Voet D., Voet Ju.G. Biochemistry

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3 GLOBULAR PROTEINS

Globular proteins comprise a highly diverse group of sub-

stances that, in their native states, exist as compact spher-

oidal molecules. Enzymes are globular proteins, as are

transport and receptor proteins. In this section we consider

the tertiary structures of globular proteins. However, since

most of our detailed structural knowledge of proteins, and

thus to a large extent their function, has resulted from X-

ray crystal structure determinations of globular proteins

and, more recently, from their nuclear magnetic resonance

(NMR) structure determinations, we begin this section

with a discussion of the capabilities and limitations of these

powerful techniques.

A. Interpretation of Protein X-Ray

and NMR Structures

X-ray crystallography is a technique that directly images

molecules. X-rays must be used to do so because, accord-

ing to optical principles, the uncertainty in locating an ob-

ject is approximately equal to the wavelength of the radia-

tion used to observe it (covalent bond distances and the

wavelengths of the X-rays used in structural studies are

both ⬃1.5 Å; individual molecules cannot be seen in a

light microscope because visible light has a minimum

wavelength of 4000 Å).There is, however, no such thing as

an X-ray microscope because there are no X-ray lenses.

Rather, a crystal of the molecule to be imaged is exposed

to a collimated beam of X-rays and the consequent dif-

fraction pattern is recorded by a radiation detector or,

now infrequently, on photographic film (Fig. 8-35). The

X-rays used in structural studies are produced by laboratory

X-ray generators or, increasingly often, by synchrotrons, a

type of particle accelerator that produces X-rays of far

greater intensity. The intensities of the diffraction maxima

(darkness of the spots on a film) are then used to construct

mathematically the three-dimensional image of the crystal

structure through methods that are beyond the scope of

this text. In what follows, we discuss some of the special

problems associated with interpreting the X-ray crystal

structures of proteins.

X-rays interact almost exclusively with the electrons in

matter,not the far more massive nuclei.An X-ray structure

is therefore an image of the electron density of the object

under study. Such electron density maps may be presented

as a series of parallel sections through the object. On each

section, the electron density is represented by contours

(Fig. 8-36a) in the same way that altitude is represented by

the contours on a topographic map. A stack of such sec-

tions, drawn on transparencies, yields a three-dimensional

electron density map (Fig. 8-36b). Modern structural analy-

sis, however, is carried out with the aid of computers that

graphically display electron density maps that are con-

toured in three dimensions (Fig. 8-36c).

Most Protein Crystal Structures Exhibit Less than

Atomic Resolution

The molecules in protein crystals, as in other crys-

talline substances, are arranged in regularly repeating

three-dimensional lattices. Protein crystals, however, dif-

fer from those of most small organic and inorganic mole-

cules in being highly hydrated; they are typically 40 to

60% water by volume. The aqueous solvent of crystalliza-

tion is necessary for the structural integrity of the protein

crystals, as J.D. Bernal and Dorothy Crowfoot Hodgkin

first noted in 1934 when they carried out the original

X-ray studies of protein crystals. This is because water is

required for the structural integrity of native proteins

themselves (Section 8-4).

The large solvent content of protein crystals gives them

a soft, jellylike consistency, so that their molecules often

lack the rigid order characteristic of crystals of small mole-

cules such as NaCl or glycine. The molecules in a protein

crystal are typically disordered by more than an angstrom,

so that the corresponding electron density map lacks infor-

mation concerning structural details of smaller size. The

crystal is therefore said to have a resolution limit of that

size. Protein crystals typically have resolution limits in the

range 1.5 to 3.0 Å, although some are better ordered (have

higher resolution, that is, a lesser resolution limit) and

many are less ordered (have lower resolution).

Since an electron density map of a protein must be in-

terpreted in terms of its atomic positions, the accuracy and

Section 8-3. Globular Proteins 241

Figure 8-35 X-ray diffraction photograph of a single crystal of

sperm whale myoglobin. The intensity of each diffraction

maximum (the darkness of each spot) is a function of the

myoglobin crystal’s electron density. The photograph contains

only a small fraction of the total diffraction information available

from a myoglobin crystal. [Courtesy of John Kendrew, Cambridge

University, U.K.]

JWCL281_c08_221-277.qxd 2/23/10 1:58 PM Page 241

even the feasibility of a crystal structure analysis depends

on the crystal’s resolution limit. Indeed, the ability to ob-

tain crystals of sufficiently high resolution is a major limit-

ing factor in determining the X-ray crystal structure of a

protein or other macromolecule. Figure 8-37 indicates how

the quality (degree of focus) of an electron density map

varies with its resolution limit. At 6-Å resolution, the pres-

ence of a molecule the size of diketopiperazine is difficult

to discern. At 2.0-Å resolution, its individual atoms cannot

yet be distinguished, although its molecular shape has be-

come reasonably evident. At 1.5-Å resolution, which

roughly corresponds to a bond distance, individual atoms

become partially resolved. At 1.1-Å resolution, atoms are

clearly visible.

Most protein crystal structures are too poorly resolved

for their electron density maps to reveal clearly the posi-

tions of individual atoms (e.g., Fig. 8-36). Nevertheless, the

distinctive shape of the polypeptide backbone usually per-

mits it to be traced, which, in turn, allows the positions and

orientations of its side chains to be deduced (e.g., Fig. 8-37c).

242 Chapter 8. Three-Dimensional Structures of Proteins

Figure 8-36 Electron density maps of proteins. (a) A section

through the 2.0-Å-resolution electron density map of sperm whale

myoglobin, which contains the heme group (red).The large peak

at the center of the map represents the electron-dense Fe atom.

[After Kendrew, J.C., Dickerson, R.E., Strandberg, B.E., Hart,

R.G., Davies, D.R., Phillips, D.C., and Shore,V.C., Nature 185, 434

(1960).] (b) A portion of the 2.4-Å-resolution electron density

map of myoglobin constructed from a stack of contoured

transparencies. Dots have been placed at the positions deduced for

the nonhydrogen atoms.The heme group is seen edge-on together

with its two associated His residues and a water molecule, W.An ␣

helix, the so-called E helix (Fig. 8-12), extends across the bottom of

the map. Another ␣ helix, the C helix, extends into the plane of the

paper on the upper right. Note the hole along its axis. [Courtesy

of John Kendrew, Cambridge University, U.K.] (c) A thin section

through the 1.5-Å-resolution electron density map of E. coli

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (which

catalyzes the first reaction in the biosynthesis of folic acid; Section

26-4D) contoured in three dimensions. Only a single contour level

(cyan) is shown,together with an atomic model of the corresponding

polypeptide segments colored according to atom type (C yellow, O

red, and N blue, with a water molecule represented by a red

sphere). [Courtesy of Xinhua Ji, NCI–Frederick Cancer Research

and Development Center, Frederick, Maryland.]

(b)

(a)

(c)

JWCL281_c08_221-277.qxd 6/3/10 9:46 AM Page 242

Yet side chains of comparable size and shape, such as those

of Leu, Ile, and Thr, cannot always be differentiated with a

reasonable degree of confidence (hydrogen atoms, having

but one electron, are only visible in the few macromolecular

X-ray structures with resolution limits less than ⬃1.2 Å), so

that a protein structure cannot be elucidated from its elec-

tron density map alone. Rather, the primary structure of

the protein must be known, thereby permitting the se-

quence of amino acid residues to be fitted to its electron

density map. Mathematical refinement can then reduce the

uncertainty in the crystal structure’s atomic positions to as

little as 0.1 Å (in contrast, positional errors in the most

accurately determined small molecule X-ray structures are

as little as 0.001 Å).

b. Most Crystalline Proteins Maintain Their

Native Conformations

What is the relationship between the structure of a pro-

tein in a crystal and that in solution, where globular pro-

teins normally function? Several lines of evidence indicate

that crystalline proteins assume very nearly the same struc-

tures that they have in solution:

1. A protein molecule in a crystal is essentially in solu-

tion because it is bathed by the solvent of crystallization

over all of its surface except for the few, generally small

patches that contact neighboring protein molecules. In fact,

the 40 to 60% water content of typical protein crystals is

similar to that of many cells (e.g., see Fig. 1-13).

2. A protein may crystallize in one of several forms or

“habits,” depending on crystallization conditions, that dif-

fer in how the protein molecules are arranged in space

relative to each other. In the numerous cases in which

different crystal forms of the same protein have been

independently analyzed, the molecules have virtually iden-

tical conformations. Similarly, in the several cases in which

both the X-ray crystal structure and the solution NMR

structure of the same protein have been determined, the

two structures are, for the most part, identical to within ex-

perimental error (see below). Evidently, crystal packing

forces do not greatly perturb the structures of protein mol-

ecules.

3. The most compelling evidence that crystalline pro-

teins have biologically relevant structures, however, is the

observation that many enzymes are catalytically active in

the crystalline state. The catalytic activity of an enzyme is

very sensitive to the relative orientations of the groups in-

volved in binding and catalysis (Chapter 15). Active crys-

talline enzymes must therefore have conformations that

closely resemble their solution conformations.

c. Protein Structure Determination by NMR

The determination of the three-dimensional structures

of small globular proteins in aqueous solution has become

possible, since the mid 1980s, through the development of

two-dimensional (2D) NMR spectroscopy (and, more re-

cently, of 3D and 4D techniques), in large part by Kurt

Wüthrich. Such NMR measurements, whose description is

beyond the scope of this text, yield the interatomic dis-

tances between specific protons that are ⬍5 Å apart in a

protein of known sequence. The interproton distances may

be either through space, as determined by nuclear Over-

hauser effect spectroscopy (NOESY, Fig. 8-38a), or

through bonds, as determined by correlated spectroscopy

(COSY). These distances, together with known geometric

constraints such as covalent bond distances and angles,

group planarity, chirality, and van der Waals radii, are used

to compute the protein’s three-dimensional structure.

However,since interproton distance measurements are im-

precise, they are insufficient to imply a unique structure.

Rather, they are consistent with an ensemble of closely re-

lated structures. Consequently, an NMR structure of a pro-

tein (or any other macromolecule with a well-defined

structure) is often presented as a representative sample of

structures that are consistent with the constraints (e.g., Fig-

ure 8-38b).The “tightness” of a bundle of such structures is

Section 8-3. Globular Proteins 243

1.1-Å resolution1.5-Å resolution2.0-Å resolution6.0-Å resolution

CHN

(a) (b) (c) (d)

Figure 8-37 Sections through the electron density map of diketopiperazine calculated at the

indicated resolution levels. Hydrogen atoms are not apparent in this map because of their low

electron density. [After Hodgkin, D.C., Nature 188, 445 (1960).]

JWCL281_c08_221-277.qxd 2/23/10 1:58 PM Page 243

indicative both of the accuracy with which the structure is

known, which in the most favorable cases is roughly com-

parable to that of an X-ray crystal structure with a resolu-

tion of 2 to 2.5 Å, and of the conformational fluctuations

that the protein undergoes (Section 9-4).Although present

NMR methods are limited to determining the structures of

macromolecules with molecular masses no greater than

⬃100 kD, advances in NMR technology suggest that this

limit may increase to ⬃1000 kD or more.

In most of the several cases in which both the NMR

and X-ray crystal structures of a particular protein have

been determined, the two structures are in good agree-

ment. There are, however, a few instances in which there

are real differences between the corresponding X-ray

and NMR structures. These, for the most part, involve

surface residues that, in the crystal, participate in inter-

molecular contacts and are thereby perturbed from their

solution conformations. NMR methods, besides provid-

ing mutual cross-checks with X-ray techniques, can de-

termine the structures of proteins and other macromole-

cules that fail to crystallize. Moreover, since NMR can

probe motions over time scales spanning 10 orders of

magnitude, it can be used to study protein folding and dy-

namics (Chapter 9).

d. Protein Molecular Structures Are Most Effectively

Illustrated in Simplified Form

The several hundred nonhydrogen atoms of even a small

protein makes understanding a protein’s detailed struc-

ture a considerable effort. This complexity makes build-

ing a skeletal or ball-and-stick model of a protein such a

time-consuming task that such models are rarely avail-

able. Moreover,a drawing of a protein showing all its non-

hydrogen atoms (e.g., Fig. 8-39a) is too complicated to be

of much use. In order to be intelligible, drawings of pro-

teins must be selectively simplified. One way of doing so

is to represent the polypeptide backbone only by its C

␣

atoms (its C

␣

backbone) and to display only a few key

side chains (e.g, Fig. 8-39b). A further level of abstraction

244 Chapter 8. Three-Dimensional Structures of Proteins

(a )

Figure 8-38 The 2D proton NMR structures of proteins. (a) A

NOESY spectrum of a protein presented as a contour plot with

two frequency axes, ␻

and ␻

.The conventional 1D-NMR

spectrum of the protein, which occurs along the diagonal of the

plot (␻

⫽␻

), is too crowded with peaks to be directly

interpretable (even a small protein has hundreds of protons).

The off-diagonal peaks, the so-called cross peaks, each arise from

the interaction of two protons that are ⬍5 Å apart in space and

whose 1D-NMR peaks are located where the horizontal and

vertical lines through the cross peak intersect the diagonal [a

nuclear Overhauser effect (NOE)]. For example, the line to the

left of the spectrum represents the extended polypeptide chain

with its N- and C-terminal ends identified by the letters N and C

and with the positions of four protons, a to d, represented by

small circles.The dashed arrows indicate the diagonal NMR

peaks to which these protons give rise. Cross peaks, such as i, j,

and k, which are each located at the intersections of the

horizontal and vertical lines through two diagonal peaks, are

indicative of an NOE between the corresponding two protons,

indicating that they are ⬍5 Å apart.These distance relationships

are schematically indicated by the three looped structures drawn

below the spectrum. Note that the assignment of a distance

relationship between two protons in a polypeptide requires that

the NMR peaks to which they give rise and their positions in the

polypeptide be known, which requires that the polypeptide’s

amino acid sequence has been previously determined. [After

Wüthrich, K., Science 243, 45 (1989).] (b) The NMR structure of

a 64-residue polypeptide comprising the Src protein SH3 domain

(Section 19-3C).The drawing represents 20 superimposed

structures that are consistent with the 2D- and 3D-NMR spectra

of the protein (each calculated from a different, randomly

generated starting structure).The polypeptide backbone, as

represented by its connected C

␣

atoms, is white and its Phe,Tyr,

and Trp side chains are yellow, red, and blue, respectively. It can

be seen that the polypeptide backbone folds into two

three-stranded antiparallel ␤ sheets that form a sandwich.

[Courtesy of Stuart Schreiber, Harvard University.]

(b)

JWCL281_c08_221-277.qxd 2/23/10 1:58 PM Page 244

may be obtained by representing the protein in a cartoon

form that emphasizes its secondary structure (e.g., Figs.

8-39c and 8-19). Computer-generated drawings of space-

filling models, such as Figs. 8-12 and 8-18, may also be em-

ployed to illustrate certain features of protein structures.

However, the most instructive way to examine a macro-

molecular structure is through the use of interactive com-

puter graphics programs. The use of such programs is dis-

cussed in Section 8-3Cc.

B. Tertiary Structure

The tertiary structure (3° structure) of a protein is its three-

dimensional arrangement; that is, the folding of its 2° struc-

tural elements, together with the spatial dispositions of its

side chains. The first protein X-ray structure, that of sperm

Section 8-3. Globular Proteins 245

Figure 8-39 Representations of the X-ray structure of sperm

whale myoglobin. (a) The protein and its bound heme are drawn

in stick form, with protein C atoms green, heme C atoms red, N

atoms blue, and O atoms red.The Fe and its bound water

molecule are shown as orange and gray spheres and hydrogen

bonds are gray. In this one-of-a-kind painting of the first known

protein structure, the artist has employed “creative distortions”

to emphasize the protein’s structural features, particularly its ␣

helices. (b) A diagram in which the protein is represented by its

computer-generated C

␣

backbone, with its C

␣

atoms, shown as

balls, consecutively numbered from the N-terminus. The

153-residue polypeptide chain is folded into eight ␣ helices

(highlighted here by hand-drawn envelopes), designated A

through H, that are connected by short polypeptide links. The

protein’s bound heme group (purple, with its Fe atom represented

by a red sphere), in complex with a water molecule (orange

sphere), is shown together with its two closely associated His side

chains (blue). One of the heme group’s propionic acid side chains

has been displaced for clarity. Hydrogen atoms are not visible in

the X-ray structure. (c) A computer-generated cartoon drawing

in an orientation similar to that of Part b, emphasizing the protein’s

secondary structure. Here helices are green and the intervening

coil regions are yellow. The heme group with its bound O

molecule and its two associated His side chains are shown in

ball-and-stick form with C magenta, N blue, O red, and Fe orange.

[Parts a and b are based on an X-ray structure by John Kendrew,

MRC Laboratory of Molecular Biology, Cambridge, U.K. PDBid

1MBN. [Illustration, Irving Geis. Image from the Irving Geis

Collection, Howard Hughes Medical Institute. Reprinted with

permission.] Part c is based on an X-ray structure by Simon

Phillips, University of Leeds, Leeds, U.K. PDBid 1MBO.]

See

Kinemage Exercise 6-1

(a)

(b)

(c)

JWCL281_c08_221-277.qxd 8/10/10 11:48 AM Page 245

whale myoglobin, was elucidated in the late 1950s by John

Kendrew and co-workers. Its polypeptide chain follows

such a tortuous, wormlike path (Fig. 8-39) that these inves-

tigators were moved to indicate their disappointment at its

lack of regularity. In the intervening years, ⬃70,000 protein

structures have been reported. Each of them is a unique,

highly complicated entity. Nevertheless, their tertiary struc-

tures have several outstanding features in common, as we

shall see below.

a. Globular Proteins May Contain Both ␣ Helices

and ␤ Sheets

The major types of secondary structural elements, ␣ he-

lices and ␤ pleated sheets, commonly occur in globular pro-

teins but in varying proportions and combinations. Some

proteins, such as myoglobin, consist only of ␣ helices

spanned by short connecting links that have coil conforma-

tions (Fig. 8-39). Others, such as concanavalin A, have a

large proportion of ␤ sheets but are devoid of ␣ helices

(Fig.8-40). Most proteins, however,have significant amounts

of both types of secondary structure (on average, ⬃31%

␣ helix and ⬃28% ␤ sheet, with nearly all of their inner cores

so arranged). Human carbonic anhydrase (Fig. 8-41) as well

as carboxypeptidase A and triose phosphate isomerase

(Fig. 8-19) are examples of such proteins.

b. Side Chain Location Varies with Polarity

The primary structures of globular proteins generally

lack the repeating or pseudorepeating sequences that are re-

sponsible for the regular conformations of fibrous proteins.

The amino acid side chains in globular proteins are, never-

theless, spatially distributed according to their polarities:

1. The nonpolar residues Val, Leu, Ile, Met, and Phe

largely occur in the interior of a protein, out of contact with

the aqueous solvent. The hydrophobic interactions that

promote this distribution, which are largely responsible for

the three-dimensional structures of native proteins, are fur-

ther discussed in Section 8-4C.

2. The charged polar residues Arg,His, Lys,Asp, and Glu

are largely located on the surface of a protein in contact with

the aqueous solvent. This is because the immersion of an

ion in the virtually anhydrous interior of a protein results

in the uncompensated loss of much of its hydration energy.

In the instances that these groups occur in the interior of a

protein, they often have a specific chemical function such

as promoting catalysis or participating in metal ion bind-

ing (e.g., the metal ion–liganding His residues in Figs. 8-39

and 8-41).

3. The uncharged polar groups Ser, Thr, Asn, Gln, Tyr,

and Trp are usually on the protein surface but frequently

246 Chapter 8. Three-Dimensional Structures of Proteins

Figure 8-40 X-ray structure of the jack bean protein

concanavalin A. This protein largely consists of antiparallel ␤

pleated sheets, here represented by flat arrows pointing toward

the polypeptide chain’s C-terminus. The polypeptide chain is

colored in rainbow order from its N-terminus (blue) to its

C-terminus (red).The spheres represent protein-bound Mn

2⫹

(magenta) and Ca

2⫹

(cyan) ions.The front sheet is shown in a

space-filling representation in Fig. 8-18 but viewed from the

opposite side as seen here. [Based on an X-ray structure by

George Reeke, Jr., Joseph Becker, and Gerald Edelman,The

Rockefeller University. PDBid 2CNA.]

Figure 8-41 X-ray structure of human carbonic anhydrase.

The polypeptide backbone is drawn in ribbon form colored in

rainbow order from its N-terminus (blue) to its C-terminus (red).

The purple sphere in the center represents a Zn

2⫹

ion that is

coordinated by three His side chains in stick form colored with C

yellow and N blue. Note that the C-terminus is tucked through

the plane of a surrounding loop of polypeptide chain, so that

carbonic anhydrase is one of the rare native proteins in which a

polypeptide chain forms a knot. [Based on an X-ray structure by

T. Alwyn Jones, Uppsala University, Uppsala, Sweden. PDBid

2CAB.]

See Interactive Exercise 3

JWCL281_c08_221-277.qxd 10/19/10 7:13 AM Page 246

occur in the interior of the molecule. In the latter case,

these residues are almost always hydrogen bonded to other

groups in the protein. In fact, nearly all buried hydrogen

bond donors form hydrogen bonds with buried acceptor

groups; in a sense, the formation of a hydrogen bond “neu-

tralizes” the polarity of a hydrogen bonding group.

Such a side chain distribution is clearly apparent in Fig. 8-42,

which displays the X-ray structure of cytochrome c. This

arrangement is also seen in Fig. 8-43, which shows the sur-

face and interior exposures of the amino acid side chains of

myoglobin’s H helix, and in Fig. 8-44, which shows one of

the antiparallel ␤ pleated sheets of concanavalin A.

c. Globular Protein Cores Are Efficiently Arranged

with Their Side Chains in Relaxed Conformations

Globular proteins are quite compact; there is very little

space inside them, so that water is largely excluded from

their interiors. The micellelike arrangement of their side

chains (polar groups outside, nonpolar groups inside) has

led to their description as “oil drops with polar coats.”This

generalization, although picturesque, lacks precision. The

packing density (ratio of the volume enclosed by the van

der Waals envelopes of the atoms in a region to the total

volume of the region) of the internal regions of globular

proteins averages ⬃0.75, which is in the same range as that

of molecular crystals of small organic molecules. In com-

parison, equal-sized close-packed spheres have a packing

density of 0.74, whereas organic liquids (oil drops) have

packing densities that are mostly between 0.60 and 0.70.

The interior of a protein is therefore more like a molecular

crystal than an oil drop; that is, it is efficiently packed.

The bonds of protein side chains, including those occu-

pying protein cores, almost invariably have low-energy

staggered torsion angles (Fig. 8-5a). Evidently, interior side

chains adopt relaxed conformations despite their profusion

of intramolecular interactions (Section 8-4).

d. Large Polypeptides Form Domains

Polypeptide chains that consist of more than ⬃200

residues usually fold into two or more globular clusters

known as domains, which often give these proteins a bi- or

multilobal appearance. Most domains consist of 100 to 200

amino acid residues and have an average diameter of ⬃25 Å.

Section 8-3. Globular Proteins 247

Figure 8-42 X-ray structure of horse heart cytochrome c. The

protein (blue) is illuminated by the Fe atom of its heme group

(orange). In Part a, the hydrophobic side chains are red, and in

Part b, the hydrophilic side chains are green. [Based on an X-ray

structure by Richard Dickerson, UCLA; [Illustration, Irving

Geis. Image from the Irving Geis Collection, Howard Hughes

Medical Institute. Reprinted with permission.]

See Kinemage

Exercise 5 and Interactive Exercise 4

(a)

(b)

JWCL281_c08_221-277.qxd 10/19/10 11:02 AM Page 247

Each subunit of the enzyme glyceraldehyde-3-phosphate

dehydrogenase, for example, has two distinct domains

(Fig. 8-45). A polypeptide chain wanders back and forth

within a domain, but neighboring domains are usually con-

nected by one, or less commonly two, polypeptide seg-

ments. Domains are therefore structurally independent units

that each have the characteristics of a small globular protein.

Indeed, limited proteolysis of a multidomain protein often

liberates its domains without greatly altering their struc-

tures or enzymatic activities. Nevertheless, the domain

structure of a protein is not always obvious since its do-

mains may make such extensive contacts with each other

that the protein appears to be a single globular entity.

An inspection of the various protein structures dia-

grammed in this chapter reveals that domains consist of

two or more layers of secondary structural elements. The

reason for this is clear:At least two such layers are required

to seal off a domain’s hydrophobic core from the aqueous

environment.

Domains often have a specific function, such as the

binding of a small molecule. In Fig. 8-45, for example, the

dinucleotide nicotinamide adenine dinucleotide (NAD

⫹

)

binds to the first domain of the enzyme glyceraldehyde-3-

phosphate dehydrogenase. Small molecule binding sites in

multidomain proteins often occur in the clefts between do-

mains; that is, the small molecules are bound by groups

from two domains. This arrangement arises, in part, from

OH OH

Nicotinamide adenine dinucleotide (NAD

ⴙ

)

⫺

Ribose Adenine

⫹

248 Chapter 8. Three-Dimensional Structures of Proteins

Figure 8-43 The H helix of sperm whale myoglobin. (a) A helical wheel representation

in which side chain positions about the ␣ helix are projected down the helix axis onto

a plane. Here each residue is identified both according to its sequence in the

polypeptide chain and according to its order in the H helix.The residues lining the

side of the helix facing the protein’s interior regions are all nonpolar (orange).The

other residues, except Leu 137, which contacts the protein segment linking helices E

and F (Fig. 8-39b), are exposed to the solvent and are all more or less polar (purple).

(b) A stick model, viewed as in Part a, in which the polypeptide backbone is gray,

nonpolar side chains are orange, and polar side chains are purple. (c) A space-filling

model, viewed from the bottom of Part b such that the helix axis is vertical and

colored as in Part b. [Parts b and c based on an X-ray structure by Ilme Schlichting,

Max Planck Institut für Molekulare Physiologie, Dortmund, Germany. PDBid 1A6M.]

See Kinemage Exercise 3-2 for another example

(b)

(c)

(a)

Asp

Ala

Asn

Arg

Gln

Leu

Ile

Met

Phe

Glu

Gly

Lys

Leu

141 H18

132 H9

134 H11

127 H4

138 H15

131 H8

130 H7

137 H14

144 H21

133 H10

139 H16

128 H5

135 H12

142 H19

143 H20

136 H13

129 H6

140 H17

Residues

exposed

to surface

Residues

facing

protein

interior

JWCL281_c08_221-277.qxd 2/23/10 1:58 PM Page 248

the need for a flexible interaction between the protein and

the small molecule that the relatively pliant covalent con-

nection between the domains can provide.

e. Supersecondary Structures Are the Building

Blocks of Proteins

Certain groupings of secondary structural elements,

named supersecondary structures or motifs, occur in many

unrelated globular proteins:

1. The most common form of supersecondary structure

is the ␤␣␤ motif (Fig. 8-46a), in which the usually right-

handed crossover connection between two consecutive

parallel strands of a ␤ sheet consists of an ␣ helix.

2. Another common supersecondary structure, the ␤

hairpin motif (Fig. 8-46b), consists of an antiparallel ␤ sheet

formed by sequential segments of polypeptide chain that

are connected by relatively tight reverse turns.

3. In an ␣␣ motif (Fig. 8-46c), two successive antiparallel

␣ helices pack against each other with their axes inclined so

Section 8-3. Globular Proteins 249

Figure 8-44 A space-filling model of an antiparallel ␤ sheet

from concanavalin A. The ␤ sheet is shown in side view with the

interior of the protein (the surface of a second antiparallel ␤

sheet; Fig. 8-40) to the right and the exterior to the left. The main

chain is gray, nonpolar side chains are orange, and polar side

chains are purple.

See Kinemage Exercise 3-3

Figure 8-45 One subunit of the enzyme glyceraldehyde-3-

phosphate dehydrogenase from Bacillus stearothermophilus.

The polypeptide folds into two distinct domains. The N-terminal

domain (light blue, residues 1–146) binds NAD

⫹

(drawn in stick

form with C green, N blue, O red, and P magenta) near the

C-terminal ends of its parallel ␤ strands, and the C-terminal

domain (orange, residues 148–333) binds glyceraldehyde-3-

phosphate (not shown). [After Biesecker, G., Harris, J.I.,Thierry,

J.C.,Walker, J.E., and Wonacott,A., Nature 266, 331 (1977).]

See Interactive Exercise 5

Greek key

4123

2 3

fold

(d)

(a) (b) (c)

Figure 8-46 Schematic diagrams of supersecondary structures.

(a) A ␤␣␤ motif, (b) a ␤ hairpin motif, (c) an ␣␣ motif, and (d) a

Greek key motif, showing how it is constructed from a folded-over

␤ hairpin.

JWCL281_c08_221-277.qxd 10/19/10 7:14 AM Page 249

as to permit their contacting side chains to interdigitate ef-

ficiently. Such energetically favorable associations stabilize

the coiled coil conformation of ␣ keratin (Section 8-2A).

4. In the Greek key motif (Fig. 8-46d; named after an

ornamental design commonly used in ancient Greece; see

inset), a ␤ hairpin is folded over to form a four-stranded an-

tiparallel ␤ sheet.Of the 10 possible ways of connecting the

strands of a four-stranded antiparallel ␤ sheet, the two that

form Greek key motifs are, by far, the most common in

proteins of known structure.

Groups of motifs combine in overlapping and nonoverlap-

ping ways to form the tertiary structure of a domain, which

is called a fold.

The number of possible different folds would, of

course, seem to be unlimited. However, comparisons of

the now large number of known protein structures have

revealed that few protein folds are unique; that is, most

proteins of known structure have folds that also occur in

unrelated proteins. Indeed, theoretical considerations sug-

gest there are less than 8000 naturally occurring folds. Of

250 Chapter 8. Three-Dimensional Structures of Proteins

Figure 8-47 X-ray structures of 4-helix bundle proteins. (a) E.

coli cytochrome b

562

and (b) human growth hormone.The proteins

are represented by their peptide backbones drawn in ribbon

form colored in rainbow order from N-terminus (red) to

C-terminus (blue). Cytochrome b

562

’s bound heme group is

shown in ball-and-stick form with C magenta, N blue, O red, and

Fe orange.The inset below each ribbon diagram is a topological

diagram indicating the connectivity of the ␣ helices in each

4-helix bundle. Cytochrome b

562

(106 residues) has up-down-up-

down topology, whereas human growth hormone (191 residues)

has up-up-down-down topology. Note that the N- and C-terminal

helices of human growth hormone are longer than its other two

helices, so that, at one end, these longer helices associate as an

␣␣ motif. [Based on X-ray structures by (a) F. Scott Matthews,

Washington University School of Medicine, and (b) Alexander

Wlodawer, National Cancer Institute, Frederick, Maryland.

PDBids (a) 256B and (b) 1HGU.]

(a)

(b)

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