Effect of Phosphatidic Acid on Biomembrane: Experimental and Molecular Dynamics Simulations Study Urszula Kwolek, † Waldemar Kulig, ‡ Pawel Wydro, † Maria Nowakowska, † Tomasz Ró g,* ,‡ and Mariusz Kepczynski* ,† † Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakó w, Poland ‡ Department of Physics, Tampere University of Technology, P.O. Box 692, FI-33101 Tampere, Finland * S Supporting Information ABSTRACT: We consider the impact of phosphatidic acid (namely, 1,2-dioleoyl-sn-glycero-3-phosphate, DOPA) on the properties of a zwitterionic (1,2-dipalmitoyl-sn-glycero-3- phosphocholine, DPPC) bilayer used as a model system for protein-free cell membranes. For this purpose, experimental measurements were performed using differential scanning calorimetry and the Langmuir monolayer technique at physiological pH. Moreover, atomistic-scale molecular dynam- ics (MD) simulations were performed to gain information on the mixed bilayer’s molecular organization. The results of the monolayer studies clearly showed that the DPPC/DOPA mixtures are nonideal and the interactions between lipid species change from attractive, at low contents of DOPA, to repulsive, at higher contents of that component. In accordance with these results, the MD simulations demonstrated that both monoanionic and dianionic forms of DOPA have an ordering and condensing effect on the mixed bilayer at low concentrations. For the DOPA monoanions, this is the result of both (i) strong electrostatic interactions between the negatively charged oxygen of DOPA and the positively charged choline groups of DPPC and (ii) conformational changes of the lipid acyl chains, leading to their tight packing according to the so-called “umbrella model”, in which large headgroups of DPPC shield the hydrophobic part of DOPA (the conical shape lipid) from contact with water. In the case of the DOPA dianions, cation-mediated clustering was observed. Our results provide a detailed molecular-level description of the lipid organization inside the mixed zwitterionic/PA membranes, which is fully supported by the experimental data. 1. INTRODUCTION Phosphatidic acid (PA, 1,2-diacyl-sn-glycero-3-phosphate) is the simplest diacyl-glycerophospholipid; it consists of glycerol, to which two fatty acids are esterified at the sn-1 and sn-2 carbons, and the phosphate group is attached at the sn-3 position. Therefore, PA is a negatively charged (anionic) molecule at physiological pH. Although PA is present only in small amounts (often less than a few mol %) in biological membranes, it is crucial for cell survival. 1 Its role in the vital processes occurring in cells has been previously discussed in several reviews. 2-5 Most significantly, PA is at the center of membrane phospholipid biosynthesis, and as a consequence, the level of PA is carefully controlled to maintain lipid homeostasis. 5 PA plays important role in regulating cell proliferation; therefore, elevated expression and/or activity of enzymes that generate PA is commonly detected in human cancer. 2 Cancer cells are known to produce PA to avoid apoptosis. 6 PA has been also identified as an important signaling molecule in both plants and animals. 3 In plants, its formation is triggered in response to various biotic and abiotic stress factors, including low temperature, drought, salinity, or wounding. Recently, it was shown that PA signaling can be dynamically regulated by changes in pH. 7 The change in pH alters the state of phosphate headgroup protonation, making the PA lipid a pH biosensor. The biological significance of PA was the motivation for researchers to carry out experimental and computing studies aimed at explaining the impact of PA on the properties of zwitterionic membranes. The miscibility of PA/phosphocholine (PC) binary mixtures containing saturated lipids of various chain lengths was studied using differential scanning calorim- etry (DSC), 8-10 and the mixtures comprising unsaturated lipids were examined directly by fluorescence microscopy. 11,12 Blume et al. showed that the miscibility of a PA/PC system (chain length n of 14 or 16 C atoms) strongly depends on the degree of ionization of the PA headgroups and thus on pH of the surrounding environment. 8,9 The authors suggested that at pH 7, at which PA is negatively charged, electrostatic repulsion between the PA molecules and hydrogen bonds between the PA and PC headgroups (complex formation) favor the mixing of the two components. 8,9 Lowering pH to 4 induced large changes in the mixing behavior due to the lower ionization degree of the PA headgroups. The reduction in electrostatic Received: April 15, 2015 Revised: July 9, 2015 Published: July 13, 2015 Article pubs.acs.org/JPCB © 2015 American Chemical Society 10042 DOI: 10.1021/acs.jpcb.5b03604 J. Phys. Chem. B 2015, 119, 10042-10051