ABSTRACT
We have reconstituted functional Na(+)/K(+)-ATPase (NKA) into giant unilamellar vesicles (GUVs) of well-defined binary and ternary lipid composition including cholesterol. The activity of the membrane system can be turned on and off by ATP. The hydrolytic activity of NKA is found to depend on membrane phase, and the water relaxation in the membrane on the presence of NKA. By collapsing and fixating the GUVs onto a solid support and using high-resolution atomic-force microscopy (AFM) imaging we determine the protein orientation and spatial distribution at the single-molecule level and find that NKA is preferentially located at lo/ld interfaces in two-phase GUVs and homogeneously distributed in single-phase GUVs. When turned active, the membrane is found to unbind from the support suggesting that the protein function leads to softening of the membrane.
Subject(s)
Lipid Bilayers , Sodium-Potassium-Exchanging ATPase/chemistry , Unilamellar LiposomesABSTRACT
Giant unilamellar vesicles (GUVs) are simple model membrane systems of cell-size, which are instrumental to study the function of more complex biological membranes involving heterogeneities in lipid composition, shape, mechanical properties, and chemical properties. We have devised a method that makes it possible to prepare a uniform sample of ternary GUVs of a prescribed composition and heterogeneity by mixing different populations of small unilamellar vesicles (SUVs). The validity of the protocol has been demonstrated by applying it to ternary lipid mixture of DOPC, DPPC, and cholesterol by mixing small unilamellar vesicles (SUVs) of two different populations and with different lipid compositions. The compositional homogeneity among GUVs resulting from SUV mixing is quantified by measuring the area fraction of the liquid ordered-liquid disordered phases in giant vesicles and is found to be comparable to that in GUVs of the prescribed composition produced from hydration of dried lipids mixed in organic solvent. Our method opens up the possibility to quickly increase and manipulate the complexity of GUV membranes in a controlled manner at physiological buffer and temperature conditions. The new protocol will permit quantitative biophysical studies of a whole new class of well-defined model membrane systems of a complexity that resembles biological membranes with rafts.
Subject(s)
Complex Mixtures , Lipids/chemistry , Microscopy, ConfocalABSTRACT
The Na(+)/K(+)-ATPase pumps three sodium ions out of and two potassium ions into the cell for each ATP molecule that is split, thereby generating the chemical and electrical gradients across the plasma membrane that are essential in, for example, signalling, secondary transport and volume regulation in animal cells. Crystal structures of the potassium-bound form of the pump revealed an intimate docking of the alpha-subunit carboxy terminus at the transmembrane domain. Here we show that this element is a key regulator of a previously unrecognized ion pathway. Current models of P-type ATPases operate with a single ion conduit through the pump, but our data suggest an additional pathway in the Na(+)/K(+)-ATPase between the ion-binding sites and the cytoplasm. The C-terminal pathway allows a cytoplasmic proton to enter and stabilize site III when empty in the potassium-bound state, and when potassium is released the proton will also return to the cytoplasm, thus allowing an overall asymmetric stoichiometry of the transported ions. The C terminus controls the gate to the pathway. Its structure is crucial for pump function, as demonstrated by at least eight mutations in the region that cause severe neurological diseases. This novel model for ion transport by the Na(+)/K(+)-ATPase is established by electrophysiological studies of C-terminal mutations in familial hemiplegic migraine 2 (FHM2) and is further substantiated by molecular dynamics simulations. A similar ion regulation is likely to apply to the H(+)/K(+)-ATPase and the Ca(2+)-ATPase.
Subject(s)
Ion Transport , Migraine with Aura/metabolism , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Crystallography, X-Ray , Humans , Migraine with Aura/genetics , Models, Molecular , Molecular Dynamics Simulation , Oocytes/metabolism , Potassium/metabolism , Protons , Squalus acanthias/metabolism , Sus scrofa/metabolism , XenopusABSTRACT
Interaction between integral membrane proteins and the lipid-bilayer component of biological membranes is expected to mutually influence the proteins and the membrane. We present quantitative evidence of a manifestation of the lipid-protein interactions in liposomal membranes, reconstituted with actively pumping Na(+),K(+)-ATPase, in terms of nonequilibrium shape fluctuations that contain a relaxation time, τ, which is robust and independent of the specific fluctuation modes of the membrane. In the case of pumping Na(+)-ions, analysis of the flicker-noise temporal correlation spectrum of the liposomes leads to τ ~/= 0.5 s, comparing favorably with an intrinsic reaction-cycle time of about 0.4 s from enzymology.
Subject(s)
Lipid Metabolism , Lipids/chemistry , Liposomes/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Adenosine Triphosphate/metabolism , Animals , Cholesterol/chemistry , Enzyme Activation , Phosphatidylcholines , Phosphatidylserines , Protein Binding , Sharks , Sodium-Potassium-Exchanging ATPase/chemistry , Time FactorsABSTRACT
Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes and the notion of lipid rafts gives specificity to this broad concept. However, the lipid raft field is now at a technical impasse because the physical tools to study biological membranes as a liquid that is ordered in space and time are still being developed. This has lead to a disconnection between the concept of lipid rafts as derived from biochemical and biophysical assays and their existence in the cell. Here, we compare the concept of lipid rafts as it has emerged from the study of synthetic membranes with the reality of lateral heterogeneity in biological membranes. Further application of existing tools and the development of new tools are needed to understand the dynamic heterogeneity of biological membranes.
Subject(s)
Biology , Cell Membrane/metabolism , Membrane Microdomains/chemistry , Physics , Cell Membrane/chemistry , Humans , Models, Molecular , Physical Phenomena , Protein Structure, Tertiary , Proteins/chemistry , Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Phosphorylation is one of the major mechanisms for posttranscriptional modification of proteins. The addition of a compact, negatively charged moiety to a protein can significantly change its function and localization by affecting its structure and interaction network. We have used all-atom Molecular Dynamics simulations to investigate the structural consequences of phosphorylating the Na(+)/K(+)-ATPase (NKA) residue Ser(936), which is the best characterized phosphorylation site in NKA, targeted in vivo by protein kinase A (PKA). The Molecular Dynamics simulations suggest that Ser(936) phosphorylation opens a C-terminal hydrated pathway leading to Asp(926), a transmembrane residue proposed to form part of the third sodium ion-binding site. Simulations of a S936E mutant form, for which only subtle effects are observed when expressed in Xenopus oocytes and studied with electrophysiology, does not mimic the effects of Ser(936) phosphorylation. The results establish a structural association of Ser(936) with the C terminus of NKA and indicate that phosphorylation of Ser(936) can modulate pumping activity by changing the accessibility to the ion-binding site.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Molecular Dynamics Simulation , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium/metabolism , Animals , Binding Sites , Female , Humans , Hydrogen Bonding , Models, Molecular , Mutation , Phosphorylation , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/genetics , Swine , Water/chemistry , Water/metabolism , Xenopus laevisABSTRACT
Catansomes, which are vesicles prepared from mixtures of oppositely charged surfactants, have been suggested as effective alternatives to phospholipid vesicles, i.e., liposomes, in applications such as drug-delivery. This is mainly due to their enhanced chemical and physical stability as well as to their relatively easy preparation, which is an advantage for large-scale productions. In this study we have investigated catansomes prepared from a perfluorinated anionic surfactant (sodium perfluorooctanoate) premixed with a hydrogenated cationic surfactant (dodecyltrimethylammonium bromide or 1-dodecylpyridinium chloride). The aim was to gain insights into the physicochemical properties of these systems, such as size, stability, surface charge, and membrane morphology, which are essential for their use in drug-delivery applications. The catansomes were mostly unilamellar and 100-200 nm in size, and were stable for more than five months at room temperature. After loading the catansomes with the fluorescent marker calcein, they were found to exhibit an appreciable encapsulation efficiency and a low calcein leakage over time. The addition of fatty acids to calcein-loaded catansomes considerably promoted the release of calcein, and the rate and efficiency of calcein release were found to be proportional to the fatty acid concentration and chain length. Our results prove the feasibility of utilizing catansomes as drug-delivery vehicles as well as provide a means to efficiently release the encapsulated load.
Subject(s)
Drug Carriers/chemistry , Drug Delivery Systems , Surface-Active Agents/chemistry , Drug Carriers/chemical synthesis , Molecular Structure , Particle Size , Surface Properties , Surface-Active Agents/chemical synthesisABSTRACT
The leaves of the annual plant Perilla frutescens are used widely as a spice and a preservative in Asian food as well as in traditional medicine. The active compounds in the leaves are the cyclic monoterpene limonene (1) and its bio-oxidation products, perillaldehyde (2), perillyl alcohol (3), and perillic acid (4). These compounds are known to be biologically active and exhibit antimicrobial, anticancer, and anti-inflammatory effects that could all be membrane mediated. In order to assess the possible biophysical effects of these compounds on membranes quantitatively, the influence of limonene and its bio-oxidation products has been investigated on a membrane model composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) using differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and electron paramagnetic resonance spectroscopy (EPR). It was found that limonene (1), perillyl alcohol (2), and perillaldehyde (3) partitioned into the DMPC membrane, whereas perillic acid (4) did not. The DSC results demonstrated that all the partitioning compounds strongly perturbed the phase transition of DMPC, whereas no perturbation of the local membrane order was detected by EPR spectroscopy. The results of the study showed that limonene (1) and its bio-oxidation products affect membranes in rather subtle ways.
Subject(s)
Cell Membrane/physiology , Membrane Lipids/physiology , Perilla frutescens/chemistry , Plants, Medicinal/chemistry , Cyclohexenes/analysis , Cyclohexenes/chemistry , Cyclohexenes/pharmacology , Dimyristoylphosphatidylcholine/chemistry , Limonene , Models, Molecular , Molecular Structure , Monoterpenes/analysis , Monoterpenes/chemistry , Monoterpenes/pharmacology , Plant Leaves/chemistry , Terpenes/analysis , Terpenes/chemistry , Terpenes/pharmacologyABSTRACT
Diet and lifestyle have an impact on the burden of ill health and non-communicable ailments such as cardiovascular disease (including hypertension), obesity, diabetes, cancer and certain mental illnesses. The consequences of malnutrition and critical unbalances in the diet with regard to sugar, salt and fat are becoming increasingly manifest in the Western world and are also gradually influencing the general health condition for populations in developing countries. In this topical mini-review I highlight the lack of deliciousness and umami (savoury) flavour in prepared meals as a possible reason for poor nutritional management and excess intake of salt, fat and sugar. I argue that a better informed use of the current scientific understanding of umami and its dependence of the synergetic relationship between monosodium glutamate and certain 5'-ribonucleotides and their action on the umami taste receptors will not only provide better-tasting and more flavoursome meals but may also help to regulate food intake, in relation to both overeating and nutritional management of elderly and sick individuals.
Subject(s)
Appetite Regulation , Flavoring Agents , Food Additives , Food Preferences , Sodium Glutamate , Taste , Dietary Fats/adverse effects , Dietary Sucrose/adverse effects , Flavoring Agents/pharmacology , Food Additives/pharmacology , Food Safety , Humans , Nutrition Disorders/prevention & control , Sodium Chloride, Dietary/adverse effects , Sodium Glutamate/pharmacology , Taste BudsABSTRACT
Foods are complex systems due to their biological origin. Biological materials are soft matter hierarchically structured on all scales from molecules to tissues. The structure reflects the biological constraints of the organism and the function of the tissue. The structural properties influence the texture and hence the mouthfeel of foods prepared from the tissue, and the presence of flavour compounds is similarly determined by biological function. Cephalopods, such as squid, cuttlefish, and octopuses, are notoriously known for having challenging texture due to their muscles being muscular hydrostats with highly cross-linked collagen. Similar with other marine animals such as fish and crustaceans, cephalopods are rich in certain compounds such as free amino acids and free 5'-ribonucleotides that together elicit umami taste. Scientific investigations of culinary applications of cephalopods as foods must therefore involve mechanical studies (texture analysis), physicochemical measurements of thermodynamic properties (protein denaturation), as well as chemical analysis (taste and aroma compounds). The combination of such basic science investigations of food as a soft material along with an exploration of the gastronomic potential has been termed gastrophysics. In this review paper, we reviewed available gastrophysical studies of cephalopod structure, texture, and taste both as raw, soft material and in certain preparations.
ABSTRACT
Secretory phospholipase A(2) (sPLA(2)) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (L(α)/L(ß)) lipid domains. In this work, we explore whether lipid domains can also direct the activity of the enzyme by inducing hydrolysis of certain lipid components due to preferential activity of the enzyme toward lipid domains susceptible to sPLA(2). Specifically, we show that the presence of L(α)/L(ß) and L(α)/P(ß') phase coexistence in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) system results in the preferential hydrolysis of the shorter-chained lipid component in the mixture, leading to an enrichment in the longer-chained component. The restructuring process is monitored by atomic force microscopy on supported single and double bilayers formed by vesicle fusion. We observe that during preferential hydrolysis of the DMPC-rich L(α) regions, the L(ß) and P(ß') regions grow and reseal, maintaining membrane integrity. This result indicates that a sharp reorganization of the membrane structure can occur during sPLA(2) hydrolysis without necessarily destroying the membrane. We confirm by high-performance liquid chromatography the preferential hydrolysis of DMPC within the phase coexistence region of the DMPC/DSPC phase diagram, showing that this preferential hydrolysis is accentuated close to the solidus phase boundary. Differential scanning calorimetry results show that this preferential hydrolysis in the presence of lipid domains leads to a membrane system with a higher-temperature melting profile due to enrichment in DSPC. Together, these results show that the presence of lipid domains can induce specificity in the hydrolytic activity of the enzyme, resulting in marked differences in the physical properties of the membrane end-product.
Subject(s)
Lipids/chemistry , Membranes, Artificial , Phospholipases A2, Secretory/metabolism , Animals , Calorimetry, Differential Scanning , Dimyristoylphosphatidylcholine/chemistry , Hydrolysis , Lipid Bilayers/chemistry , Microscopy, Atomic Force , Phase Transition , Snake Venoms/enzymologyABSTRACT
Biomembranes are unique states of soft matter that share some of their materials properties with the mesophases of liquid crystals. Although of genuinely fluid character, membranes can display ordered states under physiological conditions, and it appears that their lateral organization and the related functional properties are intimately coupled to states in-between order and disorder. Hence, the liquid-ordered state of membranes, which owes its existence to the unique ability of cholesterol to mediate between order and disorder, has moved center stage in the characterization of membranes in terms of domains or rafts.
Subject(s)
Cholesterol/chemistry , Cholesterol/metabolism , Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Membranes, Artificial , Animals , HumansABSTRACT
Our picture of lipid membranes has come a long way since Gorter and Grendel in 1925 formulated the lipid bilayer hypothesis. Most modern textbook models of membranes are based on the Singer-Nicolson model from 1972, although we have in recent years seen significant amendments to this model, not least fuelled by the finding of lipid membrane domains and the subsequent 'raft rush'. The science of lipids, lipidology, has now become an established discipline, acknowledging that lipids organize in space and time and display emergent physico-chemical properties that are beyond the chemical nature of the individual molecules and which collectively control membrane function. Recently, lipidomics has been followed as a new discipline in the omics-sequel, characterized by an explosion in detailed data for lipid profiles of tissues, cells, and subcellular components. The focus is now swinging toward enumerating individual lipid species, determining their identity, and quantitating their amount. Time is ripe to marry the two disciplines, both in order to take lipidomics beyond the stage of 'stamp collection' and in order to incorporate into the lipidology approach the new knowledge about the individual lipid species. As an important matchmaker for this marriage, the physical chemistry of lipids in lipid bilayers and membranes is entering a new era of renaissance.
Subject(s)
Cell Membrane/chemistry , Chemistry, Physical , Lipids/chemistry , Micelles , Models, BiologicalABSTRACT
The escape of encapsulated anticancer drugs from liposomes by passive diffusion often leads to suboptimal drug concentrations in the cancer tissue, therefore calling for effective trigger mechanisms to release the drug at the target. We investigated mixtures of lipid components that not only form stable liposomes, but also can be turned into active drugs by secretory phospholipase A2 (sPLA2), an enzyme that is upregulated in various cancer cells, without the necessity for conventional liposome drug loading. The liposomes are composed of a novel lipid-based retinoid prodrug premixed with saturated phospholipids. The prodrug is found to be miscible with phospholipids, and the lipid mixtures are shown to form liposomes with the desired size distribution. The preparation procedure, phase behavior, and physicochemical properties of the formed liposomes are described as a function of lipid composition. We show that the premixing of the prodrug with phospholipids can be used to modify the physicochemical properties of liposomal formulations. The results should prove useful for further exploration of the potential for using these novel lipid prodrugs in liposomal formulations for cancer treatment.
Subject(s)
Antineoplastic Agents/chemistry , Lipids/chemistry , Liposomes/chemistry , Prodrugs/chemistry , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Calorimetry, Differential Scanning , Humans , Molecular Structure , Permeability , Receptors, Retinoic Acid/agonists , Retinoids/chemical synthesis , Retinoids/chemistryABSTRACT
The free amino acid (FAA) contents of a special selection of fermented beverages have been measured by ultra-high performance liquid chromatography (UHPLC). The selection, which includes 8 sakes, 9 white, rosé, and sparkling wines, 9 genuine champagnes, as well as 5 types of beer, was made to uncover the umami potential of different types of fermented beverages, in particular whether long yeast contact and ageing may influence the contents of free glutamate that is known to elicit umami sensation. The data show that in particular sakes as well as some beers, wines and champagnes with long yeast contact contain appreciable amounts of free glutamate. The results are discussed in the context of food pairing where umami synergy can be achieved by combining fermented beverages with long yeast contact with food rich in free nucleotides.
Subject(s)
Alcoholic Beverages/analysis , Fermented Foods/analysis , Amino Acids/analysis , Beer/analysis , Chromatography, High Pressure Liquid/methods , Fermentation , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Taste , Wine/analysisABSTRACT
Seaweeds (macroalgae) are, together with microalgae, main contributors to the Earth's production of organic matter and atmospheric oxygen as well as fixation of carbon dioxide. In addition, they contain a bounty of fibres and minerals, as well as macro- and micronutrients that can serve both technical and medicinal purposes, as well as be a healthy and nutritious food for humans and animals. It is therefore natural that seaweeds and humans have had a myriad of interwoven relationships both on evolutionary timescales as well as in recent millennia and centuries all the way into the Anthropocene. It is no wonder that seaweeds have also entered and served as a saviour for humankind around the globe in many periods of severe needs and crises. Indeed, they have sometimes been the last resort, be it during times of famine, warfare, outbreak of diseases, nuclear accidents, or as components of securing the fabric of social stability. The present topical review presents testimony from the history of human interaction with seaweeds to the way humankind has, over and over again, been 'saved by seaweeds'. It remains a historical fact that in extreme conditions, such as shortage and wars, humans have turned to seaweeds in times of 'needs must' and created new opportunities for their uses in order to mitigate disasters. Lessons to be learned from this history can be used as reminders and inspiration, and as a guide as how to turn to seaweeds in current and inevitable, future times of crises, not least for the present needs of how to deal with changing climates and the pressing challenges of sustainable and healthy eating.
ABSTRACT
Squid (Loligo forbesii and Loligo vulgaris) mantles were cooked by sous vide cooking using different temperatures (46°C, 55°C, 77°C) and times (30 s, 2 min, 15 min, 1 h, 5 h, 24 h), including samples of raw tissue. Macroscopic textural properties were characterized by texture analysis (TA) conducted with Meullenet-Owens razor shear blade and compared to analysis results from differential scanning calorimetry. The collagen content of raw tissues of squid was quantified as amount of total hydroxyproline using ultra-high-performance liquid chromatography. Structural changes were monitored by Raman spectroscopy and small-angle X-ray scattering and visualized by second harmonic generation microscopy. Collagen in the squid tissue was found to be highest in arms (4.3% of total protein), then fins (3.0%), and lowest in the mantle (1.5%), the content of the mantle being very low compared to that of other species of squid. Collagen was found to be the major protein responsible for cooking loss, whereas both collagen and actin were found to be key to mechanical textural changes. A significant decreased amount of cooking loss was obtained using a lower cooking temperature of 55°C compared to 77°C, without yielding significant textural changes in most TA parameters, except for TA hardness which was significantly less reduced. An optimized sous vide cooking time and temperature around 55-77°C and 0.5-5 h deserves further investigation, preferably coupled to sensory consumer evaluation. PRACTICAL APPLICATION: The study provides knowledge about structural changes during sous vide cooking of squid mantle. The results may be translated into gastronomic use, promoting the use of an underutilized resource of delicious and nutritious protein (Loligo vulgaris and Loligo forbesii).
Subject(s)
Cooking , Decapodiformes , Animals , Hardness , Seafood , TemperatureABSTRACT
We investigate the effects of two structurally similar small cyclic molecules: salicylic acid and perillic acid on a zwitterionic model lipid bilayer, and show that both molecules might have biological activity related to membrane thinning. Salicylic acid is a nonsteroidal antiinflammatory drug, some of the pharmacological properties of which arise from its interaction with the lipid bilayer component of the plasma membrane. Prior simulations show that salicylate orders zwitterionic lipid membranes. However, this is in conflict with Raman scattering and vesicle fluctuation analysis data, which suggest the opposite. We show using extensive molecular dynamics simulations, cumulatively >2.5 µs, that salicylic acid indeed disorders membranes with concomitant membrane thinning and that the conflict arose because prior simulations suffered from artifacts related to the sodium-ion induced condensation of zwitterionic lipids modeled by the Berger force field. Perillic acid is a terpenoid plant extract that has antiinfective and anticancer properties, and is extensively used in eastern medicine. We found that perillic acid causes large-scale membrane thinning and could therefore exert its antimicrobial properties via a membrane-lytic mechanism reminiscent of antimicrobial peptides. Being more amphipathic, perillic acid is more potent in disrupting lipid headgroup packing, and significantly modifies headgroup dipole orientation. Like salicylate, the membrane thinning effect of perillic acid is masked by the presence of sodium ions. As an alternative to sodium cations, we advocate the straightforward solution of using larger countercations like potassium or tetra-methyl-ammonium that will maintain electroneutrality but not interact strongly with, and thus not condense, the lipid bilayer.
Subject(s)
Computer Simulation , Cyclohexenes/chemistry , Membranes, Artificial , Monoterpenes/chemistry , Plant Extracts/chemistry , Salicylic Acid/chemistry , Terpenes/chemistry , Dimyristoylphosphatidylcholine/chemistry , Ions , Lipid Bilayers/chemistry , Phosphates/chemistry , Quaternary Ammonium Compounds/chemistry , Sodium/chemistry , Static ElectricityABSTRACT
The dynamic compartmentalization of eukaryotic cells is a fascinating phenomenon that is not yet understood. A prominent example of this challenge is the Golgi apparatus, the central hub for protein sorting and lipid metabolism in the secretory pathway. Despite major advances in elucidating its molecular biology, the fundamental question of how the morphogenesis of this organelle is organized on a system level has remained elusive. Here, we have formulated a coarse-grained computational model that captures key features of the dynamic morphogenesis of a Golgi apparatus. In particular, our model relates the experimentally observed Golgi phenotypes, the typical turnover times, and the size and number of cisternae to three basic, experimentally accessible quantities: the rates for material influx from the endoplasmic reticulum, and the anterograde and retrograde transport rates. Based on these results, we propose which molecular factors should be mutated to alter the organelle's phenotype and dynamics.
Subject(s)
Golgi Apparatus/metabolism , Models, Biological , Animals , Cell Membrane/metabolism , Models, Molecular , Phenotype , Proteins/chemistry , Proteins/metabolism , Time FactorsABSTRACT
The thermal phase behaviors of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) were compared by fluorescence spectroscopy, using PPDPC (1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine) as a reporter, in parallel with differential scanning calorimetry (DSC). A striking difference is seen between MLVs and LUVs in the lateral organizational dynamics of PPDPC, in particular, below the main phase transition temperature T(m), with efficient clustering of PPDPC into fluid microdomains in the L(beta') and P(beta') (ripple) phases of DPPC MLVs. In the P(beta') phase of MLVs, the probe is likely to become enriched in linear line defects, restricting intermolecular collisions to occur in a quasi one-dimensional system. In contrast, fluorescence and DSC data both suggest that the P(beta') phase is not well-defined in LUVs. Fluorescence anisotropy for 1-palmitoyl-2-[3-(diphenylhexatrienyl)propanoyl]-sn-glycero-3-phosphocholine (DPH-PC) revealed similar acyl chain order for both LUVs and MLVs in the L(beta') and P(beta') phases. However, for MLVs with this probe, T(m) determined from anisotropy was elevated by 0.7 degrees, with higher anisotropy evident in the L(alpha) phase compared to LUVs. These differences in the thermal phase behavior of the two types of liposomes are likely to derive from the augmented acyl chain order due to cooperative coupling of the lamellae of DPPC MLVs, thus manifesting in new, emerging material properties in the latter type of bilayer membrane assembly, as reflected in the organizational dynamics of the pyrene-labeled analogue.