3 Биологические мембраны. Обмен веществом (1160072), страница 12
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The amino acid sequence ofbacteriorhodopsin has seven segments with about 20 hydrophobic residues, each segment just long enough to make a helix that spans thelipid bilayer. Hydrophobic interactions between the nonpolar aminoacids and the acyl side chains of the membrane lipids firmly anchor theprotein in the membrane, providing a transmembrane pathway forproton translocation.Figure 10-10 The single polypeptide chain of bacteriorhodopsin folds into seven hydrophobic a helices, each of which traverses the lipid bilayer andis roughly perpendicular to the plane of the membrane.
The seven transmembrane helices are clustered, and the space around and between them isfilled with the acyl chains of membrane lipids.Protein-glycanIntegral protein(hydrophobic domaincoated with detergent)Figure 10—9 Membrane proteins can be distinguished by the conditions required to release themfrom the membrane. Most peripheral proteins canbe released by changes in pH or ionic strength,removal of Ca2+ by a chelating agent, or addition ofurea, which breaks hydrogen bonds.
Peripheral proteins covalently attached to a membrane lipid, suchas through a phosphatidylinositol-glycan anchor(see Fig. 10-3), are released by phospholipase C orD. Integral proteins can be extracted with detergents, which disrupt the hydrophobic interactionswith the lipid bilayer, forming micelles with individual protein molecules.AminoterminusOutersurfaceInnersurfaceCarboxylterminus277278Part II Structure and CatalysisBOX 10-2Predicting the Topology of Membrane ProteinsTable 1 ResiduehydrophobicityAminoacidHePheValLeuTVpMetAlaGlyCysTVrProThrSerHisGluAsnGinAspLysArgFree energyof transfer(kJ/mol)3.12.52.32.21.51.11.00.670.170.08-0.29-0.75-1.1-1.7-2.6-2.7-2.9-3.0-4.6-7.5Source: From Eisenberg, D.,et al. (1982) Hydrophobicmoments in protein structure.
Faraday Symp. Chem.Soc. 17, 109-120.It is generally much easier to determine the aminoacid sequence of a membrane protein (by sequencing the protein itself or its gene) than to determineits three-dimensional structure. Consequently,only a few three-dimensional structures areknown, but hundreds of sequences are available formembrane proteins. The sequences of most integral proteins contain one or more regions rich inhydrophobic residues and long enough to span the3 nm thick lipid bilayer. An a-helical peptide of 20residues is just long enough to span the bilayer(the length per residue is 0.15 nm). Because a polypeptide chain surrounded by lipids has no watermolecules with which to form hydrogen bonds, itwill tend to fold into a helices or /3 sheets, in whichintrachain hydrogen bonding is maximized.
If theside chains of all amino acids in a helix are nonpolar, hydrophobic interactions with the surrounding lipids further stabilize the helices.Several simple methods of analyzing amino acidsequences have been found to yield reasonably accurate predictions of secondary structure for transmembrane proteins. The relative polarity of each ofthe 20 amino acids has been determined experimentally by measuring the free-energy change ofmoving a given residue from a hydrophobic solventinto water. This free energy of transfer rangesfrom very exergonic for charged or polar residuesto very endergonic for amino acids with aromaticor aliphatic hydrocarbon side chains (Table 1). Toestimate the overall hydrophobicity of a sequenceof amino acids, one sums the free energies of transfer for those residues, obtaining a hydropathyindex for that region.
To search a sequence for potential membrane-spanning segments, one calculates the hydropathy index for successive segmentsof a given size (a "window," which may be from 7 to20 residues). For a window of 7 residues, the indexes for residues 1 to 7, 2 to 8, 3 to 9, and so on,are plotted as in Figure 1.
A region of about 20 residues of high hydropathy index is presumed to be atransmembrane segment. When the sequences ofmembrane proteins of known three-dimensionalstructure are scanned in this way, a reasonablygood correspondence is found between predictedand known membrane-spanning segments. Hydropathy analysis predicts a single hydrophobichelix for glycophorin (Fig. la), five for the M subunit of the photosynthetic reaction center protein(Fig. lb), seven transmembrane segments for bacteriorhodopsin (Fig.
lc), and twelve segments forthe chloride—bicarbonate exchanger (Fig. Id).Many of the transport proteins described in thischapter are believed, on the basis of their aminoacid sequences and hydropathy plots, to have multiple membrane-spanning helical regions. Theseassignments of topology should be considered tentative until confirmed by direct structural determination.150200100300HydrophobicHydrophilic0Figure 1 Plots of hydropathy index againstresidue number forfour integral membraneproteins.50100150200VVvV\t300M subunit of bacterial photosyntheticreaction center(b)(a)c)20010050100Glycophorin3HydrophobicHydrophilic200250400600800iXVHydrophobicHydrophilicHydrophobicHydrophilic-3050100150200Bacteriorhodopsin(c)250200400600800Chloride-bicarbonate exchange protein(d)Chapter 10 Biological Membranes and Transport279This pattern of seven hydrophobic membrane-spanning helices hasproven to be a common motif in membrane structure, seen in at leastten other membrane proteins, all involved in signal reception.
Although no information is yet available on the three-dimensional structures of these proteins, it seems likely that they will prove to be structurally similar to bacteriorhodopsin. The presence of long hydrophobicregions along the amino acid sequence of a membrane protein is generally taken as evidence that such sequences traverse the lipid bilayer,acting as hydrophobic anchors or forming transmembrane channels;virtually all integral membrane proteins have at least one such sequence (Box 10—2).
When sequence information yields predictions consistent with chemical studies of protein localization (such as those described above for glycophorin and bacteriorhodopsin), the assumptionthat hydrophobic regions correspond to membrane-spanning domainsis better justified.The Structure of a Crystalline Integral Membrane ProteinHas Been DeterminedThe same techniques that have allowed determination of the threedimensional structures of many soluble proteins can in principle beapplied to membrane proteins. However, very few membrane proteinshave been crystallized; they tend instead to form amorphous aggregates.
One instructive exception is the photosynthetic reaction centerfrom a purple bacterium (Fig. 10-11). The protein has four subunits,three of which contain a-helical segments that span the membrane.These segments are rich in nonpolar amino acids, and their hydrophobic side chains are oriented toward the outside of the protein, interacting with the hydrocarbon side chains of membrane lipids.
The architecture of the reaction center protein is therefore the inverse of that seenin most water-soluble proteins, which have their hydrophobic residuesburied within the protein core and their hydrophilic residues on thesurface available for polar interactions with water (recall the structures of myoglobin and hemoglobin, for example).
The structures ofonly a few membrane proteins are known, but the hydrophobic exteriorof the reaction center protein seems to be typical of integral membraneproteins.Figure 10—11 Three-dimensional structure of thephotosynthetic reaction center of a purple bacterium, Rhodopseudomonas viridis. This was the firstintegral membrane protein to have its atomic structure determined by x-ray diffraction methods. Eleven of-helical segments from three of the four subunits span the lipid bilayer, forming a cylinder4.5 nm long, with hydrophobic residues on the exterior, interacting with lipids of the bilayer.
In theribbon representation at left, the residues that arepart of the transmembrane helices are shown inpurple. The high density of nonpolar residues inthe region of the bilayer is illustrated in the spacefilling model on the right, in which the four veryhydrophobic residues (Phe, Val, He, Leu) are shownin purple. In both views, the prosthetic groups(light-absorbing pigments and electron carriers; seeFig.
18-47) are shown in yellow.4). .mmii6rL.. v- -:^w280Part II Structure and CatalysisPeripheral Proteins Associate Reversibly with the MembraneHumancellMany peripheral proteins are held to the membrane by electrostaticinteractions and hydrogen bonding with the hydrophilic domains ofintegral membrane proteins, and perhaps with the polar head groupsof membrane lipids. They can be released by relatively mild treatmentsthat interfere with electrostatic interactions or break hydrogen bonds(see Fig.
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