Bastien Loubet

Pairing of cholesterol with oxidized phospholipid species in lipid bilayers.

We claim that (1) cholesterol protects bilayers from disruption caused by lipid oxidation by sequestering conical shaped oxidized lipid species such as 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PZPC) away from phospholipid, because cholesterol and the oxidized lipid have complementary shapes and (2) mixtures of cholesterol and oxidized lipids can self-assemble into bilayers much like lysolipid–cholesterol mixtures. The evidence for bilayer protection comes from molecular dynamics (MD) simulations and dynamic light scattering (DLS) measurements. Unimodal size distributions of extruded vesicles (LUVETs) made up of a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and PZPC containing high amounts of PZPC are only obtained when cholesterol is present in high concentrations. In simulations, bilayers containing high amounts of PZPC become porous, unless cholesterol is also present. The protective effect of cholesterol on oxidized lipids has been observed previously using electron paramagnetic resonance (EPR) and electron microscopy imaging of vesicles. The evidence for the pairing of cholesterol and PZPC comes mainly from correlated 2-D density and thickness plots from simulations, which show that these two molecules co-localize in bilayers. Further evidence that the two molecules can cohabitate comes from self-assembly simulations, where we show that cholesterol-oxidized lipid mixtures can form lamellar phases at specific concentrations, reminiscent of lysolipid–cholesterol mixtures. The additivity of the packing parameters of cholesterol and PZPC explains their cohabitation in a planar bilayer. Oxidized lipids are ubiquitously present in significant amounts in high- and low-density lipoprotein (HDL and LDL) particles, diseased tissues, and in model phospholipid mixtures containing polyunsaturated lipids. Therefore, our hypothesis has important consequences for cellular cholesterol trafficking; diseases related to oxidized lipids, and to biophysical studies of phase behaviour of cholesterol-containing phospholipid mixtures.

Molecular mechanism of Na(+),K(+)-ATPase malfunction in mutations characteristic of adrenal hypertension.

Mutations within ion-transporting proteins may severely affect their ability to traffic ions properly and thus perturb the delicate balance of ion gradients. Somatic gain-of-function mutations of the Na(+),K(+)-ATPase α1-subunit have been found in aldosterone-producing adenomas that are among the causes of hypertension. We used molecular dynamics simulations to investigate the structural consequences of these mutations, namely, Leu97 substitution by Arg (L97R), Val325 substitution by Gly (V325G), deletion of residues 93-97 (Del93-97), and deletion-substitution of residues 953-956 by Ser (EETA956S), which shows inward leak currents under physiological conditions. The first three mutations affect the structural context of the key ion-binding residue Glu327 at binding site II, which leads to the loss of the ability to bind ions correctly and to occlude the pump. The mutated residue in L97R is more hydrated, which ultimately leads to the observed proton leak. V325G mimics the structural behavior of L97R; however, it does not promote the hydration of surrounding residues. In Del93-97, a broader opening is observed because of the rearrangement of the kinked transmembrane helix 1, M1, which may explain the sodium leak measured with the mutant. The last mutant, EETA956S, opens an additional water pathway near the C-terminus, affecting the III sodium-specific binding site. The results are in excellent agreement with recent electrophysiology measurements and suggest how three mutations prevent the occlusion of the Na(+),K(+)-ATPase, with the possibility of transforming the pump into a passive ion channel, whereas the fourth mutation provides insight into the sodium binding in the E1 state.

Effective tension and fluctuations in active membranes

We calculate the fluctuation spectrum of the shape of a lipid vesicle or cell exposed to a nonthermal source of noise. In particular, we take constraints on the membrane area and the volume of fluid that it encapsulates into account when obtaining expressions for the dependency of the membrane tension on the noise. We then investigate three possible origins of the nonthermal noise taken from the literature: A direct force, which models an external medium pushing on the membrane, a curvature force, which models a fluctuating spontaneous curvature, and a permeation force coming from an active transport of fluid through the membrane. For the direct force and curvature force cases, we compare our results to existing experiments on active membranes.

Accelerating All-Atom MD Simulations of Lipids Using a Modified Virtual-Sites Technique.

We present two new implementations of the virtual sites technique which completely suppresses the degrees of freedom of the hydrogen atoms in a lipid bilayer allowing for an increased time step of 5 fs in all-atom simulations of the CHARMM36 force field. One of our approaches uses the derivation of the virtual sites used in GROMACS while the other uses a new definition of the virtual sites of the CH2 groups. Our methods is tested on a DPPC (no unsaturated chain), a POPC (one unsaturated chain), and a DOPC (two unsaturated chains) lipid bilayers. We calculate various physical properties of the membrane of our simulations with and without virtual sites and explain the differences and similarity observed. The best agreements are obtained for the GROMACS original virtual sites on the DOPC bilayer where we get an area per lipid of 67.3 ± 0.3 Å(2) without virtual sites and 67.6 ± 0.3 Å(2) with virtual sites. In conclusion the virtual-sites technique on lipid membranes is a powerful simulation tool, but it should be used with care. The procedure can be applied to other force fields and lipids in a straightforward manner.

Elastic moderation of intrinsically applied tension in lipid membranes

Tension in lipid membranes is often controlled externally, by pulling on the boundary of the membrane or changing osmotic pressure across a curved membrane. But modifications of the tension can also be induced in an internal fashion, for instance as a byproduct of changing a membranes electric potential or, as observed experimentally, by activity of membrane proteins. Here we develop a theory that demonstrates how such internal contributions to the tension are moderated through elastic stretching of the membrane when the membrane is initially in a low-tension floppy state.

Tension moderation and fluctuation spectrum in simulated lipid membranes under an applied electric potential

We investigate the effect of an applied electric potential on the mechanics of a coarse grained POPC bilayer under tension. The size and duration of our simulations allow for a detailed and accurate study of the fluctuations. Effects on the fluctuation spectrum, tension, bending rigidity, and bilayer thickness are investigated in detail. In particular, the least square fitting technique is used to calculate the fluctuation spectra. The simulations confirm a recently proposed theory that the effect of an applied electric potential on the membrane will be moderated by the elastic properties of the membrane. In agreement with the theory, we find that the larger the initial tension the larger the effect of the electric potential. Application of the electric potential increases the amplitude of the long wavelength part of the spectrum and the bending rigidity is deduced from the short wavelength fluctuations. The effect of the applied electric potential on the bending rigidity is non-existent within error bars. However, when the membrane is stretched there is a point where the bending rigidity is lowered due to a decrease of the thickness of the membrane. All these effects should prove important for mechanosensitive channels and biomembrane mechanics in general.

The Molecular Mechanism of Na+, K+-ATPase Malfunction in Mutations Characteristic for Adrenal Hypertension

Mutations within ion-transporting proteins may severely affect their ability to traffic ions properly and thus perturb the delicate balance of ion gradients. Somatic gain-of-function mutations of the Na+,K+-ATPase α1-subunit have been found in aldosterone-producing adenomas that are among the causes of hypertension. We used molecular dynamics simulations to investigate the structural consequences of these mutations, namely, Leu97 substitution by Arg (L97R), Val325 substitution by Gly (V325G), deletion of residues 93–97 (Del93–97), and deletion–substitution of residues 953–956 by Ser (EETA956S), which shows inward leak currents under physiological conditions. The first three mutations affect the structural context of the key ion-binding residue Glu327 at binding site II, which leads to the loss of the ability to bind ions correctly and to occlude the pump. The mutated residue in L97R is more hydrated, which ultimately leads to the observed proton leak. V325G mimics the structural behavior of L97R; however,...

Fluctuation Spectrum and Electrical Stresses in Biomembranes Probed by Molecular Dynamics Simulations

We simulate a large patch of membrane (130x10x20 nm) using the Martini coarse grained force field for lipids. The large size of the membrane enables access to long wavelength fluctuations, which allows us to compute a fluctuation spectrum (fluctuation amplitude at different wavelengths). The spectrum contains both a tension dominated and a bending rigidity dominated regime on wavelengths well above the protrusion regime [1] or molecular tilt regime [2]. The fluctuation spectrum can then be fitted to the prediction of to the standard Helfrich effective Hamiltonian [3], like in flickering experiments. The tension used in the fit of the fluctuation spectrum is calculated directly from the lateral stresses, and our only fit parameter is then the bending rigidity. We establish an overall consistency of the bending rigidity obtained from fits of the fluctuation spectrum for membranes with different tensions. We have also applied an electric field on the membrane. The electric field will change the lateral stress profile of the membrane and the change of the fluctuation spectrum is studied in detail. In particular, the response of the membrane stress profile to the applied electric field is compared to our recent theory [4].[1] G. Brannigan and F. L. H. Brown, Biophys J. 90, 1501 (2006)[2] M. C. Watson E. G. Brandt P. M. Welch and F. L. H. Brown, Phys. Rev. Lett. 109, 028102 (2012)[3] U. Seifert, Adv. Phys., 46, 13-137 (1997)[4] M. A. Lomholt, B. Loubet and J. H. Ipsen, Phys. Rev. E 83, 011913 (2011)

Ion pump activity generates fluctuating electrostatic forces in biomembranes

We study the non-equilibrium dynamics of lipid membranes with proteins that actively pump ions across the membrane. We find that the activity leads to a fluctuating force distribution due to electrostatic interactions arising from the variation in the dielectric constant across the membrane. By applying a multipole expansion we find effects on both the tension and bending rigidity dominated parts of the membranes fluctuation spectrum. We discuss how our model compares with previous studies of force-multipole models.