Biochemistry, 4th Edition P20 pptx

The framework of sheets and helices in the interior of a globular protein is typi-cally constant and conserved in both sequence and structure.. In some globular proteins Figure 6.22, it

core of the protein The extensively H-bonded nature of -helices and -sheets is

ideal for this purpose, and these structures effectively stabilize the polar groups of

the peptide backbone in the protein core

The framework of sheets and helices in the interior of a globular protein is

typi-cally constant and conserved in both sequence and structure The surface of a

glob-ular protein is different in several ways Typically, much of the protein surface is

com-posed of the loops and tight turns that connect the helices and sheets of the protein

core, although helices and sheets may also be found on the surface The result is that

the surface of a globular protein is often a complex landscape of different structural

elements These complex surface structures can interact in certain cases with small

molecules or even large proteins that have complementary structure or charge

(Fig-ure 6.20) These regions of complementary, recognizable struct(Fig-ure are formed

typi-cally from the peptide segments that connect elements of secondary structure They

are the basis for enzyme–substrate interactions, protein–protein associations in cell

signaling pathways, and antigen–antibody interactions, and more

The segments of the protein that are neither helix, sheet, nor turn have

tradi-tionally been referred to as coil or random coil Both of these terms are misleading.

Most of these “loop” segments are neither coiled nor random, in any sense of the

words These structures are every bit as organized and stable as the defined

sec-ondary structures They just don’t conform to any frequently recurring pattern

These so-called coil structures are strongly influenced by side-chain interactions

with the rest of the protein

Waters on the Protein Surface Stabilize the Structure

A globular protein’s surface structure also includes water molecules Many of the

polar backbone and side chain groups on the surface of a globular protein make

H bonds with solvent water molecules There are often several such water molecules

per amino acid residue, and some are in fixed positions (Figure 6.21) Relatively few

water molecules are found inside the protein

In some globular proteins (Figure 6.22), it is common for one face of an

-helix to be exposed to the water solvent, with the other face toward the

hydro-phobic interior of the protein The outward face of such an amphiphilic helix

con-sists mainly of polar and charged residues, whereas the inward face contains mostly

nonpolar, hydrophobic residues A good example of such a surface helix is that of

residues 153 to 166 of flavodoxin from Anabaena (Figure 6.22a) Note that the

helical wheel presentationof this helix readily shows that one face contains four

hydrophobic residues and that the other is almost entirely polar and charged

Less commonly, an -helix can be completely buried in the protein interior

or completely exposed to solvent Citrate synthase is a dimeric protein in which

-helical segments form part of the subunit–subunit interface As shown in Figure

6.22b, one of these helices (residues 260 to 270) is highly hydrophobic and contains

only two polar residues, as would befit a helix in the protein core On the other hand,

Figure 6.22c shows the solvent-exposed helix (residues 74 to 87) of calmodulin, which

consists of 10 charged residues, 2 polar residues, and only 2 nonpolar residues

Packing Considerations

The secondary and tertiary structures of ribonuclease A (Figure 6.19) and other

glob-ular proteins illustrate the importance of packing in tertiary structures Secondary

structures pack closely to one another and also intercalate with (insert between)

ex-tended polypeptide chains If the sum of the van der Waals volumes of a protein’s

con-stituent amino acids is divided by the volume occupied by the protein, packing

densi-ties of 0.72 to 0.77 are typically obtained These packing densidensi-ties are similar to those

of a collection of solid spheres This means that even with close packing, approximately

25% of the total volume of a protein is not occupied by protein atoms Nearly all of this

space is in the form of very small cavities Cavities the size of water molecules or larger

do occasionally occur, but they make up only a small fraction of the total protein

vol-FIGURE 6.20 The surfaces of proteins are complemen-tary to the molecules they bind PEP carboxykinase (shown here, pdb id  1K3D) is an enzyme from the metabolic pathway that synthesizes glucose (gluconeo-genesis; see Chapter 22) In the so-called “active site” (yel-low) of this enzyme, catalysis depends on complemen-tary binding of substrates Shown in this image are ADP (brown), a Mg 2  ion (blue), and AlF 3  (a phosphate ana-log, in green, above the Mg 2  ).

FIGURE 6.21 The surfaces of proteins are ideally suited

to form multiple H bonds with water molecules Shown here are waters (blue and white) associated with actini-din, an enzyme from kiwi fruit that cleaves polypeptide chains at arginine residues (pdb id  2ACT).The polar backbone atoms and side chain groups on the surface

of actinidin are extensively H-bonded with water.

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to examine the secondary and tertiary structure of ribonuclease.

3

4

5

6 7

8

9

10

11

12 Asp 153

13 14

Lys Lys

Ala Asp

Ser Ser Glu

Glu Arg Trp

Leu Ile

Val

(a)

-Helix from flavodoxin (residues 153–166)

(b)

(c)

2

1

3

4

5

6 7

8

9

10 11

Leu 260

Gly Ala

Ala Ser

Leu Phe

Met

Ala Ala

Asn

-Helix from citrate synthase (residues 260–270)

2

1

3

4

5

6 7

8

9

10

11

12 Arg 74

13 14

Asp Ile

Glu Lys

Arg Thr Glu

Glu Met Asp

Glu Lys

Ser

-Helix from calmodulin (residues 74–87)

2

ACTIVE FIGURE 6.22 The so-called helical wheel presentation can reveal the polar or nonpo-lar character of -helices If the helix is viewed end on, and the residues are numbered with residue 1 closest

to the viewer, it is easy to see how polar and nonpolar residues are distributed to form a wheel (a) The-helix

consisting of residues 153–166 (red) in flavodoxin from Anabaena is a surface helix and is amphipathic (pdb id

 1RCF) (b) The two helices (orange and red) in the interior of the citrate synthase dimer (residues 260–270 in

each monomer) are mostly hydrophobic (pdb id  5CSC) (c) The exposed helix (residues 74–87—red) of

calmodulin is entirely accessible to solvent and consists mainly of polar and charged residues (pdb id  1CLL).

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HUMAN BIOCHEMISTRY

Collagen-Related Diseases

Collagen provides an ideal case study of the molecular basis of

physiology and disease For example, the nature and extent of

col-lagen crosslinking depends on the age and function of the tissue

Collagen from young animals is predominantly un-crosslinked and

can be extracted in soluble form, whereas collagen from older

an-imals is highly crosslinked and thus insoluble The loss of flexibility

of joints with aging is probably due in part to increased

crosslink-ing of collagen

Several serious and debilitating diseases involving collagen

ab-normalities are known Lathyrism occurs in animals due to the

regular consumption of seeds of Lathyrus odoratus, the sweet pea,

and involves weakening and abnormalities in blood vessels, joints,

and bones These conditions are caused by ␤-aminopropionitrile

(see figure), which covalently inactivates lysyl oxidase, preventing

intramolecular crosslinking of collagen and causing abnormalities

in joints, bones, and blood vessels

Scurvy results from a dietary vitamin C deficiency and in-volves the inability to form collagen fibrils properly This is the result of reduced activity of prolyl hydroxylase, which is vitamin C–dependent, as previously noted Scurvy leads to lesions in the skin and blood vessels, and in its advanced stages, it can lead to grotesque disfiguration and eventual death Although rare in the modern world, it was a disease well known to sea-faring explorers

in earlier times who did not appreciate the importance of fresh fruits and vegetables in the diet

A number of rare genetic diseases involve collagen

abnormali-ties, including Marfan’s syndrome and the Ehlers–Danlos syndromes,

which result in hyperextensible joints and skin The formation of

atherosclerotic plaques, which cause arterial blockages in advanced stages, is due in part to the abnormal formation of collagenous structures in blood vessels

N C CH2 CH2

-Aminopropionitrile

NH+ 3

FIGURE 6.23 Ton-EBP is a DNA-binding protein consist-ing of two distinct domains The N-terminal domain is shown here on the right, with DNA (orange) in the middle, and the C-terminal domain on the left (pdb id

 1IMH).

ume It is likely that such cavities provide flexibility for proteins and facilitate

confor-mation changes and a wide range of protein dynamics (discussed later)

Protein Domains Are Nature’s Modular Strategy for Protein Design

Proteins range in molecular weight from a thousand to more than a million It is

tempting to think that the size of unique globular, folded structures would increase

with molecular weight, but this is not what has been observed Proteins composed

of about 250 amino acids or less often have a simple, compact globular shape

How-ever, larger globular proteins are usually made up of two or more recognizable and

distinct structures, termed domains or modules—compact, folded protein

struc-tures that are usually stable by themselves in aqueous solution Figure 6.23 shows a

two-domain DNA-binding protein, TonEBP, in which the two distinct domains are

joined by a short segment of the peptide chain Most domains consist of a single

continuous portion of the protein sequence, but in some proteins the domain

se-quence is interrupted by a sese-quence belonging to some other part of the protein

that may even form a separate domain (Figure 6.24) In either case, typical domain structures consist of hydrophobic cores with hydrophilic surfaces (as was the case for ribonuclease, Figure 6.19) Importantly, individual domains often possess unique functional behaviors (for example, the ability to bind a particular ligand with high affinity and specificity), and an individual domain from a larger protein often expresses its unique function within the larger protein in which it is found Multidomain proteins typically possess the sum total of functional properties and behaviors of their constituent domains

It is likely that proteins consisting of multiple domains (and thus multiple func-tions) evolved by the fusion of genes that once coded for separate proteins This would require gene duplication to be common in nature, and analysis of completed genomes has confirmed that approximately 90% of domains in eukaryotes have been duplicated Thus, the protein domain is a fundamental unit in evolution Many proteins have been “assembled” by duplicating domains and then combining them in different ways Many proteins are assemblies constructed from several in-dividual domains, and some proteins contain multiple copies of the same domain Figure 6.25 shows the tertiary structures of nine domains that are frequently dupli-cated, and Figure 6.26 presents several proteins that contain multiple copies of one

or more of these domains

FIGURE 6.24 Malonyl CoA:ACP transacylase (pdb id 

1NM2) is a metabolic enzyme consisting of two

subdo-mains The large subdomain (blue) includes residues

1–132 and 198–316 and consists of a -sheet

sur-rounded by 12 -helices.The small subdomain (gold 

residues 133–197) consists of a four-stranded

antiparal-lel-sheet and two -helices.

(a)

(f)

(e) (b)

1 nm

FIGURE 6.25 Ribbon structures of several protein modules used in the construction of complex multimodule

proteins (a) The complement control protein module (pdb id  1HCC) (b) The immunoglobulin module

(pdb id  1T89) (c) The fibronectin type I module (pdb id  1Q06) (d) The growth factor module (pdb id  1FSB) (e) The kringle module (pdb id  1HPK) (f) The GYF module (pdb id  1GYF) (g) The

-carboxygluta-mate module (pdb id  1CFI) (h) The FF module (pdb id  1UZC) (i) The DED domain (pdb id  1A1W).

Classification Schemes for the Protein Universe Are Based on Domains

The astounding diversity of properties and behaviors in living things can now be

explored through the analysis of vast amounts of genomic information Assessment

of sequence and structural data from several million proteins in both protein and

genome databases has shown that there is a relatively limited number of structurally

distinct domains in proteins Several comprehensive projects have organized the

available information in defined hierarchies or levels of protein structure

The Structural Classification of Proteins database (SCOP, http://scop.mrc-lmb

.cam.ac.uk/scop) recognizes five overarching classes, which encompass most

pro-teins SCOP is based on hierarchical levels that embody the evolutionary and

struc-tural relationships among known proteins, and protein classification in SCOP is

es-sentially a manual process using visual inspection and comparison of structures

CATH is another hierarchical classification system (http://www.cathdb.info) that

groups protein domain structures into evolutionary families and structural

group-ings, depending on sequence and structure similarities CATH differs from SCOP

Factors VII,

IX, X and

protein C

F1

F2

Factor XII

F1

tPA

Clr,Cls

C2, factor B

F1

F1

F1

F1

F1

F1

F2

F2

F1

F1

F1

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F1

F1

F1

Fibronectin

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

Twitchin

F3

F3

Plasma

membrane

FIGURE 6.26 A sampling of proteins that consist of mo-saics of individual protein modules The modules shown includeCG, a module containing -carboxyglutamate

residues; G, an epidermal growth factor–like module;

K, the “kringle” domain, named for a Danish pastry;

C, which is found in complement proteins; F1, F2, and F3, first found in fibronectin; I, the immunoglobulin superfamily domain; N, found in some growth factor receptors; E, a module homologous to the calcium-binding E–F hand domain; and LB, a lectin module found in some cell surface proteins (Adapted from Baron,

M., Norman, D., and Campbell, I., 1991 Protein modules Trends in

Biochemical Sciences 16:13–17.)

in that it combines manual analysis with automation based on quantitative algo-rithms to classify protein structures Figure 6.27 compares the hierarchical struc-tures of SCOP and CATH and defines the different levels of structure

Although the hierarchical names in SCOP and CATH differ somewhat, there are

common threads shared in these schemes Class is determined from the overall com-position of secondary structure elements in a domain A fold describes the number, arrangement, and connections of these secondary structure elements A superfamily

includes domains of similar folds and usually similar functions, thus suggesting a

common evolutionary ancestry A family usually includes domains with closely

re-lated amino acid sequences (in addition to folding similarities) Although the num-bers of unique folds, superfamilies, and families increase as more genomes are

known and analyzed, it has become apparent that the number of protein domains in na-ture is large but limited How many proteins can we expect to identify and understand

someday? There are approximately 103to 105genes per organism and approximately 13.6 million species of living organisms on earth (and this latter number is likely an underestimate) Thus, there may be approximately (103 1.36  107) or 1010to 1012 different proteins in all organisms on earth Still, this vast number of proteins may well consist of only about 105sequence domain families (Figure 6.27) and

approxi-The CATH Hierarchy

The SCOP Hierarchy

Class (4)

Class (7)

Architecture (40)

Topology (1084)

Fold (1086)

Superfamily (1777)

Family (3464)

Homologous Superfamily (2091)

Sequence Family (7794)

Domain (93885)

Domain (97178)

FIGURE 6.27 SCOP and CATH are hierarchical

classifica-tion systems for the known proteins Proteins are

classi-fied in SCOP by a manual process, whereas CATH

com-bines manual and automated procedures Numbers

indicate the population of each category.

mately 103protein folds of known structure—a remarkably small number compared

to the total number of protein-coding genes (see Table 1.6) It is anticipated that

most newly identified proteins will resemble other known proteins and that most

structures can be broken into two or more domains, which resemble tertiary

struc-tures observed in other proteins

Because structure depends on sequence, and because function depends on

struc-ture, it is tempting to imagine that all proteins of a similar structure should share a

common function, but this is not always true For example, the TIM barrel is a

com-mon protein fold consisting of eight -helices and eight -strands that alternate

along the peptide backbone to form a doughnutlike tertiary structure The TIM

barrel is named for triose phosphate isomerase, an enzyme that interconverts

ke-tone and aldehyde substrates in the breakdown of sugars (see Chapter 18)

How-ever, other TIM barrel proteins carry out very different functions (Figure 6.28a),

including the reduction of aldose sugars and hydrolysis of phosphate esters

More-over, not all proteins of similar function possess similar domains Both proteins

shown in Figure 6.28b catalyze the same reaction, but they bear little structural

sim-ilarity to each other

Denaturation Leads to Loss of Protein Structure and Function

Whereas the primary structure of proteins arises from covalent bonds, the

sec-ondary, tertiary, and quaternary levels of protein structure are maintained by weak,

noncovalent forces The environment of a living cell is exquisitely suited to

main-tain these weak forces and to preserve the structures of its many proteins However,

a variety of external stresses—for example, heat or chemical treatment—can disrupt

Aspartate aminotransferase

D -amino acid aminotransferase

Same domain type, different functions:

(a)

Same function, different structures:

(b)

FIGURE 6.28 (a) Some proteins share similar structural

features but carry out quite different functions (triose phosphate isomerase, pdb id  8TIM; aldose reductase, pdb id  1ADS; phosphotriesterase, pdb id  1DPM).

(b) Proteins with quite different structures can carry

out similar functions (yeast aspartate aminotransferase, pdb id  1YAA); D -amino acid aminotransferase, pdb id

 3DAA).

these weak forces in a process termed denaturation—the loss of protein structure

and function

An everyday example is the denaturation of the protein ovalbumin during the cooking of an egg (Figure 6.29) About 10% of the mass of an egg white is protein, and 54% of that is ovalbumin When a chicken egg is cracked open, the “egg white”

is a nearly transparent, viscous fluid Cooking turns this fluid to a solid, white mass The egg white proteins have unfolded and have precipitated out of solution, and the unfolded proteins have aggregated into a solid mass

As a typical protein solution is heated slowly, the protein remains in its native state until it approaches a characteristic melting temperature, Tm As the solution is heated further, the protein denatures over a narrow range of temperatures around

Tm(Figure 6.30) Denaturation over a very small temperature range such as this is

evidence of a two-state transition between the native and the unfolded states of the

protein, and this implies that unfolding is an all-or-none process: When weak forces are disrupted in one part of the protein, the entire structure breaks down

Most proteins can also be denatured below the transition temperature by a vari-ety of chemical agents, including acid or base, organic solvents, detergents, and par-ticular denaturing solutes Guanidine hydrochloride and urea are examples of the latter (Figure 6.31) Denaturation in all these cases involves disruption of the weak forces that stabilize proteins Covalent bonds are not affected Acids and bases cause protonation and deprotonation of dissociable groups on the protein, altering ionic interactions and hydrogen bonds Organic solvents and detergents disrupt hydro-phobic interactions that bury nonpolar groups in the protein interior The effects

of guanidine hydrochloride and urea are more complex Recent research indicates

Ovalbumin monomer

FIGURE 6.29 The proteins of egg white are denatured (as evidenced by their precipitation and aggregation) during cooking More than half of the protein in egg whites is ovalbumin Ovalbumin pdb id  1OVA.

[GdmCl] (M)

0.8 1.0

0

0.6

0.4

0.2

(b)

FIGURE 6.31 Proteins can be denatured (unfolded) by high concentrations of guanidine-HCl or urea The denaturation of chymotrypsin is plotted here.(Adapted from Fersht, A., 1999 Structure and Mechanism in Protein Science.

1.00

Temperature (°C)

0.75

0.50

0.25

0.00

FIGURE 6.30 Proteins can be denatured by heat, with

commensurate loss of function Ribonuclease A (blue)

and ribonuclease B (red) lose activity above about 55°C.

These two enzymes share identical protein structures,

but ribonuclease B possesses a carbohydrate chain

attached to Asn 34 (Adapted from Arnold, U., and

Ulbrich-Hofmann, R., 1997 Kinetic and thermodynamic thermal

stabili-ties of ribonuclease A and ribonuclease B Biochemistry 36:

2166-2172.)

NH2

H2N C O

+

NH2

H2N C

NH2

(a)

Guanidine HCl

Urea

Cl–

that these agents denature proteins by both direct effects (binding to hydrophilic

groups on the protein) and indirect effects (altering the structure and dynamics of

the water solvent) Also, both guanidine hydrochloride and urea are good H-bond

donors and acceptors

Anfinsen’s Classic Experiment Proved That Sequence

Determines Structure

As noted earlier (Section 6.2), all the information needed to fold a polypeptide into

its native structure is contained in the amino acid sequence This simple but

pro-found truth of protein structure was confirmed in the 1950s by the elegant studies

of denaturation and renaturation of proteins by Christian Anfinsen and his

co-workers at the National Institutes of Health For their pivotal studies, they chose the

small enzyme ribonuclease A from bovine pancreas, a protein with 124 residues

and four disulfide bonds (Figures 6.19 and 6.32) (Ribonuclease cleaves chains of

95

95

40

40

26

26

110 110

58

58

84 84

72

72

65 65

58

72

Hypothetical Inactive Form

(Note random formation of disulfides)

– MCE – Urea + Oxygen

Small amount of MCE

w/gentle warming

– MCE + Oxygen

+ MCE + Urea

FIGURE 6.32 Ribonuclease can be unfolded by treatment with urea, and -mercaptoethanol

(MCE) cleaves disulfide bonds If -mercaptoethanol is then removed (but not urea) under

oxidizing conditions, disulfide bonds reform in the still-unfolded protein (one possible

hypo-thetical inactive form is shown) If urea is removed in the presence of a small amount of

-mercaptoethanol with gentle warming, ribonuclease returns to its native structure (with the

correct set of disulfide bonds), and full enzymatic activity is restored This experiment

demon-strated that the information required for folding of globular proteins is contained in the

pri-mary structure.

ribonucleic acid Only ribonuclease in its native structure posseses enzyme activity,

so loss of activity in a denaturation experiment was proof of loss of structure.) They treated solutions of ribonuclease with a combination of urea, which unfolded the protein, and mercaptoethanol, which reduced the disulfide bridges This treatment destroyed all enzymatic activity of ribonuclease

Anfinsen discovered that removing the mercaptoethanol but not the urea re-stored only 1% of the enzyme activity This was attributed to the formation of ran-dom disulfide bridges by the still-denatured protein With eight Cys residues, there are 105 possible ways to make four disulfide bridges; thus, a residual activity of 1% made sense to Anfinsen (The first Cys to form a disulfide has seven possible part-ners, the next Cys has five possible partpart-ners, the next has three, and the last Cys has only one choice 7  5  3  1 = 105) However, if Anfinsen removed

mercap-toethanol and urea at the same time, the polypeptide was able to fold into its native

structure, the correct set of four disulfides reformed, and full enzyme activity was recovered (Figure 6.32) This experiment demonstrated that the information needed for protein folding resided entirely within the amino acid sequence of the protein itself Many subsequent experiments with a variety of proteins have con-firmed this fundamental postulate For his studies of the relationship of sequence and structure, Anfinsen shared the 1972 Nobel Prize in Chemistry (with William H Stein and Stanford Moore)

Is There a Single Mechanism for Protein Folding?

Christian Anfinsen’s experiments demonstrated that proteins can fold reversibly A corollary result of Anfinsen’s work is that the native structures of at least some glob-ular proteins are thermodynamically stable states But the matter of how a given pro-tein achieves such a stable state is a complex one Cyrus Levinthal pointed out in

1968 that so many conformations are possible for a typical protein that the protein does not have sufficient time to reach its most stable conformational state by

sam-pling all the possible conformations This argument, termed Levinthal’s paradox, goes

as follows: Consider a protein of 100 amino acids Assume that there are only two conformational possibilities per amino acid, or 2100 1.27  1030possibilities Allow

1013sec for the protein to test each conformational possibility in search of the over-all energy minimum:

(1013sec)(1.27 1030) 1.27  1017sec 4  109years Four billion years is the approximate age of the earth

Levinthal’s paradox led protein chemists to hypothesize that proteins must fold

by specific “folding pathways,” and many research efforts have been devoted to the search for these pathways Several consistent themes have emerged from these stud-ies Each of them may well play a role in the folding process:

• Secondary structures—helices, sheets, and turns—probably form first

• Nonpolar residues may aggregate or coalesce in a process termed a hydrophobic

collapse.

• Subsequent steps probably involve formation of long-range interactions between secondary structures or involving other hydrophobic interactions

• The folding process may involve one or more intermediate states, including

tran-sition states and what have become known as molten globules.

The folding of most globular proteins may well involve several of these themes For example, even in the denatured state, many proteins appear to possess small

amounts of residual structure due to hydrophobic interactions, with strong

inter-residue contacts between side chains that are relatively distant in the native protein structure Such interactions, together with small amounts of secondary structure,

may act as sites of nucleation for the folding process A bit further in the folding

process, the molten globule is postulated to be a flexible but compact form charac-terized by significant amounts of secondary structure, virtually no precise tertiary structure, and a loosely packed hydrophobic core Moreover, it is likely that any one