Three-Dimensional Structures 1 Secondary Structure ‘A. The Peptide Group B. Helical Structures . Beta Structures D. Nonrepetive Structures. 2 Fibrous Proteins Aa Keratin—A Helix of Helices B. Collagen-A Triple Helical Cable 3 Globular Proteins A Interpretation of Protein X Ray and NMR Structures B. Tertiary Structure C. Structural Bioinformatics 4 Protein Stability ‘A. Blectrostatic Forces B. Hydrogen Bonding Forces C. Hydrophobic Forces, D. Disulfide Bonds Protein Denaturation F, Explaining the Stability of Thermostable Proteins 5 Quaternary Structure ‘A. Subunit Interactions B. Symmetry in Proteins €. Determination of Subunit Composition Appendix: Viewing Stereo Pictures The properties of a protein are largely determined by its three-dimensional structure. One might naively suppose that since proteins are all composed of the same 20 types of amino acid residues, they would be more or less alike in their properties. Indeed, pro- tena boce hasalar cab tarinioss eet Robes geneous “average” of their randomly dangling side chains. However, the three-dimensional structure of a yrimary st ‘that it has a unique set of charac- pores In this chapter, we shall discuss the structural features of proteins, the forces that hold them together, and their hier archical organization to form complex structures. This will form the basis for understanding the structure-function re- lationships necessary to comprehend the biochemical roles of Proteins CHAPTER 8 of proteins Detailed consideration of the dynamic behav- ior of proteins and how they fold to their native structures is deferred until Chapter 9. 1 SECONDARY STRUCTURE A polymer's secondary structure (2° structure) is defined as the Igcal conformation of its backbone. For proteins. this has come to mean the specification of fegular polypeptide backbone folding patterns: helices, pleated sheets, and turns However, before we begin our discussion of these ba- sic structural motifs let us consider the geometrical proper- ties of the peptide group because its understanding is prerequisite to that of any structure containing it. ‘A. The Peptide Group In the 19305 and 1940s,'Linus Pauling and Robert Corey determined the X-ray structures of several amino acids and dipeptides in an effort to elucidate the structural constraints on the conformations of a polypeptide chain. ‘These studies indicated that the peptide group has a rigid, Planar structure (Fig. 8-1), which, Pauling pointed out, is a Figure 8-1 ‘The trans-peptide group. The standard dimensio (in angstroms, A, and degrees, ) of this planar group were derived by averaging the corresponding quantities in the X-ra crystal structures of amino acids and peptides. [After Marsh, REE. and Donohue, J, Adv, Protein Chem. 22, 249 (1967).] ‘See Kinemage Exercise 3-1 Scanned with CamScanner222 Chapter &. Thwee-Dimensional Structures of Proteins consequence of resonance interactions that give the peptide bond an ~40% double-bond character: Ro 1 1 H ‘This explanation is supported by the observations that a peptide's C—N bond js 0.13 A shorter than its N—C, single bond and that its C =O bond is 0.02 A longer than that of aldehydes and Ketones. The peptide bond's Fes nance energy has its maximum value, ~85 kJ - mol” when the peptide group is planar because Ts 7-bondIng overlap is maximized in this conformation, This overlap, and thus the resonance energy, falls to zero as the peptide bond is twisted to 90° out of planarity, thereby account ing for the planar peptide group's rigidity. (The positive ‘charge on the above resonance structure should be taken as a formal charge; quantum mechanical calculations in- dicate that the peptide N atom, in fact, has a partial neg- ative charge arising from the polarization of the CN a bond.) Peptide groups, with few exceptions, assume the trans conformation: that in which successive C, atoms are on op- posite sides of the peptide bond joining them (Fig. 8-1) ‘This is partly a result of steric interference, which causes mol"! ese difference is somewhat less in peptide bonds followed by a Pro residue and, in fact, ~10% of the Pro residues in proteins ails aie cena Gomera ca peptides are other- wise extremely rare). Polypeptide Backbone Conformations May Be Described by Their Torsion Angles ‘The above considerations are important because they indicate that the backbone of a protein is a linked se~ quence of rigid planar peptide groups (Fig. 8-3). We can therefore specify a polypeptide's backbone conforma- tion by the t tion angles or dihed: ples) about the CaN bond (6) and the G—C bond (9) ‘Main chain| Figure 8-2 "The dipeptide group. 62 See Kiem of each of its amino acid residues. These angles, and i, are both defined as 180° when the polypeptide chain is in its planar, fully extended (all-trans) conformation and in- crease for a clockwise rotation when viewed from Cy (Fig. 8-4). ‘There are several steric constraints on the torsion an- sles, and J, of a polypeptide backbone that limit its con- formational range. The electronic structure of a single (2) bond, such as a C—C bond, is cylindrically symmetrical about its bond axis, so that we might expect such a bond to ‘exhibit free rotation. If this were the case, then in ethane, for example, all torsion angles about the C—C bond would. ‘be equally likely. Yet certain conformations in ethane are favored due to quantum mechanical effects arising from the interactions of its molecular orbitals. The staggered ‘conformation (Fig. 8-5a, angle = 180°) is ethane’s most stable arrangement, whereas the eclipsed conforma- tion (Fig. 8-5b; torsion angle = 0°) is Teast stable. The en- ergy difference between the staggered and eclipsed con- formations in ethan mol, a quantity that represents an energy barrier to free rotation about the Scanned with CamScannerFigure 8-4 The torsional degrees of freedom in a peptide unit. ‘The only reasonably free movements are rotations about the C.=N bond (6) and the C,—C bond (¥). The torsion angles are ‘both 180° in the conformation shown and increase, a is indicated, in a clockwise manner when viewed from C,. [llustration, Irving Geis. Image from the Irving Geis Collection, Howard Hughes Medical Institute. Reprinted with permission.] 6 See kinemage Exercise 3-1 Section $1. Secondary Structure 223 if nH ; Om iw A i Hh W H (a) Staggered ©) Belipsed Figure 8-5 Conformations of ethane. Newman projections indicating the (a) staggered conformation and (6) eclipsed ‘conformation of ethane. Indeed, with large substituents, some conformations may be sterically forbidden. b. Allowed Contormations of Polypeptides Are Indicated by the Ramachandran Diagram The sterically allowed values of and w can be deter- ‘mined by calculating the distances between the atoms of a tripeptide at all values of ¢ and for the central peptide unit. Sterically forbidden conformations, such as that shown in Fig. 8-6,are those in which any nonbonding inter- atomic distance is less than its corresponding van der Waals, distance. Such information is summarized in a conforma- Figure 8-7 indicates that 77% of the Ramachandran di- agram (most combinations of and y) is conformationally inaccessible to a polypeptide chain. The particular regions of the Ramachandran diagram that represent allowed con- formations depend on the van der Waals radii chosen to calculate it. But with aby feu SeT OT vaTaeS-such as that in Table 8-1, only three small regions of the conformational ‘map are physically accessible to a polypeptide chain. Never- theless, as we shall see, all of the common types of regular secondary structures found in proteins fall within allowed regions of the Ramachandran diagram. Indeed, the Figure 8-6 Stericinterference between adjacent residues. Th collision between a carbonyl oxygen and the following amide hydrogen prevents the conformation ¢ = ~60°,\ = 30°. [Illustration, Irving Geis. Image from the Irving Geis Collectio Howard Hughes Medical Institute. Reprinted with permission 2 see Kinemage Exercise 3-1. Scanned with CamScanner180, 180 90 ° 00 380 (600) Figure 8-7 The Ramachandran diagram. It shows the sterically allowed 6 and angles for poly-t-alanine and was calculated ‘using the van der Waals distances in Table 8-1. Regions of “normaly allowed” ¢ and i) angles are shaded in blue, whereas sgreen-shaded regions correspond to conformations having “outer limit” van der Waals distances. The conformation angles é and, (of several secondary structures are indicated below: Secondary Structure (deg) _y (Gen) Right-handed « helix (a) = = Parallel 8 pleated sheet (Tt) 119 13 AntiparallelB pleated sheet (11) =139 BS Right-handed 3y5 helix (3) 49 -26 [Right-handed helix (x) “37 70 22, ribbon (2) 78 9 Left-handed polyglycine [1 and -79 150 ‘polyproline II helices (II) Collagen (C) st 153 ‘Left-handed « helix (a) 7 "7 (Aster Flory, PI, Statistical Mechanics of Chain Molecules. 253, Interscience (1969): and [UPAC-IUB Commission on Biochemical Nomenclature, Biochemistry 9, 475 (1970) observed conformational angles of most non-Gly residues in proteins whose X-ray structures have been determined lie in these allowed regions (Fig. 8-8). ‘Most points that fall in forbidden regions of Fig. 88 lie between its two fully allowed areas near y = 0. However, these “forbidden” conformations, which arise ftom the col- lision of successive amide groups, are allowed if twists of only a few degrees about the peptide bond are petmitted, This is not unreasonable since the peptide bond offers little resistance to small deformations from planarity. Scanned with CamScanner 180 180 -90 (cee Figure 8-8 Distribution of conformation angles in proteins. ‘The conformation angle distribution ofall residues but Gly in 207 high-resolution (=1.2 A) X-ray structures comprising 25.327 residues is Superimposed on the Ramachandran diagram (resolution is discussed in Section 8-34), [Courtesy of Scott Hollingsworth and Andrew Karplus, Oregon State University, ‘Corvallis, Oregon.] Table 8-1 yan der Waals Distances for Interatomic Contacts Contact Type Normally Allowed (A) Outer Limit (A) WH 20 19 HO 24 2 Hon 2 22 Hc 24 22 0-0 27 26 O=N 27 26 o-c 28 27 Non 27 26 Nec 29 28 cc 30 29 CCH 32 30 CH CH 32 30 ‘Source: Ramachandran, GN. and Saisekharan, V, Adv, Protein Chem. 28, 53261968). Gly, the only residue without a C, atom, is much less sterically hindered than the other amino acid residues. This is clearly apparent in comparing the Ramachandran dia- gram for Gly in a polypeptide chain (Fig. 8-9) with that of other residues (Fig. 8-7). In fact, Gly often occupies posi- tions where a polypeptide backbone makes a sharp turn180 20, widen 180, "180 90 ° 20 180 (ee Figure 8-9 The Ramachandran diagram of Gly residues in polypeptide chain, “Normally allowed” regions are shaded in blue, whereas green-shaded regions correspond to “outer limit” van der Waals distances. Gly residues have far greater ‘conformational freedom than do other (bulkier) amino acid residues, as the comparison of this figure with Fig 8-7 indicates. [After Ramachandran, G.N. and Sasisekharan, V,, Adv Provein Chem. 23, 332 (1968)] ‘Section 8-1. Secondary Structure 225 ‘which, with any other residue, would be subject to steric in- terference. Figure 8-7 was calculated for three consecutive Ala residues. Similar plots for larger residues that are un- branched at C., such as Phe, are nearly identical. In Ra- machandran diagrams of residues that are branched at p,such as Thr, the allowed regions are somewhat smaller than for Ala. The cyclic side chain of Pro limits its $ to the range ~60° + 25°, making it, not surprisingly, the most conformationally restricted amino acid residue. The con- formations of residues in chains longer than tripeptides are even more restricted than the Ramachandran diagram indicates because a polypeptide chain with all its @ and angles allowed nevertheless cannot assume a conforma- tion in which it passes through itself. We shall see, how- ever, that despite the great restrictions that peptide bond planarity and side chain bulk place on the conformations of a polypeptide chain, different unique primary struc- tures have correspondingly unique three-dimensional structures. By Helical Structures E2see Guided Exploration 7: Stable helices in proteins: The « helix Helices are the most striking elements of protein 2° struc- ate polypeptide chain is twisted by the same amount ‘about each of its C, atoms, it assumes a helical conforma- tion. As an alternative to specifying its 6 and y angles, he~ -d by the number, n, of peptide units a lix © SE ones ned Per n=2 Figure 8-10 Examples of helices. These provide definitions of values of n. For m = 2, the helix degenerates to a nonchiral ril For p = 0,the helix degenerates to a closed ring, (Ilustration Irving Geis. Image from the Irving Geis Collection, Howard Hughes Medical Institute. Reprinted with permission.] e the helical pitch, p, the number of repeating units per turn,n, and the helical rise per repeating unit, d = p/n. Right-and left-handed hilices are defined, respectively, as having positive ang negative Scanned with CamScanner226 Chapter 8 Thee-Dimensional Structures of Proteins is it may be either right handed or left handed (a right- handed helix turns in the direction that the fingers of a right hand curl when its thumb points along the helix axis, in the direction that the helix rises). In proteins moreover. ‘need not be an integer and, in fact, rarely is A polypeptide helix must, of course, have conformation angles that fall within the allowed regions of the Ra- machandran diagram. As we have seen, this greatly limits the possibilities. Furthermore. if a particular conformation is to have more than a transient existence, it must be more than just allowed, it must be stabilized. The “glue” that holds polypeptide helices and other 2” structures together part, hydrogen bonds. a Helix ‘Only one helical polypeptide conformation has simulta- neously allowed conformation angles and a favorable hy- drogen bonding pattern: th (Fig. 8-11), a particu- larly rigid arrangement of the polypeptide chain. Us discovery through model building, by. Pauling in 1951, jochemistr. ranks as one of the landmarks of structura bi For a polypeptide made from, andy with conformation angles 6 = +57" but with the same value of p.) Figure §-11 indicates that the hydrogen bonds of an a helix are arranged such that the peptide N—H bond of the nnth residue points along the helix toward the peptide C= group of the (n ~ 4)th residue. This results in a strong hydrogen bofid that has the nearly optimum N ---O. distance of 28 A. In addition, the core of the « helix is tightly packed; that is, its atoms are jn van der Waals con- t helix, thereby maximizing their association energies (Section 8-1Ab). The R groups, whose positions, as we saw, are not fully dealt with by the Ramachandran di- agram, all project backward (downward in Fig. 8-11) and tucward Gr fee Res wo as gr sere exforcoce with the polypeptide backbone Say cach otECE: ‘Such eee nTneir tes ma- jor reason why the left-handed helix has never been ob- served (its helical parameters are but mildly forbidden; Fig. 8-7) is that itgsde chainrontstitsanhnenide backbone too closely. Note, however, that 1 to 2% of the individual non-Gly residues in proteins je this conformation (Fig 8-8). —_— ‘The a helix is a common secondary structural element of both fibrous and globular proteins In globular proteins, a helices have an average span of =12 residues, wo ae responds to over three. However, « helices with re au ». Other Polypeptide Helices Figure 8-13 indicates how hydrogen bonded polypep- tide helices may be constructed. The first tw the, fete and the 3, helix, are described by the notation, Me. Figure 8-11 Theright-handed a helix. Hydrogen bonds between the N—H groups and the C—O groups that are four residues ‘back along the polypeptide chain are indicated by dashed lines. [Wustration, Irving Geis Image from the Irving Geis Collection, Howard Hughes Medical Institute. Reprinted with permission.] GU See Kinemage Exercise 32 and the Animated Figures where m, as before, is the number of residues per helical turn and m is the number of atoms, including H, in the ring that is closed by the hydrogen bond. With this notation, an a helix is Scanned with CamScannerSection 81, Seeondary Structure 227 Figure 8-12 Stereo, space-filling representation of an a helical ‘segment of sperm whale myoglobin (its E helix) as determined by Xoray erystal structure analysis, Backbone atoms are colored according to type (C green, blue, O red, and H white) and the side chain atoms are gold. Instructions for viewing stereo 2 ribbon, diagrams are given in the appendix to this chapter. (Based on an. Xray structure by Ime Schlichting, Max Planck Institut far Molekulare Physiologie, Dortmund, Germany. PDBid LA6M (for the definition of “PDBid.” see Section 8-3Ca).] @2-See Kinemage Exercise 3.2 Figure 8-13 The hydrogen bonding pattern of several polypeptide helices. In the cases shown, the polypeptide chain is Ihelically wound such that the N—H group on residue n forms a hydrogen bond with the C=O groups on fesiduesin ¢ 2,n — 3, = 4,0r n ~ 5. [llustration, Irving Geis. Image from the T Geis Collection, Howard Hughes Medical Institute. Rep with permission.) Scanned with CamScanner6 34g heli Figure 8-14 Comparison ofthe two polypeptide helices that ‘oceasionally occur in proteins withthe commonly occuring @ helix. (a) The 3,9 helix, which has 3.0 peptide units per turn and a pitch of 60 A, making it thinner and more elongated than thea helix. (b) The « helix, which has 3.6 peptide units per turn and a abelix ‘The right-handed 3,9 helix (Fig. 8-14a), which has a pitch of 6.0 A,is thinner and rises more steeply than does the « helix (Fig. 8145). Its torsion angles place it in a mildly for- biden zone of the Ramachandran diagram that is rather near the position of the « helix (Fig. 8-7), and its R groups experience some steric interference. This explains why the 3,9 helix is only occasionally observed in proteins, and then ‘mostly in short segments that are frequently distorted from the ideal 3 conformation (the longest known 3y9 helix in a protein has 15 residues). The 3,9 helix most often occurs as a single-turn transition between one end of an a helix and the adjoining portion of a polypeptide chain. ‘The a helix (4.4. helix), which also has a mildly forbid- den conformation (Fig. 8-7), has only rarely been observed and then only as segments of longer helices. This is proba- bly because its comparatively wide and flat conformation (Fig. 8-14c) results in an axial hole that is too small to admit water molecules but too wide to allow van der Waals asso- ations across the helix axis; this greatly reduces its stabil- ity relative to more closely packed conformations. The 2.2, Scanned with CamScanner hele pitch ofS A (also se Fig 11. (c) The hei, which has 44 Peide units per tum anda pitch of 5.2 A. making it wider and Shorter than the ahelx The peptide planes are indicated. [tusration, ving Ges Image from the Irving Geis Collection, Howard Hughes Medical Insitute. Reprinted with permission] ribbon, which, as Fig. 8-7 indicates, has strongly forbidden conformation angles, has never been observed. Certain synthetic homopolypeptides assume conforma- tions that are models for helices in particular proteins. Polyproline is unable to assume any common secondary structure due to the conformational constraints imposed by its cyclic pyrrolidine side chains. Furthermore, the lack of a hydrogen substituent on its backbone nitrogen pre- cludes any polyproline conformation from being knit to- gether by hydrogen bonding. Nevertheless, under the proper conditions, polyproline precipitates from solution as a left-handed helix of all-trans peptides that has 3.0 residues per helical turn and a pitch of 9.4 A (Fig. 8-15). ‘This rather extended conformation, which is known as the polyproline IT helix, permits the Pro side chains to avoid cach other. Curiously, polyglycine, the least conformation- ally constrained polypeptide, precipitates from solution as a helix whose parameters are essentially identical to those ‘of polyproline, the most conformationally constrained polypeptide (although the polyglycine helix may be eitherright or left handed because Giy isnonchiral) The structures of the polyglycine and polyproline helices are of biological sienificance because they form the basi structural motif of collagen, a structural protein that contains a remarkably high proportion of both Gly and Pro (Section 8-2B). In addi- tion, the polyproline II helical conformation is commonly assumed by polypeptide segments of up to 12 residues, even though it lacks intrahelical hydrogen bonds ¢. Beta Structures 62 se0 Guided Exploration 8: Hydrogren bonding In p sheets and Gulded Exploration 0: Secondary structures In proteins In 1951, the year that they proposed the « helix, Pauling and Corey also (a) Antiparattet © Peratel Figure 8-15 The polyprotine II helix. Polyglycine forms a nearly Section 8.1. Secondary Structure 229 postulated the existence of a different polypeptide second- fey structre the Bplesisd shes As withthe a ei the Pleated sheet’s conformation has repeating and 4 angles that fall in the allowed region of the Ramachandran dia- gram (Fig. 8-7) and utilizes the full hydrogen bonding ca~ Pacity of the polypeptide backbone. In B pleated sheets, however, hydrogen bonding occurs between neighboring: polypeptide chains rather than within one as ina helices. ABleated sheets come in two xasiti 1. The antiparallel B pleated sheet, in which neighbor- ing hydrogen bonded polypeptide chains run in opposite directions (Fig. 8-162). 2. The parallel B pleated sheet, in which the hydrogen bonded chains extend in the same direction (Fig. 8-168). ‘The conformations in which these B structures are opti- mally hydrogen bonded vary somewhat from that of a fully extended polypeptide ( £180"), as indicated in . PC+—N* . Figure 8-16 _® Pleated sheets. Hydrogen bonds are indicated by dashed side chains are omitted for clarity. (a) The antiparallel B pleated sheet. (b)* pleated sheet. [llustration, Irving Geis. Image from the Irving Geis Collec. Hughes Medical Institute. Reprinted with permission.] @2-See Kinemage ¥ the Animated Figures identical helix (polyglyeine ID). [ltustration, Irving Geis. Image from the Irving Geis Collection, Howard ‘Hughes Medical Institute, Reprinted with permission] Scanned with CamScannerFi. 8.7. hey therefore have a rippled or pleated edge-on appearance (Fig. 8-17), which accounts for the appellation “pleated sheet.” In this conformation, successive side chains ‘of a polypeptide chain extend to opposite sides of the pleated sheet with a {wo-residue repeat distance of 0 A, B Sheets are Sopuon sacral eis proteins. In lobular proteins they consist of from 210 as many a8 22 alpeptide strands the average beth 6 strands, which Fkeatapeepte wind ST S25 Ae popped chains ina sheet are known to'be up to 1S zzsidues Jane, with the average beifig 6 cesidues that have a length of ~21 47rd enipaclelB sheet for example, ooeurs in the jack bean protein goncanavalin A (Fig. 8-18). Parallel B sheets of less than five strands are rare, This observation suggests that parallel B sheets are less ‘stable than antiparallel § sheets, possibly because the hy- drogen bonds of parallel sheets are distorted in compa- rison to those of the antiparallel sheets (Fig, 8-16). Mixed parallel-antiparallel B sheets are common but, neverthe- less, only ~20% of the strands in B sheets have parallel ‘bonding on one side and antiparallel bonding on the other (vs an expected 50% for the random mixing of strand directions). . ‘The B pleated sheets in globular proteins invariably ex- bit a pronounced right-handed twist when viewed along, their polypeptide strands (e.g, Fig. 8-19). Such twisted B sheets are important architectural features of globular pro- teins since B sheets often form their central cores (Fig. 8-19). Conformational energy calculations indicate that a B sheet’ right-handed twist isa consequence of nonbonded interactions between the chiral L-amino acid residues in the she’s extended polypeptide chains. These interactions tend to give the polypeptide chains a slight right-handed helical twist (Fig. 819) which distorts and hence weakens the B sheet’s interchain hydrogen bonds. A particular sheet’ geometry is thus the result of a compromise be- ‘ween optimizing the conformational energies of its polypeptide chains and preserving its hydrogen bonds. ‘The topology (connectivity) of the polypeptide strands in aB sheet can be quite complex; the connecting links of these assemblies often consist of long runs of polypeptide chain which frequently contain helices (eg, Fig. 8-19). The link connecting two consecutive antiparallel strands is topologically equivalent toasimple hairpin turn (Fig. 8-20). However, tandem parallel strands must be linked by a crossover connection that is out of the plane of the B sheet. Such crossover connections almost always have a right- handed helical sense (Fig. 8-206), which is thought to better fit the B sheets’ inherent right-handed twist (Fig. 821). Figure 8-17 _A two-stranded 8 antiparallel pleated sheet drawn to emphasize its pleated appearance. Dashed lines indicate hydrogen bonds. Note that the R groups (purple ball), Nonrepetitive Structures ‘on each polypeptide chain alternately extend to opposite sides of ‘ the sheet and that they are in register on adjacent chains. Globular proteins consist of, on average, ~31% a helix,and [Illustration, Irving Geis. Image from the Irving Geis Collection, ~28% f-shees. The protein's remaining polypeptide seg- Howard Hughes Media lute. Repited with permision] ments at sald Bave aga. oy saat, That is €ELSee Kinemage Exercise 3-3 not to say that these nonrepetitive Secondary structures are any less ordered than are helices or B sheets; they are simply irregular and hence more difficult to describe. You should therefore not confuse the term coil conformation with the Scanned with CamScannerFigure 8-18 Stereo, ps representation of the 7-stranded antiparallel B pleated sheet in jack bean concanavalin A as determined by X-ray ‘eystal structure analysis The B strands are approximately horizontal with their backbone atoms colored according to type (C green, N blue, O red,and Hiwhite) and their side chains represented by magenta spheres Instructions for viewing stereo drawings are given in the appendix to this chapter. [Based on an X-ray structure by Gerald Edelman, The Rockefeller University. PDBid 2CNA] 2 See Kinemage wercse 33, Figure 8-19 Polypeptide chain folding in proteins illustrating the right-handed twist of 8 sheets. In these ribbon drawings, the athelices shown as cyan helices, the strands of B sheets are represented by green arrows pointing toward the C-terminus, and the remaining portions of the backbone are drawn as orange ‘worms Side chains are not shown. (a) Bovine carboxypeptidase ‘A, 307-tesidue protein, contains an 8-stranded mixed f sheet that forms a saddle-shaped curved surface witha right-handed twist. (b) Chicken muscle triase phosphate isomerase, & 247-residue enzyme, contains an 8-stranded parallel B sheet that forms a cylindrical structure known as a barrel, here viewed from the top. Note that the crossover connections between successive strands ofthe f barrel, which each consist predominantly of an « helix, are outside the ® barrel and have a right-handed helical sense. (c) Side view of triose phosphate - isomerase. Its N-terminus (N) and,C-terminus (C) are indicated. (Part a based on an X-ray structure by William Lipscomb, Harvard University. PDBid 3CPA. Parts b and c based on an X-ray structure by David Phillips, Oxford Universi PDBid 1TIM.] 62 See interactive Exercise 2 IK. Scanned with CamScannerie 282 Chapier 4 Three-Dimensional Structures of Proteins (a) a © B2Ea eo Figure 8-20 Connections between adjacent polypeptide ftrunds in p pleated sheets. (a) The hairpin connection between Antparalel strands i topologically inthe plane of the sheet. (6) ‘x right handed crossover connection betwen suceaive strands ‘ofa parallel sheet. Nearly all such crossover connections in term random coil, which refers to the totally disordered and rapidly fluctuating set of conformations assumed by dena- tured proteins and other polymers in solution. “ Globular proteins consist largely of approximately straight runs of secondary structure joined by stretches of polypeptide that abruptly change direction. Such reverse turns or B bends (so named because they often connect sutcessive strands of antiparallel B sheets) almost always occur at protein surfaces; indeed, they partially define these surfaces. Most reverse turns involve, four successive — amino acid residues more or less arranged in one of two idues 2 and 3 (Fig. 22). Both types B bends contain a hydrogen bond, although deviations from these ideal confermations often disrupt this hydrogen bond. Type I B bends may be considered to be distorted sections of 3; helix. In Type I bends, the oxygen atom of residue 2 crowds the Cy atm of residue 3, which is there fore usually Gly. Residue 2 of either type of 8 bend is often Pro since it can facilely assume the required conformation. Many proteins have regions that are truly disordered, Extended, charged surface groups such as Lys side chains or the N- or C-termini of polypeptide chains are good ex- amples: They often wave around in solution because there are few forces to hold them in place (Section 8-4). Often entire peptide chain segments are disordered. Such seg- ments may have functional roles, such as the binding of a specific molecule, so they may be disordered in one state of Figure 8-21 Origin of a right-handed crossover connection. A ‘possible folding scheme illustrates how right-handed polypeptide hain twisting favors the formation of right-handed crossover ‘connections between successive strands of a parallel B sheet. proteins have this chirality (se, ¢, Fg 8-196), eosiover connection between paralel sheet Connections with this chirality are rare. [After Richardson.JS., Ads. Prowein Chem. 34,290,295 (1981),] the protein (molecule absent) and ordered in another (molecule bound). This is one mechanism whereby a pro- {cin can interact flexibly with another molecule in the per- oa of ts biological function. 2 FIBROUS PROTEINS Fibrous proteins are highly elongated molecules whose sec- ondary structures are their dominant structural motifs. Many fibrous proteins, such as those of skin, tendon, and bbqngfunction as structural materials that have a protective, ‘connective, or supportive role in living organisms. Others, such as muscle and ciliary proteins, have motive functions In this section, we shall discuss structure-function relation- ships in two common and well-characterized fibrous pro- ins Kgratin and collagen {muscle and cary proteins are considered in Section 35-3). The structural simplicity of these proteins relative to those of globular proteins (Sec- tion 8-3) makes them particularly amenable to understand- ing how their structures suit them to their biological roles. Fibrous molecules rarely crystallize and hence are usu- ally not subject to structural determination by single-crystal X-ray structure analysis (Section 8-3A). Rather than crys- tallizing, they associate as fibers in which their long molec- Uular axes are more or less parallel to the fiber axis bul which they lack specific orientation in other directions. The X-ray diffraction pattern of such a fiber, Fig. 8-23, for Scanned with CamScannerCovalent Structures of Proteins and Nucleic Acids 4 Primary Structure Determination of Proteins [A End Group Analysis: How Many Diferent Types of Subunits? B. Cleavage ofthe Disulfide Bonds C. Separation, Purcation, and Characterization of the Polypeptide Chains . Specific Peptide Cleavage Reactions E. Separation and Purification ofthe Poptde Fragments F. Sequence Determination {G. Ordering the Peptide Fragments H. Assignment of Disulfide Bond Positions |. eptde Characterization and Sequencing by Mass Spectrometry 4. Peptide Mapping 2 Nucleic Acid Sequencing {A The Sanger Method B. Genome Sequencing . Nex! Generation DNA Sequencing Technologies 1. Nucleic Acid Sequencing versus Amino Acid Sequencing 3 Chemical Evolution {A Sick Gell Anemia: The nfiuence of Natural Selection 1. Species Variations in Homologous Proteins: The Elects of Neutral rit . Evolution through Gene Duplication 4 Bioinformatics: An Introduction {A Sequence Databases B. Sequence Afgnment C. Construction of Phylogenetic Trees 5 Chemical Synthesis of Polypeptides ‘A Synthetic Procedures B. Problems and Prospects 6 Chemical Synthesis of Oligonucleotides ‘A Synthetic Procedures BONA Microarrays ©. SELEX Proteins are at the center of the action in biological processes. They function as enzymes, which catalyze the complex set of chemical reactions that are collectively re- ferred to as life. Proteins serve as regulators of these reac- tions, both directly as components of enzymes and indi- rectly in the form of chemical messengers, known as hormones, as well asthe receptors for those hormones. They act to transport and store biologically important substances such a8 metal ions, O, glucose, lipids, and Scanned with CamScanner ETa NATO POUT TTT FTTH [cjororoxaygyarrarec CHAPTER @ many other molecules. In the form of muscle fibers and ‘other contractile assemblies, proteins generate the coor- dinated mechanical motion of numerous biological processes, such as the separation of chromosomes during cell division and the movement of your eyes as you read this page. Proteins, such as rhodopsin inthe retina of your eye, acquire sensory information that is processed through the actions of nerve cell proteins. The proteins of the immune system, such as the immunoglobulins, form an essential biological defense system in higher animals. Proteins are major active elements in, as well as products ion of genetic information. However, pro- portant passive roles, such as that of col lagen, which provides bones, tendons, and ligaments with their characteristic tensile strength. Clearly, there is con- siderable validity to the old cliché that proteins are the “building blocks” of life. The function of DNA as the genetic archive and the as- sociation of RNA with protein synthesis have been known ce the mid-twentieth century. However, it was not until the 1970s that it became clear that RNA can form struc- tures whose complexities rival those of proteins, and it was not until the mid-1980s that it was shown that RNA has biologically important catalytic functions. Protein and nucleic acid function can best be under- stood in terms of thei structures, that is the three-dimen- sional relationships between their component atoms. The structural descriptions of proteins and nucleic acids, as well as those of other polymeric materials, have been t ditionally described in terms of four levels of organization (Fig.7-1) 1. Primary structure (1° structure), which for a protein is the amino acid sequence of its polypeptide chain(s) and for a nucleic acid is its base sequence. 2, Secondary (2°) structure, which is the local spatial arrangement of a polypeptide's or a nucleic acid’s back- bone atoms without regard to the conformations of their side chains or bases. 3. Tertiary (3°) structure, which refers to the three- dimensional structure of an entire polypeptide or polynu- cleotide chain. The distinction between secondary and ter- tiary structures i of necessity, somewhat vague; in practice, the term “secondary structure” alludes to easily character- ized structural entities such as helices. 169 = AC MATT SORCANCAT SO\ WO Chagur 1 Come 1 Sp an « to ana OU ret ray wrt , tenon ere eecamote poten chin Goren ononcgen Secnday sce thn) Figure 7-1. The strectura rach in proteas() Primary structure, (b) secondary 8% (ote stricture. and (d) quaternary strctre[l ving Geis. Image fom sti Conction, Howerd Hopes Media! Insitute Reprinted wth permission) 4. Most proteins are composed of two or more polypep- tide chains loosely referred to as subunits, which associate through noncovalent interactions and, in some cases, disul- fide bonds. A protein's quaternary (4°) structure refers to the spatial arrangement ofits subunits. A nucleic acd's quaternary structure is similarly defined. {In this chapter, we discuss the 1° structures of proteins and nucleic acids: how they are elucidated and their bio- Jogical and evolutionary significance. We also survey the field of bioinformatics as well as methods of chemically synthesizing polypeptide and oligonucleotide chains The ‘and structures of proteins and nucleic acids.as we Shall se, are a consequence of their 1° structures. Fr pro- teins, these topics are treated in Chapters 8 and 9, whereas for nucleic acids, they are discussed mainly in Chapters 29, 31,and 32. 1 PRIMARY STRUCTURE DETERMINATION OF PROTEINS G2es00 cues Explosion 4: Protein sequence determination THe first determination ofthe complete amino acid sequence of 1 protein, that of the bovine polypeptide hormone insulin by Frederick Sanger in 1953, was of enormous biochemical significance in that it definitively established that proteins have unique covalent structures. Since that time, the amino Sc acid sequences often of thousands of proteins have been 1. This extensive information has been of central importance in the formulation of modern concepts of big. chemistry for several reasons: 1. The knowledge of protca’s amino acid sequence essential for an understanding of its molecular mechanism of action as well s being prerequisite for the elucidation of its three-dimensional structure by both X-ray erystal- Jography and suclear magnetic resonance (NMR) spec- troscopy (Section 834), 2, Sequence comparisons among analogous proteins from the same individual, from members of the same species, and from members of related species have yielded important insights into how protein function and have ind- ‘ated the evolutionary relationships among the proteins and the organisms that produce them, These analyses as We shall see in Section 7-3, complement and extend corresponding taxonometrc studies based on anatomical comparisons. 3. Amino acid sequence analyses have important ei cal applications because many inherited diseases are caused by mutations leading to an amino acid change in a protein, Recognition of this fact has led to the develop- ment of valuable diagnostic tests for many such diseases and, in many cases, to symptom-elevng therapy __ The elucidation ofthe Si-tesidue primary structure of insulin (Fig. 7-2) was the labor of many scientists over & anned with CamScanner