2.2.2—
Proteins

In many membranes, particularly in those of chloroplasts and mitochondria proteins make up most of the weight. The proteins found are many but as a first step in classifying them integral and peripheral proteins may be distinguished. This classification anticipates the subsequent discussion of membrane structure on page 38 but the terms clearly suggest that proteins in the two classes are associated with other components in the membrane in different ways. The recognition that certain proteins are embedded deeply in the lipid membrane represents a departure from the view, often advanced in earlier texts, that all of the membrane protein is located in the two peripheral bands which stain darkly with osmium and are visualized in the electron microscope (see Fig. 2.1). Whereas some of the protein is certainly located in this way and is probably bonded to the polar regions of the phospholipids electrostatically, it has become apparent that much protein is associated with the non-polar regions of the lipid by hydrophobic bonding. Peripherally located protein can be easily separated by washing with salt solutions or chelating agents but integral proteins are attached very strongly to the membrane and can be removed only after drastic treatment with organic solvents or detergents; even then, the isolated protein usually has some lipid attached to it.

Most of the membrane-bound enzymes, transport proteins (e.g. monovalent cation stimulated ATPase), drug and hormone receptors (in animal cells) and antigenic proteins are integral and are revealed when membranes are split open in freeze-fracturing (see p. 38). In many instances the enzymes arenonfunctional in vitro in the absence of lipid. It is thought that the non-polar parts of the polypeptide chains are associated with the hydrophobic tails of the fatty

acids and, this being so, several types of conformation are possible (see Singer, 1974 for a review). Proteins, like phospholipids, are amphipathic and their polar regions will arrange themselves so that their contacts with the hydrophobic regions of the membrane will be minimized; to ensure this, a protein could be arranged so that its polar, hydrophilic region lies among the phospholipid 'heads', or projects through them into the protein at the membrane periphery (Fig. 2.5).

Figure 2.5
Possible orientations of proteins in a membrane. (a) peripherally bound protein
with polar groups all over its surface. (b) and (c) non-polar regions of the protein
bonded hydrophobically to lipid but with different numbers of polar groups. (d) polar
groups at either end of a long molecule with a non-polar central region. (e) a pair of
proteins as in (d) making up a polar pore or channel in the membrane. (f) a hydrophobic
globular protein wholly in the lipid domain—polar groups, if any, directed inwardly.

Long polypeptide chains with charged groups at either end may actually lie across the width of the membrane, or groups of them might lie with polar groups directed inwardly to form a hydrophilic pore across the membrane. An alternative conformation would be provided by the formation of a globular structure in which all of the polar groups would be directed towards the centre of the globule so that a hydrophobic surface would be presented to the lipid. This latter kind of conformation is probably least common.

The peripheral proteins can be attached to the polar groups of either phospholipids or integral proteins; examples which might be taken include cytochrome c which is located on the outer surface of the inner mitochondrial membranes (Schneider et al., 1972; see also Chapter 5), the chromoprotein, phytochrome, which is thought to be attached to the plasmalemma (Marmé et al., 1974; see also Chapter 12) and the sulphate and other ion-binding proteins on the outer surface of bacterial membranes (see Oxender, 1972, for a review).

From a quantitative point of view, certain generalizations about the relative abundance of peripheral and integral proteins can be made. The greater the metabolic activity which centres upon a given membrane system the greater amount of protein integrated into it. Thus, it might be anticipated that chloroplast lamellae and inner mitochondrial membranes would be relatively rich in these proteins, whereas membranes whose role is more concerned with providing

a diffusion barrier, e.g. the plasmalemma and the tonoplast, would be less so; evidence from electron microscopy shows that this is so (see Table 2.5).