Molecular Biochemistry I

Lipids and Membrane Structure

Contents of this page:
Fatty acids
Glycerophospholipids
Sphingolipids
Bilayer membranes & membrane fluidity
Cholesterol
Lateral mobility & flip flop
Lipid rafts & caveolae
Membrane proteins
Integral protein structure

Lipids are non-polar (hydrophobic) compounds, soluble in organic solvents.

Most membrane lipids are amphipathic, having a non-polar end and a polar end.

There is free rotation about C-C bonds in the fatty acid hydrocarbon, except where there is a double bond. Each cis double bond causes a kink in the chain, as emphasized in the diagram above. Rotation about other C-C bonds would permit a more linear structure than is shown, but there would be a kink.

Phosphatidylinositol, with inositol as polar head group, is one glycerophospholipid. In addition to being a membrane lipid, phosphatidylinositol has roles in cell signaling, to be discussed later.

Phosphatidylcholine, with choline as polar head group, is another glycerophospholipid. It is a common membrane lipid.

Sphingomyelin has a phosphocholine or phosphoethanolamine head group. Sphingomyelins are common constituents of plasma membranes. Sphingomyelin, with a phosphocholine head group, is comparable in size and shape to the glycerophospholipid phosphatidyl choline. (Figs 12-4 & 12-6, p. 386, 387).

A cerebroside is a sphingolipid (ceramide) with a monosaccharide such as glucose or galactose as polar head group. A ganglioside is a ceramide with a polar head group that is a complex oligosaccharide, including the acidic sugar derivative sialic acid. See p. 388. Cerebrosides and gangliosides, which are collectively called glycosphingolipids, are commonly found in the outer leaflet of the plasma membrane bilayer, with their sugar chains extending out from the cell surface.

Amphipathic lipids in association with water form complexes in which their polar regions are in contact with water and their hydrophobic regions are away from water. Depending on the lipid, possible molecular arrangements include (p. 390-391):

Cholesterol, an important constituent of cell membranes, has a rigid ring system and a short branched hydrocarbon tail. Cholesterol is largely hydrophobic. But it has one polar group, a hydroxyl, making it amphipathic. See also p. 388-389.

Cholesterol viewed by Chime, in sticks & spacefill displays.

Cholesterol synthesis and transport are discussed elsewhere. Cholesterol inserts into bilayer membranes with its hydroxyl group oriented toward the aqueous phase and its hydrophobic ring system adjacent to fatty acid tails of phospholipids. The hydroxyl group of cholesterol forms hydrogen bonds with polar phospholipid head groups.

Interaction with the relatively rigid cholesterol decreases the mobility of hydrocarbon tails of phospholipids. But the presence of cholesterol in a phospholipid membrane interferes with close packing of fatty acid tails in the crystal state, and thus inhibits transition to the crystalline state. Phospholipid membranes with a high concentration of cholesterol have a fluidity intermediate between the liquid crystal and crystal states.

Two strategies by which phase changes of membrane lipids are avoided: Cholesterol is abundant in membranes, such as plasma membranes, that include many lipids with long-chain saturated fatty acids. In the absence of cholesterol, such membranes would crystallize at physiological temperatures. The inner mitochondrial membrane lacks cholesterol, but includes many phospholipids whose fatty acids have one or more double bonds, which lower the melting point to below physiological temperature.

Lateral mobility of a lipid, within the plane of a membrane, is depicted at right and in the animation provided.  High speed tracking of individual lipid molecules has shown that lateral movements are constrained within small membrane domains. Hopping from one domain to another occurs less frequently than rapid movements within a domain. The apparent constraints on lateral movements of lipids (and proteins) has been attributed to integral membrane proteins, anchored  to the cytoskeleton, functioning as a picket fence. For more information, see slide shows in a website of the Kusumi laboratory.

of lipid lateral diffusion

Flip-flop of lipids (from one half of a bilayer to the other) is normally very slow. Flip-flop would require the polar head-group of a lipid to traverse the hydrophobic core of the membrane. The two leaflets of a bilayer membrane tend to differ in their lipid composition (see p. 406). Flippases catalyze flip-flop in membranes where lipid synthesis occurs, and some membranes contain enzymes that actively transport particular lipids from one monolayer to the other.

of lipid flip-flop

Membrane Proteins may be classified as peripheral, integral, or having a lipid anchor. Peripheral proteins are on the membrane surface. They are water-soluble, with mostly hydrophilic surfaces. Often peripheral proteins can be dislodged from membranes by conditions that disrupt ionic and H-bond interactions, e.g., extraction with solutions containing high concentrations of salts, change of pH, and/or agents (chelators) that bind divalent cations.

Many proteins have a modular design, with different segments of the primary structure folding into domains with different functions.

Some cytosolic proteins have domains that bind to polar head groups of lipids that transiently exist in a membrane. The enzymes that create or degrade these lipids are subject to signal-mediated regulation, providing a mechanism for modulating affinity of a protein for a membrane surface. For example, pleckstrin homology (PH) domains bind to phosphorylated derivatives of phosphatidylinositol (PI). Some pleckstrin homology domains bind to PIP2 (PI-4,5-P2), shown at right.

Other pleckstrin homology domains recognize and bind to phosphatidylinositol derivatives with Pi esterified at the 3' hydroxyl of inositol. Examples include PI-3-P (at right), PI-3,4-P2, and PI-3,4,5-P3.

Lipid anchor: Some proteins bind to membranes via a covalently attached lipid anchor, that inserts into the bilayer (see p. 402-404). A protein may link to the cytosolic surface of the plasma membrane via a covalently attached fatty acid (e.g., palmitate or myristate) or an isoprenoid group. Palmitate is usually attached via an ester linkage to the thiol of a cysteine residue, as shown at right. A protein may be released from the plasma membrane to the cytosol via depalmitoylation, hydrolysis of the ester linkage.

An isoprenoid group, such as a farnesyl residue, is attached to some proteins via a thioether linkage to a cysteine thiol, as shown at right.

Glycosylphosphatidylinositols, abbreviated GPI, are complex glycolipids that attach some proteins to the outer surface of the plasma membrane (see p. 404). The linkage is similar to the following, although the oligosaccharide composition may vary:

protein (C-terminus) - phosphoethanolamine - mannose - mannose - mannose - N-acetylglucosamine - inositol (of membrane-embedded phosphatidylinositol)

The protein is tethered some distance out from the membrane surface by the long oligosaccharide chain. GPI-linked proteins may be released from the outer cell surface by phospholipases.

Integral proteins have domains that extend into the hydrocarbon core of the membrane. Often they span the bilayer. Intramembrane domains have largely hydrophobic surfaces, that interact with membrane lipids. See Fig. 12-18 p. 395.

Amphipathic detergents are required for solubilization of integral proteins. Hydrophobic domains of detergents substitute for lipids in coating hydrophobic protein surfaces. Polar domains of detergents interact with water. If detergents are removed, purified integral proteins tend to aggregate and come out of solution. Their hydrophobic surfaces associate to minimize contact with water.

Lipid rafts: Complex sphingolipids tend to separate out from glycerophospholipids and co-localize with cholesterol in membrane microdomains called lipid rafts. Membrane fragments assumed to be lipid rafts are found to be resistant to detergent solubilization, which has facilitated their isolation and characterization. Differences in molecular shape may contribute to the tendency for sphingolipids to separate out from glycerophospholipids in membrane microdomains. Sphingolipids usually lack double bonds in their fatty acid chains. In contrast, glycerophospholipids often include at least one fatty acid that is kinked, due to one or more double bonds. See an online diagram from an article by J. Santini & coworkers. Hydrogen bonding between the hydroxyl group of cholesterol and the amide group of sphingomyelin may in part account for the observed affinity of cholesterol for sphingomyelin in raft domains. Lipid raft domains tend to be thicker than adjacent membrane areas, in part because the saturated hydrocarbon chains of sphingolipids are more extended. Proteins involved in cell signaling often associate with lipid raft domains. Otherwise soluble signal proteins often assemble in complexes at the cytosolic surface of the plasma membrane in part via insertion of attached fatty acyl or isoprenoid lipid anchors into raft domains.  Integral proteins may concentrate in raft domains via interactions with raft lipids or with other raft proteins. Some raft domains contain derivatives of phosphatidylinositol that bind signal proteins with pleckstrin homology domains. Caveolae are invaginated lipid raft domains of the plasma membrane that have roles in cell signaling and membrane internalization. Caveolin is a protein associated with the cytosolic leaflet of the plasma membrane in caveolae. Caveolin interacts with cholesterol and self-associates as oligomers that may contribute to deforming the membrane to create the unique morphology of caveolae. Additional websites of interest: Electron micrograph and information about caveolae (home page of Deborah Brown at SUNY Stony Brook). Diagram and information about lipid rafts (website maintained by the Maciver lab at University of Edinburgh).

Integral protein structure Atomic-resolution structures have been determined for only a small number of integral membrane proteins. Integral proteins are difficult to crystallize for X-ray analysis. Because of their hydrophobic transmembrane domains, detergents must be present during crystallization. A membrane-spanning a-helix is the most common structural motif found in integral proteins.

In an a-helix, amino acid R-groups protrude out from the helically coiled polypeptide backbone. The largely hydrophobic R-groups of a membrane-spanning a-helix contact the hydrophobic membrane core, while the more polar peptide backbone is buried. In the images at right, H atoms are not visible. Colors: C  N  O  R-group

Particular amino acids tend to occur at different positions relative to the surface or interior of the bilayer in transmembrane segments of integral proteins has shown that . Residues with aliphatic side-chains (leucine, isoleucine, alanine, valine) predominate in the middle of the bilayer.

Tyrosine and tryptophan are common near the membrane surface. It has been suggested that the polar character of the tryptophan amide group and the tyrosine hydroxyl, along with their hydrophobic ring structures, suit them for localization at the polar/apolar interface. Lysine and arginine are often at the lipid/water interface, with the positively charged groups at the ends of their aliphatic side chains extending toward the polar membrane surface.

Cytochrome oxidase is an example of an integral protein whose intramembrane domains consist mainly of transmembrane a-helices. For another example, see notes on the protein rhodopsin. Explore below the transmembrane a-helix colored green at the far left in this ribbon display of cytochrome oxidase.

Display as sticks. Rotate to view the a-helix from the side & down its axis.   Identify the polar backbone atoms using CPK color. To distinguish the amino acid side-chains (R groups), select protein, sidechain and then change the display, e.g., to ball & stick. What part of such a transmembrane a-helix would mainly contact the lipid core of a membrane?  Identify each amino acid. Examples of some amino acid structures are shown below. Questions to be answered: 1. What is the location of the partly polar/partly hydrophobic residues tyrosine and tryptophan? 2. What is the location and orientation of a lysine residue and it's side-chain amino group? What is the significance of this location and orientation? 3. What types of amino acids are in the part of the transmembrane a-helix corresponding to the middle of the membrane? Why might this be? A few amino acid structures:

C   O   N   S

Hydropathy plots: A 20-amino acid a-helix just spans a lipid bilayer.  Hydropathy plots are used to search for 20-amino acid stretches of hydrophobic residues in the primary sequence of a protein for which a crystal structure is not available (Fig. 12-22 p. 397). Putative hydrophobic transmembrane a-helices have been identified this way in many membrane proteins. It should be emphasized that hydropathy plots alone are not conclusive. Protein topology studies are used to test the transmembrane distribution of protein domains predicted by hydropathy plots. E.g., if a hydropathy plot indicates one 20-amino acid hydrophobic stretch (one putative transmembrane a-helix), topology studies are expected to confirm location of N and C termini on opposite sides of the membrane. If two transmembrane a-helices are predicted, N and C termini should be on the same side of the membrane. The segment between the transmembrane a-helices should be on the other side of the membrane.

Transmembrane topology is tested with impermeant probes, added on one side of a membrane. For example:

• Protease enzymes. Degradation of a protein segment would indicate exposure to the aqueous phase on the side of the membrane to which the protease was added.
• Monoclonal antibodies raised to peptides equivalent to individual segments of the protein. Binding would indicate surface exposure of that protein segment on the side to which Ab was added.

Transmembrane topology studies have shown that all copies of a given type of integral protein have the same orientation relative to the bilayer membrane. Flip-flop of integral proteins does not occur.

Do a hydropathy plot (studio exercise).

A helical wheel diagram looks down the axis of an a helix, projecting amino acid side-chains onto a plane (see Fig. 8-43 p. 247). Transmembrane a-helices that line a water-filled channel might have polar amino acid R-groups (side-chains) facing the lumen, and non-polar amino acid R-groups facing lipids or other hydrophobic a-helices. Such mixed polarity would prevent detection by a hydropathy plot.

While transmembrane a-helices are the most common structural motif for integral proteins, a family of bacterial outer envelope channel proteins called porins have instead b barrel structures. A b barrel is a b-sheet rolled up to form a cylindrical pore. At right is shown one channel of a trimeric porin complex.

In a b-sheet, amino acid R-groups alternately point above and below the sheet. See simplified cartoon at right and diagram p. 228. Much of the primary structure of a porin consists of alternating polar and non-polar amino acids. Polar residues face the aqueous lumen. Non-polar residues are in contact with membrane lipids.

Explore below a sucrose-selective porin from Salmonella typhimurium. (PDB 1AOS, structure solved by D. Forst, W. Welte, T. Wacker & K. Diederichs in 1998.)

Copyright © 1998-2007by Joyce J. Diwan. All rights reserved.

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