2 Структура и функция белка (1160071), страница 21
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Protein crystallization is something of anempirical science, and the structures of many important proteins are not yet known simply becausethey have proven difficult to crystallize. Once acrystal is obtained, it is placed in an x-ray beambetween the x-ray source and a detector. A regulararray of spots called reflections (Fig. 1) is generated by precessional motion of the crystal. Thespots represent reflections of the x-ray beam, andeach atom in a molecule makes a contribution toeach spot. The overall pattern of spots is related tothe structure of the protein through a mathematical device called a Fourier transform. The intensityof each spot is measured from the positions andintensities of the spots in several of these diffraction patterns, and the precise three-dimensionalstructure of the protein is calculated.John Kendrew found that the x-ray diffractionpattern of crystalline myoglobin from muscles ofthe sperm whale is very complex, with nearly25,000 reflections.
Computer analysis of these reflections took place in stages. The resolution improved at each stage, until in 1959 the positions ofvirtually all the atoms in the protein could be determined. The amino acid sequence deduced fromthe structure agreed with that obtained by chemical analysis. The structures of hundreds of proteins have since been determined to a similar levelof resolution, many of them much more complexthan myoglobin.(a)(b)(c)(d)Figure 7-19 Tertiary structure of sperm whalemyoglobin.
The orientation of the protein is thesame in all panels; the heme group is shown in red.(a) The polypeptide backbone, shown in a ribbonrepresentation of a type introduced by Jane Richardson; this highlights regions of secondary structure. The a-helical regions in myoglobin are evident. Amino acid side chains are not shown, (b) Aspace-filling model, showing that the heme group islargely buried. All amino acid side chains are included, (c) A ribbon representation, including sidechains (purple) for the hydrophobic residues Leu,He, Val, and Phe. (d) A space-filling model with allamino acid side chains.
The hydrophobic residuesare again shown in purple; most are not visiblebecause they are buried in the interior of theprotein.178Figure 7-19 shows several structural representations of myoglobin, illustrating how the polypeptide chain is folded in three dimensions—its tertiary structure. The backbone of the myoglobin moleculeis made up of eight relatively straight segments of a helix interruptedby bends. The longest a helix has 23 amino acid residues and the shortest only seven; all are right-handed. More than 70% of the amino acidsin the myoglobin molecule are in these a-helical regions.
X-ray analysis also revealed the precise position of each of the R groups, whichoccupy nearly all the open space between the folded loops.Other important conclusions were drawn from the structure ofmyoglobin. The positioning of amino acid side chains reflects a structure that derives much of its stability from hydrophobic interactions.Most of the hydrophobic R groups are in the interior of the myoglobinmolecule, hidden from exposure to water.
All but two of the polar Rgroups are located on the outer surface of the molecule, and all of themare hydrated. The myoglobin molecule is so compact that in its interiorthere is room for only four molecules of water. This dense hydrophobiccore is typical of globular proteins. The fraction of space occupied byatoms in an organic liquid is 0.25 to 0.35; in a typical solid the fractionis 0.75. In a protein the fraction is 0.72 to 0.76, very comparable to thatin a solid.
In this closely packed environment weak interactionsstrengthen and reinforce each other. For example, the nonpolar sidechains in the core are so close together that short-range van der Waalsinteractions make a significant contribution to stabilizing hydrophobicinteractions. By contrast, in an oil droplet suspended in water, the vander Waals interactions are minimal and the cohesiveness of the dropletis based almost exclusively on entropy.The structure of myoglobin both confirmed some expectations andintroduced some new elements of secondary structure. As predicted byPauling and Corey, all the peptide bonds are in the planar trans configuration.
The a helices in myoglobin provided the first direct experimental evidence for the existence of this type of secondary structure.Each of the four Pro residues of myoglobin occurs at a bend (recall thatthe rigid R group of proline is largely incompatible with a-helical structure). Other bends contain Ser, Thr, and Asn residues, which areamong the amino acids that tend to be incompatible with a-helicalstructure if they are in close proximity (p. 168).The flat heme group rests in a crevice, or pocket, in the myoglobinmolecule. The iron atom in the center of the heme group has two bonding (coordination) positions perpendicular to the plane of the heme.Chapter 7 The Three-Dimensional Structure of Proteins179One of these is bound to the R group of the His residue at position 93;the other is the site to which an O2 molecule is bound.
Within thispocket, the accessibility of the heme group to solvent is highly restricted. This is important for function because free heme groups in anoxygenated solution are rapidly oxidized from the ferrous (Fe2 + ) form,which is active in the reversible binding of O2, to the ferric (Fe 3+ ) form,which does not bind O2.Proteins Differ in Tertiary StructureWith the elucidation of the tertiary structures of hundreds of otherglobular proteins by x-ray analysis, it is clear that myoglobin represents only one of many ways in which a polypeptide chain can befolded.
In Figure 7-20 the structures of cytochrome c, lysozyme, andribonuclease are compared. All have different amino acid sequencesand different tertiary structures, reflecting differences in function.Like myoglobin, cytochrome c is a small heme protein (Mr 12,400) containing a single polypeptide chain of about 100 residues and a singleheme group, which in this case is covalently attached to the polypeptide. It functions as a component of the respiratory chain of mitochondria (Chapter 18).
X-ray analysis of cytochrome c (Fig. 7-20)shows that only about 40% of the polypeptide is in a-helical segments, compared with almost 80% of the myoglobin chain. The rest ofthe cytochrome c chain contains bends, turns, and irregularly coiledand extended segments. Thus, cytochrome c and myoglobin differmarkedly in structure, even though both ar^e small heme proteins.Cytochrome cLysozymeFigure 7—20 The three-dimensional structures ofthree small proteins: cytochrome c, lysozyme, andribonuclease.
For lysozyme and ribonuclease theactive site of the enzyme faces the viewer. Keyfunctional groups (the heme in cytochrome c, andamino acid side chains in the active site of lysozyme and ribonuclease) are shown in red; disulfidebonds are shown in yellow. Two representations ofeach protein are shown: a space-filling model and aribbon representation.
In the ribbon depictions, the(3 structures are represented by flat arrows and thea helices by spiral ribbons; the orientation in eachcase is the same as that of the space-filling model,to facilitate comparison.RibonucleasePart II Structure and Catalysis180Table 7-2 Approximate iamounts of a helixand p conformation in some single-chainproteins*Protein(totalresidues)Myoglobin(153)Cytochrome c(104)Lysozyme(129)Ribonuclease(124)Chymotrypsin(247)Carboxypeptidase(307)Residues (%)a Helixp Conformation7803904012263514453817Source: Data from Cantor, C.R. & Schimmel, P.R. (1980) Biophysical Chemistry, Part I: The Conformation of BiologicalMacromolecules, p.
100, W.H. Freeman and Company, NewYork.* Portions of the polypeptide chains that are not accounted forby a helix or j3 conformation consist of bends and irregularlycoiled or extended stretches. Segments of a helix and (3 conformation sometimes deviate slightly from their normal dimensions and geometry.Lysozyme (Mr 14,600) is an enzyme in egg white and human tearsthat catalyzes the hydrolytic cleavage of polysaccharides in the protective cell walls of some families of bacteria. Lysozyme is so named because it can lyse, or degrade, bacterial cell walls and thus serve as abactericidal agent. Like cytochrome c, about 40% of its 129 amino acidresidues are in a-helical segments, but the arrangement is differentand some /3 structure is also present. Four disulfide bonds contributestability to this structure.
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