Filipin

ABSTRACT Experimental evidences have indicated that cholesterol may adapt highly regular lateral distributions (i.e., superlattices) in a phospholipid bilayer. We investigated the formations of superlattices at cholesterol mole fraction of 0.154, 0.25, 0.40, and 0.5 using Monte Carlo simulation. We found that in general, conventional pairwise-additive interactions cannot produce superlattices. Instead, a multibody (nonpairwise) interaction is required. Cholesterol superlattice formation reveals that although the overall interaction between cholesterol and phospholipids is favorable, it contains two large opposing components: an interaction favoring cholesterol-phospholipid mixing and an unfavorable acyl chain multibody interaction that increases nonlinearly with the number of cholesterol contacts. The magnitudes of interactions are in the order of kT. The physical origins of these interactions can be explained by our umbrella model. They most likely come from the requirement for polar phospholipid headgroups to cover the nonpolar cholesterol to avoid the exposure of cholesterol to water and from the sharp decreasing of acyl chain conformation entropy due to cholesterol contact. This study together with our previous work demonstrate that the driving force of cholesterol-phospholipid mixing is a hydrophobic interaction, and multibody interactions dominate others over a wide range of cholesterol concentration.

INTRODUCTION

Cholesterol is a major constituent of the mammalian plasma membranes. The molecular interactions between cholesterol and other lipid molecules have been the subjects of many studies (Finegold, 1993). We seek a general picture of cholesterol-phospholipids interaction, which can capture the key molecular interactions, and would allow us to understand a wide range of experimental results or even to predict new phenomena. In this study, we focus on the molecular interactions, which produce an interesting phenomenon in cholesterol containing lipid membranes: cholesterol superlattices.

Regular distribution of certain molecules in a lipid bilayer was initially proposed based on the observation of a series of "kinks" or "dips" in the ratio of excimer-to-monomer fluorescence of pyrene-phosphatidylcholine at some particular mole fractions (Somerharju et al., 1985; Tang and Chong, 1992; Chong et al., 1994). The bulky pyrene moieties were thought to form hexagonal superlattices to maximize separation from each other. Later, fluorescence data on cholesterol/phospholipid mixtures indicated that cholesterol molecules might also form superlattices in lipid bilayers (Chong, 1994; Virtanen et al., 1995; Liu et al., 1997). Recently, it has been suggested that lipid headgroups may also adopt superlattice distribution in phosphatidylethanolamtine/phosphatidylcholine (PE/PC) bilayers (Cheng et al., 1997, 1999).

Numerous superlattice patterns have been suggested based on geometrical symmetry arguments, such as at cholesterol mole fraction (chi^sub chol^) of 0.118, 0.154, 0.20, 0.25, 0.33, 0.40, and 0.5. Sugar et al. (1994) explored the formation of superlattice patterns using Monte Carlo simulations and introduced a long-range pairwise-additive repulsive interaction. They found that the long-range pairwise-additive repulsion can generate a superlattice pattern at chi^sub chol^ = 0.5 but cannot produce large-scale superlattices of any other compositions. Thus, some key issues about the superlattices remain unsolved: How could such crystal-like structures exist in a bilayer without rigid chemical bonds between molecules? What kinds of molecular interactions are generally required to produce cholesterol superlattices? What are the magnitudes of the interaction energies? What are the physical origins of these interactions?

Recently, using x-ray diffraction and novel sample preparation procedures, we have measured the solubility limits of cholesterol in several different phospholipid bilayers (Huang et al., 1999). Interestingly, these solubility limits occur at cholesterol concentrations that correspond to welldefined cholesterol/phospholipid mole ratios: 1/1 for PE bilayers and 2/1 for PC bilayers.

We have developed a model of cholesterol-phospholipid interaction, which explains these discrete solubility limits. Our Monte Carlo simulations showed that pairwise-additive interaction was inadequate. Instead, the data can only be explained if cholesterol molecules take part in certain type of multibody interactions. The unfavorable cholesterol-cholesterol multibody interaction can be explained by our "umbrella model": in a bilayer environment, nonpolar cholesterols must be covered by neighboring polar phospholipid headgroups to avoid the unfavorable free energy of exposing cholesterol to water. In a lattice model, this requirement can only be expressed in terms of multibody (or nonpair-- wise) interactions. At high cholesterol concentrations, this multibody interaction dominates all others. Thus, only those phospholipid/cholesterol lateral distributions, which meet this coverage requirement, would be allowed. As the concentration of cholesterol increases, fewer and fewer lateral distributions become possible. Near the solubility limit, cholesterol and phospholipid molecules can only adapt some highly regular lateral distributions (i.e., superlattices). The solubility limit is reached when surrounding phospholipid headgroups can no longer completely cover any more cholesterol: the chemical potential of cholesterol jumps steeply, which leads to cholesterol crystal precipitation. Our model predicted that depending on the ability of phospholipid headgroups covering the neighboring cholesterol, cholesterol precipitation is most likely to occur near three discrete values of cholesterol mole fraction, 0.50, 0.57, and 0.67, which correspond to cholesterol/phospholipid mole ratios of 1/1, 4/3, and 2/1, respectively. Thus, the hydrophobic interaction has been implicated as the key driving force in the lateral organization of cholesterol in biomembranes (Huang and Feigenson, 1999).

The author thanks Dr. Gerald W. Feigenson for many productive discussions. This work was supported by the National Science Foundation Grant MCB-9722818 and the Petroleum Research Fund Grant PRF-34872-AC7.

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Juyang Huang

Department of Physics, Texas Tech University, Lubbock, Texas 79409 USA

Submitted January 22, 2002, and accepted for publication April 3, 2002.

Address reprint requests to Juyang Huang, Department of Physics, Texas Tech University, Box 41051 Lubbock, TX 79409. Tel: 806-742-4780; Fax: 806-742-1182; E-mail: juhuang@ttacs.ttu.edu.

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