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October 2009 © 2009 The Biochemical Society

Lipids

Key words: detergent,

lipid bilayer, lysophos-

phatide, membrane lipid,

sphingolipid

The biophysical mastery of indentured servants

Boundary layersLipids are an extremely diverse assortment of biochemical compounds that defy a unified structural defi-nition. The physical characteristic that distinguishes them from non-lipid compounds is their solubility in solvents of low polarity. This feature is handy when it comes to isolation because they can be conveniently extracted into organic solvents virtually free of contamination by other cellular constituents.

17741. The relevant passage is reproduced below:

This description of a monomolecular film lay fallow until the early part of the last century when Irving Lang-muir2, who received the 1932 Nobel Prize in Chemistry, undertook his work on surfactant films.

The chemical origin of surfactant activity of lipids lies in their amphiphilic properties. Thus a polar group comprising a charged or hydroxylated function is located at one end of the molecule and is discretely separated from hydrocarbon at the other. The extent of the hydro-carbon domain is the reason membrane lipids are weak surfactants and have low solubility in water. They can, however, be converted into relatively strong detergents able to haemolyse red blood cells simply by removing one of the fatty acyl chains; for this reason, such lipids are referred to as lysophosphatides. The balance of hydro-phobic and hydrophilic affinity determines the structure formed by the aggregates on dispersal in water.

Membrane lipids form osmotically active barriers

The concept that lipids act as a barrier between the liv-ing cell and its environment stems from the pioneer-ing experiments performed in Zurich at the end of the 19th Century by the Englishman Ernest Overton3. He reported that plasmolysis of plant and animal cells was produced by a variety of water-soluble solutes, but not with lower alcohols, ether or chloroform. The selective permeability was related to the oil–water partition co-

Membrane lipids represent a special category of lipids because they have properties of a surfactant. They are, however, relatively weak detergents as judged by their critical micelle concentration (CMC). By way of comparison a typical domestic washing up liquid has a CMC in the millimolar range, whereas that of membrane lipids in free solution is in the nanomolar range. This means that membrane lipids are essentially insoluble in water and are overwhelmingly dispersed in the form of aggregates.

The polar lipids are almost invariably found as components of cell membranes. Notable exceptions to this rule are egg yolk, lung surfactant and the prolamel-lar bodies of chloroplasts. All these structures require a variety of special proteins to organize the lipids into organelles which release their membrane progenitors in response to specific triggers. This infers that in most cases membrane lipids are synthesized and are subject to metabolic turnover at rates synchronized with the proc-esses of membrane biosynthesis and differentiation.

Origins of surfactant activity

Historically, we need to go back to the 18th Century to find a well-documented account of the biophysical properties of lipids. The experimentalist responsible was the renowned polymath and statesman, Benjamin Franklin, who repeated the experiments of Pliny the Elder (ad77) on Clapham Pond in 1762. Figure 1 shows an etching of the location where the experi-ments were performed. In his account, he writes “…a teaspoonful [of oil] produced an instant calm over a space of several yards square which spread amazingly and extended itself gradually till it reached the lee side, making all that quarter of the pond, perhaps half an acre, as smooth as a looking glass.”

These experiments were a prelude to the profound observation he describes in a letter to Doctor Brown-rigg published in the Philosophical Transactions in

Peter J. Quinn (Biochemistry Department, King’s College London)

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Lipids

effi cient, meaning that passage of the solute through the membrane involved interaction with a membrane com-ponent rather than through a water-fi lled pore. From the nature of the permeant solutes, Overton concluded these components consisted of a mixture of cholesterol and its esters with lecithin (phosphatidylcholine).

Lipid arrangement in membranes

With the concept of a semi-permeable lipid membrane and the later studies of the surfactant properties of lipids, the next question was how were the lipids ar-ranged in the membrane? Th is challenge was taken up by Gorter and Grendel4 working at the University of Leiden. Th ey used a Langmuir trough, modifi ed by N.K. Adam in Cambridge, incorporating a surface bal-ance to measure lateral pressure in the monomolecular fi lm5 (Figure 2). Th eir experiment consisted of com-paring the area occupied at an air/water interface by the lipids extracted from a known area of membrane. Th ey examined mature erythrocytes that possess only a plasma membrane from six species, including humans. It was found that the area occupied by the lipid at the air/water interface was almost exactly twice that of the area of membrane from which it was extracted. Th is led to the conclusion that the structure of cell membrane was composed of a lipid bilayer.

Th e methods used in these experiments, however, were subsequently found to be fl awed. Th e use of acetone solvent, for example, resulted in incomplete extraction of the lipid, but this was partially compensated for by inaccuracy in the measurement of membrane area. Fur-thermore, there was also uncertainty about the packing density of lipid molecules in the surface fi lm at which the area was measured; in fact, the measurements were made on fi lms with a surface pressure of only 2 mN·m−1. It is now known, on the basis of the diff erential action of snake venoms and endogenous phospholipases on sub-strate monolayers of diff erent densities6, that phospho-lipids in a bilayer confi guration are packed at a density equivalent to a monomolecular fi lm pressure of 30–35 mN·m−1. Repeat of the Gorter and Grendel experiment to ensure complete lipid extraction and with accurate measurements of membrane area7 and calculations per-formed on the basis of molecular dimensions8 invariably showed that the area occupied by the lipids is less than required to form two layers over the entire membrane area. In this case concluding that the additional space is occupied by protein is logically fl awed because it is assumed that the lipids are arranged in a bilayer!

Th e current model of biological membranes pro-posed by Singer and Nicolson9 in 1972 incorporates these ideas and envisages the matrix of the membrane to be a bilayer of fl uid lipids with the remaining area

occupied by intrinsic proteins. Th e arguments put forward in conceptualizing the arrangement of the lipids in the model were primarily thermodynamic. Th e arrangement of lipids in a bilayer confi guration minimizes entropy by providing maximum exposure of the polar groups to water while sequestering the hydro-carbon away from the interface into the interior of the structure. Much of the experimental evidence cited was tenuous and based on calorimetry and spectroscopic methods that do not inform on structure. Th e critical piece of evidence was produced by Maurice Wilkins which more than justifi es the award of a Nobel Prize for the molecular confi guration of nucleic acids.

Wilkins10 examined X-ray scattering patterns from oriented multilayers of egg lecithin (phosphatidylcho-line) deposited on mica surfaces. Th e relative atomic positions within the structure were determined from an analysis of the intensity distribution between the various diff raction bands by means of a Fourier rela-tionship which equates electron density distribution with the X-ray scattering intensity pattern. In perform-ing these calculations, not only is the amplitude of the scattered X-rays important (amplitude is proportional to the square root of the intensity), but also knowledge of their phase is required. Th ere are no precise meth-ods for assigning the correct phase and usually one of a number of phase permutations are possible. Neverthe-less, the choice of phases giving relative electron densi-ties shown in Figure 3 gave the best fi t to the model of a lipid bilayer and so far this has remained unchal-lenged. X-ray scattering from lipid layers containing 14% water were compared with specimens hydrated with 21% water. Th is showed that the distance separat-ing the peaks of high electron density, assigned to the electron-dense phosphate groups on either side of the

Figure 1. An etching of Long Pond on Clapham Common by W.H. Urwick ca. 1870. This Arcadian view of the pond post-dated Franklin’s experiment by more than a century. The ponds were created by excavation of gravel to con-struct the turnpike road and elevate the foundations of St Paul’s Church.

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bilayer, was not significantly changed by manipulat-ing the water content, whereas the distance between the bilayers was largely responsible for the change in overall repeat spacing of the structure. Thus the model places all the water between the lipid layers from which water is excluded. The region of lowest electron density occurs in the central plane of the bilayer where the ter-minal methyl groups of the fatty acid chains reside.

Stability of lipid bilayers

Although lipid bilayers are relatively robust structures considering they are of the order of only 5–6 nm thick, the mixtures of lipids found in cell membranes do not form stable bilayers. This appears somewhat enigmatic if one conceives the function of membranes to be se-lective permeability barriers to the passage of solutes into and out of the cell and between subcellular com-partments. Membranes, of course, support a variety of biochemical processes, not least those that are medi-ated by the different membrane proteins.

Reconstitution studies in which membrane proteins are removed from endogenous lipids in order to inves-tigate the role of lipid–protein interactions in these processes almost invariably demonstrate that restora-tion of catalytic function simply requires incorporation into bilayers of pure synthetic phospholipid. This raises the question central to the role of lipids in membrane structure: why do most membranes comprise liter-ally hundreds of molecular species of lipid? In order to approach this question, it is necessary, as in defining lipids, to make some generalizations.

Membrane lipids fall broadly into two categories: those that, in pure form, assemble in a bilayer structure when dispersed in aqueous media under physiological conditions and those that do not. The classes of mem-brane lipid forming bilayers are molecular species of the choline phosphatides, phosphatidylcholine and sphingomyelin, phosphatidylserines, phosphatidyli-nositols, phosphatidylglycerols and dihexosyldiacylg-lycerols. Membrane lipids belonging to the classes of phosphatidylethanolamine and monohexosyldiacyl-glycerols form, so-called, hexagonal-II structure. This structure consists of the organization of the lipids into water-filled tubes with the polar groups lining the tubes which are then packed together into a hexagonal array.

Without exception, all cell membranes have rep-resentatives of both type of lipid. The proportion, however, is dependent on the ratio of protein to lipid in the membrane such that membranes with a high proportion of protein likewise have high proportions of lipids that prefer to form hexagonal-II structure. Indeed examination of aqueous dispersions of total polar lipid extracts of membranes show that they do

rela

tive

elec

tron

den

sity

nm

0−2−4 42

14% water

21% water

Figure 3. Fit of molecular arrangement of phospholipids to relative electron-density profiles through the unit cell of a smectic phase of phospholipid aligned on a silica substrate10. The bilayer thickness is relatively unchanged by different hydrations of the phospholipid.

Figure 2. Adam’s modified surface balance trough of the type used in the Gorter and Grendel experiment.

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the non-bilayer-forming lipids so that they remain constrained in bilayer form when the cells are sub-sequently thawed. If they are not so constrained, rewarming above their gel-transition temperature allows them to form non-bilayer structures that irreversibly destroy the selective permeability proper-ties of the membrane.

Lipid domain formation in membranes

Considerable interest has been generated over the last few years in the ability of lipids to form domains of ordered structure in a fluid bilayer matrix. Isolation of these domains by virtue of their ability to survive treatment with the detergent Triton X-100, at 4°C has proved problematic, but studies employing treat-ments at 37°C provide confidence that such domains are indeed structural entities of the membranes of living cells12. These so-called membrane rafts are be-lieved to act as signalling platforms by which recep-tors are arrayed on the cell surface and coupled with appropriate effectors on the cytoplasmic surface. The ordered structures are said to act as selective filters that include particular membrane proteins and ex-clude the remainder.

Lipid order results from the interaction of cho-lesterol and the more saturated molecular species of phospholipid, and these are found to be prominent components of membrane rafts. The condensing effect

not form homogeneous bilayers, but instead assemble into a mixture of lamellar and non-lamellar structures. Since all lipids in cell membranes are arranged in a bilayer, it follows that an interaction of non-bilayer-forming lipids with other membrane constituents must constrain them into a bilayer confi guration.

Role of non-bilayer-forming membrane lipids

As indicated above, reconstitution of catalytic activity of membrane proteins can usu-ally be achieved with pure bilayer-forming lipids, but restoration of biochemical func-tion requires both types of lipid. For example, solute pumps can translate their cargo across bilayers, but cannot create a gradient because the solute passively diff uses back across the membrane through channels that occur at the protein/lipid interface. One essential function that non-bilayer lipids perform is to seal this interface by fi lling the interstices created by the irregular protein surface with hydrocarbon of which they have a greater preponderance than their bilayer-forming counterparts.

Other essential functions attributed to non-bilayer-forming lipids are to organize the oligomeric protein complexes, such as those of mitochondria and chloroplasts, into functional assemblies and to mediate fusion between mem-branes. The process of membrane fusion is central to the conduct of subcellular traffic, secretion and membrane biogenesis, and a critical step in fusion involves an intermediate non-bilayer configuration of the lipids.

Th e function of non-bilayer-forming lipids in packaging intrinsic oligomeric pro-tein complexes is demonstrated in the experiment illustrated in Figure 411. Th is shows a freeze–fracture electron micrograph of the plasma membrane of the blue-green alga Synecoccus. When the alga is thermally quenched from the growth temperature, the membrane-associated particles, said to represent intrinsic membrane proteins, are randomly distributed in the fracture plane along the central hydrophobic domain of the membrane. Th e organism in this case, although grown at 38°C, was cooled to 15°C prior to thermal quenching, and this has produced a phase-separation of the membrane components. Th e conventional interpretation is that the high melting point lipids, of which non-bilayer-forming lipids are a prominent fraction, phase-separate into a gel phase from which intrinsic membrane proteins are excluded. By phase-separation, it is usually inferred that this is a lateral phase-separation and this is in part true as can be seen by an increase in density of particles around the periphery of the smooth regions of the membrane fracture plane. Th e additional particles in this high-density region, however, cannot account for all the particles originally present in the membrane. Th is suggests that membrane proteins have been ejected from the hydrophobic interior of the membrane into the aqueous phase or the constituents of the particles have under-gone a reorganization that enables them to accommodate into a lamellar gel phase.

Synecoccus can be cultured over a wide range of temperatures, but whatever this is, the distribution of membrane-associated particles is random if thermally quenched from the growth temperature. Analysis of the molecular species of membrane lipids at diff erent growth temperatures shows that the organism is able to adapt its mem-brane lipid composition to refl ect the particular environmental conditions in which it is growing. Because the tendency of lipids to form non-bilayer structures is temper-ature-dependent, the adaptive response acts to preserve the balance between bilayer- and non-bilayer-forming lipids under the prevailing growth conditions. Retailoring membrane lipids is a general feature of adaptation of poikilothermic organisms to changing temperature, salinity and other environmental factors.

A noteworthy aspect of the experiment depicted in Figure 4 is that the organism does not survive the temperature shift. It undergoes a physiological injury known as cold shock which is analogous in many respects to the damage that occurs to cells when they are frozen to sub-zero temperatures. The action of cryopreserva-tive agents in such events is to effectively lower the gel-transition temperature of

Figure 4. Electron micrograph of a freeze–fracture replica recovered from a longitudinal fracture plane in the plasma membrane of Synecoccus cultured at 38°C and thermally quenched from 15°C.

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of cholesterol on polar lipids was first reported in the Croonian Lectures of J.B. Leathes13, who noted from studies of mixed monolayers at the air/water inter-face that “it [the condensing effect] is observed with fatty acids even more than lecithine [sic], though still observable with this, tempts one to suppose that the action is between cholesterol and the paraffin chains rather than the complex glyceryl cholyl phosphoric acid, and therefore is again a change in physical be-haviour, an alteration in the force of cohesion between these chains, that depends upon chemical characteris-tics not capable of resulting in actual chemical union in the ordinary sense”. The examination of cholesterol/phospholipid mixtures by modern spectroscopic tech-niques conceptualizes the rigid sterol ring structure as hindering the rotational motion of the fatty acyl chains of the phospholipid.

A more recent discovery is why sphingolipids, particularly molecular species of sphingolipids with conspicuously long N-acyl fatty acids, are also promi-nent constituents of membrane rafts. There has been considerable speculation about the role of these sphin-golipids since a sizable proportion of them, especially in myelin and other tissues of the central nervous system, were shown to contain nervonic (C24:1) and similar fatty acids up to 26 carbons in length14. One idea was that the long-chain fatty acid interdigitated with the hydrocarbon chains of lipids in the appos-ing monolayer to couple the two halves of the bilayer. However, it turns out that the additional hydrocarbon forces the polar group of the sphingolipid into the aqueous phase, thereby preventing intermolecular hydrogen-bonding leading to gel-phase-separation of the sphingolipid in the plane of the membrane. Instead, the sphingolipids form a stoichiometric complex with diacyl phospholipids with properties of a liquid-ordered phase similar to that formed by the interaction of cholesterol with phospholipids15.

Peter Quinn’s main research is on biological membranes and their constituents. His primary approach in this research has been to apply a range of biophysical methods including time-resolved X-ray di� raction, di� erential scanning

calorimetry, freeze-fracture electron microscopy, nuclear magnetic resonance spectroscopy, laser � ash photolysis and Fourier transform infrared spectroscopy to address questions concerned with relationships between biomembrane structure and function. Other research includes the development of sensor devices based on nonlinear optical detection systems. He took his PhD at the University of Sydney and went on to an MSc in Immunology at University of London followed by a DSc in Biochemistry, also at London. He was appointed Professor of Biochemistry, King’s College London in 1989 and Emeritus Professor of Biochemistry in 2009. email: p.quinn@kcl.ac.uk

1. Franklin, B., Brownrigg, W. and Farish (1774) Philos. Trans. 64, 445–460

2. Langmuir, I. (1917) J. Am. Chem. Soc. 39, 1848–19063. Overton, E. (1895) Vjschr. Naturf. Ges. Zurich 40,

159–2014. Gorter, E. and Grendel, F. (1925) J. Exp. Med. 41, 439–4435. Adam, N.K. (1921) Proc. R. Soc. London Ser. A101,

452–4726. Demel, R.A., Geurts van Kessel, W.S.M., Zwaal, R.F.A.,

Roelofsen, B. and van Deenen, L.L.M. (1975) Biochim. Biophys. Acta 406, 97–107

7. Bar, R.S., Deamer, D.W. and Cornwell, D.G. (1966) Science 153, 1010–1012

8. Engelman, D.M. (1969) Nature 223, 1279–12809. Singer, S.J. and Nicolson, G.L. (1972) Science 175,

720–73110. Levine Y.K. and Wilkins, M.H.F. (1971) Nat. New Biol. 230,

69–7211. Furtado, D., Williams, W. P., Brain, A. P. and Quinn, P.J.

(1979) Biochim Biophys Acta 555 352–35712. Chen, X., Jen, A., Warley, A., Lawrence, M.J., Quinn, P.J.

and Morris, R.J. (2009) Biochem. J. 417, 525–53313. Leathes, J.B. (1925) Lancet 1, 853–85614. Klenk, E. (1927) Z. Physiol. Chem. 166, 268–28615. Quinn, P.J. (2009) Biochim. Biophys. Acta, doi:10.1016/j.

bbamem.2009.06.020

References

Prospective

What do we still need to know about these captive slaves that are indentured to form the matrix of bio-logical membranes? One obvious avenue of explora-tion is the need for a detailed understanding of the way ordered lipid domains organizes the arrangement of particular membrane proteins. Another, which has only been touched upon in this article, is the bio-chemical pathways responsible for maintaining mem-brane lipid homoeostasis and how these are regulated. What, for example, are the biophysical mechanisms for sensing the unique molecular species composition of each morphologically distinct membrane in the cell and ensuring that this is preserved within relatively narrow limits? No doubt, with the deployment of more sophisticated lipidomic techniques, the answers to these questions will be forthcoming in the not too distant future. ■