14 2.Structure of Proteins 1.Naming the peptide:By convention,the free amino end(N-terminal) A tion of the of the peptide chain is written to the left and the free carboxyl end (C-te Theretore. no aci uences ar in Figure 2.2A.the order of the amino acids is"valine. CHa Linkage of many amino acids through peptide bonds results in an HaC-CH H nbrancned cnal called a polypeptide. HgN-C-coo- C-coc atoms of water are lost in the for H CH mation of the peptide bond.When a polypeptide is named,all Valine amino acid ues have their suffixe (-in e.an.-ic.or ate H04 a tr d of ar ninal valine and a C-terminal leucine is called valvlalycvlleucine. cfge8rpopae 2.c HaC-Ch H bond has a par 'HgN- and is rigid and planar(Figure 2.2B).This prevents free rotation around the bond between the e carbonyl carbon and the nitrogen o the peptide bond.However,the bonds b een th s an by the sized character of the R-groups).This Peptide bond allows the polypeptide chain to assume a variety of possible config urations.The peptide bond is gene ally a trans bond d(instead o B Cha 3.Polarity of the peptide bond:Like all amide linkages,the -C=0 roups o h haf9ei2ananeith9 charged groups present in poly ptides consist solely of the 0 C-N-C C N-terminal (-amino)group.the C-terminal (a-carboxyl)group. C:N group in the the H 0 bond are polar.and are involved in hydrogen bonds.for example. in a-helices and B-sheet structures,described on pp.16-17. Peptide bonds in proteins B.Determination of the amino acid composition of a polypeptide double-bon .Rigid and planar sample of the polypeptide to be analyzed is first hydrolyzed by 。Trans config for 4 hours. his treatment .Uncharged but ato mixture of amino acids is applied to a column that contains a resin to Figure 2.2 which a negatively charged group is tightly atta hed.[Note:If the group is vely cna sho ogaeaiaPePebond ide ent affinities,de nding on their charo es,hydrophobicity,and other of the peptide characteristics.Each amino acid is sequentially released from the chromatography eluting with solutions of inc easing ninhydrin-a reagent that forms a purple compound with most
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 (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.” Linkage of many amino acids through peptide bonds results in an unbranched chain called a polypeptide. Each component amino acid in a polypeptide is called a “residue” because it is the portion of the amino acid remaining after the atoms of water are lost in the formation of the peptide bond. When a polypeptide is named, all amino acid residues have their suffixes (-ine, -an, -ic, or -ate) changed to -yl, with the exception of the C-terminal amino acid. For example, a tripeptide composed of an N-terminal valine, a glycine, and a C-terminal leucine is called valyl glycyl leucine. 2. Characteristics of the peptide bond: The peptide bond has a partial double-bond character, that is, it is shorter than a single bond, and is rigid and planar (Figure 2.2B). This prevents free rotation around the bond between the carbonyl carbon and the nitrogen of the peptide bond. However, the bonds between the α-carbons and 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 allows the polypeptide chain to assume a variety of possible configurations. 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. 3. Polarity of the peptide bond: Like all amide linkages, the – C=O and – NH groups of the peptide bond are uncharged, and neither accept nor release protons over the pH range of 2–12. Thus, the charged groups present in polypeptides consist solely of the N-terminal (α-amino) group, the C-terminal (α-carboxyl) group, and any ionized groups present in the side chains of the constituent amino acids. The – C=O and – NH groups of the peptide bond are polar, and are involved in hydrogen bonds, for example, in α-helices and β-sheet structures, described on pp. 16–17. B. Determination of the amino acid composition of a polypeptide The first step in determining the primary structure of a polypeptide is to identify and quantitate its constituent amino acids. A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid at 110°C for 24 hours. This treatment cleaves the peptide 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 attached group is positively charged, the column becomes an anionexchange column.] The amino acids bind to the column with different affinities, depending on their charges, hydrophobicity, and other characteristics. Each amino acid is sequentially released from the chromatography column by eluting with sol utions 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 Figure 2.2 A. Formation of a peptide bond, showing the structure of the dipeptide valylalanine. B. Characteristics of the peptide bond. C COO– H Valine Valylalanine C +H3N COO– H CH3 Alanine C C H N C COO– H O CH3 H Free carboxyl end of peptide H3C CH CH3 H2O Free amino end of peptide Peptide bond +H3N H3C CH CH3 +H3N A B Trans peptide bond C N H O Cα Cα C N O H Cα Cα Peptide bonds in proteins Partial double-bond character Rigid and planar Trans configuration Uncharged but polar Cis peptide bond R R R R Characteristics of the peptide bond Formation of the peptide bond 14 2. Structure of Proteins 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 14
ll.Primary Structure of Proteins amino acids,ammonia,and amines.The amount of each amino acid is determined spectrophotometrically by measuring the amount of light absorbed by the ninhydrin derivative.The analysis described 8- ed using an amino n automated e componen C.Sequencing of the peptide from its N-terminal end Sequencing is a stepwise process of identifying the specific amino acid n position in t the peptide chain,beginning at the N-termina ions(Fig 2.4).The resulting phenylthiohydantoin(PTH)derivative introduces an instability in the N-terminal pep be applied repeatedly to the shortened peptide obtained ous cycle. D.Cleavage of the polypeptide into smaller fragments □ph omputer from end to end Ho these l es can be clea d a specific sites,and the resulting fragments sequenced.By using eaving agent (enzym es and/or chemicals)on sepa nit th of the ments.thus providing a complete amino acid sequence of the large polypeptide(Figure 2.5).Enzymes that hydrolyze peptide bonds are Figure2.3 terme ategongtiog fthaaepa8ciing ide ptidases.Carb used in determining the C-terminal amino acid.Endopeptidases cleave within a protein.] E.Determination of a protein's primary structure by DNA sequencing edge of the genetic code(see p.431).to translate the sequence of nucleotides into the corresponding amino acid sequence of that Labeling CH Peptid t'9 Shortened peptide PTH-alanine age2aontheanmot ninal residue of a polypeptide by Edman degradation
amino acids, ammonia, and amines. The amount of each amino acid is determined spectro photo metrically by measuring the amount of light absorbed by the ninhydrin derivative. The analysis described above is performed using an amino acid analyzer—an automated machine whose components are depicted in Figure 2.3. C. Sequencing of the peptide from its N-terminal end Sequencing is a stepwise process of identifying the specific amino acid at each position in the peptide chain, beginning at the N- terminal end. Phenylisothiocyanate, known as Edman reagent, is used to label the amino-terminal residue under mildly alkaline conditions (Figure 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 the amino acid derivative can then be determined. Edman reagent can be applied repeatedly to the shortened peptide obtained in each previous cycle. D. Cleavage of the polypeptide into smaller fragments Many polypeptides have a primary structure composed of more than 100 amino acids. Such molecules cannot be sequenced directly 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 separate samples of the purified polypeptide, overlapping fragments can be generated that permit the proper ordering of the sequenced fragments, thus providing a complete amino acid sequence of the large polypeptide (Figure 2.5). Enzymes that hydrolyze peptide bonds are termed peptidases (proteases). [Note: Exopeptidases cut at the ends of proteins, and are divided into aminopeptidases and carboxy peptidases. 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 knowledge of the genetic code (see p. 431), to translate the sequence of nucleotides into the corresponding amino acid sequence of that Figure 2.4 Determination of the amino-terminal residue of a polypeptide by Edman degradation. PTH-alanine N C S NH CH C O CH3 + Shortened peptide H2N Peptide Labeled peptide His Leu Arg COOH C N S H2N C NH S HN CH C CH3 O Phenylisothiocyanate N-terminal alanine CH C CH3 O His Leu Arg COOH His Leu Arg COOH Release of amino acid derivative by acid hydrolysis Labeling Lys Lys Lys 1 2 II. Primary Structure of Proteins 15 Figure 2.3 Determination of the amino acid composition of a polypeptide using an amino acid analyzer. Photometer Light source Separated amino acids Reaction coil Ninhydrin pump Buffer pump Ion exchange column Sample injection Strip-chart recorder or computer 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 15
16 2.Structure of Proteins polypeptide.This indirect process,although routinely used to obtain tide of unknown the amino acid sequences of proteins,has the limitations of not ict the positions of dis ds in the fo osin at lysine 1 eptide(posttranslational modifica tion,see p.443).Therefore,direct protein sequencing is an extremely important tool for determining the true character of the pri Peptide A Peptide B Peptide mary sequence of many polypeptides Ill.SECONDARY STRUCTURE OF PROTEINS what is the? me a ra n thre aocuoag0etaad0earo2aeohenenero6nec2n3 anangerBeshe are termed ne secondary stuciure o the pbolypepude.mne eet,and p -turn are e 2 Thor se A.a-Helix ptide Peptide Y Several different polypeptide helices are found in nature.but the a-helix is the most common.It is a spiral structure,consisting of a Original sequenc of peptide tightly packed,coiled polypeptide ackbone core,with the side chains aoonent mino ac ending o er (F A very diverse group of proteins contains a-helices.For ample.the Figure 2.5 keratins are a family of closely related,fibrous proteins whose struc ure is rely d- are major cyanogen bromide omponen chains.In contrast to keratin,myoglobin,whose structure is also highly a-helical,is a globular,flexible molecule(see p.26). An a-helix is stabilized by extensivehy ns that are art ot the eptide back (see Fiou 2.6.The hydrogen bonds extend up and are parallel to the spiral rom the carbonyl oxygen of one pep tide ond to the -NH group but the first and las are linked to each other through intrachain hydrogen bonds. gse2athorendrduayweak,btheyolecneysone apart in the primary sequence are spatially close together when folded in the a-helix 3.Amino acids that disrupt an a-helix:Proline disrupts an a-heli le its dary amino oup is no Figure 2.6 kinkn the chain which interferes with the smooth.helical struc a-Helix showing peptide backbone. ture.Large numbers of charged amino acids(for example,gluta-
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 chain, and of not identifying any amino acids that are modified after their incorporation into the polypeptide (posttranslational modification, see p. 443). Therefore, direct protein sequencing is an extremely important tool for determining the true character of the primary sequence of many polypeptides. III. SECONDARY STRUCTURE OF PROTEINS The polypeptide backbone does not assume a random three-dimensional structure, but instead generally forms regular arrangements of amino 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 structures frequently encountered in proteins. [Note: The collagen α-chain helix, another example of secondary structure, is discussed on p. 45.] A. α-Helix Several different polypeptide helices are found in nature, but the α-helix is the most common. It is a spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side chains of the component amino acids extending outward from the central axis to avoid interfering sterically with each other (Figure 2.6). A very diverse group of proteins contains α-helices. For example, the keratins are a family of closely related, fibrous proteins whose structure is nearly entirely α-helical. They are a major component of tissues such as hair and skin, and their rigidity is determined by the number of disulfide bonds between the constituent polypeptide chains. In contrast to keratin, myoglobin, whose structure is also highly α-helical, is a globular, flexible molecule (see p. 26). 1. Hydrogen bonds: An α-helix is stabilized by extensive hydrogen bonding between the peptide-bond carbonyl oxygens and amide hydrogens that are part of the polypeptide backbone (see Figure 2.6). The hydrogen bonds extend up and are parallel to the spiral from the carbonyl oxygen of one peptide bond to the – NH – group of a peptide linkage four residues ahead in the polypeptide. This ensures that all but the first and last peptide bond components are linked to each other through intrachain hydrogen bonds. Hydrogen bonds are individually weak, but they collectively serve to stabilize the helix. 2. Amino acids per turn: Each turn of an α-helix contains 3.6 amino acids. Thus, amino acid residues spaced three or four residues apart in the primary sequence are spatially close together when folded in the α-helix. 3. Amino acids that disrupt an α-helix: Proline disrupts an α-helix because its secondary amino group is not geometrically compatible with the right-handed spiral of the α-helix. Instead, it inserts a kink in the chain, which interferes with the smooth, helical structure. Large numbers of charged amino acids (for example, glutaFigure 2.6 α-Helix showing peptide backbone. Side chains of amino acids extend outward Intrachain hydrogen bond N H C O C O N C C N H H O C C C N H O C C O O H N C C N H N H R C C C R R R 16 2. Structure of Proteins Figure 2.5 1. Cleave with trypsin at lysine and arginine Peptide of unknown sequence 2. Determine sequence of peptides using Edman's method What is the correct order? Peptide A Peptide B Peptide X Peptide Y Peptide C 1. Cleave with cyanogen bromide at methionine 2. Determine sequence of peptides using Edman's method 1 2 Original sequence of peptide A B C ? A C B ? B A C ? B C A ? C A B ? C B A ? Overlapping of peptides produced by the action of trypsin and cyanogen bromide. Peptide of unknown sequence 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 16
Ill.Secondary Structure of Proteins other.Finally,amino acids with bulky side chains,such as trypto A n as valine or is that branch Hydrogen bonds between chain present in large numbers. B.B-Sheet 0 of se re ofen vee d sheets."When illus. of polypeptide chains,which are am roheas s are per polypepti pone (se 盒 that are a eltoeach as 27B1 uel to (with all the N-termini of the B-strands together as shown in Figure 2.7C).When the hydrogen bonds are formed between the A R arate polypeptide chains. they are this case.the hydrogen bonds are intrachain bonds.In globular eets er viwep a bone. The a-helix and B-sheet structures provide maximal hydrogen bonding tor peptide bond n the inte C.B-Bends(reverse turns,B-turns) protein molecules,and often include charged residues.Note B-Bends were given this name because they often connect succes gure 2. sive strands of antiparallel B-sheets. -Benas are generally com ne.the am parallel a fr a single acid with the smallest R-group,is also frequently found in B-bends. B-Bends are stabilized by the formation of hydrogen and ionic bonds
mate, aspartate, histidine, lysine, or arginine) also disrupt the helix by forming ionic bonds, or by electrostatically repelling each other. Finally, amino acids with bulky side chains, such as tryptophan, or amino acids, such as valine or isoleucine, that branch at the β-carbon (the first carbon in the R-group, next to the α-carbon) can interfere with formation of the α-helix if they are present in large numbers. B. β-Sheet The β-sheet is another form of secondary structure in which all of the peptide bond components are involved in hydrogen bonding (Figure 2.7A). The surfaces of β-sheets appear “pleated,” and these structures are, therefore, often called “β-pleated sheets.” When illustrations are made of protein structure, β-strands are often visualized as broad arrows (Figure 2.7B). 1. Comparison of a β-sheet and an α-helix: Unlike the α-helix, β-sheets are composed of two or more peptide chains (β-strands), or segments of polypeptide chains, which are almost fully extended. Note also that in β-sheets the hydrogen bonds are perpendicular to the polypeptide backbone (see Figure 2.7A). 2. Parallel and antiparallel sheets: A β-sheet can be formed from two or more separate polypeptide chains or segments of polypeptide chains that are arranged either antiparallel to each other (with the N-terminal and C-terminal ends of the β-strands alternating as shown in Figure 2.7B), or parallel to each other (with all the N-termini of the β-strands together as shown in Figure 2.7C). When the hydrogen bonds are formed between the polypeptide backbones of separate polypeptide chains, they are termed interchain bonds. A β-sheet can also be formed by a single polypeptide chain folding back on itself (see Figure 2.7C). In this case, the hydrogen bonds are intrachain bonds. In globular proteins, β-sheets always have a right-handed curl, or twist, when viewed along the polypeptide backbone. [Note: Twisted β- sheets often form the core of globular proteins.] The α-helix and β-sheet structures provide maximal hydrogen bonding for peptide bond components within the interior of polypeptides. C. β-Bends (reverse turns, β-turns) β-Bends reverse the direction of a polypeptide chain, helping it form a compact, globular shape. They are usually found on the surface of protein molecules, and often include charged residues. [Note: β-Bends were given this name because they often connect successive strands of antiparallel β-sheets.] β-Bends are generally composed of four amino acids, one of which may be proline—the amino acid that causes a “kink” in the polypeptide chain. Glycine, the amino acid with the smallest R-group, is also frequently found in β-bends. β-Bends are stabilized by the formation of hydrogen and ionic bonds. Figure 2.7 A. Structure of a β-sheet. B. An antiparallel β-sheet with the β-strands represented as broad arrows. C. A parallel β-sheet formed from a single polypeptide chain folding back on itself. Polypeptide chains almost fully extended Antiparallel β-pleated sheet Parallel β-pleated sheet C-terminal N-terminal N-terminal C-terminal N-terminal C-terminal O R - C - H H N N C C N C C - R H C H O R - C H O H H H C A B C Hydrogen bonds between chains III. Secondary Structure of Proteins 17 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 17
18 2.Structure of Proteins M) N 修计i Helix-oop-helix B-g-B unit B-Meander B-Barrel Figure 2.8 structural motifs mbining o-helices and/or B-sheets.The names describe their schematic D.Nonrepetitive secondary structure Approximately one half of an average globular protein is organized mpetive structures,such as the a-helix and/or B-she as having a loop o random.but rather simply have a less regular structure than those described above.[Note:The term"random coil"refers to the disor dered structure obtained when proteins are denatured (see p.20).] E.Supersecondary structures(motifs) Globular proteins are constructed by combining secondary structural elements (a-helices,B-sheets,nonrepetitive sequences).These TwoSXtene are connected by oop( the CH duced by packing side chains from adjacent secondary structural B-shes to oach other.Thus,for example.a-helices and 0ac more common motifsare iustrated in Fiqure 28 wN-c AAAV Proteins that bind to numt function as transcription factors(see p.455). IV.TERTIARY STRUCTURE OF GLOBULAR PROTEINS The primary structure of a polypeptide chain determines its tertiary e folding of domains (the basic units arr of de id n The str Figure 2.9 proteninquouiniscomwithaigdensity(cls producing one cystine residue
D. Nonrepetitive secondary structure Approximately one half of an average globular protein is organized into repetitive structures, such as the α-helix and/or β-sheet. The remainder of the polypeptide chain is described as having a loop or coil conformation. These nonrepetitive secondary structures are not “random,” but rather simply have a less regular structure than those described above. [Note: The term “random coil” refers to the disordered structure obtained when proteins are denatured (see p. 20).] E. Supersecondary structures (motifs) Globular proteins are constructed by combining secondary structural elements (α-helices, β-sheets, nonrepetitive sequences). These form primarily the core region—that is, the interior of the molecule. They are connected by loop regions (for example, β-bends) at the surface of the protein. Supersecondary structures are usually produced by packing side chains from adjacent secondary structural elements close to each other. Thus, for example, α-helices and β-sheets that are adjacent in the amino acid sequence are also usually (but not always) adjacent in the final, folded protein. Some of the more common motifs are illustrated in Figure 2.8. Proteins that bind to DNA contain a limited number of motifs. The helix-loop-helix motif is an example found in a number of proteins that function as transcription factors (see p. 455). IV. TERTIARY STRUCTURE OF GLOBULAR PROTEINS The primary structure of a polypeptide chain determines its tertiary structure. “Tertiary” refers both to the folding of domains (the basic units of structure and function, see discussion below), and to the final arrangement of domains in the polypeptide. The structure of globular proteins in aqueous solution is compact, with a high-density (close packing) of the atoms in the core of the molecule. Hydrophobic side chains are buried in the interior, whereas hydro philic groups are generally found on the surface of the molecule. Figure 2.8 Some common structural motifs combining α-helices and/or β-sheets. The names describe their schematic appearance. Helix-loop-helix β-α-β unit β-Meander β-Barrel 18 2. Structure of Proteins Figure 2.9 Formation of a disulfide bond by the oxidation of two cysteine residues, producing one cystine residue. C C H CH2 N O H Two cysteine residues H N C H CH2 SH SH C C H C O CH2 N O H Polypeptide backbone Disulfide bond Oxidant (for example, O2) 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 18