Membrane Structure

The detailed structure of membranes differs considerably according to their location. However a general model for most membranes is consistent with an extended bilayer of amphiphilic lipids with hydrophobic moieties directed to the centre and hydrophilic head groups at the two surfaces. Ionic or hydrophobic interactions occur between lipids and membrane proteins. The latter are located at the surface or embedded within the membrane.

6.5.1 Early models already envisaged a bilayer of lipids but were uncertain about the location of the proteins

It has been known for a long time that membranes were composed basically of lipid and protein. The question was how were these constituents arranged in the membrane and how could their special properties be reconciled with their possible physiological functions?

In 1925, the Dutch workers Gorter and Grendel extracted the lipids from erythrocytes and calculated the area occupied by the lipid from a known number of cells when it was spread as a monolayer on a Langmuir Trough. There was sufficient, they claimed, to surround a red cell in a layer two molecules thick. (Actually these workers made two mistakes which happened to exactly cancel each other out. First, they didn't know how hard to compress the monolayer and, hence, estimate the area accurately. Secondly, they made no allowance for the contribution of proteins.) Nevertheless, the estimates made by Gorter and Grendel turned out to be quite accurate and were accepted at the time to indicate that a bimolecular lipid leaflet surrounded cells. This organization, which provides a permeability barrier between various cellular compartments, was further refined by Danielli and Davson. They were unaware of Gorter and Gren-del's paper and proposed a similar model in 1935, but realized that the measured surface tension of the membrane was too low to be accounted for by lipid and proposed that a layer of protein was present at each surface.

With the advent of electron microscopy came pictures of membranes that seemed to support the Danielli model. For the purposes of electron microscopy, the specimen has to be dehydrated, stained (usually with osmium tetroxide or potassium permanganate - so-called positive staining) and embedded in a plastic material such as an epoxy resin. The sample sections are then cut and examined under the microscope. Much of the interpretation of the structures observed depends on how much shrinkage occurs during dehydration of the sample, and what regions of the membrane take up the stain; it is now generally accepted that osmium tetroxide accumulates at the polar regions of lipid and protein. In just about every membrane examined in this way a triple-layered structure [two dark lines on either side of a light band; see Fig. 6.10(b)], was seen on the micrographs and on this basis Robertson put forward the unit membrane hypothesis, which held that every membrane had a basic structure consisting of a bimolecular lipid leaflet sandwiched between a layer of protein on one side and glycoprotein on the other. Further support for the hypothesis came from X-ray studies on myelin by Finean. Myelin is an ideal material for this purpose; it can be easily isolated in a pure state, has a simple composition and regular repeating features providing good diffraction patterns.

In Table 6.2 we have tried to indicate that from the point of view of composition at least, there is no such thing as a typical membrane. With such widely differing compositions it is unreasonable to expect a universal structure. Myelin, the structure of which lent most support to the unit membrane theory, seems to be the most atypical.

6.5.2 The lipid-globular protein mosaic model now represents the best overall picture of membrane structure

Implicit in the bimolecular leaflet model is the idea that the protein is spread as an extended sheet (P-conformation) over the ionic heads of the phos-pholipids and that the binding between phospho-lipid and protein is essentially electrostatic. Several observations about membrane lipids and proteins were not consistent with this model.

First, the protein component of many membranes represents well over 50% of the bulk of the membrane material. The average amino acid composition of membrane proteins shows no marked preponderance of ionic or hydrophobic residues. Thus, if the protein were extended as a sheet, a significant proportion of the hydrophobic groups would have to be in contact with the water.

Secondly, increasing the ionic strength of the medium surrounding the membrane does not dissociate a large fraction of membrane proteins from lipids as would be expected if electrostatic interactions were predominant.

Thirdly, when the lipid moiety is removed, the proteins are not very soluble in water; in fact they tend to interact hydrophobically with one another.

Lastly, most membrane lipids are zwitterionic rather than having a net charge and would have no strong electrostatic interactions. Those that are charged tend to be acidic and preferably interact with predominantly basic proteins, which are not common in membranes.

Chloroplast Freeze Etching

Fig. 6.10 Examples of some different electron microscopic techniques used for examining membrane preparations, (a) An electron micrograph of a 2% dispersion of dioleoyl phosphatidylcholine in water, This is an example of the 'freeze-etching' technique and illustrates the way in which phosphatidylcholine molecules take up a lamellar configuration when dispersed in water, Magnification is x98 000, (b) An electron micrograph of the chloroplast lamellae of a green narcissus petal, The specimen was fixed in glutaraldehyde osmium, embedded in epon and post-stained with lead citrate, This is an example of the 'positive staining' technique and clearly illustrates the typical 'unit membrane' feature of two dark lines separated by a light band, Magnification is x 142500, (c) An electron micrograph of beef heart mitochondria membranes, This is an example of the 'negative staining' technique and illustrates the regular array of globular lipid/protein particles (known as 'elementary particles') attached to the membrane, Some detached particles can be seen, The particles correspond to the F1 component of ATP synthase, Magnification x93 000,

Fig. 6.10 Examples of some different electron microscopic techniques used for examining membrane preparations, (a) An electron micrograph of a 2% dispersion of dioleoyl phosphatidylcholine in water, This is an example of the 'freeze-etching' technique and illustrates the way in which phosphatidylcholine molecules take up a lamellar configuration when dispersed in water, Magnification is x98 000, (b) An electron micrograph of the chloroplast lamellae of a green narcissus petal, The specimen was fixed in glutaraldehyde osmium, embedded in epon and post-stained with lead citrate, This is an example of the 'positive staining' technique and clearly illustrates the typical 'unit membrane' feature of two dark lines separated by a light band, Magnification is x 142500, (c) An electron micrograph of beef heart mitochondria membranes, This is an example of the 'negative staining' technique and illustrates the regular array of globular lipid/protein particles (known as 'elementary particles') attached to the membrane, Some detached particles can be seen, The particles correspond to the F1 component of ATP synthase, Magnification x93 000,

Subsequent observations such as the known rapid lateral diffusion of lipid and protein in the plane of the membrane and knowledge that proteins are often inserted into and through the lipid matrix have been added to the points made above. These considerations have been allowed for in the proposal by Singer and Nicholson in 1972 of their fluid mosaic model for membrane structure (Fig. 6.11). In the diagram an asymmetric lipid bilayer forms the basis of the membrane structure with proteins spanning the membrane or embedded into the hydrophobic core region.

Two techniques in the electron microscopist's armoury have helped to encourage a reappraisal of membrane structure. One is negative staining, in which the sample is not fixed and embedded but dispersed in an aqueous solution of the negative stain (phosphotungstate) and dried down on a support film [Fig. 6.10(c)]. Stain accumulates in the hydrophilic regions. The other method, drastically different from the staining methods and therefore useful to give independent corroboration, is the freeze-etching technique [Fig. 6.10(a)].

The freeze-etching technique allows a three-dimensional view of membrane surfaces to be made. Furthermore, the analogous freeze-fracture method permits visualization of the interior of the membrane bilayer. With both techniques intrinsic membrane proteins can be seen that penetrate into the bilayer or pass through it, just as proposed in the Singer/Nicholson model (Fig. 6.11).

6.5.3 Membrane structure is not static but shows rapid movement of both lipid and protein components

The ability of many membrane lipids to form the basic bilayer structure is caused by a number of properties, the most important of which is their amphipathic character. Amphipathicity is caused by the lipids having a polar or hydrophilic head

Gorter And Grendel

Fig. 6.H The Singer and Nicholson fluid-mosaic model of membrane structure. The figure shows the topography of membrane protein, lipid and carbohydrate in the fluid-mosaic model of a typical eukaryotic plasma membrane. Phospholipid asymmetry results in the preferential location of phosphatidylethanolamine and phosphatidylserine in the cytosolic monolayer. Carbohydrate moieties on lipids and proteins face the extracellular space. Reproduced with kind permission of Dr. P.R. Cullis and the Benjamin/Cummings Company, from Biochemistry of Lipids and Membranes, Benjamin/Cummings, Menlo Park, CA.

Fig. 6.H The Singer and Nicholson fluid-mosaic model of membrane structure. The figure shows the topography of membrane protein, lipid and carbohydrate in the fluid-mosaic model of a typical eukaryotic plasma membrane. Phospholipid asymmetry results in the preferential location of phosphatidylethanolamine and phosphatidylserine in the cytosolic monolayer. Carbohydrate moieties on lipids and proteins face the extracellular space. Reproduced with kind permission of Dr. P.R. Cullis and the Benjamin/Cummings Company, from Biochemistry of Lipids and Membranes, Benjamin/Cummings, Menlo Park, CA.

group region and a non-polar or hydrophobic part. Thus, such molecules will naturally orientate themselves to ensure that the polar groups associate with water molecules while the hydrophobic tails interact with each other. Provided the molecule in question is roughly cylindrical in dimension and has no net charge then bimolecular planar leaflets will be the most stable configuration in an aqueous system. However, it is also true that many major lipid components of membranes do not form bilayers when isolated and placed into aqueous systems in vitro. Some of the packing characteristics of different lipids and the structures they form are shown in Fig. 6.12.

Israelachvili, in a masterly exposition in 1978, has discussed the theoretical shapes of these structures and the consequences for cells (see Further Reading). In some naturally occurring membranes, a very high proportion of the lipid is capable of adopting the hexagonal II phase (inverted micelles) rather than bilayers. In this arrangement cylinders of lipids are formed with an aqueous interior in contact with the head groups of the lipids (Fig. 6.12). The cylinders interact with each other by hydrophobic forces and this can be clearly seen in freeze-fracture micrographs as a regular corrugated pattern.

In general, biological membranes contain an appreciable fraction (up to 40 mol%) of lipid species that, individually, prefer the hexagonal II arrangement. But it has been discovered that, depending on the acyl chain composition, temperature and head group size and charge, complete bilayer stabilization can be achieved by the addition of 10-50 mol% of bilayer-forming lipid species. Moreover, these properties are displayed without any stabilizing

Fig. 6.12 Lipid shapes and their packing characteristics.

effects from membrane proteins. Thus, many bacteria contain unsaturated phosphatidylethanola-mine and diphosphatidylglycerol in large amounts while chloroplast thylakoids contain up to 50% of their lipid as monogalactosyldiacylglycerol. Such lipids form hexagonal II phases on their own but, clearly, the bacterial and chloroplast membranes provide good permeability barriers with the vast bulk of the lipid arranged in a bilayer in vivo. Therefore, other factors such as the ionic environment, interaction with other lipids and the contributions of membrane proteins are obviously important.

For certain purposes, it seems important for non-bilayer phases to be present in membranes. A good example is in membrane fusion or in the budding of vesicles. These aspects are discussed in Section 6.5.10.

The fluidity of membranes is dependent on the nature of the hydrophobic moieties of lipids. When lipids are isolated the acyl chains of each type undergo transitions from a viscous gel to a fluid state at a certain temperature. Above the transition temperature, the molecules exist in a liquid-crystalline state where the acyl chains, but not the head group, are fluid. This is the normal state of membrane lipids and ensures proper functioning of the membrane proteins. When the temperature of a cell is lowered, three phenomena can contribute to impairment of membrane function. First, ice crystals formed in the aqueous compartments can cause physical damage. Second, transition of the acyl chains into the gel phase leads to changed enzyme activity and altered transport. Third, temperature lowering can lead to the membrane lipids becoming phase-separated. Concentration of particular lipid types by this phase separation can cause the formation of non-bilayer structures. Lipid adaptations to environmental temperatures are discussed in Section 6.5.9.

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