Protein Structure PDF

Protein Structure Protein Functions • Three examples of protein functions Alcohol dehydrogenase oxidizes alcohols to aldehydes or ketones – Catalysis: Almost all chemical reactions in a living cell are catalyzed by protein enzymes. – Transport: Some proteins transports various substances, suc...

Protein Structure Protein Functions • Three examples of protein functions Alcohol dehydrogenase oxidizes alcohols to aldehydes or ketones – Catalysis: Almost all chemical reactions in a living cell are catalyzed by protein enzymes. – Transport: Some proteins transports various substances, such as oxygen, ions, and so on. Haemoglobin carries oxygen – Information transfer: For example, hormones. Insulin controls the amount of sugar in the blood Amino Acids • When scientists first turned their attention to nutrition, early in the nineteenth century, they quickly discovered that natural products containing nitrogen were essential for the survival of animals. In 1839, the Swedish chemist Jacob Berzelius coined the term protein (Greek: proteios, primary) for this class of compounds. The physiological chemists of that time did not realize that proteins were actually composed of smaller units, amino acids, although the first amino acids had been isolated in 1830. In fact, for many years, it was believed that substances from plants—including proteins—were incorporated whole into animal tissues. This misconception was laid to rest when the process of digestion came to light. After it became clear that ingested proteins were broken down to smaller compounds containing amino acids, scientists began to consider the nutritive qualities of those compounds. Modern studies of proteins and amino acids owe a great deal to nineteenthand early twentieth-century experiments. We now understand that nitrogen containing amino acids are essential for life and that they are the building blocks of proteins. The central role of amino acids in biochemistry is perhaps not surprising: Several amino acids are among the organic compounds believed to have appeared early in the earth’s history (Section 1-1A). Amino acids, as ancient and ubiquitous molecules, have been co-opted by evolution for a variety of purposes in living systems. We begin this chapter by discussing the structures and chemical properties of the common amino acids, including their stereochemistry, and end with a brief summary of the structures and functions of some related compounds. Staphylococcus epidermidis, which grows on human skin, links the amino acid glutamate into long chains. These help protect the bacteria from changes in extracellular salt concentration that normally occur on the skin surface. Pathways to Discovery William C. Rose and the Discovery of Threonine William C. Rose (1887–1985) Identifying the aminonacid constituents of proteins was a scientific challenge that grew out of studies of animal nutrition. At the start of the twentieth century, physiological chemists (the term biochemist was not yet used) recognized that not all foods provided adequate nutrition. For example, rats fed the corn protein zein as their only source of nitrogen failed to grow unless the amino acids tryptophan and lysine were added to their diet. Knowledge of metabolism at that time was limited mostly to information gleaned from studies in which intake of particular foods in experimental subjects (including humans) was linked to the urinary excretion of various compounds. Results of such studies were consistent with the idea that compounds could be transformed into other compounds, but clearly, nutrients were not wholly interchangeable. At the University of Illinois, William C. Rose focused his research on nutritional studies to decipher the metabolic relationships of nitrogenous compounds. Among other things, his studies of rat growth and nutrition helped show that purines and pyrimidines were derived from amino acids but that those compounds could not replace dietary amino acids. To examine the nutritional requirements for individual amino acids, Rose hydrolyzed proteins to obtain their component amino acids and then selectively removed certain amino acids. In one of his first experiments, he removed arginine and histidine from a hydrolysate of the milk protein casein. Rats fed on this preparation lost weight unless the amino acid histidine was added back to the food. However, adding back arginine did not compensate for the apparent requirement for histidine. These results prompted Rose to investigate the requirements for all the amino acids. Using similar experimental approaches, Rose demonstrated that cysteine histidine, and tryptophan could not be replaced by other amino acids. From preparations based on hydrolyzed proteins, Rose moved to mixtures of pure amino acids. Thirteen of the 19 known amino acids could be purified, and the other six synthesized. However, rats fed these 19 amino acids as their sole source of dietary nitrogen lost weight. Although one possible explanation was that the proportions of the pure amino acids were not optimal, Rose concluded that there must be an additional essential amino acid, present in naturally occurring proteins and their hydrolysates but not in his amino acid mixtures. After several years of effort, Rose obtained and identified the missing amino acid. In work published in 1935, Rose showed that adding this amino acid to the other 19 could support rat growth. Thus, the twentieth and last amino acid, threonine, was discovered. Experiments extending over the next 20 years revealed that 10 of the 20 amino acids found in proteins are nutritionally essential, so that removal of one of these causes growth failure and eventually death in experimental animals. The other 10 amino acids were considered “dispensable” since animals could synthesize adequate amounts of them. Rose’s subsequent work included verifying the amino acid requirements of humans, using graduate students as subjects. Knowing which amino acids were required for normal health— and in what amounts—made it possible to evaluate the potential nutritive value of different types of food proteins. Eventually, these findings helped guide the formulations used for intravenous feeding. McCoy, R.H., Meyer, C.E., and Rose, W.C., Feeding experiments with mixtures of highly purified amino acids. VIII. Isolation and identification of a new essential amino acid, J. Biol. Chem. 112, 283– 302 (1935). [Freely available at http://www.jbc.org.] Amino Acid Structure • KEY CONCEPTS • The 20 standard amino acids share a common structure but differ in their side chains. • Peptide bonds link amino acid residues in a polypeptide. • Some amino acid side chains contain ionizable groups whose pK values may vary. The analyses of a vast number of proteins from almost every conceivable source have shown that all proteins are composed of 20 “standard” amino acids. Not every protein contains all 20 types of amino acids, but most proteins contain most, if not all, of the 20 types. The common amino acids are known as α-amino acids because they have a primary amino group (—NH2) as a substituent of the α carbon atom, the carbon next to the carboxylic acid group (—COOH; Fig. 1). The sole exception is proline, which has a secondary amino group (—NH—), although for uniformity we will refer to proline as an α-imino acid. The 20 standard amino acids differ in the structures of their side chains (R groups). Table 1 displays the names and complete chemical structures of the 20 standard. Amino acid: Basic unit of protein R NH3+ C Amino group H Different side chains, R, determine the COO properties of 20 amino Carboxylic acid group acids. An amino acid 20 Amino acids Glycine (G) Alanine (A) Valine (V) Isoleucine (I) Leucine (L) Proline (P) Methionine (M) Phenylalanine (F) Tryptophan (W) Asparagine (N) Glutamine (Q) Serine (S) Threonine (T) Tyrosine (Y) Cysteine (C) Lysine (K) Arginine (R) Histidine (H) Asparatic acid (D) Glutamic acid (E) White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic Each protein has a unique structure! Amino acid sequence NLKTEWPELVGKSVEEAK KVILQDKPEAQIIVLPVGTI VTMEYRIDRVRLFVDKLD Folding! Amino Acids Are Dipolar Ions The amino and carboxylic acid groups of amino acids ionize readily. The pK values of the carboxylic acid groups (represented by pK1 ) lie in a small range around 2.2, while the pK values of the α-amino groups (pK2) are near 9.4. At physiological pH (∼7.4), the amino groups are protonated and the carboxylic acid groups are in their conjugate base (carboxylate) form (pKR). . An amino acid can therefore act as both an acid and a base. Molecules such as amino acids, which bear charged groups of opposite polarity, are known as dipolar ions or zwitterions. Amino acids, like other ionic compounds, are more soluble in polar solvents than in nonpolar solvents. As we will see, the ionic properties of the side chains influence the physical and chemical properties of free amino acids and amino acids in proteins. Amino Acid Ionization The nonionic and zwitterionic forms of a simple amino acid such as alanine are shown in Fig. The zwitterionic form predominates at neutral pH. The nonionic form does not occur in significant amounts in aqueous solution at any pH. A zwitterion can act as either an acid (proton donor) or a base (proton acceptor). Peptide Bonds Link Amino Acids Amino acids can be polymerized to form chains. This process can be represented as a condensation reaction (bond formation with the elimination of a water molecule), as shown in Fig. The resulting CO—NH linkage, an amide linkage, is known as a peptide bond. Polymers composed of two, three, a few (3–10), and many amino acid units are known, respectively, as dipeptides, tripeptides, oligopeptides, and polypeptides. These substances, however, are often referred to simply as “peptides.” After they are incorporated into a peptide, the individual amino acids (the monomeric units) are referred to as amino acid residues. Polypeptides are linear polymers rather than branched chains; that is, each amino acid residue participates in two peptide bonds and is linked to its neighbors in a head-to-tail fashion. The residues at the two ends of the polypeptide each participate in just one peptide bond. The residue with a free amino group (by convention) is called the amino terminus or Nterminus. The residue with a free carboxylate group (at the right) is called the carboxyl terminus or C-terminus. Proteins are molecules that contain one or more polypeptide chains. Variations in the length and the amino acid sequence of polypeptides are major contributors to the diversity in the shapes and biological functions of proteins. Amino Acid Side Chains Are Nonpolar, Polar, or Charged The most useful way to classify the 20 standard amino acids is by the polarities of their side chains. According to the most common classification scheme, there are three major types of amino acids: (1) those with nonpolar R groups, (2) those with uncharged polar R groups, and (3) those with charged polar R groups. The Nonpolar Amino Acid Side Chains Have a Variety of Shapes and Sizes. Nine amino acids are classified as having nonpolar side chains. The threedimensional shapes of some of these amino acids are shown in Fig. Glycine has the smallest possible side chain, an H atom. Alanine, valine, leucine, and isoleucine have aliphatic hydrocarbon side chains ranging in size from a methyl group for alanine to isomeric butyl groups for leucine and isoleucine. Methionine has a thioether side chain that resembles an n-butyl group in many of its physical properties (C and S have nearly equal electronegativities, and S is about the size of a methylene group). Proline has a cyclic pyrrolidine side group. Phenylalanine (with its phenyl moiety) and tryptophan (with its indole group) contain aromatic side groups, which are characterized by bulk as wel as nonpolarity Isoleucine Alanine Phenylalanine Some amino acids with nonpolar side chains. The amino acids are shown as ball-and-stick models embedded in transparent space-filling models. The atoms are colored according to type, with C green, H white, N blue, and O red. Uncharged Polar Side Chains Have Hydroxyl, Amide, or Thiol Groups. Six amino acids are commonly classified as having uncharged polar side chains . Serine and threonine bear hydroxylic R groups of different sizes. Asparagine and glutamine have amide-bearing side chains of different sizes. Tyrosine has a phenolic group (and, like phenylalanine and tryptophan, is aromatic). Cysteine is unique among the 20 amino acids in that it has a thiol group that can form a disulfide bond with another cysteine through the oxidation of the two thiol groups . Serine Glutamine Some amino acids with uncharged polar side chains. Note the presence of electronegative atoms on the side chains. The thiol groups of two cysteine residues are readily oxidized to form a covalently linked dimeric amino acid known as cystine. In cystine, the two cysteines are joined by a disulfide bond (Fig.). The disulfide-linked cystine residue is strongly hydrophobic. In proteins, disulfide bonds form covalent links between different parts of a polypeptide chain, or between two different polypeptide chains Charged Polar Side Chains Are Positively or Negatively Charged. Five amino acids have charged side chains . The side chains of the basic amino acids are positively charged at physiological pH values. Lysine has a butylammonium side chain, and arginine bears a guanidino group. Histidine carries an imidazolium moiety. Note that only histidine, with a pKR of 6.04, readily ionizes within the physiological pH range. Consequently, both the neutral and cationic forms occur in proteins. In fact, the protonation–deprotonation of histidine side chains is a feature of numerous enzymatic reaction mechanisms. The side chains of the acidic amino acids, aspartic acid and glutamic acid, are negatively charged above pH 3; in their ionized state, they are often referred to as aspartate and glutamate. Asparagine and glutamine are, respectively, the amides of aspartic acid and glutamic acid. Positively Charged Amino Acids The side-chains of lysine and arginine are fully positively charged at neutral pH. In lysine, a primary amino group is attached to the e carbon of the side-chain. In arginine, the guanidinium group of the side-chain is postively charged. The histidine R group contains an aromatic imidazole group that is partially positively charged at neutral pH . Histidine residues function in many enzymecatalyzed reactions as proton donors and/or acceptors. Negatively Charged Amino Acids The R groups of aspartate and glutamate contain carboxyl groups that are fully negatively charged at neutral pH (pKRs of 3.65 and 4.25). In aspartate, the carboxyl group is attached to the ß carbon of the amino acid backbone. In glutamate, the carboxyl group is attached to the g carbon. Aspartate Lysine Some amino acids with charged polar side chains The foregoing allocation of the 20 amino acids among the three different groups is somewhat arbitrary. For example, glycine and alanine, the smallest of the amino acids, and tryptophan, with its heterocyclic ring, might just as well be classified as uncharged polar amino acids. Similarly, tyrosine and cysteine, with their ionizable side chains, might also be thought of as charged polar amino acids, particularly at higher pH values. In fact, the deprotonated side chain of cysteine (which contains the thiolate anion, S−) occurs in a variety of enzymes, where it actively participates in chemical reactions. Inclusion of a particular amino acid in one group or another reflects not just the properties of the isolated amino acid, but its behavior when it is part of a polypeptide. The structures of most polypeptides depend on a tendency for polar and ionic side chains to be hydrated and for nonpolar side chains to associate with each other rather than with water. This property of polypeptides is the hydrophobic effect in action. As we will see, the chemical and physical properties of amino acid side chains also govern the chemical reactivity of the polypeptide. It is therefore worthwhile studying the structures of the 20 standard amino acids in order to appreciate how they vary in polarity, acidity, aromaticity, bulk, conformational flexibility, ability to cross-link, ability to hydrogen bond, and reactivity toward other groups. The pK Values of Ionizable Groups Depend on Nearby Groups The α-amino acids have two or, for those with ionizable side chains, three acid–base groups. At very low pH values, these groups are fully protonated, and at very high pH values, these groups are unprotonated. At intermediate Ph values, the acidic groups tend to be unprotonated, and the basic groups tend to be protonated. Thus, for the amino acid glycine, below pH 2.35 (the pK value of its carboxylic acid group), the +H3NCH2COOH form predominates. Above pH 2.35, the carboxylic acid is mostly ionized but the amino group is still mostly protonated (+H3NCH2COO−). Above pH 9.78 (the pK value of the amino group), the H2NCH2COO− form predominates. Note that in aqueous solution, the un-ionized form (H2NCH2COOH) is present only in vanishingly small quantities. The pH at which a molecule carries no net electric charge is known as its isoelectric point, pI. For the α-amino acids, pI = 1/2 (pKi + pKj) where Ki and Kj are the dissociation constants of the two ionizations involving the neutral species. For monoamino, monocarboxylic acids such as glycine, Ki and Kj represent K1 and K2. However, for aspartic and glutamic acids, Ki and Kj are K1 and KR, whereas for arginine, histidine, and lysine, these quantities are KR and K2 . Of course, amino acid residues in the interior of a polypeptide chain do not have free α-amino and α-carboxyl groups that can ionize (these groups are joined in peptide bonds. Furthermore, the pK values of all ionizable groups, including the N- and C-termini, usually differ from the pK values listed in Table 3-1 for free amino acids. For example, the pK values of αcarboxyl groups in unfolded proteins range from 3.5 to 4.0 In the free amino acids, the pK values are much lower, because the positively charged ammonium group electrostatically stabilizes the COO– group, in effect making it easier for the carboxylic acid group to ionize. Similarly, the pK values for α-amino groups in proteins range from 7.5 to 8.5. In the free amino acids, the pK values are higher, due to the electron-withdrawing character of the nearby carboxylate group, which makes it more diffi cult for the ammonium group to become deprotonated. In addition, the three-dimensional structure of a folded polypeptide chain may bring polar side chains and the N and C-termini close together. The resulting electrostatic interactions between these groups may shift their pK values up to several pH units from the values for the corresponding free amino acids. For this reason, the pI of a polypeptide, which is a function of the pK values of its many ionizable groups, is not easily predicted and is usually determined experimentally. Titration of Simple Amino Acids The titration curves of simple amino acids such as glycine that have non-dissociable R groups, have two plateaus, which correspond to the dissociation and titration of the a-carboxyl group (pK1, left) and the aamino group (pK2, right). As shown above the curve, the predominant ionic species in solution at low pH is the fully protonated form, +H3N-CH2-COOH (net charge = +1), In between the two plateaus, the zwitterionic form, +H3N-CH2-COO- (net charge = 0) predominates. At the end of the titration, the fully dissociated species H2N-CH2-COO- (net charge -1) predominates. The curve shows that glycine has two regions of buffering power centered ±1 pH unit above pK1 and pK2. The Henderson-Hasselbalch equation can be used to calculate the amounts of the conjugate acid and conjugate base species in solution at any pH. Lastly, the pH at which the zwitterionic (0-charged) species of glycine predominates (one equivalent of OH- added) is called the isoelectric point or isoelectric pH. The isoelectric pH is exactly halfway between the two pKas for glycine. Chemical Environment and the pKa The pKas of the a-carboxyl groups of all amino acids are lower than the pKas of the carboxyl groups in methyl-substituted carboxylic acids such as acetic acid . This is due to the local chemical environment of the a-carboxyl groups in amino acids. Namely, placement near the a-amino group, which is positively charged, makes the a-carboxyl groups of amino acids more acidic than the carboxyl group of acetic acid. Similarly, the chemical environment near a-amino groups makes them more acidic than the amino groups of a methyl-substituted amino compounds such as methylamine. In this case the electron withdrawing properties of the oxygens on the a-carboxyl groups of amino acids make the aamino groups hold onto their protons less tightly than in other environments. Titration of Glutamate The acidic amino acid, glutamate, has a second carboxyl group present in its side-chain. Thus the titration curve for glutamate (and aspartate) has three plateaus, each one corresponding to the dissociation of a proton from the amino acid . Since the R group carboxyl group has a pKR between that of the pK1 and pK2, the second plateau corresponds to the titration of this group. Based on inspection of the ionic forms in solution (top) it is clear that the zwitterionic form of glutamate occurs at a pH midway between that of pK1 and pKR. Thus the pI for glutamate is 3.22. Titration of Histidine Histidine has an imidazole R group that contains a dissociable proton with a pKR of 6.0. Thus the titration curve for histidine also has three plateaus . For histidine, the 0charged zwitterionic species occurs in solution at a pH midway between pKR and pK2 (pI = 7.59). Because the histidine pKR is near neutrality, the R group of histidine plays a role in buffering the pH of solutions containing proteins. Amino Acid Names Are Abbreviated The three-letter abbreviations for the 20 standard amino acids given in Table 3-1 are widely used in the biochemical literature. Most of these abbreviations are taken from the first three letters of the name of the corresponding amino acid and are pronounced as written. The symbol Glx indicates Glu or Gln, and similarly, Asx means Asp or Asn. This ambiguous notation stems from laboratory experience: Asn and Gln are easily hydrolyzed to Asp and Glu, respectively, under the acidic or basic conditions often used to recover them from proteins. Without special precautions, it is impossible to tell whether a detected Glu was originally Glu or Gln, and likewise for Asp and Asn. The one-letter symbols for the amino acids are also given in Table 3-1. This more compact code is often used when comparing the amino acid sequences of several similar proteins. Note that the one-letter symbol is usually the first letter of the amino acid’s name. However, for sets of residues that have the same first letter, this is true only of the most abundant residue of the set. Amino acid residues in polypeptides are named by dropping the suffi x, usually -ine, in the name of the amino acid and replacing it by -yl. Polypeptide chains are described by starting at the N-terminus and proceeding to the C-terminus. The amino acid at the C-terminus is given the name of its parent amino acid. Thus, the compound is called alanyltyrosylaspartylglycine. Obviously, such names for polypeptide chains of more than a few residues are extremely cumbersome. The tetrapeptide above can also be written as Ala-Tyr-Asp-Gly using the three-letter abbreviations, or AYDG using the one-letter symbols. The structure of the pentapeptide, serylglycyltyrosylalanylleucine (Ser-Gly-Tyr-Ala-Leu, SGYAL) Note that all five amino acids are linked by peptide bonds (shaded groups). R groups are shown in red. When an amino acid sequence of a peptide, polypeptide, or protein is shown, by convention the amino-terminal (N-terminal) end is placed on the left, and the carboxyl-terminal (C-terminal) end is place on the right. The amino acid sequence is read left-toright beginning with the N-terminal end. The various atoms of the amino acid side chains are often named in sequence with the Greek alphabet, starting at the carbon atom adjacent to the peptide carbonyl group. Therefore, as Fig. indicates, the Lys residue is said to have an ε-amino group . Unfortunately, this labeling system is ambiguous for several amino acids. Consequently, standard numbering schemes for organic molecules are also employed . The conventions for labeling the carbon atoms in amino acids is illustrated using lysine in the figure. The a carbon is always carbon2 of the amino acid. The a-carboxyl group is always carbon-1 Stereochemistry KEY CONCEPTS • Amino acids and many other biological compounds are chiral molecules whose confi gurations can be depicted by Fischer projections. • The amino acids in proteins all have the L stereochemical confi guration. With the exception of glycine, all the amino acids recovered from polypeptides are optically active; that is, they rotate the plane of polarized light. The direction and angle of rotation can be measured using an instrument known as a polarimeter . Optically active molecules are asymmetric; that is, they are not superimposable on their mirror image in the same way that a left hand is not superimposable on its mirror image, a right hand. This situation is characteristic of substances containing tetrahedral carbon atoms that have four different substituents. For example, the two molecules depicted in Fig. are not superimposable; they are mirror images. The central atoms in such molecules are known as asymmetric centers or chiral centers and are said to have the property of chirality (Greek: cheir, hand). The Cα atoms of the amino acids (except glycine) are asymmetric centers. Glycine, which has two H atoms attached to its Cα atom, is superimposable on its mirror image and is therefore not optically active. Many biological molecules in addition to amino acids contain one or more chiral centers. Chiral Centers Give Rise to Enantiomers. Molecules that are nonsuperimposable mirror images are known as enantiomers of one another. Enantiomeric molecules are physically and chemically indistinguishable by most techniques. Only when probed asymmetrically, for example, by plane-polarized light or by reactants that also contain chiral centers, can they be distinguished or differentially manipulated. Unfortunately, there is no clear relationship between the structure of a molecule and the degree or direction to which it rotates the plane of polarized light. For example, leucine isolated from proteins rotates polarized light 10.4° to the left, whereas arginine rotates polarized light 12.5° to the right. (The enantiomers of these compounds rotate polarized light to the same degree but in the opposite direction.) It is not yet possible to predict optical rotation from the structure of a molecule or to derive the absolute confi guration (spatial arrangement) of chemical groups around a chiral center from optical rotation measurements.