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    BIOLOGICAL MODELING | Contents | Next

    Determining Why Nature Needs Cholesterol

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    Alexander Smondyrev
    Max Berkowitz
    University of North Carolina

    T he public has heard much about the adverse effects of too much cholesterol, but cholesterol and related molecules are required for normal function in all mammalian cells. But what does cholesterol look like, and how does it bind to cells? Prior to molecular simulations created by Alexander Smondyrev and Max Berkowitz of the University of North Carolina, little work had been done to discern, computationally, what role cholesterol plays in cellular membranes at the molecular level. Computer simulations instead had concentrated on pure membranes with no other interfering molecules. Smondyrev and Berkowitz refined their simulations to include a more complex--and more realistic--computational model with various forms of cholesterol embedded into membranes. This research is the first step toward determining why nature went to such lengths to produce cholesterol.

    Sterol molecules, such as cholesterol, play a significant role in biological membranes. The cholesterol molecule has a polar head, a rigid ring structure, and a hydrocarbon tail. Previous experimental studies found that cholesterol regulates membrane fluidity and produces a condensing effect on membranes, decreasing their permeability and making them more rigid.

    "We started with atom force fields for pure membranes--without sterols of any kind--and then we did simulations where membranes were composed of phospholipids and cholesterol membranes," said Smondyrev, a postdoctoral research associate in Berkowitz's group in the Department of Chemistry at the University of North Carolina at Chapel Hill. Phospholipids are the most prevalent type of the three classes of fatty molecules found in membranes, collectively called lipids. The other two classes are sterols and and glycolipids.

    "Prior to our studies most of the work was on one-component membranes," Smondyrev said. "We looked at sterols because they are required for normal function in all mammalian cells." It is believed that the observed effects of cholesterol arise from the rigidity of the ring system, which restricts the motion of lipid chains next to it. To understand the effects of cholesterol, Smondyrev and Berkowitz studied the dipalmitoylphosphatidylcholine (DPPC) lipid bilayer with low and high cholesterol content. The data from these simulations were compared to data from simulations on a pure DPPC membrane. They found that the structural properties of membranes change dramatically with increasing cholesterol concentrations, which is consistent with experimental results and theoretical predictions.

    Currently, Smondyrev and Berkowitz are studying dimyristoylphosphatidylcholine (DMPC) membranes with cholesterol, lanosterol, and ergosterol. The goals of these simulations are to determine the effects of bilayer thickness--DMPC has 14 carbons per chain compared to 16 in DPPC--on cholesterol packing and to study how changes in sterol structure affect membrane properties. Although chemical structures of cholesterol and lanosterol are very similar, the pathway from lanosterol to cholesterol in biological systems contains several intermediate sterol derivatives. By comparing the properties of membranes with cholesterol, lanosterol, and ergosterol, Smondyrev and Berkowitz are trying to determine why nature went to such lengths to produce cholesterol.

    CHOLESTEROL IN THE MEMBRANE

    MOLECULAR DYNAMICS SIMULATIONS

    REFINING AMBER

    REFERENCES

    Smondyrev, A.M.,
    and M.L. Berkowitz.
    1999. United atom
    force field for phospholipid
    membranes: Constant
    pressure molecular
    dynamics simulation of dipalmitoylphosphati-
    dylcholine/water system.
    Journal of Computational
    Chemistry
    20(5): 531–545.

    Smondyrev, A,M.,
    and M.L. Berkowitz.
    1999. Structure of dipalmitoylphosphatidyl-
    choline/cholesterol bilayer at low and high cholesterol concentrations: Molecular dynamics simulation. Biophysical Journal
    77: 2075–2089.

    Smondyrev, A.M., and M.L. Berkowitz. 1999. Molecular dynamics simulation of dipalmitoylphosphatidylcholine membrane with cholesterol sulfate. Biophysical Journal (submitted).


    The headgroups of DPPC-cholesterol bilayer Lipid bilayers with low cholesterol content
    Figure 1. The headgroups of DPPC-cholesterol bilayer
    In a membrane, the headgroups of DPPC molecules cover the molecules of cholesterol. Cholesterol carbon atoms are shown in green. Among DPPC atoms, phosphate is shown in orange and phosphate group oxygens are in red.
    Figure 3. Lipid bilayers with low cholesterol content
    In a side view of a lipid bilayer with low cholesterol content, the tails of DPPC molecules are disordered. Cholesterol molecules are shown in green, water as blue wires, and DPPC atoms as balls and black wires for the hydrocarbon chains. At high sterol content, the DPPC tails are more rigid.

    CHOLESTEROL IN THE MEMBRANE

    The initial placement of cholesterol molecules in lipid membranes presented a challenge to the researchers. Because they were not able to observe diffusion in time periods long enough for two molecules in a lipid membrane to exchange their positions, they needed to start with a guess about the structure of the membrane with cholesterol. Previous studies suggested two possible packing structures for equal mixtures of DMPC and cholesterol. One favors "like-like" interactions such as lipid-lipid and cholesterol-cholesterol, and another "like-unlike," such as lipid-cholesterol. Both predicted patterns have similar energies. The researchers performed two simulations with equal mixtures of DPPC and cholesterol bilayers, assuming both like-like (Figures 1 and 2) and like-unlike packing. Although certain differences in membrane structure were observed, it was clear that longer simulations were needed to test this hypothesis directly.

    Of the various simulations performed by Smondyrev and Berkowitz, probably the most compelling was the molecular dynamics simulation of a DPPC membrane with another sterol, cholesterol sulfate. A paper describing the details of this simulation will be published later this year in Biophysical Journal.

    Although the structures of cholesterol and cholesterol sulfate are similar, they have different functions in biological systems.While cholesterol regulates membrane fluidity, membrane permeability, and lateral mobility of proteins, cholesterol sulfate acts as a membrane stabilizer. This difference may be due in part to different polar headgroups of the two molecules. The headgroup of cholesterol is a small, polar hydroxyl group, while cholesterol sulfate has a large, charged, and hydrated polar headgroup. Cholesterol sulfate is found in spermatozoon plasma membrane, stratum corneum (the outermost layer of the skin), and the membranes of red blood cells.

    For example, past research shows that cholesterol sulfate helps protect the membranes of red blood cells from destruction by lysins--antibodies that disintegrate red blood cells or bacteria cells. It is also believed that cholesterol sulfate has a noticeable effect on fertilization efficiency. While cholesterol sulfate accounts for only 2% of the total sterol in the human sperm, its concentration in the membranes overlying the acrosome--the head area that contains enzymes to digest the egg cell coating--is as much as 20%. It is likely that cholesterol sulfate contributes to membrane stability in this region.

    Smondyrev and Berkowitz studied the changes occurring in a DPPC-cholesterol membrane at a high sterol content. In their previous studies, they studied low amounts versus high amounts of cholesterol, with results matching experimental data. Now, they replaced cholesterol with cholesterol sulfate. Again, the results agreed with data from experiments.

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    Lipid Bilayers with Low Cholesterol Content
    Figure 3 - Lipid Bilayers with Low Cholesterol Content
    In a side view of a pilip bilayer with low cholesterol content, the tails of the DPPC molecules are disordered. Cholesterol molecules are shown in green, water as blue wires, and DPPC atoms as balls and black wires for the hydrocarbon chains. At high sterol content, the DPPC tails are more rigid.

    MOLECULAR DYNAMICS SIMULATIONS

    Much of the study on sterol-containing membranes was done on the Cray T3E at SDSC and the Cray T3E at the Texas Advanced Computing Center at the University of Texas at Austin, an NPACI resource partner. Calculations were also performed on a Cray T3E at the North Carolina Supercomputer Center.

    Smondyrev and Berkowitz applied molecular dynamics simulation techniques to study the structural and dynamic properties of the membranes. This technique was a well-established tool in the study of chemical dynamics, electronic structure, structure of condensed matter, and structure and dynamics of such biomolecules as protein and DNA. In studying membranes, this technique allowed them to explore some of the characteristics, such as permeability or surface potentials, that are hard to obtain using other methods. They used the parallel molecular dynamics simulation package DL_POLY, which was developed at Daresbury Laboratory by W. Smith and T.R. Forester. It performs molecular dynamics in many different ensembles, using a wide variety of algorithms for time-stepping, electrostatic models, periodic images, and rigid molecular entities. The DL_POLY force field includes a range of features. The parameterization can be obtained, for example, from AMBER or GROMOS force fields. It is also easy to adapt DL_POLY to user-specific force fields.

    To reduce the computational intensity of the problem, they used the united atom model. In this model, CH2 and CH3 groups are treated as a single, "united" atom. This model is especially useful in problems concerned with the modeling of very large systems, such as proteins in lipid membranes. Force fields for united atom models are included in such packages as AMBER and GROMOS, but these were originally designed to describe proteins and nucleic acids. AMBER had to be modified to simulate sterols in membranes.

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    REFINING AMBER

    Smondyrev and Berkowitz refined the united atom AMBER force field for the simulation of phospholipid membranes. This was done by obtaining an accurate set of torsional parameters corresponding to a set of atomic partial charges calculated for DPPC and DMPC molecules. Bond and angle parameters were adopted from the AMBER force field, while torsional parameters were fitted to reproduce geometry and energy profiles obtained from ab initio calculations of model compounds. The Ryckaert-Bellemans potential was employed to describe the torsion potentials for the united atoms in lipid chains.

    To test the force field, Smondyrev and Berkowitz performed a constant pressure simulation on a DPPC bilayer in water (Figure 3). The average area per headgroup, peak-to-peak distance in the electron density, and order parameter profile for the hydrocarbon chains agreed with the experimental values and with the results of a simulation using a model with explicit hydrogens.

    "Typical cell membranes contain mixtures of phospholipid molecules with proteins and sterols," Smondyrev said. "The long-term goal of our work is to develop more realistic models of biological membranes. Then we can study mechanisms by which cholesterol influences the properties of proteins and channels in membranes. Eventually, we may shed some light not only on how cholesterol works in the membranes of the human body, but why such a potentially hazardous compound is there in the first place."
    --AV *

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