a-Keratin.collagen.and elastin provide clear examples of the relationship between protein simple st ctures,and a are insoluble in water,a property conferred by a high concentration of hydrophobic amino acid both in the interior of the protein and on the surface.These proteins represent an exception to the rule that hydrophobie groups must be buried.The hydrophobic core of the molecule therefore contributes less to structural stability,and covalent bonds assume an especially important role. 2.5 Protein Tertiary and Quaternary Structures 25.1The concepts of tertiary and quaterary structure The overall three-dimensional arrangement of all atoms in a protein is referred to as the protein's tertiary structure.Some proteins contain two or more separate polypeptide chains.or subunits,which may be identical or different The arrangement of these protein subunits in three-dimensional 2.52 Forces influ ncing protein structure Several different kinds of noncovalent interactions are of vital importance in protein structure Hydrogen bonds,hydrophobic interactions,electrostatic bonds,and van der Waals forces are all noncovalent in nature.vet are extremely important influences on protein conformations.The free energies afforded by each of may be highly dependenton the local environment within he protein but certain mad Hydrogen Bonds:Hydrogen bonds are generally made wherever possible within a giver protein structure.In most protein structures that have been examined to date.component atoms of the peptide backbone tend to form hydrogen bonds with one another.Furthermore,side chains eapable of forming H bonds are usually located on the protein surface and form such bonds primarily with the solvent.Although e ach hydrogen bond average of on about 12 kJ/mol in stabilization energy for the protein structure,the number of Hbonds formed in the typical protein is very large.For example,in ahelices,the C=O and N-H groups of every residue participate in H bonds.The importance of H bonds in protein structure cannot be overstated Hydrophobic Inte tions:Hydrophobiconds,more accurately,interactions,form because nonpolar side chains of amino acids and other nonpolar solutes prefer to cluster in nonpolar environment rather than to intercalate in a polar solvent such as water.The forming of hydrophobic bonds minimizes the interaction of nonpolar residues with water and is therefore highly favorable.Such clustering is entropically driven.The side chains of the amino acids in the interior or core of the protein structure are almost exclusively hydrophobic.Polar amino acids are most never found in the of a but the may consist of both pola and nonpolar residues Eleetrostatic Interactions:lonic interactions arise either as electrostatic attractions between opposite charges or repulsions between like charges.Amino acid side chains can carry positive charges.as in the case of lysine.arginine.and histidine.or negative charges.as in aspartate and glutamate.In addition,the NH2-te pep chain usually exist in ionized states and arry positive or negative charges,respectively.All these may experience electrostatic interactions in a protein structure.Charged residues are normally located on the protein surface,where they may interact optimally with the water solvent. It is energetically unfavorable for an ionized residue to be located in the hydrophobic core of the 29
29 α-Keratin, collagen, and elastin provide clear examples of the relationship between protein structure and biological function (Table 2-6). These proteins share properties that give strength and/or elasticity to structures in which they occur. They have relatively simple structures, and all are insoluble in water, a property conferred by a high concentration of hydrophobic amino acids both in the interior of the protein and on the surface. These proteins represent an exception to the rule that hydrophobic groups must be buried. The hydrophobic core of the molecule therefore contributes less to structural stability, and covalent bonds assume an especially important role. 2.5 Protein Tertiary and Quaternary Structures 2.5.1 The concepts of tertiary and quaternary structure The overall three-dimensional arrangement of all atoms in a protein is referred to as the protein’s tertiary structure. Some proteins contain two or more separate polypeptide chains, or subunits, which may be identical or different. The arrangement of these protein subunits in three-dimensional complexes constitutes quaternary structure. 2.5.2 Forces influencing protein structure Several different kinds of noncovalent interactions are of vital importance in protein structure. Hydrogen bonds, hydrophobic interactions, electrostatic bonds, and van der Waals forces are all noncovalent in nature, yet are extremely important influences on protein conformations. The stabilization free energies afforded by each of these interactions may be highly dependent on the local environment within the protein, but certain generalizations can still be made. Hydrogen Bonds：Hydrogen bonds are generally made wherever possible within a given protein structure. In most protein structures that have been examined to date, component atoms of the peptide backbone tend to form hydrogen bonds with one another. Furthermore, side chains capable of forming H bonds are usually located on the protein surface and form such bonds primarily with the water solvent. Although each hydrogen bond may contribute an average of only about 12 kJ/mol in stabilization energy for the protein structure, the number of Hbonds formed in the typical protein is very large. For example, in α-helices, the C﹦O and N-H groups of every residue participate in H bonds. The importance of H bonds in protein structure cannot be overstated. Hydrophobic Interactions：Hydrophobic “bonds,” or, more accurately, interactions, form because nonpolar side chains of amino acids and other nonpolar solutes prefer to cluster in a nonpolar environment rather than to intercalate in a polar solvent such as water. The forming of hydrophobic bonds minimizes the interaction of nonpolar residues with water and is therefore highly favorable. Such clustering is entropically driven. The side chains of the amino acids in the interior or core of the protein structure are almost exclusively hydrophobic. Polar amino acids are almost never found in the interior of a protein, but the protein surface may consist of both polar and nonpolar residues. Electrostatic Interactions：Ionic interactions arise either as electrostatic attractions between opposite charges or repulsions between like charges. Amino acid side chains can carry positive charges, as in the case of lysine, arginine, and histidine, or negative charges, as in aspartate and glutamate. In addition, the NH2-terminal and COOH-terminal residues of a protein or peptide chain usually exist in ionized states and carry positive or negative charges, respectively. All of these may experience electrostatic interactions in a protein structure. Charged residues are normally located on the protein surface, where they may interact optimally with the water solvent. It is energetically unfavorable for an ionized residue to be located in the hydrophobic core of the
protein.Electrostatic interactions between charged groups on a protein surface are often der Waals interactions.The atractive forees are due primarily to instantaneous dipole-induced dipole interactions that arise because of fluctuations in the electron charge distributions of adiacent nonbonded atoms Individual van der Waals interactions are weak ones (with stabilization energies of 4.0 to 1.2 kJ/mol).but many such interactions occur in a typical protein. protein.Peter Privalo and George Makhatadze have shown that for pancreatic ibonuclease hen egg white lysozyme,horse heart eytochrome c,and sperm whale myoglobin,van der Waals interactions between tightly packed groups in the interior of the protein are a major contribution to protein stability 253 Protein domain and tertiary structure Polypeptides with than a few hundred amino acid residues often fold int stable,globular units called domains.In many cases,a domain from a large protein will retain its correct three-dimensional structure even when it is separated (for example.by proteolytic cleavage) from the remainder of the polypeptide chain.A protein with multiple domains may appear to have a distinct globular lobe for each domain (Figure 2-26)but more commonly.extensive contacts een domins make individual domains hard todiscem Different have distinc tions,such as the binding of small molecules or interaction with other proteins.Small proteins usually have only one domain (the domain is the protein). Fig.2-26Structural domains in the polypeptide troponinC Myoglobin (Figure 2-27)is the oxygen-storage protein of muscle.The muscles of diving mammals such as seals and whales are especially rich in this protein,which serves as a store for O,during the animal's prolonged periods underwater.Mvoglobin is abundant in skeletal and cardiac muscle of nondiving animals as well.Myoglobin is the cause of the characteristic red coo ofmuscle. The myoglobin polypeptide chain is folded to form a cradle(4.4x4.4x2.5 nm)that nestles the heme prosthetic group(Figure 2-28).O2 binding depends on the heme's oxidation state.The iron ion in the heme of myoglobin is in the 2 oxidation state,that is,the ferrous form.This is the form that binds 2.Oxidation of the ferrous form to yields metmyoglobin,which will readily interact with also,but the oxygen quickly oxidizes the iron atom to the ferric state.Fe3protoporphyrin IX is referred to as hematin.Thus,the 30
30 protein. Electrostatic interactions between charged groups on a protein surface are often complicated by the presence of salts in the solution. Van der Waals Interactions：Both attractive forces and repulsive forces are included in van der Waals interactions. The attractive forces are due primarily to instantaneous dipole-induced dipole interactions that arise because of fluctuations in the electron charge distributions of adjacent nonbonded atoms. Individual van der Waals interactions are weak ones (with stabilization energies of 4.0 to 1.2 kJ/mol), but many such interactions occur in a typical protein, and, by sheer force of numbers, they can represent a significant contribution to the stability of a protein. Peter Privalov and George Makhatadze have shown that, for pancreatic ribonuclease A, hen egg white lysozyme, horse heart cytochrome c, and sperm whale myoglobin, van der Waals interactions between tightly packed groups in the interior of the protein are a major contribution to protein stability. 2.5.3 Protein domain and tertiary structure Polypeptides with more than a few hundred amino acid residues often fold into two or more stable, globular units called domains. In many cases, a domain from a large protein will retain its correct three-dimensional structure even when it is separated (for example, by proteolytic cleavage) from the remainder of the polypeptide chain. A protein with multiple domains may appear to have a distinct globular lobe for each domain (Figure 2–26), but, more commonly, extensive contacts between domains make individual domains hard to discern. Different domains often have distinct functions, such as the binding of small molecules or interaction with other proteins. Small proteins usually have only one domain (the domain is the protein). Fig. 2–26 Structural domains in the polypeptide troponin C. Myoglobin (Figure 2-27) is the oxygen-storage protein of muscle. The muscles of diving mammals such as seals and whales are especially rich in this protein, which serves as a store for O2 during the animal’s prolonged periods underwater. Myoglobin is abundant in skeletal and cardiac muscle of nondiving animals as well. Myoglobin is the cause of the characteristic red color of muscle. The myoglobin polypeptide chain is folded to form a cradle (4.4×4.4×2.5 nm) that nestles the heme prosthetic group (Figure 2-28). O2 binding depends on the heme’s oxidation state. The iron ion in the heme of myoglobin is in the _2 oxidation state, that is, the ferrous form. This is the form that binds O2. Oxidation of the ferrous form to the _3 ferric form yields metmyoglobin, which will not bind O2. It is interesting to note that free heme in solution will readily interact with O2 also, but the oxygen quickly oxidizes the iron atom to the ferric state. Fe3_:protoporphyrin IX is referred to as hematin. Thus, the
Fig.2-27 The myoglobin and hemoglobin molecules Myoglobin (sperm whale):one polypeptide chain of 153 aa residues (mass=17.2 kD)has one heme(mass -652 D)and binds one O2.Hemtoglobin (human):four polypeptide chains,two of 141 aa residues ()a 0of146 residues (B) 56445kD. polypeptide has a heme;the Hb tetramer binds four 02. Fig.2-28 Detailed structure of the myoglobin moThe myoglobin polypeptide chain onsists of eight helical segments,designated by the lettersA through H,counting from the N-terminus.These helices,ranging in length from 7 to 26 residues,are linked by short,unordered regions that are named for the helices they conneet,as in the AB region or the EF region. The individual amino acids in 山 polypeptide are indicated according to their position within the various segments,as in His F8,the eighth residue in helix F,or Phe CD1,the first amino acid in the interhelical CD region.Occasionally,amino acids are specified in the comvemtional way,hat is,by th relative position in the chain,as in Gly153.The heme group is cradled within the folded polypeptide chain. Hemoglobin(Figure 2-27)is an 2 tetramer.Each of the four subunits has a conformation virtually identical to that of myoglobin.Two different types of subunits,a and B. are necessary to achieve cooperative O-binding by Hb.The chain at 146 amino acid residues is shorter than the myoglobin chain(153 residues),mainly because its final helical segment(the H helix)is shorter The a-chain (14l residues)also has a shortened H helix and lacks the d helix as well (Figure -)Max has devoted of Hb. noted very early in his studies that the molecule was highly symmetrical.The actual arrangemen of the four subunits with respect to one another is shown in Figure 2-30 for horse methemoglobin. All vertebrate hemoglobins show a three-dimensional structure essentially the same as this.The subunits pack in a tetrahedral array,creating a roughly spherical molecule64x5.5x5.0nm.The
31 Hemoglobin (Figure 2-27) is an α2β2 tetramer. Each of the four subunits has a conformation virtually identical to that of myoglobin. Two different types of subunits, α and β, are necessary to achieve cooperative O2-binding by Hb. The β- chain at 146 amino acid residues is shorter than the myoglobin chain (153 residues), mainly because its final helical segment (the H helix) is shorter. The α-chain (141 residues) also has a shortened H helix and lacks the D helix as well (Figure 2-29). Max Perutz, who has devoted his life to elucidating the atomic structure of Hb, noted very early in his studies that the molecule was highly symmetrical. The actual arrangement of the four subunits with respect to one another is shown in Figure 2-30 for horse methemoglobin. All vertebrate hemoglobins show a three-dimensional structure essentially the same as this. The subunits pack in a tetrahedral array, creating a roughly spherical molecule 6.4 × 5.5 × 5.0 nm. The Fig. 2-27 The myoglobin and hemoglobin molecules. Myoglobin (sperm whale): one polypeptide chain of 153 aa residues (mass = 17.2 kD) has one heme (mass =652 D) and binds one O2. Hemoglobin (human): four polypeptide chains, two of 141 aa residues (α) and two of 146 residues (β); mass=64.45 kD. Each polypeptide has a heme; the Hb tetramer binds four O2. Fig. 2-28 Detailed structure of the myoglobin molecule. The myoglobin polypeptide chain consists of eight helical segments, designated by the letters A through H, counting from the N-terminus. These helices, ranging in length from 7 to 26 residues, are linked by short, unordered regions that are named for the helices they connect, as in the AB region or the EF region. The individual amino acids in the polypeptide are indicated according to their position within the various segments, as in His F8, the eighth residue in helix F, or Phe CD1, the first amino acid in the interhelical CD region. Occasionally, amino acids are specified in the conventional way, that is, by the relative position in the chain, as in Gly153. The heme group is cradled within the folded polypeptide chain
four heme grouns nestled within the easily recognizable cleft formed hetween the f and f helices apart,25nm separates the and B1.The subunit interactions are mostly between dissimilar chains:each of the a-chains is in contact with both chains.but there are few orinteractions. Fig.2-29 Conformational drawings of the @and Bchains of Hb and the Myoglobin chain. Fig.2-30 The arrangement of subunits in horse methemoglobin,the first hemoglobin whose structure was determined by X-ray diffraction.The iron atoms on metHb are in the oxidized,ferric (Fe)state. Review Questions 1.How Many B-Galactosidase Molecules Are Present in an E.coli Cell?E.coli is a rod-shaped bacterium 2 um long and 1 um in diameter.When grown on lactose (a sugar found in milk). the bacterium synthesizes the enzyme B-galactosidase (M.450.000).which catalyzes the reakdown of lactose.The average density of the bacterial cell is2/mand%ofits total mass protein,of which%is B-galactosidase.Calculate the number B-galactosidase molecules in an E.coli cell grown on lactose 2.The Size of Proteins What is the approximate molecular weight of a protein containing 682 amino acids in a single polypeptide chain? 32
32 four heme groups, nestled within the easily recognizable cleft formed between the E and F helices of each polypeptide, are exposed at the surface of the molecule. The heme groups are quite far apart; 2.5 nm separates the closest iron ions, those of hemes α1 and β2, and those of hemes α2 and β1. The subunit interactions are mostly between dissimilar chains: each of the α-chains is in contact with both β-chains, but there are few α–α or β–β interactions. Fig. 2-29 Conformational drawings of the α and β-chains of Hb and the Myoglobin chain. Fig. 2-30 The arrangement of subunits in horse methemoglobin, the first hemoglobin whose structure was determined by X-ray diffraction. The iron atoms on metHb are in the oxidized, ferric (Fe3+) state. Review Questions 1. How Many β-Galactosidase Molecules Are Present in an E. coli Cell? E. coli is a rod-shaped bacterium 2 μm long and 1 μm in diameter. When grown on lactose (a sugar found in milk), the bacterium synthesizes the enzyme β-galactosidase (M,. 450,000), which catalyzes the breakdown of lactose. The average density of the bacterial cell is 1.2 g/mL, and 14% of its total mass is soluble protein, of which l.O% is β-galactosidase. Calculate the number of β-galactosidase molecules in an E. coli cell grown on lactose. 2. The Size of Proteins What is the approximate molecular weight of a protein containing 682 amino acids in a single polypeptide chain?
3.Rate of Svnthesis of Hair a-Keratin In human dimensions.the growth of hair is a relatively assembled into ropelike structures (see Fig.7-13).The fundamental structural element of a-keratin is the a helix,which has 3.6 amino acid residues per turn and a rise of .56nm per turn (see Fig 7-6).Assuming that the biosynthesis of a-helical keratin chains is the rate-limiting factor in the growth of hair,calculate the rate at which peptide bonds of a-ratin chains must be synthesized (peptide bonds per second)for the observed yearly growth of hai 4.Early Obseruations on the Structure of Wool William Astbury discovered that the x-ray pattern of wool shows a repeating structural unit spaced about 0.54 nm along the direction of the wool fiber.When he steamed and stretched the wool,the x-ray pattern showed a new .Steaming and stretching the wool and ther gave an ray pattem with the orignl spacing of about 54nm Although these observations provided important clues to the molecular structure of wool Astbury was unable to interpret them at the time.Given our current understanding of the structure of wool.interpret Astbury's observations. 5.Why Does Wool Shrink?When wool sweatersor socks are washed in hot water and/or dried in an electric dryer,they shrink.From what you 40 a-keratin structure,how can you account for this?Silk,on the other hand,does not shrink under the same conditions.Explain References 1.H.R.Horton,L.A.Moran er al.Principles of Biochemistry (3ed).New York:Pearson .T.MeKeeR.MeKee,Biochemisiry:con Mer- companies,Inc,2001. 3.A.L.Lehninger,D.L.Nelson and M.M.Cox,Principles of Biochemistry(2ed),New York. Worth Publishers.Inc.1993. 4.J.M.Berg.J.L.Tymoezko and L.Stryer,Biochemistry (5hed),New York:W.H.Freeman and Company,2002 33
33 3. Rate of Synthesis of Hair α-Keratin In human dimensions, the growth of hair is a relatively slow process, occurring at a rate of 15 to 20 cm/yr. All this growth is concentrated at the base of the hair fiber, where α-keratin filaments are synthesized inside living epidermal cells and assembled into ropelike structures (see Fig. 7-13). The fundamental structural element of α-keratin is the α helix, which has 3.6 amino acid residues per turn and a rise of 0.56 nm per turn (see Fig. 7-6). Assuming that the biosynthesis of α-helical keratin chains is the rate-limiting factor in the growth of hair, calculate the rate at which peptide bonds of α-keratin chains must be synthesized (peptide bonds per second) to account for the observed yearly growth of hair. 4. Early Obseruations on the Structure of Wool William Astbury discovered that the x-ray pattern of wool shows a repeating structural unit spaced about 0.54 nm along the direction of the wool fiber. When he steamed and stretched the wool, the x-ray pattern showed a new repeating structural unit at a spacing of 0.70 nm. Steaming and stretching the wool and then letting it shrink gave an x-ray pattern consistent with the original spacing of about 0.54 nm. Although these observations provided important clues to the molecular structure of wool, Astbury was unable to interpret them at the time. Given our current understanding of the structure of wool, interpret Astbury's observations. 5. Why Does Wool Shrink? When wool sweaters or socks are washed in hot water and/or dried in an electric dryer, they shrink. From what you know of a-keratin structure, how can you account for this? Silk, on the other hand, does not shrink under the same conditions. Explain. References 1. H. R. Horton，L. A. Moran et al. Principles of Biochemistry (3rd ed), New York: Pearson Education Inc, 2002. 2. T. McKee & J. R. McKee, Biochemistry: an introduction, second edition, McGraw-Hill companies, Inc, 2001. 3. A. L. Lehninger, D. L. Nelson and M. M. Cox, Principles of Biochemistry (2nd ed), New York: Worth Publishers, Inc. 1993. 4. J. M. Berg, J. L. Tymoczko and L. Stryer, Biochemistry (5th ed), New York: W. H. Freeman and Company, 2002