1,2-diacyl-phosphatidylcholine flip-flop measured directly by sum-frequency vibrational spectroscopy

Abstract

Sum-frequency vibrational spectroscopy (SFVS) is used to measure the intrinsic rate of lipid flip-flop for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in planar-supported lipid bilayers (PSs). Asymmetric PSLBs were prepared using the Langmuir-Blodgett/Langmuir-Schaefer method by placing a perdeuterated lipid analog in one leaflet of the PSLB. SFVS was used to directly measure the asymmetric distribution of the native lipid within the membrane by measuring the decay in the CH3 v(s) intensity at 2875 cm(-1) with time and as a function of temperature. An average activation energy of 220 kJ/mol for the translocation of DMPC, DPPC, and DSPC was determined. A decrease in alkyl chain length resulted in a substantial increase in the rate of flip-flop manifested as an increase in the Arrhenius preexponential factor. The effect of lipid labeling was investigated by measuring the exchange of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n,n-Dimethyl-n-(2',2',6',6'-tetramethyl-4'-piperidyl) (TEMPO-DPPC). The rate of TEMPO-DPPC flip-flop was an order-of-magnitude slower compared to DPPC. An activation energy of 79 kJ/mol was measured which is comparable to that previously measured by electron spin resonance. The results of this study illustrate how SFVS can be used to directly measure lipid flip-flop without the need for a fluorescent or spin-labeled lipid probe, which can significantly alter the rate of lipid translocation.

FIGURE 1

Absence of dipole cancellation for the CH₃ v_s in an asymmetric bilayer with one leaflet containing CD₃ groups (left) and the illustration of the antiparallel arrangement of the transition dipole moments for the CH₃ v_s in a pure lipid bilayer resulting in dipole cancellation (right).

FIGURE 2

From the top to the bottom, SFVS spectra of DMPC/DMPC-d₆₇, DPPC/DPPC-d₇₅, DSPC/DSPC-d_83, DSPC-d₇₀/DSPC-d₈₃, and a premixed 1:1 DSPC-d₈₃ + DSPC lipid bilayer recorded at 23°C (DMPC/DMPC-d₆₇ at 5°C) with s-polarized sum-frequency, s-polarized visible and p-polarized IR.

FIGURE 3

CH₃ v_s intensity decay curves for DMPC/DMPC-d₆₇, DPPC/DPPC-d₇₅, and DSPC/DSPC-d₈₃ bilayers (top to bottom) at various temperatures (DMPC/DMPC-d₆₇ at 12.1, 9.8, 7.8, and 4.2°C; DPPC/DPPC-d₇₅ at 36.0, 32.3, 29.7, and 27.7°C; and DSPC/DSPC-d₈₃ at 50.3, 49.2, 45.7, and 41.7°C from left to right). All data were collected with s-polarized sum-frequency, s-polarized visible, and p-polarized IR. The solid lines are the fits to the data using Eq. 11.

FIGURE 4

Fluorescence measurements of bilayer desorption using 4% NBD-DPPE in a PSLB of DPPC recorded at 37°C. (a) Dependence of fluorescence intensity on the number of laser pulses for two different time intervals; one in which the time between sample excitation was 10 min (shaded squares) and the other for 60 min (solid circles). (b) The fluorescence intensity from the bilayer after correcting for photobleaching for both time intervals.

FIGURE 5

Arrhenius plot for DMPC/DMPC-d₆₇, DPPC/DPPC-d₇₅, and DSPC/DSPC-d₈₃ using the data in Table 1.

FIGURE 6

Arrhenius plot for TEMPO-DPPC in a DPPC-d₇₅ bilayer using the data in Table 1. The dashed lines represent the 95% confidence interval derived from the fit of the data.