Amino Acids, Peptides and Proteins Cheat Sheet: Key Concepts & Problems, Cheat Sheet of Biochemistry

Download Amino Acids, Peptides and Proteins Cheat Sheet: Key Concepts & Problems and more Cheat Sheet Biochemistry in PDF only on Docsity! 1 OUTLINE SPIDER SILK: A BIOSTEEL PROTEIN 5.1 AMINO ACIDS Amino Acid Classes Biologically Active Amino Acids Modified Amino Acids in Proteins Amino Acid Stereoisomers Titration of Amino Acids Amino Acid Reactions 5.2 PEPTIDES 5.3 PROTEINS Protein Structure The Folding Problem Fibrous Proteins Globular Proteins 5.4 MOLECULAR MACHINES BIOCHEMISTRY IN PERSPECTIVE Spider Silk and Biomimetics BIOCHEMISTRY IN THE LAB Protein Technology Available Online BIOCHEMISTRY IN PERSPECTIVE Protein Poisons BIOCHEMISTRY IN PERSPECTIVE Lead Poisoning BIOCHEMISTRY IN PERSPECTIVE Protein Folding and Human Disease BIOCHEMISTRY IN PERSPECTIVE Myosin: A Molecular Machine BIOCHEMISTRY IN THE LAB Protein Sequence Analysis: The Edman Degradation 5CHAPTE R Amino Acids, Peptides, and Proteins A Spider’s Web Constructed with Silk Fiber The amino acid sequence of spider silk protein and the spider’s silk fiber spinning process combine to made spider silk, one of the strongest materials on earth. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:55 PM Page 1 Overview PROTEINS ARE MOLECULAR TOOLS THAT PERFORM AN ASTONISHING VARI- ETY OF FUNCTIONS. IN ADDITION TO SERVING AS STRUCTURAL MATERIALS in all living organisms (e.g., actin and myosin in animal muscle cells), proteins are involved in such diverse functions as catalysis, metabolic regulation, transport, and defense. Proteins are composed of one or more polypeptides, unbranched polymers of 20 different amino acids. The genomes of most organisms specify the amino acid sequences of thousands or tens of thousands of proteins.  Spider Silk: A Biosteel Protein Spiders have evolved over 400 million years intoexceptionally successful predators. These invertebrate animals are a class of arthropods, called the arachnids. They have an exoskeleton, a segmented body, and jointed appendages. Although spiders possess an efficient venom in jection system, their most impressive feature is the production of silk, a multiuse protein fiber. Silk, which is spun through spinnerets at the end of the spider’s abdomen, is used in locomotion, mating, and offspring protection. The most promi- nent use of spider silk, however, is prey capture. The most sophisticated method of prey capture is the spiral, wheel-shape orb web, which is ori- ented vertically to intercept fast-moving flying prey. Spider silk’s mechanical properties ensure that the web readily absorbs impact energy so that prey is retained until the spider can subdue it. Orb webs (and the species that produce them) have fascinated humans for many thousands of years because of their dramatic visual impact. Ancient Greeks and Romans, for example, explained the occurrence of spiders and orb webs with the myth of Arachne, in which the mortal woman Arachne, an extraordinarily gifted weaver, offended Minerva (Athena in the Greek version), the goddess of weaving and other crafts, with her arrogant acceptance of a challenge to a weaving contest with the goddess. When confronted with Arachne’s flawless work, an enraged Minerva transformed her into a spider, doomed to forever weave webs. Humans have also long appreciated spider webs for their physical properties. Examples range from the ancient Greeks, who used spider webs to treat wounds, to the Australian aborigines who used spider silk to make fishing lines. In modern times spider silk has served as crosshairs in sci- entific equipment and gun sights. In the past sev- eral decades, spider silk and orb webs have attracted the attention of life scientists, bioengi- neers, and material scientists as they began to appreciate the unique mechanical properties of this remarkable protein. There are eight different types of spider silk, although no spider makes all of them. Dragline silk, a very strong fiber, is used for frame and radial lines in orb webs and as a safety line (to break a fall or escape other predators). Capture silk, an elas- tic and sticky fiber, is used in the spiral of webs. Spider silk is a lightweight fiber with impressive mechanical properties. Toughness, a combination of stiffness and strength, is a measure of how much energy is needed to rupture a fiber. Spider silk is about five times as tough as high-grade steel wire of the same weight and about twice as tough as synthetic fibers such as Kevlar (used in body armor). Spider silk’s tensile strength, the resis- tance of a material to breaking when stretched, is as great as that of Kevlar and greater than that of high-grade steel wire. Torsional resistance, the capacity of a fiber to resist twisting (an absolute requirement for draglines used as safety lines), is higher for spider silk than for all textile fibers, including Kevlar. It also has superior elasticity and resilience, the capacity of a material when it is deformed elastically to absorb and then release energy. Scientists estimate that a 2.54 cm (1 in)–thick rope made of spider silk could be sub- stituted for the flexible steel arresting wires used on aircraft carriers to rapidly stop a jet plane as it lands. 2 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:55 PM Page 2 5.1 Amino Acids 5 FIGURE 5.3 General Structure of the -Amino Acids C R O O− NH3 + CH These amino acids are referred to as standard amino acids. Common abbreviations for the standard amino acids are listed in Table 5.1. Note that 19 of the standard amino acids have the same general structure (Figure 5.3). These molecules con- tain a central carbon atom (the -carbon) to which an amino group, a carboxy- late group, a hydrogen atom, and an R (side chain) group are attached. The exception, proline, differs from the other standard amino acids in that its amino group is secondary, formed by ring closure between the R group and the amino nitrogen. Proline confers rigidity to the peptide chain because rotation about the -carbon is not possible. This structural feature has significant implications in the structure and, therefore, the function of proteins with a high proline content. Nonstandard amino acids consist of amino acid residues that have been chem- ically modified after incorporation into a polypeptide or amino acids that occur in living organisms but are not found in proteins. Nonstandard amino acids found in proteins are usually the result of posttranslational modifications (chemical changes that follow protein synthesis). Selenocysteine, an exception to this rule, is discussed in Chapter 19. At a pH of 7, the carboxyl group of an amino acid is in its conjugate base form (—COO–), and the amino group is in its conjugate acid form (—NH3). Thus each amino acid can behave as either an acid or a base. The term amphoteric is used to describe this property. Molecules that bear both positive and negative charges are called zwitterions. The R group gives each amino acid its unique properties. Amino Acid Classes Because the sequence of amino acids determines the final three-dimensional con- figuration of each protein, their structures are examined carefully in the next four subsections. Amino acids are classified according to their capacity to interact with water. By using this criterion, four classes may be distinguished: (1) nonpolar, (2) polar, (3) acidic, and (4) basic. Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Names and Abbreviations of the Standard Amino AcidsTABLE 5.1 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:56 PM Page 5 6 CHAPTER FIVE Amino Acids, Peptides, and Proteins NONPOLAR AMINO ACIDS The nonpolar amino acids contain mostly hydrocarbon R groups that do not bear positive or negative charges. Nonpolar (i.e., hydrophobic) amino acids play an important role in maintaining the three-dimensional structures of proteins, because they interact poorly with water. Two types of hydrocarbon side chains are found in this group: aromatic and aliphatic. Aromatic hydrocarbons contain cyclic structures that constitute a class of unsaturated hydrocarbons with planar conjugated π electron clouds. Benzene is one of the simplest aromatic hydrocarbons (Figure 5.4). The term aliphatic refers to nonaromatic hydrocarbons such as methane and cyclohexane. Phenylalanine and tryptophan contain aromatic ring structures. Glycine, alanine, valine, leucine, isoleucine, and proline have aliphatic R groups. A sulfur atom appears in the aliphatic side chains of methionine and cysteine. Methionine contains a thioether group (—S—CH3) in its side chain. Its derivative S-adenosyl methionine (SAM) is an important metabolite that serves as a methyl donor in numerous biochemical reactions. POLAR AMINO ACIDS Because polar amino acids have functional groups capable of hydrogen bonding, they easily interact with water. (Polar amino acids are described as hydrophilic, or “water-loving.”) Serine, threonine, tyrosine, asparagine, and glutamine belong to this category. Serine, threonine, and tyrosine contain a polar hydroxyl group, which enables them to participate in hydrogen bonding, an important factor in protein structure. The hydroxyl groups serve other functions in proteins. For example, the formation of the phosphate ester of tyrosine is a common regulatory mechanism. Additionally, the —OH groups of serine and threonine are points for attaching carbohydrates. Asparagine and glutamine are amide derivatives of the acidic amino acids aspartic acid and glutamic acid, respectively. Because the amide functional group is highly polar, the hydrogen-bonding capability of asparagine and glutamine has a significant effect on protein stability. The sulfhydryl group (—SH) of cysteine is highly reactive and is an important component of many enzymes. It also binds metals (e.g., iron and copper ions) in proteins. Additionally, the sulfhydryl groups of two cysteine molecules oxidize easily in the extracellular compartment to form a disulfide compound called cystine. (See p. 136 for a discussion of this reaction.) ACIDIC AMINO ACIDS Two standard amino acids have side chains with carboxylate groups. Because the side chains of aspartic acid and glutamic acid are negatively charged at physiological pH, they are often referred to as aspartate and glutamate. FIGURE 5.4 Benzene Classify these standard amino acids according to whether their structures are non- polar, polar, acidic, or basic. C C CH2 O H C C CH2 CH2 CH2 CH2 NH+ 3 O H C C CH2 CH2 O O H C (a) (b) (c) (d) C C CH2SH O HH3N + H3N + H3N + H3N + O– O– O– O– O– QUESTION 5.1 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:56 PM Page 6 BASIC AMINO ACIDS Basic amino acids bear a positive charge at physiological pH. They can therefore form ionic bonds with acidic amino acids. Lysine, which has a side chain amino group, accepts a proton from water to form the conjugate acid (—NH3). When lysine side chains in collagen fibrils, a vital structural component of ligaments and tendons, are oxidized and subsequently condensed, strong intramolecular and intermolecular cross-linkages are formed. Because the guanidino group of arginine has a pKa range of 11.5 to 12.5 in proteins, it is permanently protonated at physiological pH and, therefore, does not function in acid-base reactions. The imidazole side chain histidine, on the other hand, is a weak base because it is only partially ionized at pH 7 because its pKa is approximately 6. Its capacity under physiological conditions to accept or donate protons in response to small changes in pH plays an important role in the catalytic activity of numerous enzymes. Biologically Active Amino Acids In addition to their primary function as components of protein, amino acids have several other biological roles. 1. Several -amino acids or their derivatives act as chemical messengers (Figure 5.5). For example, glycine, glutamate, -amino butyric acid (GABA, a derivative of glutamate), and serotonin and melatonin (deriva- tives of try-ptophan) are neurotransmitters, substances released from one nerve cell that influence the function of a second nerve cell or a muscle cell. Thyroxine (a tyrosine derivative produced in the thyroid gland of animals) and indole acetic acid (a tryptophan derivative found in plants) are hor- mones—chemical signal mole-cules produced in one cell that regulate the function of othercells. 2. Amino acids are precursors of a variety of complex nitrogen-containing molecules. Examples include the nitrogenous base components of nucleotides and the nucleic acids, heme (the iron-containing organic group required for the biological activity of several important proteins), and chlorophyll (a pigment of critical importance in photosynthesis). 5.1 Amino Acids 7 K E Y C O N C E P T Amino acids are classified according to their capacity to interact with water. This criterion may be used to distinguish four classes: nonpolar, polar, acidic, and basic. FIGURE 5.5 Some Derivatives of Amino Acids GABA CH2 CH2 CH2 C O −H3N + O HO N CH2 CH2 + NH3 Serotonin H Thyroxine OHO I I I I CH2 CH C O − +NH3 O Indole acetic acid N H CH2 C OH O N H OH3C CH2 CH2 NH C H3C Melatonin O 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:56 PM Page 7 10 CHAPTER FIVE Amino Acids, Peptides, and Proteins Amino Acid pK1 (—COOH) pK2 (—NH3) pKR Glycine 2.34 9.60 Alanine 2.34 9.69 Valine 2.32 9.62 Leucine 2.36 9.60 Isoleucine 2.36 9.60 Serine 2.21 9.15 Threonine 2.63 10.43 Methionine 2.28 9.21 Phenylalanine 1.83 9.13 Tryptophan 2.83 9.39 Asparagine 2.02 8.80 Glutamine 2.17 9.13 Proline 1.99 10.60 Cysteine 1.71 10.78 8.33 Histidine 1.82 9.17 6.00 Aspartic acid 2.09 9.82 3.86 Glutamic acid 2.19 9.67 4.25 Tyrosine 2.20 9.11 10.07 Lysine 2.18 8.95 10.79 Arginine 2.17 9.04 12.48 pKa Values for the Ionizing Groups of the Amino AcidsTABLE 5.2 FIGURE 5.10 Titration of Two Amino Acids (a) Alanine and (b) Glutamic Acids. The ionized forms of glutamic acid are illustrated on p. xxx. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:56 PM Page 10 As more base is added, the second carboxyl group loses a proton, and the molecule has a –1 charge. Adding additional base results in the ammonium ion losing its proton. At this point, glutamate has a net charge of –2. The pI value for glutamate is the pH halfway between the pKa values for the two carboxyl groups (i.e., the pKa values that bracket the zwitterion): Problems 5.1 to 5.3 are sample titration problems. When amino acids are incorporated in polypeptides, the -amino and -carboxyl groups lose their charges. Consequently, except for the -amino and -carboxyl groups of the amino acid residues at the beginning and end, respec- tively, of a polypeptide chain all the ionizable groups of proteins are the side chain groups of seven amino acids: histidine, lysine, arginine, aspartate, glutamate, cysteine, and tyrosine. It should be noted that the pKa values of these groups can differ from those of free amino acids. The pKa values of individual R groups are affected by their positions within protein microenvironments. For example, when the side chain groups of two aspartate residues are in close proximity, the pKa of one of the carboxylate groups is raised. The significance of this phenom- enon will become apparent in the discussion of enzyme catalytic mechanisms (Section 6.4). 5.1 Amino Acids 11 Amino acids with ionizable side chains have more complex titration curves. Glutamic acid, for example, has a carboxyl side chain group (Figure 5.10b). At low pH, glutamic acid has net charge 1. As base is added, the -carboxyl group loses a proton to become a carboxylate group. Glutamate now has no net charge. pI  2.19  4.25  3.22 2 Calculate the isoelectric point of the following tripeptide: Assume that the pKa values listed for the amino acids in Table 5.2 are applica- ble to this problem. QUESTION 5.3 H3N CH + C NH O CH CH2 C O NH CH SH CH C O O– CH2 N H H3C CH3 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:56 PM Page 11 K E Y C O N C E P T S • Titration is useful in determining the rela- tive ionization potential of acidic and basic groups in an amino acid or peptide. • The pH at which an amino acid has no net charge is called its isoelectric point. WORKED PROBLEM 5.1 Consider the following amino acid and its pKa values: pKa1  2.18 pKa2  8.95 pKaR  10.79 a. Draw the structure of the amino acid as the pH of the solution changes from highly acidic to strongly basic. Solution (a) The ionizable hydrogens are lost in order of acidity, with the most acidic ioniz- ing first. b. Which form of the amino acid is present at the isoelectric point? Solution (b) The form present at the isoelectric point is electrically neutral: c. Calculate the isoelectric point. Solution (c) The isoelectric point is the average of the two pKas bracketing the zwitterion. pI  pK2 + pKR  8.95 + 10.79  9.87 2 2 H3N CH2 CH2 CH2 CH2 CH +NH3 C O O– + H3N CH2 CH2 CH2 CH2 CH +NH3 C O OH + + OH– OH–OH– H3N CH2 CH2 CH2 CH2 CH +NH3 C O O– + H2N CH2 CH2 CH2 CH2 CH NH2 C O O–H3N CH2 CH2 CH2 CH2 CH NH2 C O O H3N CH2 CH2 CH2 CH2 CH NH2 C O O– + 12 CHAPTER FIVE Amino Acids, Peptides, and Proteins 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:56 PM Page 12 FIGURE 5.12 The Peptide Bond (a) Resonance forms of the peptide bond. (b) Dimensions of a dipeptide. Because peptide bonds are rigid, the conformational degrees of freedom of a polypeptide chain are limited to rotations around the C—C and C—N bonds. The corresponding rotations are represented by  and , respectively. C C N H O C C C + N H –O C 1 2 (a) O O -Carbon Amide plane -Carbon Side group Amide plane N C N C H H R H (b) C 5.1 Amino Acids 15 Large polypeptides usually have well-defined, three-dimensional structures. This structure, referred to as the molecule’s native conformation, is a direct con- sequence of its amino acid sequence (the order in which the amino acids are linked together). Because all the linkages connecting the amino acid residues con- sist of single bonds, each polypeptide might be expected to undergo constant con- formational changes caused by rotation around the single bonds. However, most polypeptides spontaneously fold into a single biologically active form. In the early 1950s, Linus Pauling (1901–1994, 1954 Nobel Prize in Chemistry) and his col- leagues proposed an explanation. Using X-ray diffraction studies, they charac- terized the peptide bond (1.33 Å) as rigid and planar (flat) (Figure 5.12). Having discovered that the C—N bonds joining each two amino acids are shorter than other types of C—N bonds (1.45 Å), Pauling deduced that peptide bonds have a partial double-bond character. (This indicates that peptide bonds are resonance hybrids.) Because of the rigidity of the peptide bond, fully one-third of the bonds in a polypeptide backbone chain cannot rotate freely. Consequently, there are lim- its to the number of conformational possibilities. CYSTEINE OXIDATION The sulfhydryl group of cysteine is highly reactive. The most common reaction of this group is a reversible oxidation that forms a disulfide. Oxidation of two molecules of cysteine forms cystine, a molecule that contains a disulfide bond (Figure 5.13). When two cysteine residues form such a bond, it is referred to as a disulfide bridge. This bond can occur in a single chain to form a ring or between two separate chains to form an intermolecular bridge. Disulfide bridges help stabilize many polypeptides and proteins. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:56 PM Page 15 16 CHAPTER FIVE Amino Acids, Peptides, and Proteins K E Y C O N C E P T S • Polypeptides are polymers composed of amino acids linked by peptide bonds. The order of the amino acids in a polypeptide is called the amino acid sequence. • Disulfide bridges, formed by the oxidation of cysteine residues, are an important structural element in polypeptides and pro- teins. • Schiff bases are imines that form when amine groups react reversibly with carbonyl groups. In extracellular fluids such as blood (pH 7.2–7.4) and urine (pH 6.5), the sulfhydryl groups of cysteine (pKa 8.1) are subject to oxidation to form cystine. In peptides and proteins thiol groups are used to advantage in stabilizing protein structure and in thiol transfer reactions, but the free amino acid in tissue fluids can be prob - lematic because of the low solubility of cystine. In a genetic disorder known as cystinuria, defective membrane transport of cystine results in excessive excre- tion of cystine into the urine. Crystallization of the amino acid results in formation of calculi (stones) in the kidney, ureter, or urinary bladder. The stones may cause pain, infection, and blood in the urine. Cystine concentration in the kidney is reduced by massively increasing fluid intake and administering D-penicillamine. It is believed that penicillamine (Figure 5.14) is effective because penicil- lamine–cysteine disulfide, which is substantially more soluble than cystine, is formed. What is the structure of the penicillamine–cysteine disulfide? QUESTION 5.4 H3C C CH C OH NH2 OCH3 SH FIGURE 5.14 Structure of Penicillamine FIGURE 5.13 Oxidation of Two Cysteine Molecules to Form Cystine The disulfide bond in a polypeptide is called a disulfide bridge. 2H+ +2e– SCHIFF BASE FORMATION Molecules such as amino acids that possess primary amine groups can reversibly react with carbonyl groups. The imine products of this reaction are often referred to as Schiff bases. In a nucleophilic addition reaction, an amine nitrogen attacks the electrophilic carbon of a carbonyl group to form an alkoxide product. The transfer of a proton from the amine group to the oxygen to form a carbinolamine, followed by the transfer of another proton from an acid catalyst, converts the oxygen into a good leaving group (OH2). The subsequent elimination of a water molecule followed by loss of a proton from the nitrogen yields the imine product. The most important examples of Schiff base formation in biochemistry occur in amino acid metabolism. Schiff bases, referred to as aldimines, formed by the reversible reaction of an amino group with an aldehyde group, are intermediates (species formed during a reaction) in transamination reactions (pp. xxx–xxx). Cystinuria 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:56 PM Page 16 5.2 PEPTIDES Although less structurally complex than the larger protein molecules, peptides have significant biological activities. The structure and function of several inter- esting examples, presented in Table 5.3, are now discussed. The tripeptide glutathione (-glutamyl-L-cysteinylglycine) contains an unusual -amide bond. (Note that the -carboxyl group of the glutamic acid residue, not the -carboxyl group, contributes to the peptide bond.) Found in almost all organ- isms, glutathione (GSH) is involved in protein and DNA synthesis, drug and envi- ronmental toxin metabolism, amino acid transport, and other important biological processes. One group of glutathione’s functions exploits its effectiveness as a reducing agent. Glutathione protects cells from the destructive effects of oxida- tion by reacting with substances such as peroxides (R–O–O–R), by-products of O2 metabolism. For example, in red blood cells, hydrogen peroxide (H2O2) oxidizes the iron of hemoglobin to its ferric form (Fe3). Methemoglobin, the product of this reaction, is incapable of binding O2. Glutathione protects against the formation of methemoglobin by reducing H2O2 in a reaction catalyzed by 5.2 Peptides 17 Amine Alkoxide Carbinolamine CR N O O− H+ RR R H R +N H + CR N OH H2O R R C R H R H R N N +OH2 Aldimine (Schiff Base) R N + H C R R CR R R C RH H Name Amino Acid Sequence Selected Biologically Important PeptidesTABLE 5.3 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:56 PM Page 17 20 CHAPTER FIVE Amino Acids, Peptides, and Proteins shock proteins (hsps) that promote the correct refolding of damaged proteins. If such proteins are severely damaged, hsps promote their degradation. (Certain hsps function in the normal process of protein fold- ing.) Cells are protected from radiation by DNA repair enzymes. Protein research efforts in recent years have revealed that numerous proteins have multiple and often unrelated functions. Once thought to be a rare phenom- enon, multifunction proteins (sometimes referred to as moonlighting proteins) are a diverse class of molecules. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is a prominent example. As the name suggests, GAPD (p. 273) is an enzyme that catalyzes the oxidation of glyceraldehyde-3-phosphate, an inter- mediate in glucose catabolism. The GAPD protein is now known to have roles in such diverse processes as DNA replication and repair, endocytosis, and mem- brane fusion events. In addition to their functional classifications, proteins are categorized on the basis of amino acid sequence similarities and overall three-dimensional shape. Protein families are composed of protein molecules that are related by amino acid sequence similarity. Such proteins share an obvious common ancestry. Classic protein fam- ilies include the hemoglobins (blood oxygen transport proteins, pp. 168–171) and the immunoglobulins, the antibody proteins produced by the immune system in response to antigens (foreign substances). Proteins more distantly related are often classified into superfamilies. For example, the globin superfamily includes a vari- ety of heme-containing proteins that serve in the binding and/or transport of oxy- gen. In addition to the hemoglobins and myoglobins (oxygen-binding proteins in muscle cells), the globin superfamily includes neuroglobin and cytoglobin (oxygen- binding proteins in brain and other tissues, respectively) and the leghemoglobins (oxygen-sequestering proteins in the root nodules of leguminous plants). Because of their diversity, proteins are often classified in two additional ways: shape and composition. Proteins are classified into two major groups based on their shape. As the name suggests, fibrous proteins are long, rod-shaped molecules that are insoluble in water and physically tough. Fibrous proteins, such as the ker- atins found in skin, hair, and nails, have structural and protective functions. Globular proteins are compact spherical molecules that are usually water- soluble. Typically, globular proteins have dynamic functions. For example, nearly all enzymes have globular structures. Other examples include the immunoglo bulins and the transport proteins hemoglobin and albumin (a carrier of fatty acids inblood). On the basis of composition, proteins are classified as simple or conjugated. Simple proteins, such as serum albumin and keratin, contain only amino acids. In contrast, each conjugated protein consists of a simple protein combined with a non- protein component. The nonprotein component is called a prosthetic group. (A pro- tein without its prosthetic group is called an apoprotein. A protein molecule combined with its prosthetic group is referred to as a holoprotein.) Prosthetic groups typically play an important, even crucial, role in the function of proteins. Conjugated proteins are classified according to the nature of their prosthetic groups. For exam- ple, glycoproteins contain a carbohydrate component, lipoproteins contain lipid molecules, and metalloproteins contain metal ions. Similarly, phosphoproteins contain phosphate groups, and hemoproteins possess heme groups (p. xx). Protein Structure Proteins are extraordinarily complex molecules. Complete models depicting even the smallest of the polypeptide chains are almost impossible to comprehend. Simpler images that highlight specific features of a molecule are useful. Two methods of conveying structural information about proteins are presented in Figure 5.15. Another structural representation, referred to as a ball-and-stick model, is presented later (Figures 5.37 and 5.39). Biochemists have distinguished several levels of the structural organization of proteins. Primary structure, the amino acid sequence, is specified by genetic 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:56 PM Page 20 5.3 Proteins 21 FIGURE 5.15 The Enzyme Adenylate Kinase (a) This space-filling model illustrates the volume occupied by molecular components and overall shape. (b) In a ribbon model -pleated segments are represented by flat arrows. The -helices appear as spiral ribbons. information. As the polypeptide chain folds, it forms certain localized arrange- ments of adjacent (but not necessarily contiguous) amino acids that constitute secondary structure. The overall three-dimensional shape that a polypeptide assumes is called the tertiary structure. Proteins that consist of two or more polypeptide chains (or subunits) are said to have a quaternary structure. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 21 22 CHAPTER FIVE Amino Acids, Peptides, and Proteins PRIMARY STRUCTURE Every polypeptide has a specific amino acid sequence. The interactions between amino acid residues determine the protein’s three- dimensional structure and its functional role and relationship to other proteins. Polypeptides that have similar amino acid sequences and have arisen from the same ancestral gene are said to be homologous. Sequence comparisons among homologous polypeptides have been used to trace the genetic relationships of different species. For example, the sequence homologies of the mitochondrial redox protein cytochrome c have been used extensively in the study of evolution of species. Sequence comparisons of cytochrome c, an essential molecule in energy production, among numerous species reveal a significant amount of sequence conservation. The amino acid residues that are identical in all homologues of a protein, referred to as invariant, are presumed to be essential for the protein’s function. (In cytochrome c the invariant residues interact with heme, a prosthetic group, or certain other proteins involved in energy generation.) PRIMARY STRUCTURE, EVOLUTION, AND MOLECULAR DISEASES Over time, as the result of evolutionary processes, the amino acid sequences of polypeptides change. These modifications are caused by random and spontaneous alterations in DNA sequences called mutations. A significant number of primary sequence changes do not affect a polypeptide’s function. Some of these substitutions are said to be conservative because an amino acid with a chemically similar side chain is substituted. For example, at certain sequence positions leucine and isoleucine, which both contain hydrophobic side chains, may be substituted for each other without affecting function. Some sequence positions are significantly less stringent. These residues, referred to as variable, apparently perform nonspecific roles in the polypeptide’s function. Substitutions at conservative and variable sites have been used to trace evolutionary relationships. These studies assume that the longer the time since two species diverged from each other, the larger the number of differences in a certain polypeptide’s primary structure. For example, humans and chimpanzees are believed to have diverged relatively recently (perhaps only 4 million years ago). This presumption, based principally on fossil and anatomical evidence, is supported by cytochrome c primary sequence data indicating that the protein is identical in both species. Kangaroos, whales, and sheep, whose cytochrome c molecules each differ by 10 residues from the human protein, are believed to have evolved from a common ancestor that lived over 50 million years ago. It is interesting to note that quite often the overall three-dimensional structure does not change despite numerous amino acid sequence changes. The shape of pro- teins coded for by genes that diverged millions of years ago may show a remark- able resemblance to each other. Mutations, however, can also be deleterious. Such random changes in gene sequence can range from moderate to severe. Individual organisms with non- conservative, variable amino acid substitutions at the conservative, invariant residues of cytochrome c, for example, are not viable. Mutations can also have a profound effect without being immediately lethal. Sickle-cell anemia, which is caused by mutant hemoglobin, is a classic example of a group of maladies that Linus Pauling and his colleagues referred to as molecular diseases. (Dr. Pauling first demonstrated that sickle-cell patients have a mutant hemoglobin through the use of electrophoresis.) Human adult hemoglobin (HbA) is composed of two identical -chains and two identical -chains. Sickle-cell anemia results from a single amino acid substitution in the -chain of HbA. Analysis of the hemoglobin molecules of sickle-cell patients reveals that the only difference between HbA and sickle-cell hemoglobin (HbS) is at amino acid residue 6 in the -chain (Figure 5.16). Because of the substitution of a hydrophobic valine for a negatively charged glutamic acid, HbS molecules aggregate to form rigid rod- like structures in the oxygen-free state (Figure 5.17). The patient’s red blood cells K E Y C O N C E P T S • The primary structure of a polypeptide is its amino acid sequence. The amino acids are connected by peptide bonds. • Amino acid residues that are essential for the molecule’s function are referred to as invariant. • Proteins with similar amino acid sequences and functions and a common origin are said to be homologous. Molecular Diseases 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 22 (a) (b) (c) (d) (e) FIGURE 5.20 Selected Supersecondary Structures (a)  units, (b) -meander, (c)  unit, (d) -barrel, and (e) Greek key. Note that -strands are depicted as arrows. Arrow tips point toward the C-terminus. 5.3 Proteins 25 H O R-CH C C N HC-R N R-CH C N HC-R NO H HC-R C R-CH N C HC-R N C R-CH OH OH HO R-CH C C N HC-R N R-CH C N HC-R N C N C N H R-CH HC-R HC-R R-CH C Antiparallel Parallel 13.0 Å14.0 Å C N N C C NH O H O O O H O H O H O H C N FIGURE 5.19 -Pleated Sheet (a) Two forms of -pleated sheet: antiparallel and parallel. Hydrogen bonds are represented by dotted lines. (b) A more detailed view of antiparallel - pleated sheet.(b) (a) 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 25 26 CHAPTER FIVE Amino Acids, Peptides, and Proteins an organic side group permits a contiguous proline to assume a cis orientation (same side of the peptide plane), and a tight turn can form in a polypeptide strand. Proline is a helix-breaking residue that alters the direction of the polypeptide chain. The b-turn is common in proteins rich in -helical segments. In the -meander pattern, two antiparallel -sheets are connected by polar amino acids and glycines to effect a more abrupt change in direction called a reverse or hairpin turn. In  units (or helix-loop-helix units), two -helical regions separated by a nonhelical loop become aligned in a defined way because of interacting side chains. Several -barrel arrangements are formed when var- ious -sheet configurations fold back on themselves. When an antiparallel -sheet doubles back on itself in a pattern that resembles a common Greek pottery design the motif is called the Greek key. TERTIARY STRUCTURE Although globular proteins often contain significant numbers of secondary structural elements, several other factors contribute to their structure. The term tertiary structure refers to the unique three-dimensional conformations that globular proteins assume as they fold into their native (biologically active) structures and prosthetic groups, if any, are inserted. Protein folding, a process in which an unorganized, nascent (newly synthesized) molecule acquires a highly organized structure, occurs as a consequence of the interactions between the side chains in their primary structure. Tertiary structure has several important features: 1. Many polypeptides fold in such a fashion that amino acid residues that are distant from each other in the primary structure come into close proximity. 2. Globular proteins are compact because of efficient packing as the polypep- tide folds. During this process, most water molecules are excluded from the protein’s interior making interactions between both polar and nonpo- lar groups possible. 3. Large globular proteins (i.e., those with more than 200 amino acid residues) often contain several compact units called domains. Domains (Figure 5.21) are typically structurally independent segments that have specific functions (e.g., binding an ion or small molecule). The core three-dimensional struc- ture of a domain is called a fold. Well-known examples of folds include the nucleotide-binding Rossman fold and the globin fold. Domains are clas- sified on the basis of their core motif structure. Examples include , , /, and   . -Domains are composed exclusively of -helices, and -domains consist of antiparallel  strands. /-Domains contain vari- ous combinations of an -helix alternating with -strands ( motifs).    Domains are primarily -sheets with one or more outlying -helices. Most proteins contain two or more domains. 4. A number of eukaryotic proteins, referred to as modular or mosaic pro- teins, contain numerous duplicate or imperfect copies of one or more domains that are linked in series. Fibronectin (Figure 5.22) contains three repeating domains: Fl, F2, and F3. All three domains, which are found in a variety of extracellular matrix (ECM) proteins, contain binding sites for other ECM molecules such as collagen (p. xxx) and heparan sulfate (p. xxx), as well as certain cell surface receptors. Domain modules are coded for by genetic sequences created by gene duplications (extra gene copies that arise from errors in DNA replication). Such sequences are used by living organisms to construct new proteins. For example, the immunoglobulin structural domain is found not only in antibodies, but also in a variety of cell surface proteins. Several types of interactions stabilize tertiary structure (Figure 5.23): 1. Hydrophobic interactions. As a polypeptide folds, hydrophobic R groups are brought into close proximity because they are excluded from water. Then 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 26 E helix F helix (a) EF hand Ca2+ (b) Leucine zipper N HO CH3 CH3 C 6 6 1 1 8 7 7 2 2 3 3 5 5 4 4 (c) b-barrel 4 5 3 2 1 4 5 C N 3 21 (d) ATP-binding domain of hexokinase HOOC His 23 His 19 Zn Cys 3 Cys 6 NH2 1 (e) The α /β zinc-binding motif FIGURE 5.21 Selected Domains Found in Large Numbers of Proteins (a) The EF hand, a helix-loop-helix that binds specifically to Ca2+, and (b) the leucine zipper, a DNA-bind- ing domain, are two examples of -domains. (c) Human retinol-binding protein, a type of -barrel domain (retinol, a visual pigment molecule, is shown in yellow). (d) The ATP-binding domain of hexo kinase, a type of /-domain. (e) The / zinc-binding motif, a core feature of numerous DNA-binding domains. 5.3 Proteins 27 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 27 The precise nature of the forces that promote the folding of proteins (described on pp. 159–162) has not been completely resolved. It is clear, however, that pro- tein folding is a thermodynamically favorable process with an overall negative free energy change. According to the free energy equation G  H  TS a negative free energy change in a process is the result of a balance between favourable and unfavorable enthalpy and entropy changes (pp. xx –xx). As a polypeptide folds, favorable (negative) H values are the result in part of the sequestration of hydrophobic side chains within the interior of the molecule and the optimization of other noncovalent interactions. Opposing these factors is the unfavorable decrease in entropy that occurs as the disorganized polypeptide folds into its highly organized native state. The change in entropy of the water that surrounds the protein is positive because of the decreased organization of the water in going from the unfolded to the folded state of the protein. For most polypeptide molecules the net free energy change between the folded and unfolded state is relatively modest (the energy equivalent of several hydrogen bonds). The precarious balance between favorable and unfavorable forces allows proteins the flexibility they require for biological function. 30 CHAPTER FIVE Amino Acids, Peptides, and Proteins FIGURE 5.25 Structure of Immunoglobulin G IgG is an antibody molecule composed of two heavy chains (H) and two light chains (L) that together form a Y-shaped mole- cule. Each of the heavy and light chains contains constant (C) and variable (V), -barrel domains (the classic immunoglobu- lin fold). The chains are held together by disulfide bridges (yellow lines) and noncovalent interactions. The variable domains of the H and L chains form the site that binds to antigens (foreign molecules). Many antigenic proteins bind to the external surface of these sites. Note that disulfide bridges are also a structural feature within each constant domain. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 30 QUATERNARY STRUCTURE Many proteins, especially those with high molecular weights, are composed of several polypeptide chains. As mentioned, each polypeptide component is called a subunit. Subunits in a protein complex may be identical or quite different. Multisubunit proteins in which some or all subunits are identical are referred to as oligomers. Oligomers are composed of protomers, which may consist of one or more subunits. A large number of oligomeric proteins contain two or four subunit protomers, referred to as dimers and tetramers, respectively. There appear to be several reasons for the common occurrence of multisubunit proteins: 1. Synthesis of separate subunits may be more efficient than substantially increasing the length of a single polypeptide chain. 2. In supramolecular complexes such as collagen fibers, replacement of smaller worn-out or damaged components can be managed more effec- tively. 3. The complex interactions of multiple subunits help regulate a protein’s bio- logical function. Polypeptide subunits assemble and are held together by noncovalent interac- tions such as the hydrophobic and electrostatic interactions, and hydrogen bonds, as well as covalent cross-links. As with protein folding, the hydrophobic effect is clearly the most important because the structures of the complementary inter- facing surfaces between subunits are similar to those observed in the interior of globular protein domains. Although they are less numerous, covalent cross-links significantly stabilize certain multisubunit proteins. Prominent examples include the disulfide bridges in the immunoglobulins (Figure 5.25), and the desmosine and lysinonorleucine linkages in certain connective tissue proteins. Desmosine (Figure 5.26) cross-links connect four polypeptide chains in the rubberlike con- nective tissue protein elastin. They are formed as a result of a series of reactions involving the oxidation and condensation of lysine side chains. A similar process results in the formation of lysinonorleucine, a cross-linking structure that is found in elastin and collagen. Quite often the interactions between subunits are affected by the binding of ligands. In allostery, which is the control of protein function through lig- and binding, binding a ligand to a specific site in a protein triggers a confor- mational change that alters its affinity for other ligands. Ligand-induced conformational changes in such proteins are called allosteric transitions, and the ligands that trigger them are called effectors or modulators. Allosteric effects can be positive or negative, depending on whether effector binding increases or decreases the protein’s affinity for other ligands. One of the best understood examples of allosteric effects, the reversible binding of O2 and other ligands to hemoglobin, is described on pp. 169–171. (Because allosteric enzymes play a key role in the control of metabolic processes, allostery is dis- cussed further in Sections 6.3 and 6.5.) UNSTRUCTURED PROTEINS In the traditional view of proteins, a polypeptide’s function is determined by its specific and relatively stable three-dimensional structure. In recent years, however, as a result of new genomic methodologies and new applications of various forms of spectroscopy, it has become apparent that many proteins are in fact partially or completely unstructured. Unstructured proteins are referred to as IUPs (intrinsically unstructured proteins). If there is a complete lack of ordered structure, the term natively unfolded proteins is used. Most IUPs are eukaryotic. Amazingly, over 30% of eukaryotic proteins are partially or completely disordered, whereas only about 2 and 4% of archaean and bacterial proteins, respectively, can be described as unstructured. The folding of IUPs into stable three-dimensional conformations is prevented by biased 5.3 Proteins 31 Desmosine N + CH (CH2)3 C O NH (CH2)4 CH CNH O (CH2)2(CH2)2 CH C O NH HC HN C O (CH2)4 CH C O NH (CH2)4HC HN C O NH Lysinonorleucine FIGURE 5.26 Desmosine and Lysinonorleucine Linkages 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 31 32 CHAPTER FIVE Amino Acids, Peptides, and Proteins C N 1 23 4 5 6 7 8 N C C N 1 2 3 4 5 6 78 Review the following illustrations of globular proteins. Identify examples of secondary and supersecondary structure. QUESTION 5.8 Illustrate the noncovalent interactions that can occur between the following side chain groups in folded polypeptides: (a) serine and glutamate, (b) arginine and aspartate, (c) threonine and serine, (d) glutamine and aspartate, (e) phenylalanine and tryptophan. QUESTION 5.9 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 32 FIGURE 5.28 The Anfinsen Experiment Ribonuclease denatured by 8 M urea and a mercaptan (RSH, a reagent that reduces disulfides to sulfhydryl groups) can be renaturated by removing the urea and RSH and air-oxidizing the reduced disulfides. 5.3 Proteins 35 reported by Christian Anfinsen in the late 1950s. Working with bovine pancreatic RNase, Anfinsen demonstrated that under favorable conditions a denatured pro- tein could refold into its native and biologically active state (Figure 5.28). This discovery suggested that the three-dimensional structure of any protein could be predicted if the physical and chemical properties of the amino acids and the forces that drive the folding process (e.g., bond rotations, free energy considera- tions, and the behavior of amino acids in aqueous environments) were understood. Unfortunately, several decades of painstaking research with the most sophisticated tools available (e.g., X-ray crystallography and NMR in combination with site- directed mutagenesis and computer-based mathematical modeling) resulted in only limited progress. However, this work did reveal that protein folding is a step- wise process in which secondary structure formation (i.e., -helix and -pleated sheet) is an early feature. Hydrophobic interactions are an important force in fold- ing. In addition, amino acid substitutions experimentally introduced into cer- tain proteins reveal that changes in surface amino acids rarely affect the protein’s structure. In contrast, substitutions of amino acids within the hydrophobic core often lead to serious structural changes in conformation. In recent years important advances have been made by biochemists in protein- folding research. Protein-folding researchers have determined that the process does not consist, as was originally thought, of a single pathway. Instead, there are numerous routes that a polypeptide can take to fold into its native state. As illus- trated in Figure 5.29a an energy landscape with a funnel shape appears to best describe how an unfolded polypeptide with its own unique set of constraints (e.g., its amino acid sequence and posttranslational modifications, and environmental features within the cell such as temperature, pH, and molecular crowding) nego- tiates its way to a low-energy folded state. Depending largely on its size, a polypeptide may or may not form intermediates (species existing long enough to be detected) that are momentarily trapped in local energy wells (Figure 5.29b). Small molecules (fewer than 100 residues) often fold without intermediate for- mation (Figure 5.30a). As these molecules begin emerging from the ribosome, a rapid and cooperative folding process begins in which side chain interactions Visit the companion website at www.oup.com/us/mckee to read the Biochemistry in Perspective essay on protein folding and human health. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 7:33 PM Page 35 36 CHAPTER FIVE Amino Acids, Peptides, and Proteins FIGURE 5.29 The Energy Landscape for Protein Folding (a) Color is used to indicate the entropy level of the folding polypeptide. As folding progresses the polypeptide moves from a disordered stated (high entropy, red) toward a progressively more ordered conformation until its unique biologically active confor- mation is achieved (lower entropy, blue): (b) A depiction of the conformational state of a polypeptide during folding: polypep- tides can fold into their native states by sev- eral different pathways. Many molecules form transient intermediates, whereas others may become trapped in a misfolded state. facilitate the formation and alignment of secondary structures. The folding of larger polypeptides typically involves the formation of several intermediates (Figure 5.30b, c). In many of these molecules or the domains within a mole- cule, the hydrophobically collapsed shape of the intermediate is referred to as a molten globule. The term molten globule refers to a partially organized globu- lar state of a folding polypeptide that resembles the molecule’s native state. Within the interior of a molten globule, tertiary interactions among amino acid side chains are fluctuating; that is, they have not yet stabilized. It has also become increasingly clear that the folding and targeting of many proteins in living cells are aided by a group of molecules now referred to as the molecular chaperones. These molecules, most of which appear to be heat shock proteins (hsps), apparently occur in all organisms. Several classes of molecular chaperones have been found in organisms, ranging from bacteria to the higher animals and plants. They are found in several eukaryotic organelles, such as mito- chondria, chloroplasts, and ER. There is a high degree of sequence homology among the molecular chaperones of all species so far investigated. The properties of several of these important molecules are described next. MOLECULAR CHAPERONES Molecular chaperones apparently assist unfolded proteins in two ways. First, during a finite time between synthesis and folding, 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 36 proteins must be protected from inappropriate protein-protein interactions. For example, certain mitochondrial and chloroplast proteins must remain unfolded until they are inserted in an organelle membrane. Second, proteins must fold rapidly and precisely into their correct conformations. Some must be assembled into multisubunit complexes. Investigations of protein folding in a variety of organisms reveal the existence of two major molecular chaperone classes in protein folding. 1. Hsp70s. The hsp70s are a family of molecular chaperones that bind to and stabilize proteins during the early stages of folding. Numerous hsp70 monomers bind to short hydrophobic segments in unfolded polypeptides, thereby preventing molten globule formation. Each type of hsp70 possesses two binding sites, one for an unfolded protein segment and another for ATP. Release of a polypeptide from an hsp70 involves ATP hydrolysis. Mitochondrial and ER-localized hsp70s are required for transmembrane translocation of some polypeptides. 2. Hsp60s. Once an unfolded polypeptide has been released by hsp70, it is passed on to a member of a family of molecular chaperones referred to as the hsp60s (also called the chaperonins or Cpn60s), which mediate pro- tein folding. The hsp60s form a large structure composed of two stacked seven-subunit rings. The unfolded protein enters the hydrophobic cavity of the hsp60 complex (Figure 5.31). The hsp60 system, which consists of two 5.3 Proteins 37 FIGURE 5.30 Protein Folding (a) In many small proteins, folding is coop- erative with no intermediates formed. (b) In some larger proteins, folding involves the initial formation of a molten globule fol- lowed by rearrangement into the native con- formation. (c) Large proteins with multiple domains follow a more complex pathway, with each domain folding separately before the entire molecule progresses to its native conformation. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 37 40 CHAPTER FIVE Amino Acids, Peptides, and Proteins Collagen is composed of three left-handed polypeptide helices that are twisted around each other to form a right-handed triple helix (Figure 5.35). Type I col- lagen molecules, found in teeth, bone, skin, and tendons, are about 300 nm long and approximately 1.5 nm wide. Approximately 90% of the collagen found in humans is type I. The amino acid composition of collagen is distinctive. Glycine constitutes approximately one-third of the amino acid residues. Proline and 4-hydroxypro- line may account for as much as 30% of a collagen molecule’s amino acid com- position. Small amounts of 3-hydroxyproline and 5-hydroxylysine also occur. (Specific proline and lysine residues in collagen’s primary sequence are hydrox- ylated within the rough ER after the polypeptides have been synthesized. These reactions, which are discussed in Chapter 19, require ascorbic acid (p. xxx). Collagen’s amino acid sequence primarily consists of large numbers of repeat- ing triplets with the sequence of Gly—X—Y, in which X and Y are often proline and hydroxyproline. Hydroxylysine is also found in the Y position. Simple carbohydrate groups are often attached to the hydroxyl group of hydroxylysine residues. It has been suggested that collagen’s carbohydrate components are required for fibrilogenesis, the assembly of collagen fibers in their extracellular locations, such as tendons and bone. α-Helix Coiled coil of two α-helices Filament (four right-hand twisted protofilaments) Protofilament (pair of coiled coils) FIGURE 5.33 -Keratin The -helical rodlike domains of two keratin polypeptides form a coiled coil. Two stag- gered antiparallel rows of these dimers form a supercoiled protofilament. Hydrogen bonds and disulfied bridges are the principal inter- actions between subunits. Hundreds of fila- ments, each containing four protofilaments, form a macrofibril. Each hair cell, also called a fiber, contains several macrofibrils. Each strand of hair consists of numerous dead cells packed with keratin molecules. In addition to hair, the keratins are also found in wool, skin, horns, and fingernails. FIGURE 5.34 Molecular Model of Silk Fibroin In fibroin, the silk fibrous protein produced by silkworms, the polypeptide chains are arranged in fully extended antiparallel - pleated sheet conformations. Note that the R groups of alanine on one side of each - pleated sheet interdigitate with similar residues on the adjacent sheet. Silk fibers (fibroin embedded in an amorphous matrix) are flexible because the pleated sheets are loosely bonded to each other (primarily with weak van der Waals forces) and slide over each other easily. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:57 PM Page 40 5.3 Proteins 41 Collagen molecule Packing of molecules Hole zone Overlap zone FIGURE 5.35 Collagen Fibrils The bands are formed by staggered collagen molecules. Cross-striations are about 680 Å apart. Each collagen molecule is about 3000 Å long. The enzyme lysyl oxidase converts some of the lysine and hydroxylysine side groups to aldehydes through oxidative deamination, and this facilitates the spon- taneous nonenzymatic formation of strengthening aldimine, and aldol cross-links. (An aldol cross-link is formed in a reaction, called an aldol condensation, in which two aldehydes form an , -unsaturated aldehyde linkage. In condensa- tion reactions, a small molecule, in this case H2O, is removed.) Cross-linkages also occur between hydroxylysine-linked carbohydrates and the amino group of other lysine and hydroxylysine residues on adjacent molecules. Increased cross- linking with age leads to the brittleness and breakage of the collagen fibers that occur in older organisms. Glycine is prominent in collagen sequences because the triple helix is formed by interchain hydrogen bonding involving the glycine residues. Therefore every third residue is in close contact with the other two chains. Glycine is the only amino acid with an R group sufficiently small for the space available. Larger R groups would destabilize the superhelix structure. The triple helix is further strengthened by hydrogen bonding between the polypeptides (caused princi- pally by the large number of hydroxyproline residues) and lysinonorleucine link- ages that stabilize the orderly arrays of triple helices in the final collagen fibril. Globular Proteins The biological functions of globular proteins usually involve the precise bind- ing of small ligands or large macromolecules such as nucleic acids or other proteins. Each protein possesses one or more unique cavities or clefts whose struc- ture is complementary to a specific ligand. After ligand binding, a conformational change occurs in the protein that is linked to a biochemical event. For example, the binding of ATP to myosin in muscle cells is a critical event in muscle contraction. The oxygen-binding proteins myoglobin and hemoglobin are interesting and well-researched examples of globular proteins. They are both members of the hemoproteins, a specialized group of proteins that contain the prosthetic group heme. Although the heme group (Figure 5.36) in both proteins is responsible CH3 C CH C C CHC C C CH3 C C CH CH2 CCCH3 N NN Fe CHC N CHC C CH CH2 CH2 C CH2 COO− CC CH2 CH3 CH2 COO− FIGURE 5.36 Heme Heme consists of a porphyrin ring (composed of four pyrroles) with Fe2+ in the center. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:58 PM Page 41 42 CHAPTER FIVE Amino Acids, Peptides, and Proteins Covalent cross-links contribute to the strength of collagen. The first reaction in cross-link formation is catalyzed by the copper-containing enzyme lysyl oxidase, which converts lysine residues to the aldehyde allysine: Allysine then reacts with other side chain aldehyde or amino groups to form cross- linkages. For example, two allysine residues react to form an aldol cross-linked product: In a disease called lathyrism, which occurs in humans and several other ani- mals, a toxin (-aminopropionitrile) found in sweet peas (Lathyrus odoratus) inactivates lysyl oxidase. Consider the abundance of collagen in animal bodies and suggest some likely symptoms of this malady. (CH2)4CH C O NH NH2 (CH2)3CH C O NH C H OLysyl oxidase Lysine residue Allysine residue Allysine residue Allysine residue +(CH2)3CH C O NH C H (CH2)3C O CH OC NH H (CH2)2CH C O NH C CH (CH2)3 CH OC NHC H O Aldol cross-link O QUESTION 5.10 for the reversible binding of molecular oxygen, the physiological roles of myo- globin and hemoglobin are significantly different. The chemical properties of heme are dependent on the Fe2 ion in the center of the prosthetic group. Fe2, which forms six coordinate bonds, is bound to the four nitrogens in the center of the protoporphyrin ring. Two other coordinate bonds are available, one on each side of the planar heme structure. In myoglobin and hemoglobin, the fifth coor- dination bond is to the nitrogen atom in a histidine residue, and the sixth coor- dination bond is available for binding oxygen. In addition to serving as a reservoir for oxygen within muscle cells, myoglobin also facilitates the intracellular dif- fusion of oxygen. The role of hemoglobin, the primary protein of red blood cells, is to deliver oxygen to cells throughout the body. A comparison of the struc- tures of these two proteins illustrates several important principles of protein struc- ture, function, and regulation. MYOGLOBIN Myoglobin, found in high concentration in skeletal and cardiac muscle, gives these tissues their characteristic red color. The muscles of diving mammals such as whales, which remain submerged for long periods, have high myoglobin concentrations. Because of the extremely high concentrations of Lathyrism 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:58 PM Page 42 left of the hemoglobin curve, myoglobin gives up oxygen only when the mus- cle cell’s oxygen concentration is very low (i.e., during strenuous exercise). In addition, because myoglobin has a greater affinity for oxygen than does hemo- globin, oxygen moves from blood to muscle. The binding of ligands other than oxygen affects hemoglobin’s oxygen-bind- ing properties. For example, the dissociation of oxygen from hemoglobin is enhanced if pH decreases. By this mechanism, called the Bohr effect, oxygen is delivered to cells in proportion to their needs. Metabolically active cells, which require large amounts of oxygen for energy generation, also produce large amounts of the waste product CO2. As CO2 diffuses into blood, it reacts with water to form HCO3– and H. (The bicarbonate buffer was discussed on p. xx.) The subsequent binding of H to several ionizable groups on hemoglobin molecules enhances the dissociation of O2 by converting hemoglobin to its T state. (Hydrogen ions bind preferentially to deoxyHb. Any increase in H concentration stabilizes the deoxy conformation of the protein and therefore shifts the equilibrium distribution between the T and R states.) When a small number of CO2 molecules bind to ter- minal amino groups on hemoglobin (forming carbamate or —NHCOO– groups) the deoxy form (T state) of the protein is additionally stabilized. 2,3-Bisphosphoglycerate (BPG) (also called glycerate-2,3-bisphosphate) is also an important regulator of hemoglobin function. Although most cells contain only trace amounts of BPG, red blood cells contain a considerable amount. BPG is derived from glycerate-l,3-bisphosphate, an intermediate in the breakdown of the energy-rich compound glucose. In the absence of BPG, 5.3 Proteins 45 (a) Deoxyhemoglobin (b) Oxyhemoglobin 15° 15° b2 b1 b1 b2 b1 a1 a2 a1 a1 a2 FIGURE 5.40 The Hemoglobin Allosteric Transition When hemoglobin is oxygenated, the 11 and 22 dimers slide by each other and rotate 15º. 20 40 60 Partial pressure of oxygen (torr) 80 100 120 Venous pressure Arterial pressure Hemoglobin Myoglobin 0 O 2 sa tu ra tio n (% ) 80 60 40 20 100 FIGURE 5.41 Dissociation Curves Measure the Affinity of Hemoglobin and Myoglobin for Oxygen. Arterial blood, enriched in O2, delivers it to the tissues. Venous blood, which drains from tissues, is O2 depleted. K E Y C O N C E P T S • Globular protein function usually involves binding to small ligands or to other macro- molecules. • The oxygen-binding properties of myoglo- bin and hemoglobin are determined in part by the number of subunits they contain. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:58 PM Page 45 46 CHAPTER FIVE Amino Acids, Peptides, and Proteins 100 10 20 300 Partial pressure of oxygen (torr) 40 50 80 60 40 20 +BPG 60 –BPG O 2 sa tu ra tio n (% ) FIGURE 5.42 The Effect of 2,3-Bisphosphoglycerate (BPG) on the Affinity Between Oxygen and Hemoglobin In the absence of BPG (–BPG), hemoglobin has a high affinity for O2; where BPG is present and binds to hemoglobin (+BPG), its affinity for O2 decreases. Fetal hemoglobin (HbF) binds to BPG to a lesser extent than does HbA. Why do you think HbF has a greater affinity for oxygen than does maternal hemoglobin? QUESTION 5.11 Myoglobin stores O2 in muscle tissue to be used by the mitochondria only when the cell is in oxygen debt, while hemoglobin can effectively transport O2 from the lungs and deliver it discriminately to cells in need of O2. Describe the structural features that allow these two proteins to accomplish separate functions. QUESTION 5.12 hemoglobin has a very high affinity for oxygen (Figure 5.42). As with H and CO2, binding BPG stabilizes deoxyHb. A negatively charged BPG molecule binds in a central cavity within hemoglobin that is lined with positively charged amino acids. In the lungs the process is reversed. A high oxygen concentration drives the conversion from the deoxyHb configuration to that of oxyHb. The change in the protein’s three-dimensional structure initiated by the binding of the first oxygen molecule releases bound CO2, H, and BPG. The H recombines with HCO3– to form carbonic acid, which then dissociates to form CO2 and H2O. Afterward, CO2 diffuses from the blood into the alveoli. 5.4 MOLECULAR MACHINES Purposeful movement is thehallmark of living organisms. This behavior takes myriad forms that range from the record-setting 110 km/h chasing sprint of the cheetah to more subtle movements such as the migration of white blood cells in the animal body, cytoplasmic streaming in plant cells, intracellular transport of organelles, and the enzyme-catalyzed unwinding of DNA. The multisubunit pro- teins responsible for these phenomena (e.g., the muscle sarcomere and various other types of cytoskeletal components, and DNA polymerase) function as bio- logical machines. Machines are defined as mechanical devices with moving parts that perform work (the product of force and distance). When machines are used correctly, they permit the accomplishment of tasks that would often be impossi- ble without them. Although biological machines are composed of relatively fragile 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:58 PM Page 46 proteins that cannot withstand the physical conditions associated with human- made machines (e.g., heat and friction), the two types do share important features. In addition to having moving parts, all machines require energy-transducing mechanisms; that is, they convert energy into directed motion. Despite the wide diversity of motion types in living organisms, in all cases, energy- driven changes in protein conformations result in the accomplishment of work. Protein conformation changes occur when a ligand is bound. When a specific lig- and binds to one subunit of a multisubunit protein complex, the change in its con- formation will affect the shapes of adjacent subunits. These changes are reversible; that is, ligand dissociation from a protein causes it to revert to its previous confor- mation. The work performed by complex biological machines requires that the conformational and, therefore, functional changes occur in an orderly and directed manner. In other words, an energy source (usually provided by the hydrolysis of ATP or GTP) drives a sequence of conformational changes of adjacent subunits in one functional direction. The directed functioning of biological machines is possible because nucleotide hydrolysis is irreversible under physiological conditions. Motor Proteins Despite their functional diversity, all biological machines possess one or more pro- tein components that bind nucleoside triphosphates (NTP). These subunits, called NTPases, function as mechanical transducers or motor proteins. The NTP hydrolysis–driven changes in the conformation of a motor protein trigger ordered conformational changes in adjacent subunits in the molecular machine. NTP-bind- ing proteins perform a wide variety of functions in eukaryotes, most of which occur in one or more of the following categories. 1. Classical motors. Classical motor proteins are ATPases that move a load along a protein filament, as shown earlier (Figure 2.4). The best-known examples include the myosins, which move along actin filaments, and the kinesins and dyneins, which move vesicles and organelles along micro- tubules. Kinesins walk along the microtubules toward the () end, away from the centrosome (the microtubule organizing center). Dyneins walk along the microtubules toward the (–) end, toward the centrosome. 2. Timing devices. The function of certain NTP-binding proteins is to pro- vide a delay period during a complex process that ensures accuracy. The prokaryotic protein synthesis protein EF-Tu (Biochemistry in Perpective Box EF-TU: A Motor Protein, Ch. 19) is a well-known example. The rela- tively slow rate of GTP hydrolysis by EF-Tu when it is bound to an aminoa- cyl-tRNA allows sufficient time for the dissociation of the complex from the ribosome if the tRNA-mRNA base sequence binding is not correct. 3. Microprocessing switching devices. A variety of GTP-binding proteins act as on-off molecular switches in signal transduction pathways. Exam- ples include the -subunits of the trimeric G proteins. Numerous intra- cellular signal control mechanisms are regulated by G proteins. 4. Assembly and disassembly factors. Numerous cellular processes require the rapid and reversible assembly of protein subunits into larger molecu- lar complexes. Among the most dramatic examples of protein subunit poly- merization are the assembly of tubulin and actin into microtubules and microfilaments, respectively. The slow hydrolysis of GTP by tubulin and ATP by actin monomers, after the incorporation of these molecules into their respective polymeric filaments, promotes subtle conformational changes that later allow disassembly. The best-characterized motor protein is myosin. A brief overview of the struc- ture and function of myosin in the molecular events in muscle contraction is provided online in the Biochemistry in Perspective essay Myosin: A Moecular Machine. 5.4 Molecular Machines 47 Visit the companion website at www.oup.com/us/mckee to read the Biochemistry in Perspective essay on myosin: a molecular machine. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 7:34 PM Page 47 50 CHAPTER FIVE Amino Acids, Peptides, and Proteins nascent silk polymer forms as a result of increasing shear stress (force applied by the parallel duct wall) and several biochemical environment changes. Within the duct, Na+ and Cl- are extracted and phosphate and K+ are pumped in. An increase in the K+/Na+ ratio, combined with the secretion of phosphate and H+, is believed to cause the conversion of α- helical conformations to β-pleated sheets. At first randomly oriented, the β-pleated sheets are eventually forced into par- allel alignment with the long axis of the filament. In the third segment of the duct, large amounts of water, released from the silk protein as hydrophobic interactions increase, are pumped out by epithelial cells. The valve at the end of the duct is believed to act as a clamp that grips the silk and a means of restarting the spinning process if the silk breaks. The silk polymer then enters one of numerous spigots within a spinneret (Figure 5E). As the silk filament emerges and the remaining water evaporates, it is solid. The filaments from numerous spigots wrap around each other to form a cable-like fiber. The diameter and strength of the fiber depend on the muscular tension within the spinneret valve and how fast the spider draws it out. Biochemistry IN PERSPECTIVE cont Major Ampullate Gland Funnel Spinning Duct Valve Na+ K+ H+ H+ H2O H2O H2O Cl- PO4 2- Spinneret Spigots Dragline Silk Fibre Spidroin Secretion FIGURE 5D Processing of Spider Dragline Silk. After the spidroins are secreted into the lumen of the major ampullate gland they move toward the funnel, where they exit into the beginning of the spinning duct. As a result of shear stress and other forces (e.g., squeezing of the wall of the ampullate gland and the pulling of the silk fiber out of the spinneret by the spider), the spidroins in the silk dope are compressed and forced to align along their long axes. As the silk polymer progresses down the tapering duct, biochemical changes (e.g., Na+, K+, and H+) cause the conversion of α-helices into hydrophobic β-pleated sheets that expel H2O. After passing through the valve the polymer is forced through one of several spigots. Several emerging filaments are twisted together to form a silk fiber that is pulled out of the spinneret by the spider. FIGURE 5E Illustration of the Silk Spinning Spigots of a Spider Spinneret. Note that emerging filaments are twisting together to form a fiber. SUMMARY: Biodegradable, lightweight, strong spider silk has an enormous number of potential applications. Intense, and as yet unsuccessful, research efforts have focused on duplicating the natural process by which spiders produce this remarkable fiber. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:58 PM Page 50 Biochemistry in Perspective 51 L iving organisms produce a stunning variety of proteins.Consequently, it is not surprising that considerable time,effort, and funding have been devoted to investigating their properties. Since the amino acid sequence of bovine insulin was determined by Frederick Sanger in 1953, the structures of several thousand proteins have been elucidated. In contrast to the 10 years required for insulin, current tech- nologies allow protein sequence determination within a few days by mass spectrometry. The amino acid sequence of a protein can be generated from its DNA or mRNA sequence if this informa- tion is available. After a brief review of protein purification methods, mass spectrometry is described. An older means of determining the primary sequence of polypeptides, the Edman degradation method, is described in an online Biochemistry in the Lab box Protein Sequencing: The Edman Degradation Method. Note that all the techniques for isolating, purifying, and charac- terizing proteins exploit differences in charge, molecular weight, and binding affinities. Many of these technologies apply to the investigation of other biomolecules. Purification Protein analysis begins with isolation and purification. Extraction of a protein requires cell disruption and homogenization (see Biochemistry in the Lab, Cell Technology, Chapter 2). This process is often followed by differential centrifugation and, if the protein is a component of an organelle, by density gradient cen- trifugation. After the protein-containing fraction has been obtained, several relatively crude methods may be used to enhance purification. In salting out, high concentrations of salts such as ammonium sulfate [(NH4)2SO4] are used to precipitate proteins. Because each protein has a characteristic salting-out point, this technique removes many impurities. (Unwanted pro- teins that remain in solution are discarded when the liquid is decanted.) When proteins are tightly bound to membrane, organic solvents or detergents often aid in their extraction. Dialysis (Figure 5F) is routinely used to remove low-molecular-weight impurities such as salts, solvents, and detergents. As a protein sample becomes progressively more pure, more sophisticated methods are used to achieve further purification. Among the most commonly used techniques are chromatography and electrophoresis. Chromatography Originally devised to separate low-molecular-weight substances such as sugars and amino acids, chromatography has become an invaluable tool in protein purification. A wide variety of chro- matographic techniques are used to separate protein mixtures on the basis of molecular properties such as size, shape, and weight, Biochemistry IN THE LAB or certain binding affinities. Often several techniques must be used sequentially to obtain a demonstrably pure protein. In all chromatographic methods the protein mixture is dis- solved in a liquid known as the mobile phase. As the protein molecules pass across the stationary phase (a solid matrix), they separate from each other because they are differently distributed between the two phases. The relative movement of each molecule results from its capacity to remain associated with the station- ary phase while the mobile phase continues to flow. Three chromatographic methods commonly used in protein purification are gel-filtration chromatography, ion-exchange chromatography, and affinity chromatography. Gel-filtration chromatography (Figure 5G) is a form of size-exclusion chro- matography in which particles in an aqueous solution flow through a column (a hollow tube) filled with gel and are separated according to size. Molecules that are larger than the gel pores are excluded and therefore move through the column quickly. Molecules that are smaller than the gel pores diffuse in and out of the pores, so their movement through the column is retarded. Differences in the rates of particle movement separate the protein mixture into bands, which are then collected separately. Ion-exchange chromatography separates proteins on the basis of their charge. Anion-exchange resins, which consist of positively charged materials, bind reversibly with a protein’s neg- atively charged groups. Similarly, cation-exchange resins bind positively charged groups. After proteins that do not bind to the resin have been removed, the protein of interest is recovered by Protein Technology Protein molecule W ater in W ater out Small solute molecule FIGURE 5F Dialysis Proteins are routinely separated from low-molecular-weight impu- rities by dialysis. When a dialysis bag (an artificial semipermeable membrane) containing a cell extract is suspended in water or a buffered solution, small molecules pass out through the mem- brane’s pores. If the solvent outside the bag is continually renewed, all low-molecular-weight impurities are removed from the inside. ▼ 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:58 PM Page 51 Biochemistry IN THE LAB cont FIGURE 5G Gel-Filtration Chromatography In gel-filtration chromatography the stationary phase is a gelatinous polymer with pore sizes selected by the experimenter to separate molecules according to their sizes. The sample is applied to the top of the column and is eluted with buffer (the mobile phase). As elution proceeds, larger molecules travel faster through the gel than smaller molecules, whose progress is slowed because they can enter the pores. If fractions are collected, the larger molecules appear in the earlier fractions and later fractions contain smaller molecules. Small molecules can penetrate beads: passage is retarded Column of stationary porous beads Large molecules move between beads Solvent flow Buffer Buffer 1 2 3 4 5 1 2 3 4 5 1 2 Later time Later time 3 4 5 Absorbant material Sample Buffer Buffer ▼ an appropriate change in the solvent pH and/or salt concentra- tion. (A change in pH alters the protein’s net charge.) Affinity chromatography takes advantage of the unique biological properties of proteins. That is, it uses a special non- covalent binding affinity between the protein and a special mol- ecule (the ligand). The ligand is covalently bound to an insolu- ble matrix, which is placed in a column. After nonbinding protein molecules have passed through the column, the protein of interest 52 CHAPTER FIVE Amino Acids, Peptides, and Proteins 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:58 PM Page 52 O Biochemistry in the Lab 55 Small molecules can penetrate beads: passage is retarded Column of stationary porous beads Large molecules move between beads Solvent flow Absorbant material Sample Buffer Buffer FIGURE 5J Schematic Diagram of X-Ray Crystallography X-rays are useful in the analysis of biomolecules because their wavelength range is quite similar to the magnitude of chemical bonds. Consequently, the resolving power of X-ray crystallography is equivalent to interatomic distances. another enzyme. The computer uses the sequence information derived from both digests to determine the amino acid sequence of the original polypeptide. Protein Sequence-based Function Prediction Once a polypeptide has been isolated, purified, and sequenced, the next logical step is to determine its function. This endeavor usually begins with a database search of known protein sequences. BLAST (Basic Local Alignment Search Tool) is a computer program (www.ncbi.nim.nih.gov/blast) that allows fast searches of known sequences for matches to the unknown pro- tein sequence (the query sequence). Protein sequence databases (e.g., UniProt [Universal Protein resource] www.uniprot.org) are sufficiently large that about 50% of sequence comparison queries yield matched sequences that are close enough to infer function. X-Ray Crystallography Much of the three-dimensional structural information about proteins was obtained by X-ray crystallography. Because the bond distances in proteins are approximately 0.15 nm, the elec- tromagnetic radiation used to resolve protein structure must have a short wavelength. Visible light wavelengths [( )  400–700 nm] clearly does not have sufficient resolving power for biomolecules. X-rays, however, have very short wave- lengths (0.07–0.25 nm). In X-ray crystallography, highly ordered crystalline specimens are exposed to an X-ray beam (Figure 5J). As the X-rays hit the crystal, they are scattered by the atoms in the crystal. The dif- fraction pattern that results is recorded on charge-coupled device (CCD) detectors. The diffraction patterns are used to construct an electron density map. Because there is no objective lens to recom- bine the scattered X-rays, the three-dimensional image is recon- structed mathematically. Computer programs now perform these extremely complex and laborious computations. The three- dimensional structure of a polypeptide can also be determined using homologous modeling, a method that is based on the obser- vation that three-dimensional protein structure is more conserved than protein sequences. A structural model is constructed from X-ray diffraction data of one or more homologous proteins in the Protein Data Bank (www.pdb.org). Biochemistry IN THE LAB cont 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:58 PM Page 55 1. Polypeptides are amino acid polymers. Proteins may consist of one or more polypeptide chains. 2. Each amino acid contains a central carbon atom (the -carbon) to which an amino group, a carboxylate group, a hydrogen atom, and an R group are attached. In addition to comprising protein, amino acids have several other biologi- cal roles. According to their capacity to interact with water, amino acids may be separated into four classes: nonpolar, polar, acidic, and basic. 3. Titration of amino acids and peptides illustrates the effect of pH on their structures. The pH at which a molecule has no net charge is called its isoelectric point. 4. Amino acids undergo several chemical reactions. Two reac- tions are especially important: peptide bond formation and cysteine oxidation. 5. Proteins have a vast array of functions in living organisms. In addition to serving as structural materials, proteins are involved in metabolic regulation, transport, defense, and catalysis. Some proteins are multifunctional; that is, they have two or more seemingly unrelated functions. Proteins can also be classified into families and superfamilies, according to their sequence similarities as well as their shapes and composition. Fibrous proteins (e.g., collagen) are long, rod-shaped molecules that are insoluble in water and physically tough. Globular proteins (e.g., hemoglobin) are compact, spherical molecules that are usually soluble in water. 6. Biochemists have distinguished four levels of protein struc- ture. Primary structure, the amino acid sequence, is specified by genetic information. As the polypeptide chain folds, local folding patterns constitute the protein’s secondary structure. The overall three-dimensional shape that a polypeptide assumes is called the tertiary structure. Proteins that consist of two or more polypeptides have quaternary structure. The functions of numerous proteins, especially molecules that participate in eukaryotic regulatory processes, are partially or completely unstructured. Many physical and chemical conditions disrupt protein structure. Denaturing agents include strong acids or bases, reducing agents, organic sol- vents, detergents, high salt concentrations, heavy metals, temperature changes, and mechanical stress. 7. One of the most important aspects of protein synthesis is the folding of polypeptides into their biologically active confor- mations. Despite decades of investigation into the physical and chemical properties of polypetide chains, the mechanism by which a primary sequence dictates the molecule’s final conformation is unresolved. Many proteins require molecu- lar chaperones to fold into their final three-dimensional con- formations. Protein misfolding is now known to be an important feature of several human diseases, including Alzheimer’s disease and Huntington’s disease. 8. Fibrous proteins (e.g., -keratin and collagen), which con- tain high proportions of -helices or -pleated sheets, have structural rather than dynamic roles. Despite their varied functions, most globular proteins have features that allow them to bind to specific ligands or sites on certain macro- molecules. These binding events involve conformational changes in the globular protein’s structure. 9. The biological activity of complex multisubunit proteins is often regulated by allosteric interactions in which small ligands bind to the protein. Any change in the protein’s activity is due to changes in the interactions among the protein’s subunits. Effectors can increase or decrease the function of a protein. Chapter Summary Take your learning further by visiting the companion website for Biochemistry at www.oup.com/us/mckee where you can complete a multiple-choice quiz on amino acids, peptides, and proteins to help you prepare for exams. 56 CHAPTER FIVE Amino Acids, Peptides, and Proteins Bustamonte, C., Of Torques, Forces, and Protein Machines, Protein Sci. 13:306l–65, 2004. Dyson, H. J., and Wright, P. E., Intrinsically Unstructured Proteins and Their Functions, Nat. Rev. Mol. Cell Biol. 6(3):197–208, 2005. Fink, A. L., Natively Unfolded Proteins, Curr. Opin. Struct Biol. 15:35–41, 2005. Heim, M., Keerl, D., and Scheibel, T., Spider Silk: From Soluble Protein to Extraordinary Fiber, Angewandte Chem. Int. Ed. 48:3584–3596, 2009. Lindorff, K., Rogen, P., Poci, E., Vendruscolo, M., and Dobson, M., Protein Folding and the Organization of the Protein Topology Universe, Trends, Biochem. Sci. 30(l):l3–19, 2005. Mattos, C., Protein-Water Interactions in a Dynamic World, Trends Biochem. Sci. 27(4):203–208, 2002. Schnabel, J., Protein Folding: The Dark Side of Proteins, Nature 484:828–829, 2010. Tompa, P., Szasz, C., and Buday, L., Structural Disorder Throws New Light on Moonlighting. Trends Biochem. Sci. 30(9):484–489, 2005. Suggested Readings 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 7:35 PM Page 56 Suggested Readings 57 affinity chromatography, xxx aldimine, xxx aldol condensation, xxx aliphatic hydrocarbon, xxx allosteric transition, xxx allostery, xxx Alzheimer’s disease, xxx amino acid residue, xxx amphipathic molecule, xxx amphoteric molecule, xxx antigen, xxx apoprotein, xxx aromatic hydrocarbon, xxx asymmetric carbon, xxx chaperonins, xxx chiral carbon, xxx circular dichroism, xxx conjugated protein, xxx cooperative binding, xxx denaturation, xxx disulfide bridge, xxx dynein, xxx effector, xxx electrophoresis, xxx enantiomer, xxx fibrous protein, xxx fold, xxx gel-filtration chromatography, xxx globular protein, xxx glycoprotein, xxx heat shock protein, xxx hemoprotein, xxx holoprotein, xxx homologous polypeptide, xxx hormone, xxx hsp60, xxx hsp70, xxx Huntington’s disease, xxx intrinsically unstructured protein, xxx ion-exchange chromatography, xxx isoelectric point, xxx kinesin, xxx ligand, xxx lipoprotein, xxx mass spectrometry, xxx metalloprotein, xxx mobile phase, xxx modular protein, xxx modulator, xxx molecular chaperone, xxx molecular disease, xxx molten globule, xxx motif, xxx motor protein, xxx multifunction protein, xxx myosin, xxx natively unfolded protein, xxx neurotransmitter, xxx oligomer, xxx optical isomer, xxx peptide, xxx peptide bond, xxx phosphoprotein, xxx polypeptide, xxx primary structure, xxx prosthetic group, xxx protein, xxx protein family, xxx protein folding, xxx protein superfamily, xxx protomer, xxx quaternary structure, xxx response element, xxx salt bridge, xxx salting out, xxx Schiff base, xxx SDS-polyacrylamide gel electrophoresis, xxx secondary structure, xxx stationary phase, xxx stereoisomer, xxx subunit, xxx supersecondary structure, xxx tertiary structure, xxx zwitterion, xxx Key Words 1. Define the following terms: a. supersecondary structure b. protomer c. phosphoprotein d. denaturation e. ion exchange chromatography 2. Define the following terms: a. protein motif b. conjugated protein c. dynein d. zwitterion e. electrophoresis 3. Define the following terms: a. metalloprotein b. hormone c. holoprotein d. intrinsically unstructured protein e. kinesin 4. Define the following terms: a. aliphatic hydrocarbon b. neurotransmitter c. asymmetric carbon d. chiral carbon e. stereoisomer 5. Define the following terms: a. optical isomer b. isoelectric point c. peptide bond d. disulfide bridge e. Schiff base 6. Define the following terms: a. aldimine b. heat shock protein c. multifunction protein d. protein family e. protein superfamily 7. Define the following terms: a. multifunction protein b. fibrous protein c. globular protein d. prosthetic group e. apoprotein 8. Define the following terms: a. homologous polypeptide b. molecular disease c. protein fold d. mosaic protein e. ligand Review Questions These questions are designed to test your knowledge of the key concepts discussed in this chapter, before moving on to the next chapter. You may like to compare your answers to the solutions provided in the back of the book and in the accompanying Study Guide. 05-McKee-Chap05.qxd:05-McKee-Chap05 1/13/11 6:58 PM Page 57