Structure of phospholipid-cholesterol membranes: An x-ray diffraction study

We have studied the phase behavior of mixtures of cholesterol with dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), and dilauroyl phosphatidylethanolamine (DLPE), using x-ray diffraction techniques. Phosphatidylcholine (PC)-cholesterol mixtures are found to exhibit a modulated phase for cholesterol concentrations around $15\mathrm{mol}\%$ at temperatures below the chain melting transition. Lowering the relative humidity from 98% to 75% increases the temperature range over which it exists. An electron density map of this phase in DPPC-cholesterol mixtures, calculated from the x-ray diffraction data, shows bilayers with a periodic height modulation, as in the ripple phase observed in many PCs in between the main- and pretransitions. However, these two phases differ in many aspects, such as the dependence of the modulation wavelength on the cholesterol content and thermodynamic stability at reduced humidities. This modulated phase is found to be absent in DLPE-cholesterol mixtures. At higher cholesterol contents the gel phase does not occur in any of these three systems, and the fluid lamellar phase is observed down to the lowest temperature studied $(5°\mathrm{C})$.

Figure 1

The phase diagrams of DPPC-cholesterol mixtures at 98% RH (a) and 75% RH (b), determined from the diffraction data.

Figure 2

The diffraction pattern of the $P_{\beta }$ phase of DPPC-cholesterol mixtures at $\mathrm{RH}=98\%$ $(X_c=15\mathrm{mol}\%,T=6°\mathrm{C})$. The reflections can be indexed on a primitive rectangular lattice as shown. The $q$ scales are approximate and are intended only as guides.

Figure 3

(a) The diffraction pattern of the $P_{\beta }$ phase of DPPC-cholesterol mixtures at 75% RH $(X_c=12.5\mathrm{mol}\%,$ $T=21°\mathrm{C},$ $d=61.4\mathrm{\AA },$ $\lambda =67.5\mathrm{\AA })$. The inset shows the small angle region of the diffraction pattern on an expanded scale. The reflections can be indexed on a primitive rectangular lattice as shown. For comparison the diffraction pattern of the ripple $\left(P_{{\beta }^{\prime }}\right)$ phase of DMPC $(T=20°\mathrm{C},$ $\mathrm{RH}=98\%)$, which occurs in between the pre- and main transitions, is shown in (b) $(d=57.6\mathrm{\AA },$ $\lambda =158.4\mathrm{\AA })$. These reflections can be indexed on an oblique lattice as shown. The $q$ scales are approximate and are intended only as guides.

Figure 4

(a) The diffraction pattern showing the coexistence of the gel and $P_{\beta }$ phases $(X_c=10\mathrm{mol}\%,$ $T=10°\mathrm{C},\mathrm{RH}=98\%)$. (b) The diffraction pattern of the cholesterol-rich $l_o$ phase $(X_c=50\mathrm{mol}\%,$ $T=55°\mathrm{C},$ $\mathrm{RH}=98\%)$. In (b) the first-order lamellar peak is masked by the beam stop. The $q$ scales are approximate and are intended only as guides.

Figure 5

The variation of the modulation wavelength $\left(\lambda \right)$ of the $P_{\beta }$ phase of DPPC-cholesterol mixtures with temperature at $X_c=12.5\mathrm{mol}\%$ (a) and with $X_c$ at $T=15°\mathrm{C}$ (b).

Figure 6

The phase diagram of DMPC-cholesterol mixtures at 98% RH, determined from the diffraction data. The phase boundaries indicated by dotted lines have not been determined very precisely.

Figure 7

The diffraction pattern of the $L_{\beta }$ phase of DLPE $(T=10°\mathrm{C},$ $\mathrm{RH}=98\%)$. The absence of chain tilt is indicated by the on-axis $(q_z=0)$ wide-angle reflections. The shadow at the bottom is due to the absorption by the substrate. The $q$ scales are approximate and are intended only as guides.

Figure 8

The diffraction pattern of a highly ordered phase of DLPE-cholesterol mixtures observed in samples before heating to high temperatures $(X_c=10\mathrm{mol}\%)$. The $q$ scales are approximate and are intended only as guides.

Figure 9

The phase diagram of DLPE-cholesterol mixtures at 98% RH, determined from the diffraction data.

Figure 10

The electron density map of the $P_{\beta }$ phase of DPPC-cholesterol mixtures calculated from the diffraction data given in Table , using the phases obtained with the five $\delta$-function model. The solid (dotted) contours correspond to the electron rich (poor) regions of the bilayer. H, W, and C denote the head group, water, and chain regions of the bilayer. CH denotes the electron-rich band in the bilayer due to the presence of cholesterol. $X_c=15\mathrm{mol}\%$, $T=6°\mathrm{C}$, $\mathrm{RH}=98\%$, $d=66.3\mathrm{\AA }$, and $\lambda =60.7\mathrm{\AA }$.

Figure 11

The transbilayer electron density profile in the $l_o$ phase of (a) DLPE at $30\mathrm{mol}\%$ cholesterol $(T=6°\mathrm{C},$ $\mathrm{RH}=98\%)$ and (b) DPPC at $50\mathrm{mol}\%$ cholesterol $(T=24°\mathrm{C},\mathrm{RH}=98\%)$, calculated from the data given in Table . Peaks near $\pm 20\mathrm{\AA }$ and the trough at the center correspond to the head groups and terminal methyl groups of hydrocarbon chains, respectively. Peaks near $\pm 10\mathrm{\AA }$ are due to the presence of cholesterol.

Figure 12

The wide angle chain reflections of DLPE bilayers at different cholesterol concentrations indicated by the labels on the curves $(T=20°\mathrm{C},\mathrm{RH}=98\%)$.