`
`Organic Chemistr
`
`Fourth Edition
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`William H. Brown
`
`Beloit College
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`Christopher S. Foote
`University of California, Los Angeles
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`Brent L. Iverson
`
`University of Texas, Austin
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`

`1074
`
`Chapter 27 Amino Acids andProteins
`
`Solution
`
`Theisoelectric point of histidine is 7.64. At this pH, histidine has a net charge of
`and does not move from the origin. The pl of cysteine is 5.02; at pH 7.64 (mo
`than its isoelectric point), cysteine has a net negative charge and moves towar
`positive electrode. The pl oflysine is 9.74; at pH 7.64 (more acidic thanits isoele
`point), lysine has a net positive charge and moves toward the negative electrode
`
`
`
`
`Problem 27.3
`
`Describe the behavior of a mixture of glutamic acid, arginine, and valine on
`electrophoresis at pH 6.0.
`
`
`27.3 Polypeptides and Proteins
`
`
`
`In 1902, Emil Fischer proposed that proteins are long chains of aminoacids je
`together by amide bonds between the a-carboxyl group of one amino acid am
`a-amino group of another. For these amide bonds, Fischer proposed the sp
`name peptide bond. Figure 27.5 shows the peptide bond formed betweenserine
`alaninein the dipeptide serylalanine.
`Peptide is the name given to a short polymer of amino acids. Peptides are «
`fied by the number of amino acid units in the chain. A molecule containing 2 a
`acids joined by an amide bondis called a dipeptide. Those containing 3 to 10 a
`acids are called tripeptides, tetrapeptides, pentapeptides, and so on. Molecules
`taining more than 10 but fewer than 20 aminoacids are called oligopeptides. T
`
`containing several dozen or more aminoacids are called polypeptides. Proteim
`biological macromolecules of molecular weight 5000 or greater, consisting of ox
`more polypeptide chains. The distinctions in this terminology are notprecise.
`By convention, polypeptides are written from the left, beginning with the
`acid having the free —NH;* group and proceedingto the right toward the as
`
`
`
`
`
`
`
`Peptide bond Thespecial name
`given to the amide bond formed
`between the a-amino group of one
`aminoacid and the a-carboxyl
`group of another aminoacid.
`
`Dipeptide A molecule containing
`two aminoacid units joined by a
`peptide bond.
`
`Tripeptide A molecule
`containing three aminoacid
`units, each joined to the next
`by a peptide bond.
`
`Polypeptide A macromolecule
`containing many amino acid
`units, each joined to the next by a
`peptide bond.
`
`
`
`Figure 27.5
`The peptide bondin serylalanine.
`
`
`
`Peptide bom
`
`
`
`H
`|
`N
`
`HO—y
`4.
`
`H
`S
`
`O
`
`®
`
`AS
`HCH,
`
`Serylalanine
`(Ser-Ala, S-A)
`
`
`
`

`

`27.4 Primary Structure of Polypeptides and Proteins
`
`1075
`
`acid with the free —COO™ group. The amino acid with the free —NH,* group
`is called the N-terminal amino acid and that with the free —COO™groupis called
`the C-terminal amino acid. Notice the repeating pattern in the peptide chain of
`N—a-carbon—carbonyl, etc.
`
`C-terminal
`
`-F
`HN
`we”=
`N-terminal
`2
`amino acid
`
`O
`
`N
`H
`OH
`
`O amino acid
`CoHs
`Le V
`N
`_
`O
`i
`O
`E
`“Scoo7
`
`Ser-Phe-Asp
`
`N-Terminal amino acid The
`aminoacid at the end of a
`polypeptide chain having thefree
`—NH,group.
`C-Terminal amino acid The
`aminoacid at the end of a
`polypeptide chain having the free
`—COOHgroup.
`
`
`Example 27.4
`Draw a structural formula for Cys-Arg-Met-Asn. Label the Nterminal amino acid and
`the Cterminal amino acid. Whatis the net charge onthis tetrapeptide at pH 6.0?
`
`Solution
`The backboneofthis tetrapeptide is a repeating sequence of nitrogen—a-carbon—
`carbonyl. The net charge onthis tetrapeptide at pH 6.0 is +1.
`pK, 8.00 lg
`O
`: NN
`O YL
`O
`NE
`HNOSNE,*
`
`N-terminal
`amino acid
`
`TH
`;
`HN
`
`SH
`
`H
`
`z
`iia
`
`H
`
`H
`
`O
`
`C-terminal
`;
`‘
`_— amino acid
`O
`NH,ot
`
`2
`
`pK,12.48
`
`,
`
`.¥
`
`Problem 27.4
`Draw a structural formula for Lys-Phe-Ala. Label the Nterminal amino acid and the
`C-terminal aminoacid. Whatis the net charge on this tripeptide at pH 6.0?
`a
`
`27.4 Primary Structure of Polypeptides
`and Proteins
`The primary (1°) structure of a polypeptide or protein refers to the sequence of
`amino acids in its polypeptide chain. In this sense, primary structure is a complete
`description ofall covalent bonding in a polypeptide orprotein.
`- In 1953, Frederick Sanger of Cambridge University, England, reported the pri-
`mary structure of the two polypeptide chains o£ the hormoneinsulin. Not only was
`
`
`Primarystructure of proteins
`The sequence of aminoacidsin
`the polypeptide chain, read from
`the Mterminal aminoacid to the
`C-terminal aminoacid.
`
`

`

`
`
`
`
`3.2500 ——__________>»pH 4.25 —W__»
`pH
`0.2N Sodium
`citrate buffer
`0.2N Sodium citrate buffer
`
`
`
`
`0.35N Sodium citrate buffer
`
`
`
`0.30
`
`0.95}
`
`0.20
`
`0.15 |z
`
`0.10 a
`
`0.05
`
`Figure 27.6
`Analysis of a mixture of aminoacids by ion-exchange chromatography using Amberlit=
`IR-120, a sulfonated polystyrene resin. The resin contains phenyl-SO3Na* groups.
`The aminoacid mixture is applied to the columnat low pH (3.25) under which conde
`tions the acidic amino acids (Asp, Glu) are weakly boundto theresin, and the basic
`aminoacids (Lys, His, Arg) are tightly bound. Sodiumcitrate buffers at two different
`concentrations, and three different values of pH are usedto elute the aminoacids
`from the column. Cysteine is determinedas cystine, Cys-S-S-Cys, the disulfide of
`cysteine.
`
`0
`
`100 125
`75
`50
`25
`Volumeof eluant (mL)
`
`1076
`
`Chapter 27 Amino Acids and Proteins
`
`this a remarkable achievementin analytical chemistry, but it also clearly establish’
`that the molecules of a given protein all have the same amino acid composition
`the same amino acid sequence. Today, the amino acid sequences of over 20,
`different proteins are known.
`
`A. Amino Acid Analysis
`The first step for determining the primary structure of a polypeptide is hydrolysis
`quantitative analysis of its amino acid composition. Recall from Section 18.4D
`amide bonds are very resistant
`to hydrolysis. Typically, samples of protein
`hydrolyzed in 6 M HClin sealedglass vials at 110°C for 24 to 72 hours. This hydrols
`can be done in a microwave oven in a shorter time. After the polypeptideiis hydrok
`the resulting mixture of amino acids is analyzed by ion-exchange chromatogra
`
`0.30 |
`
`0.25
`
`0.20
`
`0.15
`
`0.10 |
`
`0.05|
`
`athe a“Methionine|
`isoleucine|
`Leucine
`
`Gycine.
`[Alanine
`
`_Tyrosine,
`
`0
`
`25
`
`50
`
`75.
`
`100
`
`125
`
`150
`
`175
`
`275
`225 250
`200
`Volumeof eluant (mL)
`
`300
`
`325
`
`350
`
`375
`
`400
`
`425
`
`450
`
`475
`
`
`
`

`

`
`
`27.4 Primary Structure of Polypeptides and Proteins
`
`1077
`
`Amino acids are detected as they emerge from the column by reaction with ninhydrin
`(Section 27.2D) followed by absorption spectroscopy. Current procedures for hydroly-
`sis of polypeptides and analysis of amino acid mixtures have been refined to the point
`where it is possible to obtain amino acid composition from aslittle as 50 nanomoles
`(50 X 10° mole) of polypeptide. Figure 27.6 shows the analysis of a polypeptide
`hydrolysate by ion-exchange chromatography. Note that during hydrolysis, the side-
`chain amide groups of asparagine and glutamine are hydrolyzed, and these amino
`acids are detected as aspartic acid and glutamic acid. For each glutamine or asparagine
`hydrolyzed, an equivalent amount of ammonium chloride is formed.
`
`B. Sequence Analysis
`
`After the amino acid composition of a polypeptide has been determined, the next
`step is to determine the order in which the aminoacids are joined in the polypeptide
`chain. The most common sequencingstrategy is to cleave the polypeptide at specific
`peptide bonds (using, for example, cyanogen bromide or certain proteolytic en-
`zymes), determine the sequence of each fragment (using, for example, the Edman
`degradation), and then match overlapping fragments to arrive at the sequence of the
`polypeptide.
`
`Cyanogen Bromide
`Cyanogen bromide (BrCN) is specific for cleavage of peptide bonds formed by the
`carboxyl group of methionine (Figure 27.7). The products of this cleavage are a sub-
`stituted y-lactone (Section 18.1C) derived from the Nterminal portion of the
`polypeptide and a second fragment containing the Cterminal portion of the
`polypeptide.
`A three-step mechanism can be written for this reaction. The strategy for
`cyanogen bromide cleavage depends on chemical manipulation of the leaving ability
`of the sulfur atom of methionine. Because CH,S~ is the anion of a weak acid,it is a
`very poor leaving group, just as OHis a poor leaving group (Section 9.4F). Yet, just
`as the oxygen atom of an alcohol can be transformedinto a better leaving group by
`converting it into an oxonium ion (by protonation), so too can the sulfur atom of
`methionine be transformed into a.better leaving group by converting it into a
`sulfonium ion.
`
`1"
`
`
`
`Figure 27.7
`Cleavage by cyanogen
`bromide, BrCN,of a peptide
`bond formed by the carboxyl
`group of methionine.
`
`this peptide
`bondcleaved
`O
`MW
`\aNY~coo
` f
`+
`H3Nv~™VC— NH
`
`-
`
`fe
`side chain
`of methionine
`
`
`+ HaNwrcoo- +5
`
`0.1 MHCI
`+ Br-C=N —y7
`H,O
`
`Cyanogen
`bromide
`
`A substituted y-lactone of
`the amino acid homoserine
`
`Methyl
`thiocyanate
`
`
`
`

`

`1078
`
`Chapter 27 Amino Acids and Proteins
`
`
`
`
`Cleavage of a Peptide Bond at Methionine
`by Cyanogen Bromide
`
`Step 1: Reactionis initiated by nucleophilic attack of the divalent sulfur atom of methio
`on the carbon of cyanogen bromide displacing bromide ion. The product of this nuc!
`ophilic displacementis a sulfonium ion.
`
`+
`H3N~~C—NH
`
`HN~~COO7
`O
`
`—_
`
`1
`+
`H3,N~~C—NH
`
`HN~~COO7
`Oo
`
`oSS—CH,
`BreN
`m/
`Cyanogen bromide
`
`Bro
`&
`:
`a SIGE gtsCHy
`ion; a good
`|
`leaving group C=N
`
`e
`
`2
`
`HN~-COO7
`
`:O:
`An iminolactone
`hydrobromide
`
`+
`
`CHs—S—C= [
`Methyl
`thiocyanate
`
`Step 2: An internal Sy? reaction in which the oxygen of the methionine carbonyl group
`tacks the y-carbon and displaces methyl thiocyanate gives a five-membered ring. Note
`the oxygen of a carbonyl group is at best a weak nucleophile. This displacementis fa
`tated, however, because the sulfonium ion is a very good leaving group and becauseofth
`ease with whichafive-memberedring is formed.
`
`nine,tyrosine, and tryptophan (Table 27.3).
`
`O
`aol
`H3N~-C—NH
`
`oa
`H,;N~-C—NH
`
`L
`
`es
`
`(N--COO
`SB:
`a
`GoreCH
`taN
`
`os,
`
`Step 3: Hydrolysis of the imino group gives a ylactone derived from the Mterminal en
`the original polypeptide.
`
`Oo
`|
`+
`H3N~~C—NH
`
`:
`
`Hs
`HN-~~ COO-
`
`O
`|
`+
`H3sN~~C—NH
`
`O
`
`H,O
`O a
`
`ti
`O + H,N~~CoO
`
`be
`
`A substituted y-lactone of
`the aminoacid homoserine
`
`Enzyme-Catalyzed Hydrolysis of Peptide Bonds
`A group ofproteolytic enzymes, among them trypsin and chymotrypsin, can be used
`to catalyze the hydrolysis of specific peptide bonds. Trypsin catalyzes the hydrolysis of
`peptide bonds formed by the carboxyl groups of arginine and lysine; chymotrypsin
`catalyzes the hydrolysis of peptide bonds formed by the carboxyl groups of phenylala-
`
`

`

`
`
`27.6 Three-Dimensional Shapes of Polypeptides and Proteins
`
`1087
`
`Hy
`H
`Hy
`Hy
`Cc Ce
`Cc
`Ke
`HO™ HO He ce ie’
`
`Figure 27.10
`The support used forthe
`Merrifield solid-phase synthesis
`is a chloromethylated
`polystyreneresin.
`
`cross-linking of ——————_—
`polystyrene chains
`..
`by copolymerization
`Hs
`of styrene and 2%
`p-divinylbenzene
`
`CG
`
`CH
`
`CHCl
`
`Following polymerization,
`about 5% of benzenerings
`are chloromethylated.
`
`polypeptide is released from the polymer beadsbycleavage ofthe benzyl ester. The
`stepsin solid-phase synthesis of a polypeptide are summarized in Figure 27.11.
`Thanks to automation, the synthesis of polypeptides is now a routine procedure
`in chemical research. It is commonfor researchers to orderseveral peptides at a time
`for use in fields as diverse as medicine, biology, material science, and biomedical
`. engineering.
`A dramatic illustration of the power of the solid-phase method was the synthesis
`of the enzyme ribonuclease by Merrifield in 1969. The synthesis involved 369 chemi-
`cal reactions and 11,931 operations, all of which were performed by an automated
`machine and without any intermediate isolation stages. Each of the 124 amino acids
`was added as an Mtert-butoxycarbonyl derivative and coupled using DCC. Cleavage
`from the resin and removal ofall protective groups gave a mixture that was purified
`by ion-exchange chromatography. The specific activity of the synthetic enzyme was
`13-24% of that of the natural enzyme. Thefact that the specific activity of the syn-
`thetic enzyme was lower than that of the natural enzyme was probably attributable to
`the presence of polypeptide byproducts closely related to but not identical to the nat-
`ural enzyme. Synthesizing ribonuclease (124 amino acids) requires forming 123 pep-
`tide bonds. If each peptide bondis formed in 99% yield, the yield of homogeneous
`polypeptide is 0.99'*° = 29%. If each peptide bondis formed in 98% yield,the yield
`is 8%. Thus, even with yields as high as 99% in each peptide bond-formingstep, a
`large portion of the synthetic polypeptides have one or more sequence defects. Many
`of these, nonetheless, may befully or partially active.
`
`27.6 Three-Dimensional Shapes
`of Polypeptides and Proteins
`
`A. Geometry of a Peptide Bond
`In the late 1930s, Linus Pauling begana series of studies to determine the geometry
`of a peptide bond. One ofhis first and most importantdiscoveries was that a peptide
`bonditself is planar. As shown in Figure 27.12, the four atoms of a peptide bond and
`the two a-carbonsjoinedtoit all lie in the sameplane.
`Had you been asked in Chapter 1 to describe the geometry ofa peptide bond,
`you probably would have predicted bond angles of 120° about the carbonyl carbon
`and 109.5° about the amide nitrogen. However, as fully discussed in Connections to
`Biological Chemistry: “The Unique Structure of Amide Bonds” in Chapter 18, both
`
`Grisham
`
`Charles
`
`Ml A modelofthe protein
`ribonuclease A. The purple
`segments are regionsof a-helix
`and the yellow segments are
`regions of pleated sheet,
`both of which are described in
`Section 27.6. Othercolors
`represent loop regions.
`
`
`
`
`
`
`
`:
`
`

`

`
`
`Figure 27.11
`Steps in the Merrifield
`solid-phase polypeptide
`synthesis.
`
`1088
`
` polypeptide
`
`polypeptide —NI
`
`

`

`27.6 Three-Dimensional Shapes of Polypeptides and Proteins
`
`1089
`
`120.0°
`
`1215?
`
`polypeptide chain
`(in B sheet)
`
`\
`
`
`
`
`
`Figure 27.12
`Planarity of a peptide bond. Bond angles about the carbonyl carbon and the amide nitrogen
`are approximately 120°.
`
`atomsare actually planar with approximately 120° bond angles about each because of
`resonance of the nitrogen lone pair with the carbonyl. Two configurations are pos-
`sible for the atomsof a planar peptide bond.In one, the two a-carbonsare cis to each
`other; in the other, they are trans to each other. The trans configuration is more
`favorable because the a-carbons with the bulky groups bonded to them are farther
`from each other than they are in the cis configuration. Almostall peptide bondsin
`naturally occurring proteins studied to date have the trans configuration. Proline is
`found cis most of the time, and there are some well-known examples of other cis
`peptide bonds aswell.
`
`
`
`s-trans configuration
`
`s-cis configuration
`
`B. Secondary Structure
`Secondary (2°) structure refers to ordered arrangements (conformations) of amino
`acids in localized regions of a polypeptide or protein molecule. Thefirst studies of
`polypeptide conformations were carried out by Linus Pauling and Robert Corey be-
`ginning in 1939. They assumedthat in conformationsof greatest stability, all atomsin
`a peptide bondlie in the same plane, and there is hydrogen bonding between the
`N—Hofone peptide bond and the C=O ofanother,as shownin Figure 27.13.
`On the basis of model building, Pauling proposed that two types of secondary
`structure should be particularly stable: the a-helix and the antiparallel 6-pleated
`sheet. X-ray crystallography has validated this prediction completely.
`
`
`
`-_
`
`Secondarystructure of proteins
`The ordered arrangements
`(conformations) of aminoacids in
`localized regions of a polypeptide
`or protein.
`
`
`
`
`Amide
`“&
`Hydrogen
`plane aebonding
`
`
`
`Figure 27.13
`Hydrogen bonding between
`amide groups.
`
`

`

`1090
`
`Chapter 27 Amino Acids and Proteins
`
`hydrogen bonding
`
`
`
`M1,
`
`Fy
`
`
`
`
`
`te go
`
`My
`
`My
`
`a-Helix A type of secondary
`structure in which a section of
`
`polypeptide chain coils into a
`spiral, most commonly a
`right-handedspiral.
`
`B-Pleated sheet A type of
`secondary structure in which
`sections of polypeptide chains are
`aligned parallel or antiparallel to
`one another.
`
`Figure 27.14
`An a-helix. The peptide chain is repeating units of L-alanine.
`
`The a-Helix
`
`In an a-helix pattern shown in Figure 27.14, a polypeptide chain is coiled in a bir
`As you study this section of a-helix, note the following.
`
`1. Thehelix is coiled in a clockwise, or right-handed, manner. Right-handed means&
`if you turn the helix clockwise, it twists away from you. In this sense, a right-hand
`helix is analogousto the right-handed thread of a common wood or machine sam
`
`oSNO
`. There are 3.6 aminoacids per turn of the helix.
`. Each peptide bondis trans and planar.
`
`4, The N—Hgroupofeach peptide bond points roughly downward,parallel toa
`axis of the helix, and the C—O ofeach peptide bond points roughly upward. a&
`
`parallel to the axis of the helix.
`5. The carbonyl group of each peptide bond is hydrogen-bonded to the N=
`
`group of the peptide bond four aminoacid units away from it. Hydrogen bem
`
`are shownas dashedlines.
`
`6. All R— groups point outward from the helix.
`
`Almost immediately after Pauling proposed the a-helix conformation, other
`searchers proved the presence of a-helix conformations in keratin, the protei
`hair and wool. It soon became obvious that the a-helix is one of the fundamen
`
`folding patterns of polypeptide chains.
`The B-Pleated Sheet
`An antiparallel B-pleated sheetconsists of extended polypeptide chains with neigh D0
`
`ing chains running in opposite (antiparallel) directions. In a parallel B-pleated she
`
`the polypeptide chains run in the same direction. Unlike the a-helix arrangemes
`N—H and C=O groupslie in the planeof the sheet and are roughly perpendicule
`
`to the long axis of the sheet. The C=O group of each peptide bondis hydrogeSe
`bonded to the N—Hgroup of a peptide bond of a neighboring chain (Figure 97.15a
`As you study this section of B-pleated sheet, note the following.
`
`
`1. The three polypeptide chains lie adjacent to each other and run in opposite (2
`parallel) directions.
`4
`2. Each peptide bondis planar, and the a-carbonsare trans to each other.
`
`

`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`27.6 Three-Dimensional Shapes of Polypeptides and Proteins
`
`1091
`
`Figure 27.15
`Pleated sheet conformation
`with three polypeptide
`chains running in opposite
`(antiparallel) directions.
`Hydrogen bonding between
`chainsis indicated by dashed
`
`
`
`
`
`
`
`
`
`
`
`
`
`lines.
`
`3. The C=O and N—H groups of peptide bonds from adjacent chains point at
`each other and are in the same plane so that hydrogen bondingis possible be-
`tween adjacent polypeptide chains.
`4. The R-groups on any onechain alternate,first above and then below the plane of
`the sheet, and so on.
`
`The #-pleated sheet conformation is stabilized by hydrogen bonding between
`N—Hgroupsof one chain and C=O groupsof an adjacent chain. By comparison,
`the a-helix is stabilized by hydrogen bonding between N—H and C=O groups
`within the same polypeptide chain.
`
`Tertiary structure of proteins
`Thethree-dimensional
`
`arrangementin spaceofall atoms
`in a single polypeptide chain.
`
`C. Tertiary Structure
`Tertiary (3°) structure refers to the overall folding pattern and arrangement in space
`of all atoms in a single polypeptide chain. No sharp dividing line exists between sec-
`ondary andtertiary structures. Secondary structure refers to the spatial arrangement
`of amino acids close to one another on a polypeptide chain, whereastertiary struc-
`ture refers to the three-dimensional arrangementofall atoms of a polypeptide chain.
`Among ‘the most important factors in maintaining 3° structure are disulfide bonds,
`hydrophobic interactions, hydrogen bonding,andsalt linkages.
`Disulfide bonds (Section 10.9G) play an importantrole in maintaining tertiary
`structure. Disulfide bonds are formed betweenside chainsof two cysteine units by ox-
`idation of their thiol groups (—SH) to form a disulfide bond. Treatmentof a disul-
`fide bond with a reducing agent regeneratesthe thiol groups.
`O
`O
`
`H
`4
`wN
`Bat
`of cysteine
`
`ig
`H
`
`ot
`
`N’
`H
`
`ay
`SH
`H
`Ne
`
`O
`
`H
`-N
`
`-
`
`2
`
`N’
`H
`
`_—
`reduction
`
`s
`S
`
`a disulfide bond
`
`H
`a i
`H
`O
`
`

`

`
`
`CHEMICAL
`CONNECTIONS
`
`Spider Silk
`
`Spider silk has some remarkable properties. Research is
`currently concentrated on the strong dragline silk that
`forms the spokes of a web of the Golden Orb Weaver
`(Nephila clavipes). This silk has three times the impact
`strength of Kevlar and is 30% moreflexible than nylon.
`The commercial application of spider silk is not a novel
`concept. Eighteenth-century French entrepreneur Bon de
`Saint-Hilaire attempted to mass-produce silk in his high-
`density spider farms but failed because of cannibalism
`amonghis territorial arachnid workers. In contrast, native
`New Guineans continue to successfully collect and utilize
`spidersilk for a wide range of applications including bags
`and fishing nets. Today,
`the only way to obtain large
`amounts of silk is to extract it from the abdomensof im-
`mobilized spiders, but scientific advances make the mass
`production and industrial application of spider silk in-
`creasingly possible.
`Biologically produced dragline silk is a combina-
`tion of two liquid proteins, Spidroin 1 and 2, which be-
`come oriented and solidify as they travel through a
`complex duct system in the spider’s abdomen. These
`
`
`
`TomBean/Stone/Getty
`
`proteins are composedlargely of alanine and glycine,
`the two smallest amino acids. Although glycine com-
`prises almost 42% of each protein, the short, 5 to 10 _
`peptide chains of alanine, which account for 25% of
`each protein’s composition, are more importantfor the
`properties. Nuclear magnetic resonance (NMR)
`tech-
`niques have vastly improved the level of understanding
`of spider silk’s structure, which was originally deter
`mined by x-ray crystallography. NMR data of spidroins
`containing deuterium-tagged alanine have shown that
`all alanines are configured into 6-pleated sheets. Fur-
`thermore, the NMR data suggest that 40% of the ala
`nine ®sheets are highly structured while the other 60%
`are less oriented, forming fingers that reach out from
`each individual strand. These fingers are believed to
`join the oriented alanine 6-sheets and the glycine-rich,
`amorphous “background”sectors of the polypeptide.
`is
`Currently, genetically modified Escherichia coli
`used to mass-produce Spidroin 1 and 2. However, DNA
`redundancy initially caused synthesis problems when
`the spider genes were transposed into the bacteria. The
`E. cok did not transcribe some of the codons in the
`same way that spider cells would, forcing scientists to
`modify the DNA. Whenthe proteins could be synthe-
`sized, it was necessary to develop a system to mimic the
`natural production ofspider silk while preventing the
`silk from contacting the air and subsequently harden-
`ing. After the two proteins are separated from the
`E. coli, they are drawn together into methanol through
`separate needles. Another approach is to dissolve the
`silk in formic acid or to add codons for hydrophilic
`amino acids, in this case histidine and arginine, to keep
`the artificial silk pliable. The industrial and practical
`applicationsof spider silk will not be fully known until
`it can be abiotically synthesized in large quantities.
`
`Hi Golden orb weaver.
`Based on a Chem 30H honorspaperby Paul Celestre, UCLA.
`
`
`Figure 27.16 shows the amino acid sequence of human insulin. This protem —
`consists of two polypeptide chains: an A chain of 21 amino acids and a B chain of
`30 amino acids. The A chain is bondedto the B chain by two interchain disulfide |
`bonds. An intrachain disulfide bond also connects the cysteine units at positions 6
`and 11 of the A chain.
`
`
`
`
`

`

`27.6 Three-Dimensional Shapesof Polypeptides and Proteins
`
`1093
`
`:
`A chain
`
`§ semen
`
`TpGeae C-terminal
`d
`
`i N-terminal !
`ee
`
`» C-terminal
`
`'
`
`
`
`‘omit SPISSSSTPIIIVSSSS:
`
`
`
`
`
`
`Figure 27.16
`Humaninsulin. The A chain of 21 aminoacids and B chain of 30 amino acids are connected
`by interchain disulfide bonds between A7 and B7 and between A20 and B19. In addition, a
`single intrachain disulfide bond occurs between A6 and All.
`
`As an example of 2° and 3° structure, let us look at the three-dimensionalstruc-
`ture of myoglobin—a protein foundin skeletal muscle and particularly abundantin
`diving mammals, such asseals, whales, and porpoises. Myoglobin and its structural
`relative, hemoglobin, are the oxygen storage and transport molecules ofvertebrates.
`Hemoglobin binds molecular oxygen in the lungs and transports it to myoglobin in
`muscles. Myoglobin stores molecular oxygen until
`it
`is required for metabolic
`oxidation.
`Myoglobin consists of a single polypeptide chain of 153 amino acids. Myoglobin
`also contains a single heme unit. Heme consists of one Fe?* ion coordinated in a
`square planar array with the four nitrogen atoms of a molecule of porphyrin
`(Figure 27.17).
`Determination of the three-dimensional structure of myoglobin represented a
`milestone in the study of molecular architecture. For their contribution to this
`research, John C. Kendrew and Max F.Perutz, both of Britain, shared the 1962 Nobel
`Prize for chemistry. The secondaryandtertiary structures of myoglobin are shown in
`Figure 27.18. The single polypeptide chainis folded into a complex, almost boxlike
`shape.
`Following are important structural features of the three-dimensional shape of
`myoglobin.
`1. The backboneconsists of eight relatively straight sections of a-helix, each sepa-
`rated by a bend in the polypeptide chain. The longest section of a-helix has
`94 arwino acids, the shortest has 7. Some 75% of the amino acids are found in
`these eight regions of a-helix.
`
`H3C.
`
`H,C =CH
`
`/
`
`H3C
`
`H,C=CH
`
`CH,CH,COO-
`
`CH;
`
`CH,CH,COO-
`
`CH;
`
`Figure 27.17
`Thestructure of heme, found
`in myoglobin and hemoglobin.
`
`

`

`1094
`
`Chapter 27 Amino Acids and Proteins
`
`
`
`
`
`BrentIverson,UniversityofTexas
`
`
`
`Figure 27.18
`Ribbon model of myoglobin.
`The polypeptide chain is
`shownin yellow, the heme
`ligandin red, and the Fe atom
`as a white sphere.
`
`Quaternary structure The
`arrangementof polypeptide
`monomersinto a noncovalently
`bonded aggregate.
`
`
`isoleucmm=
`2. Hydrophobic side chains of phenylalanine, alanine, valine, leucine,
`and methionine are clustered in the interior of the molecule where they
`=
`shielded from contact with water. Hydrophobicinteractions are a major factor =
`
`directing the folding of the polypeptide chain of myoglobin into this compas’
`three-dimensional shape.
`3. The outer surface of myoglobin is coated with hydrophilic side chains, such
`
`those oflysine, arginine, serine, glutamic acid, histidine, and glutamine, which =
`teract with the aqueous environment by hydrogen bonding. The only polar=
`his
`chains thatpoint to the interior of the myoglobin molecule are those of two
`
`dine units, which point inward toward the heme group.
`
`4, Oppositely charged aminoacid side chains close to each otherin the three-dimet
`sional structure interact by electrostatic attractions called salt linkages. An ex2
`ple of a salt linkageis the attraction ofthe side chains of lysine (—NH;") and
`
`tamic acid (—COO_).
`Thetertiary structures of hundredsofproteins have also been determined.It is =
`that proteins contain a-helix and f-pleated sheetstructures, butthat widevariations ==
`in the relative amounts of each. Lysozyme, with 129 aminoacidsin a single polypepa=
`
`chain, has only 25% of its amino acids in a-helix regions. Cytochrome, with 104 aaa
`acids in a single polypeptide chain, has no a-helix structure but does contain several =
`gions of pleated sheet. Yet, whatever the proportions of a-helix, Bpleated sheet
`=
`
`other periodic structure, most nonpolarside chains ofwater-soluble proteins are directs
`toward the interior of the molecule, whereas polar side chains are on the surface of =
`molecule and in contact with the aqueous environment. Note that this arrangemen® S
`
`polar and nonpolar groups in water-soluble proteins very much resemblesthe arr
`mentof polar and nonpolar groups of soap molecules in micelles (Figure 26.3). It
`resembles the arrangementof phospholipidsin lipid bilayers (Figure 26.13).
`
`
`Example 27.7
`
`With which of the following amino acid side chains can the side chain of threon™
`form hydrogen bonds?
`(a) Valine
`(b) Asparagine
`(d) Histidine
`(e) Tyrosine
`
`(c) Phenylalanine
`(f) Alanine
`
`Problem 27.7
`At pH 7.4, with what aminoacid side chains can the side chain of lysine form =
`linkages?
`
`
`Solution
`Theside chain of threonine contains a hydroxyl group that can participate in hyde
`gen bondingin two ways: Its oxygen has a partial negative charge and can function
`a hydrogen bond acceptor, and its hydrogen has a partial positive charge and &
`function as a hydrogen bond donor. Therefore,the side chain of threonine can ton
`hydrogen bonds with the side chainsof tyrosine, asparagine, and histidine.
`
`
`
`
`
`
`D. Quaternary Structure
`
`Most proteins of molecular weight greater than 50,000 consist of two or more DGS
`covalently linked polypeptide chains. The arrangement of protein monomersinte
`
`aggregation is known as quaternary (4°) structure. A good example is hemoglobin.
`
`
`

`

`
`
`protein that consists of four separate polypeptide chains: two a-chains of 141 amino
`acids each and two @-chains of 146 amino acids each. The quaternary structure of
`hemoglobin is shown in Figure 27.19.
`A majorfactorstabilizing the aggregation of protein subunits is the hydrophobic
`effect. When separate polypeptide chains fold into compact
`three-dimensional
`shapes to expose polarside chains to the aqueous environmentandshield nonpolar
`side chains from water, hydrophobic “patches”still appear on the surface, in contact
`with water. These patches can be shielded from water if two or more monomersas-
`semble so that their hydrophobic patches are in contact. The numbersof subunits of
`several proteins of known quaternary structure are shown in Table 27.4. Other