392 Chapter 8. Lipids, Membranes, and Cell Coats Five kinds of phospholipid predominate: phospha- tidylcholine, phosphatidylethanolamine, phosphatidyl- serine, phosphatidylglycerols, and sphingomyelin. Usually there are also small amounts of phosphatidyli- nositol. The major phospholipid in animal cells is phosphatidylcholine, but in bacteria it is phosphati- dylethanolamine. The phospholipids of E. coli consist of 80% phosphatidylethanolamine, 15% phosphati- dylglycerol, and 5% diphosphatidylglycerol (cardio- lipin). Significant amounts of cardiolipin are found only in bacteria and in the inner membrane of mito- chondria. Sphingomyelin is almost absent from mito- chondria, endoplasmic reticulum, or nuclear membranes. 0.5 nm Glycolipids are important constituents of the plasma membranes, of the endoplasmic reticulum, and of chloroplasts. The cerebrosides and their sulfate esters, the sulfatides, are especially abundant in myelin. Figure 8-7 Atomic force microscope image of a dimyristoyl- In plant membranes, the predominant lipids are the phosphatidylethanolamine bilayer deposited by the Langmuir galactosyl diglycerides.29,74 The previously described –Blodgett technique (see Fig. 8-8) at a specific molecular area ether phospholipids (archaebacteria), ceramide amino- of 0.41 nm2 and a surface pressure of 40 mN/m on a freshly ethylphosphonate (invertebrates), and sulfolipid cleaved mica substrate. The images were taken under water. (chloroplasts) are also important membrane components. The long, uniformly spaced rows are roughly 0.7– 0.9 nm in Cholesterol makes up 17% of myelin and is present spacing. The modulation along the rows, with rounded in plasma membranes. However, it usually does not bright spots roughly every 0.5 nm, corresponds to the indi- vidual headgroups of the phosphatidylethanolamine mole- occur in bacteria and is present only in trace amounts cules. The lattice spacing is identical to that measured by in mitochondria. Related sterols are present in plant X-ray diffraction at the air–water interface. The area per membranes. Esters of sterols occur as transport forms molecule in the AFM image is ~ 0.4 nm2. From Zasadzinski but are not found in membranes. Membrane bilayers, et al.68 likewise, contain little or no triacylglycerols, the latter being found largely as droplets in the cytoplasm. Quantitatively minor membrane components with that at temperatures suitable for growth and metabo- important biological functions include ubiquinone, lism the hydrocarbon chains are not rigidly packed in which is present in the inner mitochondrial membrane, the center of the bilayer but are “molten” (see Section 2). and the tocopherols. Plant chloroplast membranes However, at a low enough temperature they become contain chlorophyll, carotenes, and other lipid-soluble rigid and pack together in a manner similar to that of pigments. the chains in crystals of the phosphatidylcholine shown in Fig. 8-6A. These crystals consist of stacked bilayers Liquid crystals, liposomes, and artificial mem- of thickness 5.5 nm.66,69 The scanning tunneling and branes. Phospholipids dissolve in water to form true atomic force microscopes have provided direct views solutions only at very low concentrations (~ 10–10M for of a similar arrangement of side chains in a monomo- distearoyl phosphatidylcholine). At higher concentra- lecular fatty acid layer68,70,71 (Fig. 8-7). Measurements tions they exist in liquid crystalline phases in which on multilamellar vesicles of dipalmitoylphosphatidyl- the molecules are partially oriented. Phosphatidylcho- choline give bilayer thicknessess from 5.4 nm for dehy- lines (lecithins) exist almost exclusively in a lamellar drated vesicles to 6.7 nm for the biologically relevant (smectic) phase in which the molecules form bilayers. fully hydrated bilayers.72 In a warm phosphatidylcholine–water mixture con- taining at least 30% water by weight the phospholipid Lipids of membranes. Approximately 1500 formsmultilamellar vesicles, one lipid bilayer sur- different lipids have been identified in the myelin of rounding another in an “onion skin” structure. When the central nervous system of humans. About 30 of such vesicles are subjected to ultrasonic vibration they these are present in substantial amounts.73 The distri- break up, forming some very small vesicles of diameter bution of the different lipids varies markedly between down to 25 nm which are surrounded by a single membranes from different sources (Table 8-3) making bilayer. These unilamellar vesicles are often used for generalization difficult. However, phospholipids are study of the properties of bilayers. Vesicles of both apparently always present and, except in chloroplasts, types are often called liposomes.75– 77 make up from 40% to over 90% of the total lipid (Table When liposomes are stained with osmium tetroxide 8-3). or potassium permanganate, embedded, and sectioned B. Membranes 393 for electron microscopy, their membranes show a O O O O characteristic three layered structure similar to that Os Os observed for biological membranes. Two darkly stained lines ~ 2 – 2.5 nm thick are separated by a clear O O O O space ~ 2.5 – 3.5 nm wide in the center. Both myelin An osmate ester formed and the retinal rod outer segments show closely spaced from two unsaturated groups pairs of such membranes with a combined width of 18 nm. These results seemed to support the original brane which can be made from a solution of phospha- Davsen–Danielli model. However, many questions tidylcholine or of a mixture of phospholipids plus must be raised about the interpretation of these results. cholesterol in a hydrocarbon solvent. A droplet of Why does OsO stain only the outer protein layer when solution is placed on a small orifice in a plastic sheet, 4 it is known to react also with double bonds of hydro- separating two compartments filled with an aqueous carbon side chains of lipids to form osmate esters medium (Fig. 8-8). The solution in the orifice quickly which are readily reduced to a diol and osmium?77a-c,78 drains, just as does a soap bubble, and the resulting Membranes from which most of the lipid has been film eventually becomes so thin that the bright colors extracted still stain with OsO to give three-layered disappear and a “black membrane” is formed. Similar 4 electron micrographs. Perhaps little can be concluded membranes, but without a residual content of hydro- from the three-layered appearance. We have learned carbon solvent, have been created by apposition of that it is difficult to determine even the thickness, let two lipid monolayers formed at an air–water inter- alone the complete structure of an object that is only face.79,80 The thickness of such artificial membranes is 6– 10 nm thick. thought to be only 6 – 9 nm. Resilient and self-sealing, Strong support for the lipid bilayer model comes the membranes can be stained with OsO to give a 4 from the preparation of another type of artificial mem- typical three-layered pattern. TABLE 8-3 Estimated Chemical Compositions of Some Membranes Percentage of total dry weight of membranea Plasma membrane E. colib,c,d Myelin Retinal (human Mitochondrial (inner and outer Compound (bovine) rod erythrocyte) membranes membranes) Chloroplastse Protein 22 59 60 76 75 48 Total lipid 78 41 40 24 25 52f Phosphatidylcholine 7.5 13 6.9 8.8 Phosphatidylethanolamine 11.7 6.5 6.5 8.4 18 Phosphatidylserine 7.1 2.5 3.1 Phosphatidylinositol 0.6 0.4 0.3 0.75 Phosphatidylglycerol 4 Cardioliping 0.4 4.3 3 Sphingomyelin 6.4 0.5 6.5 Glycolipid 22.0 9.5 Trace Trace 23 Cholesterol 17.0 2.0 9.2 0.24 Total phospholipid 33 27 24 22.5 25 4.7 Phospholipid as a percentage 42 66 60 94 >90% 9 of total lipid a Dewey, M. M. and Barr, L. (1970) Curr. Top. Membr. Transp.1, 6. b Kaback, H. R. (1970) Curr. Top. Membr. Transp. 1, 35 – 99. c Mizushima, S. and Yamada, H. (1975) Biochim. Biophys. Acta.375, 44 – 53 . d Yamato, I. Anraku, Y. and Hirosawa, H. (1975) J. Biochem. (Tokyo)77, 705 – 718. These investigators found 67% protein, 21% lipids, 10% carbohydrate, and 2% RNA. e Lichtenthaler, H. K. and Park, R. B. (1963) Nature (London)198, 1070 –1072. f About 14% is accounted for by chlorophyll, carotenoids, and quinones.e g Diphosphatidylglycerol (Fig. 8 -2). 394 Chapter 8. Lipids, Membranes, and Cell Coats The study of monolayers formed on a water sur- pressure is higher for longer molecules as a result of the face has also provided important information. A thin greater number of van der Waals interaction between film of an amphiphilic (containing both polar and non- the chains. Langmuir–Blodgett layers are prepared polar groups) compound such as a fatty acid is prepared. by transferring one or more monolayers onto a smooth This is done by depositing a small quantity of the solid surface (Fig. 8-7).82,83 compound dissolved in a volatile solvent on a clean aqueous surface between the barriers of a Langmuir Physical properties of membrane lipids. A trough (Fig. 8-8).81,82 The difference in surface tension completely extended C fatty acid chain as shown in 18 (π) across the barriers is measured with a suitable Fig. 8-4 has a length of ~ 2.0 nm and occupies, either in device81 for different areas of the monolayer, i.e., for crystals or in monolayers, when viewed “end-on,” an different positions of the moveable barrier. The value area of ~ 0.2 nm2. The hydrocarbon layer in a lipid ofπ is low for expanded monolayers and falls to nearly bilayer containing such chains would have a thickness zero when the surface is no longer completely covered. of about 4.0 nm; that determined by X-ray diffraction The pressure reaches a plateau when a compact mono- for myelin is ~ 3.5 nm. However, for artificial black layer is formed, after which it rises again (Fig. 8-8B). membranes the thickness of the hydrocarbon layer can At very high values of π the monolayer collapses be as little as 3.1 nm when all solvent is removed.84 (buckles). Both the cross-sectional area per molecule These and many other results85 indicate that the hydro- in the monolayer and the collapse pressure can be carbon chains are to some extent folded and that the determined. For typical fatty acids, regardless of chain membrane is expanded over that expected according length, the area covered is only ~ 0.2 nm2 per molecule to the simplest model. indicating that the fatty acid chains are stacked verti- Structure determinations on crystalline alkanes cally to the surface in the monolayer. The collapse confirm that the chains exist in a completely extended conformation and that adjacent chains often pack together in the orthorhombic arrangement shown in Fig. 8-6B. As the temperature of such crystals is raised A Moveable Fixed a series of solid–solid phase transitions is observed γ0 barrier γ barrier below the melting point of the crystals.86 These can be Trough detected by changes in the infrared absorption spectrum or by small amounts of heat absorption revealed by differential scanning calorimetry (Fig. 8-9). Each Aqueous phase new phase permits a greater degree of mobility for the hydrocarbon chains. Thus, at a high enough tempera- ture but below the melting point, the chains are able to rotate freely about their own axes in a so-called hexagonal phase. Now the chains are packed in a B hexagonal array instead of the orthorhombic array of Fig. 8-6B. At intermediate temperatures, some of the Solid chains may assume nonplanar conformations and -1m) Condensed cmhaayn goecsc uinr. the tilt of the hydrocarbon chains (Fig. 8-6) N m Similar phase transitions are observed for bilay- π ( ers.88– 90 For dipalmitoyl phosphatidylcholine the first detectablesubtransition91 is centered at a temperature Expanded T of 18°C. The second, known as the pretransition, s occurs at 35°C(T ). The structure below T may be p s 0.2 0.3 0.4 0.5 0.6 0.7 0.8 described as rigid or crystalline and that above T as s nm2/molecule agel in which the hydrocarbon side chains twist and turn much more freely but in which the orthorhombic packing is maintained.86 Above T the head groups p Figure 8-8 (A) The Langmuir–Adam film balance. Ten- become disordered. Although the orthorhombic pack- sion on the moveable barrier is recorded for different areas ing of the tails may be maintained, there are several of the surface between the barriers. This gives the surface distinct phases,92,93 including one or more in which the pressure π, which is the difference between the surface gel is thought to assume a structure analogous to that tension (γo) of a clean aqueous surface and that of a spread in the hexagonal phase of hydrocarbons. At 41°C the monolayer (γ): π = γ –γ. Courtesy of Jones and Chapman.81 (B) Surface pressureo (π)– area per molecule isotherm for a main transition occurs. typical fatty acid (e.g., pentadecanoic acid C H CO H) at 14 29 2 the aqueous – air interface. From Knobler.81a B. Membranes 395 High temperature different components containing a variety of fatty acid Lα Liquid crystalline, partially ordered chains leads to a broadening of the melting range. disordered chains, biologically relevant The behavior of bilayers is strongly influenced by the lipid composition. Phospholipids containing Pβ (Gr) Rippled; Tm 41°C Gh, Hexagonal saturated, long-chain fatty acids have high transition Go, Orthorhombic, like alkanes temperatures. The presence of unsaturated fatty acyl groups with cis double bonds in membrane lipids Lβ'(Gd) Gpoella pr hhaesaed, ogrrtohuoprhs odmisboircd,ered encourages folding of the hydrocarbon chains and lowersT . Even a single double bond lowers T , the m m T 35°C decrease being greatest when the double bond is near P the center of the chain.94– 96 While T for dipalmitoyl m phosphatidylcholine is 41°C, that of 1,2-dipalmitoyl L (G) Sub-gel, interdigitated C S phosphatidyl-sn-glycerol, which lacks the phospho- choline head group, is 70°C. This falls to 11.6°C for the polyunsaturated 1-stereoyl-2-linoleoyl-sn-glycerol, T 18°C S whose melting curve is shown in Fig. 8-9.87 This lipid also shows a complex phase behavior and a melting LΒ Crystalline phase, dehydrated highly point for the stable, crystalline β’ phase higher than ordered all-trans extended chains that of the α phase. Low temperature Inclusion of other molecules of irregular shape within membranes also lowers T . However, a mole- m This is a sharper transition with a well-defined melting cule of cholesterol can pack into a bilayer with a cross- temperature designated T . Above T the lipid is in sectional area of 0.39 nm2, just equal to that of two m m the lamellar liquid crystalline or Lα state. The bilayer hydrocarbon chains.49 It tends to harden membranes continues to hold together, but the fatty acid chains aboveT but increases mobility of hydrocarbon chains m have melted and are now free to rotate and undergo belowT .97– 100 A complex of cholesterol and phospha- m twisting movements more freely than at lower temper- tidylcholine may form a separate phase within the atures (Fig. 8-11). The main transition is highly, but membrane.101,102 The ether-linked plasmalogens may not completely, cooperative. Thus, the melting of the account for over 30% of the phosphoglycerides of the membrane occurs over a range of several degrees. white matter of the brain and of heart and ether linked The presence in biological membranes of a variety of phospholipids are the major lipids of many anaerobic bacteria.103 Their T values are a few m degrees higher than those of the corre- sponding acyl phospholipids.104 Between the pretransition temper- β' Melt ature and T solid and liquid regions m may coexist within a bilayer.101 The Heat termlateral phase separation has been absorbed Heating (a) applied to this phenomenon.105,106 Since changes in the equilibrium be- Heat subα2 subα1 α Crystallization (b) tween solid and liquid can be induced evolved Cooling readily, e.g., by changes in the ionic Reheating environment surrounding the bilayer, α Melt lateral phase separation may be of absoHrbeeadt subα2 subα1 (c) significance in such phenomena as nerve conduction.107 The phase transitions in bilayers –10 0 10 20 30 Temperature (°C) can be recognized in many ways. Differential scanning colorimetry has already been mentioned. Another Figure 8-9 Differential scanning calorimetric curves for 1-stearoyl-2- approach is to measure the spacing linoleoyl-sn-glycerol. (A) Crystals of the compound grown from a hexane between molecules by X-ray diffrac- solution were heated from –10° to 35°C at a rate of 5°C per minute and the tion. The cross-sectional area occupied heat absorbed by the sample was recorded. (B) The molten lipid was by a phospholipid in a bilayer is cooled from 35° to –10°C at a rate of 5° per minute and the heat evolved always greater than the 0.40 nm2 was recorded as the lipid crystallized in the α phase and was then trans- formed through two sub-α phases. (C) The solid was reheated. From Di expected for closest packing of a pair and Small.87 Courtesy of Donald M. Small. of extended hydrocarbon chains.39,85 396 Chapter 8. Lipids, Membranes, and Cell Coats BelowT the spacing between chains is about 0.42 nm bilayer into two or more phases can be observed using m corresponding to close packing of the fatty acid chains 2H- or 31P- NMR.118 – 120 The orientation and dynamic in a hexagonal array with an area per phospholipid of behavior of various head groups has been explored,110,121 0.41 nm2. As the temperature is raised above T the as have effects of mixing into the bilayer other lipids m spacing increases85 to give an average area per phos- such as glycosphingolipids122 and cholesterol.123,124 pholipid of 0.64–0.73 nm2. Another technique (Box 8-C) Crystalline phospholipids are being investigated by is to study a spin label by EPR while yet another is to solid-state NMR.125 observe the fluorescence of a polarity-dependent Fourier transform infrared spectroscopy126,127 also fluorescence probe such as N-phenylnaphthylamine provides information about conformation of both or other fluorescent probes108 (see Chapter 23). The hydrocarbon chains and head groups. EPR spectros- compound is incorporated into the membrane and copy (Box 8-C) with doxyl probes on carbon atoms at undergoes changes in the intensity of its fluorescence different depths within the bilayer has also been em- when the state of the membrane is altered. ployed.128 A variety of NMR techniques are being applied109– 113 In recent years molecular dynamics simulations both to liposomes and to natural membranes.111,114 have been used to predict behavior of membranes. As Incorporation of 13C or 2H into various positions in the is indicated in Fig. 8-11, the molten interior of the liquid hydrocarbon chains has allowed measurements of the crystalline Lα state is portrayed clearly.129– 131 In the gel relative degree of mobility of the chains at different state the hydrocarbon chains maintain a closer packing depths in the bilayer (Fig. 8-10).109,115 – 117 The results and undergo coordinated movement.88 It is difficult to are in agreement with statistical mechanical predictions know how realistic the simulations are. To calibrate that configurational freedom increases with depth the method efforts are made to correctly predict a series toward the midplane of the bilayer. Separation of a of known properties such as density and area per lipid (0.61 nm).130 A 3-9 14 3-9 Functions of phospholipid head groups. The 10 14 10 dipolar ionic head groups of phosphatidylcholine and phosphatidylethanolamine occupy about the same 11 cross-sectional area as the two hydrocarbon tails. 1213 2 2 2 2131211 Thus, they are in rather close contact with each other. In crystals chains of hydrogen-bonded atoms may be formed. In phosphatidylethanolamine the phosphate and–NH + ions may alternate in these chains.132 3 H H H B 3-891101122132 14 14 2 122111093-8 H N+ H –O P O H N+ H –O P O H N+ H 13 O O O O Glycerol Glycerol 10 kHz In phosphatidylcholine, in which the nitrogen is sur- rounded by methyl groups and cannot form this kind Figure 8-10 2H NMR spectra of dimyristoyl phosphatidyl- of chain, water molecules bridge between the phos- choline-d27/water in lamellar phases at 40°C. One chain of phates but the positive charges still interact with the the phosphatidylcholine is fully deuterated, containing 27 adjacent negative charges. atoms of 2H. The mole ratios of water to lipid were 5.0 in (A) The chains of hydrogen bonds between the head and 25.0 in (B). The average interfacial areas per alkyl chain groups of phosphatidylethanolamine help to stabilize as measured by X-ray diffraction were 0.252 nm2 for (A) and 0.313 nm2 for (B). 2H NMR spectra are presented as “powder the bilayer and are apparently responsible for the patterns” because the lipid molecules are randomly oriented elevation of Tm by 10 – 30° above that observed for in the magnetic field of the spectrometer as if in a powder. phosphatidylcholine.132 In contrast, the negatively This gives rise to pairs of peaks symmetrically located on charged carboxyl groups of phosphatidylserine make the both sides of the origin. The separation distances are a membrane less stable. The melting point is increased measure of the quadrupole splitting of the NMR absorption if the pH is lowered, protonating these groups. Their line caused by the 2H nucleus. The various splittings of the presence also makes the membrane sensitive to the resonances of the 13 – CH – and one – CH groups reflect 2 3 concentration of cations.133 The same is true of phos- differences in mobility.109 The peaks have been assigned phatidylglycerol, whose head group contains a tentatively as indicated. From Boden, Jones, and Sixl.115 negatively charged phosphate without an attached Courtesy of N. Boden. B. Membranes 397 counterion. Addition of calcium ions increases T thought to be a result of very high curvature of a m greatly and causes either phosphatidyglycerol or bilayer that arises from the sizes and packing of their phosphatidylserine to form a separate phase with a head groups. Another phase, even though it is liquid, more crystalline-like packing of the hydrocarbon side has a three-dimensional cubic symmetry.142– 144a It chains.134 Hydrogen bonding between head groups apparently consists of a complex arrangement of also occurs with glycolipids.135 polyhedral bilayer surfaces with interpenetrating water channels between them.143 Non-bilayer structures of phospholipids. Under appropriate conditions some aqueous phospho- Membrane fluidity and life. In agreement with lipids can exist in non-bilayer phases, a fact that may the known behavior of bilayers, the lipids of most be of considerable biological importance.119,136,137 In membranes in all organisms are partially liquid at the presence of Ca2+ some pure phospholipids can be those temperatures suitable for life. Organisms have converted to the inverted hexagonal or H phase developed at least three distinct means of ensuring II (Fig. 8-12).136,138– 140 In this phase the phospholipid that membrane lipids remain liquid.145 (1) In our heads are clustered together in cylindrical “inverted” bodies (as well as in E. coli) the unsaturated fatty acids micelles which pack in a hexagonal array. The ease that are present lower the melting point. Mutants of E. with which this transition can occur is increased by the coli that are unable to synthesize unsaturated fatty acids presence of small amounts of diacylglycerols or lyso- cannot live unless these materials are supplied in the lecithins.141 Some lipids, such as the galactosyldia- medium.146 (2) In Bacillus subtilis, which contains no un- cylglycerol of chloroplasts, do not form bilayers but saturatedfatty acids when grown at 37°C, and in other prefer the hexagonal phase structure.29,32a This is gram-positive bacteria, more than 70% of membrane A 31P NMR spectrum Phospholipid phase Mol % egg PC 0 30 ? 50 25 ppm H B Figure 8-12 (A) 31P NMR spectra of different phospholipid phases. Hydrated soya phosphatidylethanolamine adopts Figure 8-11 Results of simulated motion in a lipid bilayer the hexagonal H phase at 30°C. In the presence of 50 mol% II consisting of 64 molecules of dipalmitoylphosphatidylcho- of egg phosphatidylcholine only the bilayer phase is observed. line and 23 water molecules per lipid at a pressure of 2 atm At intermediate (30%) phosphatidylcholine concentrations and 50°C. The view is that observed after 500 ps of simula- an isotropic component appears in the spectrum. (B) Inverted tion. Bold lines represent the head group and glycerol parts micelles proposed to explain “lipidic particles” seen in freeze of the structures and the thin lines the hydrocarbon chains. fracture micrographs of bilayer mixture of phospholipids, The gray spheres represent water molecules. From Berger, e.g., of phosphatidylethanolanine + phosphatidylcholine + Edholm, and Jähnig.130 Courtesy of Dr. Olle Edholm. cholesterol. From de Kruijft et al.119 Courtesy of B. de Kruijft. 398 Chapter 8. Lipids, Membranes, and Cell Coats BOX 8-C ELECTRON PARAMAGNETIC RESONANCE (EPR) SPECTRA AND “SPIN LABELS” Unpaired electrons have magnetic moments and absorption itself. Thus, for the paramagnetic nitroxide are therefore suitable objects for magnetic resonance 2,2,6,6-tetramethylpiperidine-1-oxyl the EPR spec- spectroscopy. The technique is similar to NMR spec- trum consists of three equally spaced bands whose troscopy, but microwave frequencies of ~1010 Hz are peaks are marked at the points where the steep employed, the energies being ~100 times greater than those used in NMR.a,b Unpaired electrons are found in organic free radicals and in certain transition metal This "spin" label is often ions, both of which are important to many enzymatic attached by covalent linkage at this point to larger molecules. processes. Furthermore, spin labels in the form of N stable organic free radicals, can be attached to macro- molecules at many different points.a,c– e Coupling of O such artificially introduced unpaired electrons with 2,2,6,6-Tetramethylpiperidine-1-oxyl the magnetic moments of other unpaired electrons or of magnetic nuclei can often be observed by EPR lines in the middle of the first derivative plots cross techniques. the horizontal axis. The conditions for absorption of energy in the Coupling with the 14N nuclear spin causes split- EPR spectrometer are given by the equation ting into three lines as shown in the accompanying figure. hν = gβH o which is identical in form to that for NMR spectros- copy. Here H is the external magnetic field strength o andβ is a constant called the Bohr magneton. The value of g, the spectroscopic splitting factor, is one of the major characteristics needed to describe an EPR spectrum. The value of g is exactly 2.000 for a free electron but may be somewhat different in radicals and substantially different in transition metals. One factor that causes g values to vary with environment is spin-orbit coupling which arises because the p and d orbitals of atoms have directional character. For the same reason g sometimes has three discrete values for the three different directions (g value anisotropy). The g value parallel to the direc- tion of H (g ) often differs from that in the perpen- o || dicular direction (g⊥). Both values can be ascertained experimentally. A second feature of an EPR spectrum is hyper- EPR spectrum of tetramethylpiperidine-1-oxyl dissolved fine structure which results from coupling of the in an aqueous dispersion of phospholipids. (Top) above the main bilayer transition temperature T; (center) be- magnetic moment of the unpaired electron with t tween T and pretransition temperature; (bottom) below nuclear spins. The coupling is analogous to the spin t pretransition temperature. From Shimshick and –spin coupling of NMR (Chapter 3). The hyperfine McConnell.f splitting constant A, like the coupling constant J of NMR spectroscopy, is given in Hertz. Splitting may be caused by a magnetic atomic nucleus about which This nitroxide is more soluble in liquid regions the electron is moving or by some adjacent nucleus of bilayers than it is in solid regions. As bilayers or other unpaired electron. Sometimes important are warmed in the EPR spectrometer, the solubility chemical conclusions can be drawn from the presence of this spin-labeled compound in the lipid can be or absence of splitting. Thus, the EPR spectrum of a followed (see figure). The lower of the three spectra metal ion in a complex will be split by nuclei in the approximates that of the spin label in water alone, ligand only if covalent bonding takes place. while the others are composite spectra for which It is customary in EPR spectroscopy to plot part of the spin label has dissolved in the phospho- the first derivative of the absorption rather than the lipid bilayers. B. Membranes 399 BOX 8-C (continued) Since frequencies for EPR spectroscopy are ~100 Much of the interpretation of the observed times higher than those for NMR spectroscopy, changes in EPR spectra of spin labels is empirical. correlation times (Chapter 3) must be less than For example, the spectra in the accompanying figure ~10–9s if sharp spectra are to be obtained. Sharp can be interpreted to indicate that the spin label bandsmay sometimes be obtained for solutions, but dissolves in the lipid to a greater extent at higher samples are often frozen to eliminate molecular temperatures. The ratio f (defined in the figure) is motion; spectra are taken at very low temperatures. an empirical quantity whose change can be moni- For spin labels in lipid bilayers, both the bandwidth tored as a function of temperature. Plots of f vs T and shape are sensitively dependent upon molecular have been used to identify transition and pretransi- motion, which may be either random or restricted. tion temperatures in bilayers.f Computer simulations are often used to match EPR spectroscopy is used widely in the study of observed band shapes under varying conditions with proteins and of lipid–protein interactions.c It has those predicted by theories of motional broadening often been used to estimate distances between spin of lines. Among the many spin-labeled compounds labels and bound paramagnetic metal ions.g A high- that have been incorporated into lipid bilayers are resolution EPR technique that detects NMR transi- the following: tions by a simultaneously irradiated EPR transition is known as electron-nuclear double resonance (ENDOR).h O N a Berliner, L. J., and Reuben, J., eds. (1989) Spinlabeling. Theory and C HOOC (CH2)n O Applications, Vol. 8, Plenum, New York ((Biological Magnetic Resonance Series) (CH2)m OH b Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry, H3C CH3 Freeman, San Francisco, California (pp. 525 – 536, 1352 – 1362) c Marsh, D. (1983) Trends Biochem. Sci.8, 330 – 333 d Esmann, M., Hideg, K., and Marsh, D. (1988) Biochemistry27, CH3 3913– 3917 e Millhauser, G. L. (1992) Trends Biochem. Sci.17, 448 – 452 f Shimshick, E. J., and McConnell, H. M. (1973) Biochemistry12, O 2351– 2360 N g Voss, J., Salwinski, L., Kaback, H. R., and Hubbell, W. L. (1995) O Proc. Natl. Acad. Sci. U.S.A.92, 12295 – 12299 H h Lubitz, W., and Babcock, G. T. (1987) Trends Biochem. Sci.12, 96 – 100 fatty acids contain methyl branches (Chapter 21)147,148 membranes in many vital transport processes. Biologi- which can decrease the melting point and increase the cal membranes have a relatively high permeability to monolayer surface area by a factor of as much as 1.5. neutral molecules (including H O),64,150 and it has been 2 (3) Yet another mechanism for lowering the melting suggested that above T fatty acid chains are free to m point of fats is the incorporation of cyclopropane- rotate by 120° around single bonds from trans to gauche containing fatty acids (Chapter 21). conformations. When such rotation occurs about On the other hand, as we have already seen, cho- adjacent, or nearly adjacent single bonds, kinks are lesterol tends to reduce the mobility of molecules in formed. If a kink originates near the bilayer surface, membranesand causes phospholipid molecules to as will usually be the case, a small molecule may jump occupy a smaller area than they would otherwise. into the void created. Since the kink can easily migrate Myelin is especially rich in long-chain sphingolipids through the bilayer, a small molecule may be carried and cholesterol, both of which tend to stabilize artificial through with it.151,152 The same factors may assist bilayers. Within our bodies, the bilayers of myelin tend larger protein molecules which function in membrane to be almost solid. Bilayers of some gram-positive transport. They probably also account for the substan- bacteria growing at elevated temperatures are stiffened tial degree of hydration of bilayers which involves by biosynthesis of bifunctional fatty acids with co- both the polar head groups and water diffusing through valently joined “tails” that link the opposite sides of the nonpolar interior.153 a bilayer.149 Not only can molecules diffuse through membranes Why must membrane lipids be mobile? One but also membrane lipids and proteins can move with reason is probably to be found in the participation of respect to neighboring molecules. The rates of lateral 400 Chapter 8. Lipids, Membranes, and Cell Coats diffusion of lipids in bilayers and of antigenic proteins value at an ionic strength of 10–3 M and to still greater on cell surfaces are rapid.80,154 If diffusion of phospho- distances at lower ionic strengths. lipids is assumed to occur by a pairwise exchange of The net surface charge of a cell and the associated neighboring molecules, the frequency of such exchanges electrical double layer are important in interactions can be estimated155 as ~ 107s–1. However proteins may between cells and may influence the development of meet many obstacles to free diffusion.156 Lateral diffu- extracellular structure such as basement membranes. sion is often measured by the technique of fluorescence The net negative charge on cells also gives rise to an recovery after photobleaching. One small spot in experimentally measurable electrophoretic mobility. a bilayer that contains a dye attached to a lipid or a A characteristic of living cells is the maintenance protein is bleached by a laser beam. Lateral diffusion ofionic gradients across the plasma membrane. Thus of nearby unbleached molecules into the bleached almost all cells accumulate K+, even “pumping” it from spot can then be observed.80 Lateral diffusion can also very dilute external solutions. Cells also exclude sodium, be observed by NMR spectroscopy157 and by single- pumping it out from the cytoplasm by mechanisms particle tracking.158,159 In addition to diffusion there considered in Section C,2. If a microelectrode is inserted may often be a flow of membrane constituents in through a cell membrane and the potential difference directions dictated by metabolism.160 Although lateral is measured between the inside and outside of the cell, diffusion is fast a “flip-flop” transfer of a phospholipid aresting potential which, in nerve cells, may be as from one side of the bilayer to the other may require high as 90 mV is observed. The origin of the potential many seconds.161 However, a sudden increase in the appears to lie in the concentration differences of ions. calcium ion concentration, an important intramolecular From the value of ∆G for dilution of an ion (Eq. 6-25) signal (Chapter 11), activates a “scramblase” protein and the relationship between ∆G and electrode poten- which promotes a rapid transbilayer movement of tial (Eq. 6-63), the Nernst equation (Eq. 8-2) can be phospholipids.162 derived. According to this equation, which applies to a single ion for which the membrane is permeable, Electrical properties of membranes. Biological membranes serve as barriers to the passage of ions and polar molecules, a fact that is reflected in their high RT c  0.059 c  E = ln 1 = log 1 at 25°C electrical resistance and capacitance. The electrical m nF c  n c  2 2 (8-2) resistance is usually 103 ohms cm–2, while the capaci- tance is 0.5 – 1.5 microfarad (µF) cm–2. The correspond- ing values for artificial membranes are ~ 107 ohms cm–2 a 10-fold concentration difference across the membrane and 0.6 – 0.9 µF cm–2. The lower resistance of biological for a monovalent ion (n = 1) would lead to a 59-mV membranes must result from the presence of proteins membrane potential, E . Since membranes are relatively m and other ion-carrying substances or of pores in the impermeable to sodium ions, it is generally conceded membranes. The capacitance values for the two types that for many membranes the origin of the membrane of membrane are very close to those expected for a potential lies mainly with the potassium ion concen- bilayer with a thickness of ~ 2.5 nm and a dielectric tration difference which is maintained by the Na+, K+- constant of 2.54,84,163 The electrical potential gradient ATPase (Section C). A more complete equation takes is steep. account of K+, Na+, and Cl- together with their respec- Outer cell surfaces usually carry a net negative tive permeabilities.167– 169 Note also that Eq. 6-64 is also charge, the result of phosphate groups of phospholipids, often called the Nernst equation.170 of carboxylate groups on proteins, and of sialic acids Protons are also pumped across cytoplasmic and attached to glycoproteins. This negatively charged inner mitochondrial membranes, a topic of Chapter 18. surface layer attracts ions of the opposite charge (coun- The flow of protons from inside to outside also contri- terions), including protons, and repels those of the butes to the membrane potential. The positive charges same charge. The result is development of a diffuse of H+, K+, and other cations associated with the exter- electrical double layer consisting of the fixed nal membrane surface are balanced by the negative negative charges on the surface and a positive ionic charges of protein molecules as well as Cl– and phos- atmosphere extending into the solution for a distance phate anions that are in or near to the inner surface of that depends upon the ionic strength.164– 166 This ionic the membranes. atmosphere is analogous to that postulated by the Another possibility for proton flow has intrigued Debye–Hückel theory (Chapter 6). At the physiological biophysicists for years. Membranes often display a ionic strength of 0.145 M the thickness of the double substantial electrical conductivity in a lateral direction layer, taken as the distance at which the electrical along the membrane surface.171– 173 Electrical conduction potential falls to a certain fraction of that at the cell may involve movement of protons along hydrogen- surface,164,165 is about 0.8 nm. However, the double- bonded lines, e.g., involving ethanolamine head layer thickness increases to about three times this groups or phosphate groups and bridging water as