The Composition and Architecture of Membranes:- The Topology of an Integral Membrane Protein Can Be Predicted from Its Sequence

Determination of the three-dimensional structure of a membrane protein, or its topology, is generally much more difficult than determining its amino acid sequence, which can be accomplished by sequencing the protein or its gene. Thousands of sequences are known for mem brane proteins, but relatively few three-dimensional structures have been established by crystallography or NMR spectroscopy. The presence of unbroken sequences of more than 20 hydrophobic residues in a membrane protein is commonly taken as evidence that these sequences traverse the lipid bilayer, acting as hydrophobic anchors or forming transmembrane channels. Virtually all integral proteins have at least one such sequence. Application of this logic to entire genomic sequences leads to the conclusion that in many species, 10% to 20% of all proteins are integral membrane proteins. What can we predict about the secondary structure of the membrane-spanning portions of integral proteins? An -helical sequence of 20 to 25 residues is just long enough to span the thickness (30 Å) of the lipid bilayer (recall that the length of an α helix is 1.5 Å (0.15 nm) per amino acid residue). A polypeptide chain sur rounded by lipids, having no water molecules with which to hydrogen-bond, will tend to form α helices or β sheets, in which intrachain hydrogen bonding is maximized. If the side chains of all amino acids in a helix are nonpolar, hydrophobic interactions with the surrounding lipids further stabilize the helix.

Several simple methods of analyzing amino acid sequences yield reasonably accurate predictions of secondary structure for transmembrane proteins. The relative polarity of each amino acid has been determined experimentally by measuring the free-energy change ac companying the movement of that amino acid side chain from a hydrophobic solvent into water. This free energy of transfer ranges from very exergonic for charged or polar residues to very endergonic for amino acids with aromatic or aliphatic hydrocarbon side chains. The overall hydrophobicity of a sequence of amino acids is estimated by summing the free energies of transfer for the residues in the sequence, which yields a hydropa thy index for that region (see Table 3–1). To scan a polypeptide sequence for potential membrane-spanning segments, an investigator calculates the hydropathy in dex for successive segments (called windows) of a given size, from 7 to 20 residues. For a window of seven residues, for example, the indices for residues 1 to 7, 2 to 8, 3 to 9, and so on, are plotted as in Figure 11–11 (plotted for the middle residue in each window— residue 4 for residues 1 to 7, for example). A region with more than 20 residues of high hydropathy index is presumed to be a transmembrane segment. When the sequences of membrane proteins of known three-dimensional structure are scanned in this way, we find a reasonably good correspondence between predicted and known membrane-spanning segments. Hydropathy analysis predicts a single hydrophobic helix for glycophorin (Fig. 11–11a) and seven transmembrane segments for bacteriorhodopsin (Fig. 11–11b)—in agreement with experimental studies. On the basis of their amino acid sequences and hydropathy plots, many of the transport proteins described in this chapter are believed to have multiple membrane-spanning helical regions—that is, they are type III or type IV integral proteins (Fig. 11–8). When predictions are consistent with chemical studies of protein localization (such as those described above for glycophorin and bacteriorhodopsin), the assumption that hydrophobic regions correspond to membrane spanning domains is much better justified. A further remarkable feature of many transmem brane proteins of known structure is the presence of Tyr and Trp residues at the interface between lipid and water (Fig. 11–12). The side chains of these residues apparently serve as membrane interface anchors, able to interact simultaneously with the central lipid phase and the aqueous phases on either side of the membrane.

FIGURE 11–11 Hydropathy plots. Hydropathy index (see Table 3–1) is plotted against residue number for two integral membrane proteins. The hydropathy index for each amino acid residue in a sequence of defined length (called the window) is used to calculate the average hydropathy for the residues in that window. The horizontal axis shows the residue number in the middle of the window. (a) Glycophorin from human erythrocytes has a single hydrophobic sequence between residues 75 and 93 (yellow); compare this with Figure 11–7. (b) Bacteriorhodopsin, known from independent physical studies to have seven transmembrane helices, has seven hydrophobic regions. Note, however, that the hydropathy plot is ambiguous in the region of segments 6 and 7. Physical studies have confirmed that this region has two transmembrane segments.

FIGURE 11–12 Tyr and Trp residues of membrane proteins clustering at the water-lipid interface. The detailed structures of these five integral membrane proteins are known from crystallographic studies. The K channel (PDB ID 1BL8) is from the bacterium Streptomyces lividans; maltoporin (PDB ID 1AF6), outer membrane phospholipase A (PDB ID 1QD5), OmpX (PDB ID 1QJ9), and phos-phoporin E (PDB ID 1PHO) are proteins of the outer membrane of E. coli. Residues of Tyr (orange) and Trp (red) are found predominantly where the nonpolar region of acyl chains meets the polar head group region. Charged residues (Lys, Arg, Glu, Asp) are shown in blue; they are found almost exclusively in the aqueous phases.

The hydrophobic domains of some integral mem brane proteins penetrate only one leaflet of the bilayer. Cyclooxygenase, the target of aspirin action, is an ex ample; its hydrophobic helices do not span the whole membrane but interact strongly with the acyl groups on one side of the bilayer. Not all integral membrane proteins are composed of transmembrane α helices. Another structural motif common in membrane proteins is the barrel (see Fig. 4–20d), in which 20 or more transmembrane segments form sheets that line a cylinder (Fig. 11–13). The same factors that favor -helix formation in the hydrophobic interior of a lipid bilayer also stabilize β barrels. When no water molecules are available to hydrogen-bond with the carbonyl oxygen and nitrogen of the peptide bond, maximal intrachain hydrogen bonding gives the most stable conformation. Planar β sheets do not maximize these interactions and are generally not found in the membrane interior; β barrels do allow all possible hydrogen bonds and are apparently common among membrane proteins. Porins, proteins that allow certain polar solutes to cross the outer membrane of gram-negative bacteria such as E. coli, have many-stranded β barrels lining the polar transmembrane passage. A polypeptide is more extended in the β conformation than in an helix; just seven to nine residues of β conformation are needed to span a membrane. Recall that in the β conformation, alternating side chains project above and below the sheet (see Fig. 4–7). In β strands of membrane proteins, every second residue in the membrane-spanning segment is hydrophobic and interacts with the lipid bilayer; aromatic side chains are commonly found at the lipid-protein interface. The other residues may or may not be hydrophilic. The hydropathy plot is not useful in predicting transmembrane segments for proteins with β barrel motifs, but as the database of known β barrel motifs increases, sequence-based predictions of transmembrane β conformations have become feasible. For example, a number of outer membrane proteins of gram-negative bacteria (Fig. 11–13) have been correctly predicted, by sequence analysis, to contain β barrels.

FIGURE 11–13 Membrane proteins with -barrel structure. Five ex amples are shown, viewed in the plane of the membrane; The first four are from the E. coli outer membrane. FepA (PDB ID 1FEP), in volved in iron uptake, has 22 membrane-spanning strands. OmpLA (derived from PDB ID 1QD5), a phospholipase, is a 12-stranded β barrel that exists as a dimer in the membrane. Maltoporin (derived from PDB ID 1MAL), a maltose transporter, is a trimer, each monomer constructed of 16 strands. TolC (PDB ID 1EK9), another transporter, has three separate subunits, each contributing four β strands in this 12-stranded barrel. The Staphylococcus aureus-hemolysin toxin (PDB ID 7AHL; top view below) is composed of seven identical sub units, each contributing one hairpin-shaped pair of β strands to the 14-stranded barrel.

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مواضيع ذات صلة

Membrane Dynamics:- Transbilayer Movement of Lipids Requires Catalysis

The Composition and Architecture of Membranes:- Lipids and Proteins Diffuse Laterally in the Bilayer

Membrane Dynamics:- Acyl Groups in the Bilayer Interior Are Ordered to Varying Degrees

The Composition and Architecture of Membranes:- Covalently Attached Lipids Anchor Some Membrane Proteins

The Composition and Architecture of Membranes: -Integral Proteins Are Held in the Membrane by Hydrophobic Interactions with Lipids

The Composition and Architecture of Membranes:- All Biological Membranes Share Some Fundamental Properties

The Composition and Architecture of Membranes:- Each Type of Membrane Has Characteristic Lipids and Proteins

Working with Lipids:- Mass Spectrometry Reveals Complete Lipid Structure

Working with Lipids:- Gas-Liquid Chromatography Resolves Mixtures of Volatile Lipid Derivatives

Working with Lipids:- Adsorption Chromatography Separates Lipids of Different Polarity

Working with Lipids:- Lipid Extraction Requires Organic Solvents

Lipids as Signals, Cofactors, and Pigments: -Plants Use Phosphatidylinositols, Steroids, and Eicosanoidlike Compounds in Signaling