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subunits in a molecule that are no farther apart than the

length of the cross-link (assuming, of course, that the

proper amino acid residues are present). The molecular

mass of a cross-linked protein therefore places a lower

limit on its number of subunits. Such studies can also pro-

vide some indication of the distance between subunits, par-

ticularly if a series of cross-linking agents with different

lengths is employed.

Appendix 271

Figure 8-70 Stereo drawing of a

tetrahedron inscribed in a cube. When

properly viewed, the apex of the

tetrahedron should appear to be pointing

toward the viewer.

Although we live in a three-dimensional world, the images

that we see have been projected onto the two-dimensional

plane of our retinas. Depth perception therefore involves

binocular vision: The slightly different views perceived by

each eye are synthesized by the brain into a single three-

dimensional impression.

Two-dimensional pictures of complex three-dimensional

objects are difficult to interpret because most of the informa-

tion concerning the third dimension is suppressed.This infor-

mation can be recovered by presenting each eye with the im-

age only it would see if the three-dimensional object were

actually being viewed.A stereo pair therefore consists of two

images, one for each eye. Corresponding points of stereo

pairs are generally separated by ⬃6 cm, the average distance

between human eyes. Stereo drawings are usually computer

generated because of the required precision of the geometric

relationship between the members of a stereo pair.

In viewing a stereo picture, one must overcome the vi-

sual habits of a lifetime because each eye must see its cor-

responding view independently. Viewers are commercially

available to aid in this endeavor. However, with some

training and practice, equivalent results can be obtained

without their use.

To train yourself to view stereo pictures, you should be-

come aware that each eye sees a separate image. Hold your

finger up about a foot (30 cm) before your eyes while fixing

your gaze on some object beyond it. You may realize that

you are seeing two images of your finger.If, after some con-

centration, you are still aware of only one image, try blink-

ing your eyes alternately to ascertain which of your eyes is

seeing the image you perceive. Perhaps alternately cover-

ing and uncovering this dominant eye while staring past

your finger will help you become aware of the independent

workings of your eyes.

The principle involved in seeing a stereo picture is to vi-

sually fuse the left member of the stereo pair seen by the

left eye with the right member seen by the right eye.To do

this, sit comfortably at a desk, center your eyes about a

foot over a stereo drawing such as Fig. 8-70 and stare

through it at a point about a foot (30 cm) below the draw-

ing. Try to visually fuse the central members of the four

out-of-focus images you see. When you have succeeded,

your visual system will “lock onto” it and this fused central

image will appear three-dimensional. Ignore the outer im-

ages.You may have to slightly turn the book, which should

be held perfectly flat, or your head in order to bring the

two images to the same level. It may help to place the

book near the edge of a desk, center your finger about a

foot below the drawing, and fixate on your finger while

concentrating on the stereo pair. Another trick is to hold

your flattened hand or an index card between your eyes so

that the left eye sees only the left half of the stereo pair

and the right eye sees only the right half and then fuse the

two images you see.

The final step in viewing a stereo picture is to focus on

the image while maintaining fusion. This may not be easy

because our ingrained tendency is to focus on the point at

which our gaze converges. It may help to move your head

closer to or farther from the picture. Most people (includ-

ing the authors) require a fair amount of practice to be-

come proficient at seeing stereo without a viewer. How-

ever, the three-dimensional information provided by

stereo pictures, not to mention their aesthetic appeal,

makes it worth the effort. In any case,the few stereo figures

used in this textbook have been selected for their visual

clarity without the use of stereo; stereo will simply enhance

their impression of depth.

APPENDIX Viewing Stereo Pictures

JWCL281_c08_221-277.qxd 2/23/10 1:59 PM Page 271

272 Chapter 8. Three-Dimensional Structures of Proteins

1 Secondary Structure The peptide group is constrained

by resonance effects to a planar, trans conformation. Steric in-

teractions further limit the conformations of the polypeptide

backbone by restricting the torsion angles, ␾ and ␺, of each

peptide group to three small regions of the Ramachandran di-

agram. The ␣ helix, whose conformation angles fall within the

allowed regions of the Ramachandram diagram, is held to-

gether by hydrogen bonds. The 3

helix, which is more tightly

coiled than the ␣ helix, lies in a mildly forbidden region of the

Ramachandran diagram. Its infrequent occurrences are most

often as single-turn terminations of ␣ helices. In the parallel

and antiparallel ␤ pleated sheets, two or more almost fully ex-

tended polypeptide chains associate such that neighboring

chains are hydrogen bonded. These ␤ sheets have a right-

handed curl when viewed along their polypeptide chains. The

polypeptide chain often reverses its direction through a ␤ bend.

Other arrangements of the polypeptide chain, which are col-

lectively known as coil conformations, are more difficult to

describe but are no less ordered than are ␣ or ␤ structures.

2 Fibrous Proteins The mechanical properties of fibrous

proteins can often be correlated with their structures. Keratin,

the principal component of hair, horn, and nails, forms

protofibrils that consist of two pairs of ␣ helices in which the

members of each pair are twisted together into a left-handed

coil.The pliability of keratin decreases as the content of disul-

fide cross-links between the protofibrils increases. Collagen is

the major protein component of connective tissue. Its every

third residue is Gly, and many of the others are Pro and Hyp.

This permits collagen to form a ropelike triple helical struc-

ture that has great tensile strength. Collagen molecules aggre-

gate in a staggered array to form fibrils that are covalently

cross-linked by groups derived from their His and Lys side

chains. Mutations in collagen or the inactivation of the en-

zymes that process it usually cause a loss in the structural in-

tegrity of the affected tissues.

3 Globular Proteins The accuracies of protein X-ray

structure determinations are limited by crystalline disorder to

resolutions that are mostly in the range 1.5 to 3.0 Å. This re-

quires that a protein’s structure be determined by fitting its

primary structure to its electron density map. Several lines of

evidence indicate that protein crystal structures are nearly

identical to their solution structures. The structures of small

proteins may also be determined in solution by 2D- or multi-

dimensional-NMR techniques which, for the most part, yield

results similar to those of X-ray crystal structures.

A globular protein’s 3° structure is the arrangement of its

various elements of 2° structure together with the spatial dis-

positions of its side chains. Its amino acid residues tend to

segregate according to residue polarity. Nonpolar residues

preferentially occupy the interior of a protein out of contact

with the aqueous solvent, whereas charged polar residues are

located on its surface. Uncharged polar residues may occur at

either location but, if they are internal, they form hydrogen

bonds with other protein groups.The interior of a protein mol-

ecule resembles a crystal of an organic molecule in its packing

efficiency. Larger proteins often fold into two or more do-

mains that may have functionally and structurally independ-

ent properties. Certain groupings of secondary structural ele-

ments, known as motifs or supersecondary structures,

repeatedly occur as components of globular proteins. They

combine in numerous ways to form a fold, that is, the tertiary

structure of a domain. Among the common folds in globular

proteins are the globin fold, 4-helix bundles, various ␤ barrels

(up-and-down, Greek key, and jelly roll), the ␣/␤ barrel, and

the various open ␤ sheets. The atomic coordinates of the

⬃70,000 proteins and nucleic acids whose structures have

been determined are archived in the Protein Data Bank

(PDB). These macromolecules may be visually examined

through the use of a variety of molecular graphics programs

and may be analyzed by programs that compare and classify

their structures in various ways.

4 Protein Stability Proteins have marginally stable na-

tive structures that form as a result of a fine balance among

the various noncovalent forces to which they are subject: ionic

and dipolar interactions, hydrogen bonding, and hydrophobic

forces. Ionic interactions are relatively weak in aqueous solu-

tions due to the solvating effects of water.The various interac-

tions among permanent and induced dipoles, which are collec-

tively referred to as van der Waals forces, are even weaker and

are effective only at short range. Nevertheless, because of their

large numbers, they cumulatively have an important influence

on protein structures. Hydrogen bonding forces are far more

directional than are other noncovalent forces. They add little

stability to a protein structure, however, because the H bonds

that native proteins form internally are only marginally

stronger than those that unfolded proteins form with water.

Yet, because most H bonds are local, a protein can only fold

stably in ways such that almost all of its possible internal H

bonds are formed. Hence H bonding is important in specifying

the native structure of a protein. Hydrophobic forces arise

from the unfavorable ordering of water structure that results

from the hydration of nonpolar groups. By folding such that its

nonpolar groups are out of contact with the aqueous solvent, a

protein minimizes these unfavorable interactions.The fact that

most protein denaturants interfere with the hydrophobic ef-

fect demonstrates the importance of hydrophobic forces in

stabilizing native protein structures. Disulfide bonds often sta-

bilize the native structures of extracellular proteins. The pro-

teins from (hyper)thermophilic organisms have higher T

’s

than their mesophilic counterparts. Several hyperthermophilic

proteins are stabilized by extensive surface networks of salt

bridges.

5 Quaternary Structure Many proteins consist of nonco-

valently linked aggregates of subunits in which the subunits

may or may not be identical. Most oligomeric proteins are ro-

tationally symmetric.The protomers in many fibrous proteins

are related by helical symmetry. The quaternary structures of

proteins are best elucidated using X-ray or NMR techniques.

In their absence, cross-linking studies can indicate the subunit

composition of an oligomeric protein.

CHAPTER SUMMARY

JWCL281_c08_221-277.qxd 2/23/10 1:59 PM Page 272

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274 Chapter 8. Three-Dimensional Structures of Proteins

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Problems 275

Bioinformatics Exercises are available at www.wiley.com/college/voet

Chapter 8

Visualization of Three-Dimensional Protein Structures Using Jmol and Pymol

1. Obtaining Structural Information. Compare different secondary structure predictions for a given protein sequence, then inspect its

X-ray crystallographic structure.

2. Exploring the Protein Data Bank. Learn how to locate and download specific protein structure files, sequences, and images. Explore

additional educational resources and links to additional structural biology resources. Examine a protein structure file and use Jmol

and Pymol to visualize the protein and highlight selected features.

3. Protein Families. Identify homologous proteins in other structural databases.

BIOINFORMATICS EXERCISES

1. What is the length of an ␣ helical section of a polypeptide

chain of 20 residues? What is its length when it is fully extended

(all trans)?

*2. From an examination of Fig. 8-7, it is apparent that the

polypeptide conformation angle ␾ is more constrained than is ␺.

By referring to Fig. 8-4, or better yet, by examining a molecular

model, indicate the sources of the steric interference that limit the

allowed values of ␾ when ␺⫽180°.

3. For a polypeptide chain made of ␥-amino acids, state the

nomenclature of the helix analogous to the 3

helix of ␣-amino

acids. Assume the helix has a pitch of 9.9 Å and a rise per residue

of 3.2 Å.

*4. Table 8-7 (page 276) gives the torsion angles, ␾ and ␺,of

hen egg white lysozyme for residues 24–73 of this 129-residue pro-

tein. (a) What is the secondary structure of residues 26–35? (b)

What is the secondary structure of residues 42–53? (c) What is the

probable identity of residue 54? (d) What is the secondary struc-

ture of residues 56–68? (e) What is the secondary structure of

residues 69–71? (f) What additional information besides the tor-

sion angles, ␾ and ␺, of each of its residues is required to define

the three-dimensional structure of a protein?

5. Hair splits most easily along its fiber axis, whereas finger-

nails tend to split across the finger rather than along it. What are

the directions of the keratin fibrils in hair and in fingernails? Ex-

plain your reasoning.

6. What is the growth rate, in turns per second, of the ␣ helices

in a hair that is growing 15 cm ⴢ year

⫺1

7. Can polyproline form a collagenlike triple helix? Explain.

8. As Mother Nature’s chief engineer, you have been asked to

design a five-turn ␣ helix that is destined to have half its circum-

ference immersed in the interior of a protein. Indicate the helical

wheel projection of your prototype ␣ helix and its amino acid se-

quence (see Fig. 8-43a).

9. ␤-Aminopropionitrile is effective in reducing excessive scar

tissue formation after an injury (although its use is contraindi-

cated by side effects). What is the mechanism of action of this

lathyrogen?

*10. Using your network browser, visit the Protein Data Bank

(PDB) at http://www.rcsb.org/ (for this exercise, you should use

some other browser besides Safari because it is not fully compati-

ble with CE). To explore the structure of ␥-B crystallin, click the

“Enter a PDB id or keyword” button, enter “4GCR” in the field to

its right, and click the “Site Search” button. On the left of the

“Structure Summary” window that comes up, click on “PDB File”

under “Display Files” and inspect the file that appears. (a) How

many residues does this protein have and how many water

molecules have been found to be associated with it in this crystal

structure? (b) Draw skeletal diagrams of an Arg, a Glu, and a Tyr

residue and label their atoms using the nomenclature employed in

the PDB file. (c) What are the atomic coordinates of the S atom of

Cys 32? What is the identity of atom 1556, which is labeled

“OXT”? (d) Return to the Structure Summary page and, under

“Display Options” on the right, examine the structure of the pro-

tein using the viewer of your choice. Can you see that the protein

is composed of two well-separated and apparently similar do-

mains? (e) To structurally classify this protein, return to the Struc-

ture Summary page and state the different ways that SCOP and

CATH (near the bottom of the page) classify this protein. (f) To

structurally compare the protein’s two domains, point your

browser to CE (http://cl.sdsc.edu/) and,in the subsequent window,

click on “Two Chains.” In the window that comes up, for both

Chain 1 and Chain 2, click on the “PDB:” button, enter “4GCR” in

the box to its right, and check the “Use Fragment From:” box.

Then for Chain 1 enter the range 1 to 83 and for Chain 2 enter the

range 84 to 174. Then click on the “Calculate Alignment” button

(at the top). The window that appears shows the aligned se-

quences of the two peptides based on their structure. What is the

percent identity of the two peptides (upper right)? Describe the

gaps, if any, in the alignment. Now click on the “Press to Start

Compare3D” button.The window that comes up shows the super-

imposed C

␣

backbones of the two peptide segments in blue and

magenta. Rotate this model by clicking on it and dragging your

mouse. Describe what you see. What is the “RMSD (A)” (root

mean square deviation in Å) of the superimposed backbone seg-

ments? What is the significance of the short white segment of

chain? What are the two gray segments? Click on specific residues

in the sequences below to highlight their positions in the aligned

structures.

*11. Using a molecular graphics viewer, inspect the struc-

tures of the following proteins as indicated by their PDBids.

Draw the corresponding topological diagrams and name the

fold, if it is a standard one, for each domain in the protein. (a)

1 RCP, (b) 1 RCB, (c) 1 TNF, (d) 2 CMD, (e) 1 RHD, and (f) 2 TAA.

[Note: Although all of these proteins consist of only one type of

subunit, some of the PDB files contain the coordinates for more

PROBLEMS

JWCL281_c08_221-277.qxd 2/23/10 1:59 PM Page 275

276 Chapter 8. Three-Dimensional Structures of Proteins

Table 8-7 Torsion Angles (␾, ␺) for Residues 24 to 73 of Hen Egg White Lysozyme

Residue Number Amino Acid ␾ (deg) ␺ (deg)

24 Ser ⫺60 147

25 Leu ⫺49 ⫺32

26 Gly ⫺67 ⫺34

27 Asn ⫺58 ⫺49

28 Trp ⫺66 ⫺32

29 Val ⫺82 ⫺36

30 Cys ⫺69 ⫺44

31 Ala ⫺61 ⫺44

32 Ala ⫺72 ⫺29

33 Lys ⫺66 ⫺65

34 Phe ⫺67 ⫺23

35 Glu ⫺81 ⫺51

36 Ser ⫺126 ⫺8

37 Asn 68 27

38 Phe 79 6

39 Asn ⫺100 109

40 Thr ⫺70 – 18

41 Glu –84 – 36

42 Ala –30 142

43 Thr –142 150

44 Asn ⫺154 121

45 Arg ⫺91 136

46 Asn ⫺110 174

47 Thr ⫺66 ⫺20

48 Asp ⫺96 36

49 Gly 95 ⫺75

50 Ser ⫺18 138

51 Thr ⫺131 157

52 Asp

⫺115 130

53 Tyr ⫺126 146

54 xxx 67 ⫺179

55 Ile ⫺42 ⫺37

56 Leu ⫺107 14

57 Gln 35 54

58 Ile ⫺72 133

59 Asn ⫺76 153

60 Ser ⫺93 ⫺3

61 Arg ⫺83 ⫺19

62 Trp ⫺133 ⫺37

63 Trp ⫺91 ⫺32

64 Cys ⫺151 143

65 Asn ⫺85 140

66 Asp 133 8

67 Gly 73 ⫺8

68 Arg ⫺135 17

69 Thr ⫺122 83

70 Pro ⫺39 ⫺43

71 Gly ⫺61 ⫺11

72 Ser ⫺45 122

73 Arg ⫺124 146

Source: Imoto, T., Johnson, L.N., North,A.C.T., Phillips, D.C., and Rupley, J.A., in Boyer, P.D. (Ed.), The

Enzymes (3rd ed.),Vol. 7, pp. 693–695, Academic Press (1972).

JWCL281_c08_221-277.qxd 2/23/10 1:59 PM Page 276

Problems 277

than one chain. Your task will be simplified if you display only

one of these chains (their structures are essentially identical). If

you use the stand-alone version of Jmol as your viewer (a Jmol tu-

torial is on the “Student Companion Site” at http://www.wiley.com/

college/voet/), this is easily done by typing, for example,“restrict

*a” into the Console window to restrict the display to the A

chain. For proteins consisting of more than one domain, inspect

each domain individually using the same technique (for exam-

ple, after typing “restrict *a”, typing “restrict 1-20” will display

only residues 1–20 of the A chain). A polypeptide chain is most

easily followed if it is displayed in Backbone, Cartoons, Ribbons,

Strands, or Trace form and assigned Group colors by opening the

Console and entering the command “color group all” (which

colors the chain, from N- to C-terminus, in rainbow order, blue

to red).]

12. It is often stated that proteins are quite large compared to

the molecules they bind. However, what constitutes a large mole-

cule depends on your point of view. Calculate the ratio of the vol-

ume of a hemoglobin molecule (65 kD) to that of the four O

mol-

ecules it binds. Also calculate the ratio of the volume of a typical

office (4 ⫻ 4 ⫻ 3 m) to that of a typical office worker (70 kg) that

occupies it. Assume that the molecular volumes of hemoglobin

and O

are in equal proportion to their molecular masses and that

the office worker has a density of 1.0 g ⴢ cm

⫺3

. Compare these ra-

tios. Is this the answer you expected?

13. Why are London dispersion forces always attractive?

14. Membrane-bound proteins are generally closely associ-

ated with the nonpolar groups of lipid molecules (Section 12-3A).

Explain how detergents affect the structural integrity of mem-

brane-bound proteins in comparison to their effects on normal

globular proteins.

15. The coat protein of tomato bushy stunt virus consists of

180 chemically identical subunits, each of which is composed of

⬃386 amino acid residues. The probability that a wrong amino

acid residue will be biosynthetically incorporated in a polypeptide

chain is 1 part in 3000 per residue. Calculate the average number

of coat protein subunits that would have to be synthesized in or-

der to produce a perfect viral coat. What would this number be if

the viral coat were a single polypeptide chain with the same num-

ber of residues that it actually has?

16. State the rotational symmetries of each of the following

objects: (a) a starfish, (b) a square pyramid, (c) a rectangular box,

and (d) a trigonal bipyramid.

*17. Through the use of your favorite molecular graphics

viewer, state the rotational symmetries of the proteins with the

following PDBids: (a) 1 TIM, (b) 1 TNF, (c) 6 PFK, and (d) 1 AIY.

[Note: When using Jmol, individual polypeptide chains are most

easily differentiated by displaying the protein in Backbone, Car-

toons, Ribbons, Strands, or Trace form and assigning them Chain

colors (e.g., Color ⬎ Structure ⬎ Cartoon ⬎ By Scheme ⬎ Chain),

which makes each polypeptide chain a different color).]

18. Myoglobin and the subunits of hemoglobin are polypep-

tides of similar size and structure. Compare the expected ratio of

nonpolar to polar amino acid residues in myoglobin and in hemo-

globin.

19. Sickle-cell hemoglobin (HbS) differs from normal human

adult hemoglobin (HbA) by a single mutational change, Glu

␤6 S Val, which causes the HbS molecules to aggregate under

proper conditions (Section 7-3A). Under certain conditions, the

HbS filaments that form at body temperature disaggregate when

the temperature is lowered to 0°C. Explain.

20. Indicate experimental evidence that is inconsistent with

the hypothesis that urea and guanidinium ion act to denature pro-

teins by competing for their internal hydrogen bonds.

21. Proteins in solution are often denatured if the solution is

shaken violently enough to cause foaming. Indicate the mecha-

nism of this process. (Hint: The nonpolar groups of detergents ex-

tend into the air at air–water interfaces.)

22. An oligomeric protein in a dilute buffer at pH 7 dissoci-

ates to its component subunits when exposed to the following

agents. Which of these observations would not support the con-

tention that the quaternary structure of the protein is stabilized

exclusively by hydrophobic interactions? Explain. (a) 6M guani-

dinium chloride, (b) 20% ethanol, (c) 2M NaCl, (d) temperatures

below 0°C, (e) 2-mercaptoethanol, (f) pH 3, and (g) 0.01M SDS.

*23. The SDS–polyacrylamide gel electrophoresis of a protein

yields two bands corresponding to molecular masses of 10 and 17

kD. After cross-linking this protein with dimethylsuberimidate

under sufficient dilution to eliminate intermolecular cross-linking,

SDS–polyacrylamide gel electrophoresis of the product yields 12

bands with molecular masses 10, 17, 20, 27, 30, 37, 40, 47, 54, 57, 64,

and 74 kD. Assuming that dimethylsuberimidate can cross-link

only contacting subunits, diagram the quaternary structure of the

protein.

*24. Mammals possess two genetically distinct but closely re-

lated forms of lactate dehydrogenase (LDH), the M type (which

predominates in skeletal muscle) and the H type (which predom-

inates in heart tissue). Before the X-ray structure of LDH was

known, the oligomeric state of LDH was determined by dissociat-

ing M type and H type LDH to their component subunits and then

reconstituting the mixture. This treatment yielded five elec-

trophoretically distinct isozymes (catalytically and structurally

similar but genetically distinct enzymes from the same organism),

H, M

,MH

, and H

, thereby demonstrating that LDH is

a tetramer.What are the relative amounts of each isozyme formed

when equimolar amounts of M

and H

are so hybridized?

JWCL281_c08_221-277.qxd 2/23/10 1:59 PM Page 277

278

CHAPTER 9

Protein Folding,

Dynamics, and

Structural Evolution

1 Protein Folding: Theory and Experiment

A. Protein Renaturation

B. Determinants of Protein Folding

C. Folding Pathways

2 Folding Accessory Proteins

A. Protein Disulfide Isomerase

B. Peptidyl Prolyl Cis–Trans Isomerase

C. Molecular Chaperones: The GroEL/ES System

3 Protein Structure Prediction and Design

A. Secondary Structure Prediction

B. Tertiary Structure Prediction

C. Protein Design

4 Protein Dynamics

5 Conformational Diseases: Amyloids and Prions

A. Amyloid Diseases

B. Alzheimer’s Disease

C. Prion Diseases

6 Structural Evolution

A. Structures of Cytochromes c

B. Gene Duplication

In this third of four chapters on protein structure, we

consider the temporal behavior of proteins. Specifically, we

first take up the problem of how random coil polypeptides

fold to their native structures and how this process is facil-

itated by other proteins.This is followed by a consideration

of the progress that has been made in predicting protein

structures based on their sequences and in understanding

their dynamic properties, that is, the nature and functional

significance of their internal motions. Next we consider the

diseases caused by proteins taking up alternate conforma-

tions.We end by extending the discussions we began in Sec-

tion 7-3 on protein evolution but do so in terms of the

three-dimensional structures of proteins.

1 PROTEIN FOLDING: THEORY

AND EXPERIMENT

Solving the so-called protein folding problem, that is, de-

termining how and why proteins fold to their native states,

is considered to be one of the grand challenges of biochem-

istry. Early notions of protein folding postulated the exis-

tence of “templates” that somehow caused proteins to as-

sume their native conformations. Such an explanation begs

the question of how proteins fold because, even if it were

true, one would still have to explain how the template

achieved its conformation. In fact, proteins spontaneously

fold into their native conformations under physiological

conditions. This implies that a protein’s primary structure

dictates its three-dimensional structure. In general,under the

proper conditions, biological structures are self-assembling

so that they have no need of external templates to guide

their formation.

A. Protein Renaturation

Although evidence had been accumulating since the 1930s

that proteins could be reversibly denatured, it was not un-

til 1957 that the elegant experiments of Christian Anfinsen

on bovine pancreatic RNase A put protein renaturation

on a quantitative basis. RNase A, a 124-residue single-

chain protein, is completely unfolded and its four disulfide

bonds reductively cleaved in an 8M urea solution contain-

ing 2-mercaptoethanol (Fig. 9-2). Yet dialyzing away the

urea and exposing the resulting solution to O

at pH 8

In the preceding chapters, we saw how proteins are con-

structed from their component parts. This puts us in a simi-

lar position to a mechanic who has learned to take apart

and put together an automobile engine without any inkling

of how the engine works. What we need in order to under-

stand the workings of a protein is knowledge of the types of

internal motions it can and must undergo in order to carry

out its biological function as well as how it achieves its na-

tive structure. Put in terms of our deprived auto mechanic,

we wish to understand the operations of the “gears” and

“levers” with which proteins carry out their function. This

is a problem of enormous complexity whose solution we

are only beginning to understand. We shall see in later

chapters, for example, that even though the catalytic mech-

anisms of many enzymes of known structure have been

studied in great detail, it cannot be said that we fully under-

stand any of these mechanisms. This is because our com-

prehension of the ways in which a protein’s component

groups interact is far from complete. As far as proteins are

concerned, we have not greatly surpassed our hypothetical

mechanic’s level of understanding.

JWCL281_c09_278-322.qxd 2/24/10 1:17 PM Page 278

yields a protein that is virtually 100% enzymatically active

and physically indistinguishable from native RNase A. The

protein must therefore have spontaneously renatured.Any

reservations that this occurs only because RNase A is re-

ally not totally denatured by 8M urea have been satisfied

by the chemical synthesis of enzymatically active RNase A

(Section 7-5).

The renaturation of RNase A demands that its four

disulfide bonds reform. The probability of one of the eight

Cys residues from RNase A randomly reforming a disul-

fide bond with its proper (native) mate among the other

seven Cys residues is that of one of the remaining six Cys

residues then randomly reforming its proper disulfide

bond is etc. The overall probability of RNase A reform-

ing its four native disulfide links at random is

Clearly, the disulfide bonds from RNase A do not ran-

domly reform under renaturing conditions.

If the RNase A is reoxidized in 8M urea so that its disul-

fide bonds reform while the polypeptide chain is a random

coil, then after removal of the urea, the RNase A is, as ex-

pected, only ⬃1% enzymatically active. This “scrambled”

RNase A can be made fully active by exposing it to a trace

of 2-mercaptoethanol, which, over about a 10-h period,

⫻

⫽

105

;

Section 9-1. Protein Folding: Theory and Experiment 279

Figure 9-1 [Drawing by T.A. Bramley, in Robson, B., Trends Biochem. Sci. 1, 50 (1976).

Figure 9-2 Reductive denaturation and oxidative renaturation

of RNase A. [Illustration, Irving Geis. Image from the Irving

Geis Collection, Howard Hughes Medical Institute. Reprinted

with permission.]

RNase A

native molecule

Oxidation with

at pH 8

(renaturing

conditions)

Reduction with

HOCH

(denaturing

conditions)

Reduced

denatured

molecule

110

124

110

JWCL281_c09_278-322.qxd 8/10/10 11:16 AM Page 279

catalyzes disulfide bond interchange reactions until the na-

tive structure is achieved (Fig. 9-3). The native state of

RNase A under physiological conditions is therefore, most

probably, its thermodynamically most stable conformation

(if the protein has a conformation that is more stable than

the native state, conversion to it must involve such a large

activation barrier so as to make it kinetically inaccessible;

rate processes are discussed in Section 14-1C).

The time for renaturation of “scrambled” RNase A is re-

duced to ⬃2 min through the use of an enzyme, protein

disulfide isomerase (PDI), that catalyzes disulfide inter-

change reactions. (In fact, the supposition that in vivo fold-

ing to the native state requires no more than a few minutes

prompted the search that led to this enzyme’s discovery.)

PDI itself contains two active site Cys residues, which must

be in the form for the isomerase to be active.The en-

zyme evidently catalyzes the random cleavage and refor-

mation of a protein’s disulfide bonds (Fig. 9-3), thereby in-

terchanging them as the protein progressively attains

thermodynamically more favorable conformations. PDI is

further discussed in Section 9-2A.

a. Post-Translationally Modified Proteins May Not

Readily Renature

Many “scrambled” proteins are renatured through the ac-

tion of PDI and are unaffected by it in their native state (their

PDI-cleaved disulfide bonds rapidly reform because these

native proteins are in their most stable local conformations).

In post-translationally modified proteins, however, the disul-

fide bonds may serve to hold the protein in its otherwise un-

stable native state. For instance, the 51-residue polypeptide

hormone insulin, which consists of two polypeptide chains

joined by two disulfide bonds (Fig.7-2),is inactivated by PDI.

This observation led to the discovery that insulin is derived

from a single-chain, 84-residue precursor named proinsulin

(Fig. 9-4). Only after its disulfide bonds have formed is

proinsulin converted to the two-chained active hormone

by the specific proteolytic excision of an internal 33-

residue segment known as its C chain. Nevertheless, two

¬SH

sets of observations suggest that the C chain does not di-

rect the folding of the A and B chains but, rather, simply

holds them together while they form their native disulfide

bonds: (1) Under proper renaturing conditions, native

280 Chapter 9. Protein Folding, Dynamics, and Structural Evolution

Figure 9-3 Plausible mechanism for the thiol- or enzyme-catalyzed disulfide interchange reaction

in a protein. The purple ribbon represents the polypeptide backbone of the protein. The attacking

thiol group must be in its ionized thiolate form.

Figure 9-4 Primary structure of porcine proinsulin. Its C chain

(brown) is proteolytically excised from between its A and B

chains to form the mature hormone. [After Chance, R.E., Ellis,

R.M., and Brommer,W.W., Science 161, 165 (1968).]

–

RSH

–

RSH

Proteolytic

cleavage

C Chain

A Chain

B Chain

Proteolytic

cleavage

COO

–

Asn

Tyr

Asn

Glu

Leu

Gln

Tyr

Leu

Ser

Cys

Ile

Ser

Thr

Cys

Gln

Glu

Val

Ile

Gly

Arg

Lys

Gln

Pro

Gly

Glu

Leu

Ala

Leu

Ala

Gln

Leu

Gly

Leu

Gly

Leu

Glu

Val

Ala

Gly

Ala

Gin

Pro

Asn

Gln

Ala

Glu

Arg

Ala

Lys

Pro

Thr

Tyr

Phe

Gly

Arg

Glu

Gly

Cys

Val

Leu

Tyr

Leu

Ala

Val

Leu

Glu

His

Ser

Gly

Cys

Leu

His

Gln

Asn

Val

Phe

Cys

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