Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

5.7 ● The Primary Structure of a Protein: Determining the Amino Acid Sequence

141

Step 8. Location of Disulfide Cross-Bridges

Strictly speaking, the disulfide bonds formed between cysteine residues in a protein are not a part of its primary structure. Nevertheless, information about their location can be obtained by procedures used in sequencing, provided the disulfides are not broken prior to cleaving the polypeptide chain. Because these covalent bonds are stable under most conditions used in the cleavage of polypeptides, intact disulfides link the peptide fragments containing their specific cysteinyl residues and thus these linked fragments can be isolated and identified within the protein digest.

An effective way to isolate these fragments is through diagonal electrophoresis (Figure 5.26) (the basic technique of electrophoresis is described in

(a)

(b)

+

Buffer

(c)

(d)

+

(e)

Partial protein digest of sample is smeared along one edge of paper

–

Migration of peptides toward – electrode

Sample strip is cut from electrophoretogram and treated with performic acid vapors

Performic acid

– HCOOOH-treated strip is attached to new sheet of paper and second

electrophoresis run is performed

Peptides derived from disulfide-linked protein fragments

FIGURE 5.26 ● Disulfide bridges typically are cleaved prior to determining the primary structure of a polypeptide. Consequently, the positions of disulfide links are not obvious from the sequence data. To determine their location, a sample of the polypeptide with intact SOS bonds can be fragmented and the sites of any disulfides can be elucidated from fragments that remain linked. Diagonal electrophoresis is a technique for identifying such fragments. (a) A protein digest in which any disulfide bonds remain intact and link their respective Cys-con- taining peptides is streaked along the edge of a filter paper and (b) subjected to electrophoresis. (c) A strip cut from the edge of the paper is then exposed to performic acid fumes to oxidize any disulfide bridges. (d) Then the paper strip is attached to a new filter paper so that a second electrophoresis can be run in a direction perpendicular to the first. (e) Peptides devoid of disulfides experience no mobility change, and thus their pattern of migration defines a diagonal. Peptides that had disulfides migrate off this diagonal and can be easily identified, isolated, and sequenced to reveal the location of cysteic acid residues formerly involved in disulfide bridges.

Diagonal

142 Chapter 5 ● Proteins: Their Biological Functions and Primary Structure

the Chapter Appendix). Peptides that were originally linked by disulfides now migrate as distinct species following disulfide cleavage and are obvious by their location off the diagonal (Figure 5.26e). These cysteic acid–containing peptides are then isolated from the paper and sequenced. From this information, the positions of the disulfides in the protein can be stipulated.

Sequence Databases

A database of protein sequences collected by protein chemists can be found in the Atlas of Protein Sequence and Structure. However, most protein sequence information has been derived from translating the nucleotide sequences of genes into codons and, thus, amino acid sequences (see Chapter 13). Sequencing the order of nucleotides in cloned genes is a more rapid, efficient, and informative process than determining the amino acid sequences of proteins. A number of electronic databases containing continuously updated sequence information are readily accessible by personal computer. Prominent among these are PIR (Protein Identification Resource Protein Sequence Database), GenBank (Genetic Sequence Data Bank), and EMBL (European Molecular Biology Laboratory Data Library).

5.8 ● Nature of Amino Acid Sequences

With a knowledge of the methodology in hand, let’s review the results of amino acid composition and sequence studies on proteins. Table 5.8 lists the relative frequencies of the amino acids in various proteins. It is very unusual for a globular protein to have an amino acid composition that deviates substantially from these values. Apparently, these abundances reflect a distribution of amino acid polarities that is optimal for protein stability in an aqueous milieu. Membrane proteins have relatively more hydrophobic and fewer ionic amino acids, a condition consistent with their location. Fibrous proteins may show compositions that are atypical with respect to these norms, indicating an underlying relationship between the composition and the structure of these proteins.

Proteins have unique amino acid sequences, and it is this uniqueness of sequence that ultimately gives each protein its own particular personality. Because the number of possible amino acid sequences in a protein is astronomically large, the probability that two proteins will, by chance, have similar amino acid sequences is negligible. Consequently, sequence similarities between proteins imply evolutionary relatedness.

Homologous Proteins from Different Organisms

Have Homologous Amino Acid Sequences

Proteins sharing a significant degree of sequence similarity are said to be homologous. Proteins that perform the same function in different organisms are also referred to as homologous. For example, the oxygen transport protein, hemoglobin, serves a similar role and has a similar structure in all vertebrates. The study of the amino acid sequences of homologous proteins from different organisms provides very strong evidence for their evolutionary origin within a common ancestor. Homologous proteins characteristically have polypeptide chains that are nearly identical in length, and their sequences share identity in direct correlation to the relatedness of the species from which they are derived.

Table 5.8

Frequency of Occurrence of Amino Acid Residues in Proteins

			Occurrence in
Amino Acid		Mr*	Proteins (%)†
Alanine	Ala A	71.1	9.0
Arginine	Arg R	156.2	4.7
Asparagine	Asn N	114.1	4.4
Aspartic acid	Asp D	115.1	5.5
Cysteine	Cys C	103.1	2.8
Glutamine	Gln Q	128.1	3.9
Glutamic acid	Glu E	129.1	6.2
Glycine	Gly G	57.1	7.5
Histidine	His H	137.2	2.1
Isoleucine	Ile I	113.2	4.6
Leucine	Leu L	113.2	7.5
Lysine	Lys K	128.2	7.0
Methionine	Met M	131.2	1.7
Phenylalanine	Phe F	147.2	3.5
Proline	Pro P	97.1	4.6
Serine	Ser S	87.1	7.1
Threonine	Thr T	101.1	6.0
Tryptophan	Trp W	186.2	1.1
Tyrosine	Tyr Y	163.2	3.5
Valine	Val V	99.1	6.9

*Molecular weight of amino acid minus that of water.

†Frequency of occurrence of each amino acid residue in the polypeptide chains of 207 unrelated proteins of known sequence.

Values from Klapper, M. H., 1977. Biochemical and Biophysical Research Communications 78:1018–1024.

5.8 ● Nature of Amino Acid Sequences

143

1 Gly

6 Gly

10 Phe

Heme

17 Cys

18 His

29Gly

30 Pro

32 Leu

34 Gly

38 Arg

41 Gly

45 Gly

48 Tyr

52 Asn

59 Trp

C y t o c h rome c

The electron transport protein, cytochrome c, found in the mitochondria of all eukaryotic organisms, provides the best-studied example of homology. The polypeptide chain of cytochrome c from most species contains slightly more than 100 amino acids and has a molecular weight of about 12.5 kD. Amino acid sequencing of cytochrome c from more than 40 different species has revealed that there are 28 positions in the polypeptide chain where the same amino acid residues are always found (Figure 5.27). These invariant residues apparently serve roles crucial to the biological function of this protein, and thus substitutions of other amino acids at these positions cannot be tolerated.

FIGURE 5.27 ● Cytochrome c is a small protein consisting of a single polypeptide chain	▲
of 104 residues in terrestrial vertebrates, 103 or 104 in fishes, 107 in insects, 107 to 109 in
fungi and yeasts, and 111 or 112 in green plants. Analysis of the sequence of cytochrome c
from more than 40 different species reveals that 28 residues are invariant. These invariant
residues are scattered irregularly along the polypeptide chain, except for a cluster between
residues 70 and 80. All cytochrome c polypeptide chains have a cysteine residue at position
17, and all but one have another Cys at position 14. These Cys residues serve to link the
heme prosthetic group of cytochrome c to the protein, a role explaining their invariable
presence.

68 Leu

70Asn

71Pro

72Lys

73Lys

74 Tyr

76 Pro

78Thr

79 Lys

80 Met

82 Phe

84 Gly

91 Arg

100

FIGURE 5.28

144	Chapter 5 ● Proteins: Their Biological Functions and Primary Structure
		Chimpanzee	Sheep	Rattlesnake	Carp	Snail	Moth	Yeast	Cauliflower	Parsnip
	Human	0	10	14	18	29	31	44	44	43
	Chimpanzee		10	14	18	29	31	44	44	43
	Sheep			20	11	24	27	44	46	46
	Rattlesnake				26	28	33	47	45	43
	Carp					26	26	44	47	46
	Garden snail						28	48	51	50
	Tobacco hornworm moth							44	44	41
	Baker’s yeast (iso-1)								47	47
	Cauliflower									13

● The number of amino acid differences among the cytochrome c sequences of various organisms can be compared. The numbers bear a direct relationship to the degree of relatedness between the organisms. Each of these species has a cytochrome c of at least 104 residues, so any given pair of species has more than half its residues in common. (Adapted from Creighton, T. E., 1983. Proteins: Structure and Molecular Properties.

San Francisco: W. H. Freeman and Co.)

Furthermore, as shown in Figure 5.28, the number of amino acid differences between two cytochrome c sequences is proportional to the phylogenetic difference between the species from which they are derived. The cytochrome c in humans and in chimpanzees is identical; human and another mammalian (sheep) cytochrome c differ at 10 residues. The human cytochrome c sequence has 14 variant residues from a reptile sequence (rattlesnake), 18 from a fish (carp), 29 from a mollusc (snail), 31 from an insect (moth), and more than 40 from yeast or higher plants (cauliflower).

The Phylogenetic Tree for Cytochrome c

Figure 5.29 displays a phylogenetic tree (a diagram illustrating the evolutionary relationships among a group of organisms) constructed from the sequences of cytochrome c. The tips of the branches are occupied by contemporary species whose sequences have been determined. The tree has been deduced by computer analysis of these sequences to find the minimum number of mutational changes connecting the branches. Other computer methods can be used to infer potential ancestral sequences represented by nodes, or branch points, in the tree. Such analysis ultimately suggests a primordial cytochrome c sequence lying at the base of the tree. Evolutionary trees constructed in this manner, that is, solely on the basis of amino acid differences occurring in the primary sequence of one selected protein, show remarkable agreement with phylogenetic relationships derived from more classic approaches and have given rise to the field of molecular evolution.

FIGURE 5.29 ● This phylogenetic tree depicts the evolutionary relationships among organisms as determined by the similarity of their cytochrome c amino acid sequences. The numbers along the branches give the amino acid changes between a species and a hypothetical progenitor. Note that extant species are located only at the tips of branches. Below, the sequence of human cytochrome c is compared with an inferred ancestral sequence represented by the base of the tree. Uncertainties are denoted by question marks. (Adapted from Creighton, T. E., 1983. Proteins: Structure and Molecular Properties. San Francisco:

W. H. Freeman and Co.)

▲

5.8 ● Nature of Amino Acid Sequences

145

Ancestral

1

10

20

cytochrome c

Pro

Ala

Gly

Asp

?

Lys

Lys

Gly

Ala

Lys

Ile

Phe

Lys

Thr

?

Cys

Ala

Gln

Cys

His

Thr

Val

Glu

?

Gly

Gly

?

Human

Val

Gly

Asp

Glu

Lys

Gly

Lys

Lys

Ile

Phe

Ile

Met

Lys

Cys

Ser

Gln

Cys

His

Thr

Val

Glu

Lys

Gly

Gly

Lys

cytochrome c

30

40

50

His

Lys

Val

Gly

Pro

Asn

Leu

His

Gly

Leu

Phe

Gly

Arg

Lys

?

Gly

Gln

Ala

?

Gly

Tyr

Ser

Tyr

Thr

Asp

His

Lys

Thr

Gly

Pro

Asn

Leu

His

Gly

Leu

Phe

Gly

Arg

Lys

Thr

Gly

Gln

Ala

Pro

Gly

Tyr

Ser

Tyr

Thr

Ala

60

70

Ala

Asn

Lys

Asn

Lys

Gly

?

?

Trp

?

Glu

Asn

Thr

Leu

Phe

Glu

Tyr

Leu

Glu

Asn

Pro

Lys

Lys

Tyr

Ile

Ala

Asn

Lys

Asn

Lys

Gly

Ile

Ile

Trp

Gly

Glu

Asp

Thr

Leu

Met

Gln

Tyr

Leu

Glu

Asn

Pro

Lys

Lys

Tyr

Pro

80

90

100

Pro

Gly

Thr

Lys

Met

?

Phe

?

Gly

Leu

Lys

Lys

?

?

Asp

Arg

Ala

Asp

Leu

Ile

Ala

Tyr

Leu

Lys

?

Pro

Gly

Thr

Lys

Met

Ile

Phe

Val

Gly

Ile

Lys

Lys

Lys

Glu

Glu

Arg

Ala

Asp

Leu

Ile

Ala

Tyr

Leu

Lys

Lys

Ala

Thr

Ala

Ala

Thr

Asn

Glu

146 Chapter 5 ● Proteins: Their Biological Functions and Primary Structure

Related Proteins Share a Common Evolutionary Origin

Amino acid sequence analysis reveals that proteins with related functions often show a high degree of sequence similarity. Such findings suggest a common ancestry for these proteins.

Oxygen-Binding Heme Proteins

The oxygen-binding heme protein of muscle, myoglobin, consists of a single polypeptide chain of 153 residues. Hemoglobin, the oxygen transport protein of erythrocytes, is a tetramer composed of two -chains (141 residues each) and two -chains (146 residues each). These globin polypeptides—myoglobin,-globin, and -globin—share a strong degree of sequence homology (Figure 5.30). Human myoglobin and the human -globin chain show 38 amino acid

1

10

20

Myoglobin

Gly

Leu

Ser

Asp

Gly

Glu

Trp

Gln

Leu

Val

Leu

Asn

Val

Trp

Gly

Lys

Val

Glu

Ala

Asp

Ile

Pro

Gly

His

Gly

Gln

Glu

Val

α

Ser

Ala

Asp

Thr

Asn

Lys

Ala

Gly

Ala

His

Ala

Gly

Gln

Tyr

Ala

Val

Leu

Pro

Lys

Val

Ala

Trp

Gly

Lys

Val

Gly

Glu

Ala

β

Val

His

Leu

Thr

Pro

Glu

Glu

Lys

Ser

Ala

Val

Thr

Ala

Leu

Trp

Gly

Lys

Val

Asn

Val

Asp

Glu

Val

Gly

Gly

Glu

Ala

30

40

50

60

Leu

Ile

Arg

Leu

Phe

Lys

Gly

His

Pro

Glu

Thr

Leu

Glu

Lys

Phe

Asp

Lys

Phe

Lys

His

Leu

Lys

Ser

Glu

Asp

Glu

Met

Lys

Ala

Ser

Glu

Leu

Glu

Arg

Met

Phe

Leu

Ser

Phe

Pro

Thr

Thr

Lys

Thr

Tyr

Phe

Pro

His

Phe

Asp

Leu

Ser

His

Gly

Ser

Ala

Leu

Gly

Arg

Leu

Leu

Val

Val

Tyr

Pro

Trp

Thr

Gln

Arg

Phe

Phe

Glu

Ser

Phe

Gly

Asp

Leu

Ser

Thr

Pro

Asp

Ala

Val

Met

Gly

Asn

Pro

70

80

90

Asp

Leu

Lys

Ala

Thr

Leu

Thr

Gly

Gly

Ile

Leu

Lys

Lys

Lys

Gly

His

His

Glu

Ala

Glu

Ile

Lys

Pro

Ala

Lys

His

Gly

Val

Ala

Leu

Leu

Gln

Val

Lys

Gly

His

Gly

Lys

Lys

Val

Ala

Asp

Ala

Leu

Thr

Asn

Ala

Val

Ala

His

Val

Asp

Asp

Met

Pro

Asn

Ala

Leu

Ser

Ala

Leu

Ser

Lys

Val

Lys

Ala

His

Gly

Lys

Lys

Val

Leu

Gly

Ala

Phe

Ser

Asp

Gly

Leu

Ala

His

Leu

Asp

Asn

Leu

Lys

Gly

Thr

Phe

Ala

Thr

Leu

Ser

100

110

120

Gln

Ser

His

Ala

Thr

Lys

His

Lys

Ile

Pro

Val

Lys

Tyr

Leu

Glu

Phe

Ile

Ser

Glu

Cys

Ile

Ile

Gln

Val

Leu

Gln

Ser

Lys

His

Pro

Gly

Asp

Ala

His

Arg

Val

Lys

Ser

His

Cys

Leu

His

Thr

Ala

Leu

Pro

Ala

Leu

His

Lys

Leu

Val

Asp

Pro

Asn

Phe

Leu

Leu

Leu

Leu

Ala

His

Glu

Leu

His

Cys

Asp

Lys

Leu

His

Val

Asp

Pro

Glu

Asn

Phe

Arg

Leu

Leu

Gly

Asn

Val

Leu

Val

Asn

Val

Leu

Ala

His

His

Phe

Gly

Lys

130

140

150

Asp

Phe

Gly

Ala

Asp

Ala

Gln

Gly

Ala

Met

Asn

Lys

Ala

Leu

Glu

Leu

Phe

Arg

Lys

Asp

Met

Ala

Ser

Asn

Tyr

Lys

Glu

Leu

Gly

Phe

Gln

Gly

Ala

His

Ser

Leu

Asp

Phe

Leu

Ser

Ser

Thr

Val

Thr

Ser

Arg

Glu

Phe

Thr

Pro

Val

Ala

Lys

Ala

Val

Leu

Lys

Tyr

Glu

Phe

Thr

Pro

Pro

Val

Gln

Ala

Ala

Tyr

Gln

Lys

Val

Val

Ala

Gly

Val

Ala

Asn

Ala

Leu

Ala

His

Lys

Tyr

His

α –chain of horse methemoglobin

β –chain of horse methemoglobin

Sperm whale myoglobin

FIGURE 5.30 ● Inspection of the amino acid sequences of the globin chains of human hemoglobin and myoglobin reveals a strong degree of homology. The -globin and

-globin chains share 64 residues of their approximately 140 residues in common. Myoglobin and the -globin chain have 38 amino acid sequence identities. This homology is further reflected in these proteins’ tertiary structure. (Irving Geis)

identities, whereas human -globin and human -globin have 64 residues in common. The relatedness suggests an evolutionary sequence of events in which chance mutations led to amino acid substitutions and divergence in primary structure. The ancestral myoglobin gene diverged first, after duplication of a primordial globin gene had given rise to its progenitor and an ancestral hemoglobin gene (Figure 5.31). Subsequently, the ancestral hemoglobin gene duplicated to generate the progenitors of the present-day -globin and -globin genes. The ability to bind O2 via a heme prosthetic group is retained by all three of these polypeptides.

Serine Pro t e a s e s

Whereas the globins provide an example of gene duplication giving rise to a set of proteins in which the biological function has been highly conserved, other sets of proteins united by strong sequence homology show more divergent biological functions. Trypsin, chymotrypsin (see Section 5.7), and elastase are members of a class of proteolytic enzymes called serine proteases because of the central role played by specific serine residues in their catalytic activity. Thrombin, an essential enzyme in blood clotting, is also a serine protease. These enzymes show sufficient sequence homology to conclude that they arose via duplication of a progenitor serine protease gene, even though their substrate preferences are now quite different.

Apparently Different Proteins May Share a Common Ancestry

A more remarkable example of evolutionary relatedness is inferred from sequence homology between hen egg white lysozyme and human milk - lactalbumin, proteins of quite different biological activity and origin. Lysozyme (129 residues) and -lactalbumin (123 residues) are identical at 48 positions. Lysozyme hydrolyzes the polysaccharide wall of bacterial cells, whereas - lactalbumin regulates milk sugar (lactose) synthesis in the mammary gland. Although both proteins act in reactions involving carbohydrates, their functions show little similarity otherwise. Nevertheless, their tertiary structures are strikingly similar (Figure 5.32). It is conceivable that many proteins are related in this way, but time and the course of evolutionary change erased most evidence of their common ancestry. In an interesting contrast to this case, the proteins actin and hexokinase share essentially no sequence homology, yet they have very similar three-dimensional structures, even though their biological roles and physical properties are quite different. Actin forms a filamentous polymer that is a principal component of the contractile apparatus in muscle; hexokinase is a cytosolic enzyme that catalyzes the first reaction in glucose catabolism.

Mutant Proteins

Given a large population of individuals, a considerable number of sequence variants can be found for a protein. These variants are a consequence of mutations in a gene (base substitutions in DNA) that have arisen naturally within the population. Gene mutations lead to mutant forms of the protein in which the amino acid sequence is altered at one or more positions. Many of these mutant forms are “neutral” in that the functional properties of the protein are unaffected by the amino acid substitution. Others may be nonfunctional (if loss of function is not lethal to the individual), and still others may display a range of aberrations between these two extremes. The severity of the effects on function depends on the nature of the amino acid substitution and its role in the protein. These conclusions are exemplified by the more than 300 human

5.8 ● Nature of Amino Acid Sequences		147
Myoglobin	β	α

Ancestral	Ancestral
β -globin	α -globin

Ancestral hemoglobin

Ancestral globin

FIGURE 5.31 ● This evolutionary tree is inferred from the homology between the amino acid sequences of the -globin, -glo- bin, and myoglobin chains. Duplication of an ancestral globin gene allowed the divergence of the myoglobin and ancestral hemoglobin genes. Another gene duplication event subsequently gave rise to ancestral and forms, as indicated. Gene duplication is an important evolutionary force in creating diversity.

148 Chapter 5 ● Proteins: Their Biological Functions and Primary Structure

N

N

	C	129
		C
	123
	Human milk -lactalbumin	Hen egg white lysozyme

FIGURE 5.32 ● The tertiary structures of hen egg white lysozyme and human -lactal- bumin are very similar. (Adapted from Acharya, K. R., et al., 1990. Journal of Protein Chemistry 9:549–563; and Acharya, K. R., et al., 1991. Journal of Molecular Biology 221:571–581.

-Lactalbumin

Lysozyme

Table 5.9

Some Pathological Sequence Variants of Human Hemoglobin

Abnormal Hemoglobin*	Normal Residue and Position	Substitution
Alpha chain
Torino	Phenylalanine 43	Valine
MBoston	Histidine 58	Tyrosine
Chesapeake	Arginine 92	Leucine
GGeorgia	Proline 95	Leucine
Tarrant	Aspartate 126	Asparagine
Suresnes	Arginine 141	Histidine
Beta chain
S	Glutamate 6	Valine
Riverdale–Bronx	Glycine 24	Arginine
Genova	Leucine 28	Proline
Zurich	Histidine 63	Arginine
MMilwaukee	Valine 67	Glutamate
MHyde Park	Histidine 92	Tyrosine
Yoshizuka	Asparagine 108	Aspartate
Hiroshima	Histidine 146	Aspartate

*Hemoglobin variants are often given the geographical name of their origin.

Adapted from Dickerson, R. E., and Geis, I., 1983. Hemoglobin: Structure, Function, Evolution and Pathology.

Menlo Park, CA: Benjamin-Cummings Publishing Co.

5.9 ● Synthesis of Polypeptides in the Laboratory

149

hemoglobin variants that have been discovered to date. Some of these are listed in Table 5.9.

A variety of effects on the hemoglobin molecule are seen in these mutants, including alterations in oxygen affinity, heme affinity, stability, solubility, and subunit interactions between the -globin and -globin polypeptide chains. Some variants show no apparent changes, whereas others, such as HbS, sicklecell hemoglobin (see Chapter 15), result in serious illness. This diversity of response indicates that some amino acid changes are relatively unimportant, whereas others drastically alter one or more functions of a protein.

5.9 ● Synthesis of Polypeptides in the Laboratory

Chemical synthesis of peptides and polypeptides of defined sequence can be carried out in the laboratory. Formation of peptide bonds linking amino acids together is not a chemically complex process, but making a specific peptide can be challenging because various functional groups present on side chains of amino acids may also react under the conditions used to form peptide bonds. Furthermore, if correct sequences are to be synthesized, the -COOH group of residue x must be linked to the -NH2 group of neighboring residue y in a way that prevents reaction of the amino group of x with the carboxyl group of y. Ingenious synthetic strategies are required to circumvent these technical problems. In essence, any functional groups to be excluded from reaction must be blocked while the desired coupling reactions proceed. Also, the blocking groups must be removable later under conditions in which the newly formed peptide bonds are stable. These limitations mean that addition of each amino acid requires several steps. Further, all reactions must proceed with high yield if peptide recoveries are to be acceptable. Peptide formation between amino and carboxyl groups is not spontaneous under normal conditions (see Chapter 4), so one or the other of these groups must be activated to facilitate the reaction. Despite these difficulties, biologically active peptides and polypeptides have been recreated by synthetic organic chemistry. Milestones include the pioneering synthesis of the nonapeptide posterior pituitary hormones oxytocin and vasopressin by du Vigneaud in 1953, and in later years, the blood pres- sure–regulating hormone bradykinin (9 residues), melanocyte-stimulating hormone (24 residues), adrenocorticotropin (39 residues), insulin (21 A-chain and 30 B-chain residues), and ribonuclease A (124 residues).

Solid Phase Peptide Synthesis

Bruce Merrifield and his collaborators found a clever solution to the problem of recovering intermediate products in the course of a synthesis. The carboxylterminal residues of synthesized peptide chains were covalently anchored to an insoluble resin particle large enough to be removed from reaction mixtures simply by filtration. After each new residue was added successively at the free amino-terminus, the elongated product was recovered by filtration and readied for the next synthetic step. Because the growing peptide chain was coupled to an insoluble resin bead, the method is called solid phase synthesis. The procedure is detailed in Figure 5.33. This cyclic process has been automated and computer controlled so that the reactions take place in a small cup with reagents being pumped in and removed as programmed. The 124-residue-long bovine pancreatic ribonuclease A sequence was synthesized, and the final product was enzymatically active as an RNase.

Aminoacyl-

resin particle

R1

H2N

CHC

N

N

O

CH3

R2

CH3

R2

CH3

C

O

C

NHCHCOOH +

C

CH3

C

O

C

NHCHC

O

C

NH

N

CH3

O

NH

CH3

O

O

Blocking group

Incoming

C

O

blocked

NH

amino acid

Dicyclohexyl-

Activated

carbodiimide

amino acid

Dicyclohexylurea

CH3

CH3

R2

R1

CH3

C

O

C

Cl

Amino-blocked

dipeptidyl-

CH3

C

O

C

NHCHCNHCHC

CH3

O

resin particle

BocCl

CH3

O

O

O

CH2

N

R

O

R O

N

Acid

C

H3C

CH3

+ H2N

C

C

C

OH

H2N

C

C

O C

Isobutylene

N

NH

H

H

CO2

R2

R1

Dipeptide-resin

Dicyclohexyl-

Activated

H2NCHCNHCHC

particle

carbodiimide

amino acid

O

O

CH3

R3

N

CH3

R3

N

CH3

C

O

C

NHCHCOOH +

C

CH3

C

O

C

NHCHC

O

C

NH

CH3

O

CH3

O

O

N

NH

Incoming blocked

C

O

amino acid

NH

FIGURE 5.33 ●

Solid phase synthesis of a peptide. (inset)

Activated

amino acid

Tertiary butyloxycarbonyl chloride (tBocCl) is an excellent

reagent for blocking amino groups of amino acids during

CH3

R3

R2

R1

organic synthesis. Dicyclohexylcarbodiimide (DCCD) is a pow-

Amino-blocked

erful agent for activating carboxyl groups to condense with

CH3

C

O

C

NHCHC NHCHCNHCHC

amino groups to form peptide bonds. The carboxyl group of

tripeptidyl-

resin particle

the first amino acid (the carboxyl-terminal amino acid of the

CH3

O

O

O

O

peptide to be synthesized) is attached to an insoluble resin

particle (the aminoacyl-resin particle). The next amino acid,

with its amino group blocked by a tBoc group and its carboxyl

Acid

+

Isobutylene

CO2

group activated with DCCD, is reacted with the aminoacyl-

resin particle to form a peptide linkage, with elimination of

DCCD as dicyclohexylurea. Acid treatment removes the N-ter-

R3

R2

R1

minal tBoc blocking group as the gaseous products CO2 and

Tripeptidyl-resin

isobutylene, exposing the N-terminus of the dipeptide for

H2NCHC

NHCHC

NHCHC

particle

another cycle of amino acid addition. The growing peptide

chain is easily recovered after cyclic additions of amino acids

O

O

O

simply by filtering or centrifuging the reaction mixture.

150