Energetic and Structural Insights into Phospholipid Transfer from Membranes with Different Curvatures by Time-Resolved Neutron Scattering

Abstract

Understanding how lipid dynamics change with membrane curvature is important given that biological membranes constantly change their curvature and morphology through membrane fusion and endo-/exocytosis. Here, we used time-resolved small-angle neutron scattering and time-resolved fluorescence to characterize the properties and dynamics of phospholipids in vesicles with different curvatures. Dissociation of phospholipids from vesicles required traversing an energy barrier comprising positive enthalpy and negative entropy. However, lipids in membranes with high positive curvature have dense acyl chain packing and loose headgroup packing, leading to hydrophobic hydration due to water penetration into the membrane. These properties were found to lower the hydrophobic hydration enhancement associated with phospholipid dissociation and mitigate the acyl chain packing of lipids adjacent to the space created by the lipid dissociation, resulting in an increase in activation entropy. The results of this study provide important insights into the functions of biomembranes in relation to their dynamic structural changes.

Small-Angle Neutron Scattering for Studying Lipid Bilayer Membranes

“…SANS was also used to evaluate the transition of lipid nanodisks to unilamellar vesicles [ 243 ]. Simple curvature effects can facilitate lipid exchange [ 244 ], for example, and so can methanol [ 245 ]. The transport of cholesterol between POPC vesicles and between leaflets of a single vesicle was found by SANS to be slower than previously reported [ 246 ].…”

The Nanometer-Scale Proximity of Bilayers Facilitates Intermembrane Lipid Transfer

“…Lipid exchange between vesicles reduces the SLD difference, i.e., contrast, and decreases scattering from the vesicles (Figure B). We found that the spontaneous transfer of POPCs between vesicles is very slow (Figure C), which is consistent with previous results. , The addition of PEG8000 or PEG20000 immediately decreased the contrast, suggesting enhanced lipid exchange. On the contrary, the enhancement effect of PEG200 was remarkably smaller, indicating that lipid exchange is induced by vesicle aggregation.…”

“…The values of Δ H ⧧ and − T Δ S ⧧ were positive and negative, respectively (Figure C), indicating that PEG-mediated lipid transfer is an enthalpically unfavorable but slightly entropically favorable process. Compared to the parameters without PEG, the changes in the activation free energy, enthalpy, and entropy upon the addition of PEG [Δ(Δ G ⧧ ), Δ(Δ H ⧧ ), and Δ(− T Δ S ⧧ )] were −12.2, 5.2, and −17.4 kJ/mol at 37 °C, respectively (Figure C). Thus, the decrease in the activation free energy was due to the increase in activation entropy, which is discussed below.…”

“…As the morphology of the vesicles did not change during aggregation (Figures S6 and S7), the rate-limiting step of the observed lipid transfer, with and without PEG, was likely the dissociation of lipids from the bilayer. It is well-known that the negative entropic change during membrane dissociation of lipids (in the absence of PEG) is due to the hydrophobic hydration of acyl chains . Many experimental studies − and molecular dynamics (MD) simulations − have revealed differences in the dynamics of water molecules in the vicinity of bilayers.…”

“…Polyethylene glycol (PEG) is known to induce the fusion of lipid vesicles and cells. − The presence of PEG induces depletion attraction between large obstacles owing to the excluded volume effect, resulting in their aggregation. , As PEG indirectly causes attraction between vesicles, this technique can be used to analyze the properties of bilayers adjacent to each other without introducing docking reagents into the membrane. Here, we investigate lipid transfer between vesicles brought into proximity by PEG using time-resolved small-angle neutron scattering (TR-SANS), a technique first established by our group for evaluating interbilayer and transbilayer transfer (flip-flop) of lipids. − The thermodynamic activation parameters of lipid transfer were derived from the temperature dependence of the transfer. Notably, the inclusion of PEG-conjugated lipids into the vesicles manipulated the interbilayer distance of the aggregated vesicles, which was quantitatively analyzed using SANS.…”