The cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) is one of the original synthetic cationic lipids used for the liposomal transfection of oligonucleotides in gene therapy. The key structural element of DOTAP is its quaternary ammonium headgroup that is responsible for interactions with both nucleic acids and target cell membranes. Because these interactions are fundamental to the design of a major class of transfection lipids, it is important to understand the structure of DOTAP and how it interacts with halide counterions. Here, we use x-ray and neutron diffraction techniques to examine the structure of DOTAP and how chloride (Cl-) and iodide (I-) counterions alter the hydration properties of the DOTAP headgroup. A problem of particular interest is the poor solubility of DOTAP/I- in water solutions. Our results show that the poor solubility results from very tight binding of the I- counterion to the headgroup and the consequent expulsion of water. The structural principles we report here are important for assessing the suitability of DOTAP and its quaternary ammonium derivatives for transfection.

A direct and quantitative analysis of the internal structure and dynamics of a polyunsaturated lipid bilayer composed of 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6n3-PC) containing 29 mol% cholesterol was carried out by neutron diffraction, 2H-NMR and 13C-MAS NMR. Scattering length distribution functions of cholesterol segments as well as of the sn-1 and sn-2 hydrocarbon chains of 18:0-22:6n3-PC were obtained by conducting experiments with specifically deuterated cholesterol and lipids. Cholesterol orients parallel to the phospholipids, with the A-ring near the lipid glycerol and the terminal methyl groups 3 Å away from the bilayer center. Previously, we reported that the density of polyunsaturated docosahexaenoic acid (DHA, 22:6n3) chains was higher near the lipid–water interface. Addition of cholesterol partially redistributes DHA density from near the lipid–water interface to the center of the hydrocarbon region. Cholesterol raises chain-order parameters of both stearic acid and DHA chains. The fractional order increase for stearic acid methylene carbons C8–C18 is larger, reflecting the redistribution of DHA chain density toward the bilayer center. The correlation times of DHA chain isomerization are short and mostly unperturbed by the presence of cholesterol. The uneven distribution of saturated and polyunsaturated chain densities and the cholesterol-induced balancing of chain distributions may have important implications for the function and integrity of membrane receptors, such as rhodopsin.

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly charged S1-S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated ion channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1-S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations and cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings indicate that voltage sensors have evolved to interact with the lipid membrane while keeping energetic and structural perturbations to a minimum, and that water penetrates the membrane, to hydrate charged residues and shape the transmembrane electric field.