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.

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.

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.

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.

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.

Shedding light on lipid metabolism in Kinetoplastida: A phylogenetic analysis of phospholipase D protein homologs.

Unicellular flagellates that make up the class Kinetoplastida include multiple parasites responsible for public health concerns, including Trypanosoma brucei and T. cruzi (agents of African sleeping sickness and Chagas disease, respectively), and various Leishmania species, which cause leishmaniasis. These diseases are generally difficult to eradicate, with treatments often having lethal side effects and/or being effective only during the acute phase of the diseases, when most patients are still asymptomatic. Phospholipid signaling and metabolism are important in the different life stages of Trypanosoma, including playing a role in transitions between stages and in immune system evasion, thus, making the responsible enzymes into potential therapeutic targets. However, relatively little is understood about how the pathways function in these pathogens. Thus, in this study we examined evolutionary history of proteins from one such signaling pathway, namely phospholipase D (PLD) homologs. PLD is an enzyme responsible for synthesizing phosphatidic acid (PA) from membrane phospholipids. PA is not only utilized for phospholipid synthesis, but is also involved in many other signaling pathways, including biotic and abiotic stress response. 37 different representative Kinetoplastida genomes were used for an exhaustive search to identify putative PLD homologs. The genome of Bodo saltans was the only one of surveyed Kinetoplastida genomes that encoded a protein that clustered with plant PLDs. The representatives from other Kinetoplastida species clustered together in two different clades, thought to be homologous to the PLD superfamily, but with shared sequence similarity with cardiolipin synthases (CLS), and phosphatidylserine synthases (PSS). The protein structure predictions showed that most Kinetoplastida sequences resemble CLS and PSS, with the exception of 5 sequences from Bodo saltans that shared significant structural similarities with the PLD sequences, suggesting the loss of PLD-like sequences during the evolution of parasitism in kinetoplastids. On the other hand, diacylglycerol kinase (DGK) homologs were identified for all species examined in this study, indicating that DGK could be the only pathway for the synthesis of PA involved in lipid signaling in these organisms due to genome streamlining during transition to parasitic lifestyle. Our findings offer insights for development of potential therapeutic and/or intervention approaches, particularly those focused on using PA, PLD and/or DGK related pathways, against trypanosomiasis, leishmaniasis, and Chagas disease.

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.

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.

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.

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.

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.

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.

Phosphatidylinositol-4,5-bisphosphate ionization in the presence of cholesterol, calcium or magnesium ions.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) is an important signaling lipid and plays a crucial role in a wide variety of cellular processes by interacting with protein targets and localizing proteins at the plasma membrane. These interactions are strongly influenced by the lateral distribution of PI(4,5)P2 as well as its ionization state. The characterization of the PI(4,5)P2 ionization state provides important information about how PI(4,5)P2 interacts with other membrane resident or associated chemical species. In this study we have used solid-state MAS (31)P NMR to investigate the interactions of PI(4,5)P2 with potential cluster promoting agents, divalent cations and cholesterol. Both Ca(2+) and cholesterol were found previously to promote formation of local PI(4,5)P2 clusters in vitro. The NMR approach allows us to probe independently the ionization state of PI(4,5)P2 two phosphomonoester groups. We investigated mixed phosphatidylcholine (PC)/PI(4,5)P2 multilamellar vesicles in the presence of micro and millimolar concentrations of Ca(2+) and Mg(2+). We found that both cations lead to an increased downfield chemical shift of the PI(4,5)P2 phosphomonoester peaks, indicating an increased ionization in the presence of the divalent cations. Ca(2+) has a much larger effect on PI(4,5)P2 as compared to Mg(2+) at similar concentrations. Physiological concentrations of Ca(2+) are significantly lower than those found for Mg(2+) and the comparison of the PI(4,5)P2 ionization in the presence of Ca(2+) and Mg(2+) at physiological concentrations resulted in similar charges of the phosphomonoester groups for both cations. PI(4,5)P2 was also examined with vesicles containing cholesterol since cholesterol has been shown to promote PI(4,5)P2 clustering. In the presence of 40 mol% cholesterol, the PI(4,5)P2 phosphomonoester (31)P NMR peaks shifted slightly downfield, indicating a small increase in charge. Previously published data suggest that PI(4,5)P2 is capable of forming an intra- and intermolecular hydrogen bond network, which leads to a reduction of the charge at the phosphomonoester groups through dissipation of the charge across the bilayer/water interface. We hypothesize that cholesterol participates in this intermolecular hydrogen bond network, resulting in a stabilization of PI(4,5)P2 enriched domains due an increased spacing between the PI(4,5)P2 headgroup. We also examined the cumulative effects of cholesterol combined with the divalent cations, phosphatidylethanolamine (PE), and phosphatidylinositol (PI), separately. The combination of cholesterol and divalent cations results in an additive effect on PI(4,5)P2 ionization, while the effect of cholesterol on PI(4,5)P2 ionization is reduced in the presence of PE or PI.

Ionization behavior of polyphosphoinositides determined via the preparation of pH titration curves using solid-state 31P NMR.

Detailed knowledge of the degree of ionization of lipid titratable groups is important for the evaluation of protein-lipid and lipid-lipid interactions. The degree of ionization is commonly evaluated by acid-base titration, but for lipids localized in a multicomponent membrane interface this is not a suitable technique. For phosphomonoester-containing lipids such as the polyphosphoinositides, phosphatidic acid, and ceramide-1-phosphate, this is more conveniently accomplished by (31)P NMR. Here, we describe a solid-state (31)P NMR procedure to construct pH titration curves to determine the degree of ionization of phosphomonoester groups in polyphosphoinositides. This procedure can also be used, with suitable sample preparation conditions, for other important signaling lipids. Access to a solid-state, i.e., magic angle spinning, capable NMR spectrometer is assumed. The procedures described here are valid for a Bruker instrument, but can be adapted for other spectrometers as needed.

Increased pH-sensitivity of protein binding to lipid membranes through the electrostatic-hydrogen bond switch.

The signaling lipid phosphatidic acid (PA) is believed to interact specifically with membrane-bound globular proteins through a combination of electrostatic interactions and hydrogen bond formation known as the electrostatic-hydrogen bond switch. PA, which adjusts its protonation state according to the ambient pH, is able to regulate protein binding under physiological conditions in a pH-dependent manner. We investigate the question to what extent the electrostatic-hydrogen bond switch contributes to the pH-sensitivity of protein binding. To this end, we propose a theoretical model for the adsorption of a basic protein on a zwitterionic membrane that contains phosphatidic acid as a minor component. Our model is based on an extended continuum Poisson-Boltzmann approach that accounts for zwitterionic lipids, the protonation/deprotonation equilibrium of PA, and the lateral mobility of the lipids in the membrane. The electrostatic-hydrogen bond switch enters as an additional non-electrostatic attractive interaction of deprotonated PA with basic protein residues. For a generic model protein we calculate the adsorption free energy and its pH-dependence. Our results suggest that the electrostatic-hydrogen bond switch not only increases the affinity between PA and the protein but also its sensitivity with respect to changes in pH. That is, the electrostatic-hydrogen bond switch helps enabling the membrane to use physiological pH changes in order to trigger protein adsorption/desorption.