Simple Does Not Mean Trivial: Behavior of Phosphatidic Acid in Lipid Mono- and Bilayers

Abstract

Phosphatidic acid (PA) is one of the simplest membrane phospholipids, yet it plays a crucial role in various biologically relevant processes that take place in cells. Since PA generation may be triggered by a variety of factors, very often of antagonistic character, the specific nature of physiological responses driven by PA is not clear. In order to shed more light on these issues, we carried out a systematic characterization of membranes containing one of the three biologically significant PA molecular species. The effect of these molecules on the properties of membranes composed of phosphatidylcholine and/or cholesterol was assessed in a multidisciplinary approach, including molecular dynamic simulations, flicker noise spectroscopy, and Langmuir monolayer isotherms. The first enables the determination of various macroscopic and microscopic parameters such as lateral diffusion, membrane thickness, and defect analysis. The obtained data revealed a strong interaction between unsaturated PA species and phosphatidylcholine. On the other hand, the behavior of saturated PA was greatly influenced by cholesterol. Additionally, a strong effect on mechanical properties was observed in the case of three-component systems, which could not be explained by the simple extrapolation of parameters of the corresponding two-component systems. Our data show that various PA species are not equivalent in terms of their influence on lipid mono- and bilayers and that membrane composition/properties, particularly those related to the presence of cholesterol, may strongly modulate PA behavior.

Keywords: Langmuir monolayers; area compressibility; bending rigidity; flicker noise spectroscopy; free energy of mixing; molecular dynamics; phosphatidic acid.

Figure 1

Snapshots of membrane MD systems. In panels (A) DPPA, (B) Chol/DPPA 1:1 (C) POPC/Chol/SAPA 5:3:2 (molar ratios) are shown. Molecules are colored according to the following rule: orange is PA component, red is cholesterol, and blue is POPC lipid molecules. As periodic boundary conditions were applied, the water molecules are organized with respect to periodic repetition of the system (gray/light blue).

Figure 2

Fraction of exposed acyl chains in function of probe radius for (A) POPC/PA, (B) Chol/PA, and (C) POPC/Chol/PA membrane systems. (D) A top-down probed surface grid of a frame in the POPC/SAPA bilayer simulation. On the grid, yellow represents the headgroup region, while blue represents the acyl chains region. The surface is probed with 0.2 nm radius size.

Figure 3

(A) Linear correlation of APL values from monolayer and molecular dynamics systems. (B) Effect of PA on POPC membranes showed with determined values of APL for POPC/PA systems for both simulation and experimental approaches. Theoretical values were determined based on values from single-component systems multiplied by factor corresponding to molar concentration on the membrane (Equation (3)).

Figure 4

(A) Effect of PA on Chol membranes and (B) effect of PA on POPC/Chol membranes in APL determination for both simulation and experimental approaches. Theoretical values are determined based on values from single-component systems multiplied by a factor corresponding to molar concentration on the membrane (Equation (3)).

Figure 5

Excess free energy of mixing for investigated membrane systems with insets showing mixing of the most interesting two-component monolayers—POPC/POPA, POPC/SAPA, and Chol/DPPA. The rest of the mixing is presented in Supporting Information (Section S2.3).

Figure 6

(A) Bending rigidity for investigated systems determined using statistical approach. (B) Example of recorded POPC/Chol/SAPA vesicle. (C) Correlation of bending rigidity values from flicker noise spectroscopy and molecular dynamic systems.