated using¹H-NMR spectroscopy and small unilamellar phospholipid vesicles. Transport across egg-yolk phosphatidylcholine (egg PC) and dipalmitoyl phosphatidylcholine (DPPC) vesicular membranes in the presence of the channel-forming ionophores alamethicin, melittin, and nystatin was monitored using the lanthanide probe ion Pr³⁺. In the absence of the ionophores, phosphatidic acid (PA) alone was found to have no ionophore properties, but in the presence of the ionophores the incorporation of 3 mol % phosphatidic acid in the bilayer markedly increased the rate of transport using melittin and nystatin, but decreased the rate using alamethicin, independent of the type of phosphatidylcholine used. The presence of PA in the bilayer also stimulated the production of lyric type channels, the extent of which were both ionophore- and lipid-dependent. These results are discussed in terms of possible molecular interactions between the PA, the individual ionophores, and type of lipid used.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 microF/cm(2) for DPhyPC bilayers and 0.75 and 0.77 microF/cm(2) for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 mega Ohm cm(2) were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Comparison of the conformation and orientation of alamethicin and melittin in lipid membranes

The secondary structure of alamethicin in lipid membranes below and above the lipid phase transition temperature Tt is determined by Raman spectroscopy and circular dichroism (CD) measurements. In both cases structural data are obtained by fitting the experimental spectra by a superposition of the spectra of 15 reference proteins of known three-dimensional structure. According to the Raman experiments, in a lipid bilayer above Tt alamethicin is helical from residue 1 to 12, whereas below Tt the helix extends from residue 1 to 16. The remaining C-terminal part is nonhelical up to the end residue 20 both above and below Tt. A considerable lower helix content is derived from CD, namely, 38% and 46% above and below Tt, respectively, in agreement with several reported values for CD in the literature. It is shown that the commonly used set of CD spectra of water-soluble reference proteins is unsuitable to describe the CD spectra of alamethicin correctly. Therefore the secondary structure of alamethicin as derived from CD measurements is at the present state of analysis unreliable. In contrast to the case of alamethicin, the CD spectra of melittin in lipid membranes are correctly described by the reference protein spectra. The helix content of melittin is determined thereby to be 72% in lipid membranes above Tt and 75% below Tt. The data are in accord with a structure where the hydrophobic part of melittin adopts a bent helix as determined recently by Raman spectroscopy [Vogel, H., & J?hnig, F. (1986) Biophys. J. 50, 573]. The orientational order parameters of the helical parts of alamethicin and of melittin in a lipid membrane are deduced from the difference between a corresponding CD spectrum of a polypeptide in planar multibilayers and that in lipid vesicles. The presented method for determining helix order parameters is new and may be generally applicable to other membrane proteins. The orientation of the helical part of both polypeptides depends on the physical state of the lipid bilayer at maximal membrane hydration and in the ordered lipid state furthermore on the degree of membrane hydration. Under conditions where alamethicin and melittin are incorporated in an aggregated form in a fluid lipid membrane at maximal water content the helical segments are oriented preferentially parallel to the membrane normal. Cooling such lipid membranes to a temperature below Tt changes the orientation of the helical part of alamethicin as well as melittin toward the membrane plane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the precursor of tetraether lipid biosynthesis in the thermoacidophilic archaeon<Emphasis Type="Italic"> Thermoplasma acidophilum</Emphasis>

Polar lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum was analyzed using terbinafine, an inhibitor of tetraether lipid biosynthesis. Cells of T. acidophilum were labeled with [(14)C]mevalonic acid, and their lipids were extracted and analyzed by two-dimensional thin-layer chromatography. Lipids labeled with [(14)C]mevalonic acid, [(14)C]glycerol, and [(32)P]orthophosphoric acid were extracted and hydrolyzed under different conditions to determine the structure of polar lipids. The polar lipids were estimated to be archaetidylglycerol, glycerophosphatidylcaldarchaetidylglycerol, caldarchaetidylglycerol, and beta- l-gulopyranosylcaldarchaetidylglycerol, the main polar lipid of T. acidophilum. Pulse and chase experiments with terbinafine revealed that one tetraether lipid molecule is synthesized by head-to-head condensation of two molecules of archaetidylglycerol and that a sugar group of tetraether phosphoglycolipid is expected to attach to the tetraether lipid core after head-to-head condensation in T. acidophilum. A precursor accumulated in the presence of terbinafine with a fast-atom-bombardment mass spectrometry peak m/z 806 was compatible with archaetidylglycerol. The relative height of the peak m/z 806 decreased after removal of the inhibitor. The results suggest that most of the precursor, archaetidylglycerol, is in fully saturated form.     

Lipid membrane domain formation and alamethicin aggregation studied by calorimetry, sound velocity measurements, and atomic force microscopy

An experimental study of phosphocholine membranes made from one lipid, from mixtures of DPPC and DLPC, and also from lipids and small amounts of alamethicin is presented. We used atomic force microscopy to investigate the spatial organization and structure of lipid domains and also of the defects induced by the peptide. Alamethicin was found to alter the state of lipids in the gel state in a way that domains of fluid lipids are formed close to the defects. Differential calorimetry revealed phase characteristics of the lipid mixtures and the effect of small amounts of alamethicin on the phase behavior. It was also shown that the sound velocity profiles of the membranes suspensions can be well calculated from the heat capacity traces of the samples. This result confirms the correlation between the mechanical properties and the specific heat of membrane systems.     

Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles

Morphological changes induced by the melittin tetramer on bilayers of egg phosphatidylcholine and dipalmitoylphosphatidylcholine have been studied by quasi-elastic light scattering, gel filtration and freeze-fracture electron microscopy. It is concluded that melittin similarly binds and changes the morphology of both single and multilamellar vesicles, provided that their hydrocarbon chains have a disordered conformation, i.e., at temperatures higher than that of the transition, Tm. When the hydrocarbon chains are ordered (gel phase), only small unilamellar vesicles are morphologically affected by melittin. However after incubation at T greater than Tm, major structural changes are detected in the gel phase, regardless of the initial morphology of the lipids. Results from all techniques agree on the following points. At low melittin content, phospholipid-to-peptide molar ratios, Ri greater than 30, heterogeneous systems are observed, the new structures coexisting with the original ones. For lipids in the fluid phase and Ri greater than 12, the complexes formed are large unilamellar vesicles of about 1300 +/- 300 A diameter and showing on freeze-fracture images rough fracture surfaces. For lipids in the gel phase, T less than Tm after passage above Tm, and for 5 less than Ri less than 50, disc-like complexes are observed and isolated. They have a diameter of 235 +/- 23 A and are about one bilayer thick; their composition corresponds to one melittin for about 20 +/- 2 lipid molecules. It is proposed that the discs are constituted by about 1500 lipid molecules arranged in a bilayer and surrounded by a belt of melittin in which the mellitin rods are perpendicular to the bilayer. For high amounts of melittin, Ri less than 2, much smaller and more spherical objects are observed. They are interpreted as corresponding to lipid-peptide co-micelles in which probably no more bilayer structure is left. It is concluded that melittin induces a reorganization of lipid assemblies which can involve different processes, depending on experimental conditions: vesicularization of multibilayers; fusion of small lipid vesicles; fragmentation into discs and micelles. Such processes are discussed in connexion with the mechanism of action of melittin: the lysis of biological membranes and the synergism between melittin and phospholipases.     

Molecular studies of the gel to liquid-crystalline phase transition for fully hydrated DPPC and DPPE bilayers

Molecular dynamics simulations were used for a comprehensive study of the structural properties of saturated lipid bilayers, DPPC and DPPE, near the main phase transition. Though the chemical structure of DPPC and DPPE are largely similar (they only differ in the choline and ethanolamine groups), their transformation process from a gel to a liquid-crystalline state is contrasting. For DPPC, three distinct structures can be identified relative to the melting temperature (Tm): below Tm with "mixed" domains consisting of lipids that are tilted with partial overlap of the lipid tails between leaflet; near Tm with a slight increase in the average area per lipid, resulting in a rearrangement of the lipid tails and an increase in the bilayer thickness; and above Tm with unhindered lipid tails in random motion resulting in an increase in %gauche formed and increase in the level of interdigitation between lipid leaflets. For DPPE, the structures identified were below Tm with "ordered" domains consisting of slightly tilted lipid tails and non-overlapping lipid tails between leaflets, near Tm with minimal rearrangement of the lipids as the bilayer thickness reduces slightly with increasing temperature, and above Tm with unhindered lipid tails as that for DPPC. For DPPE, most of the lipid tails do not overlap as observed to DPPC, which is due to the tight packing of the DPPE molecules. The non-overlapping behavior of DPPE above Tm is confirmed from the density profile of the terminal carbon atoms in each leaflet, which shows a narrow distribution near the center of the bilayer core. This study also demonstrates that atomistic simulations are capable of capturing the phase transition behavior of lipid bilayers, providing a rich set of molecular and structural information at and near the transition state.     

The modulation of ion channels by the inhalation general anaesthetics. A1H-NMR investigation using unilamellar phospholipid membranes

The modulation of a variety of mechanisms of channel-mediated transport across unilamellar phospholipid membranes by a range of halogenated inhalation general anaesthetics (chloroform, enflurane, halothane and methoxyflurane) was investigated using 1H-NMR spectroscopy. Transport of the probe ion Pr3+ across egg yolk phosphatidylcholine (PC) and dipalmitoyl phosphatidylcholine (DPPC) vesicular membranes in the presence of the channel forming polypeptides alamethicin 30 and melittin, and the polyene antibiotic nystatin, as well as the degree of vesicular lysis at the gel to liquid-crystal phase transition of DPPC vesicles was monitored. The observation that the inhalation general anaesthetics inhibit such membrane permeability independently of the channel system or type of lipid used, suggests that hydrogen-bonded water structure and/or hydrogen-bonding centres at dipolar lipid-polypeptide interfaces, can be likely sites of action of the general anaesthetics.     

Defect formation of lytic peptides in lipid membranes and their influence on the thermodynamic properties of the pore environment

We present an experimental study of the pore formation processes of small amphipathic peptides in model phosphocholine lipid membranes. We used atomic force microscopy to characterize the spatial organization and structure of alamethicin- and melittin-induced defects in lipid bilayer membranes and the influence of the peptide on local membrane properties. Alamethicin induced holes in gel DPPC membranes were directly visualized at different peptide concentrations. We found that the thermodynamic state of lipids in gel membranes can be influenced by the presence of alamethicin such that nanoscopic domains of fluid lipids form close to the peptide pores, and that the elastic constants of the membrane are altered in their vicinity. Melittin-induced holes were visualized in DPPC and DLPC membranes at room temperature in order to study the influence of the membrane state on the peptide induced hole formation. Also differential scanning calorimetry was used to investigate the effect of alamethicin on the lipid membrane phase behaviour.     

Defect formation of lytic peptides in lipid membranes and their influence on the thermodynamic properties of the pore environment

We present an experimental study of the pore formation processes of small amphipathic peptides in model phosphocholine lipid membranes. We used atomic force microscopy to characterize the spatial organization and structure of alamethicin- and melittin-induced defects in lipid bilayer membranes and the influence of the peptide on local membrane properties. Alamethicin induced holes in gel DPPC membranes were directly visualized at different peptide concentrations. We found that the thermodynamic state of lipids in gel membranes can be influenced by the presence of alamethicin such that nanoscopic domains of fluid lipids form close to the peptide pores, and that the elastic constants of the membrane are altered in their vicinity. Melittin-induced holes were visualized in DPPC and DLPC membranes at room temperature in order to study the influence of the membrane state on the peptide induced hole formation. Also differential scanning calorimetry was used to investigate the effect of alamethicin on the lipid membrane phase behaviour.     

Partitioning and location of Bay K 8644, 1,4-dihydropyridine calcium channel agonist, in model and biological membranes.

Several lines of evidence suggest that nonspecific drug interaction with the lipid bilayer plays an important role in subsequent recognition and binding to specific receptor sites in the membrane. The interaction of Bay K 8644, a 1,4-dihydropyridine (DHP) calcium channel agonist, with model and biological membranes was examined at the molecular level using small angle x-ray diffraction. Nonspecific drug partitioning into the membrane was examined by radiochemical assay. Nonspecific binding characteristics of [3H] Bay K 8644 were determined in both dipalmitoyl phosphatidylcholine (DPPC) vesicles above and below their thermal phase transition (Tm) and rabbit skeletal muscle light sarcoplasmic reticulum (LSR). In DPPC, the partition coefficient, Kp, was 14,000 above the Tm (55 degrees C) versus 160 in the gel phase (2 degrees C). The Kp determined in LSR membranes was 10,700. These values for both DPPC and LSR membranes can be compared with Kp = 290 in the traditional octanol/buffer system. Using small-angle x-ray diffraction, the equilibrium position of the electron-dense trifluoromethyl group of Bay K 8644 in DPPC (above Tm) and purified cardiac sarcolemmal (CSL) lipid bilayers was determined to be consistently located within the region of the first few methylene segments of the fatty acyl chains of these membranes. This position is similar to that observed for the DHP calcium channel antagonists nimodipine and Bay P 8857. We suggest this particular membrane location defines a region of local drug concentration and plane for lateral diffusion to a common receptor site. Below the DPPC membrane Tm, Bay K 8644 was shown to be excluded from this energetically favored position into the interbilayer water space. Heating the DPPC bilayer above the Tm (55 degrees C) showed that this exclusion was reversible and indicates that drug-membrane interaction is dependent on the bilayer physical state. The absence of any specific protein binding sites in these systems allows us to ascertain the potentially important role that the bulk lipid phase may play in the molecular mechanism of DHP binding to the specific receptor site associated with the calcium channel.     

Perturbation of binary phospholipid mixtures by melittin: a fluorescence and raman spectroscopy study

The effect of melittin on different binary mixtures of phospholipids has been studied by polarization of DPH fluorescence in order to determine if melittin can induce phase separation. Since the interaction between lipids and melittin is sensitive to both electrostatic and hydrophobic forces, we have studied the effect of the acyl chain length and of the polar head group of the lipids. In spite of the difference of the chain length between dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), no phase separation occurs in an equimolar mixture of these lipids in presence of melittin. However, when the charged lipid dipalmitoylphosphatidylglycerol (DPPG) is mixed with either DPPC or DSPC, the addition of melittin leads to phase separation. The DSPC/DPPG/melittin system, which shows a very complex thermotropism, has also been studied by Raman spectroscopy using DPPG with deuteriated chains in order to monitor each lipid independently. The results suggest that the higher affinity of melittin for DPPG leads to a partial phase separation. We propose the formation of DPPG-rich domains perturbed by melittin and peptide-free regions enriched in DSPC triggered by the head group charge and chain-length differences.     

Annexins V and XII alter the properties of planar lipid bilayers seen by conductance probes

Annexins are proteins that bind lipids in the presence of calcium. Though multiple functions have been proposed for annexins, there is no general agreement on what annexins do or how they do it. We have used the well-studied conductance probes nonactin, alamethicin, and tetraphenylborate to investigate how annexins alter the functional properties of planar lipid bilayers. We found that annexin XII reduces the nonactin-induced conductance to approximately 30% of its original value. Both negative lipid and approximately 30 microM Ca(2+) are required for the conductance reduction. The mutant annexin XIIs, E105K and E105K/K68A, do not reduce the nonactin conductance even though both bind to the membrane just as wild-type does. Thus, subtle changes in the interaction of annexins with the membrane seem to be important. Annexin V also reduces nonactin conductance in nearly the same manner as annexin XII. Pronase in the absence of annexin had no effect on the nonactin conductance. But when added to the side of the bilayer opposite that to which annexin was added, pronase increased the nonactin-induced conductance toward its pre-annexin value. Annexins also dramatically alter the conductance induced by a radically different probe, alamethicin. When added to the same side of the bilayer as alamethicin, annexin has virtually no effect, but when added trans to the alamethicin, annexin dramatically reduces the asymmetry of the I-V curve and greatly slows the kinetics of one branch of the curve without altering those of the other. Annexin also reduces the rate at which the hydrophobic anion, tetraphenylborate, crosses the bilayer. These results suggest that annexin greatly reduces the ability of small molecules to cross the membrane without altering the surface potential and that at least some fraction of the active annexin is accessible to pronase digestion from the opposite side of the membrane.     

Lipid solvation of the aqueous form of the myelin proteolipid apoprotein: evidence and characterization of two lipid populations by fluorescence polarization, differential calorimetry, and sucrose gradient centrifugation

The interaction between dipalmitoylphosphatidylcholine (DPPC) and the aqueous form of the myelin proteolipid apoprotein (PLA) has been investigated. Lyophilization was found to be an efficient and nonperturbing method for membrane reconstitution. Mixtures of different lipid/protein ratios were analyzed by means of differential calorimetry, fluorescence polarization, and sucrose gradient centrifugation. The presence of two coexisting lipid populations, termed "bulk" and "interacting" lipids, was demonstrated by these three techniques. By differential calorimetry, 23 DPPC molecules per molecule of protein (30 kDa) were shown to be excluded from the lipid phase transition. By fluorescence polarization, we detected above the phase-transition temperature a large perturbation of the lipid acyl chain dynamics induced by the aqueous form of PLA. Increasing the protein content above 35% by weight within the recombinants caused drastic changes in both delta H values and the fluorescence anisotropy parameter, which could stem from protein aggregation.     

Trehalose and dry dipalmitoylphosphatidylcholine revisited

Dry mixtures of sonicated vesicles of DPPC and trehalose which contained a maximum of 0.2 mol water/mol lipid were examined by differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy. Samples of dry DPPC and trehalose prepared from aqueous solution had a minimum Tm of 24 degrees C for the gel to liquid-crystalline transition provided that the vesicles were dried with trehalose while the lipid was in liquid-crystalline phase. This low transition is compared to a transition of 105-112 degrees C for dry pure DPPC and of 42 degrees C for hydrated pure DPPC. The present work is an extension of earlier work from this laboratory using both other lipids and other methods of preparation.