Current methods for studying intracellular liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) is a ubiquitous process that drives the formation of membrane-less intracellular compartments. This compartmentalization contains vastly different protein/RNA/macromolecule concentrations compared to the surrounding cytosol despite the absence of a lipid boundary. Because of this, LLPS is important for many cellular signaling processes and may play a role in their dysregulation. This chapter highlights recent advances in the understanding of intracellular phase transitions along with current methods used to identify LLPS in vitro and model LLPS in situ.

Interfacial tension and mechanism of liquid-liquid phase separation in aqueous media.

The organization of multiple subcellular compartments is controlled by liquid-liquid phase separation. Phase separation of this type occurs with the emergence of interfacial tension. Aqueous two-phase systems formed by two non-ionic polymers can be used to separate and analyze biological macromolecules, cells and viruses. Phase separation in these systems may serve as the simple model of phase separation in cells also occurring in aqueous media. To better understand liquid-liquid phase separation mechanisms, interfacial tension was measured in aqueous two-phase systems formed by dextran and polyethylene glycol and by polyethylene glycol and sodium sulfate in the presence of different additives. Interfacial tension values depend on differences between the solvent properties of the coexisting phases, estimated experimentally by parameters representing dipole-dipole, ion-dipole, ion-ion, and hydrogen bonding interactions. Based on both current and literature data, we propose a mechanism for phase separation in aqueous two-phase systems. This mechanism is based on the fundamental role of intermolecular forces. Although it remains to be confirmed, it is possible that these may underlie all liquid-liquid phase separation processes in biology.

Effect of H-Bond Donor Lipids on Phosphatidylinositol-3,4,5-Trisphosphate Ionization and Clustering.

The phosphoinositide, phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), is a key signaling lipid in the inner leaflet of the cell plasma membrane, regulating diverse signaling pathways including cell growth and migration. In this study we investigate the impact of the hydrogen-bond donor lipids phosphatidylethanolamine (PE) and phosphatidylinositol (PI) on the charge and phase behavior of PI(3,4,5)P3. PE and PI can interact with PI(3,4,5)P3 through hydrogen-bond formation, leading to altered ionization behavior and charge distribution within the PI(3,4,5)P3 headgroup. We quantify the altered PI(3,4,5)P3 ionization behavior using a multistate ionization model to obtain micro-pKa values for the ionization of each phosphate group. The presence of PE leads to a decrease in the pKa values for the initial deprotonation of PI(3,4,5)P3, which describes the removal of the first proton of the three protons remaining at the phosphomonoester groups at pH 4.0. The decrease in these micro-pKa values thus leads to a higher charge at low pH. Additionally, the charge distribution changes lead to increased charge on the 3- and 5-phosphates. In the presence of PI, the final deprotonation of PI(3,4,5)P3 is delayed, leading to a lower charge at high pH. This is due to a combination of hydrogen-bond formation between PI and PI(3,4,5)P3, and increased surface charge due to the addition of the negatively charged PI. The interaction between PI and PI(3,4,5)P3 leads to the formation of PI and PI(3,4,5)P3-enriched domains within the membrane. These domains may have a critical impact on PI(3,4,5)P3-signaling. We also reevaluate results for all phosphatidylinositol bisphosphates as well as for PI(4,5)P2 in complex lipid mixtures with the multistate ionization model.

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers.

Interaction of a model apolipoprotein, apoLp-III, with an oil-phospholipid interface.

Lipid droplets are "small" organelles that play an important role in de novo synthesis of new membrane, and steroid hormones, as well as in energy storage. The way proteins interact specifically with the oil-(phospho-)lipid monolayer interface of lipid droplets is a relatively unexplored but crucial question. Here, we use our home built liquid droplet tensiometer to mimic intracellular lipid droplets and study protein-lipid interactions at this interface. As model neutral lipid binding protein, we use apoLp-III, an amphipathic α-helix bundle protein. This domain is also found in proteins from the perilipin family and in apoE. Protein binding to the monolayer is studied by the decrease in the oil/water surface tension. Previous work used POPC (one of the major lipids found on lipid droplets) to form the phospholipid monolayer on the triolein surface. Here we expand this work by incorporating other lipids with different physico-chemical properties to study the effect of charge and lipid head-group size. This study sheds light on the affinity of this important protein domain to interact with lipids.

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins.

Insertion of perilipin 3 into a glycero(phospho)lipid monolayer depends on lipid headgroup and acyl chain species.

Lipid droplets (LDs) are organelles that contribute to various cellular functions that are vital for life. Aside from acting as a neutral lipid storage depot, they are also involved in building new membranes, synthesis of steroid hormones, and cell signaling. Many aspects of LD structure and function are not yet well-understood. Here we investigate the interaction of perilipin 3, a member of the perilipin family of LD binding proteins, and three N-terminal truncation mutants with lipid monolayers. The interaction is studied as a function of surface pressure for a series of systematically chosen lipids. We find that the C terminus of perilipin 3 has different insertion behavior from that of the longer truncation mutants and the full-length protein. Inclusion of N-terminal sequences with the C terminus decreases the ability of the protein construct to insert in lipid monolayers. Coupling of anionic lipids to negative spontaneous curvature facilitates protein interaction and insertion. The C terminus shows strong preference for lipids with more saturated fatty acids. This work sheds light on the LD binding properties and function of the different domains of perilipin 3.

Insertion of apoLp-III into a lipid monolayer is more favorable for saturated, more ordered, acyl-chains.

Neutral lipid transport in mammals is complicated involving many types of apolipoprotein. The exchangeable apolipoproteins mediate the transfer of hydrophobic lipids between tissues and particles, and bind to cell surface receptors. Amphipathic α-helices form a common structural motif that facilitates their lipid binding and exchangeability. ApoLp-III, the only exchangeable apolipoprotein found in insects, is a model amphipathic α-helix bundle protein and its three dimensional structure and function mimics that of the mammalian proteins apoE and apoAI. Even the intracellular exchangeable lipid droplet protein TIP47/perilipin 3 contains an α-helix bundle domain with high structural similarity to that of apoE and apoLp-III. Here, we investigated the interaction of apoLp-III from Locusta migratoria with lipid monolayers. Consistent with earlier work we find that insertion of apoLp-III into fluid lipid monolayers is highest for diacylglycerol. We observe a preference for saturated and more highly ordered lipids, suggesting a new mode of interaction for amphipathic α-helix bundles. X-ray reflectivity shows that apoLp-III unfolds at a hydrophobic interface and flexible loops connecting the amphipathic α-helices stay in solution. X-ray diffraction indicates that apoLp-III insertion into diacylglycerol monolayers induces additional ordering of saturated acyl-chains. These results thus shed important new insight into the protein-lipid interactions of a model exchangeable apolipoprotein with significant implications for its mammalian counterparts.

Identification and functional characterization of the Arabidopsis†Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain.

Phosphatidic acid (PA) is an important signalling lipid involved in various stress-induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA-binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate with cellular membranes in response to salt stress in Arabidopsis roots. A 42 amino acid sequence was identified as the primary PA-binding domain (PABD) of SnRK2.4. Unlike the full-length SnRK2.4, neither the PABD-YFP fusion protein nor the SnRK2.10 re-localized into punctate structures upon salt stress treatment, showing that additional domains of the SnRK2.4 protein are required for its re-localization during salt stress. Within the PABD, five basic amino acids, conserved in class 1 SnRK2s, were found to be necessary for PA binding. Remarkably, plants overexpressing the PABD, but not a non-PA-binding mutant version, showed a severe reduction in root growth. Together, this study biochemically characterizes the PA-SnRK2.4 interaction and shows that functionality of the SnRK2.4 PABD affects root development.

Controlled particle collision leads to direct observation of docking and fusion of lipid droplets in an optical trap.

As an intracellular organelle, phospholipid-coated lipid droplets have shown increasing importance due to their expanding biological functions other than the lipid storage. The growing biological significance necessitates a close scrutiny on lipid droplets, which have been proposed to mature in a cell through processes such as fusion. Unlike phospholipid vesicles that are well-known to fuse through docking and hemifusion steps, little is known on the fusion of lipid droplets. Herein, we used laser tweezers to capture two micrometer-sized 1,2,3-trioleoylglycerol (triolein) droplets coated with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) that closely resemble intracellular lipid droplets. We started the fusion processes by a well-controlled collision between the two lipid droplets in phosphate buffer at pH 7.4. By monitoring the change in the pathway of a trapping laser that captures the collided lipid droplets, docking and physical fusion events were clearly distinguished for the first time and their lifetimes were determined with a resolution of 10 µs after postsynchronization analysis. Our method revealed that the rate-limiting docking process is affected by anions according to a Hofmeister series, which sheds light on the important role of interfacial water shedding during the process. During the physical fusion, the kinetics between bare triolein droplets is faster than lipid droplets, suggesting that breaking of phospholipid coating is involved in the process. This scenario was further supported by direct observation of a short-lived hemifusion state with ∼46 ms lifetime in POPC-coated lipid droplets, but not in bare triolein droplets.

The effect of methylated phosphatidylethanolamine derivatives on the ionization properties of signaling phosphatidic acid.

Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC) are the most abundant glycerophospholipids in eukaryotic membranes. The differences in the physicochemical properties of their headgroups have contrasting modulatory effects on their interaction with intracellular macromolecules. As such, their overall impact on membrane structure and function differs significantly. Enzymatic methylation of PE's amine headgroup produces two methylated derivatives namely monomethyl PE (MMPE) and dimethyl PE (DMPE) which have physicochemical properties that generally range between that of PE and PC. Additionally, their influence on membrane properties differs from both PE and PC. Although variations in headgroup methylation have been reported to affect signaling pathways, the direct influence that these differences exert on the ionization properties of signaling phospholipids have not been investigated. Here, we briefly review membrane function and structure that are mediated by the differences in headgroup methylation between PE, MMPE, DMPE and PC. In addition, using 31P MAS NMR, we investigate the effect of these four phospholipids on the ionization properties of the ubiquitous signaling anionic lipid phosphatidic acid (PA). Our results show that PA's ionization properties are differentially affected by changes in phospholipid headgroup methylation. This could have important implications for PA-protein binding and hence physiological functions in cells where signaling events lead to changes in abundance of methylated PE derivatives in the membrane.

Ionization properties of mixed lipid membranes: a Gouy-Chapman model of the electrostatic-hydrogen bond switch.

The dissociation state of phosphatidic acid (PA) in a lipid bilayer is governed by the competition of proton binding and formation of a hydrogen bond through a mechanism termed the electrostatic-hydrogen bond switch. This mechanism has been suggested to provide the basis for the specific recognition of PA by proteins. Even in bare lipid bilayers the electrostatic-hydrogen bond switch is present if the membrane contains lipids like phosphatidylethanolamine that act as hydrogen bond donors. In this case, the dissociation state (pK(a)) of PA depends strongly on membrane composition. In the present work we incorporate the electrostatic-hydrogen bond switch mechanism into the Gouy-Chapman model for a membrane that is composed of PA and a hydrogen bond-donating zwitterionic lipid. To this end, our model integrates into the Gouy-Chapman approach a recently suggested electrostatic model for zwitterionic lipids. Hydrogen bond formation is incorporated phenomenologically as an additional non-electrostatic interaction between the phosphomonoester headgroup of PA and the zwitterionic lipid headgroup. We express the energetics of the composite membrane in terms of a free energy functional whose minimization leads to a modified non-linear Poisson-Boltzmann equation that we have solved numerically. Our calculations focus on the influence of the membrane environment on the dissociation state of PA. This influence can be expressed as a shift of the second pK(a) of PA, which we calculate as function of membrane composition, following experimental observation. The shift is large and negative if PA is the minor component in the membrane, and it changes over four pH units as function of the mole fraction of PA in the membrane. In contrast, the shift of the second pK(a) of PA remains small and is always positive if the zwitterionic lipid is unable to act as hydrogen bond donor. Hence, we find that the electrostatic-hydrogen bond switch mechanism regulates the dissociation state of PA with much greater sensitivity than would be possible based on a pure electrostatic regulation through the membrane potential.

The Electrostatic Basis of Diacylglycerol Pyrophosphate-Protein Interaction.

Diacylglycerol pyrophosphate (DGPP) is an anionic phospholipid formed in plants, yeast, and parasites under multiple stress stimuli. It is synthesized by the phosphorylation action of phosphatidic acid (PA) kinase on phosphatidic acid, a signaling lipid with multifunctional properties. PA functions in the membrane through the interaction of its negatively charged phosphomonoester headgroup with positively charged proteins and ions. DGPP, like PA, can interact electrostatically via the electrostatic-hydrogen bond switch mechanism but differs from PA in its overall charge and shape. The formation of DGPP from PA alters the physicochemical properties as well as the structural dynamics of the membrane. This potentially impacts the molecular and ionic binding of cationic proteins and ions with the DGPP enriched membrane. However, the results of these important interactions in the stress response and in DGPP's overall intracellular function is unknown. Here, using 31P MAS NMR, we analyze the effect of the interaction of low DGPP concentrations in model membranes with the peptides KALP23 and WALP23, which are flanked by positively charged Lysine and neutral Tryptophan residues, respectively. Our results show a significant effect of KALP23 on the charge of DGPP as compared to WALP23. There was, however, no significant effect on the charge of the phosphomonoester of DGPP due to the interaction with positively charged lipids, dioleoyl trimethylammonium propane (DOTAP) and dioleoyl ethyl-phosphatidylcholine (EtPC). Divalent calcium and magnesium cations induce deprotonation of the DGPP headgroup but showed no noticeable differences on DGPP's charge. Our results lead to a novel model for DGPP-protein interaction.

Phosphatidylinositol 4,5-bisphosphate stimulates alveolar epithelial fluid clearance in male and female adult rats.

Cell membrane phospholipids, like phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], can regulate epithelial Na channel (ENaC) activity. Gender differences in lung ENaC expression have also been demonstrated. However, the effects in vivo on alveolar fluid clearance are uncertain. Thus PI(4,5)P(2) effects on alveolar fluid clearance were studied in male and female rats. An isosmolar 5% albumin solution was intrapulmonary instilled; alveolar fluid clearance was studied for 1 h. Female rats had a 37 ± 19% higher baseline alveolar fluid clearance than male rats. Bilateral ovariectomy attenuated this gender difference. Compared with controls, PI(4,5)P(2) instillation (300 µM) increased alveolar fluid clearance by ∼93% in both genders. Amiloride or the specific αENaC small-interfering RNA inhibited baseline and PI(4,5)P(2)-stimulated alveolar fluid clearance in both genders, indicating a dependence on amiloride-sensitive pathways. The fraction of amiloride inhibition was greater in PI(4,5)P(2)-instilled rats (male: 64 ± 10%; female: 70 ± 11%) than in controls (male: 30 ± 6%; female: 44 ± 8%). PI(4,5)P(2) instillation lacked additional alveolar fluid clearance stimulation above that of terbutaline, nor did propranolol inhibit alveolar fluid clearance after PI(4,5)P(2) instillation, indicating that PI(4,5)P(2) stimulation was not secondary to endogenous ß-adrenoceptor activation. PI(4,5)P(2) amine instillation resulted in an intermediate alveolar fluid clearance stimulation, suggesting that, to reach maximal alveolar fluid clearance stimulation, PI(4,5)P(2) must reside in cell membranes. In summary, PI(4,5)P(2) instillation upregulated in vivo alveolar fluid clearance similar to short-term ß-adrenoceptor upregulation of alveolar fluid clearance. PI(4,5)P(2) stimulation was mediated partly by increased amiloride-sensitive Na transport. There exist important gender-related effects suggesting a female advantage that may have clinical implications for resolution of acute lung injury.

Interaction of Two Amphipathic α-Helix Bundle Proteins, ApoLp-III and ApoE 3, with the Oil-Aqueous Interface.

Protein-lipid interactions govern the structure and function of lipoprotein particles, which transport neutral lipids and other hydrophobic cargo through the blood stream. Apolipoproteins cover the surface of lipoprotein particles, including low-density (LDL) and high-density (HDL) lipoproteins, and determine their function. Previous work has focused on small peptides derived from these apolipoproteins or used such artificial lipid systems as Langmuir monolayers or the lipid disc assay to determine how apolipoproteins interact with the neutral lipid interface. Here, we focus on a recurring protein domain found in many neutral lipid-binding proteins, the amphipathic α-helix bundle. We use liquid droplet tensiometry to investigate protein-lipid interactions on an oil droplet, which mimics the real lipoprotein interface. The N-terminus of apoE 3 and full-length apoLp-III serve as model proteins. We find that each protein interacts with lipid monolayers at the oil-aqueous interface in unique ways. For the first time, we show that helix bundle unfolding is critical for proper protein insertion into the lipid monolayer at the oil-aqueous interface and that specific membrane lipids promote the rebinding of protein upon fluctuation in droplet size. These results shed new light on how amphipathic apolipoprotein α-helix bundles interact with neutral lipid particles.

The C-Terminus of Perilipin 3 Shows Distinct Lipid Binding at Phospholipid-Oil-Aqueous Interfaces.

Lipid droplets (LDs) are ubiquitously expressed organelles; the only intracellular organelles that contain a lipid monolayer rather than a bilayer. Proteins localize and bind to this monolayer as they do to intracellular lipid bilayers. The mechanism by which cytosolic LD binding proteins recognize, and bind, to this lipid interface remains poorly understood. Amphipathic α-helix bundles form a common motif that is shared between cytosolic LD binding proteins (e.g., perilipins 2, 3, and 5) and apolipoproteins, such as apoE and apoLp-III, found on lipoprotein particles. Here, we use pendant drop tensiometry to expand our previous work on the C-terminal α-helix bundle of perilipin 3 and the full-length protein. We measure the recruitment and insertion of perilipin 3 at mixed lipid monolayers at an aqueous-phospholipid-oil interface. We find that, compared to its C-terminus alone, the full-length perilipin 3 has a higher affinity for both a neat oil/aqueous interface and a phosphatidylcholine (PC) coated oil/aqueous interface. Both the full-length protein and the C-terminus show significantly more insertion into a fully unsaturated PC monolayer, contrary to our previous results at the air-aqueous interface. Additionally, the C-terminus shows a preference for lipid monolayers containing phosphatidylethanolamine (PE), whereas the full-length protein does not. These results strongly support a model whereby both the N-terminal 11-mer repeat region and C-terminal amphipathic α-helix bundle domains of perilipin 3 have distinct lipid binding, and potentially biological roles.

Lipid-protein interactions for ECA1 an N-ANTH domain protein involved in stress signaling in plants.

Epsin-like Clathrin Adaptor 1 (ECA1/ PICALM1A) is an A/ENTH domain protein that acts as an adaptor protein in clathrin-mediated endocytosis. ECA1 is recruited to the membrane during salt stress signaling in plants in a phosphatidic acid (PA)-dependent manner. PA is a lipid second messenger that rapidly and transiently increases in concentration under stress stimuli. Upon an increase in PA concentration another lipid, diacylglycerol pyrophosphate (DGPP), starts to accumulate. The accumulation of DGPP is suggested to be a cue for attenuating PA signaling during stress in plants. We showed in vitro that ECA1-PA binding is modulated as a function of membrane curvature stress and charge. In this work, we investigate ECA1 binding to DGPP in comparison with PA. We show that ECA1 has more affinity for the less charged PA, and this binding is pH dependent. Additionally, plant PA binding proteins SnRK2.10, TGD2C, and PDK1-PH2 were investigated for their interaction with DGPP, since no known DGPP binding proteins are available in the literature to date. Our results shed further light on DGPP and its interactions with membrane proteins which brings us closer toward understanding the complexity of protein interactions with anionic lipids, especially the enigmatic anionic lipid DGPP.

Structure of ceramide-1-phosphate at the air-water solution interface in the absence and presence of Ca2+.

Ceramide-1-phosphate, the phosphorylated form of ceramide, gained attention recently due to its diverse intracellular roles, in particular in inflammation mediated by cPLA(2)alpha. However, surprisingly little is known about the physical chemical properties of this lipid and its potential impact on physiological function. For example, the presence of Ca(2+) is indispensable for the interaction of Cer-1-P with the C2 domain of cPLA(2)alpha. We report on the structure and morphology of Cer-1-P in monomolecular layers at the air/water solution interface in the absence and presence of Ca(2+) using diverse biophysical techniques, including synchrotron x-ray reflectivity and grazing angle diffraction, to gain insight into the role and function of Cer-1-P in biomembranes. We show that relatively small changes in pH and the presence of monovalent cations dramatically affect the behavior of Cer-1-P. On pure water Cer-1-P forms a solid monolayer despite the negative charge of the phosphomonoester headgroup. In contrast, pH 7.2 buffer yields a considerably less solid-like monolayer, indicating that charge-charge repulsion becomes important at higher pH. Calcium was found to bind strongly to the headgroup of Cer-1-P even in the presence of a 100-fold larger Na(+) concentration. Analysis of the x-ray reflectivity data allowed us to estimate how much Ca(2+) is bound to the headgroup, approximately 0.5 Ca(2+) and approximately 1.0 Ca(2+) ions per Cer-1-P molecule for the water and buffer subphase respectively. These results can be qualitatively understood based on the molecular structure of Cer-1-P and the electrostatic/hydrogen-bond interactions of its phosphomonoester headgroup. Biological implications of our results are also discussed.

Ionization properties of phosphatidylinositol polyphosphates in mixed model membranes.

Phosphatidylinositol polyphosphate lipids (phosphoinositides) form only a minor pool of membrane phospholipids but are involved in many intracellular signaling processes, including membrane trafficking, cytoskeletal remodeling, and receptor signal transduction. Phosphoinositide properties are largely determined by the characteristics of their headgroup, which at physiological pH is highly charged but also capable of forming hydrogen bonds. Many proteins have developed special binding domains that facilitate specific binding to particular phosphoinositides, while other proteins interact with phosphoinositides via nonspecific electrostatic interactions. Despite its importance, only limited information is available about the ionization properties of phosphoinositides. We have investigated the pH-dependent ionization behavior of all three naturally occurring phosphatidylinositol bisphosphates as well as of phosphatidylinositol 3,4,5-trisphosphate in mixed phosphoinositide/phosphatidylcholine vesicles using magic angle spinning (31)P NMR spectroscopy. For phosphatidylinositol 3,5-bisphosphate, where the two phosphomonoester groups are separated by a hydroxyl group at the 4-position, the pH-dependent chemical shift variation can be fitted with a Henderson-Hasselbalch-type formalism, yielding pK(a)(2) values of 6.96 +/- 0.04 and 6.58 +/- 0.04 for the 3- and 5-phosphates, respectively. In contrast, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] as well as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] show a biphasic pH-dependent ionization behavior that cannot be explained by a Henderson-Hasselbalch-type formalism. This biphasic behavior can be attributed to the sharing of the last remaining proton between the vicinal phosphomonoester groups. At pH 7.0, the overall charge (including the phosphodiester group charge) is found to be -3.96 +/- 0.10 for PI(3,4)P(2) and -3.99 +/- 0.10 for PI(4,5)P(2). While for PI(3,5)P(2) and PI(4,5)P(2) the charges of the individual phosphate groups in the molecule differ, they are equal for PI(3,4)P(2). Differences in the charges of the phosphomonoester groups can be rationalized on the basis of the ability of the respective phosphomonoester group to form intramolecular hydrogen bonds with adjacent hydroxyl groups. Phosphatidylinositol 3,4,5-trisphosphate shows an extraordinary complex ionization behavior. While at pH 4 the (31)P NMR peak of the 4-phosphate is found downfield from the other two phosphomonoester group peaks, an increase in pH leads to a crossover of the 4-phosphate, which positions this peak eventually upfield from the other two peaks. As a result, the 4-phosphate group shows a significantly lower charge at pH values between 7 and 9.5 than the other two phosphomonoester groups. The charge of the respective phosphomonoester group in PI(3,4,5)P(3) is lower than the corresponding charge of the phosphatidylinositol bisphosphate phosphomonoester groups, leading to an overall charge of PI(3,4,5)P(3) of -5.05 +/- 0.15 at pH 7.0. The charge of all investigated phosphoinositides at pH 7.0 is equal or higher than the corresponding charge of soluble inositol polyphosphate headgroup analogues, which is the opposite of what is expected on the basis of simple electrostatic considerations. This higher than expected headgroup charge can be rationalized with mutual intermolecular hydrogen bond formation. Measurements using different concentrations of PI(4,5)P(2) in the lipid vesicles (1, 5, and 20 mol %) did not reveal any significant concentration-dependent shift of the two phosphomonoester peaks, suggesting that PI(4,5)P(2) is clustered even at 1 mol %.

Membrane organization and ionization behavior of the minor but crucial lipid ceramide-1-phosphate.

Ceramide-1-phosphate (Cer-1-P), one of the simplest of all sphingophospholipids, occurs in minor amounts in biological membranes. Yet recent evidence suggests important roles of this lipid as a novel second messenger with crucial tasks in cell survival and inflammatory responses. We present a detailed description of the physical chemistry of this hitherto little explored membrane lipid. At full hydration Cer-1-P forms a highly organized subgel (crystalline) bilayer phase (L(c)) at low temperature, which transforms into a regular gel phase (L(beta)) at approximately 45 degrees C, with the gel to fluid phase transition (L(beta)-L(alpha)) occurring at approximately 65 degrees C. When incorporated at 5 mol % in a phosphatidylcholine bilayer, the pK(a2) of Cer-1-P, 7.39 +/- 0.03, lies within the physiological pH range. Inclusion of phosphatidylethanolamine in the phosphatidylcholine bilayer, at equimolar ratio, dramatically reduces the pK(a2) to 6.64 +/- 0.03. We explain these results in light of the novel electrostatic/hydrogen bond switch model described recently for phosphatidic acid. In mixtures with dielaidoylphosphatidylethanolamine, small concentrations of Cer-1-P cause a large reduction of the lamellar-to-inverted hexagonal phase transition temperature, suggesting that Cer-1-P induces, like phosphatidic acid, negative membrane curvature in these types of lipid mixtures. These properties place Cer-1-P in a class more akin to certain glycerophospholipids (phosphatidylethanolamine, phosphatidic acid) than to any other sphingolipid. In particular, the similarities and differences between ceramide and Cer-1-P may be relevant in explaining some of their physiological roles.