Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31 P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains. Phosphatidic acid (PA)7 is a minor but important bioactive lipid involved in at least three essential and likely interrelated processes in a typical eukaryotic cell. PA is a key intermediate in the biosynthetic route of the main membrane phospholipids and triglycerides (1); it is involved in membrane dynamics, i.e. fission and fusion (2-4), and has important signaling functions (5-7). The role of PA in membrane dynamics and signaling is most likely 2-fold, either via an effect on the packing properties of the membrane lipids or via the specific binding of effector proteins (4, 7-9). The origin of the specific binding of effector proteins to PA is not known.The PA binding domains thus far identified (for review, see Ref. 10) and more recently (7)) are diverse and share no apparent sequence homology, in contrast to other lipid binding domains, such as e.g. the PH, PX, FYVE, and C2 domains (11-15). One general feature that the PA binding domains do have in common is the presence of basic amino acids (7). Where examined in detail, these basic amino acids were shown to be essential for the interaction with PA, which underscores the importance of electrostatic interactions. Indeed, the negatively charged phosphomonoester head group of PA would be expected to interact electrostatically with basic amino acids in a lipid binding domain. However, such a simple electrostatic interaction cannot explain the strong preference of PA-binding proteins for PA over other, often more abundant, negatively charged phospholipids.In a recent study we showed that the phosphomonoester head group of PA has remarkable properties (8). The phosphomonoester head group is able to form an intramolecular hydrogen bond upon initial deprotonation (when its charge is Ϫ1), which stabilizes the second proton against dissociation. We provided evidence that competing hydrogen bonds, e.g. from the primary amine of the head group of phosphatidylet...

The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown. Phosphatidic acid may recruit and activate downstream effectors, or change the biophysical properties of the membrane and directly induce membrane bending and/or destabilization. To evaluate these possibilities, we determined the phase properties of phosphatidic acid and lysophosphatidic acid at physiological conditions of pH and ion concentrations. In single-lipid systems, unsaturated phosphatidic acid behaved as a cylindrical, bilayer-preferring lipid at cytosolic conditions (37°C, pH 7.2, 0.5 mM free Mg 2+ ), but acquired a type-II shape at typical intra-Golgi conditions, a mildly acidic pH and submillimolar free Ca 2+ (pH 6.6±5.9, 0.3 mM Ca 2+ ). Lysophosphatidic acid formed type-I lipid micelles in the absence of divalent cations, but anhydrous cationlysophosphatidic acid bilayer complexes in their presence. These data suggest a similar molecular shape for phosphatidic acid and lysophosphatidic acid at cytosolic conditions; however, experiments in mixed-lipid systems indicate that their shape is not identical. Lysophosphatidic acid stabilized the bilayer phase of unsaturated phosphatidylethanolamine, while the opposite effect was observed in the presence of phosphatidic acid. These results support the hypothesis that a conversion of lysophosphatidic acid into phosphatidic acid by endophilin or BARS (50 kDa brefeldin A ribosylated substrate) may induce negative spontaneous monolayer curvature and regulate endocytic and Golgi membrane fission. Alternative models for the regulation of membrane fission based on the strong dependence of the molecular shape of (lyso)phosphatidic acid on pH and divalent cations are also discussed.

The formation of phosphatidic acid (PA) from lysophosphatidic acid (LPA), diacylglycerol, or phosphatidylcholine plays a key role in the regulation of intracellular membrane fission events, but the underlying molecular mechanism has not been resolved. A likely possibility is that PA affects local membrane curvature facilitating membrane bending and fission. To examine this possibility, we determined the spontaneous radius of curvature (R 0p ) of PA and LPA, carrying oleoyl fatty acids, using well-established X-ray diffraction methods. We found that, under physiological conditions of pH and salt concentration (pH 7.0, 150 mM NaCl), the R 0p values of PA and LPA were -46 Å and +20 Å, respectively. Thus PA has considerable negative spontaneous curvature while LPA has the most positive spontaneous curvature of any membrane lipid measured to date. The further addition of Ca 2+ did not significantly affect lipid spontaneous curvature; however, omitting NaCl from the hydration buffer greatly reduced the spontaneous curvature of PA, turning it into a cylindrically shaped lipid molecule (R 0p of -1.3 × 10 2 Å). Our quantitative data on the spontaneous radius of curvature of PA and LPA at a physiological pH and salt concentration will be instrumental in developing future models of biomembrane fission.

Phosphatidic acid and lysophosphatidic acid are minor but important anionic bioactive lipids involved in a number of key cellular processes, yet these molecules have a simple phosphate headgroup.To find out what is so special about these lipids, we determined the ionization behavior of phosphatidic acid (PA) and lysophosphatidic acid (LPA) in extended (flat) mixed lipid bilayers using magic angle spinning 31 P NMR. Our data show two surprising results. First, despite identical phosphomonoester headgroups, LPA carries more negative charge than PA when present in a phosphatidylcholine bilayer. Dehydroxy-LPA [1-oleoyl-3-(phosphoryl)propanediol] behaves in a manner identical to that of PA, indicating that the difference in negative charge between LPA and PA is caused by the hydroxyl on the glycerol backbone of LPA and its interaction with the phosphomonoester headgroup. Second, deprotonation of phosphatidic acid and lysophosphatidic acid was found to be strongly stimulated by the inclusion of phosphatidylethanolamine in the bilayer, indicating that lipid headgroup charge depends on local lipid composition and will vary between the different subcellular locations of (L)PA. Our findings can be understood in terms of a hydrogen bond formed within the phosphomonoester headgroup of (L)PA and its destabilization by competing intra-or intermolecular hydrogen bonds. We propose that this hydrogen bonding property of (L)PA is involved in the various cellular functions of these lipids.Phosphatidic acid (PA) 1 and the related lipid lysophosphatidic acid (LPA) are important minor lipid species in the cell. They are involved in many intracellular processes, and are important intermediates in lipid biosynthesis (1). For example, binding of LPA to its receptors evokes various cellular responses, and the local formation of (L)PA is part of signaling cascades, in particular in the regulation of membrane dynamics such as fusion and fission events, either indirectly through the recruitment of downstream effectors or directly by mediating (local) changes in the biophysical properties of the membrane (2-12).PA and LPA have a relatively simple chemical structure consisting of only a glycerol, one (LPA) or two (PA) acyl chains, and a phosphate, and it is interesting to note that these simple phospholipids are involved in such diverse processes, and are able to bind specifically to so many different types of proteins (3, 13). The question then is what is so special about these lipids. An obvious suggestion relates to the phosphate headgroup, which is attached to the glycerol backbone as a phosphomonoester, a unique feature of these lipids. Phosphomonoesters have two pK a 's, one of which is expected to be in the physiological pH range. As a consequence, small changes in (physiological) pH will affect the charge and influence the molecular shape and lipid phase behavior of these lipids (14,15). Under physiological conditions at neutral pH, phosphatidic acid is a cone (type II)-shaped lipid with a negative spontaneous curvature clos...

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 phosphoinos...

We present measurements of the ratio of the proton elastic electromagnetic form factors, p G Ep /G M p . The Jefferson Lab Hall A Focal Plane Polarimeter was used to determine the longitudinal and transverse components of the recoil proton polarization in ep elastic scattering; the ratio of these polarization components is proportional to the ratio of the two form factors. These data reproduce the observation of Jones et al. ͓Phys. Rev. Lett. 84, 1398 ͑2000͔͒, that the form factor ratio decreases significantly from unity above Q 2 ϭ1 GeV 2 .

Understanding the role of lipids in drug transport is critical in cancer chemotherapy to overcome drug resistance. In this study, we isolated lipids from doxorubicin-sensitive (MCF-7) and -resistant (MCF-7/ADR) breast cancer cells to characterize the biophysical properties of membrane lipids (particularly lipid packing and membrane fluidity) and to understand the role of the interaction of cell membrane lipids with drug/nanocarrier on drug uptake and efficacy. Resistant cell membrane lipids showed significantly different composition and formed more condensed, less fluid monolayers than did lipids from sensitive cells. Doxorubicin, used as a model anticancer agent, showed a strong hydrophobic interaction with resistant cell membrane lipids but significantly less interaction, as well as a different pattern of interaction (i.e., ionic), with sensitive ones. The threshold intracellular doxorubicin concentration required to produce an antiproliferative effect was similar for both sensitive and resistant cell lines, suggesting that drug transport is a major barrier in determining drug efficacy in resistant cells. In addition to the biophysical characteristics of resistant cell membrane lipids, lipid-doxorubicin interactions appear to decrease intracellular drug transport via diffusion as the drug is trapped in the lipid bilayer. The rigid nature of resistant cell membranes also seems to influence endosomal functions that inhibit drug uptake when a liposomal formulation of doxorubicin is used. In conclusion, biophysical properties of resistant cell membrane lipids significantly influence drug transport, and hence drug efficacy. A better understanding of the mechanisms of cancer drug resistance is vital to developing more effective therapeutic interventions. In this regard, biophysical interaction studies with cell membrane lipids might be helpful to improve drug transport and efficacy through drug discovery and/or drug delivery approaches by overcoming the lipid barrier in resistant cells.

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