Biomolecules: Amino Acids, Peptides,

and Proteins
 Proteins – Amides from Amino Acids
❑ Amino acids contain a basic amino group and an acidic carboxyl group

❑ Joined as amides between the ⎯NH2 of one amino acid and the
⎯CO2H the next

❑ Chains with fewer than 50 units are called peptides

❑ Protein: large chains that have structural or catalytic functions in biology

 Structures of Amino Acids

Each amino acid has 4 different

groups, attached to α- carbon
(which is C-atom next to COOH
group).
These 4 groups are :
a) amino group (—NH2 ),
b) Carboxyl group ( -COOH),
c) Hydrogen atom
d) Side Chain (R)

3
 Structures of Amino Acids
❑ In neutral solution, the COOH is ionized and the NH2 is protonated

❑ The resulting structures have “+” and “-” charges (a dipolar ion, or
zwitterion)

❑ They are like ionic salts in solution

 The Common Amino Acids
❑ 20 amino acids form amides in proteins (standard amino acids)

❑ All are -amino acids - the amino and carboxyl are connected to the
same C

❑ They differ by the other substituent attached to the  carbon, called the
side chain, with H as the fourth substituent except for proline

❑ Proline, is a five-membered secondary amine, with N and the  C part

of a five-membered ring
 Numbering of Carbons in Amino Acids

The conventions for labeling the carbon atoms in amino

acids is illustrated using lysine in the figure. The  carbon is
always carbon-2 of the amino acid. The -carboxyl group is
always carbon-1

6
 New Amino Acids
• Selenocysteine and pyrrolysine—are found in
some organisms

• More than 700 non-protein amino acids are

also found in nature
 Abbreviations and Codes
• The names are not systematic so you learn them by using them (They
become your friends)

• One letter and three letter codes – learn both of them

Alanine A, Ala Leucine L, Leu

Arginine R, Arg Lysine K, Lys
Asparagine N, Asn Methionine M, Met
Aspartic acid D, Asp Phenylalanine F, Phe
Cysteine C, Cys Proline P, Pro
Glutamine Q, Gln Serine S, Ser
Glutamic Acid E, Glu Threonine T, Thr
Glycine G, Gly Tryptophan W, Trp
Histidine H, His Tyrosine Y, Tyr
Isoleucine I, Ile Valine V, Val
His (Imidazole ring)
Trp (Indole ring)
Arg (Guanidino group)
Cys (thiol group)
Cys, thr, Tyr (-OH group)

Cys, Met (S containing)

Lys (ε amino group)
Ser, Thr (-OH group containing)

Pro (2ndary amine)

(All other primary amine)
 Chirality of Amino Acids
❑ Glycine, 2-amino-acetic acid, is achiral

❑ In all the others, the  carbons of the amino acids are centers of
chirality

❑ The stereochemical reference for amino acids is the Fischer projection

of L-serine

❑ Proteins are derived exclusively from L-amino acids

 Types of side chains
❑ Neutral: Fifteen of the twenty have neutral side chains
(neutral aa maintain buffering capacity at physiological pH)

❑ Asp and Glu have a second -COOH and are acidic

❑ Lys, Arg, His have additional basic amino groups side chains
(the N in tryptophan is a very weak base, simply non-basic)

❑ Cys and Tyr (OH and SH) are weak acids

 Why is histidine a basic amino acid and C5:55M
tryptophan a non-basic?
❑ Contains an imidazole ring that is partially protonated in neutral
solution

❑ Only the pyridine-like, doubly bonded nitrogen in histidine is basic. The

pyrrole-like singly bonded nitrogen is nonbasic because its lone pair of
electrons is part of the 6  electron aromatic imidazole ring

❑ Only lone pair electron in Trp contribute to 6  electron aromatic ring

Tryptophan

Histidine
 Essential Amino Acids
❑All 20 of the amino acids
are necessary for protein
Essential Nonessential **
Isoleucine Alanine
synthesis Leucine Arginine*
Lysine Aspartate
❑Humans can synthesize Methionine Cysteine*
Phenylalanine Glutamate
only 11 of the 20 Threonine Glutamine*
Tryptophan Glycine*
Valine Proline*
❑The other 9 must be Histidine Serine*
Tyrosine*
obtained from food; these Asparagine*
Selenocysteine

are called essential aa

MTTVILPHLy
 C5:45

weakly acidic
amino acid

pH<Pka=proto
nated/
Conjugated
pH>Pka acid form/
proton charge positive
chere pH>Pka=Dep
rotonated/con
dibe jugated
base/charge
negative
 Isoelectric Points C5:40

❑ In acidic solution, the carboxylate and amine are in their conjugate

acid forms, an overall cation

❑ In basic solution, the groups are in their base forms, an overall

anion

❑ In neutral solution cation and anion forms are present

❑ This pH where the overall charge is 0 is the isoelectric point, pI

 pI Depends on Side Chain
• The 15 amino acids thiol, hydroxyl groups or pure hydrocarbon
side chains have pI = 5.0 to 6.5 (average of the pKa’s)

• D and E have acidic side chains and a lower pI

• H, R, K have basic side chains and higher pI

 pI Depends on Side Chain
• The pI of any amino acid is the average of the two acid-dissociation
constants that involve the neutral zwitterion.
• For the 13 amino acids with a neutral side chain, pI is the average of
pKal and pka2
• For the 4 amino acids with either a strongly or weakly acidic side
chain, pI is the average of the two lowest pKa values
• For the three amino acids with a basic side chain, pI is the average of
the two highest pKa values.
 Electrophoresis C5:29

• Proteins have an overall pI that depends on the net acidity/basicity of the side
chains

– The enzyme lysozyme, has a high isoelectric point (pI = 11.0)

– Pepsin, has a low isoelectric point (pI = 1.0)

• The differences in pI can be used for separating proteins on a solid phase

permeated with liquid

• Solubility is usually lowest at the isoelectric point, where the protein has no net
charge, and is higher both above and below the pi, where the protein is charged

• Different amino acids migrate at different rates, depending on their isoelectric points
and on the pH of the aqueous buffer
 Electrophoresis
❑ A mixture of proteins is placed near the center of a strip of paper or gel.
❑ The paper or gel is moistened with an aqueous buffer of a given pH, and
electrodes are connected to the ends of the strip
❑ Proteins with negative charges (those that are deprotonated because the pH
of the buffer is above their isoelectric point) migrate slowly toward the positive
electrode (anode)
❑ Amino acids with positive charges (those that are protonated because the pH
of the buffer is below their isoelectric point) migrate toward the negative
electrode (cathod)
❑ Different proteins migrate at different rates, depending on their isoelectric
points and on the pH of the aqueous buffer, thereby separating the mixture
into its pure components
 Electrophoresis

Anode + _
Cathode

Anode + _ Cathode
_ _ _ _ + + + +

20
 Titration Curves of Amino Acids
• If pKa values for an amino acid are known

• The fractions of each protonation state can be calculated (Henderson-

Hasselbach Equation) if pKa and pH are both known

• pH = pKa + log [A-]/[HA]

Titration Curves of Amino Acids
 c5:7

pI calculation:

Migration in electrophoresis:
• At pH 7, the amino acid will migrate towards anode (positive electrode)
• At pH 3.0, the amino acid will migrate towards cathode (negative electrode)
• At pH 5.65?
 Ion charge distribution:

Titration graph:
 C7:80

Structure:

pI calculation:

Migration in electrophoresis: At pH 8.0, pH 4.0 or pH 5.9

• At pH 8: the peptide will migrate towards anode (positive electrode)
• At pH 4.0: the peptide will migrate towards cathode (negative electrode)
• At pH 5.9?: No migration
Ion charge distribution:

Titration graph:
 C7:84

Structure:

pI calculation:
total basic AA+1= total charge in highly acidic condition

Migration in electrophoresis: At pH 6.0, 3.0 or 4.825?

At pH 6.0: the peptide will migrate towards anode (positive electrode)
At pH 3.0: the peptide acid will migrate towards cathode (negative electrode)
At pH 4.825: No movement
Ion charge distribution:

Titration graph:
 book:22.4 Synthesis of Amino Acids
C7:28:40
Hell-Volhard-Zelinskii reaction

alphabromo carboxylic acid

Carboxylic
acid
Prochiral Alpha bromination/
free radical reaction

R group theke carboxylic acid 2 carbon higher

Racemic mixture

• The bromination is a free radical reaction; involves formation of a free radical in

the intermediate step.
• Free radical is sp2 hybridized and planner where bromine can add from top and
bottom of the plane. Therefore, racemic mixture is formed.
 The Amidomalonate Synthesis C7:22
Carboxylic acid synthesis Sodium ethoxide(base)

Malonic acid

(Bi functional carboxylic acid)

Amino acid synthesis

Conjugated acid form

Racemic mixture
 The Amidomalonate Synthesis

Example:

side chain as it is no
need to add extra carbo
 Home Task

(a) (b)

(c) (d)
 Strecker’s Synthesis C7:12.30

Aldehyde α-amino nitrile Amino acid

Mechanism:

Note: The carbonyl carbon is converted to α-carbon of amino acid.

 cyanite

Example:

side chain as it is no
need to add extra carbon
 Why are Asn and Gln non-basics? C8:87

Asparagine (Asn)

Glutamine (Gln)

Peptide bond is also non-basic

 Reductive Amination of -Keto Acids
❑Reaction of an -keto acid with NH3 and a
reducing agent produces an -amino acid
 C8:80
Enantioselective Synthesis of Amino Acids
❑ The synthesis of an α-amino acid from an achiral precursor yields
a racemic mixture, with equal amounts of S and R enantiomers

❑ Amino acids (except glycine) are chiral and pure enantiomers are
required for any protein or peptide synthesis

❑ Resolution of racemic mixtures is inherently inefficient since at

least half the material is discarded

❑ An efficient alternative is enantioselective synthesis

❑ Enantioselective synthesis requires a chiral reaction catalyst that

will temporarily hold a substrate molecule in an unsymmetrical
environment
 Chemical Resolution of R,S Amino Acids
❑ Convert the amino group into an amide and react with a
chiral amine to form diastereomeric salts

❑ Salts are separated and converted back to the amino acid by

hydrolysis of the amide
How to separate racemic mixture react with optically active pure
enantiomer.
 Enzymatic Resolution
C8:62

❑ Enzymes selectively catalyze the hydrolysis of amides

formed from an L amino acid (S chirality center)
 Peptides and Proteins C8:56
• Proteins and peptides are amino acid polymers in which the individual
amino acid units, called residues, are linked together by amide bonds,
or peptide bonds
• An amino group from one residue forms an amide bond with the
carboxyl of a second residue
• Serylalanine??

Gly-Ala-Tyr = Glycylalanyltyrosine
 Peptide Linkages
• Two dipeptides can result from reaction between A and S, depending on
which COOH reacts with which NH2 we get AS or SA

• The long, repetitive sequence of ⎯NH⎯CH⎯CO⎯ atoms that make up

a continuous chain is called the protein’s backbone

• Peptides are always written with the N-terminal amino acid (the one with
the free ⎯NH2 group) on the left and the C-terminal amino acid (the one
with the free ⎯CO2H group) on the right

• Alanylserine is abbreviated Ala-Ser (or A-S), and serylalanine is

abbreviated Ser-Ala (or S-A)
 Covalent Bonding in Peptides C8:50

• The amide bond that links different amino

acids together in peptides is no different
from any other amide bond

• Amide nitrogens are nonbasic because their

unshared electron pair is delocalized by
interaction with the carbonyl group.

• This overlap of the nitrogen p orbital with

the p orbitals of the carbonyl group imparts
a certain amount of double-bond character
to the C–N bond and restricts rotation
around it. Thus, peptide bond is planner.

• The amide bond is planar, and the N–H is

oriented 180° to the C=O.
 Covalent Bonding in Peptides: Disulfides
C8:46
❑ Thiols in adjacent chains can form a disulfide RS–SR through
spontaneous oxidation
❑ A disulfide bond between cysteine residues in different
peptide chains links the otherwise separate chains together,
while a disulfide bond between cysteine residues in the same
chain forms a loop
 Total acid hydrolysis of peptide (TAH)
C8:43
6 M HCl, 12 hrs
Peptide or Protein Individual amino acids
Heat

❑ Here, H2O is used to break the peptide bonds (everything gets

protonated)
*How can we determine the composition of protein by using
❑ Non specifictotal acid hydrolysis of peptide

Disadvantages:
1. TAH destroy tryptophan and cysteine; they will not show up as product of
hydrolysis
2. Asn------→ Asp + NH3 (thus Asn will not show as the product of
hydrolysis)
3. Gln------→ Glu + NH3 (thus Gln will not show as the product of hydrolysis)
 Enzymatic hydrolysis C8:39

Specific enzyme
Large peptide or protein Small peptides
❑ Specific peptidase selectively hydrolyses peptide bonds
❑ Trypsin cleaves –COOH side of basic amino acids, Arg or Lys
❑ Chymotrypsin cleaves –COOH side of aromatic amino acids, Phe, Tyr
and Tryptophan

Trypsin Ala-Phe-Lys-Pro-Met-Tyr-Gly-Arg-Ser-Trp-Leu-His

Chymotrypsin Ala-Phe-Lys-Pro-Met-Tyr-Gly-Arg-Ser-Trp-Leu-His

Trypsin Fragments Chymotrypsin Fragments

Ala-Phe-Lys Ala-Phe
Pro-Met-Tyr-Gly-Arg Lys-Pro-Met-Tyr
Ser-Trp-Leu-His Gly-Arg-Ser-Trp
Leu-His
 Arrange the peptide sequence
Trypsin Fragments Chymotrypsin Fragments

Ala-Ser-Ala-Gly-Phe-Lys Ile-Trp
Pro-Cys Lys-Pro-Cys
Ile-Trp-Met-His-Phe-Met-Cys-Arg Met-His-Phe
Met-Cys-Arg-Ala-Ser-Ala-Gly-Phe

Pro-Cys
Lys-Pro-Cys
Ala-Ser-Ala-Gly-Phe-Lys
Met-Cys-Arg-Ala-Ser-Ala-Gly-Phe
Ile-Trp-Met-His-Phe-Met-Cys-Arg

The sequence of the peptide:

Ile-Trp-Met-His-Phe-Met-Cys-Arg-Ala-Ser-Ala-Gly-Phe-Lys-Pro-Cys
 Arrange the peptide sequence
Home task
Trypsin Fragments Chymotrypsin Fragments

Trp-His-Phe-Met-Cys-Arg Lys-Pro-Val-Ile-Leu-Arg-Trp
Met-Phe-Val-Ala-Tyr-Lys Val-Ala-Tyr
Pro-Val-Ile-Leu-Arg Met-Cys-Arg-Gly-Pro-Phe
Gly-Pro-Phe-Ala-Val His-Phe
Ala-Val
Met-Phe

Gly-Pro-Phe-Ala-Val
Met-Cys-Arg-Gly-Pro-Phe
Trp-His-Phe-Met-Cys-Arg
Lys-Pro-Val-Ile-Leu-Arg-Trp
Met-Phe-Val-Ala-Tyr-Lys

The sequence of the peptide:

Met-Phe-Val-Ala-Tyr-Lys-Pro-Val-Ile-Leu-Arg-Trp-His-Phe-Met-Cys-Arg-Gly-Pro-Phe-Ala-Val
c8:25
Ninhydrin test

Note:
• Proline gives yellow-orange color
• This is a test for amines, including
α-amino acids
Purple Color complex
Structure Determination of Peptides: Amino Acid Analysis
• Includes determination of amino acid composition and sequencing

The composition of amino acids can be obtained by the following steps:

1. Breaking disulfide bonds (if any) by reduction and capping with

iodoacetate

2. Acid Hydrolysis of peptides into individual aa

3. Separation of amino acids by chromatography and quantitative

measurement of eluted materials using a reaction with ninhydrin that
produces an intense purple color (Proline gives yellow-orange colour)
Amino Acid Analysis Chromatogram
❑For a given amino acid elution time is
constant in a Standard Column
❑Amount of each AA depends on the intensity
of the purple colour
 Peptide Sequencing: The Edman Degradation
❑ The Edman degradation cleaves amino acids one at a time from the
N-terminus and forms a detectable, separable derivative for each
amino acid
❑ Involves treatment of a peptide with phenyl isothio-cyanate (PITC),
C6H5—N=C=S, followed by treatment with trifluoroacetic acid
The Edman Degradation
 Peptide Sequencing: The Edman Degradation

❑ Complete sequencing of large proteins by Edman degradation is

impractical (because of unwanted by-products)

What would be the solution for large proteins?

❑ The solution could be partial hydrolysis of large proteins into smaller

peptide segments followed by Edman degradation in each of the
fragments

❑ Generate complete sequence from the overlapping sequences

Peptide Sequencing: C-Terminal Residue Determination

❑ Carboxypeptidase enzymes cleave the C-terminal amide

bond

❑ Analysis determines the appearance of the first free amino

acid, which must be at the carboxy terminus of the peptide
 Peptide Synthesis
• Peptide synthesis requires that different amide bonds must be formed in a
desired sequence

• The carboxyl and amino groups are protected depending on desired

peptide bond formation

• After peptide bond formation, protection is removed

 Carboxyl Protecting Groups
❑ Usually converted into methyl or benzyl esters

❑ Removed by mild hydrolysis with aqueous NaOH

❑ Benzyl esters are cleaved by catalytic hydrogenolysis of the weak

benzylic C–O bond

2
 Amino Group Protection
❑ An amide that is less stable than the protein amide is formed and then
removed

❑ The tert-butoxycarbonyl amide (BOC) protecting group is introduced

with di-tertbutyl dicarbonate

❑ Removed by brief treatment with trifluoroacetic acid

 Peptide Coupling
• Amides are formed by treating a mixture of an acid
and amine with dicyclohexylcarbodiimide (DCC)

Here, DCC makes the –OH group a better leaving group and facilitates peptide
bond formation
 Five steps to synthesize a dipeptide

These steps can be repeated to add one amino acid at a time to the growing chain
or to link two peptide chains together
Ala-Leu-Met-Phe
 Automated Peptide Synthesis
The Merrifield Solid-Phase Technique
• Use of the solid-phase method
• Peptide synthesis is carried out
with the growing amino acid
chain covalently bonded to
small beads of a polymer resin
• Polystyrene resin is used:
• 1 of every 100 or so benzene
rings contained a chloromethyl
group
• A BOC-protected C-terminal
amino acid is then bonded to
the resin through an ester bond
formed by SN2 reaction
 The Merrifield Solid-Phase Technique
• With the first amino acid bonded to the resin, a repeating series of four
steps is then carried out to build a peptide
The Merrifield: Leu-Val-Phe-Ala
 Automated Synthesis
• The solid-phase technique has been automated, and computer-controlled
peptide synthesizers are available for automatically repeating the coupling
and deprotection steps with different amino acids

Applied Biosystems® Synthesizer

 Protein Classification
❑ Simple proteins yield only amino acids on hydrolysis

❑ Conjugated proteins, which are much more common than

simple proteins, yield other compounds such as carbohydrates,
fats, or nucleic acids in addition to amino acids on hydrolysis.

❑ Fibrous proteins consist of polypeptide chains arranged side

by side in long filaments

❑ Globular proteins are coiled into compact, roughly spherical

shapes
❑ Most enzymes are globular proteins
Some Common Fibrous and Globular
Proteins
 Protein Structure
• The primary structure of a protein is simply the amino acid
sequence.

• The secondary structure of a protein describes how segments

of the peptide backbone orient into a regular pattern.

• The tertiary structure describes how the entire protein

molecule coils into an overall three-dimensional shape.

• The quaternary structure describes how different protein

molecules come together to yield large aggregate structures
 Secondary Structure
The most common secondary structures are the
1. α helix
2. the β -pleated sheet.
-helix
• A protein coiled into a right-handed helical secondary structure

• Each turn of the helix contains 3.6 amino acid residues, with a distance
between coils of 540 pm, or 5.4 Å.

• The structure is stabilized by hydrogen bonds between amide N–H groups

and C=O groups four residues away, with an N–H····O distance of 2.8 Å.

• The helix is an extremely common secondary structure, and almost all

globular proteins contain many helical segments.

• Myoglobin, a small globular protein containing 153 amino acid residues in

a single chain (mainly  helix )
 β-pleated sheet
▪ A β-pleated sheet differs from an α helix in that
▪ the peptide chain is fully extended rather than coiled and
forms sheet-like structure
▪ The alternate R groups are arranged above and down the
plane or sheet.
▪ the hydrogen bonds occur between residues in adjacent
chains
• The neighboring chains can run either in the same
direction (parallel) or in opposite directions (antiparallel)
– The antiparallel arrangement is more common and
energetically somewhat more favorable
 The β-pleated sheet secondary structure
of proteins is stabilized by hydrogen
bonds between parallel or antiparallel
chains.
The structure of concanavalin A, a protein
with extensive regions of antiparallel
sheets, shown as ribbons.

Concanavalin A is a lectin (carbohydrate-binding protein)

 Internal and External Forces in tertiary
structure
❑ Acidic or basic amino acids with charged side chains congregate
on the exterior of the protein where they can be solvated by water

❑ Amino acids with neutral, nonpolar side chains congregate on the

hydrocarbon-like interior of a protein molecule

❑ Also important for stabilizing a protein's tertiary structure are

❑ The formation of disulfide bridges between cysteine residues,

❑ The formation of hydrogen bonds between nearby amino acid

residues, and

❑ The development of ionic attractions, called salt bridges,

between positively and negatively charged sites on various
amino acid side chains within the protein