The effect of ergosterol on dipalmitoylphosphatidylcholine bilayers: a deuterium NMR and calorimetric study

Abstract

We have studied the effect of ergosterol, an important component of fungal plasma membranes, on the physical properties of dipalmitoylphosphatidylcholine (DPPC) multibilayers using deuterium nuclear magnetic resonance ((2)H NMR) and differential scanning calorimetry (DSC). For the (2)H NMR experiments the sn-1 chain of DPPC was perdeuterated and NMR spectra were taken as a function of temperature and ergosterol concentration. The phase diagram, constructed from the NMR spectra and the DSC thermograms, exhibits both solid-ordered (so) + liquid-ordered (lo) and liquid-disordered (ld) + lo phase coexistence regions with a clear three-phase line. This is the first demonstration that lo domains exist in liquid crystalline membranes containing ergosterol. The domain sizes in the ld+lo phase coexistence region were estimated by analyzing the exchange of labeled DPPC between the two regions, and depend on ergosterol concentration. The DPPC-ergosterol phase diagram is similar to that of the DPPC-cholesterol multibilayer system except that the so+lo and ld+lo phase coexistence regions are considerably broader.

FIGURE 1

²H NMR spectra of (A) pure DPPC-d31 and (B) 95:5 DPPC-d31/ergosterol as a function of temperature.

FIGURE 2

²H NMR spectra of DPPC-d31/ergosterol as a function of ergosterol concentration at T = 42°C.

FIGURE 3

²H NMR spectra of DPPC-d31/ergosterol as a function of ergosterol concentration at T = 38°C.

FIGURE 4

²H NMR spectra as a function of temperature for 84:16 DPPC-d31/ergosterol.

FIGURE 5

The temperature dependence of M₁ for DPPC-d31/ergosterol at various ergosterol concentrations: 0 mol% (▾); 5 mol% (▵); 10 mol% (▪); 13 mol% (○); 16 mol% (♦); 20 mol% (∇); 25 mol% (•); 27.5 mol% (⋄); 30 mol% (▴); 35 mol% (□); 42 mol% (⊕).

FIGURE 6

M₁ as a function of ergosterol concentration for DPPC-d31/ergosterol at: 41°C (•); 42°C (⋄); 43°C (▪); 44°C (⊗); 45°C (▴); 47°C (○); 48°C (▾); 49°C (□); 50°C (♦); 53°C (▵); 57°C (⊕). The lines are guides to the eye.

FIGURE 7

The depaked spectra of 75:25 DPPC-d31/ergosterol as a function of temperature. Because the spectra are symmetrical, we show only the high-frequency half of each.

FIGURE 8

The depaked spectra of DPPC-d31/ergosterol as a function of ergosterol concentration at T = 45°C.

FIGURE 9

DSC scans of DPPC/ergosterol. Note that to compare DSC with NMR data one must shift the DSC temperature by −2°C, given the fact that DPPC chain deuteration reduces the transition temperature by 2°C.

FIGURE 10

Partial phase diagram of the DPPC/ergosterol membrane. Midpoint of the transition from M₁(T) curves (Fig. 5) (•); onset or end of transition in M₁(T) curves (□); obtained by inspection of the depaked spectra versus ergosterol concentration (Fig. 8) (⋄); end of transition in DSC data (shifted by −2°C) (♦); obtained by inspection of the depaked spectra versus temperature (Fig. 7) (▴); obtained from M₁(ergosterol) curves (Fig. 6) (○); obtained from spectral subtraction (▪); the onset of transition in M₁(T) curves for MLDs having ergosterol concentrations of 25, 27.5, or 30 mol% (▵).

FIGURE 11

The root mean squared distance (〈Δx²〉)^1/2 diffused by DPPC-d31 as a function of ergosterol concentration at T = 45°C. The lines are guides to the eye.

FIGURE 12

The quadrupolar splitting of the CD₃ doublet as a function of sterol concentration. DPPC-d31/ergosterol at 42°C (▪); DPPC-d62/cholesterol at 40°C (□). The lines are guides to the eye.