Energetics of the Mixing of Phospholipids in Bilayers Determined Using Vesicle Solubilization

Here, we report an experimental approach for determining the change in the free energy and the enthalpy that accompanies the mixing of the anionic phosphatidylglycerol and the zwitterionic phosphatidylcholine. The enthalpy change originates in the thermal changes of disrupting lipid bilayer vesicles titrated into a surfactant micelle solution and is monitored using isothermal titration calorimetry. The difference in the solubilization enthalpies between pure and mixed lipid vesicles yields the lipid mixing enthalpy. The Gibbs free energy changes are estimated by determining the thermodynamic equilibrium constants of forming a molecular complex between phospholipids and methyl-β-cyclodextrin. We provide direct experimental evidence that mixing of the anionic lipid and the zwitterionic lipid is explained well by the entropic term of the electrostatic free energy of a charged surface in the Gouy–Chapman model. The present strategy enables us to determine the precise energetics of lipid–lipid interactions in near-native environments such as liposomes without any chemical modification to lipid molecules

Self-Reproduction of Nanoparticles through Synergistic Self-Assembly

We describe a self-reproduction mechanism of nanometer-sized particles (i.e., nanodiscs) through chemical ligation of the precursors and self-assembly of the building blocks. The ligation reaction was accelerated on lipid bilayer surfaces, and the products spontaneously assembled into nanodiscs with lipid molecules. With the increase in the number of nanodiscs, a rapid proliferation of the nanodiscs occurred through the spatial rearrangements of the molecules between the pre-existing nanodiscs and the unreacted materials, rather than template- or complex-enhanced ligation of the precursors. The subsequent process of surface-enhanced ligation of integrated precursors matured the nanoparticles into identical copies of the pre-existing assembly. Our study showed that the synergistic self-assembly mechanism probably underlie the self-replication principles for heterogeneous multimolecular systems

High Membrane Curvature Enhances Binding, Conformational Changes, and Fibrillation of Amyloid‑β on Lipid Bilayer Surfaces

Aggregation of the amyloid-β (Aβ) protein and the formation of toxic aggregates are the possible pathogenic pathways in Alzheimer’s disease. Accumulating evidence suggests that lipid membranes play key roles in protein aggregation, although the intermolecular forces that drive the interactions between Aβ-(1–40) and the membranes vary in different membrane systems. Here, we observed that a high positive curvature of lipid vesicles with diameters of ∼30 nm enhanced the association of Aβ with anionic phosphatidylglycerol membranes in the liquid-crystalline phase and with zwitterionic phosphatidylcholine membranes in the gel phase. The binding modes of Aβ to these membranes differ in terms of the location of the protein on the membrane and of the protein secondary structure. The fibrillation of Aβ was accelerated in the presence of the vesicles and at high protein-to-lipid ratios. Under these conditions, the protein accumulated on the surfaces, as demonstrated by a high (10⁷ M^–1) binding constant. Our findings suggest that packing defects on membranes with high curvatures, such as the intraluminal vesicles in multivesicular bodies and the exosomes, might result in the accumulation of toxic protein aggregates

Kinetic and Thermodynamic Analysis of Cholesterol Transfer between Phospholipid Vesicles and Nanodiscs

We investigated interparticle transfer of cholesterol (Chol) between large unilamellar vesicles (LUVs) and phospholipid bilayer nanodiscs. The Chol transfer rate from LUVs to nanodiscs was decreased by an increase in the Chol content or incorporation of sphingomyelin in donor phosphatidylcholine/Chol LUVs but was not influenced by the lipid composition of acceptor particles. These results suggest that Chol dissociation from the lipid bilayer into aqueous phase is the rate-limiting step of the transfer and that the process depends on the fluidity of the donor membranes. The Chol dissociation rate from nanodiscs was faster than that from LUVs with similar lipid composition. Chol preferably partitioned to LUVs rather than nanodiscs, which is consistent with the faster dissociation rate from nanodiscs. The activation energy of Chol dissociation from nanodiscs was 1.7 kJ/mol lower than that from LUV, which was brought by increased (less negative) activation entropy and enthalpy. In addition, fluorescence lifetime and anisotropy data revealed that the lipid bilayer of nanodiscs is more tightly packed than that of LUVs. These results suggest that the tighter lipid packing in nanodiscs destabilizes the Chol-containing bilayer by reducing the entropy, which facilitates Chol dissociation

Nanodisc-to-Nanofiber Transition of Noncovalent Peptide–Phospholipid Assemblies

We report a novel molecular architecture of peptide–phospholipid coassemblies. The amphiphilic peptide Ac-18A-NH₂ (18A), which was designed to mimic apolipoprotein α-helices, has been shown to form nanodisc structures with phospholipid bilayers. We show that an 18A peptide cysteine substitution at residue 11, 18A­[A11C], forms fibrous assemblies with 1-palmitoyl-2-oleoyl-phosphatidylcholine at a lipid-to-peptide (L/P) molar ratio of 1, a fiber diameter of 10–20 nm, and a length of more than 1 μm. Furthermore, 18A­[A11C] can form nanodiscs with these lipid bilayers at L/P ratios of 4–6. The peptide adopts α-helical structures in both the nanodisc and nanofiber assemblies, although the α-helical bundle structures were evident only in the nanofibers, and the phospholipids of the nanofibers were not lamellar. Fluorescence spectroscopic analysis revealed that the peptide and lipid molecules in the nanofibers exhibited different solvent accessibility and hydrophobicity from those of the nanodiscs. Furthermore, the cysteine substitution at residue 11 did not result in disulfide bond formation, although it was responsible for the nanofiber formation, suggesting that this free sulfhydryl group has an important functional role. Alternatively, the disulfide dimer of 18A­[A11C] preferentially formed nanodiscs, even at an L/P ratio of 1. Interconversions of these discoidal and fibrous assemblies were induced by the stepwise addition of free 18A­[A11C] or liposomes into the solution. Furthermore, these structural transitions could also be induced by the introduction of oxidative and reductive stresses to the assemblies. Our results demonstrate that heteromolecular lipid–peptide complexes represent a novel approach to the construction of controllable and functional nanoscale assemblies