Determination of Interbilayer and Transbilayer Lipid Transfers by Time-Resolved Small-Angle Neutron Scattering

We applied a time-resolved small-angle neutron scattering technique to the vesicle system of dimyristoylphosphatidylcholine for the first time to determine lipid kinetics. The observed kinetics could be explicitly represented by a simple model that includes two independent kinetic parameters, i.e., the rates of transbilayer and interbilayer exchange. This technique is perfectly suited for the determination of lipid exchange kinetics in equilibrium and applicable to evaluation of the activity of the factors relevant to lipid migration, such as translocase and lipid transfer proteins.

Figure 1

Principle of the detection of lipid dynamics by TR-SANS. (a) Calculated SLD’s of LUV’s and solvents are graded, where high and low SLD are represented by black and white, respectively. SLD of $\mathrm{D}/\mathrm{H}$-LUV corresponds to that of water with 50 vol % ${\mathrm{D}}_2\mathrm{O}$ (contrast matching). (b) In 50 vol % ${\mathrm{D}}_2\mathrm{O}$, D-LUV (top), and H-LUV (bottom) are “visible,” while $\mathrm{D}/\mathrm{H}$-LUV (middle) is invisible by neutrons under contrast-matching conditions. (c) Lipid exchange between D- and H-LUV and flip-flop in the membranes reduces the contrast of LUV’s with solvent and LUV’s become less visible, which can be detected as a decrease in the scattering intensity by TR-SANS experiment.

Figure 2

(a) SANS profiles of D-LUV, H-LUV, and $\mathrm{D}/\mathrm{H}$-LUV in buffer with 50% ${\mathrm{D}}_2\mathrm{O}$ at $27.1°\mathrm{C}$. (b) SANS profiles of $1:1$ mixture of D- and H-LUV 2, 400, 1580, and 5035 min after mixing at $27.1°\mathrm{C}$.

Figure 3

Contrast decays of LUV’s after mixing D- and H-LUV at four different temperatures. Solid curves are fitting curves according to Eq. (5). Inset shows the contrast decay over a longer period at $27.1°\mathrm{C}$.

Figure 4

Arrhenius plots of the rates of intervesicular exchange ($k_{\mathrm{ex}}$) and flip-flop ($k_f$).