Proteins are linear polymers formed by linking the a-carboxyl group of one amino acid to the a -amino group of another amino acid. This type of linkage is called a peptide bond or an amide bond. The formation of a di-pep-tide from two amino acids is accompanied by the loss of a water molecule (Figure 2.13). The equilibrium of this reaction lies on the side of hydrolysis rather than synthesis under most conditions. Hence, the biosynthesis of peptide bonds requires an input of free energy. Nonetheless, peptide bonds are quite stable kinetically because the rate of hydrolysis is extremely slow; the lifetime of a peptide bond in aqueous solution in the absence of a catalyst approaches 1000 years. A polypeptide chain consists of a regularly repeating part, called the main chain or backbone, and a variable part, comprising the distinctive side chains (Figure 2.15). The polypeptide backbone is rich in hydrogen-bonding potential. Each residue contains a carbonyl group (CPO), which is a good hydrogen-bond acceptor, and, with the exception of proline, an NH group, which is a good hydrogen-bond donor. These groups interact with each other and with functional groups from side chains to stabilize particular structures, as will be discussed in Section 2.3. In 1953, Frederick Sanger determined the amino acid sequence of insulin, a protein hormone (Figure 2.17). This work is a landmark in biochemistry because it showed for the first time that a protein has a precisely defined amino acid sequence consisting only of L amino acids linked by peptide bonds. This accomplishment stimulated other scientists to carry out sequence studies of a wide variety of proteins. Currently, the complete amino acid sequences of more than 2,000,000 proteins are known. The striking fact is that each protein has a unique, precisely defined amino acid sequence. The amino acid sequence of a protein is referred to as its primary structure. Polypeptide chains are flexible yet conformationally restricted Examination of the geometry of the protein backbone reveals several important features. First, the peptide bond is essentially planar (Figure 2.18). Thus, for a pair of amino acids linked by a peptide bond, six atoms lie in the same plane: the ? -carbon atom and CO group of the first amino acid and the NH group and ? -carbon atom of the second amino acid. The nature of the chemical bonding within a peptide accounts for the bond?s planarity. The bond resonates between a single bond and a double bond. Because of this double-bond character, rotation about this bond is prevented and thus the conformation of the peptide backbone is constrained. Two configurations are possible for a planar peptide bond. In the trans configuration, the two a-carbon atoms are on opposite sides of the peptide bond. In the cis configuration, these groups are on the same side of the peptide bond. Almost all peptide bonds in proteins are trans. This preference for trans over cis can be explained by the fact that steric clashes between groups attached to the a-carbon atoms hinder the formation of the cis form but do not arise in the trans configuration (Figure 2.20). By far the most common cis peptide bonds are X-Pro linkages. Such bonds show less preference for the trans configuration because the nitrogen of proline is bonded to two tetrahedral carbon atoms, limiting the steric differences between the trans and cis forms (Figure 2.21) ~ In contrast with the peptide bond, the bonds between the amino group and the a -carbon atom and between the a -carbon atom and the carbonyl group are pure single bonds. The two adjacent rigid peptide units can rotate about these bonds, taking on various orientations. This freedom of rotation about two bonds of each amino acid allows proteins to fold in many different ways. The rotations about these bonds can be specified by torsion angles (Figure 2.22). The angle of rotation about the bond between the nitrogen and the a -carbon atoms is called phi ( ). The angle of rotation about the bond between the a-carbon and the carbonyl carbon atoms is called psi ( ). A clockwise rotation about either bond as viewed from the nitrogen atom toward the a -carbon atom or from the carbonyl group toward the a ?carbon atom corresponds to a positive value. The and angles determine the path of the polypeptide chain. ~ The alpha helix is a coiled structure stabilized by intrachain hydrogen bonds In evaluating potential structures, Pauling and Corey considered which conformations of peptides were sterically allowed and which most fully exploited the hydrogen-bonding capacity of the backbone NH and CO groups. The first of their proposed structures, the ?-helix, is a rodlike structure (Figure 2.24). A tightly coiled backbone forms the inner part of the rod and the side chains extend outward in a helical array. The a helix is stabilized by hydrogen bonds between the NH and CO groups of the main chain ~ The Ramachandran diagram reveals that both the right-handed and the left-handed helices are among allowed conformations(Figure 2.26). However, right-handed helices are energetically more favor-able because there is less steric clash between the side chains and the back-bone. ~ The a -helical content of proteins ranges widely, from none to almost 100%. For example, about 75% of the residues in ferritin, a protein that helps store iron, are in a helices (Figure 2.28). Beta sheets are stabilized by hydrogen bonding between polypeptide strands Pauling and Corey proposed another periodic structural motif, which they named the ? pleated sheet (b because it was the second structure that they elucidated, the a helix having been the first). The b pleated sheet (or, more simply, the b sheet) differs markedly from the rodlike a helix. It is composed of two or more polypeptide chains called ? strands. A b strand is almost fully extended rather than being tightly coiled as in the a helix. A range of extended structures are sterically allowed (Figure 2.29). A ? sheet is formed by linking two or more ? strands lying next to one another through hydrogen bonds. Adjacent chains in a b sheet can run in opposite directions (antiparallel b sheet) or in the same direction (parallel b sheet). In the antiparallel arrangement, the NH group and the CO group of each amino acid are respectively hydrogen bonded to the CO group and the NH group of a partner on the adjacent chain (Figure 2.31). In the parallel arrangement, the hydrogen-bonding scheme is slightly more complicated. For each amino acid, the NH group is hydrogen bonded to the CO group of one amino acid on the adjacent strand, whereas the CO group is hydrogen bonded to the NH group on the amino acid two residues farther along the chain (Figure 2.32). Many strands, typically 4 or 5 but as many as 10 or more, can come together in b sheets. Such b sheets can be purely antiparallel, purely parallel, or mixed (Figure 2.33). More structurally diverse than a helices, b sheets can be almost flat but most adopt a somewhat twisted shape (Figure 2.34). The b sheet is an important structural element in many proteins. The b sheet is an important structural element in many proteins. For example, fatty acid-binding proteins, important for lipid metabolism, are built almost entirely from b sheets (Figure 2.35). Polypeptide chains can change direction by making reverse turns and loops. Most proteins have compact, globular shapes owing to reversals in the direction of their polypeptide chains. Many of these reversals are accomplished by a common structural element called the reverse turn (also known as the ? turn or hairpin turn), illustrated in Figure 2.36. In many reverse turns, the CO group of residue i of a polypeptide is hydrogen bonded to the NH group of residue i + 3. This interaction stabilizes abrupt changes in direction of the polypeptide chain. Fibrous proteins provide structural support for cells and tissues Special types of helices are present in the two proteins a -keratin and collagen. These proteins form long fibers that serve a structural role. a -Keratin, which is the primary component of wool, hair, and skin, consists of two right-handed a helices intertwined to form a type of left-handed superhelix called an a ?helical coiled coil. a -Keratin is a member of a super-family of proteins referred to as coiled-coil proteins (Figure 2.38) A different type of helix is present in collagen, the most abundant protein of mammals. Collagen is the main fibrous component of skin, bone, tendon, cartilage, and teeth. This extracellular protein is a rod-shaped molecule, about 3000 ┼ long and only 15 ┼ in diameter. It contains three helical polypeptide chains, each nearly 1000 residues long. Glycine appears at every third residue in the amino acid sequence, and the sequence glycine-proline-hydroxyproline recurs frequently (Figure 2.40). Hydroxyproline is a derivative of proline that has a hydroxyl group in place of one of the hydrogen atoms on the pyrrolidine rings. The inside of the triple-stranded helical cable is very crowded and accounts for the requirement that glycine be present at every third position on each strand (Figure 2.42A). The only residue that can fit in an interior position is glycine. The amino acid residue on either side of glycine is located on the outside of the cable, where there is room for the bulky rings of proline and hydroxyproline residues (Figure 2.42B). Myoglobin, the oxygen carrier in muscle, is a single polypeptide chain of 153 amino acids (see Chapter 7). The capacity of myoglobin to bind oxygen epends on the presence of heme, a nonpolypeptide prosthetic (helper) group consisting of protoporphyrin IX and a central iron atom. Myoglobin is an extremely compact molecule. Its overall dimensions are 45 3 35 3 25 ┼, an order of magnitude less than if it were fully stretched out (Figure 2.43). About 70% of the main chain is folded into eight a helices, and much of the rest of the chain forms turns and loops between helices. The folding of the main chain of myoglobin, like that of most other proteins, is complex and devoid of symmetry. The overall course of the poly-peptidechain of a protein is referred to as its tertiary structure. A unifying principle emerges from the distribution of side chains. The striking fact is that the interior consists almost entirely of nonpolar residues such as leucine, aline, methionine, and phenylalanine (Figure 2.44). Charged residues such as aspartate, glutamate, lysine, and arginine are absent from the inside of myoglobin. The only polar residues inside are two histidine residues, which play critical roles in binding iron and oxygen. The outside of myoglobin, on the other hand, consists of both polar and nonpolar residues. The space-filling model shows that there is very little empty space inside Some proteins that span biological membranes are ?the exceptions that prove the rule? because they have the reverse distribution of hydrophobic and hydrophilic amino acids. For example, consider porins, proteins found in the outer membranes of many bacteria (Figure 2.45). Membranes are built largely of hydrophobic alkane chains (Section 12.2). Thus, porins are covered on the outside largely with hydrophobic residues that interact with he neighboring alkane chains. In contrast, the center of the protein contain many charged and polar amino acids that surround a water-filled channel running through the middle of the protein. Thus, because porins function in hydrophobic environments, they are ?inside out? relative to proteins that function in aqueous solution. Some polypeptide chains fold into two or more compact regions that may be connected by a flexible segment of polypeptide chain, rather like pearls on a string. These compact globular units, called domains, range in size from about 30 to 400 amino acid residues. For example, the extracellular part of CD4, the cell-surface protein on certain cells of the immune system to which the human immunodeficiency virus (HIV) attaches itself, comprises four similar domains of approximately 100 amino acids each (Figure 2.47). Proteins may have domains in common even if their overall tertiary structures are different. More-complicated quaternary structures also are common. More than one type of subunit can be present, often in variable numbers. For example, human hemoglobin, the oxygen-carrying protein in blood, consists of two subunits of one type (designated a ) and two subunits of another type (designated b ), as illustrated in Figure 2.49. Viruses make the most of a limited amount of genetic information by forming coats that use the same kind of subunit repetitively in a symmetric array. The coat of rhinovirus, the virus that causes the common cold, includes 60 copies of each of four subunits (Figure 2.50). The subunits come together to form a nearly spherical shell that encloses the viral genome. How is the elaborate three-dimensional structure of proteins attained? The classic work of Christian Anfinsen in the 1950s on the enzyme ribonuclease revealed the relation between the amino acid sequence of a protein and its conformation. Ribonuclease is a single polypeptide chain consisting of 124 amino acid residues cross-linked by four disulfide bonds (Figure 2.51). Anfinsen?s plan was to destroy the three-dimensional structure of the enzyme and to then determine what conditions were required to restore the structure. Most polypeptide chains devoid of cross-links assume a random-coil conformation in 8 M urea or 6 M guanidinium chloride. When ribonuclease was treated with b-mercaptoethanol in 8 M urea, the product was a fully reduced, randomly coiled polypeptide chain devoid of enzymatic activity. When a protein is converted into a randomly coiled peptide without its normal activity, it is said to be denatured (Figure 2.53). Anfinsen then made the critical observation that the denatured ribonuclease, freed of urea and b-mercaptoethanol by dialysis, slowly regained enzymatic activity Anfinsen found that scrambled ribonuclease spontaneously converted into fully active, native ribonuclease when trace amounts of b -mercaptoethanol were added to an aqueous solution of the protein (Figure 2.54). The added b-mercaptoethanol catalyzed the rearrangement of disulfide pairings until the native structure was regained in about 10 hours. This process was driven by the decrease in free energy as the scrambled conformations were converted into the stable, native conformation of the enzyme. The native disulfide pairings of ribonuclease thus contribute to the stabilization of the thermodynamically preferred structure. Similar refolding experiments have been performed on many other pro-teins. In many cases, the native structure can be generated under suitable conditions. For other proteins, however, refolding does not proceed efficiently. In these cases, the unfolding protein molecules usually become tangled up with one another to form aggregates. Inside cells, proteins called chaperones block such illicit interactions. Additionally, it is now evident that some proteins do not assume a defined structure until they interact with molecular partners, as we will see shortly
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