Chapter 27: Amino Acids, Peptides, and Proteins.

monomer unit: -amino acids

H NH2

R = sidechain

CO2H

- Amino Acid

Biopolymer: the monomeric amino acids are linked through an

amide bond (the carboxylic acids of one AA with the -amino
group of a second)
+

R1
H3N

CO2

R2
H3N

H
N
R1

CO2

R2
N
H

R1

- H2O

N-terminus

H
N
O

H3N

R3

R4
N
H

H
N
O

R5

R6
N
H

H
N

CO2
R2

H
N
O

C-terminus

R7

N
H

Peptide or protein (polypeptide)

peptide (< 50 amino acids)

protein (> 50 amino acids)

27.1: Classification of Amino Acids. AAs are classified

according to the location of the amino group.
H H
H2N C C CO2H
H H

H
H2N C CO2H
H
-amino acid
(2-amino carboxylic acid)

-amino acid
(3-amino carboxylic acid)

H H H
H2N C C C CO2H
H H H
-amino acid
(4-amino carboxylic acid)

There are 20 genetically encoded-amino acids found in peptides

and proteins
19 are primary amines, 1 (proline) is a secondary amine
19 are chiral, 1 (glycine) is achiral; the natural configuration of
the -carbon is L.
CHO
OH
H
CH2OH

CHO
H
HO
CH2OH

CO2H
H2N
H
CH3

CO2H
H
H2N
R

D-glyceraldehyde

L-glyceraldehyde

CHO
H
HO
H
OH
CH2OH

CHO
OH
H
HO
H
CH2OH

CO2H
H2N
H
H
OH
CH3

CO2H
H
H2N
H3C
H
CH2CH3

D-erythrose

L-erythrose

L-theronine
(2S,3R)

L-isoleucine
(2S,3S)

L-alanine

-Amino acids are classified by the properties of their sidechains.

Nonpolar:
COO
COO
COO

NH3

NH3

NH3

Glycine (Gly, G)

(S)-(+)-Alanine (Ala, A)

COO

(S)-()-Leucine (Leu, L)

Polar but non-ionizable:

HO

COO

COO

NH3

NH3

(2S,3S)-(+)-Isoleucine (Ile, I)

(S)-()-Methionine (Met, M)

COO

COO

(S)-()-Proline (Pro, P)

(S)-(+)-Valine (Val, V)

COO

NH3

COO

NH3

N
H

(S)-()-Phenylalanine (Phe, F)

OH

NH3

(S)-()-Tryptophan (Trp, W)

COO

COO

NH3

HO

NH3

NH3

(S)-()-Serine (Ser, S)

(2S,3R)-()-Threonine (Thr, T)

(S)-()-Tyrosine (Tyr, Y)

pKa ~ 13

pKa ~ 13

pKa ~ 10.1

HS

COO
NH3

(R)-()-Cysteine (Cys, C)
pKa ~ 8.2

COO

H2N
O

NH3

(S)-()-Asparagine (Asn, N)

O
H2N

COO
NH3

(S)-(+)-Glutamine (Gln, Q)

Acidic:

-O

COO
O

NH3

NH3

(S)-(+)-Aspartic Acid (Asp, D)

(S)-(+)-Glutamic Acid (Glu, E)

pKa ~ 3.6

Basic:
H 3N

COO
NH3

COO

-O

pKa ~ 4.2

N
H
N
H

COO
NH3

(S)-(+)-Lysine (Lys, K)

(S)-()-Histidine (His, H)

pKa ~ 10.5

pKa ~ 6.0

H
H2N

H
N
H

COO
NH3

(S)-(+)-Arginine (Arg, R)
pKa ~ 12.5

27.2: Stereochemistry of Amino Acids: The natural

configuration of the -carbon is L. D-Amino acids are found in
the cell walls of bacteria. The D-amino acids are not genetically
encoded, but derived from the epimerization of L-isomers

27.3: Acid-Base Behavior of Amino Acids. Amino acids exist

as a zwitterion: a dipolar ion having both a formal positive and
formal negative charge (overall charge neutral).
+ R
_
H3N
CO2
H

R
H2N

CO2H

pKa ~ 5

pKa ~ 9

Amino acids are amphoteric: they can react as either an acid or a

base. Ammonium ion acts as an acid, the carboxylate as a base.
Isoelectric point (pI): The pH at which the amino acid exists
largely in a neutral, zwitterionic form (influenced by the nature
of the sidechain)
+ R
H3N
CO2H
H
low pH

H3O+
pKa1

+ R
_
H3N
CO2
H

Table 27.2 (p. 1115) & 27.2 (p. 1116)

HO

pKa2

R
H2N

CO2

H
high pH

pKax + pKay

pI =

+ CH3
H3N
CO2H
H
low pH

H3N

pKa1
(2.3)

+ CH3
H3N
CO2
H

CH3
H2N
CO2
H

pKa2
(9.7)

high pH

CO2H

CO2H

CO2

CH2

CO2

CH2

CH2

CH2

CO2H

pKa1
(1.9)

H3N

CO2

pKa3
(3.6)

H3N

CO2

low pH

(CH2)4
H

CO2H

low pH

H2N

CO2

high pH

NH3
H3N

pKa2
(9.6)

pKa1
(2.2)

H3N

NH3

NH3

(CH2)4

(CH2)4

CO2

pKa2
(9.0)

H2N

CO2

NH2
pKa3
(10.5)

H2N

(CH2)4
H

CO2

high pH

Electrophoresis: separation of polar compounds based on their

mobility through a solid support. The separation is based on
charge (pI) or molecular mass.
+

_ _

+ +

27.5: Synthesis of Amino Acids:

Br

Br2, PBr3

R-CH2-CO2H

NH2

NH3

R C CO2H

R C CO2H
H

Ch. 19.16

Strecker Synthesis: recall reductive amination

O

NH2

NH3

R C CO2H

NH2

NaB(CN)H3

R C CO2H

R C CO2H

H
O
R C H

NH3

NH2

NaCN

NH2

R C CN

R C H

NH2

H3O+
-orNaOH, H2O

R C CO2H
H

NC:

Amidomalonate Synthesis: recall the malonic acid synthesis

O

O
HN CO2Et
C
H
CO2Et

EtO

Na

RCH2X

HN CO2Et
C
RCH2
CO2Et

H3O
- CO2

H2N H
C
RCH2
CO2H

27.5: Reactions of Amino Acids. Amino acids will undergo

reactions characteristic of the amino (amide formation) and
carboxylic acid (ester formation) groups.
H2N
R

HOCH2CH3

H3N

H+

CO2CH2CH3

H3C

CO2

H3C

O
O

base

CH3

HN

O
H

CO2H

27.6: Some Biochemical Reactions of Amino Acids. Many

enzymes involved in amino acid biosynthesis, metabolism and
catabolism are pyridoxal phosphate (vitamin B6) dependent
(please read)
R
CO
R

H
CO2NH3

L-amino acid

OH

2-

O3PO
N

pyridoxal
phosphate (PLP)

racemase,
epimerase

NH3

OH

PO
N
H

decarboxylase

D-amino acid
R

transaminase
CO2-

R
O

CO2H

H
H

H3N

27.7: Peptides. Proteins and peptides are polymers made up of

amino acid units (residues) that are linked together through the
formation of amide bonds (peptide bonds) from the amino
group of one residue and the carboxylate of a second residue
H2N

HO

CO2H

- H2O

H2N

CO2H

N-terminus

Serine

Alanine

H2N

N-terminus

H
N

H2N

CO2H

O
R1

R2
N
H

H
N
O

R3

C-terminus

OH

By convention, peptide sequences

are written left to right from the
N-terminus to the C-terminus

C-terminus

Ser - Ala
(S - A)

H
N

CO2H

Ala - Ser
(A - S)

- H2O
HO

H
N

R4
N
H

H
N
O

R5

R6
N
H

H
N
O

R7

N
H

backbone
10

The amide (peptide) bond has C=N double bond character due
to resonance resulting in a planar geometry
O

H
N
R1

R2
N
H

H
N

H
N

R1

amide bond

R2

+
N
H

restricts rotations
resistant to hydrolysis

H
N
O

The N-H bond of one amide linkage can form a hydrogen bond
with the C=O of another.
H N
O
H N

N H

N-O distance 2.85 - 3.20

R
N H

O
H N

N H
R

optimal N-H-O angle is 180

Disulfide bonds: the thiol groups of cysteine can be oxidized to

form disulfides (Cys-S-S-Cys)
2

NH2
HO2C

SH

NH2

H2O

1/2 O2

HO2C
H2

CO2H
NH2

11

R6
N
H

R1
N
H

O
HS

H
N
R2

H
N

N
H

R8
N
H

SH
H
N
O

O
R4

R9

R5
N
H

H
N

H
N
O

R10
N
H

R9

H
N

N
H

1/2 O2
R1

H2
N
H

R2

N
H

R11
N
H

H
N
R4

R12

R5
N
H

H
N

S
S

H
N

H
N

R13
N
H

H
N

H
N
O

Epidermal Growth Factor (EGF): the miracle of mothers spit

53 amino acid, 3 disulfide linkages

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

1986 Nobel Prize in Medicine:

Stanley Cohen
Rita Levi-Montalcini

12

27.8: Introduction to Peptide Structure Determination.

Protein Structure:
primary (1) structure: the amino acid sequence
secondary (2): frequently occurring substructures or folds
tertiary (3): three-dimensional arrangement of all atoms in a
single polypeptide chain
quaternary (4): overall organization of non-covalently linked
subunits of a functional protein.
1. Determine the amino acids present and their relative ratios
2. Cleave the peptide or protein into smaller peptide fragments
and determine their sequences
3. Cleave the peptide or protein by another method and
determine their sequences. Align the sequences of the
peptide fragments from the two methods
13

E-A-Y-L-V-C-G-E-R
F-V-N-Q-H-L-F-S-H-L-K
G-C-F-L-P-K
L-G-A

F-V-N-Q-H-L-F
S-H-L-K-E-A-Y
L-V-C-G-E-R-G-C-F
L-P-K-L-G-A

F-V-N-Q-H-L-F
F-V-N-Q-H-L-F-S-H-L-K
S-H-L-K-E-A-Y
E-A-Y-L-V-C-G-E-R
L-V-C-G-E-R-G-C-F
G-C-F-L-P-K
L-P-K-L-G-A
L-G-A
F-V-N-Q-H-L-F-S-H-L-K-E-A-Y-L-V-C-G-E-R-G-C-F-L-P-K-L-G-A
14

27.9: Amino Acid Analysis. automated method to determine the

amino acid content of a peptide or protein
Reaction of primary amines with ninhydrin
O

NH3
R

CO2

peptide
-orprotein

Enzymatic
digestion
-orH3O+,

RCHO

CO2

reduce any
disulfide
bonds

liquid
chromatography

O
Ninhydrin

[H]

NH3
R

derivatize w/
ninhydrin

Different amino
acids have different
chromatographic
mobilities (retention
times)

CO2

individual
amino acids
Detected w/
UV-vis

1972 Nobel Prize in Chemistry

William Stein
Stanford Moore
15

27.10: Partial Hydrolysis of Peptides. Acidic hydrolysis of

peptides cleave the amide bonds indiscriminately.
Proteases (peptidases): Enzymes that catalyzed the hydrolysis
of the amide bonds of peptides and proteins.
Enzymatic cleavage of peptides and proteins at defined sites:
trypsin: cleaves at the C-terminal side of basic residues,
Arg, Lys but not His
O

R1
N
H

H3N

H
N

R3
N
H

H
N

CO2

trypsin

R1
N
H

H3N

H2O

R3

H
N

H3N

H
N

CO2

NH3

NH3

chymotrypsin:cleavesattheCterminalsideofaromaticresidues
Phe,Tyr,Trp
R1

O
H3N

N
H

H
N
O

R3
N
H

H
N
O

CO2

chymotrypsin
H2O

O
H3N

R1
N
H

H
N
O

R3
O

H3N

H
N

CO2

16

Trypsin and chymotrypsin are endopeptidases

Carboxypeptidase: Cleaves the amide bond of the C-terminal
amino acid (exopeptidase)
27.11: End Group Analysis. The C-terminal AA is identified
by treating with peptide with carboxypeptidase, then analyzing
by liquid chormatography (AA Analysis).
N-labeling: The peptide is first treated with 1-fluoro-2,4-dinitro
benzene (Sangers reagent), which selectively reacts with the
N-terminal amino group. The peptide is then hydrolyzed to their
amino acids and the N-terminal amino acid identified as its
N-(2,4-dinitrophenyl) derivative (DNP).
NO2

O2N

enzymatic
digestion
-orH3O+,

R1
H3N

H
N

CO2

O2N

R1
NO2

N
H

NH2
O

nucleophilic
aromatic
substitution

O2N

R1
NO2

plus other unlabeled

amino acids

N
H

H
N

CO2

17

27.12: Insulin. (please read) Insulin has two peptide chains

(the A chain has 21 amino acids and the B chain has 30 amino
acids) held together by two disulfide linkages
Pepsin: cleaves at the C-terminal side of Phe, Tyr, Leu; but
not at Val or Ala
Pepsin cleavage
Trypsin cleavage
H3O+ cleavage

18

27.13: The Edman Degradation and Automated Peptide

Sequencing. Chemicalmethodforthesequentialcleavageand
identificationoftheaminoacidsofapeptide,oneatatimestartingfrom
theNterminus.Reagent:PhN=C=S,phenylisothiocyanate(PITC)

Ph

S
C
N

Ph N
HN

R1

H2 N

H
N

CO2

pH 9.0

Ph

N
H

R1
N
H

H+
S
R1

H
N

CO2

Ph

HN

OH

R1

H+

N-phenylthiohydantoin:

separated by liquid chromatography

(based of the R group) and detected
by UV-vis

Ph
N

S
HN

H
N

CO2

H+

O
H+

H2N

CO2

-1 peptide with a new

N-terminal amino acid
(repeat degradation cycle)

O
R1

19

Peptide sequencing by Edman degradation:

Cycle the pH to control the cleavage of the N-terminal amino
acid by PITC.
Monitor the appearance of the new N-phenylthiohydantoin for
each cycle.
Good for peptides up to ~ 25 amino acids long.
Longer peptides and proteins must be cut into smaller fragments
before Edman sequencing.
Tandem mass spectrometry has largely replaced Edman
degradation for peptide sequencing
27.14: The Strategy for Peptide Synthesis:
Chemical synthesis of peptide:
1. Solution phase synthesis
2. Solid-phase synthesis
20

H
N

H2N

- H2O

CO2H

H2N

CO2H

- H2O
H2N

H2N

CO2H

Val

Ala

Val - Ala
(V - A)

H
N

CO2H

O
Ala - Val
(A - V)

The need for protecting groups

Pn

OH

N
H

OPc

H2N

Pn

- H2O

H
N
O
Ala - Val
(A - V)

OPc

Pn

Ph
Pn

OPc

selectively
remove Pn

Ala - Val
(A - V)
peptide
coupling
(-H2O)

H
N

N
H

Val

Ala

H2N

peptide
coupling

N
H

OH

O
Phe (F)

H
N
Ph

O
N
H

H
N
O

O
OPc

Repeat

peptide
synthesis

Phe - Ala - Val

(F - A - V)

Orthogonal protecting group strategy: the carboxylate protecting

group must be stable to the reaction conditions for the removal
of the -amino protecting group and ( vice versa)
21

27.15: Amino Group Protection. The -amino group is

protected as a carbamate.
NH3

Base

RO

RO

Cl

NH
OH
O

O
C6H5

NH

NH

NH
R

tert-butoxycarbonyl
(t-BOC)

benzyloxycarbonyl
(cBz)

fluorenylmethylcarbonyl
(FMOC)

removed with
mild acid

removed with mild acid

or by hydrogenolysis

removed with mild base

(piperidine)

27.16: Carboxyl Group Protection. Protected as a benzyl

ester; removed by hydrogenolysis
O
C6H5

H2N

H
N
O

N
H

OH

H2N

C6H5

peptide
coupling
- H2O

O
C6H5

N
H

Ph
O

O
O

C6H5

C6H5

peptide
coupling

N
H

OH
O

- H2O

C6H5

H
N
O
Ph

O
N
H

H
N
O

H
N

mild acid
O

C6H5

H2, Pd/C
O

C6H5

O
H3N
Ph

N
H

H
N
O

22

O
O

27.17: Peptide Bond Formation. Amide formation from the

reaction of an amine with a carboxylic acid is slow. Amide bond
formation (peptide coupling) can be accelerated if the carboxylic
acid is activated. Reagent: dicyclohexylcarbodiimide (DCC)
O

(DCC)

C6H11

NH

O C
NH
HN
R'
+ C6H11

N
H

OH
O

Ala

OBn

H2N

cBz

peptide
coupling

C6H11
"activated acid"

O
N
H

R'

N
H

H
N

Ph
cBz

N
H

cBz
OH

O
Phe (F)

H
N
Ph

O
N
H

C6H11

N
H

H
N

OBn

O
OBn

N
H

H2, Pd/C

C6H11

DCU

Val

DCC

O C
+N
N
R'
H
H
C6H11

Amide

DCC

NH

cBz

NH
O C

R'-NH2

C6H11

+
C6H11 N C N C6H11
H

C6H11 N C N C6H11

C6H11

CF3CO2H

H2N

selectively
remove Nprotecting
group

Ph

N
H

OBn

O
H2N

H
N

H
N

O
OH

Phe - Ala - Val

(F - A - V)

23

 In order to practically synthesize peptides and proteins, time

consuming purifications steps must be avoided until the very
end of the synthesis.
Large excesses of reagents are used to drive reactions forward
and accelerate the rate of reactions.
How are the excess reagents and by-products from the reaction,
which will interfere with subsequent coupling steps, removed
without a purification step?
27.18: Solid-Phase Peptide Synthesis: The Merrifield Method.
Peptides and proteins up to ~ 100 residues long are synthesized
on a solid, insoluble, polymer support. Purification is conveniently
accomplished after each step by a simple wash and filtration.

24

The solid support (Merrifield resin): polystyrene polymer

Ph
styrene

initiator

Ph

polymerization

Ph

Ph

Ph

Ph

Ph

BOC

Ph

CF3CO2H

O
R

CH2Cl

Ph
Ph

H
N

H3COCH2Cl
ZnCl2

Ph
Ph

Ph

divinylbenzene
(crosslinker, ~1 %)

Ph

NH
BOC

NH2

Solid-phase peptide synthesis

FMOC

H2N

DCC

Ph

DCC
FMOC

Ph
N
H

FMOC

H
N

peptide
coupling

Val

FMOC

OH

N
H

OH

O
Phe (F)

N
H

H
N
O

O
N
H

O
O

O
N
H

purify:
wash & filter

purify:
wash & filter

N
H
remove Nprotecting
group

N
H
remove Nprotecting
group

HF
remove Nprotecting
group and cleave
from solid-support

purify:
wash & filter

O
H2N

N
H

O
O

purify by liquid
chromatograrphy
or electrophoresis

Ph
H2N

H
N

O
N
H

25

OH
O

RibonucleaseA124aminoacids,catalyzesthehydrolysisofRNA
SolidphasesynthesisofRNaseA:
SyntheticRNaseA:78%activity

0.4mgwassynthesized
2.9%overallyield
averageyield~97%percouplingstep
His119A

LYS
GLN
SER
LYS
LYS
LEU
LYS
ASN
ILE
LYS
GLN
GLU
ASP

GLU
HIS
SER
SER
PRO
ALA
ASN
CYS
THR
TYR
ALA
GLY
ALA

THR
MET
SER
ARG
VAL
ASP
VAL
TYR
ASP
PRO
ASN
ASN
SER

ALA
ASP
ASN
ASN
ASN
VAL
ALA
GLN
CYS
ASN
LYS
PRO
VAL

ALA
SER
TYR
LEU
THR
GLN
CYS
SER
ARG
CYS
HIS
TYR

ALA
SER
CYS
THR
PHE
ALA
LYS
TYR
GLU
ALA
ILE
VAL

LYS
THR
ASN
LYS
VAL
VAL
ASN
SER
THR
TYR
ILE
PRO

PHE
SER
GLN
ASP
HIS
CYS
GLY
THR
GLY
LYS
VAL
VAL

GLU
ALA
MET
ARG
GLU
SER
GLN
MET
SER
THR
ALA
HIS

ARG
ALA
MET
CYS
SER
GLN
THR
SER
SER
THR
CYS
PHE

His12A

His12B

His119B

pdbcode:1AFL

R.BruceMerrifield,RockefellerUniversity,1984NobelPrizeinChemistry:
for his development of methodology for chemical synthesis on a solid matrix.

26

27.19: Secondary Structures of Peptides and Proteins.

-sheet: Two or more extended peptide chain, in which the amide
backbones are associated by hydrogen bonded
anti-parallel
NC

loop
or
turn

N
H

O
R

R
O

N
R

N
O

N O C

N
R

CN

H
N

CN

parallel
NC

NC
O
H
N

H
O

H
N

NC

H
O

N
O

R
N

N
R

N
R

crossover

NC

H
R

N
R

R
H

N
R

O
R

27

-helix: 3.6 amino acids per coil, 5.4

3.6 AA
5.4

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

28

myoglobin
pdbcode:1WLA

Bacteriorhodopsin
pdb code: 1AP9

Parallel -sheets
carbonic anhydrase
pdb code: 1QRM

Anti-parallel
-sheets
of lectin
pdbcode:2LAL

29

27.20: Tertiary Structure of polypeptides and Proteins.

Fibrous. Polypeptides strands that bundle to form elongated
fibrous assemblies; insoluble;
Globular. Proteins that fold into a spherical conformation .
Hydrophobic effect. Proteins will fold so that hydrophobic amino
acids are on the inside (shielded from water) and hydrophilic
amino acids are on the outside (exposed to water).

ProIleLysTyrLeuGluPheIleSerAspAlaIleIleHisValHisSerLys

30

Enzymes: proteins that catalyze biochemical reactions.

by bringing the reactive atoms together in the optimal geometry
for the reaction.
lowering the activation energy (G) by stabilizing the
transition state and/or high energy intermediate.
many enzymes use the functional groups of the amino acid
sidechain to carry out the reactions
Proteases (peptidases): catalyzes the hydrolysis of peptide bonds
H3N

R
N
H

H
N
O

R
N
H

H
N
O

protease
N
H

CO2

H2O

H 3N

R
N
H

H
N
O

R
O

+ H N
3

H
N
O

N
H

CO2

Four classes of proteases:

Serine (trypsin): aspartate-histidine-serine
Aspartyl (HIV protease, renin): two aspartates
Cysteine (papain, caspase): histidine-cysteine
Metallo (Zn2+) (carboxypeptidase, ACE): glutamate
31

Mechanism of carboxylpeptidase, metalloprotease (p. 1151)

Mechanism of a serine protease (trypsin, chymotrypsin):
oxy-anion
hole
NH

Ser195

His57

Asp102 CO2-

R1
O
H

NH

HN
N
H

NH

R2

R1
Ser195

O
O

His57

N
N
H

Asp102 CO2NH
Ser192O

His57

Asp102 CO2-

H
N
N
H

O
O

HN

Ser192O

NHR2

H
N

H
- R2-NH2
His57

N
H

Asp102 CO2-

O
H

HN
R1
acyl-enzyme
intermediate

N
N
H

HN
R1

Ser195

H
His57

Asp102 CO2-

O
H

RCO2H

N
N
H

32

27.21: Coenzymes. Some reactions require additional organic

molecules or metal ions. These are referred to as cofactors or
coenzymes.
N
N

NH2

N
+

HO

O
OH
P
OH

HO
HO

OH

H2 N

Pyridoxal Phophates
(vitamin B6)

Thiamin Diphosphate
(vitamin B1)

NH2

O H

HN

N
Fe

HN

H
N

CO2-

O
O

Folic Acid
(vitamin B9)

OH
Heme

NH2
O

OH

O
HN

HO

NH
S
Biotin
(vitamin B7)

H
HO
O
-O P
O
O
H

CO2-

CO2H

OHO

H
O
H
OH

Vitamin B12
(cyanocobalamin)

P O P
O-

OH

N
NH

N
HO

NH2
O

O
NH2

N
N C N
Co
N
N

H2N
H2N

NH2

OH

NH

N
O

Flavin Adenine Diphosphate (FAD)

(Vitamin B2)

27.22: Protein Quaternary Structure. (please read)

33