3:  3.4 Membrane Proteins

Proteins Interact with Membranes in Different Ways

Membrane proteins can be classified into two broad categories—integral (intrinsic) and peripheral (extrinsic)—based on the nature of the membrane-protein interactions (see Figure 3-32). Most biomembranes contain both types of membrane proteins.

Integral membrane proteins, also called intrinsic proteins, have one or more segments that are embedded in the phospholipid bilayer. Most integral proteins contain residues with hydrophobic side chains that interact with fatty acyl groups of the membrane phospholipids, thus anchoring the protein to the membrane. Most integral proteins span the entire phospholipid bilayer. These transmembrane proteins contain one or more membrane-spanning domains as well as domains, from four to several hundred residues long, extending into the aqueous medium on each side of the bilayer. In all the transmembrane proteins examined to date, the membrane-spanning domains are α helices or multiple β strands. In contrast, some integral proteins are anchored to one of the membrane leaflets by covalently bound fatty acids, as discussed later. In these proteins, the bound fatty acid is embedded in the membrane, but the polypeptide chain does not enter the phospholipid bilayer.

Peripheral membrane proteins, or extrinsic proteins, do not interact with the hydrophobic core of the phospholipid bilayer. Instead they are usually bound to the membrane indirectly by interactions with integral membrane proteins or directly by interactions with lipid polar head groups. Peripheral proteins localized to the cytosolic face of the plasma membrane include the cytoskeletal proteins spectrin and actin in erythrocytes (Chapter 18) and the enzyme protein kinase C. This enzyme shuttles between the cytosol and the cytosolic face of the plasma membrane and plays a role in signal transduction (Chapter 20). Other peripheral proteins, including certain proteins of the extracellular matrix, are localized to the outer (exoplasmic) surface of the plasma membrane.

Hydrophobic α Helices in Transmembrane Proteins Are Embedded in the Bilayer

Integral proteins containing membrane-spanning α-helical domains are embedded in membranes by hydrophobic interactions with the lipid interior of the bilayer and probably also by ionic interactions with the polar head groups of the phospholipids. Glycophorin, a major erythrocyte membrane protein, exhibits both types of interaction. As shown in Figure 3-33, glycophorin contains a membrane-embedded α helix composed entirely of hydrophobic (or uncharged) amino acids. The predicted length of this α helix (3.75 nm) is just sufficient to span the hydrocarbon core of a phospholipid bilayer. The hydrophobic side chains form van der Waals interactions with the fatty acyl chains and shield the polar carbonyl (C=O) and imino (NH) groups of the peptide bond, which are all hydrogen-bonded to one another. This hydrophobic helix is prevented from slipping across the membrane by a flanking set of positively charged amino acids (lysine and arginine) that are thought to interact with negatively charged phospholipid head groups. In glycophorin, most of these charged residues lie adjacent to the cytosolic leaflet.

Many Integral Proteins Contain Multiple Transmembrane α Helices

Although Figure 3-33 depicts glycophorin as a monomer with a single α helix spanning the bilayer, this protein is present in erythrocyte membranes as a dimer of two identical polypeptide chains. The two membrane-spanning α helices of glycophorin are thought to form a coiled-coil structure (see Figure 3-9a) stabilized by specific interactions between the amino acid side chains at the interface of the two helices. It is now known that many other transmembrane proteins contain two or more membrane-spanning α helices. For instance, the bacterial photosynthetic reaction center (PRC) comprises four subunits and several prosthetic groups, including four chlorophyll molecules. In this complex protein, three of the four subunits span the membrane; two of these subunits (L and M) each contain five membrane-spanning α helices (see Figure 16-40).

A large and important family of integral proteins is defined by the presence of seven membrane-spanning α helices. More than 150 such “seven-spanning” membrane proteins have been identified. This class of integral proteins is typified by bacteriorhodopsin, a protein found in a photosynthetic bacterium (Figure 3-34). Absorption of light by the retinal group attached to bacteriorhodopsin causes a conformational change in the protein that results in pumping of protons from the cytosol across the bacterial membrane to the extracellular space. The proton concentration gradient thus generated across the membrane is used to synthesize ATP, as discussed in Chapter 16. Both the overall arrangement of the seven α helices in bacteriorhodopsin and the identity of most of the amino acids can be resolved by computer analysis of micrographs of two-dimensional crystals of the membrane-embedded protein taken at various angles to the electron beam.

Other seven-spanning membrane proteins include the opsins (eye proteins that absorb light), cell-surface receptors for many hormones, and receptors for odorous molecules. Amino acid sequence analysis of these proteins has shown that no amino acids are found in the same position in all of them, and only a few residues are conserved in even a substantial number of them. Nonetheless, each of these proteins contains seven stretches of hydrophobic amino acids long enough (>22 amino acids) to span the phospholipid bilayer. Though direct evidence is lacking, it is thought that all of these proteins adopt a conformation in the membrane similar to that of bacteriorhodopsin. This is one of several examples of how investigators can predict the orientation of proteins in a membrane from the amino acid sequence alone.

Multiple β Strands in Porins Form Membrane-Spanning “Barrels”

The porins are a class of transmembrane proteins whose structure differs radically from that of other integral proteins. Several types of porin are found in the outer membrane of gram-negative bacteria such as E. coli(see Figure 1-7a). The outer membrane protects an intestinal bacterium from harmful agents (e.g., antibiotics, bile salts, and proteases) but permits the uptake and disposal of small hydrophilic molecules including nutrients and waste products. The porins in the outer membrane of an E. coli cell provide channels for passage of disaccharides, phosphate, and similar molecules.

The amino acid sequences of porins are predominantly polar and contain no long hydrophobic segments typical of integral proteins with α-helical membrane-spanning domains. X-ray crystallography has revealed that porins are trimers of identical subunits. In each subunit 16 β strands form a barrel-shaped structure with a pore in the center (Figure 3-35). As noted earlier, half the amino acid side groups of a β strand point in one direction, and the other half point in the opposite direction (see Figure 3-8). Unlike a typical globular protein, porins have an inside-out arrangement. In a porin monomer, the outward-facing side groups on each of the β strands are hydrophobic and thus can interact with the fatty acyl groups of the membrane lipids or with other porin monomers. The side groups facing the inside of a porin monomer are predominantly hydrophilic; these line the pore through which small water-soluble molecules cross the membrane.

Covalently Attached Hydrocarbon Chains Anchor Some Proteins to the Membrane

In eukaryotic cells, as noted earlier, the polypeptide chain of some integral membrane proteins does not enter the bilayer but rather is anchored in one leaflet by a covalently attached hydrocarbon chain. Several common lipid anchors are shown in Figure 3-36.

Some cell-surface proteins are anchored to the exoplasmic face of the plasma membrane by a complex glycosylated phospholipid that is linked to the C-terminus. A common example of this type of anchor is glycosylphosphatidylinositol, which contains two fatty acyl groups, N-acetylglucosamine, mannose, and inositol (see Figure 3-36a). Several enzymes, including alkaline phosphatase, fall into this class. Various experiments have shown that the phospholipid anchor is both necessary and sufficient for binding these cell-surface proteins to the membrane. For instance, the enzyme phospholipase C cleaves the phosphate-glycerol bond in phospholipids as well as in glycosylphosphatidylinositol anchors, and treatment of cells with phospholipase C releases glycosylphosphatidylinositol-anchored proteins such as Thy-1 protein and alkaline phosphatase from the cell surface.

Some cytosolic proteins are anchored to the cytosolic face of membranes by a hydrocarbon moiety covalently attached to a cysteine near the C-terminus. The most common anchors are prenyl, farnesyl, and geranylgeranyl groups. These proteins undergo a chemical modification involving several steps. First, the anchor moiety forms a thioether bond with the thiol group of a cysteine that is four residues from the C-terminus of the protein. The modified protein then undergoes proteolysis and methylation; these reactions remove the three terminal residues and add a methyl to the new C-terminus. In some cases, fatty acyl palmitate groups form thioester bonds to nearby cysteine residues, providing additional anchors that are thought to reinforce the attachment of the protein to the membrane (see Figure 3-36b).

In another group of lipid-anchored cytosolic proteins, a fatty acyl group (e.g., myristate or palmitate) is linked by an amide bond to the N-terminal glycine residue (see Figure 3-36c). In these proteins, the N-terminal anchor is necessary for retention at the membrane and may play an important role in a membrane-associated function. For example, v-Src, a mutant form of a cellular tyrosine kinase, is oncogenic and can transform cells only when it retains a myristylated N-terminus.

Some Peripheral Proteins Are Soluble Enzymes That Act on Membrane Components

An important group of peripheral membrane proteins are water-soluble enzymes that associate with the polar head groups of membrane phospholipids. One well-understood group of such enzymes are the phospholipases, which hydrolyze various bonds in the head groups of phospholipids (Figure 3-37). These enzymes have an important role in the degradation of damaged or aged cell membranes.

The mechanism of action of phospholipase A₂ illustrates how such water-soluble enzymes can reversibly interact with membranes and catalyze reactions at the interface of an aqueous solution and lipid surface. When this enzyme is in aqueous solution, its Ca²⁺-containing active site is buried in a channel lined with hydrophobic amino acids. Binding of the enzyme to a phospholipid bilayer induces a small conformational change that fixes the protein to the phospholipid heads and opens the hydrophobic cleft. As a phospholipid molecule moves from the bilayer into the channel, the enzyme-bound Ca²⁺ binds to the phosphate in the head group and positions the ester bond to be cleaved next to the catalytic site.

SUMMARY

• Biological membranes usually contain both integral and peripheral membrane proteins (see Figure 3-32).

• Integral membrane proteins include transmembrane proteins and lipid-anchored proteins.

• Two types of membrane-spanning domains are found in transmembrane proteins: one or more α helices or, less commonly, multiple β strands (as in porins). Proteins containing seven membrane-spanning α helices form a major class that includes bacteriorhodopsin and many cell-surface receptors.

• When the polypeptide chain of a transmembrane protein spans the membrane multiple times, the core of the protein generally is hydrophilic, permitting passage of water-soluble molecules, and the surface is hydrophobic, permitting interaction with the interior of the lipid bilayer.

• Amino acid residues modified with long-chain hydrocarbons anchor some integral proteins to one membrane leaflet (see Figure 3-36).

• Peripheral membrane proteins interact with integral membrane proteins or with the polar head groups of membrane phospholipids. They do not enter the hydrophobic core of the membrane.