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Biochemistry of Metabolism

Lipids & Membranes

Copyright 1999-2003 by Joyce J. Diwan. All rights reserved.

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


Most membrane lipids are amphipathic, having a non-polar end and a polar end. Fatty acids consist of a hydrocarbon chain with a carboxylic acid at one end. A 16-C fatty acid: CH3(CH2)14-COONon-polar polar

Double bonds in fatty acids are usually have the cis configuration. Most naturally occurring fatty acids have an even number of carbon atoms.

A 16-C fatty acid with a 3 1 O one cis double bond 4 2 between C atoms 9-10 may be represented as fatty acid with a cis-D9 16:1 cis D9. double bond Some fatty acids: 14:0 myristic acid 16:0 palmitic acid 18:0 stearic acid 18:1 cisD9 oleic acid 18:2 cisD9,12 linoleic acid 18:3 cisD9,12,15 a-linonenic acid 20:4 cisD5,8,11,14 arachidonic acid 20:5 cisD5,8,11,14,17 eicosapentaenoic acid (an omega-3)

O C

a 2

O C
1

fatty acid with a cis-D9 double bond

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. Rotation about other C-C bonds would permit a more linear structure overall than is shown, but there would be a kink.

Glycerophospholipids
Glycerophospholipids (phosphoglycerides), are common constituents of cellular membranes. They have a glycerol backbone.
CH2OH H C OH

Hydroxyls at C1 & C2 are esterified to fatty acids.


An ester forms when a hydroxyl reacts with a carboxylic acid, with loss of H2O.
Formation of an ester:
O R'OH + HO-C-R"

CH2OH

glycerol

O R'-O-C-R'' + H2O

Phosphatidate
O O R1 C O H2C CH H2C O O C O P O O R2

phosphatidate

In phosphatidate:
fatty acids are esterified to hydroxyls on C1 & C2 the C3 hydroxyl is esterified to Pi.

O O R1 C O H2C CH H2C O O C O P O O X R2

glycerophospholipid

In most glycerophospholipids (phosphoglycerides), Pi is in turn esterified to OH of a polar head group (X), e.g.: serine, choline, ethanolamine, glycerol, or inositol. The 2 fatty acids tend to be non-identical. They may differ in length and/or the presence/absence of double bonds.

O O R1 C O H2C CH H2C O O C O P O O CH2 CH2 R2 CH3 + N CH3 CH3

phosphatidylcholine

Phosphatidylcholine, with choline as polar head group, is an example of a glycerophospholipid.


It is a common membrane lipid.

O O R1 C O H2 C CH H2 C O O C O P O OH H OH OH H H H OH O H OH R2

phosphatidylinositol

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

Each glycerophospholipid includes a polar region: glycerol, carbonyl of fatty acids, Pi, & the polar head group (X) 2 non-polar hydrocarbon tails of fatty acids (R1, R2). Such an amphipathic lipid may be represented as at right.

O O R1 C O H2C CH H2C O O C O P O O X R2

glycerophospholipid

polar "kink" due to double bond

non-polar

OH

OH H C CH CH HC (CH2 )12

Sphingolipids are derivatives of the


lipid sphingosine, which has a long hydrocarbon tail, and a polar domain that includes an amino group.
OH H2C H C NH O C R OH CH CH HC (CH2 )12 CH3

H2C

H3N+

sphingosine

CH3

ceramide

The amino group of sphingosine can form an amide bond with a fatty acid carboxyl to yield a ceramide. Ceramides usually include a polar head group, esterified to the terminal OH of the sphingosine.

CH3 H3C N
+

O H2 C H2 C O P O H2C H C NH C R

O
OH CH CH HC (CH2 )12 CH3

CH3

Sphingomyelin, a ceramide with a phosphocholine or phosphethanolamine head group, is a common constituent of plasma membranes

phosphocholine

sphingosine
O

fatty acid

Sphingomyelin

Sphingomyelin, with a phosphocholine head group, is similar in size and shape to the glycerophospholipid phosphatidyl choline.

Some sphingolipids (ceramides):

Sphingolipid sphingomyelin cerebroside ganglioside

Polar head group phosphocholine or phosphoethanolamine a monosaccharide such as glucose or galactose a complex oligosaccharide, including the acidic sugar sialic acid

Cerebrosides and gangliosides are collectively called glycosphingolipids. They are commonly found in the outer leaflet of the plasma membrane bilayer, with their sugar chains extending out from the cell surface.

OPO3

OH CH CH HC

In addition to being a precursor for synthesis of sphingolipids, sphingosine and its phosphorylated derivative sphingosine-1-phosphate (S1P) have antagonistic roles as cell signals, in regulation of cell growth.

H2C

CH H3N+

S1P (CH2 )12 sphingosine-1phosphate CH3

Phosphorylation and dephosphorylation of sphingosine is highly regulated. S1P is a ligand for plasma membrane G-protein coupled receptors (GPCRs, to be discussed later).

Amphipathic lipids in association with water form complexes in which polar regions are in contact with water and hydrophobic regions away from water.

Bilayer

Spherical Micelle

Depending on the lipid, possible molecular arrangements:


Various micelle structures. E.g., a spherical micelle is a stable configuration for amphipathic lipids with a conical shape, such as fatty acids. A bilayer. This is the most stable configuration for amphipathic lipids with a cylindrical shape, such as phospholipids.

Membrane fluidity:
The interior of a lipid bilayer is normally highly fluid.
liquid crystal crystal

In the liquid crystal state, hydrocarbon chains of phospholipids are disordered and in constant motion. At low temperature, phospholipids may undergo transition to a crystalline state in which fatty acid tails are fully extended, packing is highly ordered, & van der Waals interactions between adjacent chains are maximal. Kinks in fatty acid chains, due to cis double bonds, interfere with packing of lipids in the crystalline state. Double bonds inhibit transition to the crystalline state, & lower the phase transition temperature.

Cholesterol has a
rigid ring system and a short branched hydrocarbon tail.
HO

Cholesterol

Cholesterol is largely hydrophobic. But it has one polar group, a hydroxyl, making it amphipathic. Cholesterol is an essential constituent of cell membranes. It is also the precursor for synthesis of steroid hormones & vitamin D.

Cholesterol inserts into bilayer membranes with its OH oriented toward the aqueous phase & its hydrophobic ring system adjacent to fatty acid chains of phospholipids.

The hydroxyl group of cholesterol forms hydrogen bonds with polar phospholipid head groups.

Cholesterol in membrane

Incorporation of cholesterol into a phospholipid membrane inhibits transition from liquid crystal to crystal state. However interaction with the relatively rigid cholesterol decreases the mobility of hydrocarbon tails of phospholipids. Phospholipid membranes that include 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. Cholesterol blocks transition to the crystalline state. Some membranes that lack cholesterol, e.g., inner mitochondrial membrane, mainly consist of phospholipids whose fatty acids include one or more double bonds. The double bonds lower the melting point to below physiological temperature.

Lateral Mobility

Lateral mobility of a lipid, within the plane of a membrane, is depicted above and in an animation.
Lateral diffusion of membrane lipids and proteins is assayed by the FRAP technique: Fluorescence Recovery After Photobleaching.

FRAP technique:

A membrane lipid or protein is tagged with a fluorescent dye. Proteins may be labeled with fluorescent antibodies.
A laser bleaches (destroys fluorescence of) label in a region of membrane. Fluorescence recovers as undamaged label diffuses into the region. Generally lipids diffuse faster than proteins, which are larger. Protein diffusion may be constrained by proteinprotein interactions, e.g. association with elements of the cytoskeleton.

Flip Flop

Flip-flop of lipids (from one half of a bilayer to the other) is very slow.
Flip-flop would require the polar head group of a lipid to traverse the hydrophobic core of the membrane. Flippase enzymes catalyze flip-flop in membranes where lipid synthesis occurs. Otherwise, flip-flop of lipids is rare.

Consistent with low rates of flip-flop, the 2 leaflets of a bilayer tend to differ in lipid composition. E.g.:
Sphingolipids are mainly in the outer leaflet of the plasma membrane. Phosphatidylserine & phosphatidylethanolamine are mainly in the cytosolic leaflet of the plasma membrane.

Lipid rafts:
Sphingolipids in the plasma membrane outer leaflet may separate out from glycerophospholipids, and colocalize with cholesterol in membrane microdomains called lipid rafts. Lipid rafts are resistant to detergent solubilization, which has facilitated their isolation & characterization. The close packing of sphingolipids in association with cholesterol has been attributed to the lack of double bonds in sphingolipid hydrocarbon chains. Glycerophospholipids often include at least one fatty acid that is kinked due to one or more double bonds.

Caveolae are invaginated


lipid raft domains of the plasma membrane.
caveolae cytosol

A protein caveolin is associated with the cytosolic leaflet of the plasma membrane in caveolae. Caveolin interacts with cholesterol, and there is evidence for a role of caveolin in transfer of cholesterol into lipid raft domains of the plasma membrane. Signal proteins that attach to the plasma membrane via lipid anchors tend to be concentrated in caveolae. Caveolae may be important sites of assembly of complexes of signal proteins. Electron micrograph of caveolae.

peripheral

Membrane proteins may be classified as: peripheral integral having a lipid anchor

lipid anchor

lipid bilayer

integral

Membrane Proteins

Peripheral proteins are on the membrane surface. They are water-soluble, with mostly hydrophilic surfaces. They may adhere to exposed domains of integral proteins. They often can be dislodged by conditions that disrupt ionic & H-bond interactions, e.g., extraction with solutions containing high concentrations of salts, change of pH, and/or chelators that bind divalent cations.

Some cytosolic proteins include domains that bind to polar head groups of particular lipids that may transiently exist in a membrane. For example, pleckstrin homology domains (PH) bind to phosphorylated derivatives of phosphatidylinositol (PI). Some pleckstrin homology domains bind to PIP2 (PI-4,5-P2).
O O R1 C O H2C CH H2C O O C O P O OH
2 1 6

R2

O H OPO32
5

H OH
3

OH H
4

PIP2 H phosphatidylinositol4,5-bisphosphate

OPO32

O O R1 C O H2C CH H2C O O C O P O O H
1 6

R2

phosphatidylinositol3-phosphate

OH
2

OH
5

OH H OPO32 H
3 4

OH

Other pleckstrin homology domains recognize and bind phosphatidylinositol derivatives with Pi esterified at the 3' OH of inositol. E.g., PI-3-P, PI-3,4-P2, PI-3,4,5-P3.

Some signal proteins include pleckstrin homology domains that mediate binding to the cytosolic surface of the plasma membrane, where they cooperate in signal cascades.

O O R1 C O H2C CH H2C O O C O P O O H
1 6

R2

phosphatidylinositol3-phosphate

OH
2

OH
5

OH H 2 H OPO3
3 4

OH

Kinases (e.g., PI-3 Kinase) that catalyze Pi transfer from ATP to inositol of phosphatidylinositol, e.g., creating ligands for pleckstrin homology domains, are themselves signal-activated.

lipid anchor
H3C (CH2)14

O C S

O CH2

C CH NH

membrane

cysteine residue

palmitate

Some proteins have a covalently attached lipid anchor, that inserts into the bilayer, providing a mechanism for membrane binding. The attached lipid may be a fatty acid such as palmitate or myristate.

O H3C (CH2)14 C S

O CH2

C CH NH

cysteine residue

palmitate

For example, palmitate is usually attached via an ester linkage to the thiol of a cysteine residue. Integral as well as peripheral membrane proteins may be palmitoylated. Depalmitoylation, via hydrolysis of the ester linkage, may be a mechanism for reversing attachment of a cytosolic protein to the plasma membrane.

CH3 H3C C CH CH2 CH2

CH3 C CH CH2

CH3 CH2 C CH CH2 S

Protein

farnesyl residue linked to protein via cysteine S

Some proteins have an attached isoprenoid such as a farnesyl residue, which may attach via a thioether linkage to a cysteine thiol. Fatty acid or isoprenoid chains link proteins to the cytosolic surface of the plasma membrane.
GPI (glycosylphosphatidylinositol) is a complex glycolipid that can mediate reversible attachment of a protein to the outer surface of the plasma membrane. Saturated fatty acid and GPI anchors preferentially insert in lipid raft domains.

peripheral

Integral proteins have domains that extend into the hydrocarbon core of the membrane. Often they span the bilayer.
integral

lipid anchor

lipid bilayer

Membrane Proteins

Intramembrane domains have largely hydrophobic surfaces. A layer of relatively immobilized lipid surrounds the transmembrane portion of an integral protein.

membrane

Amphipathic detergents are required for solubilization of integral proteins from membranes.

detergent solubilization

polar non-polar Protein with bound detergent

Hydrophobic domains of detergents substitute for lipids, coating hydrophobic surfaces of integral proteins. Polar domains of detergents interact with water allowing solubilization. If detergents are removed, purified integral proteins tend to aggregate & come out of solution. Their hydrophobic surfaces come together to minimize contact with water.

Membrane 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.
C

A membrane-spanning a-helix is the most common structural motif found in integral proteins.

membrane

a-helix R-groups in magenta

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

membrane

Cytochrome oxidase dimer

(PDB file 1OCC)

An example of an integral protein whose intra-membrane domains consist mainly of transmembrane a-helices is cytochrome oxidase, shown here in ribbons display.

alanine (Ala, A)
H H3N+ C CH3

isoleucine (Ile, I)
H C COO

leucine (Leu, L)
H H3N+ C CH2 CH CH3 CH3 COO

valine (Val, V)
H H3N+ C COO

COO H3N+

CH CH3 CH2 CH3

CH CH3 CH3

amino acids: non-polar aliphatic R-groups

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

tryptophan
H H2N C CH2 COO

tyrosine
H H3N+ C CH2 COO

Tyrosine and tryptophan are common near the membrane surface.

HN
OH

It has been suggested that the polar character of the amide group of tryptophan and the hydroxyl of tyrosine, in addition to their hydrophobic ring structures, make them ideally suited for localization at the polar/apolar interface.

lysine
H H3N+ C CH2 CH2 COO

arginine
H H3N+ C CH2 CH2 CH2 NH

COO

Lysine & arginine are often located just outside the partly polar aromatics.

CH2 CH2

NH3

C NH2

NH2

The positively charged groups at the ends of their aliphatic side chains typically extend toward the membrane surface where they may interact with polar lipid head groups.

membrane

Cytochrome oxidase dimer

(PDB file 1OCC)

Explore with Chime the a-helix colored green at the far left in this view of cytochrome oxidase.

A 20-amino acid a-helix just spans a lipid bilayer.

Hydropathy plots are used to search for 20-amino


acid stretches of hydrophobic amino acids in the primary sequence of a protein for which a crystal structure is not available.

Putative hydrophobic transmembrane a-helices have been identified this way in many membrane proteins.
Hydropathy plots alone are not conclusive. Protein topology studies test the transmembrane distribution of protein domains predicted by hydropathy plots.

C membrane

E.g., if a hydropathy plot indicates one 20-amino acid hydrophobic segment (1 putative transmembrane a-helix), N & C termini are expected to be on opposite sides of the membrane. If a hydropathy plot predicts that a protein has two transmembrane a-helices, N & C termini should be on the same side. The segment between 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: Protease enzymes (Degradation a protein segment indicates exposure to the aqueous phase on the side of the membrane to which a protease is added.) Monoclonal antibodies raised to peptides equivalent to individual segments of the protein. (Binding indicates surface exposure of a protein segment on the side to which the Ab is added.) Enzymes that attach labels to particular amino acids or sugars (Labeling indicates surface exposure.) Such studies have shown that all copies of a given type of integral protein have the same orientation relative to the membrane. Flip-flop of integral proteins does not occur.

Simplified helical wheel diagram of four a-helices lining the lumen of an ion channel.

A helical wheel looks down the axis of an a-helix, projecting sidechains onto a plane.

Polar amino acid R-group Non-polar amino acid R-group

An a-helix lining a water-filled channel might have polar amino acid R-groups facing the lumen, & non-polar R-groups facing lipids or other hydrophobic a-helices. Such mixed polarity would prevent detection by a hydropathy plot.

Porin -barrel
While transmembrane ahelices are the most common structural motif for integral proteins, a family of bacterial outer envelope channel proteins called porins have instead barrel structures. A barrel is a sheet rolled up to form a cylindrical pore.

Porin Monomer

At right is shown one channel of a trimeric porin complex.

PDB 1AOS

polar R group,

non-polar R group

In a -sheet, amino acid R-groups alternately point above & below the sheet. Much of porin primary structure consists of alternating polar & non-polar amino acids. Polar residues face the aqueous lumen. Non-polar residues are in contact with membrane lipids. Explore an example of a bacterial porin with Chime.