The condensing effect of cholesterol in lipid bilayers

Abstract

The condensing effect of cholesterol on phospholipid bilayers was systematically investigated for saturated and unsaturated chains, as a function of cholesterol concentration. X-ray lamellar diffraction was used to measure the phosphate-to-phosphate distances, PtP, across the bilayers. The measured PtP increases nonlinearly with the cholesterol concentration until it reaches a maximum. With further increase of cholesterol concentration, the PtP remains at the maximum level until the cholesterol content reaches the solubility limit. The data in all cases can be quantitatively explained with a simple model that cholesterol forms complexes with phospholipids in the bilayers. The phospholipid molecules complexed with cholesterol are lengthened and this lengthening effect extends into the uncomplexed phospholipids surrounding the cholesterol complexes. This long-range thickening effect is similar to the effect of gramicidin on the thickness of lipid bilayers due to hydrophobic matching.

FIGURE 1

Diffraction patterns by multilayers of cholesterol/phospholipid mixtures in series of varying cholesterol mol fraction x = C/(C+L). Separate patterns are displaced vertically for clarity. The top of each panel is the pattern of pure cholesterol. The patterns were measured by θ–2θ scan and an attenuator was used below 2θ = 2.5° (the attenuation factor is 10.5). The patterns above x = 0.44 for chol/DMPC, x = 0.47 for chol/SOPC, and x = 0.40 for chol/DOPC all contain pure cholesterol peaks.

FIGURE 2

Diffraction patterns of cholesterol-containing phospholipid bilayers do not show the membrane undulation effect as the hydration approaches 100% RH, contrary to what would happen to pure phospholipid bilayers (23). (Separate patterns are displaced vertically for clarity.)

FIGURE 3

Comparison of chol/DMPC at x = 0.09 and x = 0.17; x = 0.09 exhibits two series of lamellar patterns at 95.1% RH, 30°C and at 85.1% RH, 34°C indicating two-phase coexistence, hence a phase transition. No phase transitions were found in the patterns of x = 0.17 at 30°C and 38°C. (Separate patterns are displaced vertically for clarity.)

FIGURE 4

An example of phasing diagram. Diffraction by a sample was measured over a range of RH so as to obtain the scattering amplitudes at a series of different lamellar spacings. The scattering amplitudes are either positive or negative. The phases are chosen such that the continuous form factor (solid curve) constructed from one set of data at one lamellar spacing goes through all other sets of data (24).

FIGURE 5

Electron density profiles constructed from the diffraction data plotted over one unit cell. Separate profiles are displaced vertically for clarity; z is the coordinate normal to the plane of the bilayer. The origin is chosen at the center of the bilayer. The highest peak on each side is the location of the phosphate group. The phosphate peak-to-phosphate peak distance can be measured very accurately.

FIGURE 6

PtP vs. x and theoretical fit. The error bars in panels A1 and B1 are comparable to the size of the symbols used. (A1) Equation 1 is used to fit (solid line) the data from x = 0 to the first data point that reaches the maximum thickness, defined as x_o. The dotted lines are the first two terms of Eq. 1. The arrows indicate the solubility limits. Panel B1 is the same as A1 except that Eq. 1 is replaced by Eq. 3. (A2) δPtP is the data minus the first two terms of Eq. 1. The solid line is the fit by the third term of Eq. 1. Panel B2 is the same as A2 except that Eq. 1 is replaced by Eq. 3. Inset shows the values of and the fitting parameter γ, and also to compare the goodness of fit.

FIGURE 7

Area per molecule as a function of cholesterol concentration. The averaged cross section area of phospholipid is calculated by where V_c is the chain volume of the lipid (36), and the thickness of the hydrocarbon region is PtP minus twice the length of the glycerol region (from the phosphate to the first methylene of the hydrocarbon chains); the latter is very close to 10 Å (27,33,36). The average area per molecule for the cholesterol-phospholipid mixtures is calculated by The area per cholesterol A_chol is assumed to be constant of x. A value of was taken from monolayer measurements on pure cholesterol (3,37).

FIGURE 8

Electron density profiles of cholesterol-containing phospholipid bilayers are independent of hydration from ∼95% RH to 100% RH, in contrast to pure phospholipids whose profiles vary significantly in this range of RH (23). Separate profiles are displaced vertically for clarity. The dotted lines connecting the peak positions are vertical, indicating no change in the peak positions with hydration.