Ch 1-2 Amino acid and Structure of Protein.pdf

168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 1 UNIT I: Protein Structure...

168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 1 UNIT I: Protein Structure and Function Amino Acids I. OVERVIEW 1 Proteins are the most abundant and functionally diverse molecules in A Free amino acid living systems. Virtually every life process depends on this class of Common to all α-amino molecules. For example, enzymes and polypeptide hormones direct and acids of proteins regulate metabolism in the body, whereas contractile proteins in muscle permit movement. In bone, the protein collagen forms a framework for the deposition of calcium phosphate crystals, acting like the steel C OH CO COOH cables in reinforced concrete. In the bloodstream, proteins, such as +H hemoglobin and plasma albumin, shuttle molecules essential to life, 3N Cα H whereas immunoglobulins fight infectious bacteria and viruses. In short, proteins display an incredible diversity of functions, yet all share the Amino group R Carboxyl group common structural feature of being linear polymers of amino acids. This chapter describes the properties of amino acids. Chapter 2 explores how these simple building blocks are joined to form proteins that have Side chain α-Carbon is unique three-dimensional structures, making them capable of perform- is distinctive between the for each amino carboxyl and the ing specific biologic functions. acid. amino groups. Amino acids combined B through II. STRUCTURE OF THE AMINO ACIDS peptide linkages Although more than 300 different amino acids have been described in NH-CH-CO-NH-CH-CO nature, only 20 are commonly found as constituents of mammalian pro- teins. [Note: These are the only amino acids that are coded for by DNA, R R the genetic material in the cell (see p. 395).] Each amino acid (except for proline, which has a secondary amino group) has a carboxyl group, a primary amino group, and a distinctive side chain (“R-group”) bonded Side chains determine to the α-carbon atom (Figure 1.1A). At physiologic pH (approximately properties of proteins. pH 7.4), the carboxyl group is dissociated, forming the negatively charged carboxylate ion (– COO–), and the amino group is protonated Figure 1.1 (– NH3+). In proteins, almost all of these carboxyl and amino groups are Structural features of amino acids combined through peptide linkage and, in general, are not available for (shown in their fully protonated form). chemical reaction except for hydrogen bond formation (Figure 1.1B). Thus, it is the nature of the side chains that ultimately dictates the role 1 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 2 2 1. Amino Acids an amino acid plays in a protein. It is, therefore, useful to classify the amino acids according to the properties of their side chains, that is, whether they are nonpolar (have an even distribution of electrons) or polar (have an uneven distribution of electrons, such as acids and bases; Figures 1.2 and 1.3). A. Amino acids with nonpolar side chains Each of these amino acids has a nonpolar side chain that does not gain or lose protons or participate in hydrogen or ionic bonds (Figure 1.2). The side chains of these amino acids can be thought of as “oily” or lipid-like, a property that promotes hydrophobic inter- actions (see Figure 2.10, p. 19). 1. Location of nonpolar amino acids in proteins: In proteins found in aqueous solutions––a polar environment––the side chains of the nonpolar amino acids tend to cluster together in the interior of the protein (Figure 1.4). This phenomenon, known as the hydrophobic NONPOLAR SIDE CHAINS COOH pK1 = 2.3 COOH COOH + + + H3N C H H3N C H H3N C H H CH3 CH pK2 = 9.6 H3C CH3 Glycine Alanine Valine COOH COOH COOH + + H3N C H H3N C H + H3N C H CH2 H C CH3 CH2 CH CH2 H3C CH3 CH3 Leucine Isoleucine Phenylalanine COOH COOH + H3N C H + H3N C H COOH CH2 +H CH2 2N C H C CH2 H2C CH2 CH2 CH S N H CH3 Tryptophan Methionine Proline Figure 1.2 Classification of the 20 amino acids commonly found in proteins, according to the charge and polarity of their side chains at acidic pH is shown here and continues in Figure 1.3. Each amino acid is shown in its fully protonated form, with dissociable hydrogen ions represented in red print. The pK values for the α-carboxyl and α-amino groups of the nonpolar amino acids are similar to those shown for glycine. (Continued in Figure 1.3.) 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 3 II. Structure of the Amino Acids 3 UNCHARGED POLAR SIDE CHAINS COOH pK1 = 2.2 + H3N C H COOH COOH CH2 + + H3N C H H3N C H pK2 = 9.1 H C OH H C OH H CH3 OH pK3 = 10.1 Serine Threonine Tyrosine COOH COOH +H 3N C H +H N 3 C H COOH pK1 = 1.7 + CH2 CH2 H3N C H C CH2 CH2 O NH2 C pK3 = 10.8 SH pK2 = 8.3 O NH2 Asparagine Glutamine Cysteine ACIDIC SIDE CHAINS pK1 = 2.1 COOH COOH +H N C +H pK3 = 9.8 3 H pK3 = 9.7 3N C H CH2 CH2 C CH2 O OH pK2 = 3.9 C O OH pK2 = 4.3 Aspartic acid BASIC SIDE CHAINS pK1 = 1.8 pK1 = 2.2 pK3 = 9.2 pK2 = 9.2 pK2 = 9.0 COOH COOH COOH +H + + H3N C H H3N C H H3N C H CH2 CH2 CH2 C CH CH2 CH2 +HN NH CH2 CH2 C H CH2 N H pK2 = 6.0 NH3+ pK3 = 10.5 C NH2+ pK3 = 12.5 NH2 Histidine Lysine Arginine Figure 1.3 Classification of the 20 amino acids commonly found in proteins, according to the charge and polarity of their side chains at acidic pH (continued from Figure 1.2). 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 4 4 1. Amino Acids effect, is the result of the hydrophobicity of the nonpolar R-groups, Nonpolar amino Nonpolar amino which act much like droplets of oil that coalesce in an aqueous acids ( ) cluster acids ( ) cluster in the interior of on the surface of environment. The nonpolar R-groups thus fill up the interior of the soluble proteins. membrane proteins. folded protein and help give it its three-dimensional shape. However, for proteins that are located in a hydrophobic environ- ment, such as a membrane, the nonpolar R-groups are found on the outside surface of the protein, interacting with the lipid envi- ronment (see Figure 1.4). The importance of these hydrophobic Cell interactions in stabilizing protein structure is discussed on p. 19. membrane Polar amino acids Sickle cell anemia, a sickling disease of red ( ) cluster on blood cells, results from the substitution of polar the surface of soluble proteins. glutamate by nonpolar valine at the sixth position Soluble protein Membrane protein in the β subunit of hemoglobin (see p. 36). Figure 1.4 Location of nonpolar amino acids 2. Proline: Proline differs from other amino acids in that proline’s in soluble and membrane proteins. side chain and α-amino N form a rigid, five-membered ring struc- ture (Figure 1.5). Proline, then, has a secondary (rather than a pri- mary) amino group. It is frequently referred to as an imino acid. The unique geometry of proline contributes to the formation of the Secondary amino Primary amino group group fibrous structure of collagen (see p. 45), and often interrupts the α-helices found in globular proteins (see p. 26). COOH COOH B. Amino acids with uncharged polar side chains +H N 2 C H +H N C H 3 H2C These amino acids have zero net charge at neutral pH, although the CH2 CH3 CH2 side chains of cysteine and tyrosine can lose a proton at an alkaline Proline Alanine pH (see Figure 1.3). Serine, threonine, and tyrosine each contain a polar hydroxyl group that can participate in hydrogen bond formation Figure 1.5 (Figure 1.6). The side chains of asparagine and glutamine each Comparison of the secondary contain a carbonyl group and an amide group, both of which can amino group found in proline with also participate in hydrogen bonds. the primary amino group found in other amino acids, such as 1. Disulfide bond: The side chain of cysteine contains a sulfhydryl alanine. group (–SH), which is an important component of the active site of many enzymes. In proteins, the –SH groups of two cysteines COOH can become oxidized to form a dimer, cystine, which contains a +H N 3 C H covalent cross-link called a disulfide bond (–S–S–). (See p. 19 for CH2 a further discussion of disulfide bond formation.) Tyrosine Many extracellular proteins are stabilized by O disulfide bonds. Albumin, a blood protein that H functions as a transpor ter for a variety of Hydrogen molecules, is an example. bond Carbonyl O group C 2. Side chains as sites of attachment for other compounds: The polar hydroxyl group of serine, threonine, and, rarely, tyrosine, can Figure 1.6 serve as a site of attachment for structures such as a phosphate Hydrogen bond between the phenolic hydroxyl group of tyrosine group. In addition, the amide group of asparagine, as well as the and another molecule containing a hydroxyl group of serine or threonine, can serve as a site of attach- carbonyl group. ment for oligosaccharide chains in glycoproteins (see p. 165). 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 5 II. Structure of the Amino Acids 5 C. Amino acids with acidic side chains The amino acids aspartic and glutamic acid are proton donors. At 1 Unique first letter: physiologic pH, the side chains of these amino acids are fully ionized, Cysteine = Cys = C containing a negatively charged carboxylate group (–COO–). They are, Histidine = His = H therefore, called aspartate or glutamate to emphasize that these amino Isoleucine = Ile = I Methionine = Met = M acids are negatively charged at physiologic pH (see Figure 1.3). Serine = Ser = S Valine = Val = V D. Amino acids with basic side chains The side chains of the basic amino acids accept protons (see Figure Most commonly occurring 2 amino acids have priority: 1.3). At physiologic pH the side chains of lysine and arginine are fully ionized and positively charged. In contrast, histidine is weakly basic, Alanine = Ala = A and the free amino acid is largely uncharged at physiologic pH. Glycine = Gly = G Leucine = Leu = L However, when histidine is incorporated into a protein, its side chain Proline = Pro = P can be either positively charged or neutral, depending on the ionic Threonine = Thr = T environment provided by the polypeptide chains of the protein. This is an important property of histidine that contributes to the role it plays in the functioning of proteins such as hemoglobin (see p. 31). 3 Similar sounding names: Arginine = Arg = R (“aRginine”) E. Abbreviations and symbols for commonly occurring amino acids Asparagine = Asn = N (contains N) Each amino acid name has an associated three-letter abbreviation Aspartate = Asp = D ("asparDic") Glutamate = Glu = E ("glutEmate") and a one-letter symbol (Figure 1.7). The one-letter codes are deter- Glutamine = Gln = Q (“Q-tamine”) mined by the following rules: Phenylalanine = Phe = F (“Fenylalanine”) Tyrosine = Tyr = Y (“tYrosine”) 1. Unique first letter: If only one amino acid begins with a particular Tryptophan = Trp = W (double ring in letter, then that letter is used as its symbol. For example, I = the molecule) isoleucine. 4 Letter close to initial letter: 2. Most commonly occurring amino acids have priority: If more than one amino acid begins with a particular letter, the most com- Aspartate or = Asx = B (near A) mon of these amino acids receives this letter as its symbol. For asparagine example, glycine is more common than glutamate, so G = glycine. Glutamate or = Glx = Z glutamine 3. Similar sounding names: Some one-letter symbols sound like the Lysine = Lys = K (near L) Undetermined = X amino acid they represent. For example, F = phenylalanine, or W amino acid = tryptophan (“twyptophan” as Elmer Fudd would say). 4. Letter close to initial letter: For the remaining amino acids, a one- Figure 1.7 letter symbol is assigned that is as close in the alphabet as possi- Abbreviations and symbols for the ble to the initial letter of the amino acid, for example, K = lysine. commonly occurring amino acids. Furthermore, B is assigned to Asx, signifying either aspartic acid or asparagine, Z is assigned to Glx, signifying either glutamic acid or glutamine, and X is assigned to an unidentified amino acid. F. Optical properties of amino acids HO OH OC CO H C The α-carbon of an amino acid is attached to four different chemical +H 3N C H N H C H 3+ groups and is, therefore, a chiral or optically active carbon atom. CH3 3 Glycine is the exception because its α-carbon has two hydrogen lan ine D-A lan ine substituents and, therefore, is optically inactive. Amino acids that L-A have an asymmetric center at the α-carbon can exist in two forms, designated D and L, that are mirror images of each other (Figure 1.8). The two forms in each pair are termed stereoisomers, optical Figure 1.8 isomers, or enantiomers. All amino acids found in proteins are of the D and L forms of alanine L-configuration. However, D-amino acids are found in some antibi- are mirror images. otics and in plant and bacterial cell walls. (See p. 253 for a discus- sion of D-amino acid metabolism.) 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 6 6 1. Amino Acids III. ACIDIC AND BASIC PROPERTIES OF AMINO ACIDS Amino acids in aqueous solution contain weakly acidic α-carboxyl groups and weakly basic α-amino groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain. Thus, both free amino acids and some amino acids combined in peptide linkages can act as buffers. Recall that acids may be defined as proton donors and bases as proton acceptors. Acids (or bases) described as “weak” ionize to only a limited extent. The concentration of protons in aqueous solution is expressed as pH, where pH = log 1/[H+] or –log [H+]. The quantitative relationship between the pH of the solu- tion and concentration of a weak acid (HA) and its conjugate base (A–) is described by the Henderson-Hasselbalch equation. OH– H20 A. Derivation of the equation CH3COOH CH3COO– Consider the release of a proton by a weak acid represented by HA: FORM I FORM II H+ (acetate, A– ) → H+ A– (acetic acid, HA) HA ← + weak proton salt form acid or conjugate base Buffer region [II] > [I] 1.0 The “salt” or conjugate base, A–, is the ionized form of a weak acid. By definition, the dissociation constant of the acid, Ka, is Equivalents OH– added [H+] [A–] [I] = [II] pKa = 4.8 Ka 0.5 [HA] [Note: The larger the Ka, the stronger the acid, because most of the HA has dissociated into H+ and A–. Conversely, the smaller the Ka, [I] > [II] 0 the less acid has dissociated and, therefore, the weaker the acid.] 0 3 4 5 6 7 By solving for the [H+] in the above equation, taking the logarithm of pH both sides of the equation, multiplying both sides of the equation by –1, and substituting pH = – log [H+ ] and pKa = – log Ka, we obtain Figure 1.9 the Henderson-Hasselbalch equation: Titration curve of acetic acid. [A– ] pH pKa + log [HA] B. Buffers A buffer is a solution that resists change in pH following the addition of an acid or base. A buffer can be created by mixing a weak acid (HA) with its conjugate base (A–). If an acid such as HCl is then added to such a solution, A– can neutralize it, in the process being converted to HA. If a base is added, HA can neutralize it, in the process being converted to A–. Maximum buffering capacity occurs at a pH equal to the pKa, but a conjugate acid/base pair can still serve as an effective buffer when the pH of a solution is within approximately ±1 pH unit of the pKa. If the 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 7 III. Acidic and Basic Properties of Amino Acids 7 OH– H20 OH– H20 – COOH COO COO– +H N C H +H N C H H2N C H 3 3 CH3 CH3 CH3 H+ H+ FORM I FORM II FORM III pK1 = 2.3 pK2 = 9.1 Alanine in acid solution Alanine in neutral solution Alanine in basic solution (pH less than 2) (pH approximately 6) (pH greater than 10) Net charge = +1 Net charge = 0 Net charge = –1 (isoelectric form) Figure 1.10 Ionic forms of alanine in acidic, neutral, and basic solutions. amounts of HA and A– are equal, the pH is equal to the pKa. As shown in Figure 1.9, a solution containing acetic acid (HA = CH3 – COOH) and acetate (A– = CH3 – COO–) with a pKa of 4.8 resists a change in pH from pH 3.8 to 5.8, with maximum buffering at pH 4.8. At pH values less than the pKa, the protonated acid form (CH3 – COOH) is the predominant species. At pH values greater than the pKa, the deprotonated base form (CH3 – COO–) is the predominant species in solution. C. Titration of an amino acid 1. Dissociation of the carboxyl group: The titration curve of an amino acid can be analyzed in the same way as described for acetic acid. Consider alanine, for example, which contains both an α-carboxyl and an α-amino group. At a low (acidic) pH, both of these groups are protonated (shown in Figure 1.10). As the pH of the solution is raised, the – COOH group of Form I can dissociate by donating a proton to the medium. The release of a proton results in the formation of the carboxylate group, – COO–. This structure is shown as Form II, which is the dipolar form of the molecule (see Figure 1.10). This form, also called a zwitterion, is the isoelectric form of alanine, that is, it has an overall (net) charge of zero. 2. Application of the Henderson-Hasselbalch equation: The dissoci- ation constant of the carboxyl group of an amino acid is called K1, rather than Ka, because the molecule contains a second titratable group. The Henderson-Hasselbalch equation can be used to analyze the dissociation of the carboxyl group of alanine in the same way as described for acetic acid: [H+] [II] K1 [I] where I is the fully protonated form of alanine, and II is the iso- electric form of alanine (see Figure 1.10). This equation can be rearranged and converted to its logarithmic form to yield: 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 8 8 1. Amino Acids [II] pH pK1 + log [I] 3. Dissociation of the amino group: The second titratable group of alanine is the amino (– NH3+) group shown in Figure 1.10. This is a much weaker acid than the – COOH group and, therefore, has a much smaller dissociation constant, K2. [Note: Its pKa is therefore larger.] Release of a proton from the protonated amino group of Form II results in the fully deprotonated form of alanine, Form III COO– (see Figure 1.10). H2N C H CH3 4. pKs of alanine: The sequential dissociation of protons from the FORM III carboxyl and amino groups of alanine is summarized in Figure 1.10. Each titratable group has a pKa that is numerically equal to Region of Region of buffering buffering the pH at which exactly one half of the protons have been removed from that group. The pK a for the most acidic group [II] = [III] (–COOH) is pK1, whereas the pKa for the next most acidic group 2.0 (– NH3+) is pK2. Equivalents OH– added 1.5 pI = 5.7 5. Titration curve of alanine: By applying the Hender son- [I] = [II] Hasselbalch equation to each dissociable acidic group, it is possi- 1.0 pK p K2 = 9. 9.1 ble to calculate the complete titration curve of a weak acid. Figure pK1 = 2.3 1.11 shows the change in pH that occurs during the addition of 0.5 base to the fully protonated form of alanine (I) to produce the completely deprotonated form (III). Note the following: 0 0 2 4 6 8 10 pH p a. Buffer pairs: The – COOH/– COO– pair can serve as a buffer in the pH region around pK 1, and the – NH 3+/– NH 2 pair can buffer in the region around pK2. COOH COO– +H N C H +H 3 3N C H b. When pH = pK: When the pH is equal to pK 1 (2.3), equal CH3 CH3 amounts of Forms I and II of alanine exist in solution. When FORM I FORM II the pH is equal to pK2 (9.1), equal amounts of Forms II and III are present in solution. Figure 1.11 c. Isoelectric point: At neutral pH, alanine exists predominantly The titration curve of alanine. as the dipolar Form II in which the amino and carboxyl groups are ionized, but the net charge is zero. The isoelectric point (pI) is the pH at which an amino acid is electrically neutral, that is, in which the sum of the positive charges equals the sum of the negative charges. For an amino acid, such as alanine, that has only two dissociable hydrogens (one from the α-carboxyl and one from the α-amino group), the pI is the average of pK1 and pK2 (pI = [2.3 + 9.1]/2 = 5.7, see Figure 1.11). The pI is thus midway between pK1 (2.3) and pK2 (9.1). pI corresponds to the pH at which the Form II (with a net charge of zero) pre- dominates, and at which there are also equal amounts of Forms I (net charge of +1) and III (net charge of –1). 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 9 III. Acidic and Basic Properties of Amino Acids 9 Separation of plasma proteins by charge typically A BICARBONATE AS A BUFFER is done at a pH above the pI of the major pro- – teins, thus, the charge on the proteins is negative. [HCO3 ] pH = pK + log [CO2] In an electric field, the proteins will move toward the positive electrode at a rate determined by An increase in HCO3– causes the pH to rise. their net negative charge. Variations in the mobil- ity pattern are suggestive of certain diseases. Pulmonary obstruction causes an increase in carbon dioxide and causes the pH to fall, resulting 6. Net charge of amino acids at neutral pH: At physiologic pH, in respiratory acidosis. amino acids have a negatively charged group (– COO–) and a positively charged group (– NH3+), both attached to the α-carbon. LUNG ALVEOLI [Note: Glutamate, aspartate, histidine, arginine, and lysine have additional potentially charged groups in their side chains.] Substances, such as amino acids, that can act either as an acid CO2 + H2O H2CO3 H+ + HCO3- or a base are defined as amphoteric, and are referred to as ampholytes (amphoteric electrolytes). B DRUG ABSORPTION – D. Other applications of the Henderson-Hasselbalch equation pH = pK + log [Drug ] [Drug-H] The Henderson-Hasselbalch equation can be used to calculate how At the pH of the stomach (1.5), a the pH of a physiologic solution responds to changes in the concen- drug like aspirin (weak acid, tration of a weak acid and/or its corresponding “salt” form. For exam- pK = 3.5) will be largely protonated ple, in the bicarbonate buffer system, the Henderson-Hasselbalch (COOH) and, thus, uncharged. equation predicts how shifts in the bicarbonate ion concentration, Uncharged drugs generally cross [HCO3–], and CO2 influence pH (Figure 1.12A). The equation is also membranes more rapidly than useful for calculating the abundance of ionic forms of acidic and charged molecules. basic drugs. For example, most drugs are either weak acids or weak bases (Figure 1.12B). Acidic drugs (HA) release a proton (H+), caus- STOMACH ing a charged anion (A–) to form. HA → ← H+ + A– Weak bases (BH+) can also release a H+. However, the protonated Lipid form of basic drugs is usually charged, and the loss of a proton pro- membrane duces the uncharged base (B). H+ A- BH + → ← B+ H + H+ HA H+ A- A drug passes through membranes more readily if it is uncharged. Thus, for a weak acid such as aspirin, the uncharged HA can per- H+ meate through membranes and A– cannot. For a weak base, such HA as morphine, the uncharged form, B, penetrates through the cell membrane and BH+ does not. Therefore, the effective concentration of the permeable form of each drug at its absorption site is deter- LUMEN OF BLOOD STOMACH mined by the relative concentrations of the charged and uncharged forms. The ratio between the two forms is determined by the pH at the site of absorption, and by the strength of the weak acid or base, Figure 1.12 which is represented by the pK a of the ionizable group. The The Henderson-Hasselbalch Henderson-Hasselbalch equation is useful in determining how much equation is used to predict: A, drug is found on either side of a membrane that separates two com- changes in pH as the concentrations partments that differ in pH, for example, the stomach (pH 1.0–1.5) of HCO3– or CO2 are altered; and blood plasma (pH 7.4). or B, the ionic forms of drugs. 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 10 10 1. Amino Acids IV. CONCEPT MAPS A Linked concept boxes Students sometimes view biochemistry as a blur of facts or equations to Amino acids be memorized, rather than a body of concepts to be understood. Details (fully protonated) provided to enrich understanding of these concepts inadvertently turn can into distractions. What seems to be missing is a road map—a guide that Release H+ provides the student with an intuitive understanding of how various top- ics fit together to make sense. The authors have, therefore, created a series of biochemical concept maps to graphically illustrate relationships B Concepts cross-linked within a map between ideas presented in a chapter, and to show how the information can be grouped or organized. A concept map is, thus, a tool for visualiz- ing the connections between concepts. Material is represented in a hier- archic fashion, with the most inclusive, most general concepts at the top Degradation Amino of the map, and the more specific, less general concepts arranged is produced by of body acid protein pool beneath. The concept maps ideally function as templates or guides for organizing information, so the student can readily find the best ways to integrate new information into knowledge they already possess. Simultaneous Protein synthesis and leads to turnover A. How is a concept map constructed? degradation 1. Concept boxes and links: Educators define concepts as “per- Amino ceived regularities in events or objects.” In our biochemical maps, Synthesis acid of body is consumed by pool concepts include abstractions (for example, free energy), pro- protein cesses (for example, oxidative phosphorylation), and compounds (for example, glucose 6-phosphate). These broadly defined con- cepts are prioritized with the central idea positioned at the top of the page. The concepts that follow from this central idea are then C Concepts cross-linked drawn in boxes (Figure 1.13A). The size of the type indicates the to other chapters and relative importance of each idea. Lines are drawn between con- to other books in the cept boxes to show which are related. The label on the line Lippincott Series defines the relationship between two concepts, so that it reads as a valid statement, that is, the connection creates meaning. The lines with arrowheads indicate in which direction the connection should be read (Figure 1.14).... how the... how altered protein folds protein folding 2. Cross-links: Unlike linear flow charts or outlines, concept maps into its native leads to prion conformation disease, such may contain cross-links that allow the reader to visualize complex as Creutzfeldt- relationships between ideas represented in different parts of the Jakob disease map (Figure 1.13B), or between the map and other chapters in this book or companion books in the series (Figure 1.13C). Cross- links can thus identify concepts that are central to more than one discipline, empowering students to be effective in clinical situa- Structure Lippincott's tions, and on the United States Medical Licensure Examination of Proteins 2 Illustrated Reviews (USMLE) or other examinations, that bridge disciplinary bound- aries. Students learn to visually perceive nonlinear relationships ogy between facts, in contrast to cross-referencing within linear text. l bio ro ic V. CHAPTER SUMMARY M Each amino acid has an α-carboxyl group and a primary α-amino Figure 1.13 group (except for proline, which has a secondary amino group). At Symbols used in concept maps. physiologic pH, the α-carboxyl group is dissociated, forming the nega- tively charged carboxylate ion (– COO–), and the α-amino group is pro- tonated (– NH3+). Each amino acid also contains one of 20 distinctive 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 11 V. Chapter Summary 11 Amino acids are composed of when protonated can α-Carboxyl group α-Amino group Side chains Release H + (–COOH) (–NH2) (20 different ones) and act as is is Deprotonated (COO–) Protonated (NH3+ ) grouped as Weak acids at physiologic pH at physiologic pH described by Henderson-Hasselbalch equation: [A–] pH = pKa + log [HA] Nonpolar Uncharged polar Acidic Basic predicts side chains side chains side chains side chains Alanine Asparagine Aspartic acid Arginine Glycine Cysteine Glutamic acid Histidine Buffering capacity Isoleucine Glutamine Lysine Leucine Serine Methionine Threonine characterized by characterized by predicts Phenylalanine Tyrosine Proline Tryptophan Side chain dissociates Side chain is pro- Buffering occurs Valine to –COO– at tonated and ±1 pH unit of pKa physiologic pH generally has a positive charge at physiologic pH predicts found found found found Maximal buffer On the outside of proteins that function in an aqueous environment when pH = pKa and in the interior of membrane-associated proteins predicts In the interior of proteins that function in an aqueous environment and on the surface of proteins (such as membrane proteins) that interact with lipids pH = pKa when [HA] = [A– ] Structure of Proteins 2 In proteins, most Thus, the chemical α-COO– and nature of the side... how the Therefore, these chain determines α-NH3+ of amino groups are not protein folds acids are the role that the into its native available for amino acid plays combined through chemical reaction. conformation. peptide bonds. in a protein, particularly... Figure 1.14 Key concept map for amino acids. 168397_P001-012.qxd7.0:02 Protein structure 5-20-04 2010.4.4 9:45 AM Page 12 12 1. Amino Acids side chains attached to the α-carbon atom. The chemical nature of this side chain determines the function of an amino acid in a protein, and provides the basis for classification of the amino acids as nonpolar , uncharged polar, acidic, or basic. All free amino acids, plus charged amino acids in peptide chains, can serve as buffers. The quantitative relationship between the pH of a solution and the concentration of a weak acid (HA) and its conjugate base (A–) is described by the Henderson-Hasselbalch equation. Buffering occurs within ±1pH unit of the pKa, and is maximal when pH = pKa, at which [A–] = [HA]. The α-carbon of each amino acid (except glycine) is attached to four different chemical groups and is, therefore, a chiral or optically active carbon atom. Only the L-form of amino acids is found in proteins synthesized by the human body. Study Questions Choose the ONE correct answer. 1.1 The letters A through E designate certain regions on the titration curve for glycine (shown below). Which one of the following statements concerning this curve is correct? 2.0 E Equivalents OH– added D Correct answer = C. C represents the isoelectric 1.5 point or pI, and as such is midway between pK1 1.0 and pK 2 for this monoamino monocarboxylic C acid. Glycine is fully protonated at Point A. Point 0.5 B B represents a region of maximum buffering, as does Point D. Point E represents the region A where glycine is fully deprotonated. 0 0 2 4 6 8 10 pH A. Point A represents the region where glycine is deprotonated. B. Point B represents a region of minimal buffering. C. Point C represents the region where the net charge on glycine is zero. D. Point D represents the pK of glycine’s carboxyl group. E. Point E represents the pI for glycine. 1.2 Which one of the following statements concerning the Correct answer = D. The two cysteine residues peptide shown below is correct? can, under oxidizing conditions, form a disulfide Gly-Cys-Glu-Ser-Asp-Arg-Cys bond. Glutamine’s 3-letter abbreviation is Gln. A. The peptide contains glutamine. Proline (Pro) contains a secondary amino group. B. The peptide contains a side chain with a secondary Only one (Arg) of the seven would have a posi- tively charged side chain at pH 7. amino group. C. The peptide contains a majority of amino acids with side chains that would be positively charged at pH 7. D. The peptide is able to form an internal disulfide bond. Correct answer = negative electrode. When the pH is less than the pI, the charge on glycine is 1.3 Given that the pI for glycine is 6.1, to which electrode, positive because the α-amino group is fully pro- positive or negative, will glycine move in an electric tonated. (Recall that glycine has H as its R field at pH 2? Explain. group). 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 13 Structure of Proteins 2 H H H H N C C N C 1 Primary C structure I. OVERVIEW H O CH3 The 20 amino acids commonly found in proteins are joined together by N C peptide bonds. The linear sequence of the linked amino acids contains H O C the information necessary to generate a protein molecule with a unique O N C three-dimensional shape. The complexity of protein structure is best CH C R O C N analyzed by considering the molecule in terms of four organizational H levels, namely, primary, secondary, tertiary, and quaternary (Figure 2.1). An examination of these hierarchies of increasing complexity has C R C H N O 2 Secondary structure O N C revealed that certain structural elements are repeated in a wide variety H C of proteins, suggesting that there are general “rules” regarding the ways O NC in which proteins achieve their native, functional form. These repeated H C R C structural elements range from simple combinations of α-helices and N β–sheets forming small motifs, to the complex folding of polypeptide R C H domains of multifunctional proteins (see p. 18). II. PRIMARY STRUCTURE OF PROTEINS Tertiary The sequence of amino acids in a protein is called the primary structure 3 structure of the protein. Understanding the primary structure of proteins is impor- tant because many genetic diseases result in proteins with abnormal amino acid sequences, which cause improper folding and loss or impairment of normal function. If the primary structures of the normal and the mutated proteins are known, this information may be used to diagnose or study the disease. A. Peptide bond In proteins, amino acids are joined covalently by peptide bonds, which 4 Quaternary structure are amide linkages between the α-carboxyl group of one amino acid and the α-amino group of another. For example, valine and alanine can form the dipeptide valylalanine through the formation of a peptide bond (Figure 2.2). Peptide bonds are not broken by conditions that denature proteins, such as heating or high concentrations of urea (see p. 20). Prolonged exposure to a strong acid or base at elevated Figure 2.1 temperatures is required to hydrolyze these bonds nonenzymically. Four hierarchies of protein structure. 13 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 14 14 2. Structure of Proteins 1. Naming the peptide: By convention, the free amino end (N-terminal) of the peptide chain is written to the left and the free carboxyl end A Formation of the peptide bond (C-terminal) to the right. Therefore, all amino acid sequences are read from the N- to the C-terminal end of the peptide. For example, in Figure 2.2A, the order of the amino acids is “valine, alanine.” CH3 Linkage of many amino acids through peptide bonds results in an H3C CH H unbranched chain called a polypeptide. Each component amino – acid in a polypeptide is called a “residue” because it is the portion of +H 3N C COO +H 3N C COO– the amino acid remaining after the atoms of water are lost in the for- H CH3 mation of the peptide bond. When a polypeptide is named, all Valine Alanine amino acid residues have their suffixes (-ine, -an, -ic, or -ate) changed to -yl, with the exception of the C-terminal amino acid. For H2O example, a tripeptide composed of an N-terminal valine, a glycine, Free amino Free carboxyl and a C-terminal leucine is called valylglycylleucine. end of peptide end of peptide CH3 2. Characteristics of the peptide bond: The peptide bond has a par- H3C CH H H tial double-bond character, that is, it is shorter than a single bond, + H3N C C N C COO – and is rigid and planar (Figure 2.2B). This prevents free rotation H O CH3 around the bond between the carbonyl carbon and the nitrogen of the peptide bond. However, the bonds between the α-carbons and Valylalanine the α-amino or α-carboxyl groups can be freely rotated (although they are limited by the size and character of the R-groups). This Peptide bond allows the polypeptide chain to assume a variety of possible config- urations. The peptide bond is generally a trans bond (instead of cis, see Figure 2.2B), in large part because of steric interference of the R-groups when in the cis position. B Characteristics peptide bond of the 3. Polarity of the peptide bond: Like all amide linkages, the – C =O and – NH groups of the peptide bond are uncharged, and neither Trans peptide Cis peptide accept nor release protons over the pH range of 2–12. Thus, the bond R R bond R charged groups present in polypeptides consist solely of the N-terminal (α-amino) group, the C-terminal (α-carboxyl) group, O Cα Cα Cα and any ionized groups present in the side chains of the con- C N C N Cα H stituent amino acids. The – C=O and – NH groups of the peptide O H R bond are polar, and are involved in hydrogen bonds, for example, in α-helices and β-sheet structures, described on pp. 16–17. Peptide bonds B. Determination of the amino acid composition of a polypeptide in proteins Partial double-bond The first step in determining the primary structure of a polypeptide is character to identify and quantitate its constituent amino acids. A purified Rigid and planar sample of the polypeptide to be analyzed is first hydrolyzed by Trans configuration strong acid at 110°C for 24 hours. This treatment cleaves the pep- Uncharged but polar tide bonds and releases the individual amino acids, which can be separated by cation-exchange chromatography. In this technique, a mixture of amino acids is applied to a column that contains a resin to which a negatively charged group is tightly attached. [Note: If the Figure 2.2 attached group is positively charged, the column becomes an anion- A. Formation of a peptide bond, exchange column.] The amino acids bind to the column with differ- showing the structure of the dipeptide valylalanine. ent affinities, depending on their charges, hydrophobicity, and other B. Characteristics of the peptide characteristics. Each amino acid is sequentially released from the bond. chromatography column by eluting with solutions of increasing ionic strength and pH (Figure 2.3). The separated amino acids contained in the eluate from the column are quantitated by heating them with ninhydrin—a reagent that forms a purple compound with most 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 15 II. Primary Structure of Proteins 15 amino acids, ammonia, and amines. The amount of each amino acid is determined spectrophotometrically by measuring the amount of Buffer pump light absorbed by the ninhydrin derivative. The analysis described Sample injection above is performed using an amino acid analyzer—an automated machine whose components are depicted in Figure 2.3. Ion exchange C. Sequencing of the peptide from its N-terminal end column Sequencing is a stepwise process of identifying the specific amino Separated acid at each position in the peptide chain, beginning at the N-terminal amino acids end. Phenylisothiocyanate, known as Edman reagent, is used to label the amino-terminal residue under mildly alkaline conditions (Figure Ninhydrin pump 2.4). The resulting phenylthiohydantoin (PTH) derivative introduces an instability in the N-terminal peptide bond that can be selectively hydrolyzed without cleaving the other peptide bonds. The identity of Reaction the amino acid derivative can then be determined. Edman reagent can coil be applied repeatedly to the shortened peptide obtained in each previ- ous cycle. Photometer D. Cleavage of the polypeptide into smaller fragments Light source Strip-chart Many polypeptides have a primary structure composed of more than recorder 100 amino acids. Such molecules cannot be sequenced directly or computer from end to end. However, these large molecules can be cleaved at specific sites, and the resulting fragments sequenced. By using more than one cleaving agent (enzymes and/or chemicals) on sepa- rate samples of the purified polypeptide, overlapping fragments can be generated that permit the proper ordering of the sequenced frag- ments, thus providing a complete amino acid sequence of the large Figure 2.3 polypeptide (Figure 2.5). Enzymes that hydrolyze peptide bonds are Determination of the amino acid termed peptidases (proteases). [Note: Exopeptidases cut at the composition of a polypeptide using ends of proteins, and are divided into aminopeptidases and an amino acid analyzer. carboxypeptidases. Carboxypeptidases are used in determining the C-terminal amino acid. Endopeptidases cleave within a protein.] E. Determination of a protein’s primary structure by DNA sequencing The sequence of nucleotides in a protein-coding region of the DNA specifies the amino acid sequence of a polypeptide. Therefore, if the nucleotide sequence can be determined, it is possible, from knowl- edge of the genetic code (see p. 431), to translate the sequence of nucleotides into the corresponding amino acid sequence of that Release of amino acid 1 Labeling 2 derivative by acid hydrolysis O O H2N CH C Lys His Leu Arg COOH HN CH C Lys His Leu Arg COOH H2N Lys His Leu Arg COOH CH3 CH3 Peptide S Labeled peptide Shortened peptide N-terminal C S C alanine N NH + S N C C NH O CH Phenyl- CH3 isothiocyanate PTH-alanine Figure 2.4 Determination of the amino-terminal residue of a polypeptide by Edman degradation. 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 16 16 2. Structure of Proteins Peptide of unknown sequence polypeptide. This indirect process, although routinely used to obtain the amino acid sequences of proteins, has the limitations of not being able to predict the positions of disulfide bonds in the folded 1. Cleave with trypsin at lysine chain, and of not identifying any amino acids that are modified after and arginine 1 2. Determine sequence of their incorporation into the polypeptide (posttranslational modifica- peptides using Edman's tion, see p. 443). Therefore, direct protein sequencing is an method extremely important tool for determining the true character of the pri- Peptide A Peptide B Peptide C mary sequence of many polypeptides. A B C? A C B? III. SECONDARY STRUCTURE OF PROTEINS What is the B A C? correct order? B C A? The polypeptide backbone does not assume a random three-dimensional C A B? C B A? structure, but instead generally forms regular arrangements of amino Peptide of unknown sequence acids that are located near to each other in the linear sequence. These arrangements are termed the secondary structure of the polypeptide. The α-helix, β-sheet, and β-bend (β-turn) are examples of secondary struc- 1. Cleave with cyanogen tures frequently encountered in proteins. [Note: The collagen α-chain 2 bromide at methionine 2. Determine sequence of helix, another example of secondary structure, is discussed on p. 45.] peptides using Edman's method A. α-Helix Peptide X Peptide Y Several different polypeptide helices are found in nature, but the