Although fibrous proteins generally have only one type of secondary structure, globular proteins can incorporate several types of secondary structure in the same molecule. Globular proteins-including enzymes, transport proteins, some peptide hormones, and immunoglobulins-are folded structures much more compact than α or β conformations (as shown for serum albumin in Figure 7-17).
The three-dimensional arrangement of all atoms in a protein is referred to as the tertiary structure, and this now becomes our focus. Whereas the secondary structure of polypeptide chains is determined by 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 and reside in different types of secondary structure may interact when the protein is folded. The formation of bends in the polypeptide chain during folding and the direction and angle of these bends are determined by the number and location of specific bend-producing amino acids, such as Pro, Thr, Ser, and Gly residues. Moreover, loops of the highly folded polypeptide chain are held in their characteristic tertiary positions by different kinds of weak-bonding interactions (and sometimes by covalent bonds such as disulfide cross-links) between R groups of adjacent loops.
We will now consider how secondary structures contribute to the tertiary folding of a polypeptide chain in a globular protein, and how this structure is stabilized by weak interactions, in particular by hydrophobic interactions involving nonpolar amino acid side chains in the tightly packed core of the protein.
Other important conclusions were drawn from the structure of myoglobin. 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 myoglobin molecule, hidden from exposure to water. All but two of the polar R groups are located on the outer surface of the molecule, and all of them are hydrated. The myoglobin molecule is so compact that in its interior there is room for only four molecules of water. This dense hydrophobic core is typical of globular proteins. The fraction of space occupied by atoms in an organic liquid is 0.25 to 0.35; in a typical solid the fraction is 0.75. In a protein the fraction is 0.72 to 0.76, very comparable to that in a solid. In this closely packed environment weak interactions strengthen and reinforce each other. For example, the nonpolar side chains in the core are so close together that short-range van der Waals interactions make a significant contribution to stabilizing hydrophobic interactions. By contrast, in an oil droplet suspended in water, the van der Waals interactions are minimal and the cohesiveness of the droplet is based almost exclusively on entropy.
The structure of myoglobin both confirmed some expectations and introduced some new elements of secondary structure. As predicted by Pauling and Corey, all the peptide bonds are in the planar trans configuration. The α 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 that the rigid R group of proline is largely incompatible with α-helical structure). Other bends contain Ser, Thr, and Asn residues, which are among the amino acids that tend to be incompatible with α-helical structure if they are in close proximity (p. 168).
The flat heme group rests in a crevice, or pocket, in the myoglobin molecule. The iron atom in the center of the heme group has two bonding (coordination) positions perpendicular to the plane of the heme.One 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 this pocket, the accessibility of the heme group to solvent is highly restricted. This is important for function because free heme groups in an oxygenated solution are rapidly oxidized from the ferrous (Fe2+) form, which is active in the reversible binding of O2, to the ferric (Fe3+) form, which does not bind O2.
Lysozyme (Mr 14,600) is an enzyme in egg white and human tear; that 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 a bactericidal agent. Like cytochrome c, about 40% of its 129 amino acie residues are in α-helical segments, but the arrangement is differenl and some (3 structure is also present. Four disulfide bonds contribute stability to this structure. The a helices line a long crevice in the side oi the molecule (Fig. 7-20), called the active site, which is the site o1 substrate binding and action. The bacterial polysaccharide that is the substrate for lysozyme fits into this crevice.
Ribonuclease, another small globular protein (Mr 13,700), is ar enzyme secreted by the pancreas into the small intestine, where i1 catalyzes the hydrolysis of certain bonds in the ribonucleic acids present in ingested food. Its tertiary structure, determined by x-ray analysis, shows that little of its 124 amino acid polypeptide chain is in α-helical conformation, but it contains many segments in the β conformation. Like lysozyme, ribonuclease has four disulfide bonds between loops of the polypeptide chain (Fig. 7-20).
The way to demonstrate the importance of a specific protein structure for biological function is to alter the structure and determine the effect on function. One extreme alteration is the total loss or randomization of three-dimensional structure, a process called denaturation. This is the familiar process that occurs when an egg is cooked. The white of the egg, which contains the soluble protein egg albumin, coagulates to a white solid on heating. It will not redissolve on cooling to yield a clear solution of protein as in the original unheated egg white. Heating of egg albumin has therefore changed it, seemingly in an irreversible manner. This effect of heat occurs with virtually all globular proteins, regardless of their size or biological function, although the precise temperature at which it occurs may vary and it is not always irreversible. The change in structure brought about by denaturation is almost invariably associated with loss of function. This is an expected consequence of the principle that the specific three-dimensional structure of a protein is critical to its function.The three-dimensional arrangement of all atoms in a protein is referred to as the tertiary structure, and this now becomes our focus. Whereas the secondary structure of polypeptide chains is determined by 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 and reside in different types of secondary structure may interact when the protein is folded. The formation of bends in the polypeptide chain during folding and the direction and angle of these bends are determined by the number and location of specific bend-producing amino acids, such as Pro, Thr, Ser, and Gly residues. Moreover, loops of the highly folded polypeptide chain are held in their characteristic tertiary positions by different kinds of weak-bonding interactions (and sometimes by covalent bonds such as disulfide cross-links) between R groups of adjacent loops.
We will now consider how secondary structures contribute to the tertiary folding of a polypeptide chain in a globular protein, and how this structure is stabilized by weak interactions, in particular by hydrophobic interactions involving nonpolar amino acid side chains in the tightly packed core of the protein.
X-Ray Analysis of Myoglobin R.evealed Its Tertiary Structure
The breakthrough in understanding globular protein structure came from x-ray diffraction studies of the protein myoglobin carried out by John Kendrew and his colleagues in the 1950s (Box 7-3). Myoglobin is a relatively small (Mr 16,700), oxygen-binding protein of muscle cells that functions in the storage and transport of oxygen for mitochondrial oxidation of cell nutrients. Myoglobin contains a single polypeptide chain 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 mammals such as the whale, seal, and porpoise, whose muscles are so rich in this protein that they are brown. Storage of oxygen by muscle myoglobin permits these animals to remain submerged for long periods of time.Other important conclusions were drawn from the structure of myoglobin. 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 myoglobin molecule, hidden from exposure to water. All but two of the polar R groups are located on the outer surface of the molecule, and all of them are hydrated. The myoglobin molecule is so compact that in its interior there is room for only four molecules of water. This dense hydrophobic core is typical of globular proteins. The fraction of space occupied by atoms in an organic liquid is 0.25 to 0.35; in a typical solid the fraction is 0.75. In a protein the fraction is 0.72 to 0.76, very comparable to that in a solid. In this closely packed environment weak interactions strengthen and reinforce each other. For example, the nonpolar side chains in the core are so close together that short-range van der Waals interactions make a significant contribution to stabilizing hydrophobic interactions. By contrast, in an oil droplet suspended in water, the van der Waals interactions are minimal and the cohesiveness of the droplet is based almost exclusively on entropy.
The structure of myoglobin both confirmed some expectations and introduced some new elements of secondary structure. As predicted by Pauling and Corey, all the peptide bonds are in the planar trans configuration. The α 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 that the rigid R group of proline is largely incompatible with α-helical structure). Other bends contain Ser, Thr, and Asn residues, which are among the amino acids that tend to be incompatible with α-helical structure if they are in close proximity (p. 168).
The flat heme group rests in a crevice, or pocket, in the myoglobin molecule. The iron atom in the center of the heme group has two bonding (coordination) positions perpendicular to the plane of the heme.One 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 this pocket, the accessibility of the heme group to solvent is highly restricted. This is important for function because free heme groups in an oxygenated solution are rapidly oxidized from the ferrous (Fe2+) form, which is active in the reversible binding of O2, to the ferric (Fe3+) form, which does not bind O2.
Proteins Differ in Tertiary Structure
With the elucidation of the tertiary structures of hundreds of other globular proteins by x-ray analysis, it is clear that myoglobin represents only one of many ways in which a polypeptide chain can be folded. In Figure 7-20 the structures of cytochrome c, lysozyme, and ribonuclease are compared. All have different amino acid sequences and 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 single heme 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 of the cytochrome c chain contains bends, turns, and irregularly coiled and extended segments. Thus, cytochrome c and myoglobin differ markedly in structure, even though both are small heme proteins.Lysozyme (Mr 14,600) is an enzyme in egg white and human tear; that 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 a bactericidal agent. Like cytochrome c, about 40% of its 129 amino acie residues are in α-helical segments, but the arrangement is differenl and some (3 structure is also present. Four disulfide bonds contribute stability to this structure. The a helices line a long crevice in the side oi the molecule (Fig. 7-20), called the active site, which is the site o1 substrate binding and action. The bacterial polysaccharide that is the substrate for lysozyme fits into this crevice.
Ribonuclease, another small globular protein (Mr 13,700), is ar enzyme secreted by the pancreas into the small intestine, where i1 catalyzes the hydrolysis of certain bonds in the ribonucleic acids present in ingested food. Its tertiary structure, determined by x-ray analysis, shows that little of its 124 amino acid polypeptide chain is in α-helical conformation, but it contains many segments in the β conformation. Like lysozyme, ribonuclease has four disulfide bonds between loops of the polypeptide chain (Fig. 7-20).
Proteins Lose Structure and Function on Denaturation
Proteins can be denatured not only by heat, but also by extremes of pH, by certain miscible organic solvents such as alcohol or acetone, by certain solutes such as urea, or by exposure of the protein to detergents. Each of these denaturing agents represents a relatively mild treatment in the sense that no covalent bonds in the polypeptide chain are broken. Boiling a protein solution disrupts a variety of weak interactions. Organic solvents, urea, and detergents act primarily by disrupting the hydrophobic interactions that make up the stable core of globular proteins; extremes of pH alter the net charge on the protein, causing electrostatic repulsion and disruption of some hydrogen bonding. Remember that the native structure of most proteins is only marginally stable. It is not necessary to disrupt all of the stabilizing weak interactions to reduce the thermodynamic stability to a level that is insufficient to keep the protein conformation intact.
Amino Acid Sequence Determines Tertiary Structure
The most important proof that the tertiary structure of a globular protein is determined by its amino acid sequence came from experiments showing that denaturation of some proteins is reversible. Some globular proteins denatured by heat, extremes of pH, or denaturing reagents will regain their native structure and their biological activity, a process called renaturation, if they are returned to conditions in which the native conformation is stable.A classic example is the denaturation and renaturation of ribonuclease. Purified ribonuclease can be completely denatured by exposure to a concentrated urea solution in the presence of a reducing agent. The reducing agent cleaves the four disulfide bonds to yield eight Cys residues, and the urea disrupts the stabilizing hydrophobic interactions, thus freeing the entire polypeptide from its folded conformation. Under these conditions the enzyme loses its catalytic activity and undergoes complete unfolding to a randomly coiled form (Fig. 7-21). When the urea and the reducing agent are removed, the randomly coiled, denatured ribonuclease spontaneously refolds into its correct tertiary structure, with full restoration of its catalytic activity (Fig. 7-21). The refolding of ribonuclease is so accurate that the four intrachain disulfide bonds are reformed in the same positions in the renatured molecule as in the native ribonuclease. In theory, the eight Cys residues could have recombined at random to form up to four disulfide bonds in 105 different ways. This classic experiment, carried out by Christian Animsen in the 1950s, proves that the amino acid sequence of the polypeptide chain of proteins contains all the information required to fold the chain into its native, three-dimensional structure.
| The study of homologous proteins has strengthened this conclusion. We have seen that in a series of homologous proteins, such as cytochrome c, from different species, the amino acid residues at certain positions in the sequence are invariant, whereas at other positions the amino acids may vary (see Fig. 6-15). This is also true for myoglobins isolated from different species of whales, from the seal, and from some terrestrial vertebrates. The similarity of the tertiary structures and amino acid sequences of myoglobins from different sources led to the conclusion that the amino acid sequence of myoglobin somehow must determine its three-dimensional folding pattern, an idea substantiated by the similar structures fcund by x-ray analysis of myoglobins from different species. Other sets of homologous proteins also show this relationship; in each case there are sequence homologies as well as similar tertiary structures. |
Proteins Lose Structure and Function on Denaturation
The way to demonstrate the importance of a specific protein structure for biological function is to alter the structure and determine the effect on function. One extreme alteration is the total loss or randomization of three-dimensional structure, a process called denaturation. This is the familiar process that occurs when an egg is cooked. The white of the egg, which contains the soluble protein egg albumin, coagulates to a white solid on heating. It will not redissolve on cooling to yield a clear solution of protein as in the original unheated egg white. Heating of egg albumin has therefore changed it, seemingly in an irreversible manner. This effect of heat occurs with virtually all globular proteins, regardless of their size or biological function, although the precise temperature at which it occurs may vary and it is not always irreversible. The change in structure brought about by denaturation is almost invariably associated with loss of function. This is an expected consequence of the principle that the specific three-dimensional structure of a protein is critical to its function.Proteins can be denatured not only by heat, but also by extremes of pH, by certain miscible organic solvents such as alcohol or acetone, by certain solutes such as urea, or by exposure of the protein to detergents. Each of these denaturing agents represents a relatively mild treatment in the sense that no covalent bonds in the polypeptide chain are broken. Boiling a protein solution disrupts a variety of weak interactions. Organic solvents, urea, and detergents act primarily by disrupting the hydrophobic interactions that make up the stable core of globular proteins; extremes of pH alter the net charge on the protein, causing electrostatic repulsion and disruption of some hydrogen bonding. Remember that the native structure of most proteins is only marginally stable. It is not necessary to disrupt all of the stabilizing weak interactions to reduce the thermodynamic stability to a level that is insufficient to keep the protein conformation intact.
Amino Acid Sequence Determines Tertiary Structure
The most important proof that the tertiary structure of a globular protein is determined by its amino acid sequence came from experiments showing that denaturation of some proteins is reversible. Some globular proteins denatured by heat, extremes of pH, or denaturing reagents will regain their native structure and their biological activity, a process called renaturation, if they are returned to conditions in which the native conformation is stable.A classic example is the denaturation and renaturation of ribonuclease. Purified ribonuclease can be completely denatured by exposure to a concentrated urea solution in the presence of a reducing agent. The reducing agent cleaves the four disulfide bonds to yield eight Cys residues, and the urea disrupts the stabilizing hydrophobic interactions, thus freeing the entire polypeptide from its folded conformation. Under these conditions the enzyme loses its catalytic activity and undergoes complete unfolding to a randomly coiled form (Fig. 7-21). When the urea and the reducing agent are removed, the randomly coiled, denatured ribonuclease spontaneously refolds into its correct tertiary structure, with full restoration of its catalytic activity (Fig. 7-21). The refolding of ribonuclease is so accurate that the four intrachain disulfide bonds are reformed in the same positions in the renatured molecule as in the native ribonuclease. In theory, the eight Cys residues could have recombined at random to form up to four disulfide bonds in 105 different ways. This classic experiment, carried out by Christian Animsen in the 1950s, proves that the amino acid sequence of the polypeptide chain of proteins contains all the information required to fold the chain into its native, three-dimensional structure.
| The study of homologous proteins has strengthened this conclusion. We have seen that in a series of homologous proteins, such as cytochrome c, from different species, the amino acid residues at certain positions in the sequence are invariant, whereas at other positions the amino acids may vary (see Fig. 6-15). This is also true for myoglobins isolated from different species of whales, from the seal, and from some terrestrial vertebrates. The similarity of the tertiary structures and amino acid sequences of myoglobins from different sources led to the conclusion that the amino acid sequence of myoglobin somehow must determine its three-dimensional folding pattern, an idea substantiated by the similar structures fcund by x-ray analysis of myoglobins from different species. Other sets of homologous proteins also show this relationship; in each case there are sequence homologies as well as similar tertiary structures. |
Proteins Lose Structure and Function on Denaturation
The way to demonstrate the importance of a specific protein structure for biological function is to alter the structure and determine the effect on function. One extreme alteration is the total loss or randomization of three-dimensional structure, a process called denaturation. This is the familiar process that occurs when an egg is cooked. The white of the egg, which contains the soluble protein egg albumin, coagulates to a white solid on heating. It will not redissolve on cooling to yield a clear solution of protein as in the original unheated egg white. Heating of egg albumin has therefore changed it, seemingly in an irreversible manner. This effect of heat occurs with virtually all globular proteins, regardless of their size or biological function, although the precise temperature at which it occurs may vary and it is not always irreversible. The change in structure brought about by denaturation is almost invariably associated with loss of function. This is an expected consequence of the principle that the specific three-dimensional structure of a protein is critical to its function.Proteins can be denatured not only by heat, but also by extremes of pH, by certain miscible organic solvents such as alcohol or acetone, by certain solutes such as urea, or by exposure of the protein to detergents. Each of these denaturing agents represents a relatively mild treatment in the sense that no covalent bonds in the polypeptide chain are broken. Boiling a protein solution disrupts a variety of weak interactions. Organic solvents, urea, and detergents act primarily by disrupting the hydrophobic interactions that make up the stable core of globular proteins; extremes of pH alter the net charge on the protein, causing electrostatic repulsion and disruption of some hydrogen bonding. Remember that the native structure of most proteins is only marginally stable. It is not necessary to disrupt all of the stabilizing weak interactions to reduce the thermodynamic stability to a level that is insufficient to keep the protein conformation intact.
Amino Acid Sequence Determines Tertiary Structure
The most important proof that the tertiary structure of a globular protein is determined by its amino acid sequence came from experiments showing that denaturation of some proteins is reversible. Some globular proteins denatured by heat, extremes of pH, or denaturing reagents will regain their native structure and their biological activity, a process called renaturation, if they are returned to conditions in which the native conformation is stable.A classic example is the denaturation and renaturation of ribonuclease. Purified ribonuclease can be completely denatured by exposure to a concentrated urea solution in the presence of a reducing agent. The reducing agent cleaves the four disulfide bonds to yield eight Cys residues, and the urea disrupts the stabilizing hydrophobic interactions, thus freeing the entire polypeptide from its folded conformation. Under these conditions the enzyme loses its catalytic activity and undergoes complete unfolding to a randomly coiled form (Fig. 7-21). When the urea and the reducing agent are removed, the randomly coiled, denatured ribonuclease spontaneously refolds into its correct tertiary structure, with full restoration of its catalytic activity (Fig. 7-21). The refolding of ribonuclease is so accurate that the four intrachain disulfide bonds are reformed in the same positions in the renatured molecule as in the native ribonuclease. In theory, the eight Cys residues could have recombined at random to form up to four disulfide bonds in 105 different ways. This classic experiment, carried out by Christian Animsen in the 1950s, proves that the amino acid sequence of the polypeptide chain of proteins contains all the information required to fold the chain into its native, three-dimensional structure.
| The study of homologous proteins has strengthened this conclusion. We have seen that in a series of homologous proteins, such as cytochrome c, from different species, the amino acid residues at certain positions in the sequence are invariant, whereas at other positions the amino acids may vary (see Fig. 6-15). This is also true for myoglobins isolated from different species of whales, from the seal, and from some terrestrial vertebrates. The similarity of the tertiary structures and amino acid sequences of myoglobins from different sources led to the conclusion that the amino acid sequence of myoglobin somehow must determine its three-dimensional folding pattern, an idea substantiated by the similar structures fcund by x-ray analysis of myoglobins from different species. Other sets of homologous proteins also show this relationship; in each case there are sequence homologies as well as similar tertiary structures. |
Looking at naturally occurring amino acid substitutions has an important limitation. Any change that abolishes the function of an essential protein (e.g., a change in an invariant residue) usually results in death of the organism very early in development. This severe form of natural selection eliminates many potentially informative changes from study. Fortunately, biochemists have devised methods to specifically alter amino acid sequences in the laboratory and examine the effects of these changes on protein structure and function. These methods are derived from recombinant DNA technology (Chapter 28) and rely on altering the genetic material encoding the protein. By this process, called site-directed mutagenesis, specific amino acid sequences can be changed by deleting, adding, rearranging, or substituting amino acid residues. The catalytic roles of certain amino acids lining the active sites of enzymes such as triose phosphate isomerase and chymotrypsin have been elucidated by substituting different amino acids in their place. The importance of certain amino acids in protein folding and structure is being addressed in the same way.
Tertiary Structures Are Not Rigid
Although the native tertiary conformation of a globular protein is the thermodynamically most stable form its polypeptide chain can assume, this conformation must not be regarded as absolutely rigid. Globular proteins have a certain amount of flexibility in their backbones and undergo short-range internal fluctuations. Many globular proteins also undergo small conformational changes in the course of their biological function. In many instances, these changes are associated with the binding of a ligand. The term ligand in this context refers to a specific molecule that is bound by a protein (from Latin, ligare, "to tie" or "bind"). For example, the hemoglobin molecule, which we shall examine later in this chapter, has one conformation when oxygen is bound, and another when the oxygen is released. Many enzyme molecules also undergo a conformational change on binding their substrates, a process that is part of their catalytic actionPolypeptides Fold Rapidly by a Stepwise Process
In living cells, proteins are made from amino acids at a very high rate. For example, Escherichia coli cells can make a complete, biologically active protein molecule containing 100 amino acid residues in about 5 s at 37°C. Yet calculations show that at least 1050yr would be required for a polypeptide chain of 100 amino acid residues to fold itself spontaneously by a random process in which it tries out all possible conformations around every single bond in its backbone until it finds its native, biologically active form. Thus protein folding cannot be a completely random, trial-and-error process. There simply must be shortcuts.The folding pathway of a large polypeptide chain is unquestionably complicated, and the principles that guide this process have not yet been worked out in detail. For several proteins, however, there is evidence that folding proceeds through several discrete intermediates, and that some of the earliest steps involve local folding of regions of secondary structure. In one model (Fig. 7-22), the process is envisioned as hierarchical, following the levels of structure outlined at the beginning of this chapter. Local secondary structures would form first, followed by longer-range interactions between, say, two α helices with compatible amino acid side chains, a process continuing until folding was complete. In an alternative model, folding is initiated by a spontaneous collapse of the polypeptide into a compact state mediated by hydrophobic interactions among nonpolar residues. The state resulting from this "hydrophobic collapse" may have a high content of secondary structure, but many amino acid side chains are not entirely fixed. Either or both models (and perhaps others) may apply to a given protein.
A number of structural constraints help to guide the interaction of regions of secondary structure. The most common patterns are sometimes referred to as supersecondary structures. A prominent one is a tendency for extended β conformations to twist in a right-handed sense (Fig. 7-23a). This influences both the arrangement of β sheets relative to one another and the path of the polypeptide segment connecting two β strands. Two parallel β strands, for example, must be connected by a crossover strand (Fig. 7-23b). In principle, this crossover could have a right- or left-handed conformation, but only the right-handed form is found in proteins. The twisting of β sheets also leads to a characteristic twisting of the structure formed when many sheets are put together. Two examples of resulting structures are the β barrel and saddle shapes (Fig. 7-23d), which form the core of many larger structures.
| Weak-bonding interactions represent the ultimate thermodynamic constraint on the interaction of different regions of secondary structure. The R groups of amino acids project outward from α-helical and a structures, and thus the need to bury hydrophobic residues means that water-soluble proteins must have more than one layer of secondary structure. One simple structural method for burying hydrophobic residues is a supersecondary structural unit called α- β-α loop (Fig. 7-24), a structure often repeated multiple times in larger proteins. More elaborate structures are domains made up of facing (3 sheets (with hydrophobic residues sandwiched between), and β sheets covered on one side with several a helices, as described later. |
It becomes more difficult to bury hydrophobic residues in smaller structures, and the number of potential weak interactions available for stabilization decreases. For this reason, smaller proteins are often held together with a number of covalent bonds, principally disulfide linkages. Recall the multiple disulfide bonds in the small proteins insulin (see Fig. 6-10) and ribonuclease (Fig. 7-21). Other types of covalent bonds also occur. The heme group in cytochrome c, for example, is covalently linked to the protein on two sides, providing a significant stabilization of the entire protein structure.
Not all proteins fold spontaneously as they are synthesized in the cell. Proteins that facilitate the folding of other proteins have been found in a wide variety of cells. These are called polypeptide chain binding proteins or molecular chaperones. Several of these proteins can bind to polypeptide chains, preventing nonspecific aggregation of weak-bonding side chains. They guide the folding of some polypeptides, as well as the assembly of multiple polypeptides into larger structures. Dissociation of polypeptide chain binding proteins from polypeptides is often coupled to ATP hydrolysis. One family of such proteins has structures that are highly conserved in organisms ranging from bacteria to mammals. These proteins (Mr 70,000), as well as several other families of polypeptide chain binding proteins, were originally identied as "heat shock" proteins because they are induced in many cells when heat stress is applied, and apparently help stabilize other proteins.
Some proteins have also been found that promote polypeptide folding by catalyzing processes that otherwise would limit the rate of folding, such as the reversible formation of disulfide bonds or proline isomerization (the interconversion of the cis and trans isomers of peptide bonds involving the imino nitrogen of proline; see Fig. 7-10).
Following the folding patterns outlined above and others yet to be discovered, a newly synthesized polypeptide chain quickly assumes its most stable tertiary structure. Although each protein has a unique structure, several patterns of tertiary structure seem to occur repeatedly in proteins that differ greatly in biological function and amino acid sequence (Fig. 7-25). This may reflect an unusual degree of stability and/or functional flexibility conferred by these particular tertiary structures. It also demonstrates that biological function is determined not only by the overall three-dimensional shape of the protein, but also by the arrangement of amino acids within that shape.
One structural motif is made up of eight β strands arranged in a circle with each β strand connected to its neighbor by an α helix. The (3 regions are arranged in the barrel structure described in Figure 7-23, and they influence the overall tertiary structure, giving rise to the name α/β barrel (Fig. 7-25a). This structure is found in many enzymes; a binding site for a cofactor or substrate is often found in a pocket formed near an end of the barrel.
Another structural motif is the four-helix bundle (Fig. 7-25b), in which four α helices are connected by three peptide loops. The helices are slightly tilted to form a pocket in the middle, which often contains a binding site for a metal or other cofactors essential for biological function. A somewhat similar structure in which seven helices are arranged in a barrel-like motif is found in some membrane proteins (see Fig. 10-10). The seven helices often surround a channel that spans the membrane.
A third motif has a β sheet in the "saddle" conformation forming a stable core, often surrounded by a number of α-helical regions (Fig. 7-25c). Structures of this kind are found in many enzymes. The location of the substrate binding site varies, determined by the placement of the a helices and other variable structural elements.
One final motif makes use of a sandwich of β sheets, layered so that the strands of the sheets form a quiltlike cross-hatching when viewed from above (Fig. 7-25d). This creates a hydrophobic pocket between the β sheets that is often a binding site for a planar hydrophobic molecule.
