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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 12.5Proteins Carry Out Most Membrane Processes

We now turn to membrane proteins, which are responsible for most of the dynamic processes carried out by membranes. Membrane lipids form a permeability barrier and thereby establish compartments, whereas specific proteins mediate nearly all other membrane functions. In particular, proteins transport chemicals and information across a membrane. Membrane lipids create the appropriate environment for the action of such proteins.

Membranes differ in their protein content. Myelin, a membrane that serves as an insulator around certain nerve fibers, has a low content of protein (18%). Relatively pure lipids are well suited for insulation. In contrast, the plasma membranes or exterior membranes of most other cells are much more active. They contain many pumps, channels, receptors, and enzymes. The protein content of these plasma membranes is typically 50%. Energy-transduction membranes, such as the internal membranes of mitochondria and chloroplasts, have the highest content of protein, typically 75%.

The protein components of a membrane can be readily visualized by SDS-polyacrylamide gel electrophoresis. As discussed earlier (Section 4.1.4), the electrophoretic mobility of many proteins in SDS-containing gels depends on the mass rather than on the net charge of the protein. The gel-electrophoresis patterns of three membranes—the plasma membrane of erythrocytes, the photoreceptor membrane of retinal rod cells, and the sarcoplasmic reticulum membrane of muscle—are shown in Figure 12.16. It is evident that each of these three membranes contains many proteins but has a distinct protein composition. In general, membranes performing different functions contain different repertoires of proteins.

Figure 12.16. Sds-Acrylamide Gel Patterns of Membrane Proteins.

Figure 12.16

Sds-Acrylamide Gel Patterns of Membrane Proteins. (A) The plasma membrane of erythrocytes. (B) The photoreceptor membranes of retinal rod cells. (C) The sarcoplasmic reticulum membrane of muscle cells. [Courtesy of Dr. Theodore Steck (Part A) and Dr. (more...)

12.5.1. Proteins Associate with the Lipid Bilayer in a Variety of Ways

The ease with which a protein can be dissociated from a membrane indicates how intimately it is associated with the membrane. Some membrane proteins can be solubilized by relatively mild means, such as extraction by a solution of high ionic strength (e.g., 1 M NaCl). Other membrane proteins are bound much more tenaciously; they can be solubilized only by using a detergent or an organic solvent. Membrane proteins can be classified as being either peripheral or integral on the basis of this difference in dissociability (Figure 12.17). Integral membrane proteins interact extensively with the hydrocarbon chains of membrane lipids, and so only agents that compete for these nonpolar interactions can release them. In fact, most integral membrane proteins span the lipid bilayer. In contrast, peripheral membrane proteins are bound to membranes primarily by electrostatic and hydrogen-bond interactions with the head groups of lipids. These polar interactions can be disrupted by adding salts or by changing the pH. Many peripheral membrane proteins are bound to the surfaces of integral proteins, on either the cytosolic or the extracellular side of the membrane. Others are anchored to the lipid bilayer by a covalently attached hydrophobic chain, such as a fatty acid.

Figure 12.17. Integral and Peripheral Membrane Proteins.

Figure 12.17

Integral and Peripheral Membrane Proteins. Integral membrane proteins (a, b, and c) interact extensively with the hydrocarbon region of the bilayer. Nearly all known integral membrane proteins traverse the lipid bilayer. Peripheral membrane proteins ( (more...)

12.5.2. Proteins Interact with Membranes in a Variety of Ways

Although membrane proteins are more difficult to purify and crystallize than are water-soluble proteins, researchers using x-ray crystallographic or electron microscopic methods have determined the three-dimensional structures of more than 20 such proteins at sufficiently high resolution to discern the molecular details. As noted in Chapter 3, the structures of membrane proteins differ from those of soluble proteins with regard to the distribution of hydrophobic and hydrophilic groups. We will consider the structures of three membrane proteins in some detail.

Proteins Can Span the Membrane with Alpha Helices

The first membrane protein that we consider is the archaeal protein bacteriorhodopsin, shown in Figure 12.18. This protein acts in energy transduction by using light energy to transport protons from inside the cell to outside. The proton gradient generated in this way is used to form ATP. Bacteriorhodopsin is built almost entirely of α helices; seven closely packed α helices, arranged almost perpendicularly to the plane of the membrane, span its 45-Å width. Examination of the primary structure of bacteriorhodopsin reveals that most of the amino acids in these membrane-spanning α helices are nonpolar and only a very few are charged (Figure 12.19). This distribution of nonpolar amino acids is sensible because these residues are either in contact with the hydrocarbon core of the membrane or with one another. Membrane-spanning α helices are the most common structural motif in membrane proteins. As will be discussed in Section 12.5.4, such regions can often be detected from amino acid sequence information alone.

Figure 12.18. Structure of Bacteriorhodopsin.

Figure 12.18

Structure of Bacteriorhodopsin. Image mouse.jpg Bacteriorhodopsin consists largely of membrane-spanning α helices. (A) View through the membrane bilayer. The interior of the membrane is green and the head groups are red. (B) A view from the cytoplasmic side (more...)

Figure 12.19. Amino Acid Sequence of Bacteriorhodopsin.

Figure 12.19

Amino Acid Sequence of Bacteriorhodopsin. The seven helical regions are highlighted in yellow and the charged residues in red.

A Channel Protein Can Be Formed from Beta Strands

Porin, a protein from the outer membranes of bacteria such as E. coli and Rhodo-bacter capsulatus, represents a class of membrane proteins with a completely different type of structure. Structures of this type are built from β strands and contain essentially no α helices (Figure 12.20).

Figure 12.20. Structure of Bacterial Porin (from Rhodopseudomonas blastica).

Figure 12.20

Structure of Bacterial Porin (from Rhodopseudomonas blastica). Image mouse.jpg Porin is a membrane protein built entirely of beta strands. (A) Side view. (B) View from the periplasmic space. Only one monomer of the trimeric protein is shown.

The arrangement of β strands is quite simple: each strand is hydrogen bonded to its neighbor in an antiparallel arrangement, forming a single β sheet. The β sheet curls up to form a hollow cylinder that functions as the active unit. As its name suggests, porin forms pores, or channels, in the membranes. A pore runs through the center of each cylinder-like protein. The outside surface of porin is appropriately nonpolar, given that it interacts with the hydrocarbon core of the membrane. In contrast, the inside of the channel is quite hydrophilic and is filled with water. This arrangement of nonpolar and polar surfaces is accomplished by the alternation of hydrophobic and hydrophilic amino acids along each β strand (Figure 12.21).

Figure 12.21. Amino Acid Sequence of a Porin.

Figure 12.21

Amino Acid Sequence of a Porin. Some membrane proteins such as porins are built from β strands that tend to have hydrophobic and hydrophilic amino acids in adjacent positions. The secondary structure of Rhodopseudomonas blastica is shown, with (more...)

Embedding Part of a Protein in a Membrane Can Link It to the Membrane Surface

The structure of the membrane-bound enzyme prostaglandin H2 synthase-1 reveals a rather different role for α helices in protein-membrane associations. This enzyme catalyzes the conversion of arachidonic acid into prostaglandin H2 in two steps: a cyclooxygenase reaction and a peroxidase reaction (Figure 12.22). Prostaglandin H2 promotes inflammation and modulates gastric acid secretion. The enzyme that produces prostaglandin H2 is a homodimer with a rather complicated structure consisting primarily of α helices. Unlike bacteriorhodopsin, this protein is not largely embedded in the membrane but, instead, lies along the outer surface of the membrane firmly bound by a set of α helices with hydrophobic surfaces that extend from the bottom of the protein into the membrane (Figure 12.23). This linkage is sufficiently strong that only the action of detergents can release the protein from the membrane. Thus, this enzyme is classified as an integral membrane protein, although it is not a membrane-spanning protein.

Figure 12.22. Formation of Prostaglandin H2.

Figure 12.22

Formation of Prostaglandin H2. Prostaglandin H2 synthase-1 catalyzes the formation of prostaglandin H2 from arachidonic acid in two steps.

Figure 12.23. Attachment of Prostaglandin H2 Synthase-1 to the Membrane.

Figure 12.23

Attachment of Prostaglandin H2 Synthase-1 to the Membrane. Image mouse.jpg Prostaglandin H2 synthase-1 is held in the membrane by a set of α helices coated with hydrophobic side chains. One monomer of the dimeric enzyme is shown.

Image caduceus.jpg The localization of prostaglandin H2 synthase-l in the membrane is crucial to its function. The substrate for this enzyme, arachidonic acid, is a hydrophobic molecule generated by the hydrolysis of membrane lipids. Arachidonic acid reaches the active site of the enzyme from the membrane without entering an aqueous environment by traveling through a hydrophobic channel in the protein (Figure 12.24). Indeed, nearly all of us have experienced the importance of this channel: drugs such as aspirin and ibuprofen block the channel and prevent prostaglandin synthesis by inhibiting the cyclooxygenase activity of the synthase. In the case of aspirin, the drug acts through the transfer of an acetyl group from the aspirin to a serine residue (Ser 530) that lies along the path to the active site (Figure 12.25).

Figure 12.24. HYdrophobic Channel of Prostaglandin H2 Synthase.

Figure 12.24

HYdrophobic Channel of Prostaglandin H2 Synthase. Image mouse.jpg A view of prostaglandin H2 synthase from the membrane, showing the hydrophobic channel that leads to the active site. The membrane-anchoring helices are shown in orange.

Figure 12.25. Aspirin's Effects On Prostaglandin H2 Synthase-1.

Figure 12.25

Aspirin's Effects On Prostaglandin H2 Synthase-1. Aspirin acts by transferring an acetyl group to a serine residue in prostaglandin H2 synthase-1.

Two important features emerge from our examination of these three examples of membrane protein structure. First, the parts of the protein that interact with the hydrophobic parts of the membrane are coated with nonpolar amino acid side chains, whereas those parts that interact with the aqueous environment are much more hydrophilic. Second, the structures positioned within the membrane are quite regular and, in particular, all backbone hydrogen-bond donors and acceptors participate in hydrogen bonds. Breaking a hydrogen bond within a membrane is quite unfavorable, because little or no water is present to compete for the polar groups.

12.5.3. Some Proteins Associate with Membranes Through Covalently Attached Hydrophobic Groups

The membrane proteins considered thus far associate with the membrane through surfaces generated by hydrophobic amino acid side chains. However, even otherwise soluble proteins can associate with membranes if the association is mediated by hydrophobic groups attached to the proteins. Three such groups are shown in Figure 12.26: (1) a palmitoyl group attached to a specific cysteine residue by a thioester bond, (2) a farnesyl group attached to a cysteine residue at the carboxyl terminus, and (3) a glycolipid structure termed a glycosyl phosphatidyl inositol (GPI) anchor attached to the carboxyl terminus. These modifications are attached by enzyme systems that recognize specific signal sequences near the site of attachment.

Figure 12.26. Membrane Anchors.

Figure 12.26

Membrane Anchors. Membrane anchors are hydrophobic groups that are covalently attached to proteins (in blue) and tether the proteins to the membrane. The red and blue hexagons correspond to mannose and GlcNAc, respectively. R groups represent points of (more...)

12.5.4. Transmembrane Helices Can Be Accurately Predicted from Amino Acid Sequences

Many membrane proteins, like bacteriorhodopsin, employ α helices to span the hydrophobic part of a membrane. As noted earlier, typically most of the residues in these α helices are nonpolar and almost none of them are charged. Can we use this information to identify putative membrane-spanning regions from sequence data alone? One approach to identifying transmembrane helices is to ask whether a postulated helical segment is likely to be most stable in a hydrocarbon milieu or in water. Specifically, we want to estimate the free-energy change when a helical segment is transferred from the interior of a membrane to water. Free-energy changes for the transfer of individual amino acid residues from a hydrophobic to an aqueous environment are given in Table 12.2. For example, the transfer of a poly-l-arginine helix, a homopolymer of a positively charged amino acid, from the interior of a membrane to water would be highly favorable [-12.3 kcal mol-1 (-51.5 kJ mol-1) per arginine residue in the helix], whereas the transfer of a poly-l-phenylalanine helix, a homopolymer of a hydrophobic amino acid, would be unfavorable [+3.7 kcal mol-1 (+15.5 kJ mol-1) per phenylalanine residue in the helix].

Table 12.2. Polarity scale for identifying transmembrane helices.

Table 12.2

Polarity scale for identifying transmembrane helices.

The hydrocarbon core of a membrane is typically 30 Å wide, a length that can be traversed by an α helix consisting of 20 residues. We can take the amino acid sequence of a protein and estimate the free-energy change that takes place when a hypothetical α helix formed of residues 1 through 20 is transferred from the membrane interior to water. The same calculation can be made for residues 2 through 21, 3 through 22, and so forth, until we reach the end of the sequence. The span of 20 residues chosen for this calculation is called a window. The free-energy change for each window is plotted against the first amino acid at the window to create a hydropathy plot. Empirically, a peak of +20 kcal mol-1 (+84 kJ mol-1) or more in a hydropathy plot based on a window of 20 residues indicates that a polypeptide segment could be a membrane-spanning α helix. For example, glycophorin, a protein found in the membranes of red blood cells, is predicted by this criterion to have one membrane-spanning helix, in agreement with experimental findings (Figure 12.27). It should be noted, however, that a peak in the hydropathy plot does not prove that a segment is a transmembrane helix. Even soluble proteins may have highly nonpolar regions. Conversely, some membrane proteins contain transmembrane structural features (such as membrane-spanning β strands) that escape detection by these plots (Figure 12.28).

Figure 12.27. Locating the Membrane-Spanning Helix of Glycophorin.

Figure 12.27

Locating the Membrane-Spanning Helix of Glycophorin. (A) Amino acid sequence and transmembrane disposition of glycophorin A from the red-cell membrane. Fifteen O-linked carbohydrate units are shown as diamond shapes, and an N-linked unit is shown as a (more...)

Figure 12.28. Hydropathy Plot for Porin.

Figure 12.28

Hydropathy Plot for Porin. No strong peaks are observed for this intrinsic membrane protein because it is constructed from membrane-spanning β strands rather than α helices.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22495


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