2 Структура и функция белка (1160071), страница 20
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A solution of areducing agent, usually a compound containing athiol or sulfhydryl group (—SH), is then appliedwith heat. The reducing agent cleaves the disulfldecross-linkages by reducing each cystine to two cysteine residues, one in each adjacent chain. Themoist heat breaks hydrogen bonds and causes thea-helical structure of the polypeptide chains touncoil and stretch.
After a time the reducing solution is removed, and an oxidizing agent is added toestablish new disulflde bonds between pairs of Cysresidues of adjacent polypeptide chains, but notthe same pairs that existed before the treatment.On washing and cooling the hair, the polypeptidechains revert to their a-helical conformation. Thehair fibers now curl in the desired fashion becausenew disulflde cross-linkages have been formedwhere they will exert some torsion or twist on thebundles of a-helical coils in the hair fibers.174Part II Structure and CatalysisFigure 7-15 The structure of collagen fibers.
Tropocollagen (Mr 300,000) is a rod-shaped molecule,about 300 nm long and only 1.5 nm thick. The threehelically intertwined polypeptides are of equallength, each having about 1,000 amino acid residues.In some collagens all three chains are identical inamino acid sequence, but in others two chains areidentical and the third differs. The heads of adjacent molecules are staggered, and the alignment ofthe head groups of every fourth molecule producescharacteristic cross-striations 64 nm apart that areevident in an electron micrograph.Heads of tropocollagenmoleculesCross-striations64 nmSection of tropocollagenmoleculeCollagen fibrils consist of recurring three-stranded polypeptide unitscalled tropocollagen, arranged head to tail in parallel bundles (Fig.7-15). The rigid, brittle character of the connective tissue in older people is the result of an accumulation of covalent cross-links in collagenas we age.Human genetic defects involving collagen illustrate the close relationship between amino acid sequence and three-dimensional structure in this protein.
Osteogenesis imperfecta results in abnormal boneformation in human babies. Ehlers-Danlos syndrome is characterizedby loose joints. Both can be lethal and both result from the substitutionof a Cys or Ser residue, respectively, for a Gly (a different Gly residuein each case) in the amino acid sequence of collagen. These seeminglysmall substitutions have a catastrophic effect on collagen function because they disrupt the Gly-X-Pro repeat that gives collagen its uniquehelical structure.Elastic connective tissue contains the fibrous protein elastin,which resembles collagen in some of its properties but is very differentin others.
The polypeptide subunit of elastin fibrils is tropoelastin (Mr72,000), containing about 800 amino acid residues. Like collagen, it isrich in Gly and Ala residues. Tropoelastin differs from tropocollagen inhaving many Lys but few Pro residues; it forms a special type of helix,different from the a helix and the collagen helix.
Tropoelastin consistsof lengths of helix rich in Gly residues separated by short regions containing Lys and Ala residues. The helical portions stretch on applyingtension but revert to their original length when tension is released.Chapter 7 The Three-Dimensional Structure of ProteinsFigure 7-16 Tropoelastin molecules and theirlinkage to form a network of polypeptide chains inelastin. Elastin consists of tropoelastin moleculescross-linked to give two-dimensional or threedimensional elasticity. In addition to desmosine residues (in red), which can link two, three, or fourtropoelastin molecules, as shown, elastin containsother kinds of cross-linkages, such as lysinonorleucine, also designated in red.175These Lysresiduesare precursorsof desmosineresidues, whichcan link fourpolypeptidechainsElastica-helicalcoils ofpolypeptide,rich in GlyresiduesThe regions containing Lys residues form covalent cross-links.
FourLys side chains come together and are enzymatically converted intodesmosine (see Fig. 5-8) and a related compound, isodesmosine; theseamino acids are found only in elastin. Lysinonorleucine (p. 173) alsooccurs in elastin. These nonstandard amino acids are capable of joiningtropoelastin chains into arrays that can be stretched reversibly in alldirections (Fig.
7-16).DesmosineresidueProtein Tertiary StructureAlthough fibrous proteins generally have only one type of secondarystructure, globular proteins can incorporate several types of secondarystructure in the same molecule. Globular proteins—including enzymes, transport proteins, some peptide hormones, and immunoglobulins—are folded structures much more compact than a or /3 conformations (as shown for serum albumin in Figure 7-17).The three-dimensional arrangement of all atoms in a protein isreferred to as the tertiary structure, and this now becomes our focus.Whereas the secondary structure of polypeptide chains is determinedby the short-range structural relationship of amino acid residues, tertiary structure is conferred by longer-range aspects of amino acid sequence.
Amino acids that are far apart in the polypeptide sequence andreside in different types of secondary structure may interact when theLysinonorleucinecross-linkCross-linking two chainsLinking three chains2Linking four chainsP Conformation200 x 0.5 nma Helix90 x 1.1 nmNative globular form13 x 3 nmFigure 7-17 Bovine serum albumin (Mr 64,500)has 584 residues in a single chain. Shown aboveare the approximate dimensions its single polypeptide chain would have if it occurred entirely in extended p conformation or as an a helix.
Also shown(left) is the actual size of native serum albumin inits native globular form, as determined by physicochemical measurements; the polypeptide chainmust be very compactly folded to fit into these dimensions.176Part II Structure and Catalysisprotein is folded. The formation of bends in the polypeptide chain during folding and the direction and angle of these bends are determinedby the number and location of specific bend-producing amino acids,such as Pro, Thr, Ser, and Gly residues. Moreover, loops of the highlyfolded polypeptide chain are held in their characteristic tertiary positions by different kinds of weak-bonding interactions (and sometimesby covalent bonds such as disulfide cross-links) between R groups ofadjacent loops.We will now consider how secondary structures contribute to thetertiary folding of a polypeptide chain in a globular protein, and howthis structure is stabilized by weak interactions, in particular by hydrophobic interactions involving nonpolar amino acid side chains inthe tightly packed core of the protein.X-Ray Analysis of Myoglobin Revealed Its Tertiary StructureThe breakthrough in understanding globular protein structure camefrom x-ray diffraction studies of the protein myoglobin carried out byJohn Kendrew and his colleagues in the 1950s (Box 7-3).
Myoglobin isa relatively small (Mr 16,700), oxygen-binding protein of muscle cellsthat functions in the storage and transport of oxygen for mitochondrialoxidation of cell nutrients. Myoglobin contains a single polypeptidechain of 153 amino acid residues of known sequence and a single ironporphyrin, or heme, group (Fig. 7-18), identical to that of hemoglobin,the oxygen-binding protein of erythrocytes. The heme group is responsible for the deep red-brown color of both myoglobin and hemoglobin.Myoglobin is particularly abundant in the muscles of diving mammalssuch as the whale, seal, and porpoise, whose muscles are so rich in thisprotein that they are brown. Storage of oxygen by muscle myoglobinpermits these animals to remain submerged for long periods oftime.vVOO"yCH2CH2CH2CH3-CHN-C£CH/H-CCH2C-N//N -Fe-O 2CH2yyHisresidueCHCH 2CH 3(a)Figure 7—18 The heme group, present in myoglobin, hemoglobin, cytochrome b, and many otherheme proteins, consists of a complex organic ringstructure, protoporphyrin, to which is bound aniron atom in its ferrous (Fe 2 ") state.
Two representations are shown in (a) and (b). (c) The iron atomhas six coordination bonds, four in the plane of,Plane ofporphyrin(b)(c)and bonded to, the flat porphyrin molecule and twoperpendicular to it. (d) In myoglobin and hemoglobin, one of the perpendicular coordination bonds isbound to a nitrogen atom of a His residue. Theother is "open" and serves as the binding site foran O 2 molecule, as shown here in the edge view.(d)Chapter 7 The Three-Dimensional Structure of ProteinsBOX 7-3177X-Ray DiffractionThe spacing of atoms in a crystal lattice can be determined by measuring the angles and the intensities at which a beam of x rays of a given wavelength is diffracted by the electron shells aroundthe atoms. For example, x-ray analysis of sodiumchloride crystals shows that Na + and Cl~ ions arearranged in a simple cubic lattice.
The spacing ofthe different kinds of atoms in complex organicmolecules, even very large ones such as proteins,can also be analyzed by x-ray diffraction methods.However, this is far more difficult than for simplesalt crystals because the very large number ofatoms in a protein molecule yields thousands ofdiffraction spots that must be analyzed by computer.The process may be understood at an elementary level by considering how images are generatedin a light microscope. Light from a point source isfocused on an object. The light waves are scatteredby the object, and these scattered waves are recombined by a series of lenses to generate an enlargedimage of the object.
The limit to the size of an object whose structure can be determined by such asystem (i.e., its resolving power) is determined bythe wavelength of the light. Objects smaller thanhalf the wavelength of the incident light cannot beresolved. This is why x rays, with wavelengths inthe range of a few tenths of a nanometer (oftenmeasured in angstroms, A; lA=0.1nm), must beused for proteins. There are no lenses that can recombine x rays to form an image; the pattern ofdiffracted light is collected directly and convertedinto an image by computer analysis.Operationally, there are several steps in x-raystructural analysis.
The amount of informationobtained depends on the degree of structural orderFigure 1 Photograph of the x-ray diffraction pattern of crystalline sperm whale myoglobin.in the sample. Some important structural parameters were obtained from early studies of the diffraction patterns of the fibrous proteins that occur infairly regular arrays in hair and wool. More detailed three-dimensional structural information,however, requires a highly ordered crystal of a protein.
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