Lipids & Membranes

8/9/2019 Lipids & Membranes

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Lipids & Membranes

Prepared by LLT

Biochemistry of Metabolism


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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-COO-

Non-polar polar

A 16-C fatty acid with one cis double bond between

C atoms 9-10 may be represented as 16:1 cis (9.


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Some fatty acids and their common names:

14:0 myristic acid; 16:0 palmitic acid; 18:0 stearic acid;

18:1 cis(9 oleic acid

18:2 cis(9,12 linoleic acid

18:3 cis(9,12,15 E-linonenic acid

20:4 cis(5,8,11,14 arachidonic acid

20:5 cis(5,8,11,14,17

eicosapentaenoic acid (an omega-3)

Double bonds in fattyacids usually have the

cis configuration.

Most naturallyoccurring fatty acidshave an even number

of carbon atoms.

C

O

O

1

2

34

EF

K

atty acid ith a cis-(9

double bond


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There is free rotation about C-Cbonds in the fatty acidhydrocarbon, except where there is a double bond.

Each cis double bond causes a kinkin the chain.

Rotation about otherC-Cbonds would permit a morelinear structure than shown, but there would be a kink.

C 1234

EF

fatty acid with a cis-(9

double bond


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Glycerophospholipids

Glycerophospholipids

(phosphoglycerides), are commonconstituents of cellular membranes.

They have a glycerolbackbone.Hydroxyls at C1 & C2 are esterifiedto fatty acids.

C HH

CH2 H

CH2 H

glycerol

An ester formswhen a hydroxylreacts with acarboxylic acid,with loss of H2O.

Formation of an ester:

O O

R'OH + HO-C-R" R'-O-C-R'' + H2O


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Phosphatidate

In phosphatidate:

fatty acids are esterified to hydroxyls on C1 & C2

the C3 hydroxyl is esterified to Pi.

O P O

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

phosphatidate


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In most glycerophospholipids (phosphoglycerides),

Pi is in turn esterified to OH of a polar head group (X):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 P O

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

X

glycerophospholipid


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Phosphatidylcholine, with choline as polar head

group, is an example of a glycerophospholipid.

It is a common membrane lipid.

O P O

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

CH2 CH2 N CH3

CH3

CH3

+

phosphatidylcholine


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Phosphatidylinositol, with inositol as polar head group,is another glycerophospholipid.

In addition to being a membrane lipid,

phosphatidylinositol has roles in cell signaling.

O P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OH

H

H

OHH

OH

H

O

H OH

phosphatidyl-inositol


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polar

non-polar

"kink" due to

double bond

O P O

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

X

glycerophospholipid

Each glycerophospholipidincludes

a polar region:

glycerol, carbonyl of

fatty acids, Pi, & thepolar head group (X)

2 non-polar hydrocarbon

tails of fatty acids (R1, R2).

Such an amphipathic lipid

may be represented as at

right.


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The amino group of sphingosine canform an amide bond with a fatty acid

carboxyl, to yield a ceramide.

Ceramides usually include a polarhead group, esterified to the terminalOH of the sphingosine.

H2CHC

OH

CH

N+ CH

C

CH2

CH3

H

H3

OH

( )12

sphingosine

H2CHC

OH

CH

NH CH

C

CH2

CH3

H

OH

( )12

C

R

O

ceramide

Sphingolipids are derivatives of thelipid sphingosine, which has a long

hydrocarbon tail, and a polar domainthat includes an amino group.


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Sphingomyelin, with a phosphocholine head group, is

similar in size and shape to the glycerophospholipid

phosphatidyl choline.

Sphingomyelin, a

ceramide with a

phosphocholine or

phosphethanolamine

head group, is a

common constituent

of plasma membranes

H2CHC

O

CH

NH CH

C

CH2

CH3

H

OH

( )12

C

R

O

PO O

O

H2C

H2CN

+

CH3

H3C

CH3

Sphi li

phosphocholi

sphi osi

tt ci


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head group that is a complex oligosaccharide, including

the acidic sugar derivative sialic acid.Cerebrosides and gangliosides, collectively calledglycosphingolipids, are commonly found in the outerleaflet of the plasma membrane bilayer, with their sugarchains extending out from the cell surface.

cerebroside withF-galactose head group

H2CHC CH

NH CH

C

CH2

CH3

OH

C

R

O

OH O

H H

H

OHH

OH

CH2OH

HO

H

( )12

A cerebroside is asphingolipid

(ceramide) with amonosaccharide

such as glucose orgalactose as polar

head group.A ganglioside is aceramide with a polar


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liquid crystal crystal

In the liquid crystal state, hydrocarbon chains of

phospholipids are disordered and in constant motion.At lower temperature, a membrane containing a singlephospholipid type undergoes transition to a crystallinestate in which fatty acid tails are fully extended, packingis highly ordered, & van der Waals interactions betweenadjacent chains are maximal.

Kinks in fatty acid chains, due to cis double bonds,interfere with packing in the crystalline state, and lowerthe phase transition temperature.

Membrane fluidity:

The interior of a lipid bilayeris normally highly fluid.


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Cholesterol is largely hydrophobic.But it has one polar group, a hydroxyl, making itamphipathic.

CholesterolHO

Cholesterol, animportant constituentof cell membranes,has a rigid ring

system and a shortbranchedhydrocarbon tail.


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Cholesterolin membrane

Cholesterol inserts into bilayer

membranes with its OH oriented

toward the aqueous phase & its

hydrophobic ring system adjacent tofatty acid chains of phospholipids.

The hydroxyl group of cholesterol

forms hydrogen bonds with polarphospholipid head groups.


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The presence ofcholesterol in a phospholipid

membrane inhibits transition to the crystalline state.

However interaction with the relatively rigidcholesterol decreases the mobility of hydrocarbon tails

of phospholipids.

Phospholipid membranes that include cholesterol have

a fluidity intermediatebetween the liquid crystal and

crystal states.


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Two strategiesby which phase changes of membranelipids are avoided:

Cholesterol is abundant in membranes, such asplasma membranes, that include many lipids with

long-chain saturated fatty acids.In the absence of cholesterol, such membranes wouldcrystallize at physiological temperatures.

The inner mitochondrial membrane lacks cholesterol,

but includes many phospholipids whose fatty acidshave one or more double bonds, which lower themelting point to below physiological temperature.


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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 isassayed by the FRAP technique:

Fluorescence Recovery AfterPhotobleaching.

Lateral obility


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FRAP technique:

A membrane lipid or protein is tagged with afluorescent dye. Proteins may be labeled withfluorescent antibodies.

A laser bleaches (destroys fluorescence of) the dye

in a small region of membrane.

Fluorescence recovers as undamaged label diffusesinto the region.

Generally lipids diffuse faster than proteins.Lateral diffusion of some proteins is constrainedby protein-protein interactions, e.g. formation oflarge complexes or association with the cytoskeleton.


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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 totraverse the hydrophobic core of the membrane.

Flippases catalyze flip-flop in membranes where lipidsynthesis occurs. Otherwise, flip-flop of lipids is rare.

Consistent with low rates of flip-flop, the 2 leaflets of abilayer tend to differ in lipid composition.

Flip Flop


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Lipid rafts:

Biological membranes typically include a mix of lipids.

Sphingolipids in the plasma membrane outer leaflet tendto separate out from glycerophospholipids, & co-localizewith cholesterol in microdomains called lipid rafts.

Lipid rafts are resistant to detergent solubilization,which has facilitated their isolation and characterization.

Close packing of sphingolipids in association with

cholesterol has been attributed to lack of double bondsin sphingolipid hydrocarbon chains.

Glycerophospholipids often include at least one fatty acidthat is kinked, due to having one or more double bonds.


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Caveolae are invaginated lipid raft domains of theplasma membrane.

Caveolin, a protein associated with the cytosolic

leaflet of the plasma membrane in caveolae, interactswith cholesterol.

Electron micrograph of caveolae (on home page of D. Brown)

caveolae

cytosol


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Peripheral proteins are on the membrane surface.

They are water-soluble, with mostly hydrophilic surfaces.

Often peripheral proteins can be dislodged by conditionsthat disrupt ionic & H-bond interactions, e.g., extractionwith solutions containing high concentrations of salts,change of pH, and/or chelators that bind divalent cations.

Membrane

proteins may beclassified as:

peripheral

integral

having alipid anchor

integral

lipid

anchor

peripheral

lipid bilayer

embraneroteins


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Some cytosolic proteins have domains that bind to polarhead groups oflipids that transiently exist in a membrane.

The enzymes that create or degrade these lipids are subjectto signal-mediated regulation, providing a mechanism formodulating affinity of a protein for a membrane surface.

O P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OPO32

H

H

OPO32

H

OH

H

O

H OH

1 6

5

43

2

PIP2

phosphatidylinositol-

4,5-bisphosphate

E.g., pleckstrinhomology (PH)domains bind tophosphorylated

derivatives ofphosphatidylinositol.

Some PH domainsbind PIP2 (PI-4,5-P2).


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Otherpleckstrin homology domains recognize and bindphosphatidylinositol derivatives with Pi esterified at the

3' OH of inositol. E.g., PI-3-P, PI-3,4-P2, PI-3,4,5-P3.

O P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OH

H

H

OHH

OPO32

H

O

H OH

1 6

52

3 4

phosphatidyl-inositol-3-phosphate


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Some proteins bind to membranes via a covalentlyattached lipid anchor, that inserts into the bilayer.

The attached lipid may be a fatty acid such as palmitate

ormyristate.

Palmitate is usually attached via an ester linkage to thethiol of a cysteine residue.

A protein may be released from plasma membrane tocytosol via depalmitoylation, hydrolysis of the ester link.

lipid

anchor

membrane

H3C (CH2)14 C

O

S CH2 CH

C

NH

O

palmitate

cysteineresidue


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An isoprenoid such as a farnesyl residue, is attached tosome proteins via a thioether linkage to a cysteine thiol.

Fatty acid orisoprenoid chains link proteins to thecytosolic surface of the plasma membrane.

Many signal proteinsbind via lipid anchors and/orpleckstrin homology domains to the cytosolic surface ofthe plasma membrane. They often bind in regions of lipidrafts, where they cooperate in signal cascades.

CH CH2CH3C

CH3

CH CH2CCH2

CH3

CH CH2 S ProteinCCH2

CH3

farnesyl residue linked to protein via cysteine S


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Glycosylphosphatidylinositols (GPI) are complex

glycolipids that attach some proteins to the outer surfaceof the plasma membrane.

The linkage is similar to the following, although theoligosaccharide composition may vary:

protein (C-term.) - phosphoethanolamine mannose - mannose -

mannose - N-acetylglucosamine inositol (of PI in membrane)

The protein is tethered some distance out from the

membrane surface by the long oligosaccharide chain.GPI-linked proteins may be released from the outercell surface by phospholipases.


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Hydrophobic domains of detergents substitute forlipids, coating hydrophobic surfaces of integral proteins.

Polar domains of detergents interact with water.If detergents are removed, purified integral proteins tendto aggregate & come out of solution. Their hydrophobicsurfaces associate to minimize contact with water.

Amphipathic

detergents arerequired forsolubilization ofintegral proteins

from membranes.

detergent

solubilization

rotein with

bound detergent

polarnon-polar

membrane


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A membrane-spanning E-helix isthe most common structural motiffound in integral proteins.

membrane

N

C

Integral protein structure

Atomic-resolution structures have been determinedfor a small (but growing) number of integral membraneproteins.

Integral proteins are difficult to crystallize for X-ray

analysis.Because of theirhydrophobictransmembrane domains,detergents must be present

during crystallization.


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In an E-helix, amino acid R-groups protrude out from thehelically coiled polypeptide backbone.

The largely hydrophobic R-groups of a membrane-spanning E-helix contact the hydrophobic membrane core,while the more polar peptide backbone is buried.

Colors: C N O R-group (H atoms not shown).

E-helix

R-groups in magenta


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Particularamino acids tend to occur at different

positions relative to the surface or interior of the bilayer

in transmembrane segments of integral proteins.

Residues with aliphatic side-chains (leucine, isoleucine,

alanine, valine) predominate in the middle of the bilayer.

H3N+ C COO

CH3

H

H3N+ C COO

CH CH3

CH2

CH3

H

H3N+ C COO

CH2

CH CH3

CH3

H

H3N+ C COO

CH CH3

CH3

H

alanine (Ala, A) isoleucine (Ile, I) leucine (Leu, L) valine (Val, V)

aminoacids:non-polaraliphatic R-groups


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It has been suggested that the polar character of thetryptophan amide group and the tyrosine hydroxyl, alongwith their hydrophobic ring structures, suit them forlocalization at the polar/apolar interface.

tryptophan tyrosine

H2N C C

CH2

HN

H

H N+

C C

CH2

H

H

Tyrosine and

tryptophan arecommon near themembrane surface.


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Cytochrome oxidase is an integral protein whose intra-membrane domains are mainly transmembrane E-helices.

Explore with Chime the E-helix colored green at far left.

membrane

Cytochrome oxidase dimer (PDB file 1 CC)


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If a hydropathy plot indicates one 20-amino acidhydrophobic stretch (1 putative transmembrane E-helix),topology studies are expected to confirm location of

N & C termini on opposite sides of membrane. Iftwo transmembrane E-helices are predicted, N & C

termini should be on the same side. The segmentbetween the E-helices should be on the other side.

N

membrane

N


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An E-helix lining a water-filled channel might have

polar amino acid R-groups facing the lumen, & non-polarR-groups facing lipids or other hydrophobic E-helices.

Such mixed polarity would prevent detection by ahydropathy plot.

A helicalwheel looksdown the axisof an E-helix,

projecting side-chains onto aplane.

Simpli ied helical heel diagram o ourE-helices lining the lumen o an ion channel.

Polar amino acid -group

on-polar amino acid -group


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Porin F-barrel

While transmembrane E-helices are the most commonstructural motif for integralproteins, a family of bacterialouter envelope channelproteins called porins haveinsteadF barrel structures.

AFbarrel is a F sheet rolledup to form a cylindrical pore.

At right is shown one channelof a trimeric porin complex.

Porin onomer

PDB 1A S


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In aF-sheet, amino acid R-groups alternately point above& below the sheet.

Much of porin primary structure consists ofalternating

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.

!polar R group, !non-polar R group

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