Filling the Gap with Long n-Alkanes: Incorporation of C20 and C30 into Phospholipid Membranes.

Investigating how hydrophobic molecules mix with phospholipid bilayers and how they affect membrane properties is commonplace in biophysics. Despite this, a molecular-level empirical description of a membrane model as simple as a phospholipid bilayer with long linear hydrophobic chains incorporated is still missing. Here, we present an unprecedented molecular characterization of the incorporation of two long n-alkanes, n-eicosane (C20) and n-triacontane (C30) with 20 and 30 carbons, respectively, in phosphatidylcholine (PC) bilayers using a combination of experimental techniques (2H NMR, 31P NMR, 1H-13C dipolar recoupling solid-state NMR, X-ray scattering, and cryogenic electron microscopy) and atomistic molecular dynamics (MD) simulations. At low hydration, deuterated C20 and C30 yield 2H NMR spectra evidencing anisotropic-motion, which demonstrates their miscibility in PC membranes up to a critical alkane-to-acyl-chain volume fraction, ϕc. The acquired 2H NMR spectra of C20 and C30 have notably different lineshapes. At low alkane volume fractions below ϕc, CHARMM36 MD simulations predict such 2H NMR spectra qualitatively and thus enable an atomistic-level interpretation of the spectra. Above ϕc, the 2H NMR lineshapes become characteristic of motions in the intermediate-regime that, together with the MD simulation results, suggest the onset of immiscibility between the alkane molecules and the acyl chains. For all the systems investigated, the phospholipid molecular structure is unperturbed by the presence of the alkanes. However, at conditions of excess hydration and at surprisingly low alkane fractions below ϕc, a peak characteristic of isotropic motion is observed in both the 2H spectra of the alkanes and 31P spectra of the phospholipids, strongly indicating that the incorporation of the alkanes induces a reduction on the average radius of the lipid vesicles.

Rotational decoupling between the hydrophilic and hydrophobic regions in lipid membranes.

Cells use homeostatic mechanisms to ensure an optimal composition of distinct types of lipids in cellular membranes. The hydrophilic region of biological lipid membranes is mainly composed of several types of phospholipid headgroups that interact with incoming molecules, nanoparticles, and viruses, whereas the hydrophobic region consists of a distribution of acyl chains and sterols affecting membrane fluidity/rigidity related properties and forming an environment for membrane-bound molecules such as transmembrane proteins. A fundamental open question is to what extent the motions of these regions are coupled and, consequently, how strongly the interactions of phospholipid headgroups with other molecules depend on the properties and composition of the membrane hydrophobic core. We combine advanced solid-state nuclear magnetic resonance spectroscopy with high-fidelity molecular dynamics simulations to demonstrate how the rotational dynamics of choline headgroups remain nearly unchanged (slightly faster) with incorporation of cholesterol into a phospholipid membrane, contrasting the well-known extreme slowdown of the other phospholipid segments. Notably, our results suggest a new paradigm in which phospholipid dipole headgroups interact as quasi-freely rotating flexible dipoles at the interface, independent of the properties in the hydrophobic region.

Improved Cation Binding to Lipid Bilayers with Negatively Charged POPS by Effective Inclusion of Electronic Polarization.

Phosphatidylserine (PS) lipids are important signaling molecules and the most common negatively charged lipids in eukaryotic membranes. The signaling can be often regulated by calcium, but its interactions with PS headgroups are not fully understood. Classical molecular dynamics (MD) simulations can potentially give detailed description of lipid-ion interactions, but the results strongly depend on the used force field. Here, we apply the electronic continuum correction (ECC) to the Amber Lipid17 parameters of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) lipid to improve its interactions with K+, Na+, and Ca2+ ions. The partial charges of the headgroup, glycerol backbone, and carbonyls of POPS, bearing a unit negative charge, were scaled with a factor of 0.75, derived for monovalent ions, and the Lennard-Jones σ parameters of the same segments were scaled with a factor of 0.89. The resulting ECC-POPS model gives more realistic interactions with Na+ and Ca2+ cations than the original Amber Lipid17 parameters when validated using headgroup order parameters and the "electrometer concept". In ECC-lipids simulations, populations of complexes of Ca2+ cations with more than two PS lipids are negligible, and interactions of Ca2+ cations with only carboxylate groups are twice more likely than with only phosphate groups, while interactions with carbonyls almost entirely involve other groups as well. Our results pave the way for more realistic MD simulations of biomolecular systems with anionic membranes, allowing signaling processes involving PS and Ca2+ to be elucidated.

Headgroup Structure and Cation Binding in Phosphatidylserine Lipid Bilayers.

Phosphatidylserine (PS) is a negatively charged lipid type commonly found in eukaryotic membranes, where it interacts with proteins via nonspecific electrostatic interactions as well as via specific binding. Moreover, in the presence of calcium ions, PS lipids can induce membrane fusion and phase separation. Molecular details of these phenomena remain poorly understood, partly because accurate models to interpret the experimental data have not been available. Here we gather a set of previously published experimental NMR data of C-H bond order parameter magnitudes, |SCH|, for pure PS and mixed PS:PC (phosphatidylcholine) lipid bilayers and augment this data set by measuring the signs of SCH in the PS headgroup using S-DROSS solid-state NMR spectroscopy. The augmented data set is then used to assess the accuracy of the PS headgroup structures in, and the cation binding to, PS-containing membranes in the most commonly used classical molecular dynamics (MD) force fields including CHARMM36, Lipid17, MacRog, Slipids, GROMOS-CKP, Berger, and variants. We show large discrepancies between different force fields and that none of them reproduces the NMR data within experimental accuracy. However, the best MD models can detect the most essential differences between PC and PS headgroup structures. The cation binding affinity is not captured correctly by any of the PS force fields-an observation that is in line with our previous results for PC lipids. Moreover, the simulated response of the PS headgroup to bound ions can differ from experiments even qualitatively. The collected experimental data set and simulation results will pave the way for development of lipid force fields that correctly describe the biologically relevant negatively charged membranes and their interactions with ions. This work is part of the NMRlipids open collaboration project ( nmrlipids.blogspot.fi ).

Molecular conformation and bilayer pores in a nonionic surfactant lamellar phase studied with 1H-13C solid-state NMR and molecular dynamics simulations.

The structure of the lamellar phase of aqueous pentaethylene glycol mono-n-dodecyl ether (C12E5) surfactant at various temperatures and molar fractions is studied by using united atom molecular dynamics simulations and nuclear magnetic resonance measurements. Namely, the simulation model is used to interpret the magnitude and temperature dependence of experimental C-H order parameter profiles in terms of the molecular conformation and orientation. Our simulations suggest that the low order parameters that are generally measured in poly(ethylene oxide) surfactant bilayers are due to the presence of bilayer pores throughout the entire lamellar phase region.