Piotr Jurkiewicz

Effects of Alkali Cations and Halide Anions on the DOPC Lipid Membrane

By means of molecular dynamics simulations with an all-atom force field, we investigated the affinities of alkali cations and halide anions for the dioleoylphosphatidylcholine lipid membrane in aqueous salt solutions. In addition, changes in phospholipid lateral diffusion and in headgroup mobility upon adding NaCl were observed using fluorescence spectroscopy. The simulations revealed that sodium is attracted to the headgroup region with its concentration being maximal in the vicinity of the phosphate groups. Potassium and cesium, however, do not preferentially adsorb to the membrane. Similarly, halide anions do not exhibit a strong affinity for the lipid headgroups but merely compensate for the positive charge of the sodium countercations. Nevertheless, larger halides such as bromide and iodide penetrate deeper into the headgroup region toward the boundary with the hydrophobic alkyl chain, this effect being likely underestimated within the present nonpolarizable force field. Addition of alkali halide salts modifies physical properties of the bilayer including the electronic density profiles, the electrostatic potential, and the area per lipid headgroup.

Mechanism of interaction of monovalent ions with phosphatidylcholine lipid membranes.

Interactions of different anions with phospholipid membranes in aqueous salt solutions were investigated by molecular dynamics simulations and fluorescence solvent relaxation measurements. Both approaches indicate that the anion-membrane interaction increases with the size and softness of the anion. Calculations show that iodide exhibits a genuine affinity for the membrane, which is due to its pairing with the choline group and its propensity for the nonpolar region of the acyl chains, the latter being enhanced in polarizable calculations showing that the iodide number density profile is expanded toward the glycerol level. Solvent relaxation measurements using Laurdan confirm the influence of large soft ions on the membrane organization at the glycerol level. In contrast, chloride exhibits a peak at the membrane surface only in the presence of a surface-attracted cation, such as sodium but not potassium, suggesting that this behavior is merely a counterion effect.

Solvent Relaxation in Phospholipid Bilayers: Principles and Recent Applications

Although there exist a number of methods, such as NMR, X-ray, e.g., which explore the hydration of phospholipid bilayers, the solvent relaxation (SR) method has the advantage of simple instrumentation, easy data treatment and possibility of measuring fully hydrated samples. The main information gained from SR by the analysis of recorded “time-resolved emission spectra” (TRES) is micro-viscosity and micro-polarity of the dye microenvironment. Based on these parameters, one can draw conclusions about water structure in the bilayer. In this review, we focus on physical background of this method, on all the procedures that are needed in order to obtain relevant parameters, and on the requirements on the fluorescence dyes. Furthermore, a few recent applications (the effect of curvature, binding of antibacterial peptides and phase transition) illustrating the versatility of this method are mentioned. Moreover, limitations and potential problems are discussed.

Structure, dynamics, and hydration of POPC/POPS bilayers suspended in NaCl, KCl, and CsCl solutions

Effects of alkali metal chlorides on the properties of mixed negatively charged lipid bilayers are experimentally measured and numerically simulated. Addition of 20mol% of negatively charged phosphatidylserine to zwitterionic phosphatidylcholine strengthens adsorption of monovalent cations revealing their specificity, in the following order: Cs(+)<K(+)<Na(+). Time-resolved fluorescence solvent relaxation shows significant decrease both in mobility and hydration of the lipid carbonyls probed by Laurdan upon addition of the cations. The experimental findings are supported by molecular dynamics simulations, which show deep penetration of the cations down to the glycerol level of the lipid bilayer where they pair with oxygen atoms of carbonyl groups (with pairing with sn-2 carbonyl being about twice stronger than pairing with the sn-1 one). Moreover, the cations bridge neighboring lipids forming clusters of up to 4 lipid molecules, which decreases the area per lipid, thickens the membrane, causes rising of lipid headgroups, and hinders lipid dynamics. All these effects follow the same Hofmeister ordering as the cationic adsorption to the bilayer.

Oxidized Phosphatidylcholines Facilitate Phospholipid Flip-Flop in Liposomes

Lipid asymmetry is a ubiquitous property of the lipid bilayers in cellular membranes and its maintenance and loss play important roles in cell physiology, such as blood coagulation and apoptosis. The resulting exposure of phosphatidylserine on the outer surface of the plasma membrane has been suggested to be caused by a specific membrane enzyme, scramblase, which catalyzes phospholipid flip-flop. Despite extensive research the role of scramblase(s) in apoptosis has remained elusive. Here, we show that phospholipid flip-flop is efficiently enhanced in liposomes by oxidatively modified phosphatidylcholines. A combination of fluorescence spectroscopy and molecular dynamics simulations reveal that the mechanistic basis for this property of oxidized phosphatidylcholines is due to major changes imposed by the oxidized phospholipids on the biophysical properties of lipid bilayers, resulting in a fast cross bilayer diffusion of membrane phospholipids and loss of lipid asymmetry, requiring no scramblase protein.

Biophysics of lipid bilayers containing oxidatively modified phospholipids: insights from fluorescence and EPR experiments and from MD simulations.

This review focuses on the influence of oxidized phosphatidylcholines (oxPCs) on the biophysical properties of model membranes and is limited to fluorescence, EPR, and MD studies. OxPCs are divided into two classes: A) hydroxy- or hydroperoxy-dieonyl phospatidylcholines, B) phospatidylcholines with oxidized and truncated chains with either aldehyde or carboxylic group. It was shown that the presence of the investigated oxPCs in phospholipid model membranes may have the following consequences: 1) decrease of the lipid order, 2) lowering of phase transition temperatures, 3) lateral expansion and thinning of the bilayer, 4) alterations of bilayer hydration profiles, 5) increased lipid mobility, 6) augmented flip-flop, 7) influence on the lateral phase organisation, and 8) promotion of water defects and, under extreme conditions (i.e. high concentrations of class B oxPCs), disintegration of the bilayer. The effects of class A oxPCs appear to be more moderate than those observed or predicted for class B. Many of the abovementioned findings are related to the ability of the oxidized chains of certain oxPCs to reorient toward the water phase. Some of the effects appear to be moderated by the presence of cholesterol. Although those biophysical alternations are found at oxPC concentrations higher than the total oxPC concentrations found under physiological conditions, certain organelles may reach such elevated oxPC concentrations locally. It is a challenge for the future to correlate the biophysics of oxidized phospholipids to metabolic studies in order to define the significance of the findings presented herein for pathophysiology. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.

A Rotational BODIPY Nucleotide: An Environment‐Sensitive Fluorescence‐Lifetime Probe for DNA Interactions and Applications in Live‐Cell Microscopy

Fluorescent probes for detecting the physical properties of cellular structures have become valuable tools in life sciences. The fluorescence lifetime of molecular rotors can be used to report on variations in local molecular packing or viscosity. We used a nucleoside linked to a meso-substituted BODIPY fluorescent molecular rotor (dC(bdp)) to sense changes in DNA microenvironment both in vitro and in living cells. DNA incorporating dC(bdp) can respond to interactions with DNA-binding proteins and lipids by changes in the fluorescence lifetimes in the range 0.5-2.2 ns. We can directly visualize changes in the local environment of exogenous DNA during transfection of living cells. Relatively long fluorescence lifetimes and extensive contrast for detecting changes in the microenvironment together with good photostability and versatility for DNA synthesis make this probe suitable for analysis of DNA-associated processes, cellular structures, and also DNA-based nanomaterials.

Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations.

Fluorescence solvent relaxation experiments are based on the characterization of time-dependent shifts in the fluorescence emission of a chromophore, yielding polarity and viscosity information about the chromophores immediate environment. A chromophore applied to a phospholipid bilayer at a well-defined location (with respect to the z-axis of the bilayer) allows monitoring of the hydration and mobility of the probed segment of the lipid molecules. Specifically, time-resolved fluorescence experiments, fluorescence quenching data and molecular dynamic (MD) simulations show that 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) probes the hydration and mobility of the sn-1 acyl groups in a phosphatidylcholine bilayer. The time-dependent fluorescence shift (TDFS) of Laurdan provides information on headgroup compression and expansion induced by the addition of different amounts of cationic lipids to phosphatidylcholine bilayers. Those changes were predicted by previous MD simulations. Addition of truncated oxidized phospholipids leads to increased mobility and hydration at the sn-1 acyl level. This experimental finding can be explained by MD simulations, which indicate that the truncated chains of the oxidized lipid molecules are looping back into aqueous phase, hence creating voids below the glycerol level. Fluorescence solvent relaxation experiments are also useful in understanding salt effects on the structure and dynamics of lipid bilayers. For example, such experiments demonstrate that large anions increase hydration and mobility at the sn-1 acyl level of phosphatidylcholine bilayers, an observation which could not be explained by standard MD simulations. If polarizability is introduced into the applied force field, however, MD simulations show that big soft polarizable anions are able to interact with the hydrophilic/hydrophobic interface of the lipid bilayer, penetrating to the level probed by Laurdan, and that they expand and destabilize the bilayer making it more hydrated and mobile.

The complex nature of calcium cation interactions with phospholipid bilayers

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

Numerical studies of the membrane fluorescent dyes dynamics in ground and excited states.

Fluorescence methods are widely used in studies of biological and model membranes. The dynamics of membrane fluorescent markers in their ground and excited electronic states and correlations with their molecular surrounding within the fully hydrated phospholipid bilayer are still not well understood. In the present work, Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations are used to characterize location and interactions of two membrane polarity probes (Prodan; 6-propionyl-2-dimethylaminonaphthalene and its derivative Laurdan; 2-dimethylamino-6-lauroylnaphthalene) with the dioleoylphosphatidylcholine (DOPC) lipid bilayer model. MD simulations with fluorophores in ground and excited states are found to be a useful tool to analyze the fluorescent dye dynamics and their immediate vicinity. The results of QM calculations and MD simulations are in excellent agreement with available experimental data. The calculation shows that the two amphiphilic dyes initially placed in bulk water diffuse within 10 ns towards their final location in the lipid bilayer. Analysis of solvent relaxation process in the aqueous phase occurs on the picoseconds timescale whereas it takes nanoseconds at the lipid/water interface. Four different relaxation time constants, corresponding to different relaxation processes, where observed when the dyes were embedded into the membrane.