Chapter 6

Proteins: Secondary, Tertiary, and Quaternary Structure

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Chapter Outline
v Protein conformation: Three-dimensional shape Amino acid sequence contains information to fold protein into conformation Weak forces act on amino acids to stabilize conformation: Hydrogen bonding, hydrophobic interactions, electrostatic interactions, van der Waals forces v Secondary Structure: Structural elements formed by peptide plane interactions and angles: Describe orientation of peptide planes about alpha carbons Peptide Plane Carbonyl oxygen -H bond acceptor - Amide hydrogen -H bond donor Alpha Helix (Pauling and Corey) - Peptide carbonyl of ith residue H bonded to peptide H of i+4th residue - 3.613: 3.6 residues per turn, 13 atoms between carbonyl oxygen and amide hydrogen - 0.54 nm per turn, 0.15 nm per residue along helix (Z) axis - H bonds parallel to helix axis Other helices - 310: 3 residues per turn 10 atoms between carbonyl oxygen and amide hydrogen - 27 ribbon - 4.416 or helix Beta pleated sheets - Polypeptide chain fully extended - Chains or strands joined by H bonds - Parallel sheets - Antiparallel sheets Beta turn - Carbonyl of ith residue H bonded to amide nitrogen of i+3 - Peptide plane in cis - Gly or Pro common Beta bulge: Structural irregularity in beta sheets due to extra or additional residue in one strand v Tertiary structure principles Secondary structures formed when possible Secondary structures associate and pack to form layers Elements between secondary structures are short and direct Stability of final fold - Large number of intramolecular H bonds - Reduction is surface area v Fibrous Proteins: Proteins whose polypeptide chains are parallel to a single axis Keratin: helical coiled coils

Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure Subunit: 311-314 residue-long helix-rich region flanked by nonhelical regions - Two-stranded coiled coils with helix repeat of 0.51 nm - 7-residue quasi repeat with 1st and 4th residues nonpolar - Disulfide bonds: Rigid, inextensible, and insoluble structure Fibroin and -keratin: Stacked sheets Collagen: A triple helix: Connective tissue protein in animals - Tropocollagen: three intertwined chains - Amino acid composition and amino acid sequence One-third Gly and one-third Pro or Hyp Modified amino acids: Hydroxyproline and hydroxylysine (vitamin Cdependent reaction) Gly-Pro-Hyp repeat with G placed in center of three-stranded coil Globular proteins Core composed of hydrophobic amino acids Peptide groups often H bonded as helices or sheets - Surface helices amphiphilic - Internal helices or sheets hydrophobic - Solvent exposed helices hydrophilic Packing of elements results in formation of small cavities imparting flexibility to structure Ordered regions with well-defined, nonrepetitive structure Disordered, flexible regions Molecular motion: Proteins are dynamic structures Atomic fluctuations: Movement over small distances Collective motions: Movement of groups of atoms Conformational changes: Movement of large domains Chain folding Structural stability Right-handed structures - Right-handed twists in sheets - Right-handed cross-overs joining secondary structural elements -strand connections - Antiparallel strands connected by hairpins - Parallel strands connected by cross-overs forming loop Hydrophobic amino acids sequestered in interior: Layers - Two layers of backbone form a single hydrophobic core - Three layers of backbone form two hydrophobic cores - Four-layered and five-layered structures Globular protein classification based on secondary structure Antiparallel helix Parallel or mixed sheet Antiparallel sheet Metal- and Disulfide-rich Molecular chaperones: Protein complexes that catalyze process of protein folding Protein design: Domains: Regions 40-100 amino acids long that form stable tertiary structures Quaternary structure Subunits typically fold into independent globular structures Subunits interactions - Isologous interactions: Identical faces of identical subunits - Heterologous interactions: Interacting surfaces not identical Symmetry - Cyclic symmetry: Single rotational axis - Dihedral symmetry: 2-fold axis perpendicular to n-fold axis Subunit association forces 78

v v v

Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure - Hydrophobic interactions between faces - Disulfide bond stabilized Polymers: Open quaternary structures v Structural and functional advantages to quaternary associations Protein stabilized by reduction in surface-to-volume ratio Genetic economy: Encode self-assembling subunits rather than single complex protein Catalytic sites brought together v Cooperativity: Influence on catalytic sites by neighboring sites

Chapter Objectives
The key to understanding protein structure is to understand the weak forces responsible for maintaining a protein in the folded state: hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces. It will be helpful to review the structures of the amino acids and to recall the kinds of weak interactions each amino acid side chain is capable of making. Peptide Bond Know the characteristics of the peptide bond: the fact that it is planar because of -bonding and resonance stabilization and the fact that it has a hydrogen bond donor and an acceptor. Secondary Structure Protein secondary structures are regular structures formed by hydrogen bonds between amide planes. Understand the structure of the -helix, the 2 7 ribbon and the -helix. If you understand how the -helix is stabilized by hydrogen bonds, the other two helices are simply the structures formed when hydrogen bonds form between groups one residue closer or one residue further along the polypeptide chain. Sheet structures form between fully extended peptide chains. Beta-turns are tight turns stabilized by a hydrogen bond and requiring cis orientation of amide planes. The -helix and -sheet are shown in Figures 6.6 and 6.11. Tertiary Structure The tertiary structure of a protein is its three-dimensional structure. Fibrous proteins have secondary structure elements arranged parallel to a single axis. Examples include -keratin and collagen. Be familiar with the structures of these two proteins. Globular proteins are by far the most abundant class of proteins. As additional protein structures are solved by x-ray crystallography and nuclear magnetic resonance, a greater understanding of the rules governing protein folding and structure stabilization will unfold. Currently a majority of globular proteins are classified into four broad groups: antiparallel -helix, parallel or mixed -sheet, antiparallel -sheet, and the small metal- and disulfide-rich proteins. Quaternary Structure Quaternary structure is reserved for proteins composed of multiple subunits. The subunits represent distinct structural domains that interact in specific ways to form the native protein. Protein Denaturation The three-dimensional shape of a functional protein is called the conformational change to a state or states lacking activity is known denaturation may occur when the weak forces, responsible for the disrupted. Denaturing agents or conditions include: heat, high guanidine HCl, high salt concentrations, low ionic strength solutions, extremes in pH.

native conformation. A as denaturation. Protein native conformation, are concentrations of urea, SDS, organic solvents, or

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

Figure 6.6 Four different graphic representations of the -helix. (a) As it originally appeared in Pauling's 1960 The Nature of the Chemical Bond . (b) Showing the arrangement of peptide planes in the helix. (c) A space-filling computer graphic presentation. (d) A "ribbon structure" with an inlaid stick figure, showing how the ribbon indicates the path of the polypeptide backbone. (Irving Geis)

Figure 6.11 The arrangement of hydrogen bonds in (a) parallel and (b) antiparallel pleated sheet.

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

Problems and Solutions

1. The central rod domain of a keratin protein is approximately 312 residues in length. What is the length (in ) of the keratin rod domain? If this same peptide segment were a true -helix, how long would it be? If the same segment were a -sheet, what would its length be? Answer: -keratin has extensively distorted -helical secondary structure. The helix repeats every 3.6 residues but has a pitch of 0.51 nm (compared to 0.54 nm for a true -helix).
312 residues = 86.7 turns residues 3.6 turn nm 86.7 turns 0.51 = 44.2 nm = 442 turn

For an -helix of the same number of residues: nm 86.7 turns 0.54 = 46.8 nm = 468 turn For pleated sheets, the distance between residues is 0.347 nm for antiparallel sheets and 0.325 nm for parallel sheets. Thus, nm 312 residues antiparallel sheet 0.347 =108.3 nm =1083 residue nm 312 residues parallel sheet 0.325 =101.4 nm =1014 residue 2. A teenager can grow 4 in. in a year during a "growth spurt". Assuming that the increase in height is due to v ertical growth of collagen fibers (in bone), calculate the number of collagen helix turns synthesized per strand per minute. Answer: Four inches of growth corresponds to
cm =10.16 cm in How many collagen helix turns does 10.16 cm represent? The collagen helix has the following parameters: 0.29 nm per residue; 3.3 residues per turn; and 0.96 nm/turn. How many turns in 10.16 cm. (Note: 1cm = 10 -2m, 1nm = 10 -9m 1cm = 10 7nm) 10.16 10 7 nm =1.06 10 8 turns nm 0.96 turn 4 in 2.54 1.06 10 8 turns turns = 201 365days 24hr 60min min 1yr yr day hr

3. Discuss the potential contributions to hydrophobic and van der Waals interactions and ionic and hydrogen bonds for the side chains of Asp, Leu, Tyr and His in a protein. Answer: Aspartic acid has a relatively small side chain composed of a -CH2- group and a carboxyl group. The presence of the ionizable carboxyl group indicates that aspartic acid can participate in ionic bonds. In addition, lone-pair electrons on the oxygen of the carboxyl group can participate in H bonds, as can the hydrogen when the carboxyl group is protonated. Hydrophobic interactions and van der Waals interactions are negligible. The leucine side chain is an alkane and as such will not participate in hydrogen bonds or ionic bonds. The side chain is hydrophobic and relatively bulky, indicating that it will participate in hydrophobic interactions and is capable of entering numerous van der Waals interactions. Tyrosine has a phenolic group attached to the carbon by a methylene bridge (i.e., -CH2-). The phenolic group is weakly ionizing with a pKa of 10. Thus, only under special conditions or environments would it be expected to participate in ionic bonds. The hydroxyl group can both donate and accept H bonds. When protonated, tyrosine is capable of

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

hydrophobic interactions, and, because it is a bulky amino acid, it is expected to participate in numerous van der Waals interactions. The imidazole side chain of histidine has an ionizable nitrogen and, when protonated, allows histidine to participate in ionic bonds. In addition, hydrogen bond donor and acceptor groups support participation in hydrogen bonds. Thus, the ability to form both hydrogen bonds and ionic bonds precludes hydrophobic bonding. The large side chain is expected to participate in van der Waals interaction. 4. Figure 6.38 shows that Pro is the amino acid least commonly found in -helices but most commonly found in -turns. Discuss the reasons for this behavior. Answer: Proline is an imido acid; it has a secondary nitrogen with only one hydrogen. When participating in a peptide bond, the nitrogen no longer has hydrogen bound to it. Thus, peptide bonds in which the nitrogen is supplied by proline are incapable of functioning as hydrogenbond donors. A prolyl residue will interrupt -helices when located on the C-terminal end of a helix. If located within 3 residues of the N terminus, proline is capable of hydrogen bonding through its carbonyl oxygen. The -bend requires a cis peptide bond and proline stabilizes the cis conformation. Recall a -bend involves hydrogen bonding of the carbonyl oxygen of a peptide bond with an amide hydrogen three residues away. Usually, adjacent peptide bonds are trans but for -bend formation a cis conformation is required. 5. For the flavodoxin in Figure 6.31 identify the right-handed cross-overs and the lefthanded cross-overs in the parallel -sheet. Answer: For a right-handed cross-over, moving in the N-terminal to C-terminal direction, the cross-over moves in a clockwise direction. The reverse is true for a left-handed cross-over, namely, movement from N-terminus to C-terminus is accompanied by counterclockwise rotation. Although not necessary it is helpful to identify the N- and C-terminus of the protein. For the Nterminus this is accomplished by starting on any strand of -sheet and moving along the opposite direction of the arrow until an end is encountered. This locates the N-terminus at the bottom of the second strand of -sheet. Starting on the N-terminus the chain connects to the first strand making a left-handed cross-over. The first strand is connected to the third, the third to the forth, and the forth to the fifth all with right-handed cross-overs. The diagram below illustrates these points.

Right

Left

Right

Right

6. Choose any three regions in the Ramachandran plot and discuss the likelihood of observing that combination of and in a peptide or protein. Defend your answer using suitable molecular models of a peptide. Answer: The Ramachandran plot reveals allowable values of and . The plots consider steric hindrance and will be somewhat specific for individual amino acids. For example, glycine has more allowable and angles in -helical conformations than do bulky amino acid like phenylalanine. 7. A new protein of unknown structure has been purified. Gel filtration chromatography reveals that the native protein has a molecular weight of 240,000. Chromatography in the presence of 6 M guanidine hydrochloride yields only a peak for a protein of Mr 60,000. Chromatography in the presence of 6 M guanidine hydrochloride

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

and 10 mM -mercaptoethanol yields peaks for proteins of M r 34,000 and 26,000. Explain what can be determined about the structure of this protein from these data. Answer: Guanidine hydrochloride is a powerful denaturing agent that disrupts tertiary and quaternary structure by disrupting hydrogen bonds. In the presence of 6M guanidine-HCl, only a 60 kD species is observed, indicating that the native protein may in fact be a tetramer of four 60 kD subunits. Upon denaturation in the presence of -mercaptoethanol (a disulfide reducing agent), two protein peaks are observed, one at 34 kD and one at 26 kD. This indicates that the 60 kD subunit is a heterodimer of two chains, 34 kD and 26 kD, held together by a disulfide bond. 8. Two polypeptides, A and B, have similar tertiary structures, but A normally exists as a monomer, whereas B exists as a tetramer, B 4. What differences might be expected in the amino acid composition of A versus B? Answer: Oligomeric proteins are held together by a number of forces including hydrogen bonds, ionic bonds, hydrophobic interactions, van der Waals interactions, and covalent, disulfide bonds. Of these interactions, we might expect hydrophobic interactions to play a major part in subunit associations. (Subunit associations involve interactions of two or more surfaces.) In comparing a monomeric protein with a homologous, polymeric protein, interacting regions in the polymer may reveal themselves as sequences of hydrophobic amino acid residues. 9. The hemagglutinin protein in influenza virus contains a remarkably long -helix with 53 residues. a. How long is this -helix (in nm)? b. How many turns does this helix have? c. Each residue in an -helix is involved in two H bonds. How many H bonds are present in this helix? Answer: The helix repeats every 0.54 nm (3.6 amino acid residues) and the distance along the helical axis between residues is 0.15 nm. An -helix of 53 residues is: 53 residues nm 0.54 = 7.95nm or, residues turn 3.6 turn nm 53 residues 0.15 = 7.95nm residue The number of turns is: 53 residues = 14 .7 turns residues 3.6 turn To calculate the number of H bonds, the ith residue is H-bonded to the (i + 4)th residue to form a helix. For a 53-residue helix, the carbonyl groups of the first 49 residues are involved in H bonding (i + 4 = 53; , i = 49). The amide H for the four residues on the N terminal side are not hydrogen-bonded nor are the carbonyl groups of the last 4 residues, on the C-terminal side. Thus, 49 hydrogen bonds are made. 10. It is often observ ed that Gly residues are conserved in proteins to a greater degree than other amino acids. From what you hav e learned in this chapter, suggest a reason for this observation. Answer: In considering protein structure we learned that globular proteins are compact structures composed of short helices and sheets. In order to form a compact structure the polypeptide chain must make sharp bends and this is often accomplished by having a glycine residue in the bend. Because glycine has a small side chain, it is easily accommodated in a tight bend. 11. Which amino acids would be capable of forming H bonds with a lysine residue in a protein?

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

Answer: Lysine has an amino group in its side chain, which at neutral pH is positively charged. Further, the amino hydrogens are capable of functioning as hydrogen-bond donors. So, amino acid residues with hydrogen bond acceptors should be able to form hydrogen bonds with a lysine residue. This would include aspartic and glutamic acid, which could also interact via ionic bonding. Serine and threonine both contain hydroxyl groups, which can function as a Hbond acceptors. Asparagine and glutamine would also be expected to participate in H-bonding as H-bond acceptors through their carbonyl carbon on their side chains. Other possibilities include unprotonated histidine, unprotonated lysine and arginine. The side chains cysteine and tyrosine might also interact as hydrogen bond donors. 12. Poly-L-glutamate adopts an -helical structure at low pH but becomes a random coil above pH 5. Explain this behavior. Answer: The side chain of glutamic acid contains a carboxylic acid group with a pKa of 4.25. At pH 5.0 glutamic acids side chain is largely ionized with a 1- charge. Thus, polyglutamic acid at this pH has negative charges, which will repel each. At pH 2.0, the side chain is protonated and thus uncharged making it possible for the peptide chain to form an -helix, which brings amino acids closer together than in random coil. 13. Imagine that the dimensions of t he alpha helix were such that there were exactly 3.5 amino acids per turn, instead of 3.6. What wo uld be the consequences for coiled-coil structures? Answer: In a helix with 3.5 residues per turn, after two turns each additional residue would line up with a residue in the first two turns. (In other words, in a wheel plot one would see only seven angles occupied by residues.) Thus, two helices could interact by simply lining up sideby-side, provided the helices are longer than two turns. For the -helix the helices would have to be longer than 5 turns for this to occur. Thus, significant numbers of interactions between side chains in a coiled-coil made up of -helices would only occur if the helices wrapped around each other. 14. Consider the following peptide sequences: EANQIDEMLYNVQSLTTLEDTVPW LGVHLDITVPLSWTWTLYVKL QQNWGGLVVILTLVWFLM CNMKHGDSQCDERTYP YTREQSDGHIPKMNCDS AGPFGPDGPTIGPK Which of the preceding sequences would be likely to be found in each of the following: a. A parallel -sheet b. An antiparallel -sheet c. A tropocollagen molecule d. The helical portions of a protein found in your hair? Answer: To help identify -sheets we can look at distributions of hydrophobic amino acids, which are typically distributed on one side of an antiparallel sheet and both sides of a parallel sheet. To help we will display hydrophobic amino acids in bold. (The font is changed to Courier, a non-proportional font, to allow for alignment of the sequence with the residue numbers.)

123456789012345678901234 EANQIDEMLYNVQSLTTLEDTVPW LGVHLDITVPLSWTWTLYVKL QQNWGGLVVILTLVWFLM CNMKHGDSQCDERTYP YTREQSDGHIPKMNCDS AGPFGPDGPTIGPK

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

The second sequence has hydrophobic amino acids at nearly every other residue. If this sequence were in -sheet structure it would have a hydrophobic surface and a hydrophilic surface. Thus, sequence two is likely to be an antiparallel sheet. The first and third sequences also contain significant numbers of hydrophobic amino acids. For the third sequence, if it were in -sheet structure it would have hydrophobic amino acids on both sides of the sheet consistent with a parallel sheet. The first sequence also has nearly an equal distribution of hydrophobic amino acids at oddnumbered and even-numbered positions, which would be expected to support parallel sheet formation. However, the first sequence seems to have hydrophobic amino acids distributed evenly throughout its length. If fact, starting at the alanine in position 2, one sees hydrophobic amino acids at positions 5 (an I) and 8 (an M), which is reminiscent of the primary structure of -keratins with quasi repeating segments, seven-residues long and containing hydrophobic amino acids at positions 1, 4 and 7. If arranged in an -helix, this would put hydrophobic amino acids on one face of the helix. Thus, the first sequence is a good candidate for the helical portions of a protein found in hair. Of the remaining sequences, the best fit for a tropocollagen sequence is the sixth sequence. Tropocollagen is a triple helix of a special type formed by intertwining three chains together. The chains have a three-residue repeat, Gly-x-y, with x and y often being proline (and or hydroxyproline). Programs designed to identify parallel and antiparallel sheets have been developed and several may be found at SwissProt (http://us.expasy.org/sprot/) under Proteomics tools (http://us.expasy.org/tools/). Under Primary Structure Analysis the link to ProtScale gives several useful programs to analyze primary protein structure. The figure below was constructed by analyzing each of the sequences using beta-sheet /Chou & Fasman. There are also programs listed that will analyze parallel and antiparallel sheets. Sequences are entered into the text box. The programs calculate the probability of a particular structure using a scoring matrix, which gives the likelihood of finding a particular amino acid is a structure. Average scores are calculated over a window of amino acids. Before activating the program, change the window size to the smallest allowable. Because several of the sequences are quite small it may help to add glycines to each end of the sequences. Data for the figure was generated using ten Gs on each end of each of the sequences. After the program was activated the results were reported b oth graphically and numerically by activating the link at the bottom of the results page. The numerical results were pasted into a Microsoft Word document as unformatted text using Paste Special. The data were converted from text to table and the values were then transferred to Excel. Sequences 1, 2 and 3 all score high, relative to 4, 5 and 6 in the Chou and Fasman analysis in addition to antiparallel and parallel sheet programs. Sequence 3 scores highest as a parallel sheet.

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 10

Chou and Fasman Beta Sheet

20

30

40

50

Sequence Position
15. To fully appreciate the elements of secondary structure in proteins, it is useful to have a practical sense of their structures. On a piece of paper, draw a simple but large zigzag pattern to represent a -strand. Then fill in the structure, drawing the locations of the atoms of the chain on this zigzag pattern. Then draw a simple, large coil on a piece of paper to represent an a-helix. Then fill in the structure, drawing the backbone atoms in the correct locations along the coil and indicating the locations of the R groups in your drawing. Answer: For the beta-pleated sheet, the zigzag below represents alpha carbons of the polypeptide backbone. The lines connecting the alpha carbons contain the elements of the peptide plane with the carbonyl oxygen alternating out of or into the plane of the paper (because the most stable conformation of adjacent peptide planes is trans). The R group and hydrogen are added to the alpha carbons as shown. To make an extended sheet, strands would be aligned side-byside in parallel or antiparallel fashion.

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

H C C O N C

C H2 N C COOH

R1

R3

R5

R7

R9

R11

H 2N R2 R4 R6 R8 R 10

COOH

To visualize how strands of a sheet are joined by hydrogen bonds, antiparallel and parallel extended peptide chains are drawn below. The peptide planes alternate (trans conformation).

R1
+

O C N H

R2 C H C O

H N

R3 C H

O C N H

R4 C H C O

H N

R5 C H

O C N H

R6 C H C O

H N

R7 C H COO-

H3N

C H

H
-

O N H C

H C R6

H N C O

H C R5 N H

O C

H C R4

H N C O

H C R3 N H

O C

H C R2

H N C O

H C R1 NH 3
+

OOC

C R7

R1
+

O C N H

R2 C H C O

H N

R3 C H

O C N H

R4 C H C O

H N

R5 C H

O C N H

R6 C H C O

H N

R7 C H COO -

H 3N

C H

R1
+

O C N H

R2 C H C O

H N

R3 C H

O C N H

R4 C H C O

H N

R5 C H

O C N H

R6 C H C O

H N

R7 C H COO -

H 3N

C H

Draw hydrogen bonds between H-bond donors and acceptors to see how the strands of a sheet are held together. For an alpha helix, the carbonyl oxygen of the ith amino acid residue is hydrogen bonded to the hydrogen on the nitrogen of the (i + 4)th amino residue. Use the template below to show this connection. Then, count atoms moving from the oxygen of the carbonyl group to the hydrogen. You should count 13 atoms. Do the same on the remaining templates for other helices ( i to i + 3, i + 2 and i = 5).

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

R1 O
+

R2 N C H H C O

R3 O C

R4 N C C H H O

R5 O C

R6 N C C H H O

R7

O C

R8 N C C H H O

R9 O C

R10 N C H H C O

R11

H3N C H

N C H

N C H

N C H

N C H

N C COO H

R1 O
+

R2 N C C H H O

R3 O C

R4 N C H H C O

R5 O C

R6 N C C H H O

R7

O C

R8 N C C H H O

R9 O C

R10 N C C H H O

R11

H3N C H

N C H

N C H

N C H

N C H

N C COO H

R1 O
+

R2 N C C H H O

R3 O C

R4 N C H H C O

R5 O C

R6 N C C H H O

R7

O C

R8 N C C H H O

R9 O C

R10 N C H H C O

R11

H3N C H

N C H

N C H

N C H

N C H

N C COO H

R1 O
+

R2 N C H H C O

R3 O C

R4 N C H H C O

R5 O C

R6 N C H H C O

R7

O C

R8 N C H H C O

R9 O C

R1 0 N C H H C O

R 11 COO
-

H 3N C H

N C H

N C H

N C H

N C H

N C H

Questions for Self Study

1. List the weak interactions responsible for maintaining protein conformation. 2. For the structures shown below indicate by D, A, D&A, or B if the structure contains only a hydrogen bond donor, only a hydrogen bond acceptor, a donor and separately an acceptor, or a group that functions both as donor or acceptor.

O a. C O b. NH2 c. OH d. C N H
3. Fill in the blanks. The -helix is an example of a structural element. It is a helical structure formed by between groups in peptide bonds. In particular, the of the ith residue of an helix is bonded to the of the (i+4)th residue. The result is a helical structure that repeats every residues. The of the helix, or distance along the z-axis per turn, is 0.54 nm. This arrangement orients the amide planes to the helix axis and the side chains ____ to the helix axis. 4. Proteins composed predominantly of -pleated sheets may be expected to form structures that are flexible yet inextensible. Please explain. 5. What two amino acids are most suited to beta-turns? 6. What posttranslational modification is necessary to produce collagen and what water soluble vitamin is this modification dependent on? 7. Give two examples of fibrous proteins. 8. What are the four globular protein groups found in nature? 9. The agents listed below are all expected to affect protein tertiary structure. For each give a brief explanation of how they affect protein structure. a. Urea b. SDS c. High temperature d. -mercaptoethanol e. Distilled water

e.

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

f. Organic solvents 10. What are the advantages to quaternary associations?

Answers
1. Hydrogen bonds, hydrophobic interactions, electrostatic interactions, van der Waals interactions. 2. a./A; b./D; c./B; d./D&A; e./A 3. Secondary; hydrogen; bonding; carbonyl; oxygen; amide; nitrogen; 3.6; pitch; parallel; perpendicular. 4. The polypeptide chain in a -pleated sheet is already fully extended; however, the chains are free to bend. 5. Proline and glycine. 6. Hydroxylation; vitamin C. 7. -Keratin, silk fibroin proteins, collagen. 8. Antiparallel -helix; parallel or mixed disulfide-rich proteins.
-sheet; antiparallel -sheet; small metal- and

9. Urea will disrupt hydrogen bonds. SDS is a detergent that will disrupt the hydrophobic cores of proteins. mercaptoethanol will reduce disulfide bonds. Distilled water will perturb ionic interactions. Organic solvents will interact with hydrophobic amino acid side chains. 10. Genetic economy and efficiency; structural stability due to reduction of surface-to-volume ratio; formation of catalytic centers; cooperativity.

Additional Problems
1. For the following conditions or agents, explain how they may denature proteins: heat, urea, guanidine HCl, distilled water, SDS, liquid phenol. 2. An often-cited example of protein denaturation is the change accompanying the heating of egg-whites. Clearly, this process is irreversible yet the classic experiments of Anfinsen on RNase A showed that denaturation is reversible. Rectify these two seemingly conflicting facts. 3. In the movie Papillon, a criminal, played by the late Steve MacQueen, is imprisoned on a remote tropical island for many years. Needless to say, conditions in prison are not idyllic; the prisoners are served only enough gruel to keep them alive. In one scene, the main character reaches into his mouth and pulls out a tooth. What disease might the prisoner have suffered from? 4. Gelatin desserts are prepared by adding hot water to a dry powder. This popular dessert is made from collagen from pig skins. Given your understanding of the structure of collagen explain the gel-like consistency of gelatin. 5. Myoglobin was the first globular protein whose structure was solved by x-ray crystallography. The protein contains a hydrophobic core formed by the sides of several helices. For human myoglobin, the sequence of helix E is given below. For this helix, label the hydrophobic residues likely to contribute to the hydrophobic core. E Helix Sequence: S E D L K K H G A T V L T A L G G I L

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

Abbreviated Answers
1. Gentle heating will disrupt hydrogen bonds. The structures of urea and guanidine HCl are shown below:

O H2N C Urea NH2 H2 N

NH2+ Cl C

NH2

Guanidinium HCl

Urea and guanidine HCl are both very soluble in water and may be used to form highly concentrated solutions. These agents denature proteins by disrupting hydrogen bonds. The low ionic strength and high dielectric constant of distilled water cause changes in ionic bonds that may lead to protein denaturation. SDS is an ionic detergent that disrupts hydrophobic interactions. In solution, SDS forms micelle structures by association of their hydrocarbon tails. Micelles will form around hydrophobic amino acid side chains, disrupting the normal hydrophobic interactions these side chains make. L iquid phenol will disrupt hydrophobic interactions. 2. Egg whites contain about 10% by weight of ovalbumin and, while it is true the heat of a hot frying pan is sufficient to denature the protein, the real problem comes subsequently. The denatured protein readily aggregates in this highly concentrated protein solution and the aggregation is for all intents and purposes irreversible. Anfinsen's experiments were done at much lower protein concentrations (and with a considerably smaller protein). 3. The disease was probably scurvy caused by a deficiency of vitamin C. Cross-linking of collagen fibers is catalyzed by prolyl hydroxylase in a vitamin C-dependent reaction. A deficiency of vitamin C results in incomplete cross-linking leading to defective collagen fibers. Collagen fibers are found in connective tissues, which are thus weakened in scurvy. 4. Collagen fibers hydrate and interact to form a tangled mass of crisscrossed fibers. 5. The residues in helix E are plotted in a helical wheel plot to identify the location of amino acids on the helix surface. To construct a helical wheel plot, a projection of the residues in a helix is made along the helical axis (Z-axis) onto the X-Y plane. In an -helix, the helix repeats every 3.6 residues. Thus, each residues is 100 apart. The helical wheel plot is shown below with hydrophobic amino acids highlighted.

Leu 19
Gly 8 Ser 1 0 330 30

Leu 12
Lys 5 Gly 16 60 Ala 9

Leu 15 Leu 4 Val 11

270

300

90 Glu 2

Ile 18
His 7 240 210 150 180 Thr 10 Gly 17

120 Thr 13

Ala 14

Lys 6

Asp 3

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

The plot clearly shows that hydrophobic amino acids (in bold face) are concentrated along one side of the helix. This is the side of the helix that faces the hydrophobic core.

Summary
Proteins are composed of linear chains of amino acids held together by peptide bonds. The sequence of amino acids is the primary structure of the protein. It contains all the information necessary to fold up the chains into a functional protein. This folding involves weak forces such as hydrogen bonds, electrostatic interactions, hydrophobic interactions and van der Waals interactions. The peptide bond itself is capable of participating in hydrogen bonds with both hydrogen-bond donor and acceptor groups. For example, the carbonyl group has two pairs of lone-pair electrons capable of accepting hydrogen bonds. Also, the electronegative nitrogen induces a partial positive charge on its attached hydrogen, allowing the hydrogen to function as a hydrogen-bond donor. Hydrogen bonding between peptide bond groups is the basis of protein secondary structure elements, namely, helices, pleated sheets and turns. The side chains of the amino acids are capable of a variety of interactions including, hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions. These interactions and secondary structural elements are responsible for the tertiary structure of proteins, their actual three-dimensional shape. The experiments of Anfinsen and White showed that the information to fold a peptide chain into a protein is contained in the primary structure. Working with ribonuclease, a small protein capable of hydrolyzing phosphodiester bonds in RNA, they showed that enzymatic activity is dependent on the native conformation of the protein. The protein contains numerous disulfide bridges that stabilize the native state. By reducing these bonds and treating the protein with a denaturing agent, activity is lost. However, the activity is regained if the denaturant is first removed, allowing the chain to fold into its native conformation before disulfide bonds are allowed to reform. Secondary structure elements include helices, pleated sheets and -turns. Stable helices are a result of hydrogen bond formation between donor and acceptor group s in peptide bonds. The -helix is a right-handed helix (in moving from the N terminus to the C terminus, rotation is clockwise) formed by the carbonyl oxygen of the ith residue hydrogen bonded to the amide hydrogen of the (i + 4)th residue. The helix makes one turn every 0.54 nm and has 3.6 amino acid residues per turn. Amino acids are spaced 0.15 nm along the helix axis The hydrogen bonds are parallel to the helical axis, with the peptide-bond planes and the amino acid side chains perpendicular. The -helix is also referred to as a 3.6 13 helix; it has 3.6 residues per turn with the hydrogen-bonded groups separated by 13 atoms. Other helical structures are known that involve formation of hydrogen bonds between the carbonyl of the ith amino acid and the amide nitrogen at i + 2, (2 7 ribbon), i + 3, (3 10), and i + 5, (4.4 16 or helix). When the polypeptide chain is in an extended state, -pleated sheets may form by hydrogen bonding between parallel chains (parallel -pleated sheets) or antiparallel chains (-pleated sheets). The disposition of carbons in these structures forms a pleated pattern. The peptide chain is often required to make sharp turns (as for example when antiparallel pleated sheets are formed) and the -turn is often used to accomplish this. In this secondary structure element, a sharp turn is stabilized by hydrogen bonding between the carbonyl of a peptide bond with the amide hydrogen 2 residues away. In a polypeptide chain, peptide bonds are usually trans but in the -turn a cis arrangement is required. Proline and glycine are often found in -turns because they can assume the cis configuration. Some proteins are dominated by secondary structure. In -keratin, found in hair, wool, claws, fingernails, and horns of animals, two-stranded coiled coils composed of distorted helices predominate. Connective tissue, including tendons, cartilage, bones, teeth, skin, and blood vessels, contains collagen, whose structure is dominated by a triple helix. The collagen helix has 3.3 residues per turn, a fact reflected in its amino acid sequence. The primary structure is dominated by repeats of Gly-Pro-Hyp. Glycines are located on the helix interior, making contact with each other to form the three stranded structure. In contrast to these helical proteins, the fibroin protein of silk fibers is composed of stacked antiparallel -sheets. The largest class of proteins is that of the globular proteins. Here, helices, sheets, and turns are used to form a globular structure held together by weak forces. Hydrophobic interactions play a major role in globular protein structure; the core is often formed by hydrophobic amino

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Chapter 6 . Proteins: Secondary, Tertiary, and Quaternary Structure

acid side chains. Structural motifs consisting of secondary structure elements are often components contributing to protein structure, as for example -sheet arrays with right-handed twists and -loops. The globular proteins can be classified based on the type and arrangement of secondary structure. In antiparallel helix proteins, the core structure is a bundle composed of antiparallel helices. Parallel or mixed -sheet proteins have an extended sheet of structure. The sheet may be internal, in which case hydrophobic amino acids are distributed on both sides of the sheet. Alternatively, the sheet may be arranged in a barrel shape. Antiparallel -sheet proteins have two sheets, each composed of antiparallel -sheets. Finally, metal-rich proteins and disulfide-rich proteins rely on either metal binding sites or numerous disulfide bonds to maintain a stable globular structure. Some proteins appear to be composed of more than one structural domain and each of the domains serves a particular role in protein function. Often, a distinct structural domain is a protein module dedicated to a particular function. Quaternary structure is reserved for oligomeric proteins, proteins composed of distinct subunits. When the subunits are identical, the protein is a homomultimer; when the subunits are different, the protein is a heteromultimer. In multimeric proteins, the subunits typically fold into independent structures and make isologous (same surface) or heterologous (different surface) interactions with other subunits in the protein. The driving forces for these interactions are hydrophobic in nature and the interactions may be stabilized by disulfide bonds.

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