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, 999–1007. Westhof, E. (ed.) (1993) Water and Biological Macromolecules, CRC Press, Inc., Boca Raton, FL. Fourteen chapters, each by a different author, cover (at an advanced level) the structure of water and its interactions with proteins, nucleic acids, polysaccharides, and lipids. 8885d_c02_47-74 7/25/03 10:05 AM Page 72 mac76 mac76:385_reb: 72 Part I Structure and Catalysis Wiggins, P.M. (1990) Role of water in some biological processes. Microbiol. Rev. 54, 432–449. Martin, T.W. & Derewenda, Z.S. (1999) The name is bondOH bond. Nat. Struct. Biol. 6, 403–406. A review of water in biology, including discussion of the physical structure of liquid water, its interaction with biomolecules, and the state of water in living cells. Weak Interactions in Aqueous Systems Fersht, A.R. (1987) The hydrogen bond in molecular recognition. Trends Biochem. Sci. 12, 301–304. A clear, brief, quantitative discussion of the contribution of hydrogen bonding to molecular recognition and enzyme catalysis. Frieden, E. (1975) Non-covalent interactions: key to biological flexibility and specificity. J. Chem. Educ. 52, 754–761. Review of the four kinds of weak interactions that stabilize macromolecules and confer biological specificity, with clear examples. Jeffrey, G.A. (1997) An Introduction to Hydrogen Bonding, Oxford University Press, New York. A detailed, advanced discussion of the structure and properties of hydrogen bonds, including those in water and biomolecules. Problems 1. Simulated Vinegar One way to make vinegar (not the preferred way) is to prepare a solution of acetic acid, the sole acid component of vinegar, at the proper pH (see Fig. 2–15) and add appropriate flavoring agents. Acetic acid (Mr 60) is a liquid at 25 C, with a density of 1.049 g/mL. Calculate the volume that must be added to distilled water to make 1 L of simulated vinegar (see Fig. 2–16). In a hospital laboratory, 2. Acidity of Gastric HCl a 10.0 mL sample of gastric juice, obtained several
hours after a meal, was titrated with 0.1 M NaOH to neutrality; 7.2 mL of NaOH was required. The patient’s stomach contained no ingested food or drink, thus assume that no buffers were present. What was the pH of the gastric juice? 3. Measurement of Acetylcholine Levels by pH Changes The concentration of acetylcholine (a neurotransmitter) in a sample can be determined from the pH changes that accompany its hydrolysis. When the sample is incubated with the enzyme acetylcholinesterase, acetylcholine is quantitatively converted into choline and acetic acid, which dissociates to yield acetate and a hydrogen ion: O CH3 CH3 C O CH2 CH2 N CH3 H2O CH3 Acetylcholine CH3 HO CH2 CH2 N CH3 CH3 C O H CH3 Choline O Acetate Brief review of the evidence that hydrogen bonds have some covalent character. Schwabe, J.W.R. (1997) The role of water in protein-DNA interactions. Curr. Opin. Struct. Biol. 7, 126–134. An examination of the important role of water in both the specificity and the affinity of protein-DNA interactions. Tanford, C. (1978) The hydrophobic effect and the organization of living matter. Science 200, 1012–1018. A review of the chemical and energetic bases for hydrophobic interactions between biomolecules in aqueous solutions. Weak Acids, Weak Bases, and Buffers: Problems for Practice Segel, I.H. (1976) Biochemical Calculations, 2nd edn, John Wiley & Sons, Inc., New York. In a typical analysis, 15 mL of an aqueous solution containing an unknown amount of acetylcholine had a pH of 7.65. When incubated with acetylcholinesterase, the pH of the solution decreased to 6.87. Assuming that there was no buffer in the assay mixture, determine the number of moles of acetylcholine in the 15 mL sample. 4. Osmotic Balance in a Marine Frog The crab-eating frog of Southeast Asia, Rana cancrivora, develops and matures in fresh water but searches for its food in coastal mangrove swamps (composed of 80% to full-strength seawater). When the frog moves from its
freshwater home to seawater it experiences a large change in the osmolarity of its environment (from hypotonic to hypertonic). (a) Eighty percent seawater contains 460 mM NaCl, 10 mM KCl, 10 mM CaCl2, and 50 mM MgCl2. What are the concentrations of the various ionic species in this seawater? Assuming that these salts account for nearly all the solutes in seawater, calculate the osmolarity of the seawater. (b) The chart below lists the cytoplasmic concentrations of ions in R. cancrivora. Ignoring dissolved proteins, amino acids, nucleic acids, and other small metabolites, calculate the osmolarity of the frog’s cells based solely on the ionic concentrations given below. Na (mM) K (mM) R. cancrivora 122 10 Cl (mM) 100 Ca2 (mM) Mg2 (mM) 2 1 (c) Like all frogs, the crab-eating frog can exchange gases through its permeable skin, allowing it to stay underwater for long periods of time without breathing. How does the high permeability of frog skin affect the frog’s cells when it moves from fresh water to seawater? 8885d_c02_47-74 7/25/03 10:05 AM Page 73 mac76 mac76:385_reb: (d) The crab-eating frog uses two mechanisms to maintain its cells in osmotic balance with its environment. First, it allows the Na and Cl concentrations in its cells to increase slowly as the ions diffuse down their concentration gradients. Second, like many elasmobranchs (sharks), it retains the waste product urea in its cells. The addition of both NaCl and urea increases the osmolarity of the cytosol to a level nearly equal to that of the surrounding environment. NI A H Pyridine ion pKa ≈ 5 (a) Chapter 2 Water 73 O H -Naphthol pKa ≈ 10 (b) OH O C H2N NH2 Urea (CH4N2O) Assuming the volume of water in a typical frog is 100 mL, calculate how many grams of NaCl (formula weight (FW) 58.44) the frog must take up to make its tissues isotonic with seawater. (e) How many grams of urea (FW 60) must it
retain to accomplish the same thing? 5. Properties of a Buffer The amino acid glycine is often used as the main ingredient of a buffer in biochemical experiments. The amino group of glycine, which has a pKa of ) or as 9.6, can exist either in the protonated form (ONH3 the free base (ONH2), because of the reversible equilibrium R NH3 R NH2 H (a) In what pH range can glycine be used as an effec- tive buffer due to its amino group? (b) In a 0.1 M solution of glycine at pH 9.0, what frac- tion of glycine has its amino group in the ONH3 form? (c) How much 5 M KOH must be added to 1.0 L of 0.1 M glycine at pH 9.0 to bring its pH to exactly 10.0? (d) When 99% of the glycine is in its ONH3 form, what is the numerical relation between the pH of the solution and the pKa of the amino group? 6. The Effect of pH on Solubility The strongly polar, hydrogen-bonding properties of water make it an excellent solvent for ionic (charged) species. By contrast, nonionized, nonpolar organic molecules, such as benzene, are relatively insoluble in water. In principle, the aqueous solubility of any organic acid or base can be increased by converting the molecules to charged species. For example, the solubility of benzoic acid in water is low. The addition of sodium bicarbonate to a mixture of water and benzoic acid raises the pH and deprotonates the benzoic acid to form benzoate ion, which is quite soluble in water. O B C OOH O B COO Benzoic acid pKa ≈ 5 Benzoate ion Are the following compounds more soluble in an aqueous solution of 0.1 M NaOH or 0.1 M HCl? (The dissociable protons are shown in red.) O B C G D D CH3 H N H A OC A C J G OCH2 O OOCH3 N-Acetyltyrosine methyl ester pKa ≈ 10 (c) 7. Treatment of Poison Ivy Rash The components of poison ivy and poison oak that produce the characteristic itchy rash are catechols substituted with longchain al
kyl groups. OH OH (CH2)nOCH3 pKa ≈ 8 If you were exposed to poison ivy, which of the treatments below would you apply to the affected area? Justify your choice. (a) Wash the area with cold water. (b) Wash the area with dilute vinegar or lemon juice. (c) Wash the area with soap and water. (d) Wash the area with soap, water, and baking soda (sodium bicarbonate). 8. pH and Drug Absorption Aspirin is a weak acid with a pKa of 3.5. O B C D CH3 G O O B C G OH It is absorbed into the blood through the cells lining the stomach and the small intestine. Absorption requires passage through the plasma membrane, the rate of which is determined by the polarity of the molecule: charged and highly polar molecules pass slowly, whereas neutral hydrophobic ones pass rapidly. The pH of the stomach contents is about 1.5, and the pH of the contents of the small intestine is about 6. Is more aspirin absorbed into the bloodstream from the stomach or from the small intestine? Clearly justify your choice. 8885d_c02_47-74 7/25/03 10:05 AM Page 74 mac76 mac76:385_reb: 74 Part I Structure and Catalysis 9. Preparation of Standard Buffer for Calibration of a pH Meter The glass electrode used in commercial pH meters gives an electrical response proportional to the concentration of hydrogen ion. To convert these responses into pH, glass electrodes must be calibrated against standard solutions of known H concentration. Determine the weight in grams of sodium dihydrogen phosphate (NaH2PO4 H2O; FW 138.01) and disodium hydrogen phosphate (Na2HPO4; FW 141.98) needed to prepare 1 L of a standard buffer at pH 7.00 with a total phosphate concentration of 0.100 M (see Fig. 2–16). 10. Calculating pH from Hydrogen Ion Concentration What is the pH of a solution that has an H concentration of (a) 1.75 105 mol/L; (b) 6.50 1010 mol/L; (c) 1.0 104 mol/L; (d) 1.50 105 mol/L? 11. Calculating Hydrogen Ion Concentration from pH What is the H concentration of a solution with pH of (a) 3.82;
(b) 6.52; (c) 11.11? 12. Calculating pH from Molar Ratios Calculate the pH of a dilute solution that contains a molar ratio of potassium acetate to acetic acid (pKa 4.76) of (a) 2:1; (b) 1:3; (c) 5:1; (d) 1:1; (e) 1:10. 13. Working with Buffers A buffer contains 0.010 mol of lactic acid (pKa 3.86) and 0.050 mol of sodium lactate per liter. (a) Calculate the pH of the buffer. (b) Calculate the change in pH when 5 mL of 0.5 M HCl is added to 1 L of the buffer. (c) What pH change would you expect if you added the same quantity of HCl to 1 L of pure water? 14. Calculating pH from Concentrations What is the pH of a solution containing 0.12 mol/L of NH4Cl and 0.03 mol/L of NaOH (pKa of NH4 /NH3 is 9.25)? 15. Calculating pKa An unknown compound, X, is thought to have a carboxyl group with a pKa of 2.0 and another ionizable group with a pKa between 5 and 8. When 75 mL of 0.1 M NaOH was added to 100 mL of a 0.1 M solution of X at pH 2.0, the pH increased to 6.72. Calculate the pKa of the second ionizable group of X. 16. Control of Blood pH by Respiration Rate (a) The partial pressure of CO2 in the lungs can be varied rapidly by the rate and depth of breathing. For example, a common remedy to alleviate hiccups is to increase the concentration of CO2 in the lungs. This can be achieved by holding one’s breath, by very slow and shallow breathing (hypoventilation), or by breathing in and out of a paper bag. Under such conditions, the partial pressure of CO2 in the air space of the lungs rises above normal. Qualitatively explain the effect of these procedures on the blood pH. (b) A common practice of competitive short-distance runners is to breathe rapidly and deeply (hyperventilate) for about half a minute to remove CO2 from their lungs just before running in, say, a 100 m dash. Blood pH
may rise to 7.60. Explain why the blood pH increases. (c) During a short-distance run the muscles produce a large amount of lactic acid (CH3CH(OH)COOH, Ka 1.38 104) from their glucose stores. In view of this fact, why might hyperventilation before a dash be useful? 8885d_c03_075 12/23/03 10:16 AM Page 75 mac111 mac111:reb: – – + + chapter 3 AMINO ACIDS, PEPTIDES, AND PROTEINS 3.1 Amino Acids 75 Peptides and Proteins 85 3.2 3.3 Working with Proteins 89 3.4 3.5 The Covalent Structure of Proteins 96 Protein Sequences and Evolution 106 The word protein that I propose to you... I would wish to derive from proteios, because it appears to be the primitive or principal substance of animal nutrition that plants prepare for the herbivores, and which the latter then furnish to the carnivores. —J. J. Berzelius, letter to G. J. Mulder, 1838 Proteins are the most abundant biological macromol- ecules, occurring in all cells and all parts of cells. Proteins also occur in great variety; thousands of different kinds, ranging in size from relatively small peptides to huge polymers with molecular weights in the millions, may be found in a single cell. Moreover, proteins exhibit enormous diversity of biological function and are the most important final products of the information pathways discussed in Part III of this book. Proteins are the molecular instruments through which genetic information is expressed. Relatively simple monomeric subunits provide the key to the structure of the thousands of different proteins. All proteins, whether from the most ancient lines of bacteria or from the most complex forms of life, are constructed from the same ubiquitous set of 20 amino acids, covalently linked in characteristic linear sequences. Because each of these amino acids has a side chain with distinctive chemical properties, this group of 20 precursor molecules may be regarded as the alphabet in which the language of protein structure is written. What is most remarkable is that cells can produce proteins with strikingly different properties and activities by joining the same 20 amino acids in many different combinations and sequences. From these building blocks different organisms can make such widely diverse products as enzymes, hormones, antibodies, transporters, muscle fibers, the lens protein of the eye,
feathers, spider webs, rhinoceros horn, milk proteins, antibiotics, mushroom poisons, and myriad other substances having distinct biological activities (Fig. 3–1). Among these protein products, the enzymes are the most varied and specialized. Virtually all cellular reactions are catalyzed by enzymes. Protein structure and function are the topics of this and the next three chapters. We begin with a description of the fundamental chemical properties of amino acids, peptides, and proteins. 3.1 Amino Acids Protein Architecture—Amino Acids Proteins are polymers of amino acids, with each amino acid residue joined to its neighbor by a specific type of covalent bond. (The term “residue” reflects the loss of the elements of water when one amino acid is joined to another.) Proteins can be broken down (hydrolyzed) to their constituent amino acids by a variety of methods, and the earliest studies of proteins naturally focused on 75 8885d_c03_076 12/23/03 10:20 AM Page 76 mac111 mac111:reb: 76 Chapter 3 Amino Acids, Peptides, and Proteins (a) (b) (c) FIGURE 3–1 Some functions of proteins. (a) The light produced by fireflies is the result of a reaction involving the protein luciferin and ATP, catalyzed by the enzyme luciferase (see Box 13–2). (b) Erythrocytes contain large amounts of the oxygen-transporting protein hemoglobin. (c) The protein keratin, formed by all vertebrates, is the chief structural component of hair, scales, horn, wool, nails, and feath- ers. The black rhinoceros is nearing extinction in the wild because of the belief prevalent in some parts of the world that a powder derived from its horn has aphrodisiac properties. In reality, the chemical properties of powdered rhinoceros horn are no different from those of powdered bovine hooves or human fingernails. the free amino acids derived from them. Twenty different amino acids are commonly found in proteins. The first to be discovered was asparagine, in 1806. The last of the 20 to be found, threonine, was not identified until 1938. All the amino acids have trivial or common names, in some cases derived from the source from which they were first isolated. Asparagine was first found in asparagus
, and glutamate in wheat gluten; tyrosine was first isolated from cheese (its name is derived from the Greek tyros, “cheese”); and glycine (Greek glykos, “sweet”) was so named because of its sweet taste. Amino Acids Share Common Structural Features All 20 of the common amino acids are -amino acids. They have a carboxyl group and an amino group bonded to the same carbon atom (the carbon) (Fig. 3–2). They differ from each other in their side chains, or R groups, which vary in structure, size, and electric charge, and which influence the solubility of the amino acids in water. In addition to these 20 amino acids there are many less common ones. Some are residues modified after a protein has been synthesized; others are amino acids present in living organisms but not as constituents of proteins. The common amino acids of proteins have been assigned three-letter abbreviations and one-letter H3N COO H C R FIGURE 3–2 General structure of an amino acid. This structure is common to all but one of the -amino acids. (Proline, a cyclic amino acid, is the exception.) The R group or side chain (red) attached to the carbon (blue) is different in each amino acid. symbols (Table 3–1), which are used as shorthand to indicate the composition and sequence of amino acids polymerized in proteins. Two conventions are used to identify the carbons in an amino acid—a practice that can be confusing. The additional carbons in an R group are commonly designated,,,, and so forth, proceeding out from the carbon. For most other organic molecules, carbon atoms are simply numbered from one end, giving highest priority (C-1) to the carbon with the substituent containing the atom of highest atomic number. Within this latter convention, the carboxyl carbon of an amino acid would be C-1 and the carbon would be C-2. In some cases, such as amino acids with heterocyclic R groups, the Greek lettering system is ambiguous and the numbering convention is therefore used. e 6 CH2 NH3 g 4 d 5 CH2 CH2 CH2 CH a 2 b 3 1 COO NH3 Lysine For all the common amino acids except glycine, the carbon is bonded to four different groups: a carboxyl group, an amino group, an R group, and a hydrogen
atom (Fig. 3–2; in glycine, the R group is another hydrogen atom). The -carbon atom is thus a chiral center (p. 17). Because of the tetrahedral arrangement of the bonding orbitals around the -carbon atom, the four different groups can occupy two unique spatial arrangements, and thus amino acids have two possible stereoisomers. Since they are nonsuperimposable mirror images of each other (Fig. 3–3), the two forms represent a class of stereoisomers called enantiomers (see Fig. 1–19). All molecules with a chiral center are also optically active—that is, they rotate plane-polarized light (see Box 1–2). 8885d_c03_077 12/23/03 10:20 AM Page 77 mac111 mac111:reb: Special nomenclature has been developed to specify the absolute configuration of the four substituents of asymmetric carbon atoms. The absolute configurations of simple sugars and amino acids are specified by the D, L system (Fig. 3–4), based on the absolute configuration of the three-carbon sugar glyceraldehyde, a convention proposed by Emil Fischer in 1891. (Fischer knew what groups surrounded the asymmetric carbon of glyceraldehyde but had to guess at their absolute configuration; his guess was later confirmed by x-ray diffraction analysis.) For all chiral compounds, stereoisomers having a configuration related to that of L-glyceraldehyde are designated L, and stereoisomers related to D-glyceraldehyde are designated D. The functional groups of L-alanine are matched with those of Lglyceraldehyde by aligning those that can be interconverted by simple, one-step chemical reactions. Thus the carboxyl group of L-alanine occupies the same position about the chiral carbon as does the aldehyde group of L-glyceraldehyde, because an aldehyde is readily converted to a carboxyl group via a one-step oxidation. Historically, the similar l and d designations were used for levorotatory (rotating light to the left) and dextrorotatory (rotating light to the right). However, not all COO COO H3N C H H C NH3 CH3 (a) L-Alanine CH3 D-Alanine COO C H H3N CH3 L-Alanine C
OO C H H3N CH3 L-Alanine (b) (c) COO N H3 H C CH3 D-Alanine COO H C N H3 CH3 D-Alanine FIGURE 3–3 Stereoisomerism in -amino acids. (a) The two stereoisomers of alanine, L- and D-alanine, are nonsuperimposable mirror images of each other (enantiomers). (b, c) Two different conventions for showing the configurations in space of stereoisomers. In perspective formulas (b) the solid wedge-shaped bonds project out of the plane of the paper, the dashed bonds behind it. In projection formulas (c) the horizontal bonds are assumed to project out of the plane of the paper, the vertical bonds behind. However, projection formulas are often used casually and are not always intended to portray a specific stereochemical configuration. 3.1 Amino Acids 77 1CHO HO C 2 H 3CH2OH CHO H C OH CH2OH L-Glyceraldehyde D-Glyceraldehyde COO C H H3N CH3 L-Alanine COO H C N H3 CH3 D-Alanine FIGURE 3–4 Steric relationship of the stereoisomers of alanine to the absolute configuration of L- and D-glyceraldehyde. In these perspective formulas, the carbons are lined up vertically, with the chiral atom in the center. The carbons in these molecules are numbered beginning with the terminal aldehyde or carboxyl carbon (red), 1 to 3 from top to bottom as shown. When presented in this way, the R group of the amino acid (in this case the methyl group of alanine) is always below the carbon. L-Amino acids are those with the -amino group on the left, and D-amino acids have the -amino group on the right. L-amino acids are levorotatory, and the convention shown in Figure 3–4 was needed to avoid potential ambiguities about absolute configuration. By Fischer’s convention, L and D refer only to the absolute configuration of the four substituents around the chiral carbon, not to optical properties of the molecule. Another system of specifying configuration around a chiral center is the RS system, which is used in the systematic nomenclature of organic chemistry and describes more precisely the
configuration of molecules with more than one chiral center (see p. 18). The Amino Acid Residues in Proteins Are L Stereoisomers Nearly all biological compounds with a chiral center occur naturally in only one stereoisomeric form, either D or L. The amino acid residues in protein molecules are exclusively L stereoisomers. D-Amino acid residues have been found only in a few, generally small peptides, including some peptides of bacterial cell walls and certain peptide antibiotics. It is remarkable that virtually all amino acid residues in proteins are L stereoisomers. When chiral compounds are formed by ordinary chemical reactions, the result is a racemic mixture of D and L isomers, which are difficult for a chemist to distinguish and separate. But to a living system, D and L isomers are as different as the right hand and the left. The formation of stable, repeating substructures in proteins (Chapter 4) generally requires that their constituent amino acids be of one stereochemical series. Cells are able to specifically synthesize the L isomers of amino acids because the active sites of enzymes are asymmetric, causing the reactions they catalyze to be stereospecific. 8885d_c03_078 12/23/03 10:20 AM Page 78 mac111 mac111:reb: 78 Chapter 3 Amino Acids, Peptides, and Proteins TABLE 3–1 Properties and Conventions Associated with the Common Amino Acids Found in Proteins pKa values Abbreviation/ symbol pK1 (OCOOH) pK2 ) (ONH3 pKR (R group) Mr Amino acid Nonpolar, aliphatic R groups Glycine Alanine Proline Valine Leucine Isoleucine Methionine Aromatic R groups Phenylalanine Tyrosine Tryptophan Polar, uncharged R groups Serine Threonine Cysteine Asparagine Glutamine Positively charged R groups Lysine Histidine Arginine Negatively charged R groups Aspartate Glutamate Gly G Ala A Pro P Val V Leu L Ile I Met M Phe F Tyr Y Trp W Ser S Thr T Cys C Asn N Gln Q Lys K His H Arg R Asp D Glu E 75 89 115 117 131 131 149 165 181 204 105 119 121 132 146 146 155 174 133 147 2.34 2.34 1.99 2
.32 2.36 2.36 2.28 1.83 2.20 2.38 2.21 2.11 1.96 2.02 2.17 2.18 1.82 2.17 1.88 2.19 9.60 9.69 10.96 9.62 9.60 9.68 9.21 9.13 9.11 9.39 9.15 9.62 10.28 8.80 9.13 8.95 9.17 9.04 9.60 9.67 pI 5.97 6.01 6.48 5.97 5.98 6.02 5.74 5.48 5.66 5.89 5.68 5.87 5.07 5.41 5.65 10.07 8.18 10.53 6.00 12.48 9.74 7.59 10.76 3.65 4.25 2.77 3.22 Hydropathy index* Occurrence in proteins (%)† 0.4 1.8 1.6 4.2 3.8 4.5 1.9 2.8 1.3 0.9 0.8 0.7 2.5 3.5 3.5 3.9 3.2 4.5 3.5 3.5 7.2 7.8 5.2 6.6 9.1 5.3 2.3 3.9 3.2 1.4 6.8 5.9 1.9 4.3 4.2 5.9 2.3 5.1 5.3 6.3 *A scale combining hydrophobicity and hydrophilicity of R groups; it can be used to measure the tendency of an amino acid to seek an aqueous environment ( values) or a hydrophobic environment ( values). See Chapter 11. From Kyte, J. & Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132. †Average occurrence in more than 1,150 proteins. From Doolittle, R.F. (1989) Redundancies in protein sequences. In Prediction of Protein Structure and the Principles of Protein Conformation (Fasman, G.D., ed.), pp. 599–623, Plenum Press, New York. Amino Acids Can Be Classified by R Group Knowledge of the chemical properties of the common amino acids is
central to an understanding of biochemistry. The topic can be simplified by grouping the amino acids into five main classes based on the properties of their R groups (Table 3–1), in particular, their polarity, or tendency to interact with water at biological pH (near pH 7.0). The polarity of the R groups varies widely, from nonpolar and hydrophobic (water-insoluble) to highly polar and hydrophilic (water-soluble). The structures of the 20 common amino acids are shown in Figure 3–5, and some of their properties are listed in Table 3–1. Within each class there are gradations of polarity, size, and shape of the R groups. Nonpolar, Aliphatic R Groups The R groups in this class of amino acids are nonpolar and hydrophobic. The side chains of alanine, valine, leucine, and isoleucine tend to cluster together within proteins, stabilizing protein structure by means of hydrophobic interactions. Glycine has the simplest structure. Although it is formally nonpolar, its very small side chain makes no real contribution to hydrophobic interactions. Methionine, one of the two sulfur-containing amino acids, has a nonpolar thioether group in its side chain. Proline has an 8885d_c03_079 12/23/03 10:20 AM Page 79 mac111 mac111:reb: Nonpolar, aliphatic R groups Aromatic R groups 3.1 Amino Acids 79 COO H H3N H3N C H COO C H CH3 COO H C C H 2 CH 2 H 2N H 2C Glycine Alanine Proline H3N COO C H C H2 C H CH3 CH3 COO C H N H3 H C C 3 H C H2 CH3 Leucine Isoleucine H3N COO C H C H CH3 CH3 Valine COO C H H3N C H2 C H2 S CH3 Methionine H3N COO C H CH2 H3N COO C H CH2 H3N COO C H C H2 C CH NH OH Phenylalanine Tyrosine Tryptophan Positively charged R groups COO C H COO C H H3N H3N H3N Polar, uncharged R groups COO C
H H3N COO C H H3N H3N CH2OH H C OH Serine CH3 Threonine C H2 C H2 C H2 C H2 N H3 COO C H C H2 SH C H2 C H2 C H2 N H C N H2 NH2 Arginine Cysteine Lysine Histidine COO C H CH 2 C NH C H N C H H3N COO C H C H2 C H2N O COO C H H3N C H2 C H2 C H2N O Negatively charged R groups COO COO H3N C H C H2 COO H3N C H C H2 H2 C COO Glutamate Asparagine Glutamine Aspartate FIGURE 3–5 The 20 common amino acids of proteins. The structural formulas show the state of ionization that would predominate at pH 7.0. The unshaded portions are those common to all the amino acids; the portions shaded in red are the R groups. Although the R group of histidine is shown uncharged, its pKa (see Table 3–1) is such that a small but significant fraction of these groups are positively charged at pH 7.0. aliphatic side chain with a distinctive cyclic structure. The secondary amino (imino) group of proline residues is held in a rigid conformation that reduces the structural flexibility of polypeptide regions containing proline. Aromatic R Groups Phenylalanine, tyrosine, and tryptophan, with their aromatic side chains, are relatively nonpolar (hydrophobic). All can participate in hydrophobic interactions. The hydroxyl group of tyrosine can form hydrogen bonds, and it is an important func- tional group in some enzymes. Tyrosine and tryptophan are significantly more polar than phenylalanine, because of the tyrosine hydroxyl group and the nitrogen of the tryptophan indole ring. Tryptophan and tyrosine, and to a much lesser extent phenylalanine, absorb ultraviolet light (Fig. 3–6; Box 3–1). This accounts for the characteristic strong absorbance of light by most proteins at a wavelength of 280 nm, a property exploited by researchers in the characterization of proteins. 8885d_c03_080 12/23/03 10:20 AM Page
80 mac111 mac111:reb: 80 Chapter 3 Amino Acids, Peptides, and Proteins Polar, Uncharged R Groups The R groups of these amino acids are more soluble in water, or more hydrophilic, than those of the nonpolar amino acids, because they contain functional groups that form hydrogen bonds with water. This class of amino acids includes serine, threonine, cysteine, asparagine, and glutamine. The polarity of serine and threonine is contributed by their hydroxyl groups; that of cysteine by its sulfhydryl group; and that of asparagine and glutamine by their amide groups. Asparagine and glutamine are the amides of two other amino acids also found in proteins, aspartate and glutamate, respectively, to which asparagine and glutamine are easily hydrolyzed by acid or base. Cysteine is readily oxidized to form a covalently linked dimeric amino acid called cystine, in which two cysteine molecules or residues are joined by a disulfide bond (Fig. 3–7). The disulfide-linked residues are strongly hydrophobic (nonpolar). Disulfide bonds play a special role in the structures of many proteins by forming covalent links between parts of a protein molecule or between two different polypeptide chains. Positively Charged (Basic) R Groups The most hydrophilic R groups are those that are either positively or negatively charged. The amino acids in which the R groups have significant positive charge at pH 7.0 are lysine, which has a second primary amino group at the posi- H3N Cysteine Cysteine COO CH CH2 SH SH CH2 CH NH3 COO H3N COO CH CH2 S S 2H 2e 2H 2e Cystine CH2 CH NH3 COO FIGURE 3–7 Reversible formation of a disulfide bond by the oxidation of two molecules of cysteine. Disulfide bonds between Cys residues stabilize the structures of many proteins. tion on its aliphatic chain; arginine, which has a positively charged guanidino group; and histidine, which has an imidazole group. Histidine is the only common amino acid having an ionizable side chain with a pKa near neutrality. In many enzyme-catalyzed reactions, a His residue facilitates the
reaction by serving as a proton donor/acceptor. Negatively Charged (Acidic) R Groups The two amino acids having R groups with a net negative charge at pH 7.0 are aspartate and glutamate, each of which has a second carboxyl group. Tryptophan Uncommon Amino Acids Also Have Important Functions Tyrosine 0 230 240 250 260 270 280 290 300 310 Wavelength (nm) 3 FIGURE 3–6 Absorption of ultraviolet light by aromatic amino acids. Comparison of the light absorption spectra of the aromatic amino acids tryptophan and tyrosine at pH 6.0. The amino acids are present in equimolar amounts (10 M) under identical conditions. The measured absorbance of tryptophan is as much as four times that of tyrosine. Note that the maximum light absorption for both tryptophan and tyrosine occurs near a wavelength of 280 nm. Light absorption by the third aromatic amino acid, phenylalanine (not shown), generally contributes little to the spectroscopic properties of proteins. In addition to the 20 common amino acids, proteins may contain residues created by modification of common residues already incorporated into a polypeptide (Fig. 3–8a). Among these uncommon amino acids are 4-hydroxyproline, a derivative of proline, and 5-hydroxylysine, derived from lysine. The former is found in plant cell wall proteins, and both are found in collagen, a fibrous protein of connective tissues. 6-NMethyllysine is a constituent of myosin, a contractile protein of muscle. Another important uncommon amino acid is -carboxyglutamate, found in the bloodclotting protein prothrombin and in certain other proteins that bind Ca2 as part of their biological function. More complex is desmosine, a derivative of four Lys residues, which is found in the fibrous protein elastin. Selenocysteine is a special case. This rare amino acid residue is introduced during protein synthesis rather than created through a postsynthetic modification. It contains selenium rather than the sulfur of cysteine. Actually derived from serine, selenocysteine is a constituent of just a few known proteins. Some 300 additional amino acids have been found in cells. They have a variety of functions but are not constituents of proteins. Ornithine and citrulline 8885d_c
03_081 12/23/03 10:21 AM Page 81 mac111 mac111:reb: H HO C CH2 H2C N H COO C H H 4-Hydroxyproline H3N CH2 C H CH2 CH2 C H COO OH 5-Hydroxylysine NH3 CH3 NH CH2 CH2 CH2 CH2 CH COO 6-N-Methyllysine NH3 COO OOC C H CH2 C H COO H3N OOC NH3 -Carboxyglutamate H3N COO C H (CH2)3 N H3 C H (CH2)2 (CH2)2 C H COO N (C H2)4 C H COO H3N Desmosine HSe CH2 C H COO NH3 Selenocysteine (a) H3N CH2 CH2 CH2 C H COO NH3 Ornithine CH2 CH2 CH2 C H COO NH3 H2N C O N H (b) Citrulline 3.1 Amino Acids 81 FIGURE 3–8 Uncommon amino acids. (a) Some uncommon amino acids found in proteins. All are derived from common amino acids. Extra functional groups added by modification reactions are shown in red. Desmosine is formed from four Lys residues (the four carbon backbones are shaded in yellow). Note the use of either numbers or Greek letters to identify the carbon atoms in these structures. (b) Ornithine and citrulline, which are not found in proteins, are intermediates in the biosynthesis of arginine and in the urea cycle. O C HO H2N C H R Nonionic form O H3N O C C R H Zwitterionic form FIGURE 3–9 Nonionic and zwitterionic forms of amino acids. The nonionic form does not occur in significant amounts in aqueous solutions. The zwitterion predominates at neutral pH. Amino Acids Can Act as Acids and Bases When an amino acid is dissolved in water, it exists in solution as the dipolar ion, or zwitterion (German for “hybrid ion”), shown in Figure 3–9. A zwitterion can act as either an acid (proton donor): H H R C COO NH3 Zwitterion R C COO H NH2
or a base (proton acceptor): H H R C COO H NH3 Zwitterion R C COOH NH3 Substances having this dual nature are amphoteric and are often called ampholytes (from “amphoteric electrolytes”). A simple monoamino monocarboxylic amino acid, such as alanine, is a diprotic acid when fully protonated—it has two groups, the OCOOH group and the ONH3 group, that can yield protons: H H H H H (Fig. 3–8b) deserve special note because they are key intermediates (metabolites) in the biosynthesis of arginine (Chapter 22) and in the urea cycle (Chapter 18). R Net charge: R C COOH C COO NH3 NH3 NH2 1 0 1 R C COO 8885d_c03_082 12/23/03 10:21 AM Page 82 mac111 mac111:reb: 82 Chapter 3 Amino Acids, Peptides, and Proteins BOX 3–1 WORKING IN BIOCHEMISTRY Absorption of Light by Molecules: The Lambert-Beer Law A wide range of biomolecules absorb light at characteristic wavelengths, just as tryptophan absorbs light at 280 nm (see Fig. 3–6). Measurement of light absorption by a spectrophotometer is used to detect and identify molecules and to measure their concentration in solution. The fraction of the incident light absorbed by a solution at a given wavelength is related to the thickness of the absorbing layer (path length) and the concentration of the absorbing species (Fig. 1). These two relationships are combined into the Lambert-Beer law, I0 I cl log where I0 is the intensity of the incident light, I is the intensity of the transmitted light, is the molar extinction coefficient (in units of liters per mole-centimeter), c is the concentration of the absorbing species (in moles per liter), and l is the path length of the lightabsorbing sample (in centimeters). The Lambert-Beer law assumes that the incident light is parallel and monochromatic (of a single wavelength) and that the solvent and solute molecules are randomly oriented. The expression log (I0/I) is called the absorbance, designated A. It is important to note that each successive millimeter of path length of absorbing solution in
a 1.0 cm cell absorbs not a constant amount but a constant fraction of the light that is incident upon it. However, with an absorbing layer of fixed path length, the absorbance, A, is directly proportional to the concentration of the absorbing solute. The molar extinction coefficient varies with the nature of the absorbing compound, the solvent, and the wavelength, and also with pH if the light-absorbing species is in equilibrium with an ionization state that has different absorbance properties. FIGURE 1 The principal components of a spectrophotometer. A light source emits light along a broad spectrum, then the monochromator selects and transmits light of a particular wavelength. The monochro- matic light passes through the sample in a cuvette of path length l and is absorbed by the sample in proportion to the concentra- tion of the absorbing species. The transmit- ted light is measured by a detector. Lamp Monochromator Intensity of incident light I0 Intensity of transmitted light I A = 0.012 Detector l Sample cuvette with c moles/liter of absorbing species Amino Acids Have Characteristic Titration Curves Acid-base titration involves the gradual addition or removal of protons (Chapter 2). Figure 3–10 shows the titration curve of the diprotic form of glycine. The plot has two distinct stages, corresponding to deprotonation of two different groups on glycine. Each of the two stages resembles in shape the titration curve of a monoprotic acid, such as acetic acid (see Fig. 2–17), and can be analyzed in the same way. At very low pH, the predominant ionic species of glycine is the fully protonated form, H3NOCH2 OCOOH. At the midpoint in the first stage of the titration, in which the OCOOH group of glycine loses its proton, equimolar concentrations of the proton-donor (H3NOCH2OCOOH) and proton-acceptor (H3NOCH2OCOO) species are present. At the midpoint of any titration, a point of inflection is reached where the pH is equal to the pKa of the protonated group being titrated (see Fig. 2–18). For glycine, the pH at the midpoint is 2.34, thus its OCOOH group has a pKa (labeled pK1 in Fig.
3–10) of 2.34. (Recall from Chapter 2 that pH and pKa are simply convenient notations for proton concentration and the equilibrium constant for ionization, respectively. The pKa is a measure of the tendency of a group to give up a proton, with that tendency decreasing tenfold as the pKa increases by one unit.) As the titration proceeds, another important point is reached at pH 5.97. Here there is another point of inflection, at which removal of the first proton is essentially complete and removal of the second has just begun. At this pH glycine is present largely as the dipolar ion H3NOCH2OCOO. We shall return to the significance of this inflection point in the titration curve (labeled pI in Fig. 3–10) shortly. The second stage of the titration corresponds to the group of glycine. removal of a proton from the ONH3 The pH at the midpoint of this stage is 9.60, equal to group. the pKa (labeled pK2 in Fig. 3–10) for the ONH3 The titration is essentially complete at a pH of about 12, at which point the predominant form of glycine is H2NOCH2OCOO. 8885d_c03_083 12/23/03 10:21 AM Page 83 mac111 mac111:reb: N H3 C H2 COOH pK1 N H3 C H2 COO pK2 N H2 C H2 COO 13 Glycine pK2 9.60 7 pH pI 5.97 pK1 2.34 0 0 0.5 1 1.5 2 OH (equivalents) FIGURE 3–10 Titration of an amino acid. Shown here is the titration curve of 0.1 M glycine at 25 C. The ionic species predominating at key points in the titration are shown above the graph. The shaded 9.60, indicate the reboxes, centered at about pK1 gions of greatest buffering power. 2.34 and pK2 3.1 Amino Acids 83 From the titration curve of glycine we can derive several important pieces of information. First, it gives a quantitative measure of the pKa of each of the two ionizing groups: 2.34 for the OCOOH group and 9.60 for group. Note that the car
boxyl group of the ONH3 glycine is over 100 times more acidic (more easily ionized) than the carboxyl group of acetic acid, which, as we saw in Chapter 2, has a pKa of 4.76—about average for a carboxyl group attached to an otherwise unsubstituted aliphatic hydrocarbon. The perturbed pKa of glycine is caused by repulsion between the departing proton and the nearby positively charged amino group on the -carbon atom, as described in Figure 3–11. The opposite charges on the resulting zwitterion are stabilizing, nudging the equilibrium farther to the right. Similarly, the pKa of the amino group in glycine is perturbed downward relative to the average pKa of an amino group. This effect is due partly to the electronegative oxygen atoms in the carboxyl groups, which tend to pull electrons toward them, increasing the tendency of the amino group to give up a proton. Hence, the -amino group has a pKa that is lower than that of an aliphatic amine such as methylamine (Fig. 3–11). In short, the pKa of any functional group is greatly affected by its chemical environment, a phenomenon sometimes exploited in the active sites of enzymes to promote exquisitely adapted reaction mechanisms that depend on the perturbed pKa values of proton donor/acceptor groups of specific residues. pKa 2 4 6 8 10 12 Methyl-substituted carboxyl and amino groups CH3 COOH H H CH3 COO CH3 NH3 H H CH3 NH2 Acetic acid The normal pKa for a carboxyl group is about 4.8. Methylamine The normal pKa for an amino group is about 10.6. Carboxyl and amino groups in glycine NH3 H C COOH H H H NH3 CH COO H -Amino acid (glycine) pKa 2.34 Repulsion between the amino group and the departing proton lowers the pKa for the carboxyl group, and oppositely charged groups lower the pKa by stabilizing the zwitterion. H NH2 CH COO H H -Amino acid (glycine) pKa 9.60 Electronegative oxygen atoms in the carboxyl group pull electrons away from the amino group, lowering its pKa. FIGURE 3–11 Effect of
the chemical environment on pKa. The pKa values for the ionizable groups in glycine are lower than those for simple, methyl-substituted amino and carboxyl groups. These downward perturbations of pKa are due to intramolecular interactions. Similar effects can be caused by chemical groups that happen to be positioned nearby—for example, in the active site of an enzyme. 8885d_c03_084 12/23/03 10:21 AM Page 84 mac111 mac111:reb: 84 Chapter 3 Amino Acids, Peptides, and Proteins The second piece of information provided by the titration curve of glycine is that this amino acid has two regions of buffering power. One of these is the relatively flat portion of the curve, extending for approximately 1 pH unit on either side of the first pKa of 2.34, indicating that glycine is a good buffer near this pH. The other buffering zone is centered around pH 9.60. (Note that glycine is not a good buffer at the pH of intracellular fluid or blood, about 7.4.) Within the buffering ranges of glycine, the Henderson-Hasselbalch equation (see Box 2–3) can be used to calculate the proportions of proton-donor and proton-acceptor species of glycine required to make a buffer at a given pH. Titration Curves Predict the Electric Charge of Amino Acids Another important piece of information derived from the titration curve of an amino acid is the relationship between its net electric charge and the pH of the solution. At pH 5.97, the point of inflection between the two stages in its titration curve, glycine is present predominantly as its dipolar form, fully ionized but with no net electric charge (Fig. 3–10). The characteristic pH at which the net electric charge is zero is called the isoelectric point or isoelectric pH, designated pI. For glycine, which has no ionizable group in its side chain, the isoelectric point is simply the arithmetic mean of the two pKa values: (pK1 pK2) 1 pI 1 (2.34 9.60) 5.97 2 2 As is evident in Figure 3–10, glycine has a net negative charge at any pH above its pI and will thus move toward the positive electrode (the anode) when placed in an electric
field. At any pH below its pI, glycine has a net positive charge and will move toward the negative electrode (the cathode). The farther the pH of a glycine solution is from its isoelectric point, the greater the net electric charge of the population of glycine molecules. At pH 1.0, for example, glycine exists almost entirely as the form H3NOCH2OCOOH, with a net positive charge of 1.0. At pH 2.34, where there is an equal mixture of H3NOCH2OCOOH and H3NOCH2OCOO, the average or net positive charge is 0.5. The sign and the magnitude of the net charge of any amino acid at any pH can be predicted in the same way. Amino Acids Differ in Their Acid-Base Properties The shared properties of many amino acids permit some simplifying generalizations about their acid-base behaviors. First, all amino acids with a single -amino group, a single -carboxyl group, and an R group that does not ionize have titration curves resembling that of glycine (Fig. 3–10). These amino acids have very similar, although not identical, pKa values: pKa of the OCOOH group in the range of 1.8 to 2.4, and pKa of the ONH3 group in the range of 8.8 to 11.0 (Table 3–1). Second, amino acids with an ionizable R group have more complex titration curves, with three stages corresponding to the three possible ionization steps; thus they have three pKa values. The additional stage for the titration of the ionizable R group merges to some extent with the other two. The titration curves for two amino acids of this type, glutamate and histidine, are shown in Figure 3–12. The isoelectric points reflect the nature of the ionizing R groups present. For example, glutamate H3N N H3 COOH C H C H2 C H2 pK1 COO C H C H2 C H2 pKR COOH COOH N H3 COO C H C H2 C H2 COO pK2 COO H2N C H C H2 C H2 COO Glutamate 10 pK2 9.67 pH 8 6 4 2 0 (a) pKR 4.25 pK1 2.19 1
.0 OH (equivalents) 2.0 3.0 COOH COO COO COO H3N H3N CH pK1 C H C H2 C C H H N N H H3N CH pKR C H CH2 C C H H N N H C H CH2 C C H H N N H2N C H CH2 C C H H N N CH CH pK2 pK2 9.17 Histidine pK1 1.82 10 8 pH 6 4 2 0 (b) pKR 6.0 1.0 2.0 OH (equivalents) 3.0 FIGURE 3–12 Titration curves for (a) glutamate and (b) histidine. The pKa of the R group is designated here as pKR. 8885d_c03_085 12/23/03 10:22 AM Page 85 mac111 mac111:reb: has a pI of 3.22, considerably lower than that of glycine. This is due to the presence of two carboxyl groups, which, at the average of their pKa values (3.22), contribute a net charge of 1 that balances the 1 contributed by the amino group. Similarly, the pI of histidine, with two groups that are positively charged when protonated, is 7.59 (the average of the pKa values of the amino and imidazole groups), much higher than that of glycine. Finally, as pointed out earlier, under the general condition of free and open exposure to the aqueous environment, only histidine has an R group (pKa 6.0) providing significant buffering power near the neutral pH usually found in the intracellular and extracellular fluids of most animals and bacteria (Table 3–1). SUMMARY 3.1 Amino Acids ■ The 20 amino acids commonly found as residues in proteins contain an -carboxyl group, an -amino group, and a distinctive R group substituted on the -carbon atom. The -carbon atom of all amino acids except glycine is asymmetric, and thus amino acids can exist in at least two stereoisomeric forms. Only the L stereoisomers, with a configuration related to the absolute configuration of the reference molecule L-glyceraldehyde, are found in proteins. ■ Other, less common amino acids also occur, either as constituents of proteins (through modification of common amino acid residues after protein
synthesis) or as free metabolites. ■ Amino acids are classified into five types on the basis of the polarity and charge (at pH 7) of their R groups. ■ Amino acids vary in their acid-base properties and have characteristic titration curves. Monoamino monocarboxylic amino acids (with nonionizable R groups) are diprotic acids (H3NCH(R)COOH) at low pH and exist in several different ionic forms as the pH is increased. Amino acids with ionizable R groups have additional ionic species, depending on the pH of the medium and the pKa of the R group. 3.2 Peptides and Proteins We now turn to polymers of amino acids, the peptides and proteins. Biologically occurring polypeptides range in size from small to very large, consisting of two or three to thousands of linked amino acid residues. Our focus is on the fundamental chemical properties of these polymers. 3.2 Peptides and Proteins 85 Peptides Are Chains of Amino Acids Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptide. Such a linkage is formed by removal of the elements of water (dehydration) from the -carboxyl group of one amino acid and the -amino group of another (Fig. 3–13). Peptide bond formation is an example of a condensation reaction, a common class of reactions in living cells. Under standard biochemical conditions, the equilibrium for the reaction shown in Figure 3–13 favors the amino acids over the dipeptide. To make the reaction thermodynamically more favorable, the carboxyl group must be chemically modified or activated so that the hydroxyl group can be more readily eliminated. A chemical approach to this problem is outlined later in this chapter. The biological approach to peptide bond formation is a major topic of Chapter 27. Three amino acids can be joined by two peptide bonds to form a tripeptide; similarly, amino acids can be linked to form tetrapeptides, pentapeptides, and so forth. When a few amino acids are joined in this fashion, the structure is called an oligopeptide. When many amino acids are joined, the product is called a polypeptide. Proteins may have thousands of amino acid residues. Although the terms “protein”
and “polypeptide” are sometimes used interchangeably, molecules referred to as polypeptides generally have molecular weights below 10,000, and those called proteins have higher molecular weights. Figure 3–14 shows the structure of a pentapeptide. As already noted, an amino acid unit in a peptide is often called a residue (the part left over after losing a hydrogen atom from its amino group and the hydroxyl moiety from its carboxyl group). In a peptide, the amino acid residue at the end with a free -amino group is the amino-terminal (or N-terminal) residue; the residue R1 H R2 H3N C H C OH H N H COO C O H2O R1 H3N C H C O H2O R2 H COO C H N FIGURE 3–13 Formation of a peptide bond by condensation. The amino group of one amino acid (with R2 group) acts as a nucleophile to displace the hydroxyl group of another amino acid (with R1 group), forming a peptide bond (shaded in yellow). Amino groups are good nucleophiles, but the hydroxyl group is a poor leaving group and is not readily displaced. At physiological pH, the reaction shown does not occur to any appreciable extent. 8885d_c03_086 12/23/03 10:22 AM Page 86 mac111 mac111:reb: 86 Chapter 3 Amino Acids, Peptides, and Proteins OH CH3 CH3 H C H3N H N CH2OH CH2 C H C O H N CH3 C H C O H N C H2 COO C H Aminoterminal end Carboxylterminal end FIGURE 3–14 The pentapeptide serylglycyltyrosylalanylleucine, or Ser–Gly–Tyr–Ala–Leu. Peptides are named beginning with the aminoterminal residue, which by convention is placed at the left. The peptide bonds are shaded in yellow; the R groups are in red. at the other end, which has a free carboxyl group, is the carboxyl-terminal (C-terminal) residue. Although hydrolysis of a peptide bond is an exergonic reaction, it occurs slowly because of its high activation energy. As
a result, the peptide bonds in proteins are quite stable, with an average half-life (t1/2) of about 7 years under most intracellular conditions. Peptides Can Be Distinguished by Their Ionization Behavior Peptides contain only one free -amino group and one free -carboxyl group, at opposite ends of the chain (Fig. 3–15). These groups ionize as they do in free amino acids, although the ionization constants are different because an oppositely charged group is no longer linked to the carbon. The -amino and -carboxyl groups of all nonterminal amino acids are covalently joined in the peptide bonds, which do not ionize and thus do not contribute to the total acid-base behavior of peptides. How- N H3 Ala C H CH3 O C NH Glu C H CH2 CH2 COO Gly O C NH C H2 O C NH Lys H CH2 CH2 CH2 CH2 N C H3 COO FIGURE 3–15 Alanylglutamylglycyllysine. This tetrapeptide has one free -amino group, one free -carboxyl group, and two ionizable R groups. The groups ionized at pH 7.0 are in red. ever, the R groups of some amino acids can ionize (Table 3–1), and in a peptide these contribute to the overall acid-base properties of the molecule (Fig. 3–15). Thus the acid-base behavior of a peptide can be predicted from its free -amino and -carboxyl groups as well as the nature and number of its ionizable R groups. Like free amino acids, peptides have characteristic titration curves and a characteristic isoelectric pH (pI) at which they do not move in an electric field. These properties are exploited in some of the techniques used to separate peptides and proteins, as we shall see later in the chapter. It should be emphasized that the pKa value for an ionizable R group can change somewhat when an amino acid becomes a residue in a peptide. The loss of charge in the -carboxyl and -amino groups, the interactions with other peptide R groups, and other environmental factors can affect the pKa. The pKa values for R groups listed in Table 3–1 can be a useful guide to the pH range in which a given group will ionize, but they cannot
be strictly applied to peptides. Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes No generalizations can be made about the molecular weights of biologically active peptides and proteins in relation to their functions. Naturally occurring peptides range in length from two to many thousands of amino acid residues. Even the smallest peptides can have biologically important effects. Consider the commercially synthesized dipeptide L-aspartyl-L-phenylalanine methyl ester, the artificial sweetener better known as aspartame or NutraSweet. COO C H2 O CH2 O H3N C C H C H C N H L-Aspartyl-L-phenylalanine methyl ester (aspartame) OCH3 Many small peptides exert their effects at very low concentrations. For example, a number of vertebrate hormones (Chapter 23) are small peptides. These include oxytocin (nine amino acid residues), which is secreted by the posterior pituitary and stimulates uterine contractions; bradykinin (nine residues), which inhibits inflammation of tissues; and thyrotropin-releasing factor (three residues), which is formed in the hypothalamus and stimulates the release of another hormone, thyrotropin, from the anterior pituitary gland. Some extremely toxic mushroom poisons, such as amanitin, are also small peptides, as are many antibiotics. Slightly larger are small polypeptides and oligopeptides such as the pancreatic hormone insulin, which contains two polypeptide chains, one having 30 amino acid 8885d_c03_087 12/23/03 10:22 AM Page 87 mac111 mac111:reb: residues and the other 21. Glucagon, another pancreatic hormone, has 29 residues; it opposes the action of insulin. Corticotropin is a 39-residue hormone of the anterior pituitary gland that stimulates the adrenal cortex. How long are the polypeptide chains in proteins? As Table 3–2 shows, lengths vary considerably. Human cytochrome c has 104 amino acid residues linked in a single chain; bovine chymotrypsinogen has 245 residues. At the extreme is titin, a constituent of vertebrate muscle, which has nearly 27,000 amino acid residues and a molecular weight of about 3,000,000. The vast majority of naturally occurring proteins are much
smaller than this, containing fewer than 2,000 amino acid residues. Some proteins consist of a single polypeptide chain, but others, called multisubunit proteins, have two or more polypeptides associated noncovalently (Table 3–2). The individual polypeptide chains in a multisubunit protein may be identical or different. If at least two are identical the protein is said to be oligomeric, and the identical units (consisting of one or more polypeptide chains) are referred to as protomers. Hemoglobin, for example, has four polypeptide subunits: two identical chains and two identical chains, all four held together by noncovalent interactions. Each subunit is paired in an identical way with a subunit within the structure of this multisubunit protein, so that hemoglobin can be considered either a tetramer of four polypeptide subunits or a dimer of protomers. A few proteins contain two or more polypeptide chains linked covalently. For example, the two polypeptide chains of insulin are linked by disulfide bonds. In such cases, the individual polypeptides are not considered subunits but are commonly referred to simply as chains. We can calculate the approximate number of amino acid residues in a simple protein containing no other 3.2 Peptides and Proteins 87 chemical constituents by dividing its molecular weight by 110. Although the average molecular weight of the 20 common amino acids is about 138, the smaller amino acids predominate in most proteins. If we take into account the proportions in which the various amino acids occur in proteins (Table 3–1), the average molecular weight of protein amino acids is nearer to 128. Because a molecule of water (Mr 18) is removed to create each peptide bond, the average molecular weight of an amino acid residue in a protein is about 128 18 110. Polypeptides Have Characteristic Amino Acid Compositions Hydrolysis of peptides or proteins with acid yields a mixture of free -amino acids. When completely hydrolyzed, each type of protein yields a characteristic proportion or mixture of the different amino acids. The 20 common amino acids almost never occur in equal amounts in a protein. Some amino acids may occur only once or not at all in a given type of protein; others may occur in large numbers. Table 3–3 shows the composition of the amino acid mixtures obtained on complete hydrolysis of b
ovine cytochrome c and chymotrypsinogen, the inactive precursor of the digestive enzyme chymotrypsin. These two proteins, with very different functions, also differ significantly in the relative numbers of each kind of amino acid they contain. Complete hydrolysis alone is not sufficient for an exact analysis of amino acid composition, however, because some side reactions occur during the procedure. For example, the amide bonds in the side chains of asparagine and glutamine are cleaved by acid treatment, yielding aspartate and glutamate, respectively. The side chain of tryptophan is almost completely degraded by acid hydrolysis, and small amounts of serine, threonine, TABLE 3–2 Molecular Data on Some Proteins Molecular weight Number of residues Number of polypeptide chains Cytochrome c (human) Ribonuclease A (bovine pancreas) Lysozyme (chicken egg white) Myoglobin (equine heart) Chymotrypsin (bovine pancreas) Chymotrypsinogen (bovine) Hemoglobin (human) Serum albumin (human) Hexokinase (yeast) RNA polymerase (E. coli) Apolipoprotein B (human) Glutamine synthetase (E. coli) Titin (human) 13,000 13,700 13,930 16,890 21,600 22,000 64,500 68,500 102,000 450,000 513,000 619,000 2,993,000 104 124 129 153 241 245 574 609 972 4,158 4,536 5,628 26,926 12 1 8885d_c03_088 12/23/03 10:22 AM Page 88 mac111 mac111:reb: 88 Chapter 3 Amino Acids, Peptides, and Proteins TABLE 3–3 Two Proteins Amino Acid Composition of TABLE 3–4 Conjugated Proteins Class Prosthetic group Example Number of residues per molecule of protein* Lipoproteins Lipids Amino acid Bovine cytochrome c Bovine chymotrypsinogen Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Total 6 2 5 3 2 3 9 14 3 6 6 18 2 4 4 1 8 1 4 3 104 22 4
15 8 10 10 5 23 2 10 19 14 2 6 9 28 23 8 4 23 245 *In some common analyses, such as acid hydrolysis, Asp and Asn are not readily distinguished from each other and are together designated Asx (or B). Similarly, when Glu and Gln cannot be distinguished, they are together designated Glx (or Z). In addition, Trp is destroyed. Additional procedures must be employed to obtain an accurate assessment of complete amino acid content. and tyrosine are also lost. When a precise amino acid composition is required, biochemists use additional procedures to resolve the ambiguities that remain from acid hydrolysis. Some Proteins Contain Chemical Groups Other Than Amino Acids Many proteins, for example the enzymes ribonuclease A and chymotrypsinogen, contain only amino acid residues and no other chemical constituents; these are considered simple proteins. However, some proteins contain permanently associated chemical components in addition to amino acids; these are called conjugated proteins. The non–amino acid part of a conjugated protein is usually called its prosthetic group. Conjugated proteins are classified on the basis of the chemical nature of their prosthetic groups (Table 3–4); for example, lipoproteins contain lipids, glycoproteins contain sugar groups, and metalloproteins contain a specific Glycoproteins Phosphoproteins Hemoproteins Flavoproteins Carbohydrates Phosphate groups Heme (iron porphyrin) Flavin nucleotides Metalloproteins Iron Zinc Calcium Molybdenum Copper 1-Lipoprotein of blood Immunoglobulin G Casein of milk Hemoglobin Succinate dehydrogenase Ferritin Alcohol dehydrogenase Calmodulin Dinitrogenase Plastocyanin metal. A number of proteins contain more than one prosthetic group. Usually the prosthetic group plays an important role in the protein’s biological function. There Are Several Levels of Protein Structure For large macromolecules such as proteins, the tasks of describing and understanding structure are approached at several levels of complexity, arranged in a kind of conceptual hierarchy. Four levels of protein structure are commonly defined (Fig. 3–16). A description of all covalent bonds (mainly peptide bonds and disulfide bonds) linking amino acid residues in a polypeptide chain is its primary structure. The most important element of primary structure
is the sequence of amino acid residues. Secondary structure refers to particularly stable arrangements of amino acid residues giving rise to recurring structural patterns. Tertiary structure describes all aspects of the three-dimensional folding of a polypeptide. When a protein has two or more polypeptide subunits, their arrangement in space is referred to as quaternary structure. Primary structure is the focus of Section 3.4; the higher levels of structure are discussed in Chapter 4. SUMMARY 3.2 Peptides and Proteins ■ Amino acids can be joined covalently through peptide bonds to form peptides and proteins. Cells generally contain thousands of different proteins, each with a different biological activity. ■ Proteins can be very long polypeptide chains of 100 to several thousand amino acid residues. However, some naturally occurring peptides have only a few amino acid residues. Some proteins are composed of several noncovalently 8885d_c03_089 12/23/03 11:06 AM Page 89 mac111 mac111:reb: Primary structure Secondary structure Tertiary structure Quaternary structure 3.3 Working with Proteins 89 Lys Lys Gly Gly Leu Val Ala His Amino acid residues Helix Polypeptide chain Assembled subunits FIGURE 3–16 Levels of structure in proteins. The primary structure consists of a sequence of amino acids linked together by peptide bonds and includes any disulfide bonds. The resulting polypeptide can be coiled into units of secondary structure, such as an helix. The he- lix is a part of the tertiary structure of the folded polypeptide, which is itself one of the subunits that make up the quaternary structure of the multisubunit protein, in this case hemoglobin. associated polypeptide chains, called subunits. Simple proteins yield only amino acids on hydrolysis; conjugated proteins contain in addition some other component, such as a metal or organic prosthetic group. ■ The sequence of amino acids in a protein is characteristic of that protein and is called its primary structure. This is one of four generally recognized levels of protein structure. 3.3 Working with Proteins Our understanding of protein structure and function has been derived from the study of many individual proteins. To study a protein in detail, the researcher must be able to separate it from other proteins and must have the techniques to determine its properties. The necessary methods come from protein chemistry, a discipline
as old as biochemistry itself and one that retains a central position in biochemical research. Proteins Can Be Separated and Purified A pure preparation is essential before a protein’s properties and activities can be determined. Given that cells contain thousands of different kinds of proteins, how can one protein be purified? Methods for separating proteins take advantage of properties that vary from one protein to the next, including size, charge, and binding properties. The source of a protein is generally tissue or microbial cells. The first step in any protein purification procedure is to break open these cells, releasing their proteins into a solution called a crude extract. If necessary, differential centrifugation can be used to pre- pare subcellular fractions or to isolate specific organelles (see Fig. 1–8). Once the extract or organelle preparation is ready, various methods are available for purifying one or more of the proteins it contains. Commonly, the extract is subjected to treatments that separate the proteins into different fractions based on a property such as size or charge, a process referred to as fractionation. Early fractionation steps in a purification utilize differences in protein solubility, which is a complex function of pH, temperature, salt concentration, and other factors. The solubility of proteins is generally lowered at high salt concentrations, an effect called “salting out.” The addition of a salt in the right amount can selectively precipitate some proteins, while others remain in solution. Ammonium sulfate ((NH4)2SO4) is often used for this purpose because of its high solubility in water. A solution containing the protein of interest often must be further altered before subsequent purification steps are possible. For example, dialysis is a procedure that separates proteins from solvents by taking advantage of the proteins’ larger size. The partially purified extract is placed in a bag or tube made of a semipermeable membrane. When this is suspended in a much larger volume of buffered solution of appropriate ionic strength, the membrane allows the exchange of salt and buffer but not proteins. Thus dialysis retains large proteins within the membranous bag or tube while allowing the concentration of other solutes in the protein preparation to change until they come into equilibrium with the solution outside the membrane. Dialysis might be used, for example, to remove ammonium sulfate from the protein preparation. The most powerful methods for fractionating proteins make use of column chromatography, which takes advantage of
differences in protein charge, size, 8885d_c03_090 12/23/03 10:23 AM Page 90 mac111 mac111:reb: 90 Chapter 3 Amino Acids, Peptides, and Proteins binding affinity, and other properties (Fig. 3–17). A porous solid material with appropriate chemical properties (the stationary phase) is held in a column, and a buffered solution (the mobile phase) percolates through it. The protein-containing solution, layered on the top of the column, percolates through the solid matrix as an ever-expanding band within the larger mobile phase (Fig. 3–17). Individual proteins migrate faster or more slowly through the column depending on their properties. For example, in cation-exchange chromatography (Fig. 3–18a), the solid matrix has negatively charged groups. In the mobile phase, proteins with a net positive charge migrate through the matrix more slowly than those with a net negative charge, because the migration of the former is retarded more by interaction with the stationary phase. The two types of protein can separate into two distinct bands. The expansion of the protein band in the mobile phase (the protein solution) is caused both by separation of proteins with different properties and by diffusional spreading. As the length of the column increases, the resolution of two types of protein with different net charges generally improves. However, the rate at which the protein solution can flow through the column usually decreases with column Reservoir Protein sample (mobile phase) Solid porous matrix (stationary phase) Porous support Effluent Proteins A B C length. And as the length of time spent on the column increases, the resolution can decline as a result of diffusional spreading within each protein band. Figure 3–18 shows two other variations of column chromatography in addition to ion exchange. Sizeexclusion chromatography separates proteins according to size. In this method, large proteins emerge from the column sooner than small ones—a somewhat counterintuitive result. The solid phase consists of beads with engineered pores or cavities of a particular size. Large proteins cannot enter the cavities, and so take a short (and rapid) path through the column, around the beads. Small proteins enter the cavities, and migrate through the column more slowly as a result (Fig. 3–18b). Affinity chromatography is based on the binding affinity of a protein. The beads in the column have a covalently attached chemical group. A protein with affinity for
this particular chemical group will bind to the beads in the column, and its migration will be retarded as a result (Fig. 3–18c). A modern refinement in chromatographic methods is HPLC, or high-performance liquid chromatography. HPLC makes use of high-pressure pumps that speed the movement of the protein molecules down the column, as well as higher-quality chromatographic materials that can withstand the crushing force of the pressurized flow. By reducing the transit time on the column, HPLC can limit diffusional spreading of protein bands and thus greatly improve resolution. The approach to purification of a protein that has not previously been isolated is guided both by established precedents and by common sense. In most cases, several different methods must be used sequentially to purify a protein completely. The choice of method is FIGURE 3–17 Column chromatography. The standard elements of a chromatographic column include a solid, porous material supported inside a column, generally made of plastic or glass. The solid material (matrix) makes up the stationary phase through which flows a solution, the mobile phase. The solution that passes out of the column at the bottom (the effluent) is constantly replaced by solution supplied from a reservoir at the top. The protein solution to be separated is layered on top of the column and allowed to percolate into the solid matrix. Additional solution is added on top. The protein solution forms a band within the mobile phase that is initially the depth of the protein solution applied to the column. As proteins migrate through the column, they are retarded to different degrees by their different interactions with the matrix material. The overall protein band thus widens as it moves through the column. Individual types of proteins (such as A, B, and C, shown in blue, red, and green) gradually separate from each other, forming bands within the broader protein band. Separation improves (resolution increases) as the length of the column increases. However, each individual protein band also broadens with time due to diffusional spreading, a process that decreases resolution. In this example, protein A is well separated from B and C, but diffusional spreading prevents complete separation of B and C under these conditions. 8885d_c03_091 12/23/03 10:23 AM Page 91 mac111 mac111:reb: Large net positive charge Net positive charge Net negative charge Large net negative charge Polymer beads with negatively charged functional groups Protein mixture is added to column containing cation exchangers.
Porous polymer beads Protein mixture is added to column containing cross-linked polymer. 1 2 3 4 5 6 (b) Proteins move through the column at rates determined by their net charge at the pH being used. With cation exchangers, proteins with a more negative net charge move faster and elute earlier. (a) Protein molecules separate by size; larger molecules pass more freely, appearing in the earlier fractions. 1 2 3 4 5 6 Protein of interest Ligand FIGURE 3–18 Three chromatographic methods used in protein purification. (a) Ion-exchange chromatography exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH. The column matrix is a synthetic polymer containing bound charged groups; those with bound anionic groups are called cation exchangers, and those with bound cationic groups are called anion exchangers. Ion-exchange chromatography on a cation exchanger is shown here. The affinity of each protein for the charged groups on the column is affected by the pH (which determines the ionization state of the molecule) and the concentration of competing free salt ions in the surrounding solution. Separation can be optimized by gradually changing the pH and/or salt concentration of the mobile phase so as to create a pH or salt gradient. (b) Size-exclusion chromatography, also called gel filtration, separates proteins according to size. The column matrix is a cross-linked polymer with pores of selected size. Larger proteins migrate faster than smaller ones, because they are too large to enter the pores in the beads and hence take a more direct route through the column. The smaller proteins enter the pores and are slowed by their more labyrinthine path through the column. (c) Affinity chromatography separates proteins by their binding specificities. The proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads. (In biochemistry, the term “ligand” is used to refer to a group or molecule that binds to a macromolecule such as a protein.) After proteins that do not bind to the ligand are washed through the column, the bound protein of particular interest is eluted (washed out of the column) by a solution containing free ligand. Mixture of proteins Solution of ligand Protein mixture is added to column containing a polymer-bound ligand specific for protein of interest. (c) 21 3 4 5 6543 7 8 Unwanted proteins
are washed through column. Protein of interest is eluted by ligand solution. 8885d_c03_092 12/23/03 10:23 AM Page 92 mac111 mac111:reb: 92 Chapter 3 Amino Acids, Peptides, and Proteins TABLE 3–5 A Purification Table for a Hypothetical Enzyme Procedure or step 1. Crude cellular extract 2. Precipitation with ammonium sulfate 3. Ion-exchange chromatography 4. Size-exclusion chromatography 5. Affinity chromatography Fraction volume (ml) Total protein (mg) 1,400 280 90 80 6 10,000 3,000 400 100 3 Activity (units) 100,000 96,000 80,000 60,000 45,000 Specific activity (units/mg) 10 32 200 600 15,000 Note: All data represent the status of the sample after the designated procedure has been carried out. Activity and specific activity are defined on page 94. somewhat empirical, and many protocols may be tried before the most effective one is found. Trial and error can often be minimized by basing the procedure on purification techniques developed for similar proteins. Published purification protocols are available for many thousands of proteins. Common sense dictates that inexpensive procedures such as salting out be used first, when the total volume and the number of contaminants are greatest. Chromatographic methods are often impractical at early stages, because the amount of chromatographic medium needed increases with sample size. As each purification step is completed, the sample size generally becomes smaller (Table 3–5), making it feasible to use more sophisticated (and expensive) chromatographic procedures at later stages. Proteins Can Be Separated and Characterized by Electrophoresis Another important technique for the separation of proteins is based on the migration of charged proteins in an electric field, a process called electrophoresis. These procedures are not generally used to purify proteins in large amounts, because simpler alternatives are usually available and electrophoretic methods often adversely affect the structure and thus the function of proteins. Electrophoresis is, however, especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of different proteins in a mixture or the degree of purity of a particular protein preparation. Also, electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight.
Electrophoresis of proteins is generally carried out in gels made up of the cross-linked polymer polyacrylamide (Fig. 3–19). The polyacrylamide gel acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio. Migration may also be affected by protein shape. In electrophoresis, the force moving the macromolecule is the electrical potential, E. The electrophoretic mobility of the molecule,, is the ratio of the velocity of the par- ticle molecule, V, to the electrical potential. Electrophoretic mobility is also equal to the net charge of the molecule, Z, divided by the frictional coefficient, f, which reflects in part a protein’s shape. Thus: V Z E f The migration of a protein in a gel during electrophoresis is therefore a function of its size and its shape. An electrophoretic method commonly employed for estimation of purity and molecular weight makes use of the detergent sodium dodecyl sulfate (SDS). Na O O S O O (CH2)11CH3 Sodium dodecyl sulfate (SDS) SDS binds to most proteins in amounts roughly proportional to the molecular weight of the protein, about one molecule of SDS for every two amino acid residues. The bound SDS contributes a large net negative charge, rendering the intrinsic charge of the protein insignificant and conferring on each protein a similar charge-to-mass ratio. In addition, the native conformation of a protein is altered when SDS is bound, and most proteins assume a similar shape. Electrophoresis in the presence of SDS therefore separates proteins almost exclusively on the basis of mass (molecular weight), with smaller polypeptides migrating more rapidly. After electrophoresis, the proteins are visualized by adding a dye such as Coomassie blue, which binds to proteins but not to the gel itself (Fig. 3–19b). Thus, a researcher can monitor the progress of a protein purification procedure as the number of protein bands visible on the gel decreases after each new fractionation step. When compared with the positions to which proteins of known molecular weight migrate in the gel, the position of an unidentified protein can provide an excellent measure of its molecular weight (Fig. 3–20). If the protein has two or more different subunits, the subunits will generally be separated by the SDS treatment and
a separate band will appear for each. SDS Gel Electrophoresis 8885d_c03_093 1/16/04 6:48 AM Page 93 mac76 mac76:385_reb: 3.3 Working with Proteins 93 Sample – + Well Direction of migration (a) (b) FIGURE 3–19 Electrophoresis. (a) Different samples are loaded in wells or depressions at the top of the polyacrylamide gel. The proteins move into the gel when an electric field is applied. The gel minimizes convection currents caused by small temperature gradients, as well as protein movements other than those induced by the electric field. (b) Proteins can be visualized after electrophoresis by treating the gel with a stain such as Coomassie blue, which binds to the proteins but not to the gel itself. Each band on the gel represents a different pro- tein (or protein subunit); smaller proteins move through the gel more rapidly than larger proteins and therefore are found nearer the bottom of the gel. This gel illustrates the purification of the enzyme RNA polymerase from E. coli. The first lane shows the proteins present in the crude cellular extract. Successive lanes (left to right) show the proteins present after each purification step. The purified protein contains four subunits, as seen in the last lane on the right. Isoelectric focusing is a procedure used to determine the isoelectric point (pI) of a protein (Fig. 3–21). A pH gradient is established by allowing a mixture of low molecular weight organic acids and bases (ampholytes; p. 81) to distribute themselves in an electric field generated across the gel. When a protein mix- ture is applied, each protein migrates until it reaches the pH that matches its pI (Table 3–6). Proteins with different isoelectric points are thus distributed differently throughout the gel. Combining isoelectric focusing and SDS electrophoresis sequentially in a process called two-dimensional 1 2 – Myosin 200,000 b-Galactosidase Glycogen phosphorylase b 116,250 97,400 Bovine serum albumin 66,200 Ovalbumin 45,000 Carbonic anhydrase 31,000 Soybean trypsin inhibitor Lysozyme 21,500 14,400 FIGURE 3–20 Estimating the molecular weight of a protein. The
electrophoretic mobility of a protein on an SDS polyacrylamide gel is related to its molecular weight, Mr. (a) Standard proteins of known molecular weight are subjected to electrophoresis (lane 1). These marker proteins can be used to estimate the molecular weight of an unknown protein (lane 2). (b) A plot of log Mr of the marker proteins versus relative migration during electrophoresis is linear, which allows the molecular weight of the unknown protein to be read from the graph. Unknown protein r M g o l (a) + Mr standards Unknown protein (b) Relative migration 8885d_c03_094 12/23/03 10:24 AM Page 94 mac111 mac111:reb: 94 Chapter 3 Amino Acids, Peptides, and Proteins An ampholyte solution is incorporated into a gel. pH pH 3 + A stable pH gradient is established in the gel after application of an electric field. – + – + Protein solution is added and electric field is reapplied. After staining, proteins are shown to be distributed along pH gradient according to their pI values. FIGURE 3–21 Isoelectric focusing. This technique separates proteins according to their isoelectric points. A stable pH gradient is established in the gel by the addition of appropriate ampholytes. A protein mixture is placed in a well on the gel. With an applied electric field, proteins enter the gel and migrate until each reaches a pH equivalent to its pI. Remember that when pH pI, the net charge of a protein is zero. electrophoresis permits the resolution of complex mixtures of proteins (Fig. 3–22). This is a more sensitive analytical method than either electrophoretic method alone. Two-dimensional electrophoresis separates proteins of identical molecular weight that differ in pI, or proteins with similar pI values but different molecular weights. Unseparated Proteins Can Be Quantified To purify a protein, it is essential to have a way of detecting and quantifying that protein in the presence of many other proteins at each stage of the procedure. Often, purification must proceed in the absence of any information about the size and physical properties of the protein or about the fraction of the total protein mass it represents in the extract. For proteins that are enzymes, the amount in a given solution or tissue extract can be measured, or assayed, in terms of the catalytic effect the enzyme produces—that is, the
increase in the rate at which its substrate is converted to reaction products when the enzyme is present. For this purpose one must know (1) the overall equation of the reaction catalyzed, (2) an analytical procedure for determining the disappearance of the substrate or the appearance of a reaction product, (3) whether the enzyme requires cofactors such as metal ions or coenzymes, (4) the dependence of the enzyme activity on substrate concentration, (5) the optimum pH, and (6) a temperature zone in which the enzyme is stable and has high activity. Enzymes are usually assayed at their optimum pH and at some convenient temperature within the range 25 to 38 C. Also, very high substrate concentrations are generally used so that the initial reaction rate, measured experimentally, is proportional to enzyme concentration (Chapter 6). By international agreement, 1.0 unit of enzyme activity is defined as the amount of enzyme causing transformation of 1.0 mol of substrate per minute at 25 C under optimal conditions of measurement. The term activity refers to the total units of enzyme in a solution. The specific activity is the number of enzyme units per milligram of total protein (Fig. 3–23). The specific activity is a measure of enzyme purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure (Table 3–5). TABLE 3–6 of Some Proteins The Isoelectric Points Protein Pepsin Egg albumin Serum albumin Urease -Lactoglobulin Hemoglobin Myoglobin Chymotrypsinogen Cytochrome c Lysozyme pI 1.0 4.6 4.9 5.0 5.2 6.8 7.0 9.5 10.7 11.0 8885d_c03_095 12/23/03 10:24 AM Page 95 mac111 mac111:reb: 3.3 Working with Proteins 95 First dimension Isoelectric focusing Decreasing pI Isoelectric focusing gel is placed on SDS polyacrylamide gel. Second dimension SDS polyacrylamide gel electrophoresis Decreasing Mr (a) Decreasing pI After each purification step, the activity of the preparation (in units of enzyme activity) is assayed, the total amount of protein is determined independently, and the ratio of the two gives the specific activity. Activity and total protein generally decrease with each step. Activity decreases because some loss always occurs due to inactivation
or nonideal interactions with chromatographic materials or other molecules in the solution. Total protein decreases because the objective is to remove as much unwanted or nonspecific protein as possible. In a successful step, the loss of nonspecific protein is much greater than the loss of activity; therefore, specific activity increases even as total activity falls. The data are then assembled in a purification table similar to Table 3–5. A protein is generally considered pure (b) FIGURE 3–22 Two-dimensional electrophoresis. (a) Proteins are first separated by isoelectric focusing in a cylindrical gel. The gel is then laid horizontally on a second, slab-shaped gel, and the proteins are separated by SDS polyacrylamide gel electrophoresis. Horizontal separation reflects differences in pI; vertical separation reflects differences in molecular weight. (b) More than 1,000 different proteins from E. coli can be resolved using this technique. when further purification steps fail to increase specific activity and when only a single protein species can be detected (for example, by electrophoresis). For proteins that are not enzymes, other quantification methods are required. Transport proteins can be assayed by their binding to the molecule they transport, and hormones and toxins by the biological effect they produce; for example, growth hormones will stimulate the growth of certain cultured cells. Some structural proteins represent such a large fraction of a tissue mass that they can be readily extracted and purified without a functional assay. The approaches are as varied as the proteins themselves. FIGURE 3–23 Activity versus specific activity. The difference between these two terms can be illustrated by considering two beakers of marbles. The beakers contain the same number of red marbles, but different numbers of marbles of other colors. If the marbles represent proteins, both beakers contain the same activity of the protein represented by the red marbles. The second beaker, however, has the higher specific activity because here the red marbles represent a much higher fraction of the total. 8885d_c03_096 12/23/03 10:24 AM Page 96 mac111 mac111:reb: 96 Chapter 3 Amino Acids, Peptides, and Proteins SUMMARY 3.3 Working with Proteins ■ Proteins are separated and purified by taking advantage of differences in their properties. Proteins can be selectively precipitated by the addition of certain salts. A wide range
of chromatographic procedures makes use of differences in size, binding affinities, charge, and other properties. These include ionexchange, size-exclusion, affinity, and highperformance liquid chromatography. ■ Electrophoresis separates proteins on the basis of mass or charge. SDS gel electrophoresis and isoelectric focusing can be used separately or in combination for higher resolution. ■ All purification procedures require a method for quantifying or assaying the protein of interest in the presence of other proteins. Purification can be monitored by assaying specific activity. 3.4 The Covalent Structure of Proteins Purification of a protein is usually only a prelude to a detailed biochemical dissection of its structure and function. What is it that makes one protein an enzyme, another a hormone, another a structural protein, and still another an antibody? How do they differ chemically? The most obvious distinctions are structural, and these distinctions can be approached at every level of structure defined in Figure 3–16. The differences in primary structure can be especially informative. Each protein has a distinctive number and sequence of amino acid residues. As we shall see in Chapter 4, the primary structure of a protein determines how it folds up into a unique three-dimensional structure, and this in turn determines the function of the protein. Primary structure is the focus of the remainder of this chapter. We first consider empirical clues that amino acid sequence and protein function are closely linked, then describe how amino acid sequence is determined; finally, we outline the many uses to which this information can be put. The Function of a Protein Depends on Its Amino Acid Sequence The bacterium Escherichia coli produces more than 3,000 different proteins; a human produces 25,000 to 35,000. In both cases, each type of protein has a unique three-dimensional structure and this structure confers a unique function. Each type of protein also has a unique amino acid sequence. Intuition suggests that the amino acid sequence must play a fundamental role in determining the three-dimensional structure of the protein, and ultimately its function, but is this supposition cor- rect? A quick survey of proteins and how they vary in amino acid sequence provides a number of empirical clues that help substantiate the important relationship between amino acid sequence and biological function. First, as we have already noted, proteins with different functions always have different amino acid sequences. Second, thousands of human genetic diseases have been traced to the production of defective proteins. Perhaps one-
third of these proteins are defective because of a single change in their amino acid sequence; hence, if the primary structure is altered, the function of the protein may also be changed. Finally, on comparing functionally similar proteins from different species, we find that these proteins often have similar amino acid sequences. An extreme case is ubiquitin, a 76-residue protein involved in regulating the degradation of other proteins. The amino acid sequence of ubiquitin is identical in species as disparate as fruit flies and humans. Is the amino acid sequence absolutely fixed, or invariant, for a particular protein? No; some flexibility is possible. An estimated 20% to 30% of the proteins in humans are polymorphic, having amino acid sequence variants in the human population. Many of these variations in sequence have little or no effect on the function of the protein. Furthermore, proteins that carry out a broadly similar function in distantly related species can differ greatly in overall size and amino acid sequence. Although the amino acid sequence in some regions of the primary structure might vary considerably without affecting biological function, most proteins contain crucial regions that are essential to their function and whose sequence is therefore conserved. The fraction of the overall sequence that is critical varies from protein to protein, complicating the task of relating sequence to three-dimensional structure, and structure to function. Before we can consider this problem further, however, we must examine how sequence information is obtained. The Amino Acid Sequences of Millions of Proteins Have Been Determined Two major discoveries in 1953 were of crucial importance in the history of biochemistry. In that year James D. Watson and Francis Crick deduced the double-helical structure of DNA and proposed a structural basis for its precise replication (Chapter 8). Their proposal illuminated the molecular reality behind the idea of a gene. In that same year, Frederick Sanger worked out the sequence of amino acid residues in the polypeptide chains of the hormone insulin (Fig. 3–24), surprising many researchers who had long thought that elucidation of the amino acid sequence of a polypeptide would be a hopelessly difficult task. It quickly became evident that the nucleotide sequence in DNA and the amino acid sequence in proteins were somehow related. Barely a decade after these discoveries, the role of the nucleotide 8885d_c03_097 12/23/03 10:24 AM Page 97 mac111 mac111:reb: sequence of DNA in determining the amino acid sequence of protein molecules was revealed (Chapter 27).
An enormous number of protein sequences can now be derived indirectly from the DNA sequences in the rapidly growing genome databases. However, many are still deduced by traditional methods of polypeptide sequencing. The amino acid sequences of thousands of different proteins from many species have been determined using principles first developed by Sanger. These methods are still in use, although with many variations and improvements in detail. Chemical protein sequencing now 3.4 The Covalent Structure of Proteins 97 A chain NH3 Gly Ile Val Gln NH3 Phe Val Asn Gln 5 Gln 5 His Cys Cys Ala Ser S S S S Leu Cys Gly Ser 10 Val 10 His Cys Ser Leu Tyr Leu Val Glu Ala 15 Gln 15 Leu Leu Glu Asn Tyr Tyr Leu Val Cys S 20 Cys S 20 Gly Asn COO FIGURE 3–24 Amino acid sequence of bovine insulin. The two polypeptide chains are joined by disulfide crosslinkages. The A chain is identical in human, pig, dog, rabbit, and sperm whale insulins. The B chains of the cow, pig, dog, goat, and horse are identical. Glu Arg Gly Phe 25 Phe Tyr Thr Pro Lys 30 Ala COO Frederick Sanger B chain complements a growing list of newer methods, providing multiple avenues to obtain amino acid sequence data. Such data are now critical to every area of biochemical investigation. Short Polypeptides Are Sequenced Using Automated Procedures Various procedures are used to analyze protein primary structure. Several protocols are available to label and identify the amino-terminal amino acid residue (Fig. 3–25a). Sanger developed the reagent 1-fluoro-2,4dinitrobenzene (FDNB) for this purpose; other reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield derivatives that are more easily detectable than the dinitrophenyl derivatives. After the amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed to its constituent amino acids and the labeled amino acid is identified. Because the hydrolysis stage destroys the polypeptide, this procedure cannot be used to sequence a polypeptide beyond its amino-terminal residue. However, it can help determine the number of chemically distinct polypeptides in a protein, provided each
has a different amino-terminal residue. For example, two residues—Phe and Gly—would be labeled if insulin (Fig. 3–24) were subjected to this procedure. CH3 G N CH3 D O2Cl Dansyl chloride S CH3 G N D CH3 NPN S O2Cl Dabsyl chloride 8885d_c03_098 12/23/03 10:25 AM Page 98 mac111 mac111:reb: 98 Chapter 3 Amino Acids, Peptides, and Proteins (a) (b) Polypeptide NO2 NO2 NO2 F FDNB NO2 2,4-Dinitrophenyl derivative of polypeptide NO2 NO2 6 M HCl NH R1 C H COO 2,4-Dinitrophenyl derivative of amino-terminal residue NH R1 C H C O HN R2 C H C O Free amino acids Identify amino-terminal residue of polypeptide. phenylisothiocyanate N C S OH N H S C HN: R1 C H C O +NH2 R2 C H C O PTC adduct NH C S O C N CH R1 CF3COOH N S C C O H+ NH HC R1 Anilinothiazolinone derivative of amino acid residue Phenylthiohydantoin derivative of amino acid residue H3N 2 R C H R3 C H C O N H C O Shortened peptide Identify amino-terminal residue; purify and recycle remaining peptide fragment through Edman process. FIGURE 3–25 Steps in sequencing a polypeptide. (a) Identification of the amino-terminal residue can be the first step in sequencing a polypeptide. Sanger’s method for identifying the amino-terminal residue is shown here. (b) The Edman degradation procedure reveals the entire sequence of a peptide. For shorter peptides, this method alone readily yields the entire sequence, and step (a) is often omitted. Step (a) is useful in the case of larger polypeptides, which are often fragmented into smaller peptides for sequencing (see Fig. 3–27). To sequence an entire polypeptide, a chemical method devised by Pehr Edman is usually employed. The Edman degradation procedure labels and removes only the amino-terminal residue from a pept
ide, leaving all other peptide bonds intact (Fig. 3–25b). The peptide is reacted with phenylisothiocyanate under mildly alkaline conditions, which converts the aminoterminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The peptide bond next to the PTC adduct is then cleaved in a step carried out in anhydrous trifluoroacetic acid, with removal of the amino-terminal amino acid as an anilinothiazolinone derivative. The derivatized amino acid is extracted with organic solvents, converted to the more stable phenylthiohydantoin derivative by treatment with aqueous acid, and then identified. The use of sequential reactions carried out under first basic and then acidic conditions provides control over labeled, removed, and the entire process. Each reaction with the aminoterminal amino acid can go essentially to completion without affecting any of the other peptide bonds in the peptide. After removal and identification of the aminoterminal residue, the new amino-terminal residue so exposed can be identified through the same series of reactions. This procedure is repeated until the entire sequence is determined. The Edman degradation is carried out on a machine, called a sequenator, that mixes reagents in the proper proportions, separates the products, identifies them, and records the results. These methods are extremely sensitive. Often, the complete amino acid sequence can be determined starting with only a few micrograms of protein. The length of polypeptide that can be accurately sequenced by the Edman degradation depends on the 8885d_c03_099 12/23/03 10:25 AM Page 99 mac111 mac111:reb: efficiency of the individual chemical steps. Consider a peptide beginning with the sequence Gly–Pro–Lys– at its amino terminus. If glycine were removed with 97% efficiency, 3% of the polypeptide molecules in the solution would retain a Gly residue at their amino terminus. In the second Edman cycle, 97% of the liberated amino acids would be proline, and 3% glycine, while 3% of the polypeptide molecules would retain Gly (0.1%) or Pro (2.9%) residues at their amino terminus. At each cycle, peptides that did not react in earlier cycles would contribute amino acids to an ever-increasing background,
eventually making it impossible to determine which amino acid is next in the original peptide sequence. Modern sequenators achieve efficiencies of better than 99% per cycle, permitting the sequencing of more than 50 contiguous amino acid residues in a polypeptide. The primary structure of insulin, worked out by Sanger and colleagues over a period of 10 years, could now be completely determined in a day or two. Large Proteins Must Be Sequenced in Smaller Segments The overall accuracy of amino acid sequencing generally declines as the length of the polypeptide increases. The very large polypeptides found in proteins must be broken down into smaller pieces to be sequenced efficiently. There are several steps in this process. First, the protein is cleaved into a set of specific fragments by chemical or enzymatic methods. If any disulfide bonds 3.4 The Covalent Structure of Proteins 99 are present, they must be broken. Each fragment is purified, then sequenced by the Edman procedure. Finally, the order in which the fragments appear in the original protein is determined and disulfide bonds (if any) are located. Breaking Disulfide Bonds Disulfide bonds interfere with the sequencing procedure. A cystine residue (Fig. 3–7) that has one of its peptide bonds cleaved by the Edman procedure may remain attached to another polypeptide strand via its disulfide bond. Disulfide bonds also interfere with the enzymatic or chemical cleavage of the polypeptide. Two approaches to irreversible breakage of disulfide bonds are outlined in Figure 3–26. Cleaving the Polypeptide Chain Several methods can be used for fragmenting the polypeptide chain. Enzymes called proteases catalyze the hydrolytic cleavage of peptide bonds. Some proteases cleave only the peptide bond adjacent to particular amino acid residues (Table 3–7) and thus fragment a polypeptide chain in a predictable and reproducible way. A number of chemical reagents also cleave the peptide bond adjacent to specific residues. Among proteases, the digestive enzyme trypsin catalyzes the hydrolysis of only those peptide bonds in which the carbonyl group is contributed by either a Lys or an Arg residue, regardless of the length or amino acid sequence of the chain. The number of smaller peptides produced by trypsin cleavage can thus be predicted CH2SH CHOH CHOH CH
2SH Dithiothreitol (DTT) Disulfide bond (cystine) NH O C HC CH2 S S CH2 C H C O HN NH O O O C HC CH2 S O O S CH2 C H C O O O HN Cysteic acid residues reduction by dithiothreitol NH O C HC CH2 SH HS CH2 C H C O HN acetylation by iodoacetate FIGURE 3–26 Breaking disulfide bonds in proteins. Two common methods are illustrated. Oxidation of a cystine residue with performic acid produces two cysteic acid residues. Reduction by dithiothreitol to form Cys residues must be followed by further modification of the reactive OSH groups to prevent re-formation of the disulfide bond. Acetylation by iodoacetate serves this purpose. NH O C HC CH2 S CH2 COO OOC CH2 S CH2 C H C O HN Acetylated cysteine residues 8885d_c03_100 12/23/03 10:25 AM Page 100 mac111 mac111:reb: 100 Chapter 3 Amino Acids, Peptides, and Proteins TABLE 3–7 Methods for Fragmenting Polypeptide Chains The Specificity of Some Common Reagent (biological source)* Cleavage points† Trypsin (bovine pancreas) Submaxillarus protease (mouse submaxillary gland) Chymotrypsin (bovine pancreas) Lys, Arg (C) Arg (C) Phe, Trp, Tyr (C) Staphylococcus aureus V8 protease Asp, Glu (C) (bacterium S. aureus) Asp-N-protease (bacterium Pseudomonas fragi) Pepsin (porcine stomach) Endoproteinase Lys C (bacterium Lysobacter enzymogenes) Cyanogen bromide Asp, Glu (N) Phe, Trp, Tyr (N) Lys (C) Met (C) *All reagents except cyanogen bromide are proteases. All are available from commercial sources. †Residues furnishing the primary recognition point for the protease or reagent; peptide bond cleavage occurs on either the carbonyl (C) or
the amino (N) side of the indicated amino acid residues. from the total number of Lys or Arg residues in the original polypeptide, as determined by hydrolysis of an intact sample (Fig. 3–27). A polypeptide with five Lys and/or Arg residues will usually yield six smaller peptides on cleavage with trypsin. Moreover, all except one of these will have a carboxyl-terminal Lys or Arg. The fragments produced by trypsin (or other enzyme or chemical) action are then separated by chromatographic or electrophoretic methods. Sequencing of Peptides Each peptide fragment resulting from the action of trypsin is sequenced separately by the Edman procedure. Ordering Peptide Fragments The order of the “trypsin fragments” in the original polypeptide chain must now be determined. Another sample of the intact polypeptide is cleaved into fragments using a different enzyme or reagent, one that cleaves peptide bonds at points other than those cleaved by trypsin. For example, cyanogen bromide cleaves only those peptide bonds in which the carbonyl group is contributed by Met. The fragments resulting from this second procedure are then separated and sequenced as before. The amino acid sequences of each fragment obtained by the two cleavage procedures are examined, with the objective of finding peptides from the second procedure whose sequences establish continuity, be- cause of overlaps, between the fragments obtained by the first cleavage procedure (Fig. 3–27). Overlapping peptides obtained from the second fragmentation yield the correct order of the peptide fragments produced in the first. If the amino-terminal amino acid has been identified before the original cleavage of the protein, this information can be used to establish which fragment is derived from the amino terminus. The two sets of fragments can be compared for possible errors in determining the amino acid sequence of each fragment. If the second cleavage procedure fails to establish continuity between all peptides from the first cleavage, a third or even a fourth cleavage method must be used to obtain a set of peptides that can provide the necessary overlap(s). Locating Disulfide Bonds If the primary structure includes disulfide bonds, their locations are determined in an additional step after sequencing is completed. A sample of the protein is again cleaved with a reagent such as trypsin, this time without first breaking the
disulfide bonds. The resulting peptides are separated by electrophoresis and compared with the original set of peptides generated by trypsin. For each disulfide bond, two of the original peptides will be missing and a new, larger peptide will appear. The two missing peptides represent the regions of the intact polypeptide that are linked by the disulfide bond. Amino Acid Sequences Can Also Be Deduced by Other Methods The approach outlined above is not the only way to determine amino acid sequences. New methods based on mass spectrometry permit the sequencing of short polypeptides (20 to 30 amino acid residues) in just a few minutes (Box 3–2). In addition, with the development of rapid DNA sequencing methods (Chapter 8), the elucidation of the genetic code (Chapter 27), and the development of techniques for isolating genes (Chapter 9), researchers can deduce the sequence of a polypeptide by determining the sequence of nucleotides in the gene that codes for it (Fig. 3–28). The techniques used to determine protein and DNA sequences are complementary. When the gene is available, sequencing the DNA can be faster and more accurate than sequencing the protein. Most proteins are now sequenced in this indirect way. If the gene has not been isolated, direct sequencing of peptides is necessary, and this can provide information (the location of disulfide bonds, for example) not available in a DNA sequence. In addition, a knowledge of the amino acid sequence of even a part of a polypeptide can greatly facilitate the isolation of the corresponding gene (Chapter 9). The array of methods now available to analyze both proteins and nucleic acids is ushering in a new disci- 8885d_c03_101 12/23/03 10:26 AM Page 101 mac111 mac111:reb: 3.4 The Covalent Structure of Proteins 101 SS Procedure Result Conclusion hydrolyze; separate amino acids Polypeptide Polypeptide has 38 amino acid residues. Trypsin will cleave three times (at one R (Arg) and two K (Lys)) to give four fragments. Cyanogen bromide will cleave at two M (Met) to give three fragments. react with FDNB; hydrolyze; separate amino acids reduce disulfide bonds (if present) HS SH 2,4-Dinitrophenylglutamate detected E (Glu) is
aminoterminal residue. cleave with trypsin; separate fragments; sequence by Edman degradation cleave with cyanogen bromide; separate fragments; sequence by Edman degradation T-1 GASMALIK T-2 EGAAYHDFEPIDPR T-3 DCVHSD T-4 YLIACGPMTK placed at amino T-2 terminus because it begins with E (Glu). placed at carboxyl T-3 terminus because it does not end with R (Arg) or K (Lys). C-1 EGAAYHDFEPIDPRGASM C-3 overlaps with C-2 TKDCVHSD C-3 ALIKYLIACGPM and T-4, allowing T-1 them to be ordered. establish sequence Amino terminus T-2 T-1 T-4 T-3 EGAAYHDFEPIDPRGASMALIKYLIACGPMTKDCVHSD Carboxyl terminus C-1 C-3 C-2 FIGURE 3–27 Cleaving proteins and sequencing and ordering the peptide fragments. First, the amino acid composition and aminoterminal residue of an intact sample are determined. Then any disulfide bonds are broken before fragmenting so that sequencing can proceed efficiently. In this example, there are only two Cys (C) residues and thus only one possibility for location of the disulfide bond. In polypeptides with three or more Cys residues, the position of disulfide bonds can be determined as described in the text. (The one-letter symbols for amino acids are given in Table 3–1.) pline of “whole cell biochemistry.” The complete sequence of an organism’s DNA, its genome, is now available for organisms ranging from viruses to bacteria to multicellular eukaryotes (see Table 1–4). Genes are being discovered by the millions, including many that encode proteins with no known function. To describe the entire protein complement encoded by an organism’s DNA, researchers have coined the term proteome. As described in Chapter 9, the new disciplines of genomics and proteomics are complementing work carried out on cellular intermediary metabolism and nucleic acid metabolism to provide a new and increasingly complete picture of biochemistry at the level of cells and even organisms. Amino acid sequence (protein) Gln–Tyr–
Pro–Thr–Ile–Trp DNA sequence (gene) CAGTATCCTACGATTTGG FIGURE 3–28 Correspondence of DNA and amino acid sequences. Each amino acid is encoded by a specific sequence of three nucleotides in DNA. The genetic code is described in detail in Chapter 27. 8885d_c03_102 12/23/03 10:26 AM Page 102 mac111 mac111:reb: 102 Chapter 3 Amino Acids, Peptides, and Proteins BOX 3–2 WORKING IN BIOCHEMISTRY Investigating Proteins with Mass Spectrometry The mass spectrometer has long been an indispensable tool in chemistry. Molecules to be analyzed, referred to as analytes, are first ionized in a vacuum. When the newly charged molecules are introduced into an electric and/or magnetic field, their paths through the field are a function of their mass-to-charge ratio, m/z. This measured property of the ionized species can be used to deduce the mass (M) of the analyte with very high precision. Although mass spectrometry has been in use for many years, it could not be applied to macromolecules such as proteins and nucleic acids. The m/z measurements are made on molecules in the gas phase, and the heating or other treatment needed to transfer a macromolecule to the gas phase usually caused its rapid decomposition. In 1988, two different techniques were developed to overcome this problem. In one, proteins are placed in a light-absorbing matrix. With a short pulse of laser light, the proteins are ionized and then desorbed from the matrix into the vacuum system. This process, known as matrix-assisted laser desorption/ionization mass spectrometry, or MALDI MS, has been successfully used to measure the mass of a wide range of macromolecules. In a second and equally successful method, macromolecules in solution are forced directly from the liquid to gas phase. A solution of analytes is passed through a charged needle that is kept at a high electrical potential, dispersing the solution into a fine mist of charged microdroplets. The solvent surrounding the macromolecules rapidly evaporates, and the resulting multiply charged macromolecular ions are thus introduced nondestructively into the gas phase. This technique is called electrospray ionization mass spectrometry, or ESI MS
. Protons added during passage through the needle give additional charge to the macromolecule. The m/z of the molecule can be analyzed in the vacuum chamber. Mass spectrometry provides a wealth of information for proteomics research, enzymology, and protein chemistry in general. The techniques require only miniscule amounts of sample, so they can be readily applied to the small amounts of protein that can be extracted from a two-dimensional electrophoretic gel. The accurately measured molecular mass of a protein is one of the critical parameters in its identification. Once the mass of a protein is accurately known, mass spectrometry is a convenient and accurate method for detecting changes in mass due to the presence of bound cofactors, bound metal ions, covalent modifications, and so on. The process for determining the molecular mass of a protein with ESI MS is illustrated in Figure 1. As it is injected into the gas phase, a protein acquires a variable number of protons, and thus positive charges, from the solvent. This creates a spectrum of species with different mass-to-charge ratios. Each successive peak corresponds to a species that differs from that Glass capillary Sample solution Mass spectrometer + High voltage 100 75 50 25 0 800 Vacuum interface (a) 50+ 47,342 100 50 0 47,000 40+ 48,000 Mr 30+ 1,000 1,200 1,400 1,600 m/z (b FIGURE 1 Electrospray mass spectrometry of a protein. (a) A protein solution is dispersed into highly charged droplets by passage through a needle under the influence of a high-voltage electric field. The droplets evaporate, and the ions (with added protons in this case) enter the mass spectrometer for m/z measurement. The spec- trum generated (b) is a family of peaks, with each successive peak (from right to left) corresponding to a charged species increased by 1 in both mass and charge. A computer-generated transformation of this spectrum is shown in the inset. 8885d_c03_103 12/23/03 10:26 AM Page 103 mac111 mac111:reb: of its neighboring peak by a charge difference of 1 and a mass difference of 1 (1 proton). The mass of the protein can be determined from any two neighboring peaks. The measured m/z of one peak is (m/z)2 M n2X n2 where M is
the mass of the protein, n2 is the number of charges, and X is the mass of the added groups (protons in this case). Similarly for the neighboring peak, (m/z)1 M (n2 1)X n2 1 We now have two unknowns (M and n2) and two equations. We can solve first for n2 and then for M: n2 (m/z)2 X (m/z)2 (m/z)1 M n2 [(m/z)2 X] This calculation using the m/z values for any two peaks in a spectrum such as that shown in Figure 1b usually provides the mass of the protein (in this case, aerolysin k; 47,342 Da) with an error of only 0.01%. Generating several sets of peaks, repeating the calculation, and averaging the results generally provides an even more accurate value for M. Computer algorithms can transform the m/z spectrum into a single peak that FIGURE 2 Obtaining protein sequence information with tandem MS. (a) After proteolytic hydrolysis, a protein solution is injected into a mass spectrometer (MS-1). The different peptides are sorted so that only one type is selected for further analysis. The selected peptide is further fragmented in a chamber between the two mass spectrometers, and m/z for each fragment is measured in the sec- ond mass spectrometer (MS-2). Many of the ions generated during this second fragmentation result from breakage of the peptide bond, as shown. These are called b-type or y-type ions, depending on whether the charge is retained on the amino- or carboxyl-terminal 3.4 The Covalent Structure of Proteins 103 also provides a very accurate mass measurement (Fig. 1b, inset). Mass spectrometry can also be used to sequence short stretches of polypeptide, an application that has emerged as an invaluable tool for quickly identifying unknown proteins. Sequence information is extracted using a technique called tandem MS, or MS/MS. A solution containing the protein under investigation is first treated with a protease or chemical reagent to hydrolyze it to a mixture of shorter peptides. The mixture is then injected into a device that is essentially two mass spectrometers in tandem (Fig. 2a, top). In the first, the peptide mixture is sorted and the ionized fragments are manipulated so that only one
of the several types of peptides produced by cleavage emerges at the other end. The sample of the selected (continued on next page) MS-1 Collision cell MS-2 Detector Electrospray ionization Separation Breakage R1 C H O C H2N H N C H R2 C O b O C H N R3 C H R5 C H C O O– H N H C R4 C O N H y R1 C H O C H2N H N O C R3 C H H N C H R2 C O N H H N H C R4 C O R5 C H C O O– side, respectively. (b) A typical spectrum with peaks representing 100 the peptide fragments generated from a sample of one small pep- tide (10 residues). The labeled peaks are y-type ions. The large peak is a doubly charged ion and is not part of the y set. The next to y5 successive peaks differ by the mass of a particular amino acid in the original peptide. In this case, the deduced sequence was Phe–Pro–Gly–Gln–(Ile/Leu)–Asn–Ala–Asp–(Ile/Leu)–Arg. Note the ambiguity about Ile and Leu residues, because they have the same molecular mass. In this example, the set of peaks derived from y-type ions predominates, and the spectrum is greatly simplified as a result. This is because an Arg residue occurs at the carboxyl terminus of the peptide, and most of the positive charges are retained on this residue 75 50 25 0 (a) y2 y1 y3 200 400 y8 y4 y6 y7 y5 y9 800 1,000 600 m/z (b) 8885d_c03_104 1/16/04 6:08 AM Page 104 mac76 mac76:385_reb: 104 Chapter 3 Amino Acids, Peptides, and Proteins BOX 3–2 WORKING IN BIOCHEMISTRY (continued from previous page) peptide, each molecule of which has a charge somewhere along its length, then travels through a vacuum chamber between the two mass spectrometers. In this collision cell, the peptide is further fragmented by high-energy impact with a “collision gas,” a small amount of a noble gas such as helium or
argon that is bled into the vacuum chamber. This procedure is designed to fragment many of the peptide molecules in the sample, with each individual peptide broken in only one place, on average. Most breaks occur at peptide bonds. This fragmentation does not involve the addition of water (it is done in a near-vacuum), so the products may include molecular ion radicals such as carbonyl radicals (Fig. 2a, bottom). The charge on the original peptide is retained on one of the fragments generated from it. The second mass spectrometer then measures the m/z ratios of all the charged fragments (uncharged fragments are not detected). This generates one or more sets of peaks. A given set of peaks (Fig. 2b) consists of all the charged fragments that were generated by breaking the same type of bond (but at different points in the peptide) and are derived from the same side of the bond breakage, either the carboxyl- or amino-terminal side. Each successive peak in a given set has one less amino acid than the peak before. The difference in mass from peak to peak identifies the amino acid that was lost in each case, thus revealing the sequence of the peptide. The only ambiguities involve leucine and isoleucine, which have the same mass. The charge on the peptide can be retained on either the carboxyl- or amino-terminal fragment, and bonds other than the peptide bond can be broken in the fragmentation process, with the result that multiple sets of peaks are usually generated. The two most prominent sets generally consist of charged fragments derived from breakage of the peptide bonds. The set consisting of the carboxyl-terminal fragments can be unambiguously distinguished from that consisting of the amino-terminal fragments. Because the bond breaks generated between the spectrometers (in the collision cell) do not yield full carboxyl and amino groups at the sites of the breaks, the only intact amino and -carboxyl groups on the peptide fragments are those at the very ends (Fig. 2a). The two sets of fragments can thereby be identified by the resulting slight differences in mass. The amino acid sequence derived from one set can be confirmed by the other, improving the confidence in the sequence information obtained. Even a short sequence is often enough to permit unambiguous association of a protein with its gene, if the gene sequence is known. Sequencing by mass spectrometry cannot replace
the Edman degradation procedure for the sequencing of long polypeptides, but it is ideal for proteomics research aimed at cataloging the hundreds of cellular proteins that might be separated on a two-dimensional gel. In the coming decades, detailed genomic sequence data will be available from hundreds, eventually thousands, of organisms. The ability to rapidly associate proteins with genes using mass spectrometry will greatly facilitate the exploitation of this extraordinary information resource. Small Peptides and Proteins Can Be Chemically Synthesized Many peptides are potentially useful as pharmacologic agents, and their production is of considerable commercial importance. There are three ways to obtain a peptide: (1) purification from tissue, a task often made difficult by the vanishingly low concentrations of some peptides; (2) genetic engineering (Chapter 9); or (3) direct chemical synthesis. Powerful techniques now make direct chemical synthesis an attractive option in many cases. In addition to commercial applications, the synthesis of specific peptide portions of larger proteins is an increasingly important tool for the study of protein structure and function. The complexity of proteins makes the traditional synthetic approaches of organic chemistry impractical for peptides with more than four or five amino acid residues. One problem is the difficulty of purifying the product after each step. The major breakthrough in this technology was provided by R. Bruce Merrifield in 1962. His innovation involved synthesizing a peptide while keeping it attached at one end to a solid support. The support is an insoluble polymer (resin) contained within a column, similar to that used for chromatographic procedures. The peptide is built up on this support one amino acid at a time using a standard set of reactions in a repeating cycle (Fig. 3–29). At each successive step in the cycle, protective chemical groups block unwanted reactions. The technology for chemical peptide synthesis is now automated. As in the sequencing reactions already considered, the most important limitation of the process is the efficiency of each chemical cycle, as can be seen by calculating the overall yields of peptides of various 8885d_c03_105 12/23/03 10:27 AM Page 105 mac111 mac111:reb: 3.4 The Covalent Structure of Proteins 105 lengths when the yield for addition of each new amino acid is 96.0% versus 99.8% (Table 3–8). Incomplete reaction at one stage can lead to formation of an impurity (in the form of a shorter peptide) in the next
. The chemistry has been optimized to permit the synthesis of proteins of 100 amino acid residues in a few days in reasonable yield. A very similar approach is used to synthesize nucleic acids (see Fig. 8–38). It is worth noting that this technology, impressive as it is, still pales when compared with biological processes. The same FIGURE 3–29 Chemical synthesis of a peptide on an insoluble polymer support. Reactions 1 through 4 are necessary for the formation of each peptide bond. The 9-fluorenylmethoxycarbonyl (Fmoc) group (shaded blue) prevents unwanted reactions at the -amino group of the residue (shaded red). Chemical synthesis proceeds from the carboxyl terminus to the amino terminus, the reverse of the direction of protein synthesis in vivo (Chapter 27). CH2 O R1 CH O C O O C N H Fmoc Amino acid residue Amino acid 1 with by Fmoc group -amino group protected Cl CH2 R1 O C H C O Fmoc N H R1 O C H C O CH2 Fmoc N H Fmoc N C N Dicyclohexylcarbodiimide (DCC) N H 3 R2 O C H C O Amino acid 2 with protected -amino group is activated at carboxyl group by DCC. R1 O N H3 C H C OOCH2 Fmoc R2 O NH C H C O C N N H Fmoc R2 O C H C N H N H R1 O C H C O CH2 Insoluble polystyrene bead 1 Attachment of carboxyl-terminal amino acid to reactive group on resin. Cl 2 Protecting group is removed by flushing with solution containing a mild organic base. 4 -Amino group of amino acid 1 attacks activated carboxyl group of amino acid 2 to form peptide bond. O C N H N H Dicyclohexylurea byproduct Reactions repeated as necessary to 2 4 R. Bruce Merrifield 2 R O C H C H3N N H 11 R O C H C O F CH2 HF 5 Completed peptide is deprotected as in reaction 2 ester linkage between peptide and resin. ; HF cleaves 8885d_c03_106 12/23/03 10:27 AM Page 106 mac111 mac111:reb:
106 Chapter 3 Amino Acids, Peptides, and Proteins TABLE 3–8 Yield in Peptide Synthesis Effect of Stepwise Yield on Overall Number of residues in the final polypeptide Overall yield of final peptide (%) when the yield of each step is: 96.0% 99.8% 11 21 31 51 100 66 44 29 13 1.7 98 96 94 90 82 100-amino-acid protein would be synthesized with exquisite fidelity in about 5 seconds in a bacterial cell. A variety of new methods for the efficient ligation (joining together) of peptides has made possible the assembly of synthetic peptides into larger proteins. With these methods, novel forms of proteins can be created with precisely positioned chemical groups, including those that might not normally be found in a cellular protein. These novel forms provide new ways to test theories of enzyme catalysis, to create proteins with new chemical properties, and to design protein sequences that will fold into particular structures. This last application provides the ultimate test of our increasing ability to relate the primary structure of a peptide to the three-dimensional structure that it takes up in solution. Amino Acid Sequences Provide Important Biochemical Information Knowledge of the sequence of amino acids in a protein can offer insights into its three-dimensional structure and its function, cellular location, and evolution. Most of these insights are derived by searching for similarities with other known sequences. Thousands of sequences are known and available in databases accessible through the Internet. A comparison of a newly obtained sequence with this large bank of stored sequences often reveals relationships both surprising and enlightening. Exactly how the amino acid sequence determines three-dimensional structure is not understood in detail, nor can we always predict function from sequence. However, protein families that have some shared structural or functional features can be readily identified on the basis of amino acid sequence similarities. Individual proteins are assigned to families based on the degree of similarity in amino acid sequence. Members of a family are usually identical across 25% or more of their sequences, and proteins in these families generally share at least some structural and functional characteristics. Some families are defined, however, by identities involving only a few amino acid residues that are critical to a certain function. A number of similar substructures (to be defined in Chapter 4 as “domains”) occur in many functionally unrelated proteins. These domains often fold into structural configurations that have an unusual degree of stability or that are specialized for a certain environment. Evolutionary relationships can also be inferred
from the structural and functional similarities within protein families. Certain amino acid sequences serve as signals that determine the cellular location, chemical modification, and half-life of a protein. Special signal sequences, usually at the amino terminus, are used to target certain proteins for export from the cell; other proteins are targeted for distribution to the nucleus, the cell surface, the cytosol, and other cellular locations. Other sequences act as attachment sites for prosthetic groups, such as sugar groups in glycoproteins and lipids in lipoproteins. Some of these signals are well characterized and are easily recognized in the sequence of a newly characterized protein (Chapter 27). SUMMARY 3.4 The Covalent Structure of Proteins ■ Differences in protein function result from differences in amino acid composition and sequence. Some variations in sequence are possible for a particular protein, with little or no effect on function. ■ Amino acid sequences are deduced by fragmenting polypeptides into smaller peptides using reagents known to cleave specific peptide bonds; determining the amino acid sequence of each fragment by the automated Edman degradation procedure; then ordering the peptide fragments by finding sequence overlaps between fragments generated by different reagents. A protein sequence can also be deduced from the nucleotide sequence of its corresponding gene in DNA. ■ Short proteins and peptides (up to about 100 residues) can be chemically synthesized. The peptide is built up, one amino acid residue at a time, while remaining tethered to a solid support. 3.5 Protein Sequences and Evolution The simple string of letters denoting the amino acid sequence of a given protein belies the wealth of information this sequence holds. As more protein sequences have become available, the development of more powerful methods for extracting information from them has become a major biochemical enterprise. Each protein’s function relies on its three-dimensional structure, which 8885d_c03_107 12/23/03 10:27 AM Page 107 mac111 mac111:reb: in turn is determined largely by its primary structure. Thus, the biochemical information conveyed by a protein sequence is in principle limited only by our own understanding of structural and functional principles. On a different level of inquiry, protein sequences are beginning to tell us how the proteins evolved and, ultimately, how life evolved on this planet. Protein Sequences Can Elucidate the History of Life on Earth The field of molecular evolution is often traced to Emile Zuckerkandl and Linus Pauling, whose
work in the mid1960s advanced the use of nucleotide and protein sequences to explore evolution. The premise is deceptively straightforward. If two organisms are closely related, the sequences of their genes and proteins should be similar. The sequences increasingly diverge as the evolutionary distance between two organisms increases. The promise of this approach began to be realized in the 1970s, when Carl Woese used ribosomal RNA sequences to define archaebacteria as a group of living organisms distinct from other bacteria and eukaryotes (see Fig. 1–4). Protein sequences offer an opportunity to greatly refine the available information. With the advent of genome projects investigating organisms from bacteria to humans, the number of available sequences is growing at an enormous rate. This information can be used to trace biological history. The challenge is in learning to read the genetic hieroglyphics. Evolution has not taken a simple linear path. Complexities abound in any attempt to mine the evolutionary information stored in protein sequences. For a given protein, the amino acid residues essential for the activity of the protein are conserved over evolutionary time. The residues that are less important to function may vary over time—that is, one amino acid may substitute for another—and these variable residues can provide the information used to trace evolution. Amino acid substitutions are not always random, however. At some positions in the primary structure, the need to maintain protein function may mean that only particular amino acid substitutions can be tolerated. Some proteins have more variable amino acid residues than others. For these and other reasons, proteins can evolve at different rates. Another complicating factor in tracing evolutionary history is the rare transfer of a gene or group of genes from one organism to another, a process called lateral gene transfer. The transferred genes may be quite sim- 3.5 Protein Sequences and Evolution 107 ilar to the genes they were derived from in the original organism, whereas most other genes in the same two organisms may be quite distantly related. An example of lateral gene transfer is the recent rapid spread of antibiotic-resistance genes in bacterial populations. The proteins derived from these transferred genes would not be good candidates for the study of bacterial evolution, because they share only a very limited evolutionary history with their “host” organisms. The study of molecular evolution generally focuses on families of closely related proteins. In most cases, the families chosen for analysis have essential functions in cellular metabolism that must have been present in the earliest viable cells, thus greatly reducing the chance that they were introduced relatively recently by lateral
gene transfer. For example, a protein called EF-1 (elongation factor 1) is involved in the synthesis of proteins in all eukaryotes. A similar protein, EF-Tu, with the same function, is found in bacteria. Similarities in sequence and function indicate that EF-1 and EF-Tu are members of a family of proteins that share a common ancestor. The members of protein families are called homologous proteins, or homologs. The concept of a homolog can be further refined. If two proteins within a family (that is, two homologs) are present in the same species, they are referred to as paralogs. Homologs from different species are called orthologs (see Fig. 1–37). The process of tracing evolution involves first identifying suitable families of homologous proteins and then using them to reconstruct evolutionary paths. Homologs are identified using increasingly powerful computer programs that can directly compare two or more chosen protein sequences, or can search vast databases to find the evolutionary relatives of one selected protein sequence. The electronic search process can be thought of as sliding one sequence past the other until a section with a good match is found. Within this sequence alignment, a positive score is assigned for each position where the amino acid residues in the two sequences are identical—the value of the score varying from one program to the next—to provide a measure of the quality of the alignment. The process has some complications. Sometimes the proteins being compared match well at, say, two sequence segments, and these segments are connected by less related sequences of different lengths. Thus the two matching segments cannot be aligned at the same time. To handle this, the computer program introduces “gaps” in one of the sequences to bring the matching segments into register (Fig. 3–30). E. coli B. subtilis Gap FIGURE 3–30 Aligning protein sequences with the use of gaps. Shown here is the sequence alignment of a short section of the EF-Tu protein from two well-studied bacterial species, E. coli and Bacillus subtilis. Introduction of a gap in the B. subtilis sequence allows a better alignment of amino acid residues on either side of the gap. Identical amino acid residues are shaded. 8885d_c03_108 12/23/03 10:27 AM Page 108 mac111 mac111:reb: 108 Chapter 3 Amino Acids, Peptides, and Proteins
Of course, if a sufficient number of gaps are introduced, almost any two sequences could be brought into some sort of alignment. To avoid uninformative alignments, the programs include penalties for each gap introduced, thus lowering the overall alignment score. With electronic trial and error, the program selects the alignment with the optimal score that maximizes identical amino acid residues while minimizing the introduction of gaps. Identical amino acids are often inadequate to identify related proteins or, more importantly, to determine how closely related the proteins are on an evolutionary time scale. A more useful analysis includes a consideration of the chemical properties of substituted amino acids. When amino acid substitutions are found within a protein family, many of the differences may be conservative—that is, an amino acid residue is replaced by a residue having similar chemical properties. For example, a Glu residue may substitute in one family member for the Asp residue found in another; both amino acids are negatively charged. Such a conservative substitution should logically garner a higher score in a sequence alignment than does a nonconservative substitution, such as the replacement of the Asp residue with a hydrophobic Phe residue. To determine what scores to assign to the many different amino acid substitutions, Steven Henikoff and Jorja Henikoff examined the aligned sequences from a variety of different proteins. They did not analyze entire protein sequences, focusing instead on thousands of short conserved blocks where the fraction of identical amino acids was high and the alignments were thus reliable. Looking at the aligned sequence blocks, the Henikoffs analyzed the nonidentical amino acid residues within the blocks. Higher scores were given to nonidentical residues that occurred frequently than to those that appeared rarely. Even the identical residues were given scores based on how often they were replaced, such that amino acids with unique chemical properties (such as Cys and Trp) received higher scores than those more conservatively replaced (such as Asp and Glu). The result of this scoring system is a Blosum (blocks substitution matrix) table. The table in Figure 3–31 was generated from sequences that were identical in at least 62% of their amino acid residues, and it is thus referred to as Blosum62. Similar tables have been generated for blocks of homologous sequences that are 50% or 80% identical. When higher levels of identity are required, the most conservative amino acid substitutions can be A Ala 4 C A C Cys 0 9 D D Asp 2 3 6 E E Glu 1 4 2 5 F
F Phe 2 2 3 3 6 G G Gly 0 3 1 2 3 6 H H His 2 3 1 0 1 2 8 I I Ile Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp 11 Y Y Tyr FIGURE 3–31 The Blosum62 table. This blocks substitution matrix was created by comparing thousands of short blocks of aligned sequences that were identical in at least 62% of their amino acid residues. The nonidentical residues were assigned scores based on how frequently they were replaced by each of the other amino acids. Each substitution contributes to the score given to a particular alignment. Positive numbers (shaded yellow) add to the score for a particular alignment; negative numbers subtract from the score. Identical residues in sequences being compared (the shaded diagonal from top left to bottom right in the matrix) receive scores based on how often they are replaced, such that amino acids with unique chemical properties (e.g., Cys and Trp) receive higher scores (9 and 11, respectively) than those more easily replaced in conservative substitutions (e.g., Asp (6) and Glu (5)). Many computer programs use Blosum62 to assign scores to new sequence alignments. 8885d_c03_109 12/23/03 10:27 AM Page 109 mac111 mac111:reb: 3.5 Protein Sequences and Evolution 109 Archaebacteria Eukaryotes Gram-positive bacterium Gram-negative bacterium Halobacterium halobium Sulfolobus solfataricus Saccharomyces cerevisiae Homo sapiens Bacillus subtilis Escherichia coli Signature sequence FIGURE 3–32 A signature sequence in the EF-1/EF-Tu protein family. The signature sequence (boxed) is a 12-amino-acid insertion near the amino terminus of the sequence. Residues that align in all species are shaded yellow. Both archaebacteria and eukaryotes have the signature, although the sequences of the insertions are quite distinct for the two groups. The variation in the signature sequence reflects the significant evolutionary divergence that has occurred at this site since it first appeared in a common ancestor of both groups. overrepresented, which limits the usefulness of the matrix in identifying homologs that are somewhat distantly related. Tests have shown that the Blosum62 table provides the most reliable alignments over a wide range of protein families, and it is the default table
in many sequence alignment programs. For most efforts to find homologies and explore evolutionary relationships, protein sequences (derived either directly from protein sequencing or from the sequencing of the DNA encoding the protein) are superior to nongenic nucleic acid sequences (those that do not encode a protein or functional RNA). For a nucleic acid, with its four different types of residues, random alignment of nonhomologous sequences will generally yield matches for at least 25% of the positions. Introduction of a few gaps can often increase the fraction of matched residues to 40% or more, and the probability of chance alignment of unrelated sequences becomes quite high. The 20 different amino acid residues in proteins greatly lower the probability of uninformative chance alignments of this type. The programs used to generate a sequence alignment are complemented by methods that test the reliability of the alignments. A common computerized test is to shuffle the amino acid sequence of one of the proteins being compared to produce a random sequence, then instruct the program to align the shuffled sequence with the other, unshuffled one. Scores are assigned to the new alignment, and the shuffling and alignment process is repeated many times. The original alignment, before shuffling, should have a score significantly higher than any of those within the distribution of scores generated by the random alignments; this increases the confidence that the sequence alignment has identified a pair of homologs. Note that the absence of a significant alignment score does not necessarily mean that no evolutionary relationship exists between two proteins. As we shall see in Chapter 4, three-dimensional structural similarities sometimes reveal evolutionary relationships where sequence homology has been wiped away by time. Using a protein family to explore evolution requires the identification of family members with similar molecular functions in the widest possible range of organ- isms. Information from the family can then be used to trace the evolution of those organisms. By analyzing the sequence divergence in selected protein families, investigators can segregate organisms into classes based on their evolutionary relationships. This information must be reconciled with more classical examinations of the physiology and biochemistry of the organisms. Certain segments of a protein sequence may be found in the organisms of one taxonomic group but not in other groups; these segments can be used as signature sequences for the group in which they are found. An example of a signature sequence is an insertion of 12 amino acids near the amino terminus of the EF1/EF-Tu proteins in all archaebacteria and eukaryotes but not in other types of bacteria (Fig. 3–
32). The signature is one of many biochemical clues that can help establish the evolutionary relatedness of eukaryotes and archaebacteria. For example, the major taxa of bacteria can be distinguished by signature sequences in several different proteins. The and proteobacteria have signature sequences in the Hsp70 and DNA gyrase protein families (families of proteins involved in protein folding and DNA replication, respectively) that are not present in any other bacteria, including the other proteobacteria. The other types of proteobacteria (,, ), along with the and proteobacteria, have a separate Hsp70 signature sequence and a signature in alanyl-tRNA synthetase (an enzyme of protein synthesis) that are not present in other bacteria. The appearance of unique signatures in the and proteobacteria suggests the,, and proteobacteria arose before their and cousins. By considering the entire sequence of a protein, researchers can now construct more elaborate evolutionary trees with many species in each taxonomic group. Figure 3–33 presents one such tree for bacteria, based on sequence divergence in the protein GroEL (a protein present in all bacteria that assists in the proper folding of proteins). The tree can be refined by basing it on the sequences of multiple proteins and by supplementing the sequence information with data on the unique biochemical and physiological properties of each species. There are many methods for generating trees, each with its own advantages and shortcomings, and 8885d_c03_110 12/24/03 6:49 AM Page 110 mac76 mac76:385_reb: 110 Chapter 3 Amino Acids, Peptides, and Proteins many ways to represent the resulting evolutionary relationships. In Figure 3–33, the free end points of lines are called “external nodes”; each represents an extant species, and each is so labeled. The points where two lines come together, the “internal nodes,” represent extinct ancestor species. In most representations (including Fig. 3–33), the lengths of the lines connecting the nodes are proportional to the number of amino acid substitutions separating one species from another. If we trace two extant species to a common internal node (representing the common ancestor of the two species), the length of the branch connecting each external node to the internal node represents the number of amino acid substitutions separating one extant species from this ancestor. The sum of the lengths of all the line segments that connect an extant species to another extant species through a common ancestor
reflects the number of substitutions separating the two extant species. To determine how much time was needed for the various species to diverge, the tree must be calibrated by comparing it with information from the fossil record and other sources. As more sequence information is made available in databases, we can generate evolutionary trees based on a variety of different proteins. Some proteins evolve faster than others, or change faster within one group of species than another. A large protein, with many vari- Bacteroides Chlamydia trachomatis Chlamydia Porphyromonas gingivalis Chlamydia psittaci Helicobacter pylori Legionella pneumophila Pseudomonas aeruginosa Yersinia enterocolitica Salmonella typhi Escherichia coli Neisseria gonorrhoeae Rickettsia tsutsugamushi Bradyrhizobium japonicum Agrobacterium tumefaciens Zymomonas mobilis able amino acid residues, may exhibit a few differences between two closely related species. Another, smaller protein may be identical in the same two species. For many reasons, some details of an evolutionary tree based on the sequences of one protein may differ from those of a tree based on the sequences of another protein. Increasingly sophisticated analyses using the sequences of many different proteins can provide an exquisitely detailed and accurate picture of evolutionary relationships. The story is a work in progress, and the questions being asked and answered are fundamental to how humans view themselves and the world around them. The field of molecular evolution promises to be among the most vibrant of the scientific frontiers in the twenty-first century. SUMMARY 3.5 Protein Sequences and Evolution ■ Protein sequences are a rich source of information about protein structure and function, as well as the evolution of life on this planet. Sophisticated methods are being developed to trace evolution by analyzing the resultant slow changes in the amino acid sequences of homologous proteins. Borrelia burgdorferi Leptospira interrogans Spirochaetes Thermophilic bacterium PS-3 Bacillus subtilis Staphylococcus aureus Clostridium acetobutylicum Clostridium perfringens low G + C Streptomyces coelicolor Mycobacterium leprae Mycobacterium tuberculosis Streptomyces albus [gene] high Cyanidium caldarium chl. Synechocystis Ricinus commun
is chl. Cyanobacteria and chloroplasts 0.1 substitutions/site Triticum aestivum chl. Brassica napus chl. Arabidopsis thaliana chl. FIGURE 3–33 Evolutionary tree derived from amino acid sequence comparisons. A bacterial evolutionary tree, based on the sequence divergence observed in the GroEL family of proteins. Also included in this tree (lower right) are the chloroplasts (chl.) of some nonbacterial species. 8885d_c03_111 1/16/04 6:08 AM Page 111 mac76 mac76:385_reb: Key Terms Chapter 3 Further Reading 111 75 Terms in bold are defined in the glossary. amino acids 76 R group chiral center enantiomers absolute 76 76 configuration 77 78 D, L system 77 polarity zwitterion absorbance, A 82 isoelectric pH (isoelec- 81 tric point, pI) 84 peptide 85 85 85 85 85 protein peptide bond oligopeptide polypeptide oligomeric protein protomer conjugated protein 87 88 prosthetic group primary structure secondary structure 88 tertiary structure quaternary structure crude extract fractionation 89 dialysis column 88 89 89 chromatography 89 high-performance liquid chromatography (HPLC) 90 92 electrophoresis sodium dodecyl sulfate 87 88 88 88 (SDS) 92 isoelectric focusing 93 99 101 Edman degradation proteases proteome lateral gene transfer homologous proteins 107 98 107 homolog paralog ortholog signature sequence 107 107 107 109 Further Reading Amino Acids Dougherty, D.A. (2000) Unnatural amino acids as probes of protein structure and function. Curr. Opin. Chem. Biol. 4, 645–652. Greenstein, J.P. & Winitz, M. (1961) Chemistry of the Amino Acids, 3 Vols, John Wiley & Sons, New York. Kreil, G. (1997) D-Amino acids in animal peptides. Annu. Rev. Biochem. 66, 337–345. An update on the occurrence of these unusual stereoisomers of amino acids. Meister, A. (1965) Biochemistry of the Amino Acids, 2nd edn, Vols 1 and 2, Academic Press, Inc., New York. Encyclopedic treatment of the properties, occurrence, and metabolism of amino acids
. Peptides and Proteins Creighton, T.E. (1992) Proteins: Structures and Molecular Properties, 2nd edn, W. H. Freeman and Company, New York. Very useful general source. Working with Proteins Dunn, M.J. & Corbett, J.M. (1996) Two-dimensional polyacrylamide gel electrophoresis. Methods Enzymol. 271, 177–203. A detailed description of the technology. Kornberg, A. (1990) Why purify enzymes? Methods Enzymol. 182, 1–5. The critical role of classical biochemical methods in a new age. Scopes, R.K. (1994) Protein Purification: Principles and Practice, 3rd edn, Springer-Verlag, New York. A good source for more complete descriptions of the principles underlying chromatography and other methods. Covalent Structure of Proteins Andersson, L., Blomberg, L., Flegel, M., Lepsa, L., Nilsson, B., & Verlander, M. (2000) Large-scale synthesis of peptides. Biopolymers 55, 227–250. A discussion of approaches used to manufacture peptides as pharmaceuticals. Dell, A. & Morris, H.R. (2001) Glycoprotein structure determination by mass spectrometry. Science 291, 2351–2356. Glycoproteins can be complex; mass spectrometry is a method of choice for sorting things out. Dongre, A.R., Eng, J.K., & Yates, J.R. III (1997) Emerging tandem-mass-spectrometry techniques for the rapid identification of proteins. Trends Biotechnol. 15, 418–425. A detailed description of mass spectrometry methods. Gygi, S.P. & Aebersold, R. (2000) Mass spectrometry and proteomics. Curr. Opin. Chem. Biol. 4, 489–494. Uses of mass spectrometry to identify and study cellular proteins. Koonin, E.V., Tatusov, R.L., & Galperin, M.Y. (1998) Beyond complete genomes: from sequence to structure and function. Curr. Opin. Struct. Biol. 8, 355–363. A good discussion about the possible uses of the tremendous amount of protein sequence information becoming available. Mann
, M. & Wilm, M. (1995) Electrospray mass spectrometry for protein characterization. Trends Biochem. Sci. 20, 219–224. An approachable summary of this technique for beginners. Mayo, K.H. (2000) Recent advances in the design and construction of synthetic peptides: for the love of basics or just for the technology of it. Trends Biotechnol. 18, 212–217. 8885d_c03_112 12/30/03 7:11 AM Page 112 mac76 mac76:385_reb: 112 Chapter 3 Amino Acids, Peptides, and Proteins Miranda, L.P. & Alewood, P.F. (2000) Challenges for protein chemical synthesis in the 21st century: bridging genomics and proteomics. Biopolymers 55, 217–226. This and the Mayo article describe how to make peptides and splice them together to address a wide range of problems in protein biochemistry. Sanger, F. (1988) Sequences, sequences, sequences. Annu. Rev. Biochem. 57, 1–28. A nice historical account of the development of sequencing methods. Protein Sequences and Evolution Gupta, R.S. (1998) Protein phylogenies and signal sequences: a reappraisal of evolutionary relationships among Archaebacteria, Eubacteria, and Eukaryotes. Microbiol. Mol. Biol. Rev. 62, 1435–1491. An almost encyclopedic but very readable report of how protein sequences are used to explore evolution, introducing many in- teresting ideas and supporting them with detailed sequence comparisons. Li, W.-H. & Graur, D. (2000) Fundamentals of Molecular Evolution, 2nd edn, Sinauer Associates, Inc., Sunderland, MA. A very readable text describing methods used to analyze protein and nucleic acid sequences. Chapter 5 provides one of the best available descriptions of how evolutionary trees are constructed from sequence data. Rokas, A., Williams, B.L., King, N., & Carroll, S.B. (2003) Genome-scale approaches to resolving incongruence in molecular phylogenies. Nature 425, 798–804. How sequence comparisons of multiple proteins can yield accurate evolutionary information. Zuckerkandl, E. & Pauling, L. (1965) Molecules as documents of evolutionary history. J. Theor.
Biol. 8, 357–366. Considered by many the founding paper in the field of molecular evolution. Problems 1. Absolute Configuration of Citrulline The citrulline isolated from watermelons has the structure shown below. Is it a D- or L-amino acid? Explain. C H (CH NH ) 2 2 2 H C N COO H3 NH2 C P O 2. Relationship between the Titration Curve and the Acid-Base Properties of Glycine A 100 mL solution of 0.1 M glycine at pH 1.72 was titrated with 2 M NaOH solution. The pH was monitored and the results were plotted on a graph, as shown at right. The key points in the titration are designated I to V. For each of the statements (a) to (o), identify the appropriate key point in the titration and justify your choice. (a) Glycine is present predominantly as the species H3NOCH2OCOOH. (b) The average net charge of glycine is 1 2. (c) Half of the amino groups are ionized. (d) The pH is equal to the pKa of the carboxyl group. (e) The pH is equal to the pKa of the protonated amino group. (f) Glycine has its maximum buffering capacity. (g) The average net charge of glycine is zero. (h) The carboxyl group has been completely titrated (first equivalence point). (i) Glycine is completely titrated (second equivalence point). (j) The predominant species is H3NOCH2OCOO. (k) The average net charge of glycine is 1. (l) Glycine is present predominantly as a 50:50 mixture of H3NOCH2OCOOH and H3NOCH2OCOO. (m) This is the isoelectric point. (n) This is the end of the titration. (o) These are the worst pH regions for buffering power. 12 10 11.30 9.60 (V) (IV) pH 8 6 4 2 0 5.97 (III) 2.34 (II) (I) 0.5 1.0 1.5 2.0 OH (equivalents) 3. How Much Alanine Is Present as the Completely Uncharged Species? At a pH equal to the isoelectric point of al
anine, the net charge on alanine is zero. Two structures can be drawn that have a net charge of zero, but the predominant form of alanine at its pI is zwitterionic. CH3 C C O H3N O H Zwitterionic CH3 O H2N C C OH H Uncharged (a) Why is alanine predominantly zwitterionic rather than completely uncharged at its pI? (b) What fraction of alanine is in the completely un- charged form at its pI? Justify your assumptions. 8885d_c03_113 1/16/04 6:09 AM Page 113 mac76 mac76:385_reb: 4. Ionization State of Amino Acids Each ionizable group of an amino acid can exist in one of two states, charged or neutral. The electric charge on the functional group is determined by the relationship between its pKa and the pH of the solution. This relationship is described by the HendersonHasselbalch equation. (a) Histidine has three ionizable functional groups. Write the equilibrium equations for its three ionizations and assign the proper pKa for each ionization. Draw the structure of histidine in each ionization state. What is the net charge on the histidine molecule in each ionization state? (b) Draw the structures of the predominant ionization state of histidine at pH 1, 4, 8, and 12. Note that the ionization state can be approximated by treating each ionizable group independently. (c) What is the net charge of histidine at pH 1, 4, 8, and 12? For each pH, will histidine migrate toward the anode () or cathode () when placed in an electric field? 5. Separation of Amino Acids by Ion-Exchange Chromatography Mixtures of amino acids are analyzed by first separating the mixture into its components through ionexchange chromatography. Amino acids placed on a cationexchange resin containing sulfonate groups (see Fig. 3–18a) flow down the column at different rates because of two factors that influence their movement: (1) ionic attraction be residues on the column and positively tween the OSO3 charged functional groups on the amino acids, and (2) hydrophobic interactions between amino acid side chains and the strongly hydrophobic backbone of the polystyrene resin. For each pair of amino acids listed, determine which will be eluted
first from an ion-exchange column using a pH 7.0 buffer. (a) Asp and Lys (b) Arg and Met (c) Glu and Val (d) Gly and Leu (e) Ser and Ala 6. Naming the Stereoisomers of Isoleucine The structure of the amino acid isoleucine is COO C H H H3N H C CH3 CH2 CH3 (a) How many chiral centers does it have? (b) How many optical isomers? (c) Draw perspective formulas for all the optical isomers of isoleucine. 7. Comparing the pKa Values of Alanine and Polyalanine The titration curve of alanine shows the ionization of two functional groups with pKa values of 2.34 and 9.69, corresponding to the ionization of the carboxyl and the protonated amino groups, respectively. The titration of di-, tri-, and larger oligopeptides of alanine also shows the ionization of only two functional groups, although the experimental pKa values are different. The trend in pKa values is summarized in the table. Chapter 3 Problems 113 Amino acid or peptide Ala Ala–Ala Ala–Ala–Ala Ala–(Ala)n–Ala, n 4 pK1 2.34 3.12 3.39 3.42 pK2 9.69 8.30 8.03 7.94 (a) Draw the structure of Ala–Ala–Ala. Identify the func- tional groups associated with pK1 and pK2. (b) Why does the value of pK1 increase with each addition of an Ala residue to the Ala oligopeptide? (c) Why does the value of pK2 decrease with each ad- dition of an Ala residue to the Ala oligopeptide? 8. The Size of Proteins What is the approximate molecular weight of a protein with 682 amino acid residues in a single polypeptide chain? 9. The Number of Tryptophan Residues in Bovine Serum Albumin A quantitative amino acid analysis reveals that bovine serum albumin (BSA) contains 0.58% tryptophan (Mr 204) by weight. (a) Calculate the minimum molecular weight of BSA (i.e., assuming there is only one tryptophan residue per
protein molecule). (b) Gel filtration of BSA gives a molecular weight estimate of 70,000. How many tryptophan residues are present in a molecule of serum albumin? 10. Net Electric Charge of Peptides A peptide has the sequence Glu–His–Trp–Ser–Gly–Leu–Arg–Pro–Gly (a) What is the net charge of the molecule at pH 3, 8, and 11? (Use pKa values for side chains and terminal amino and carboxyl groups as given in Table 3–1.) (b) Estimate the pI for this peptide. 11. Isoelectric Point of Pepsin Pepsin is the name given to several digestive enzymes secreted (as larger precursor proteins) by glands that line the stomach. These glands also secrete hydrochloric acid, which dissolves the particulate matter in food, allowing pepsin to enzymatically cleave individual protein molecules. The resulting mixture of food, HCl, and digestive enzymes is known as chyme and has a pH near 1.5. What pI would you predict for the pepsin proteins? What functional groups must be present to confer this pI on pepsin? Which amino acids in the proteins would contribute such groups? 12. The Isoelectric Point of Histones Histones are proteins found in eukaryotic cell nuclei, tightly bound to DNA, which has many phosphate groups. The pI of histones is very high, about 10.8. What amino acid residues must be present in relatively large numbers in histones? In what way do these residues contribute to the strong binding of histones to DNA? 13. Solubility of Polypeptides One method for separating polypeptides makes use of their differential solubilities. The solubility of large polypeptides in water depends upon the relative polarity of their R groups, particularly on the number of ionized groups: the more ionized groups there are, the more soluble the polypeptide. Which of each pair of the polypeptides that follow is more soluble at the indicated pH? 8885d_c03_114 12/23/03 10:29 AM Page 114 mac111 mac111:reb: 114 Chapter 3 Amino Acids, Peptides, and Proteins (a) (Gly)20 or (Glu)20 at pH 7.0 (b)
(Lys–Ala)3 or (Phe–Met)3 at pH 7.0 (c) (Ala–Ser–Gly)5 or (Asn–Ser–His)5 at pH 6.0 (d) (Ala–Asp–Gly)5 or (Asn–Ser–His)5 at pH 3.0 14. Purification of an Enzyme A biochemist discovers and purifies a new enzyme, generating the purification table below. Procedure 1. Crude extract 2. Precipitation (salt) 3. Precipitation (pH) 4. Ion-exchange chromatography 5. Affinity chromatography 6. Size-exclusion chromatography Total protein (mg) 20,000 5,000 4,000 200 50 45 Activity (units) 4,000,000 3,000,000 1,000,000 800,000 750,000 675,000 (a) From the information given in the table, calculate the specific activity of the enzyme solution after each purification procedure. (b) Which of the purification procedures used for this enzyme is most effective (i.e., gives the greatest relative increase in purity)? (c) Which of the purification procedures is least effective? (d) Is there any indication based on the results shown in the table that the enzyme after step 6 is now pure? What else could be done to estimate the purity of the enzyme preparation? 15. Sequence Determination of the Brain Peptide Leucine Enkephalin A group of peptides that influence nerve transmission in certain parts of the brain has been isolated from normal brain tissue. These peptides are known as opioids, because they bind to specific receptors that also bind opiate drugs, such as morphine and naloxone. Opioids thus mimic some of the properties of opiates. Some researchers consider these peptides to be the brain’s own pain killers. Using the information below, determine the amino acid sequence of the opioid leucine enkephalin. Explain how your structure is consistent with each piece of information. (a) Complete hydrolysis by 6 M HCl at 110 C followed by amino acid analysis indicated the presence of Gly, Leu, Phe, and Tyr, in a 2:1:1:1 molar ratio. (b) Treatment of the peptide with 1-fluoro-2,4-dinitrobenzene followed by
complete hydrolysis and chromatography indicated the presence of the 2,4-dinitrophenyl derivative of tyrosine. No free tyrosine could be found. (c) Complete digestion of the peptide with pepsin followed by chromatography yielded a dipeptide containing Phe and Leu, plus a tripeptide containing Tyr and Gly in a 1:2 ratio. 16. Structure of a Peptide Antibiotic from Bacillus brevis Extracts from the bacterium Bacillus brevis contain a peptide with antibiotic properties. This peptide forms complexes with metal ions and apparently disrupts ion transport across the cell membranes of other bacterial species, killing them. The structure of the peptide has been determined from the following observations. (a) Complete acid hydrolysis of the peptide followed by amino acid analysis yielded equimolar amounts of Leu, Orn, Phe, Pro, and Val. Orn is ornithine, an amino acid not present in proteins but present in some peptides. It has the structure H3N CH2 CH2 CH2 C COO H NH3 (b) The molecular weight of the peptide was estimated as about 1,200. (c) The peptide failed to undergo hydrolysis when treated with the enzyme carboxypeptidase. This enzyme catalyzes the hydrolysis of the carboxyl-terminal residue of a polypeptide unless the residue is Pro or, for some reason, does not contain a free carboxyl group. (d) Treatment of the intact peptide with 1-fluoro-2,4dinitrobenzene, followed by complete hydrolysis and chromatography, yielded only free amino acids and the following derivative: NO2 O2N NH CH2 CH2 CH2 H COO C NH3 (Hint: Note that the 2,4-dinitrophenyl derivative involves the amino group of a side chain rather than the -amino group.) (e) Partial hydrolysis of the peptide followed by chromatographic separation and sequence analysis yielded the following di- and tripeptides (the amino-terminal amino acid is always at the left): Leu–Phe Phe–Pro Orn–Leu Val–Orn Val–Orn–Leu Phe–Pro–Val Pro–Val–Orn Given the above information, deduce the amino acid sequence of the peptide
antibiotic. Show your reasoning. When you have arrived at a structure, demonstrate that it is consistent with each experimental observation. 17. Efficiency in Peptide Sequencing A peptide with the primary structure Lys–Arg–Pro–Leu–Ile–Asp–Gly–Ala is sequenced by the Edman procedure. If each Edman cycle is 96% efficient, what percentage of the amino acids liberated in the fourth cycle will be leucine? Do the calculation a second time, but assume a 99% efficiency for each cycle. 18. Biochemistry Protocols: Your First Protein Purification As the newest and least experienced student in a biochemistry research lab, your first few weeks are spent washing glassware and labeling test tubes. You then graduate to making buffers and stock solutions for use in various laboratory procedures. Finally, you are given responsibility for purifying a protein. It is a citric acid cycle enzyme, citrate synthase, located in the mitochondrial matrix. Following a protocol for the purification, you proceed through the steps below. As you work, a more experienced student questions you about the rationale for each procedure. Supply the answers. (Hint: See Chapter 2 for information about osmolarity; see p. 6 for information on separation of organelles from cells.) (a) You pick up 20 kg of beef hearts from a nearby slaughterhouse. You transport the hearts on ice, and perform 8885d_c03_115 1/16/04 6:09 AM Page 115 mac76 mac76:385_reb: each step of the purification on ice or in a walk-in cold room. You homogenize the beef heart tissue in a high-speed blender in a medium containing 0.2 M sucrose, buffered to a pH of 7.2. Why do you use beef heart tissue, and in such large quantity? What is the purpose of keeping the tissue cold and suspending it in 0.2 M sucrose, at pH 7.2? What happens to the tissue when it is homogenized? (b) You subject the resulting heart homogenate, which is dense and opaque, to a series of differential centrifugation steps. What does this accomplish? (c) You proceed with the purification using the supernatant fraction that contains mostly intact mitochondria. Next you osmotically lyse the mitochondria. The lysate, which is less dense than the homogenate, but still opaque,
consists primarily of mitochondrial membranes and internal mitochondrial contents. To this lysate you add ammonium sulfate, a highly soluble salt, to a specific concentration. You centrifuge the solution, decant the supernatant, and discard the pellet. To the supernatant, which is clearer than the lysate, you add more ammonium sulfate. Once again, you centrifuge the sample, but this time you save the pellet because it contains the protein of interest. What is the rationale for the two-step addition of the salt? (d) You solubilize the ammonium sulfate pellet containing the mitochondrial proteins and dialyze it overnight against large volumes of buffered (pH 7.2) solution. Why isn’t ammonium sulfate included in the dialysis buffer? Why do you use the buffer solution instead of water? Chapter 3 Problems 115 (e) You run the dialyzed solution over a size-exclusion chromatographic column. Following the protocol, you collect the first protein fraction that exits the column, and discard the rest of the fractions that elute from the column later. You detect the protein by measuring UV absorbance (at 280 nm) in the fractions. What does the instruction to collect the first fraction tell you about the protein? Why is UV absorbance at 280 nm a good way to monitor for the presence of protein in the eluted fractions? (f) You place the fraction collected in (e) on a cationexchange chromatographic column. After discarding the initial solution that exits the column (the flowthrough), you add a washing solution of higher pH to the column and collect the protein fraction that immediately elutes. Explain what you are doing. (g) You run a small sample of your fraction, now very reduced in volume and quite clear (though tinged pink), on an isoelectric focusing gel. When stained, the gel shows three sharp bands. According to the protocol, the protein of interest is the one with the pI of 5.6, but you decide to do one more assay of the protein’s purity. You cut out the pI 5.6 band and subject it to SDS polyacrylamide gel electrophoresis. The protein resolves as a single band. Why were you unconvinced of the purity of the “single” protein band on your isoelectric focusing gel? What did the results of the SDS gel tell
you? Why is it important to do the SDS gel electrophoresis after the isoelectric focusing? 8885d_c04_116 12/23/03 7:43 AM Page 116 mac111 mac111:reb: chapter 4 THE THREE-DIMENSIONAL STRUCTURE OF PROTEINS 4.1 Overview of Protein Structure 116 Protein Secondary Structure 120 4.2 Protein Tertiary and Quaternary Structures 125 4.3 Protein Denaturation and Folding 147 4.4 Perhaps the more remarkable features of [myoglobin] are its complexity and its lack of symmetry. The arrangement seems to be almost totally lacking in the kind of regularities which one instinctively anticipates, and it is more complicated than has been predicted by any theory of protein structure. —John Kendrew, article in Nature, 1958 The covalent backbone of a typical protein contains hundreds of individual bonds. Because free rotation is possible around many of these bonds, the protein can assume an unlimited number of conformations. However, each protein has a specific chemical or structural function, strongly suggesting that each has a unique three-dimensional structure (Fig. 4–1). By the late 1920s, several proteins had been crystallized, including hemoglobin (Mr 64,500) and the enzyme urease (Mr 483,000). Given that the ordered array of molecules in a crystal can generally form only if the molecular units are identical, the simple fact that many proteins can be crystallized provides strong evidence that even very large proteins are discrete chemical entities with unique structures. This conclusion revolutionized thinking about proteins and their functions. In this chapter, we explore the three-dimensional structure of proteins, emphasizing five themes. First, the three-dimensional structure of a protein is determined by its amino acid sequence. Second, the function 116 of a protein depends on its structure. Third, an isolated protein usually exists in one or a small number of stable structural forms. Fourth, the most important forces stabilizing the specific structures maintained by a given protein are noncovalent interactions. Finally, amid the huge number of unique protein structures, we can recognize some common structural patterns that help us organize our understanding of protein architecture. These themes should not be taken to imply that proteins have static, unchanging three-dimensional structures. Protein function often entails an interconversion between two or more structural forms. The dynamic aspects of protein structure will be explored in Chapters 5 and 6. The relationship between the amino acid sequence of a protein and
its three-dimensional structure is an intricate puzzle that is gradually yielding to techniques used in modern biochemistry. An understanding of structure, in turn, is essential to the discussion of function in succeeding chapters. We can find and understand the patterns within the biochemical labyrinth of protein structure by applying fundamental principles of chemistry and physics. 4.1 Overview of Protein Structure The spatial arrangement of atoms in a protein is called its conformation. The possible conformations of a protein include any structural state that can be achieved without breaking covalent bonds. A change in conformation could occur, for example, by rotation about single bonds. Of the numerous conformations that are theoretically possible in a protein containing hundreds of single bonds, one or (more commonly) a few generally predominate under biological conditions. The need for multiple stable conformations reflects the changes that must occur in most proteins as they bind to other 8885d_c04_117 12/23/03 7:43 AM Page 117 mac111 mac111:reb: 4.1 Overview of Protein Structure 117 can theoretically assume countless different conformations, and as a result the unfolded state of a protein is characterized by a high degree of conformational entropy. This entropy, and the hydrogen-bonding interactions of many groups in the polypeptide chain with solvent (water), tend to maintain the unfolded state. The chemical interactions that counteract these effects and stabilize the native conformation include disulfide bonds and the weak (noncovalent) interactions described in Chapter 2: hydrogen bonds, and hydrophobic and ionic interactions. An appreciation of the role of these weak interactions is especially important to our understanding of how polypeptide chains fold into specific secondary and tertiary structures, and how they combine with other polypeptides to form quaternary structures. About 200 to 460 kJ/mol are required to break a single covalent bond, whereas weak interactions can be disrupted by a mere 4 to 30 kJ/mol. Individual covalent bonds that contribute to the native conformations of proteins, such as disulfide bonds linking separate parts of a single polypeptide chain, are clearly much stronger than individual weak interactions. Yet, because they are so numerous, it is weak interactions that predominate as a stabilizing force in protein structure. In general, the protein conformation with the lowest free energy (that is, the most stable conformation) is the one with the maximum number of weak interactions. The stability of a protein is not simply the
sum of the free energies of formation of the many weak interactions within it. Every hydrogen-bonding group in a folded polypeptide chain was hydrogen-bonded to water prior to folding, and for every hydrogen bond formed in a protein, a hydrogen bond (of similar strength) between the same group and water was broken. The net stability contributed by a given weak interaction, or the difference in free energies of the folded and unfolded states, may be close to zero. We must therefore look elsewhere to explain why the native conformation of a protein is favored. We find that the contribution of weak interactions to protein stability can be understood in terms of the properties of water (Chapter 2). Pure water contains a network of hydrogen-bonded H2O molecules. No other molecule has the hydrogen-bonding potential of water, and other molecules present in an aqueous solution disrupt the hydrogen bonding of water. When water surrounds a hydrophobic molecule, the optimal arrangement of hydrogen bonds results in a highly structured shell, or solvation layer, of water in the immediate vicinity. The increased order of the water molecules in the solvation layer correlates with an unfavorable decrease in the entropy of the water. However, when nonpolar groups are clustered together, there is a decrease in the extent of the solvation layer because each group no longer presents its entire surface to the solution. The result is a favorable increase in entropy. As described in FIGURE 4–1 Structure of the enzyme chymotrypsin, a globular protein. Proteins are large molecules and, as we shall see, each has a unique structure. A molecule of glycine (blue) is shown for size comparison. The known three-dimensional structures of proteins are archived in the Protein Data Bank, or PDB (www.rcsb.org/pdb). Each structure is assigned a unique four-character identifier, or PDB ID. Where appropriate, we will provide the PDB IDs for molecular graphic images in the figure captions. The image shown here was made using data from the PDB file 6GCH. The data from the PDB files provide only a series of coordinates detailing the location of atoms and their connectivity. Viewing the images requires easy-to-use graphics programs such as RasMol and Chime that convert the coordinates into an image and allow the viewer to manipulate the structure in three dimensions. You will find instructions for downloading Chime with the structure tutorials on the textbook website (www.
whfreeman. com/lehninger). The PDB website has instructions for downloading other viewers. We encourage all students to take advantage of the resources of the PDB and the free molecular graphics programs. molecules or catalyze reactions. The conformations existing under a given set of conditions are usually the ones that are thermodynamically the most stable, having the lowest Gibbs free energy (G). Proteins in any of their functional, folded conformations are called native proteins. What principles determine the most stable conformations of a protein? An understanding of protein conformation can be built stepwise from the discussion of primary structure in Chapter 3 through a consideration of secondary, tertiary, and quaternary structures. To this traditional approach must be added a new emphasis on supersecondary structures, a growing set of known and classifiable protein folding patterns that provides an important organizational context to this complex endeavor. We begin by introducing some guiding principles. A Protein’s Conformation Is Stabilized Largely by Weak Interactions In the context of protein structure, the term stability can be defined as the tendency to maintain a native conformation. Native proteins are only marginally stable; the G separating the folded and unfolded states in typical proteins under physiological conditions is in the range of only 20 to 65 kJ/mol. A given polypeptide chain 8885d_c04_118 12/23/03 7:43 AM Page 118 mac111 mac111:reb: 118 Chapter 4 The Three-Dimensional Structure of Proteins Chapter 2, this entropy term is the major thermodynamic driving force for the association of hydrophobic groups in aqueous solution. Hydrophobic amino acid side chains therefore tend to be clustered in a protein’s interior, away from water. Under physiological conditions, the formation of hydrogen bonds and ionic interactions in a protein is driven largely by this same entropic effect. Polar groups can generally form hydrogen bonds with water and hence are soluble in water. However, the number of hydrogen bonds per unit mass is generally greater for pure water than for any other liquid or solution, and there are limits to the solubility of even the most polar molecules as their presence causes a net decrease in hydrogen bonding per unit mass. Therefore, a solvation shell of structured water will also form to some extent around polar molecules. Even though the energy of formation of an intramolecular hydrogen bond or ionic interaction between two polar groups in a macromolecule is largely canceled out by the
elimination of such interactions between the same groups and water, the release of structured water when the intramolecular interaction is formed provides an entropic driving force for folding. Most of the net change in free energy that occurs when weak interactions are formed within a protein is therefore derived from the increased entropy in the surrounding aqueous solution resulting from the burial of hydrophobic surfaces. This more than counterbalances the large loss of conformational entropy as a polypeptide is constrained into a single folded conformation. Hydrophobic interactions are clearly important in stabilizing a protein conformation; the interior of a protein is generally a densely packed core of hydrophobic amino acid side chains. It is also important that any polar or charged groups in the protein interior have suitable partners for hydrogen bonding or ionic interactions. One hydrogen bond seems to contribute little to the stability of a native structure, but the presence of hydrogen-bonding or charged groups without partners in the hydrophobic core of a protein can be so destabilizing that conformations containing these groups are often thermodynamically untenable. The favorable freeenergy change realized by combining such a group with a partner in the surrounding solution can be greater than the difference in free energy between the folded and unfolded states. In addition, hydrogen bonds between groups in proteins form cooperatively. Formation of one hydrogen bond facilitates the formation of additional hydrogen bonds. The overall contribution of hydrogen bonds and other noncovalent interactions to the stabilization of protein conformation is still being evaluated. The interaction of oppositely charged groups that form an ion pair (salt bridge) may also have a stabilizing effect on one or more native conformations of some proteins. Most of the structural patterns outlined in this chapter reflect two simple rules: (1) hydrophobic residues are largely buried in the protein interior, away from water; and (2) the number of hydrogen bonds within the protein is maximized. Insoluble proteins and proteins within membranes (which we examine in Chapter 11) follow somewhat different rules because of their function or their environment, but weak interactions are still critical structural elements. The Peptide Bond Is Rigid and Planar Protein Architecture—Primary Structure Covalent bonds also place important constraints on the conformation of a polypeptide. In the late 1930s, Linus Pauling and Robert Corey embarked on a series of studies that laid the foundation for our present understanding of protein structure. They began with a careful analysis of the peptide bond. The carbons of adjacent amino
acid residues are separated by three covalent bonds, arranged as COCONOC. X-ray diffraction studies of crystals of amino acids and of simple dipeptides and tripeptides demonstrated that the peptide CON bond is somewhat shorter than the CON bond in a simple amine and that the atoms associated with the peptide bond are coplanar. This indicated a resonance or partial sharing of two pairs of electrons between the carbonyl oxygen and the amide nitrogen (Fig. 4–2a). The oxygen has a partial negative charge and the nitrogen a partial positive charge, setting up a small electric dipole. The six atoms of the peptide group lie in a single plane, with the oxygen atom of the carbonyl group and the hydrogen atom of the amide nitrogen trans to each other. From these findings Pauling and Corey concluded that the peptide CON bonds are unable to rotate freely because of their partial double-bond character. Rotation is permitted about the NOC and the COC bonds. The backbone of a polypeptide chain can thus be pictured as a series of rigid planes with consecutive planes sharing a common point of rotation at C (Fig. 4–2b). The rigid peptide bonds limit the range of conformations that can be assumed by a polypeptide chain. By convention, the bond angles resulting from rotations at C are labeled (phi) for the NOC bond and (psi) for the COC bond. Again by convention, both and are defined as 180 when the polypeptide is in its fully extended conformation and all peptide groups are in the same plane (Fig. 4–2b). In principle, and can have any value between 180 and 180, but many values are prohibited by steric interference between atoms in the polypeptide backbone and amino acid side chains. The conformation in which both and are 0 (Fig. 4–2c) is prohibited for this reason; this conformation is used merely as a reference point for describing the angles of rotation. Allowed values for and are graphically revealed when is plotted versus in a Ramachandran plot (Fig. 4–3), introduced by G. N. Ramachandran. 8885d_c04_119 12/30/03 2:13 PM Page 119 mac76 mac76:385_reb: 4.1 Overview of Protein Structure 119 The carbonyl oxygen has a partial negative charge and the amide nitrogen
a partial positive charge, setting up a small electric dipole. Virtually all peptide bonds in proteins occur in this trans configuration; an exception is noted in Figure 4–8b. (a) (b) O 1.24 Å 1.46 Å 1.53 Å C Ca 1.32 Å N Amino terminus H R Ca f w N–Ca Ca–C C–N w f f w Carboxyl terminus FIGURE 4–2 The planar peptide group. (a) Each peptide bond has some double-bond character due to resonance and cannot rotate. (b) Three bonds separate sequential carbons in a polypeptide chain. The NOC and COC bonds can rotate, with bond angles designated and, respectively. The peptide CON bond is not free to rotate. Other single bonds in the backbone may also be rotationally hindered, depending on the size and charge of the R groups. In the conformation shown, and are 180 (or 180). As one looks out from the carbon, the and angles increase as the carbonyl or amide nitrogens (respectively) rotate clockwise. (c) By convention, both and are defined as 0 when the two peptide bonds flanking that carbon are in the same plane and positioned as shown. In a protein, this conformation is prohibited by steric overlap between an -carbonyl oxygen and an -amino hydrogen atom. To illustrate the bonds between atoms, the balls representing each atom are smaller than the van der Waals radii for this scale. 1 Å 0.1 nm. FIGURE 4–3 Ramachandran plot for L-Ala residues. The conformations of peptides are defined by the values of and. Conformations deemed possible are those that involve little or no steric interference, based on calculations using known van der Waals radii and bond angles. The areas shaded dark blue reflect conformations that involve no steric overlap and thus are fully allowed; medium blue indicates conformations allowed at the extreme limits for unfavorable atomic contacts; the lightest blue area reflects conformations that are permissible if a little flexibility is allowed in the bond angles. The asymmetry of the plot results from the L stereochemistry of the amino acid residues. The plots for other L-amino acid residues with unbranched side chains are nearly identical. The allowed ranges for branched amino acid residues such as Val, Ile
, and Thr are somewhat smaller than for Ala. The Gly residue, which is less sterically hindered, exhibits a much broader range of allowed conformations. The range for Pro residues is greatly restricted because is limited by the cyclic side chain to the range of 35 to 85. Ca O N Ca C O C w N H Ca f H R (c 180 120 60 0 60 120 180 180 0 f (degrees) 180 8885d_c04_120 12/23/03 7:44 AM Page 120 mac111 mac111:reb: 120 Chapter 4 The Three-Dimensional Structure of Proteins ing polar chemical groups such as the CPO and NOH groups of the peptide bond. They also had the experimental results of William Astbury, who in the 1930s had conducted pioneering x-ray studies of proteins. Astbury demonstrated that the protein that makes up hair and porcupine quills (the fibrous protein -keratin) has a regular structure that repeats every 5.15 to 5.2 Å. (The angstrom, Å, named after the physicist Anders J. Ångström, is equal to 0.1 nm. Although not an SI unit, it is used universally by structural biologists to describe atomic distances.) With this information and their data on the peptide bond, and with the help of precisely constructed models, Pauling and Corey set out to determine the likely conformations of protein molecules. The simplest arrangement the polypeptide chain could assume with its rigid peptide bonds (but other single bonds free to rotate) is a helical structure, which Pauling and Corey called the helix (Fig. 4–4). In this structure the polypeptide backbone is tightly wound around an imaginary axis drawn longitudinally through the middle of the helix, and the R groups of the amino acid residues protrude outward from the helical backbone. The repeating unit is a single turn of the helix, which extends about 5.4 Å along the long axis, slightly greater than the periodicity Astbury observed on x-ray analysis of hair keratin. The amino acid residues in an helix have conformations with 45 to 50 and 60, and each helical turn includes 3.6 amino acid residues. The helical twist of the helix found in all proteins is right-handed (Box 4–1). The helix proved to be the predominant structure in -keratins. More generally, about one-fourth
of all amino acid residues in polypeptides are found in helices, the exact fraction varying greatly from one protein to the next. Why does the helix form more readily than many other possible conformations? The answer is, in part, that an helix makes optimal use of internal hydrogen bonds. The structure is stabilized by a hydrogen bond between the hydrogen atom attached to the electronegative nitrogen atom of a peptide linkage and the electronegative carbonyl oxygen atom of the fourth amino acid on the amino-terminal side of that peptide bond (Fig. 4–4b). Within the helix, every peptide bond (except those close to each end of the helix) participates in such hydrogen bonding. Each successive turn of the helix is held to adjacent turns by three to four hydrogen bonds. All the hydrogen bonds combined give the entire helical structure considerable stability. Further model-building experiments have shown that an helix can form in polypeptides consisting of either L- or D-amino acids. However, all residues must be of one stereoisomeric series; a D-amino acid will disrupt a regular structure consisting of L-amino acids, and vice versa. Naturally occurring L-amino acids can form either right- or left-handed helices, but extended lefthanded helices have not been observed in proteins. Linus Pauling, 1901–1994 Robert Corey, 1897–1971 SUMMARY 4.1 Overview of Protein Structure ■ Every protein has a three-dimensional structure that reflects its function. ■ Protein structure is stabilized by multiple weak interactions. Hydrophobic interactions are the major contributors to stabilizing the globular form of most soluble proteins; hydrogen bonds and ionic interactions are optimized in the specific structures that are thermodynamically most stable. ■ The nature of the covalent bonds in the polypeptide backbone places constraints on structure. The peptide bond has a partial doublebond character that keeps the entire six-atom peptide group in a rigid planar configuration. The NOC and COC bonds can rotate to assume bond angles of and, respectively. 4.2 Protein Secondary Structure The term secondary structure refers to the local conformation of some part of a polypeptide. The discussion of secondary structure most usefully focuses on common regular folding patterns of the polypeptide backbone. A few types of secondary structure are particularly stable and occur widely in proteins. The most prominent are the helix and
conformations described below. Using fundamental chemical principles and a few experimental observations, Pauling and Corey predicted the existence of these secondary structures in 1951, several years before the first complete protein structure was elucidated. The Helix Is a Common Protein Secondary Structure Protein Architecture— Helix Pauling and Corey were aware of the importance of hydrogen bonds in orient- 8885d_c04_121 12/23/03 7:44 AM Page 121 mac111 mac111:reb: 4.2 Protein Secondary Structure 121 Amino terminus Carbon Hydrogen Oxygen Nitrogen R group 5.4 Å (3.6 residues) Carboxyl terminus (a) (b) (c) (d) FIGURE 4–4 Four models of the helix, showing different aspects of its structure. (a) Formation of a right-handed helix. The planes of the rigid peptide bonds are parallel to the long axis of the helix, depicted here as a vertical rod. (b) Ball-and-stick model of a righthanded helix, showing the intrachain hydrogen bonds. The repeat unit is a single turn of the helix, 3.6 residues. (c) The helix as viewed from one end, looking down the longitudinal axis (derived from PDB ID 4TNC). Note the positions of the R groups, represented by purple spheres. This ball-and-stick model, used to emphasize the helical arrangement, gives the false impression that the helix is hollow, because the balls do not represent the van der Waals radii of the individual atoms. As the space-filling model (d) shows, the atoms in the center of the helix are in very close contact. Amino Acid Sequence Affects Helix Stability Not all polypeptides can form a stable helix. Interactions between amino acid side chains can stabilize or destabilize this structure. For example, if a polypeptide chain has a long block of Glu residues, this segment of the chain will not form an helix at pH 7.0. The negatively charged carboxyl groups of adjacent Glu residues repel each other so strongly that they prevent formation of the helix. For the same reason, if there are many adjacent Lys and/or Arg residues, which have positively charged R groups at pH 7.0, they will also repel each other and prevent formation of the helix. The bulk and shape of Asn, Ser, Thr,
and Cys residues can also destabilize an helix if they are close together in the chain. The twist of an helix ensures that critical interactions occur between an amino acid side chain and the side chain three (and sometimes four) residues away on either side of it (Fig. 4–5). Positively charged amino acids are often found three residues away from negatively charged amino acids, permitting the formation of an ion pair. Two aromatic amino acid residues are often similarly spaced, resulting in a hydrophobic interaction. FIGURE 4–5 Interactions between R groups of amino acids three residues apart in an helix. An ionic interaction between Asp100 and Arg103 in an -helical region of the protein troponin C, a calciumbinding protein associated with muscle, is shown in this space-filling model (derived from PDB ID 4TNC). The polypeptide backbone (carbons, -amino nitrogens, and -carbonyl oxygens) is shown in gray for a helix segment 13 residues long. The only side chains represented here are the interacting Asp (red) and Arg (blue) side chains. 8885d_c04_122 12/23/03 7:44 AM Page 122 mac111 mac111:reb: 122 Chapter 4 The Three-Dimensional Structure of Proteins BOX 4–1 WORKING IN BIOCHEMISTRY Knowing the Right Hand from the Left There is a simple method for determining whether a helical structure is right-handed or left-handed. Make fists of your two hands with thumbs outstretched and pointing straight up. Looking at your right hand, think of a helix spiraling up your right thumb in the direction in which the other four fingers are curled as shown (counterclockwise). The resulting helix is right-handed. Your left hand will demonstrate a lefthanded helix, which rotates in the clockwise direction as it spirals up your thumb. A constraint on the formation of the helix is the presence of Pro or Gly residues. In proline, the nitrogen atom is part of a rigid ring (see Fig. 4–8b), and rotation about the NOC bond is not possible. Thus, a Pro residue introduces a destabilizing kink in an helix. In addition, the nitrogen atom of a Pro residue in peptide linkage has no substituent hydrogen to participate in hydrogen bonds with other residues. For these reasons, proline is only
rarely found within an helix. Glycine occurs infrequently in helices for a different reason: it has more conformational flexibility than the other amino acid residues. Polymers of glycine tend to take up coiled structures quite different from an helix. A final factor affecting the stability of an helix in a polypeptide is the identity of the amino acid residues near the ends of the -helical segment. A small electric dipole exists in each peptide bond (Fig. 4–2a). These dipoles are connected through the hydrogen bonds of the helix, resulting in a net dipole extending along the helix that increases with helix length (Fig. 4–6). The four amino acid residues at each end of the helix do not participate fully in the helix hydrogen bonds. The partial positive and negative charges of the helix dipole actually reside on the peptide amino and carbonyl groups near the amino-terminal and carboxyl-terminal ends of the helix, respectively. For this reason, negatively charged amino acids are often found near the amino terminus of the helical segment, where they have a stabilizing interaction with the positive charge of the helix dipole; a positively charged amino acid at the aminoterminal end is destabilizing. The opposite is true at the carboxyl-terminal end of the helical segment. Thus, five different kinds of constraints affect the stability of an helix: (1) the electrostatic repulsion (or attraction) between successive amino acid residues with charged R groups, (2) the bulkiness of adjacent R groups, (3) the interactions between R groups spaced three (or four) residues apart, (4) the occurrence of Pro and Gly residues, and (5) the interaction between amino acid residues at the ends of the helical segment and the electric dipole inherent to the helix. The tendency of a given segment of a polypeptide chain to fold up as an helix therefore depends on the identity and sequence of amino acid residues within the segment. Amino terminus d– Carboxyl terminus FIGURE 4–6 Helix dipole. The electric dipole of a peptide bond (see Fig. 4–2a) is transmitted along an -helical segment through the intrachain hydrogen bonds, resulting in an overall helix dipole. In this illustration, the amino and carbonyl constituents of each peptide bond are indicated by
and symbols, respectively. Non-hydrogenbonded amino and carbonyl constituents in the peptide bonds near each end of the -helical region are shown in red. 8885d_c04_123 12/23/03 7:45 AM Page 123 mac111 mac111:reb: The Conformation Organizes Polypeptide Chains into Sheets (a) Antiparallel 4.2 Protein Secondary Structure 123 Protein Architecture— Sheet Pauling and Corey predicted a second type of repetitive structure, the conformation. This is a more extended conformation of polypeptide chains, and its structure has been confirmed by x-ray analysis. In the conformation, the backbone of the polypeptide chain is extended into a zigzag rather than helical structure (Fig. 4–7). The zigzag polypeptide chains can be arranged side by side to form a structure resembling a series of pleats. In this arrangement, called a sheet, hydrogen bonds are formed between adjacent segments of polypeptide chain. The individual segments that form a sheet are usually nearby on the polypeptide chain, but can also be quite distant from each other in the linear sequence of the polypeptide; they may even be segments in different polypeptide chains. The R groups of adjacent amino acids protrude from the zigzag structure in opposite directions, creating the alternating pattern seen in the side views in Figure 4–7. The adjacent polypeptide chains in a sheet can be either parallel or antiparallel (having the same or opposite amino-to-carboxyl orientations, respectively). The structures are somewhat similar, although the repeat period is shorter for the parallel conformation (6.5 Å, versus 7 Å for antiparallel) and the hydrogenbonding patterns are different. Some protein structures limit the kinds of amino acids that can occur in the sheet. When two or more sheets are layered close together within a protein, the R groups of the amino acid residues on the touching surfaces must be relatively small. -Keratins such as silk fibroin and the fibroin of spider webs have a very high content of Gly and Ala residues, the two amino acids with the smallest R groups. Indeed, in silk fibroin Gly and Ala alternate over large parts of the sequence. Turns Are Common in Proteins Protein Architecture— Turn In globular proteins, which have a compact folded structure, nearly one-third
of the amino acid residues are in turns or loops where the polypeptide chain reverses direction (Fig. 4–8). These are the connecting elements that link successive runs of helix or conformation. Particularly common are turns that connect the ends of two adjacent segments of an antiparallel sheet. The structure is a 180 turn involving four amino acid residues, with the carbonyl oxygen of the first residue forming a hydrogen bond with the amino-group hydrogen of the fourth. The peptide groups of the central two residues do not participate in any interresidue hydrogen bonding. Gly and Pro residues often occur in turns, the former because it is small and flexible, the latter because peptide bonds Top view Side view (b) Parallel Top view Side view FIGURE 4–7 The conformation of polypeptide chains. These top and side views reveal the R groups extending out from the sheet and emphasize the pleated shape described by the planes of the peptide bonds. (An alternative name for this structure is -pleated sheet.) Hydrogen-bond cross-links between adjacent chains are also shown. (a) Antiparallel sheet, in which the amino-terminal to carboxylterminal orientation of adjacent chains (arrows) is inverse. (b) Parallel sheet. involving the imino nitrogen of proline readily assume the cis configuration (Fig. 4–8b), a form that is particularly amenable to a tight turn. Of the several types of turns, the two shown in Figure 4–8a are the most common. Beta turns are often found near the surface of a protein, where the peptide groups of the central two amino acid residues in the turn can hydrogen-bond with water. Considerably less common is the turn, a threeresidue turn with a hydrogen bond between the first and third residues. 8885d_c04_124 12/23/03 7:45 AM Page 124 mac111 mac111:reb: 124 Chapter 4 The Three-Dimensional Structure of Proteins (a) b Turns 3 R Cα 4 R Cα 3 Glycine 4 R Cα 2 Cα 1 Type I 2 1 Type II FIGURE 4–8 Structures of turns. (a) Type I and type II turns are most common; type I turns occur more than twice as frequently as type II. Type II turns always have Gly as the third residue. Note the hydrogen bond between the peptide groups of the first and
fourth residues of the bends. (Individual amino acid residues are framed by large blue circles.) (b) The trans and cis isomers of a peptide bond involving the imino nitrogen of proline. Of the peptide bonds between amino acid residues other than Pro, over 99.95% are in the trans configuration. For peptide bonds involving the imino nitrogen of proline, however, about 6% are in the cis configuration; many of these occur at turns. (b) Proline isomers trans cis Common Secondary Structures Have Characteristic Bond Angles and Amino Acid Content The helix and the conformation are the major repetitive secondary structures in a wide variety of proteins, although other repetitive structures do exist in some specialized proteins (an example is collagen; see Fig. 4–13 on page 128). Every type of secondary structure can be completely described by the bond angles and at each residue. As shown by a Ramachandran plot, the helix and conformation fall within a relatively restricted range of sterically allowed structures (Fig. 4–9a). Most values of and taken from known protein structures fall into the expected regions, with high concentrations near the helix and conformation values as predicted (Fig. 4–9b). The only amino acid residue often found in a conformation outside these regions is glycine. Because its side chain, a single hydrogen atom, is small, a Gly residue can take part in many conformations that are sterically forbidden for other amino acids. Some amino acids are accommodated better than others in the different types of secondary structures. An overall summary is presented in Figure 4–10. Some biases, such as the common presence of Pro and Gly residues in turns and their relative absence in helices, are readily explained by the known constraints on the different secondary structures. Other evident biases may be explained by taking into account the sizes or charges of side chains, but not all the trends shown in Figure 4–10 are understood. SUMMARY 4.2 Protein Secondary Structure ■ Secondary structure is the regular arrangement of amino acid residues in a segment of a polypeptide chain, in which each residue is spatially related to its neighbors in the same way. ■ The most common secondary structures are the helix, the conformation, and turns. ■ The secondary structure of a polypeptide segment can be completely defined if the and angles are known for all amino acid residues in that segment. 8885d_c04_125 12/23/03 7
:46 AM Page 125 mac111 mac111:reb: 4.3 Protein Tertiary and Quaternary Structures 125 Antiparallel b sheets Parallel b sheets Collagen triple helix Right-twisted b sheets 180 120 60 0 60 120 ) s e e r g e d ( w Left-handed a helix Right-handed a helix 180 180 (a) 0 f (degrees) 180 180 120 60 0 60 120 180 180 ) b) 0 f (degrees) 180 FIGURE 4–9 Ramachandran plots for a variety of structures. (a) The values of and for various allowed secondary structures are overlaid on the plot from Figure 4–3. Although left-handed helices extending over several amino acid residues are theoretically possible, they have not been observed in proteins. (b) The values of and for all the amino acid residues except Gly in the enzyme pyruvate kinase (isolated from rabbit) are overlaid on the plot of theoretically allowed conformations (Fig. 4–3). The small, flexible Gly residues were excluded because they frequently fall outside the expected ranges (blue). a Helix b Conformation b Turn Glu Met Ala Leu Lys Phe Gln Trp Ile Val Asp His Arg Thr Ser Cys Asn Tyr Pro Gly FIGURE 4–10 Relative probabilities that a given amino acid will occur in the three common types of secondary structure. 4.3 Protein Tertiary and Quaternary Structures Protein Architecture—Introduction to Tertiary Structure The overall three-dimensional arrangement of all atoms in a protein is referred to as the protein’s tertiary structure. Whereas the term “secondary structure” refers to the spatial arrangement of amino acid residues that are adjacent in the primary structure, tertiary structure includes longer-range aspects of amino acid sequence. Amino acids that are far apart in the polypeptide sequence and that reside in different types of secondary structure may interact within the completely folded structure of a protein. The location of bends (including turns) in the polypeptide chain and the direction and angle of these bends are determined by the number and location of specific bend-producing residues, such as Pro, Thr, Ser, and Gly. Interacting segments of polypeptide chains are held in their characteristic tertiary positions by different kinds of weak bonding interactions (and sometimes by covalent bonds such as disulfide cross-links) between the segments. Some proteins
contain two or more separate polypeptide chains, or subunits, which may be identical or different. The arrangement of these protein subunits in three-dimensional complexes constitutes quaternary structure. In considering these higher levels of structure, it is useful to classify proteins into two major groups: fibrous proteins, having polypeptide chains arranged in long strands or sheets, and globular proteins, having polypeptide chains folded into a spherical or globular shape. The two groups are structurally distinct: fibrous proteins usually consist largely of a single type of secondary structure; globular proteins often contain several types of secondary structure. The two groups differ functionally in that the structures that provide support, shape, and external protection to vertebrates are made of fibrous proteins, whereas most enzymes and regulatory proteins are globular proteins. Certain fibrous proteins played a key role in the development of our modern understanding of protein structure and provide particularly clear examples of the relationship between structure and function. We begin our discussion with fibrous proteins, before turning to the more complex folding patterns observed in globular proteins. 8885d_c04_126 12/23/03 7:46 AM Page 126 mac111 mac111:reb: 126 Chapter 4 The Three-Dimensional Structure of Proteins Fibrous Proteins Are Adapted for a Structural Function Protein Architecture—Tertiary Structure of Fibrous Proteins -Keratin, collagen, and silk fibroin nicely illustrate the relationship between protein structure and biological function (Table 4–1). Fibrous proteins share properties that give strength and/or flexibility to the structures in which they occur. In each case, the fundamental structural unit is a simple repeating element of secondary structure. All fibrous proteins are insoluble in water, a property conferred by a high concentration of hydrophobic amino acid residues both in the interior of the protein and on its surface. These hydrophobic surfaces are largely buried by packing many similar polypeptide chains together to form elaborate supramolecular complexes. The underlying structural simplicity of fibrous proteins makes them particularly useful for illustrating some of the fundamental principles of protein structure discussed above. -Keratin The -keratins have evolved for strength. Found in mammals, these proteins constitute almost the entire dry weight of hair, wool, nails, claws, quills, horns, hooves, and much of the outer layer of skin. The -keratins are part of a broader family of proteins called intermediate filament (IF)
proteins. Other IF proteins are found in the cytoskeletons of animal cells. All IF proteins have a structural function and share structural features exemplified by the -keratins. The -keratin helix is a right-handed helix, the same helix found in many other proteins. Francis Crick and Linus Pauling in the early 1950s independently suggested that the helices of keratin were arranged as a coiled coil. Two strands of -keratin, oriented in parallel (with their amino termini at the same end), are wrapped about each other to form a supertwisted coiled coil. The supertwisting amplifies the strength of the overall structure, just as strands are twisted to make a strong rope (Fig. 4–11). The twisting of the axis of an helix to form a coiled coil explains the discrepancy between the 5.4 Å per turn predicted for an helix by Pauling and Corey and the 5.15 to 5.2 Å repeating structure observed in the x-ray diffraction of hair (p. 120). The helical path of the supertwists is left-handed, opposite in sense to the helix. The surfaces where the two helices touch are made up of hydrophobic amino acid residues, their R groups meshed together in a regular interlocking pattern. This permits a close packing of the polypeptide chains within the left-handed supertwist. Not surprisingly, -keratin is rich in the hydrophobic residues Ala, Val, Leu, Ile, Met, and Phe. An individual polypeptide in the -keratin coiled coil has a relatively simple tertiary structure, dominated by an -helical secondary structure with its helical axis twisted in a left-handed superhelix. The intertwining of the two -helical polypeptides is an example of quaternary structure. Coiled coils of this type are common structural elements in filamentous proteins and in the muscle protein myosin (see Fig. 5–29). The quaternary structure of -keratin can be quite complex. Many coiled coils can be assembled into large supramolecular complexes, such as the arrangement of -keratin to form the intermediate filament of hair (Fig. 4–11b). Keratin a helix Two-chain coiled coil Protofilament 20–30 Å Cells Intermediate filament Protofibril Protofilament Protofibril (
a) FIGURE 4–11 Structure of hair. (a) Hair -keratin is an elongated helix with somewhat thicker elements near the amino and carboxyl termini. Pairs of these helices are interwound in a left-handed sense to form two-chain coiled coils. These then combine in higher-order structures called protofilaments and protofibrils. About four protofibrils—32 strands of -keratin altogether—combine to form an intermediate filament. The individual two-chain coiled coils in the various substructures also appear to be interwound, but the handedness of the interwinding and other structural details are unknown. (b) A hair is an array of many -keratin filaments, made up of the substructures shown in (a). Two-chain coiled coil Helix (b) Cross section of a hair 8885d_c04_127 1/16/04 6:13 AM Page 127 mac76 mac76:385_reb: 4.3 Protein Tertiary and Quaternary Structures 127 TABLE 4–1 Secondary Structures and Properties of Fibrous Proteins Structure Characteristics Examples of occurrence Helix, cross-linked by disulfide bonds Conformation Collagen triple helix Tough, insoluble protective structures of varying hardness and flexibility -Keratin of hair, feathers, and nails Soft, flexible filaments Silk fibroin High tensile strength, without stretch Collagen of tendons, bone matrix The strength of fibrous proteins is enhanced by covalent cross-links between polypeptide chains within the multihelical “ropes” and between adjacent chains in a supramolecular assembly. In -keratins, the cross-links stabilizing quaternary structure are disulfide bonds (Box 4–2). In the hardest and toughest -keratins, such as those of rhinoceros horn, up to 18% of the residues are cysteines involved in disulfide bonds. Collagen Like the -keratins, collagen has evolved to provide strength. It is found in connective tissue such as tendons, cartilage, the organic matrix of bone, and the cornea of the eye. The collagen helix is a unique secondary structure quite distinct from the helix. It is left-handed and has three amino acid residues per turn (Fig. 4–12). Collagen is also a coiled
coil, but one with distinct tertiary and quaternary structures: three separate polypeptides, called chains (not to be confused with helices), are supertwisted about each other (Fig. 4–12c). The superhelical twisting is right-handed in collagen, opposite in sense to the left-handed helix of the chains. There are many types of vertebrate collagen. Typically they contain about 35% Gly, 11% Ala, and 21% Pro and 4-Hyp (4-hydroxyproline, an uncommon amino acid; see Fig. 3–8a). The food product gelatin is derived BOX 4–2 THE WORLD OF BIOCHEMISTRY Permanent Waving Is Biochemical Engineering When hair is exposed to moist heat, it can be stretched. At the molecular level, the helices in the -keratin of hair are stretched out until they arrive at the fully extended conformation. On cooling they spontaneously revert to the -helical conformation. The characteristic “stretchability” of -keratins, and their numerous disulfide cross-linkages, are the basis of permanent waving. The hair to be waved or curled is first bent around a form of appropriate shape. A solution of a reducing agent, usually a compound containing a thiol or sulfhydryl group (OSH), is then applied with heat. The reducing agent cleaves the cross-linkages by reducing each disulfide bond to form two Cys residues. The moist heat breaks hydrogen bonds and causes the -helical structure of the polypeptide chains to uncoil. After a time the reducing solution is removed, and an oxidizing agent is added to establish new disulfide bonds between pairs of Cys residues of adjacent polypeptide chains, but not the same pairs as before the treatment. After the hair is washed and cooled, the polypeptide chains revert to their -helical conformation. The hair fibers now curl in the desired fashion because the new disulfide cross-linkages exert some torsion or twist on the bundles of -helical coils in the hair fibers. A permanent wave is not truly permanent, because the hair grows; in the new hair replacing the old, the -keratin has the natural, nonwavy pattern of disulfide bonds. reduce curl HS SH HS SH HS SH HS SH HS SH HS SH HS HS SH HS SH HS SH oxidize HS HS S S S S SH S S

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