1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers

Abstract

We measured the transbilayer diffusion of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in large unilamellar vesicles, in both the gel (L_β') and fluid (L_α) phases. The choline resonance of headgroup-protiated DPPC exchanged into the outer leaflet of headgroup-deuterated DPPC-d13 vesicles was monitored using ¹H NMR spectroscopy, coupled with the addition of a paramagnetic shift reagent. This allowed us to distinguish between the inner and outer bilayer leaflet of DPPC, to determine the flip-flop rate as a function of temperature. Flip-flop of fluid-phase DPPC exhibited Arrhenius kinetics, from which we determined an activation energy of 122 kJ mol^-1. In gel-phase DPPC vesicles, flip-flop was not observed over the course of 250 h. Our findings are in contrast to previous studies of solid-supported bilayers, where the reported DPPC translocation rates are at least several orders of magnitude faster than those in vesicles at corresponding temperatures. We reconcile these differences by proposing a defect-mediated acceleration of lipid translocation in supported bilayers, where long-lived, submicron-sized holes resulting from incomplete surface coverage are the sites of rapid transbilayer movement.

Figure 1

Structure of symmetric and asymmetric DPPC vesicles. Experimental small-angle X-ray scattering (SAXS) form factors (open circles) and fits (solid colored lines) for DPPC LUVs prepared using standard extrusion (a) and aLUVs (DPPC-dH^inner/DPPC-dC^outer) prepared by cyclodextrin-mediated exchange (b), both at 55 °C. (c) Vesicle diameter of acceptor LUVs (white bars) and aLUVs (solid bars) immediately after preparation. (d) aLUV diameter before (white bars) and after (solid bars) incubation at different temperatures (incubation times are indicated on the bars).

Figure 2

Differential scanning calorimetry of DPPC vesicles in D₂O. Upper: exotherms of DPPC MLVs composed of different isotopic variants. Lower: exotherms for DPPC LUVs and DPPC-dHⁱⁿ/DPPCdC^out aLUVs. Data were collected at a scan rate of 0.5 °C/s.

Figure 3

DPPC flip-flop kinetics. (a) Asymmetric lipid distribution determined by ¹H NMR. Shown are NMR data (black line) and fitted components (filled peaks) from DPPC-dHⁱⁿ/DPPC-dC^out aLUVs in the presence of ∼70 μM Pr³⁺ shift reagent, measured immediately following aLUV preparation (upper panel) and after 24 h incubation at 65 °C (lower panel). Spectra were modeled as the sum of outer leaflet (red) and inner leaflet (green) choline resonances and residual aqueous cyclodextrin (gray). (b,c) Time decay of normalized bilayer asymmetry at various temperatures in the gel (blue symbols), gel + fluid (green symbols), and fluid (orange symbols) phases and fits to eq 3 (solid lines). ΔC is defined as the difference in inner and outer leaflet NMR peak areas normalized to their initial (time zero) difference, see eq 3. Inset to (c) shows the Arrhenius behavior of fluid-phase translocation (solid gray line, with dashed lines indicating the 95% confidence interval). (d) DSC exotherm for DPPC-dHⁱⁿ/DPPCdC^out aLUVs. Roman numerals denote different bilayer phase states and are color-coded to the data in (b) and (c): I, gel (blue); II, gel + fluid coexistence (green); and III, fluid (orange). (e) Translocation rate constants (k_f) for gel phase (blue, SSB values reproduced from Liu and Conboy), gel + fluid coexistence (green), and fluid-phase (orange) DPPC. Horizontal lines represent the minimum k_f for SSB fluid-phase DPPC and the maximum gel-phase k_f for LUVs.

Figure 4

Monte Carlo simulations of defect-mediated flip-flop. (a) Schematic illustration of an SSB, showing a defect site in cross-section. Transbilayer movement is assumed to occur via unhindered lateral diffusion through the pore formed by lipid headgroups. (b) MC simulation snapshot of a particle trajectory that includes segments in the top (black) and bottom (gray) leaflets. The inset shows an expanded view in the vicinity of a circular defect, revealing multiple translocation events. (c) Top–down view of simulation snapshots (defects shown as black circles; scale bar, 1 μm) for different bilayer surface coverages: 99.0% (gray), 99.5% (blue) and 99.9% (yellow). (d) Simulated asymmetry decay curves (open symbols) and fits (solid lines) corresponding to the surface coverages in (c), for a lateral lipid diffusion coefficient of 10^–3 μm² s^–1. (e) Lipid translocation rate k_f vs lateral diffusion coefficient D_T calculated from decay curves corresponding to the surface coverages in (c).