Chapter 12 TranslationalDiffusionof MembraneProteins 411

Michael Edidin

Chapter 13 InorganicAnionTransporterAEI 435

Michael L. Jennings

Chapter 14 The Structuresof G-ProteinCoupledReceptors 479

Philip L. Yeagle

Chapter 15 Role of Membrane Lipids in Modulating the Activity of Membrane-BoundEnzymes 499

Richard M. Epand

Chapter 16 ViralFusionMechanisms 511

Aditya Millal and Joe Benlz

Index ... 529

1 Lipid Structure

Helmut Hauser and Guy Poupart

CONTENTS

1.1 LipidClassification 11.1.1 Nonhydrolyzable(Nonsaponifiable)Lipids 2

1.1.1.1 Hydrocarbons 21.1.1.2 SubstitutedHydrocarbons 3

1.1.2 SimpleEsters 81.1.2.1 Acylglycerols. ..91.1.2.2 CholesterylEsters 101.1.2.3 Waxes 11

1.1.3 ComplexLipids 111.1.3.1 Glycerophospholipids 111.,1.3.2 Sphingolipids 14

1.1.4 Glycolipids 141.1.4.1 Glycoglycerolipids 141.1.4.2 Glycosphingolipids 171.1.4.3 Lipopolysaccharides 17

1.2 The Stereochemistryof Lipids 201.2.1 Glycerol-BasedLipids.. ..201.2.2 Sphingolipids 241.2.3 Steroids ..24

1.3 The LipidCompositionof BiologicalMembranes 251.4 X-rayCrystallographyof Lipids 27

1.4.1 CrystalStructuresof SimpleLipids 281.4.2 CrystalStructuresof Cholesterol 291.4.3 CrystalStructuresof Acylglycerols 301.4.4 CrystalStructuresof Glycerophospholipids 35

1.4.4.1 MolecularPacking 351.4.4.2 MolecularConformation ...40

, 1.4.5 CrystalStructuresof Glycosphingolipids 47!.5 Summaryof Single-CrystalX-ray CrystallographicAnalysis 48\'\cknowledgments.. 48References 49~

'<' .1.1 LIPID CLASSIFICATION.c~~:

Using the broadest possible definition, a lipid (Greek lipos = fat) may be defined as a compound

'9poW or intermediate molecular weight (5000), a substantial proportion of which is made up of

~ydrOCarbons. Included are diverse compounds such as fatty acids; soaps; detergents; steroids;

f:},?DO-.di-, and triacylglycerols; and more complex compounds such as phospholipids, sphingolip-'I(\S, glycolipids, and lipopolysaccharides. Because this volume addresses the structure and function

D:849).I4QJ.8IOSISO,OO+S 130

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2 The Structure of Biological Membranes, Second Edition

TABLE 1.1Classification of Lipids

Nonhydrolyzable (Nonsaponifiable) Lipids

Hydrocarbons

Simple alkanes

Terpenes (isoprenoid compounds)

Substituted hydrocarbons

Long-chain alcohols

Long-chain fatty acids

DetergentsSteroids

Vitamins

Simple Esters

AcylglycerolsCholesteryl estersWaxes

Complex Lipids

Glycerophospholipids

Sphingolipids

Glycolipids

Glycoglycerolipids

GlycosphingolipidsCerebrosides

Gangliosides

Lipopolysaccharides

of biological membranes, we are primarily concerned with the more complex lipids that areconstituents of biological membranes.

Membrane lipids consist of a wide and still expanding range of amphipathic (amphiphilic)

compounds containing a nonpolar (hydrocarbon) and a polar region. It is clear that such diversemolecules as lipids will differ widely in their physicochemical behavior. Nevertheless, solubility

is a property that is used as a unifying criterion. Lipids are soluble in organic solvents such asalkanes, benzene, ether, chlorinated alkanes (e.g., chloroform, tetrachlorcarbon), methanol, andmixtures of these solvents. For instance, mixtures of chloroform and methanol are used as universal

lipid solvents. Lipids with long hydrocarbon chains are practically insoluble in water. Dependingon their chemical strUcture, they are either completely immiscible in water, such as hydrocarbons,

or they interact with water, forming colloidal dispersions (Small, 1986).The classification of lipids is arbitrary. In many texts, lipids are ranked in order of increasing

complexity. Sometimes, however, practical criteria are used. As mentioned above, lipids differ widely

in their physicochemical properties, e.g., they vary considerably in their interaction with water and

in their spreading properties at the air-water interface. Hence, lipid classification may be based on

hydration, swelling of lipid in the presence of water, or spreading of lipid at the air-water interface

(Small, 1986). In Table 1.1, lipid classes are listed in order of increasing complexity.

1.1.1 NONHVDROl VZABlE (NONSAPONIFIABlE) LIPIDS

1.1.1.1 Hydrocarbons

1.1.1.1.1 Simple Alkanes

Pure hydrocarbons - saturated or unsaturated, aliphatic or aromatic - are usually classified aslipids. They are rare in the animal kingdom and, with few exceptions, are not particularly important.

They are, however, major constituents of petroleum deposits.

Lipid Structure 3

1.1. 1.1.2 Terpenes (Isoprenoid Compounds)

Many plant odors are due to volatile C'a and C's compounds termed lerpenes. Isolation of these

compounds from various parts of plants by steam distillation or ether extraction yields the so-called

essential oils. They can be regarded as derivatives of isoprene, 2-methyl-I,3-butadiene (CsHs, see

Figure 1.IA). Essential oils are obtained from cloves, roses, lavender, citronella, eucalyptus, pep-

permint, camphor, sandalwood, cedar, and turpentine. They are widely used in perfumery, as food

flavorings, medicines, and solvents (Roberts and Caserio, 1979). The essential oils, such as these

C'a and C's compounds, may be regarded as members of a much larger class of substances with

carbon skeletons that are multiples of the isoprene (C,Hs)-unit.

These compounds are categorized as isoprenoid compounds and are widespread in both plants

and animals. They comprise open-chain (acyclic) and cyclic compounds, and, in addition to the

C'a compounds that are customarily designated terpenes, they comprise CIS (sesquiterpenes), C20(diterpenes), CJOcompounds (triterpenes), and so on (Figure 1.1). Important examples of isoprenoid

compounds are II-carotene, vitamin A, and squalene. Carotenoids in general are tetraterpenes (C40

compounds) and are' widespread as yellow or red pigments of plants. II-carotene (Figure 1.1A) is

a precursor of vitamin A. It is oxidized in the liver at the central double-bond to yield vitamin A,

which is a diterpene alcohol. Squalene is a triterpene (C~so) and an example of an important

isoprenoid compound of animal origin. It occurs in fish liver oil and is a precursor of the steroidslanosterol and cholesterol (see Figure 1.6). Also, terpene hydrocarbons and oxygenated terpenes

have been isolated from insects and have hormonal and pheromonal activity.

1.1.1.2 Substituted Hydrocarbons

Substitution in position I of hydrocarbons with electronegative atoms or groups leads to amphipathic

molecules, such as long-chain alcohols, fatty acids, and detergents. Long-chain is defined as a

hydrocarbon chain with 12 or more carbon atoms.

1.1.1.2.1 Long-Chain Alcohols

Long-chain alcohols, e.g., octadecanol or stearyl alcohol (Figure 1.2) are amphipathic and surface

active. Monolayers of long-chain alcohols spread at the air-water interface prevent water evapora-

tion and have been widely used as antievaporants in water reservoirs. Furthermore, they are used

in water-in-oil emulsions and in cosmetics. Long-chain alcohols are important constituents of

complex lipids, in which they are covalently linked to glycerol via ether bonds. Such ether linkages

are found in plasmalogens and in membrane lipids of bacteria (see below). These long-chain, ether-

linked alcohols can be branched and of the isoprenoid-type, like the phytanyl residue (Figure 1.1B).

A great profusion of oxygenated isoprenoid compounds exist: of importance are alcohols and

aldehydes, which occur in plant oils and flower essences. 1\vo important diterpene alcohols are

vitamin A, mentioned previously, and phytol (Figure I.IB). The latteris a constituent of chlorophyll,

the major pigment of chloroplasts and of central importance in photosynthesis. In chlorophylls,

phytol is esterified to the propanoic acid side chain of the Mg2+ -porphyrin ring. The phytyl-group

..is also a side chain of vitamin K (Figure 1.7). Isoprenoid alcohols and their cyclopentane phytanyl

,derivatives with an OH-group on one end (monopolar) or both ends (bipolar) of the branched

,.hydrocarbon chains are found in the plasma membranes of archaebacteria (Figure 1.1B) (Luzzati

et al., 1987; BlOCher et aI., 1985). The bipolar C.. hydrocarbon chain that occurs in the diglyceryl

t{tetraether compounds of archaebacteria can contain 0-4 cyclopentane rings.

.} .1.1.2.2 Long-Chain Fatty Acids":~,ong-chain fatty acids are constituents of all esterified and complex lipids. They are covalently

linked via ester or amide bonds, and in this form they are major constituents of membrane lipids.Free fatty acids rarely make up more than a few percent of membrane lipids. Long-chain fattyacids are widely used in industry as precursors of emulsifiers, food additives, and detergents. Ineukaryotes, the fatty acids are straight with an even number of carbon atoms, typically 14 to 24

lipid Structure 5

/,/'.../,./ / /'-/' /,_,9'H Octadecanol or stearyl alcohol

~O

o

~

Octadecanal

g-Octadecanone

~ 11'613

o

(1IZ)-Retlnal or "-cis-Retlnal

o

~ (all E)-Retinal or all-/tans-RetlnaJ

FIGURE 1.2 Long-chainalcohols, aldehydes, and ketones.The lUPAC-lUBCommissionon BiochemicalNomenclature (CBN) recommends the use of the EIZ system to designate the configuration of double bonds.Although the system has advantages, it has not received widespread use in lipid nomenclature and has not

replaced the old cis-lrans convention. Z (for zusammen) corresponds to cis, and E (for enlgegen) to Irans.

and with zero to six double bonds (Table 1.2 and Figure 1.3). The double bonds of mono- and

polyunsaturated fatty acyl chains usually have the cis-configuration, unless stated otherwise (cf.Table 1.2). It should be stressed that polyunsaturated chains are not normally conjugated. Unusual

fatty acids with branched chains, cyclopropane, cyclohexyl-, and ~-OH groups are found in bacterial

membrane lipids (Figure 1.4) (Gennis, 1989; Jain, 1988).

I. I. 1.2.3 Detergents

Substitution of hydrocarbons in the I-position with polar groups, such as carboxylates, sulfonates,

phosphates, amines, aIkyl-amines, sugar residues, etc., generates the important lipid class of deter-

gents. They are widely used in everyday life and also in biology. The sodium and potassium salts

of fatty acids are called soaps and represent perhaps the oldest detergent. They are not only used

in cosmetics but also as emulsifiers and lubricants. Soaps of alkaline earths (e.g., calcium distearate)are water-insoluble and used as insoluble lubricants and constituents of water-in-oil emulsions.

Some of the most commonly used detergents in biology are shown in Figure 1.5. Sodium dode-

cylsulfate (SDS) is universally used to solubilize proteins and to determine their approximatemolecular weight by electrophoresis on polyacrylamide gels. It is also a major constituent of nearly

all shampoos. Detergents in biology are used to solubilize macromolecular assemblies, including

proteins, DNA and RNA, lipid aggregates such as bilayers, and complexes of these different classes

of compounds, e.g., biological membranes. The products of solubilization are usually small, mixed

~\celies containing the macromolecule(s), e.g., protein, and the detergent. In this micellar form,

thi:' macromolecule can be purified by subjecting the micellar dispersion to various separation

pi)X:esses, e.g., chromatography, electrophoresis, sedimentation, etc. In general, nonionic and zwit-

terj\>nit detergents are milder and have proved more successful than charged ones in solubilizing

~~lurifying membrane proteins. The purified membrane protein may then be reconstituted to aslmple'and well-defined membrane system using established methods of reconstitution (Lodish andRotluitah, 1979).

".:.))(11.I..J..~.4 Steroids

'Qte .fJ1~damental ring system common to all steroids is the cyclopentanophenanthrene or, more

precisely, its perhydrogenated form (Figure 1.6). Most steroids are alcohols, and accordingly named

4 TheStructureof BiologicalMembranes,SecondEdition

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6 The Structure of Biological Membranes, Second Edition

TABLE1.2Names of Straight FattyAcids

. Key to abbreviations: The first number is n =number of C-atoms; the number after the colon givesthe number of double

bonds; the position of Ibe double bond(s) is indicated by the superscript(s) \0 the symbol. In this shorthand notation. Ibe

position of the double bond is detennined by counting from the carboxyl group (=<:1). The confisuration of double bonds

is defined by both the E-Z and cis-Irons conventions(seeFigure 1.2). Unless indicated by the leuer I (for Irons

configuration), the cis configuration is implied. In a related system widely used for fatty acids of animal lipids, the position

of the double bond is detennined by (n-x). where x is the position of the first double bond encountered when counting

from the terminal melbyl group of the fauy acid.

as sterols. As is evident from their structures, most have the same ring skeleton but differ consid-

erably in their side chain and peripheral structural features, in stereochemistry, and in the numberof double bonds in the rings.

Steroids are widely distributed in both plants and animals and comprise a diversity of com-

pounds, such as cholesterol, bile acids, vitamin D (Figure 1.7), sex and corticoid hormones, andsaponins. Many of these compounds are of vital importance in physiology. Others are valuablemedicinals, such as cardiac glycosides, hormones, and steroidal antibiotics. The most common

sterol is cholesterol (cholest-5-en-3p-ol), which is an unsaturated alcohol (for the stereochemistryof sterols, see Section 1.2). Cholesterol is found in mammalian plasma membranes to about 30%

of the total lipid mass, and also in Iysosomes, endosomes, and Golgi. The sterols found in higherplants are P-sitosterol and stigmasterol. These plant sterols (phytosterols) frequently have an

additional side chain at position C-24 andlor an additional double bond at position C-22 (Figure

1.6). Ergoslerol is .often found in eukaryotic microorganisms, e.g., yeast.

Lipid Structure

~C,OH .o

~ C,OH.o

~C,OH(,

~C,OH .o

~ C,OH.o

~C,OH .o

~C,OH .o

7

Systematic name TrMalname

Octadecanoicacid Stearicacid

(9E).Octadecanolc acid ortrans-9-Octadecenolc acid

Elaldlc acid

(9Z)-Octadecanok: acid orci9-9.Qctadecenolc acid

Oleic acid

(9Z.12Z)-Octadecadlenolc orcJs.9.cJs.12-Octadecadlenolcacid

9.12-Linolelcacid

(9Z.12Z.15Z)-OctadecatMenolc orall ci9-9.12.15-OctadecatMenok:add

u.unolenlcacid

(6Z.9Z,12Z)-OctadecatMenoic or

all ci9-6.9.12-OctadecatMenolcacid

)'LInolenlcacid

(5Z.8Z.11Z.14Z)-Elcosatetraenolc or IArachidonicall C/9-5.8.11.14-Elcosatetraenolc acidacid

FIGURE 1.3 Fattyacids.

In the last two decades, a new class of penlacyclic sterollike compounds termed hopanoids(Figure 1.6) was discovered. Hopanoids are very widespread in bacteria but are also found in some

plants. Hopanoids were also detected in sediments and crude oils, where they account for more

than 5% of the soluble organic matter. This extrapolates to 10" to 10'2 tons of hopanoids, suggestingthat this class of compounds represents the most abundant one on our planet (Ourisson, 1987;Prince, 1987). Bacteriohopanetetrol is the most abundant microbial hopanoid.

~ I, Other compounds are diplopterol and tetrahymenol, which are found in Tetrahymena species.'J:Iiese compounds occur in bacterial membranes, and their function has been proposed to be the

,stabilization of the membrane structure, rather similar to that of membrane sterols in eukaryotes.

, ~oth sterolsand hopanoidsare rigid and rather flat amphipathicmolecules.The amphiphilicgW!lp.s'are, however, on opposite sides of the molecules: it is the 3-hydroxyl group in sterols and

.,'J.!.IS/JJ,oIYolside chain in hopanoids. The biosynthesis of sterols and hopanes shares a common

;})a~Way from acetate to mevalonate and squalene, but differs thereafter. Direct cyclization leads

.'tQhopane; lanosterol (Figure 1.6) is produced as an intermediate in the sterol pathway.T:1. 7.2.5 Vitamins

A.:n,bmber of vitamins are fat-soluble and have solubility properties characteristic of lipids. Based~J:(lltis, they are frequently classified as lipids. Fat-soluble vitamins contain alicyclic or aromatic

!}!1gs, which are usualIy oxygenated and attached to an isoprenoid hydrocarbon chain or isoprenoid

I!lcohol. Aforementioned examples are retinol (vitamin A,) and ergocalciferol (vitamin D2). Other

Carbon Melting Point,Atoms Systematic Designation Trivial Name Abbreviation(s)' .C

Saturated12 Dodecanoicacid Lauricacid 12:0 44.214 Tetradecanoicacid Myristicacid 14:0 53.9

16 Hexadecanoicacid Palmiticacid 16:0 63.1

18 Octadecanoicacid Stearicacid 18:0 69.620 Eicosanoicacid Arachidicacid 20:0 76.522 Docosanoic acid Behenic acid 22:0 79.924 Tctracosanoicacid Ugnoceric acid 24:0 S6.0

Monocnaic16 9-Hexadecenoicacid Palmitoleicacid 16:1' or 16:1 (n-7) -0.5

18 9.()ctadecenoicacid Oleic acid 18:1' or IS:1 (n-9) 13.4

IS (II E)-Octadecenoic acid or II-trans- Vaccenicacid 18:1"\ or IS:1 (n-7)t 44.0

Octadecenoic acidIS (9E)-Octadecenoic acid or 9-trans- Elaidicacid 18:1't or 18:1 (n-9)t 46.5

Octadecenoicacid24 15-Tetracosenoic acid Nervonicacid 24: I" or 24:I (n-9) 42.5-43

DienoicIS 9.12-Octadecadienoic acid Linoleic acid 18:2'.12or 18:2 (n-6) -5

TrienoicIS 9,12,15-Qctadecatrienoic acid a-Linolenic acid 18:3'.'''' or IS:3 (n-3) -11IS 6.9.12-Octadecatrienoic acid y..Unolenicacid 18:3....12or IS:3 (n-6)

Tetraenoic20 S.8,11,14.Eicosatetraenoicacid Arachidonicacid 20:4'.7.1'''.'''' or 20:4 (n-6) -49.5

Hexaenoic22 4,7,1O.13,16.19-Docosahexaenoicacid Cervonic acid 22:6'.7.""."" or 22:6 (n-3)

8 The Structure of Biological Membranes, Second Edition

HO-~~o

HO-C~o

HO-~~o

HO-C~o

OHHO-C~

o

HO-~~o OH

OH

HO-b~OH

HO-~~o OH

HO-~-(CH,).-oo

HO-C~oOH

HO-C~o

HO-~~o

FIGURE 1.4 Bacterial fatty acids.

ise>branched falty acid

anteiso-branched fatty acid

15.methyt fatty acid

Z-unsaturatedorcis-unsaturatedfatty acid

a-hydroxyt falty acid

~-hydroxyt latty acid

a-hydroxyt-jl-methyt falty acid

a.ll-dihydroxyt latty acid

",""ydohexyt falty acid (n=10, 12,main components of B. acldocaldarius)

(Z, Z)-unsaturated orcis,cis-unsaturated fatty acid

a.hydroxyt-(~E)'ene

2.Hexytcyclopropanedecanolc acid,Lactobacillic acid

examples offat-soluble vitamins are a-tocopherol (vitamin E) and vitamins K, and K2(Figure 1.7).Both vitamins Kt and K2 are substituted naphthoquinones with isoprenoid side chains differing inlength. They are abundant in most higher plants.

1.1.2 SIMPLEESTERS

The lipids discussed under this heading are saponifiable, that is, they are hydrolyzed by heatingwith alkali to yield soaps.

Lipid Structure 9

o~O-~-o~Na+

oSodium dodecyl sullate (SOS)

cH, 0

N0o~a+~ N.Lauroyisarcosinate(SarkosylNL)

o

~.HO R;.

3a,7a,12a.Trihydroxy-5/1-Cholan'24-oate,

sodium cholate (R, = OH, R, =OH)

3a,12a.Oihydroxy-5~-cholan.24-oate,

sodium deoxycholate (R, = 00, R, = H)3a.Hydroxy-5~-cholan-24-oate,

sodium lithochotate (R, = H, R, = H)

£0 CH,?

OH ~~~~-o-H CH3 0

3-[(3-cholamidopropyl)-dimethyt.ammoniol'l-propanesulfonate (CHAPS)

HO OH

~O~,OHHO OH OH

1-0-Qctyl'I\-O-glucopyranoside,~.O-Qctylglucoside

o

~o lysophosphatidylcholine

HO~O-~-o~ CH,0- ~+-c~

CH,

FIGURE 1,5 Detergents. Lysophospholipids are a class of detergents that is biologically important. A repre-sentative of this class is Iysophosphatidylcholine, which has one fatty acyl chain per polar group. The genericterm lysophosplwlipid indicates the detergentlike properties and the lytic activity of this class of lipds.

~ ,

.! ~,1.1.2.1 Acylglycerols;~ ';

"0 ~,~. number of complex lipids have, as a central part of their structure, the glycerol group, Glycerol.~ ~~(1,2,3-propanetriol) has three reactive OH-groups and forms esters (ethers) with fatty acids (alco-.~ 1...~:'bols).Depending on the number of fatty acids (alcohols) reacting with glycerol, one obtains

,d~1p0noacYI-(monoalkyl-), diacyl- (dialkyl-), and triacyl- (trialkyl-) glycerols (Figure \.8). Triacyl-glycerols, frequently also called triglycerides,. are the major components of dietary fats. They are.

e/~,.~ :....

'~y~s tenn should be avoided because it is misleading.

~jH: