Melittin-solid phospholipid mixed films trigger amyloid-like nano-fibril arrangements at air-water interface

We used the Langmuir monolayers technique to study the surface properties of melittin toxin mixed with either liquid-condensed DSPC or liquid-expanded POPC phospholipids. Pure melittin peptide forms stable insoluble monolayers at the air-water interface without interacting with Thioflavin T (Th-T), a sensitive probe to detect protein amyloid formation. When melittin peptide is mixed with DSPC lipid at 50 % of peptide area proportion at the surface, we observed the formation of fibril-like structures detected by Brewster angle microscopy (BAM), but they were not observable with POPC. The nano-structures in the melittin-DSPC mixtures became Th-T positive labeling when the arrangement was observed with fluorescence microscopy. In this condition, Th-T undergoes an unexpected shift in the typical emission wavelength of this amyloid marker when a 2D fluorescence analysis is conducted.Even when reflectivity analysis of BAM imaging evidenced that these structures would correspond to the DSPC lipid component of the mixture, the interpretation of ATR-FTIR and Th-T data suggested that both components were involved in a new lipid-peptide rearrangement. These nano-fibril arrangements were also evidenced by scanning electron and atomic force microscopy when the films were transferred to a mica support. The fibril formation was not detected when melittin was mixed with the liquid-expanded POPC lipid. We postulated that DSPC lipids can dynamically trigger the process of amyloid-like nano-arrangement formation at the interface. This process is favored by the relative peptide content, the quality of the interfacial environment, and the physical state of the lipid at the surface.     

Interaction of soluble and membrane proteins with monolayers of glycosphingolipids.

1. The interactions of four proteins (albumin, myelin basic protein, melittin and glycophorin) with eight neutral or acidic glycosphingolipids, including sulphatides and gangliosides, five zwitterionic or anionic phospholipids and some of their mixtures, were studied in lipid monolayers at the air/145 mM-NaCl interface. 2. In lipid-free interfaces, the surface pressure and surface potential reached by either soluble or integral membrane proteins did not reveal marked differences. 3. All the proteins studied showed interactions with each of the lipids but the maximal interactions were found for basic proteins with acidic glycosphingolipids. 4. Surface-potential measurements indicated that different dipolar organizations at the interface can be adopted by lipid-protein interactions showing the same value for surface free energy. 5. The individual surface properties of either the lipid of protein component are modified as a consequence of the lipid-protein interaction. 6. In mixed-lipid monolayers, the composition of the interface may affect the lipid-protein interactions in a non-proportional manner with respect to the relative amount of the individual lipid components.     

Aggregation of puroindoline in phospholipid monolayers spread at the air-liquid interface

Puroindolines, cationic and cystine-rich low molecular weight lipid binding proteins from wheat seeds, display unique foaming properties and antimicrobial activity. To unravel the mechanism involved in these properties, the interaction of puroindoline-a (PIN-a) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers was studied by coupling Langmuir-Blodgett and imaging techniques. Compression isotherms of PIN-a/phospholipid monolayers and adsorption of PIN-a to lipid monolayers showed that the protein interacted strongly with phospholipids, especially with the anionic DPPG. The electrostatic contribution led to the formation of a highly stable lipoprotein monolayer. Confocal laser scanning microscopy and atomic force microscopy showed that PIN-a was mainly inserted in the liquid-expanded phase of the DPPC, where it formed an aggregated protein network and induced the fusion of liquid-condensed domains. For DPPG, the protein partitioned in both the liquid-expanded and liquid-condensed phases, where it was aggregated. The extent of protein aggregation was related both to the physical state of phospholipids, i.e., condensed or expanded, and to the electrostatic interactions between lipids and PIN-a. Aggregation of PIN-a at air-liquid and lipid interfaces could account for the biological and technological properties of this wheat lipid binding protein.     

Lipid-like behavior of signal sequence peptides at air–water interface

Several protein transport processes in the cell are mediated by signal sequence peptides located at the N-terminal side of the mature protein sequence. To date, the specific interaction and the stability of these peptides at the amphipathic interface of biological membranes and the relevance of the peptide conformation when they interact with lipids is not clear. We report the surface properties and the peptide–lipid interaction of three signal sequence peptides at the air–NaCl 145 mM interface by using the Langmuir monolayer approach. These synthetic peptides have a natural sequence with a non-periodic amphiphilicity, where hydrophobic and hydrophilic residues are located on opposed sides of the peptide primary sequence. We show that signal sequence peptides form insoluble monolayers of high stability against lateral compression. At close packing, peptide molecular area, surface potential and the high stability of the peptide monolayer are indicative that signal sequence peptides are compatible with a β-sheet conformation at the interface. Structure was confirmed with PM-IRRAS and transmission FT-IR studies. The peptides show lateral miscibility with either POPC (a liquid-expanded lipid) or DPPC (a liquid-condensed lipid) in mixed peptide–lipid monolayers. This indicates that signal sequence peptides studied are laterally miscible with phospholipids independent of the phase state of the lipid.     

Monolayer and calorimetric studies of phosphatidylcholines containing branched-chain fatty acids and of their interactions with cholesterol and with a bacterial hopanoid in model membranes

Although methyl iso- and anteiso-branched fatty acids occur widely in the membrane lipids of prokaryotic microorganisms, relatively little is known about the physical properties of phospholipids containing these fatty acids. We report here a monolayer and differential scanning calorimetric characterization of several synthetic phosphatidylcholines containing branched-chain fatty acids, and describe the interactions of these phospholipids with cholesterol and with a bacterial hopanoid. We find that monolayers as well as bilayers of methyl isobranched- and especially of methyl anteisobranched-fatty-acid-containing phosphatidylcholines exhibit a reduced solid-to-fluid phase transition temperature in comparison with linear saturated fatty acid-containing phosphatidylcholines of comparable chain length. We also find that the liquid-condensed or gel states of branched-chain fatty acid-containing phosphatidylcholines are partially disordered relative to those of phospholipids containing linear saturated fatty acids, although the presence of a methyl branch has only a small effect on hydrocarbon chain packing in the liquid-expanded or liquid-crystalline states. The presence of cholesterol was found to produce a marked condensation of liquid-expanded films and a small condensation of liquid-condensed films, whether the phosphatidylcholine contained linear or branched-chain fatty acyl constituents. The presence of a bacterial hopanoid produced similar, although slightly smaller, monolayer-condensing effects, indicating that these compounds may perform a cholesterol-like function in bacterial membranes.     

Regulation of EGFR activation and signaling by lipids on the plasma membrane

Lipids on the plasma membrane are not only components of the membrane biophysical structures but also regulators of receptor functions. Recently, the critical roles of lipid-protein interactions have been intensively highlighted. Epidermal growth factor receptor (EGFR) is one of the most extensively studied receptors exhibiting various lipid interactions, including interactions with phosphatidylcholine, phosphatidylserine, phosphatidylinositol phosphate, cholesterol, gangliosides, and palmitate. Here, we review recent findings on how direct interaction with these lipids regulates EGFR activation and signaling, providing unprecedented insight into the comprehensive roles of various lipids in the control of EGFR functions. Finally, the current limitations in investigating lipid-protein interactions and novel technologies to potentially overcome these limitations are discussed.     

Ganglioside-protein interactions: spin-label electron spin resonance studies with (Na+,K+)-ATPase membranes

Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from Squalus acanthias have been studied using spin-labeled derivatives of the mono- and disialogangliosides GM1, GM2, GM3, and GD1b, in conjunction with electron spin resonance (ESR) spectroscopy. Ganglioside-protein interactions are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component that corresponds closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled gangliosides whose chain motion is significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A small selectively for the motionally restricted component associated with the protein is found in the order GD1b greater than GM1 approximately equal to GM2 approximately equal to GM3. Comparison with previous results from spin-labeled phospholipids in the same system [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] shows that the spin-labeled monosialogangliosides GM1, GM2, and GM3 display little selectivity in the lipid-protein interaction relative to spin-labeled phosphatidylcholine. The spectral characteristics of both the fluid and motionally restricted spin-labeled components differ very significantly, however, between the gangliosides and the phospholipids. The outer hyperfine splitting of the motionally restricted component is smaller for the gangliosides than for the phospholipids, indicating a smaller degree of motional restriction on interaction of the ganglioside lipid chains with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

31P and19F NMR studies of glycophorin-reconstituted membranes: Preferential interaction of glycophorin with phosphatidylserine

Summary Glycophorin A, a major glycoprotein of the erythrocyte membrane, has been incorporated into small unilamellar vesicles composed of a variety of pure and mixed phospholipids. Nuclear spin labels including³¹P and¹⁹F have been used at natural abundance or have been synthetically incorporated in lipids to act as probes of lipid-protein interaction. Interactions produce broadening of resonances in several cases and it can be used to demonstrate preferential interaction of certain lipids with glycophorin.³¹P and¹⁹F probes show a strong preferential interaction of glycophorin with phosphatidylserine over phosphatidylcholine. There is some evidence that interactions are more pronounced at the inner surface of the bilayer and these results are rationalized in terms of the asymmetric distribution of protein and lipid.     

Effect of the state of association of melittin and phospholipids on their reciprocal binding

In solutions of increasing ionic strength, the molecular weight of melittin varies from 2840 (monomeric melittin) to 11 200. This polymerization, concomitant with an important change in conformation (Talbot, J.C., Dufourcq, J., De Bony, J., Faucon, J.F. and Lussan, C. (1979) FEBS Lett. 102, 191–193), is accompanied by a significant alteration in the partial specific volume of the molecule. The binding of melittin to phospholipids (phosphatidylserine, lysolecithin, dihexanoyl-, dioctanoyl- and lysolauroylphosphatidylcholine) depends on the state of association of the toxin and on the critical micelle concentration of lipids. No interaction is observed between monomeric melittin and free lipids, whereas tetrameric melittin can bind free lipids to form mixed micelles. At phospholipid concentrations above the critical micelle concentration, melittin in any state of self-association can bind lipids. The mixed micelles formed at saturation appear to be independent of the initial state of association of melittin.     

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.     

Effect of Pulmonary Surfactant Protein SP-B on the Micro- and Nanostructure of Phospholipid Films

Monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3, w/w) in the absence or in the presence of 2, 5, 10, or 20 weight percent of porcine surfactant protein SP-B were spread at the air-liquid interface of a surface balance, compressed up to surface pressures in the liquid-expanded/liquid-condensed (LE-LC) plateau of the isotherm, transferred onto mica supports, and analyzed by scanning force microscopy. In the absence of protein, the films showed micrometer-sized condensed domains with morphology and size that were analogous to those observed in situ at the air-liquid interface by epifluorescence microscopy. Scanning force microscopy permits examination of the coexisting phases at a higher resolution than previously achieved with fluorescent microscopy. Both LE and LC regions of DPPC films were heterogeneous in nature. LC microdomains contained numerous expanded-like islands whereas regions apparently liquid-expanded were covered by a condensed-like framework of interconnected nanodomains. Presence of increasing amounts of pulmonary surfactant protein SP-B affected the distribution of the LE and LC regions of DPPC and DPPC/DPPG films both at the microscopic and the nanoscopic level. The condensed microdomains became more numerous but their size decreased, resulting in an overall reduction of the amount of total LC phase in both DPPC and DPPC/DPPG films. At the nanoscopic level, SP-B also caused a marked reduction of the size of the condensed-like nanodomains in the LE phase and an increase in the length of the LE/LC interface. SP-B promotes a fine nanoscopic framework of lipid and lipid-protein nanodomains that is associated with a substantial mechanical resistance to film deformation and rupture as observed during film transference and manipulation. The effect of SP-B on the nanoscopic structure of the lipid films was greater in DPPC/DPPG than in pure DPPC films, indicating additional contributions of electrostatic lipid-protein interactions. The alterations of the nanoscopic structures of phospholipid films by SP-B provide the structural framework for the protein simultaneously sustaining structural stability as well as dynamical flexibility in surfactant films at the extreme conditions imposed by the respiratory mechanics. SP-B also formed segregated two-dimensional clusters that were associated with the boundaries between LC microdomains and the LE regions of DPPC and DPPC/DPPG films. The presence of these clusters at protein-to-lipid proportions above 2% by weight suggests that the concentration of SP-B in the surfactant lipid-protein complexes may be close to the solubility limit of the protein in the lipid films.     

Phase separations induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry

Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical state of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C₁₄ lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10–20°C. This second group of lipids is constituted by monoacid C₁₆ lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C₁₆ aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C₁₄ chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.     

Fluorescent probes in model membranes. II. monolayer studies of 2,2′-(vinylenedi-p-phenylene)bisbenzoxazole, d-3-aminodesoxyequilenin and n-octadecylnaphthyl-2-amino-6-sulfonic acid with host-lipid tetradecanoic acid

Film studies at the air-water interface have been carried out for pure films of 2,2′-(vinylenedi-p-phenylene)bisbenzoxazole (VPBO), d-3-aminodesoxy-equlenin (EQ) and N-octadecylnapthyl-2-amino-6-sulfonic acid (ONS), and for mixed films with tetradecanoic acid for the first two fluorescent probes. Pure film isotherms indicate highly rigid non-monomolecular films for both VPBO and EQ, revealing the presence of strong intermolecular forces. In mixed films with tetradecanoic acid VPBO rapidly segregates with resultant film loss over a wide concentration range. EQ, however, can be stabilized by the host-lipid at low concentrations. This, coupled with an ability to only slightly affect the host-lipid liquid-condensed/liquid-expanded phase change, suggests that EQ can be regarded as “non-perturbing” and should be retained in condensed lipid phases.ONS, because of its unusual polar headgroup, resembled hexadecanoic acid more than octadecanoic acid. While difficulties in spreading ONS precluded the study of mixed films, the indications are that it would be a satisfactory expanded lipid state probe if mixing can be brought about.     

Effects of melittin on lipid-protein interactions in sarcoplasmic reticulum membranes.

To investigate the physical mechanism by which melittin inhibits Ca-adenosine triphosphatase (ATPase) activity in sarcoplasmic reticulum (SR) membranes, we have used electron paramagnetic resonance spectroscopy to probe the effect of melittin on lipid-protein interactions in SR. Previous studies have shown that melittin substantially restricts the rotational mobility of the Ca-ATPase but only slightly decreases the average lipid hydrocarbon chain fluidity in SR. Therefore, in the present study, we ask whether melittin has a preferential effect on Ca-ATPase boundary lipids, i.e., the annular shell of motionally restricted lipid that surrounds the protein. Paramagnetic derivatives of stearic acid and phosphatidylcholine, spin-labeled at C-14, were incorporated into SR membranes. The electronic paramagnetic resonance spectra of these probes contained two components, corresponding to motionally restricted and motionally fluid lipids, that were analyzed by spectral subtraction. The addition of increasing amounts of melittin, to the level of 10 mol melittin/mol Ca-ATPase, progressively increased the fraction of restricted lipids and increased the hyperfine splitting of both components in the composite spectra, indicating that melittin decreases the hydrocarbon chain rotational mobility for both the fluid and restricted populations of lipids. No further effects were observed above a level of 10 mol melittin/mol Ca-ATPase. In the spectra from control and melittin-containing samples, the fraction of restricted lipids decreased significantly with increasing temperature. The effect of melittin was similar to that of decreased temperature, i.e., each spectrum obtained in the presence of melittin (10:1) was nearly identical to the spectrum obtained without melittin at a temperature approximately 5 degrees C lower. The results suggest that the principal effect of melittin on SR membranes is to induce protein aggregation and this in turn, augmented by direct binding of melittin to the lipid, is responsible for the observed decreases in lipid mobility. Protein aggregation is concluded to be the main cause of inactivation of the Ca-ATPase by melittin, with possible modulation also by the decrease in mobility of the boundary layer lipids.     

Thermodynamics of lipid interactions in complex bilayers

The mutual interactions between lipids in bilayers are reviewed, including mixtures of phospholipids, and mixtures of phospholipids and cholesterol (Chol). Binary mixtures and ternary mixtures are considered, with special emphasis on membranes containing Chol, an ordered phospholipid, and a disordered phospholipid. Typically the ordered phospholipid is a sphingomyelin (SM) or a long-chain saturated phosphatidylcholine (PC), both of which have high phase transitions temperatures; the disordered phospholipid is 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC). The unlike nearest-neighbor interaction free energies (ω_AB) between lipids (including Chol), obtained by an variety of unrelated methods, are typically in the range of 0-400 cal/mol in absolute value. Most are positive, meaning that the interaction is unfavorable, but some are negative, meaning it is favorable. It is of special interest that favorable interactions occur mainly between ordered phospholipids and Chol. The interpretation of domain formation in complex mixtures of Chol and phospholipids in terms of phase separation or condensed complexes is discussed in the light of the values of lipid mutual interactions.     

Biophysical properties of a synthetic transit peptide from wheat chloroplast ribulose 1,5-bisphosphate carboxylase.

The surface properties of pure RuBisCo transit peptide (RTP) and its interaction with zwitterionic, anionic phospholipids and chloroplast lipids were studied by using the Langmuir monolayer technique. Pure RTP is able to form insoluble films and the observed surface parameters are compatible with an alpha-helix perpendicular to the interface. The alpha-helix structure tendency was also observed by using transmission FT-IR spectroscopy in bulk system of a membrane mimicking environment (SDS). On the other hand, RTP adopts an unordered structure in either aqueous free interface or in the presence of vesicles composed of a zwitterionic phospholipid (POPC). Monolayer studies show that in peptide/lipid mixed monolayers, RTP shows no interaction with zwitterionic phospholipids, regardless of their physical state. Also, with the anionic POPG at high peptide ratios RTP retains its individual surface properties and behaves as an immiscible component of the peptide/lipid mixed interface. This behaviour was also observed when the mixed films were composed by RTP and the typical chloroplast lipids MGDG or DGDG (mono- and di-galactosyldiacylglycerol). Conversely, RTP establishes a particular interaction with phosphatidylglycerol and cardiolipin at low peptide to lipid area covered relation. This interaction takes place with an increase in surface stability and a reduction in peptide molecular area (intermolecular interaction). Data suggest a dynamic membrane modulation by which the peptide fine-tunes its membrane orientation and its lateral stability, depending on the quality (lipid composition) of the interface.     

Adrenocorticotropic hormone (ACTH)-lipid interactions. Implication for involvement of amphipathic helix formation

ACTH-lipid interactions were investigated by: (1) lipid-monolayer studies using several zwitterionic and anionic phospholipids and gangliosides, (2) permeability experiments by following the swelling rate of liposomes in isotonic glycerol solutions by light scattering, using liposomes of synthetic lipids and liposomes made of lipids extracted from light synaptic plasma membranes, and (3) by steady-state fluorescence anisotropy measurements on liposomes derived from light synaptic plasma membranes employing 1,6-diphenyl-1,3,5-hexatriene as fluorescent probe. (1) The monolayer experiments demonstrated an interaction with gangliosides G_T1, G_M1, dioleoylphosphatidic acid and phosphatidylserine, but little or no interaction with phosphatidylcholine or sphingomyelin. The interaction with monolayers of G_T1 or phosphatidic acid decreased for ACTH_1-13-NH₂ and ACTH_1-10. (2) The liposome experiments showed that $\text{2·10}{}^{\mathrm{?5}}$ M ACTH_1–24 increased the glycerol permeability by 20% and decreased the activation energy only when liposomes derived from light synaptic plasma membranes were used. Treatment of the liposomes with neuraminidase abolished the ACTH-induced permeability increase. (3) Steady-state fluorescence depolarization measurements revealed that ACTH_1–24, ACTH_1-16-NH₂ and ACTH_1–10 did not change the fluidity of liposomes derived from light synaptic plasma membranes as sensed by diphenylhexatriene. It is concluded that ACTH_1–24 can bind to negatively charged lipids and can form an amphipathic helix aligned parallel to the membrane surface involving the N-terminal residues 1 to 12, possibly to 16. Polysialogangliosides will favorably meet the condition of a high local surface charge density under physiological circumstances. It is suggested that ACTH-ganglioside interactions will participate in ACTH-receptor interactions.     

Force-field dependence on the liquid-expanded to liquid-condensed transition in DPPC monolayers

We have investigated using molecular dynamics simulations, the influence of the interaction cut-off and water model on the surface-pressure area isotherms of dipalmitoylphosphatidylcholine monolayers, where the phospholipids and the water molecules are modelled atomistically. We find that both the cut-off employed and the water model, influence the pressure area isotherms and the location of the liquid-expanded to liquid-condensed transitions. The combination of the Berger's et al. force field, with the TIP4P/2005 water model, and a long cut-off for the pair interactions ( ≥ 1.7 nm) provides a more accurate prediction of the surface pressure–area isotherm and reproduces the liquid condensed–liquid expanded transition observed in the experiments at 310 K.     

Modulatory effects of different temperatures and Ca2+ concentrations on gangliosides and phospholipids in monolayers at air/water interfaces and their possible functional role

Gangliosides are neuraminic acid-containing glycolipids preferently localized in nervous membranes and showing physicochemical peculiarities, e.g., drastically changing amphiphilic properties by Ca2+ binding. On account of this they are favorite compounds to act as modulators of membraneous organization and functions during synaptic transmission. Lipid monolayers are suitable experimental systems for the study of the surface behavior of amphipatic molecules and therefore are useful to interpret membraneous organization. The surface pressure/area isotherms of monolayers of different individual gangliosides (GM1, GD1a, GD1b, GT1b) of an artificial reconstituted and a natural ganglioside mixture from bovine brain and of ganglioside mixtures from different brain parts of summer- and winter-adapted dsungarian hamsters were compared at three temperatures (11, 20, and 37 degrees C) with egg phosphatidylcholine (PC) and phosphatidylserine (PS) monolayers. The monolayers were formed in a Teflon trough on a triethanolamine/HCl-buffered (pH 7.4) subphase, in some cases containing different amounts of CaCl2. The surface pressure/area isotherms of ganglioside monolayers, in contrast to phospholipids, generally showed slowly rising slopes, with transitions from the liquid-expanded to the liquid-condensed state at a surface pressure of 20-30 mN/m. Ganglioside monolayers, in particular from GD1a or GT1b versus GD1b or from mixtures from summer- versus winter-adapted hamster brain, were differently affected by temperature and/or by Ca2+. PS monolayers were slightly condensed only by Ca2+. PC monolayers, however, were influenced neither by temperature nor by Ca2+. In mixed monolayers of the unpolar natural lipid cholesterol (Ch) and the disialoganglioside GD1a, intermolecular interactions were indicated. Ganglioside monolayers, in contrast to phospholipids, were shown to be easily modulated by temperature and/or Ca2+ ions, thus enabling gangliosides to act as possible membrane modulators, e.g., during synaptic transmission. In particular, the differences concerning the influences of temperature and/or Ca2+ on the surface behavior of ganglioside mixtures from the brain of summer- compared with winter-adapted hamsters are correlated with other physiologically relevant data.