Amino Acids

&
Proteins
1

 Proteins are linear copolymers built from monomeric

units called amino acids.
acids
Twenty amino acids are commonly found in proteins.
These amino acids contain a variety of different
functional groups:

Alcohols
(R-OH)
(R
OH)
Phenols
(Ph-OH)
Carboxylic acids
(R-COOH)
Thiols
(R-SH)
Amines
(R-NH2)
and others

 Protein function depends on both

amino acid content, and
amino
i acid
id sequence.

Protein fold into diverse shapes such as

spherical
elipsoidal
long strands, etc.

All information for 3-D structure is contained

in the linear sequence of amino acids.
3

 To understand protein function

function, we must first
understand the nature of amino acids.
Amino
A i acids
id are essentially
ti ll -amino
i acids:
id
alpha carbon (IUPAC #2 position)

H2N C COOH
|
R
When R is not H, the alpha carbon
is asymetric, giving rise to isomers.
4

Only L
L-amino
amino acids are constituents of proteins.
proteins
L and D isomeric nomenclature is similar to the R and
S utilized in modern organic chemistry.
5

 Carboxylic acids are traditional Bronsted-Lowry

acids,, donatingg a pproton in aqueous
q
solution.
The pKa for carboxylic acids is normally around
2 to 5. That is, the pH at which these acids are
50% ionized:
R-COOH

pH= [less than 2]

R-COO- + H+
[above 5]
6

 Amino groups function as bases, accepting a

proton.
proton
The pKa for amino groups is usually around 9
10.
10 Again
Again, at the pKa these groups are 50%
ionized:

pH=

R-NH3+

[below 8]

R-NH2 + H+
[above 9]
7

 Even though both acids and amines are present in the same
molecule, they mostly behave as though they were separate
entities:

 Summary:
At low pH, proton concentration [H+]is high.
Therefore, both amines and carboxylic acids
are protonated. (-NH3+ & -COOH)
At high pH, proton concentration is low.
Therefore, both amines and carboxylic acids
are deprotonated. (-NH2 & -COO-)
At neutral pH, amines are protonated(-NH3+) and
carboxylates are deprotonated(-COO-)

10

 Zwitter
Zwitter Ions:
Ions bearing two charges were named zwitter
ions by German scientists; the name still
applies today, especially for amino acids at
neutral pH:

+H

3N CH2 COO
11

Acid-Base Properties of Amino Acids

Draw the following chemical structures for glycine:
(Non-existent form:)

H2N CH2 - COOH

pH=1:
pH=7:
ppH=12:
12

Acid-Base Properties of Amino Acids

Draw the following chemical structures for glycine:
(Non-existent form:)

pH=1:

H2N CH2 - COOH

+H

3N

CH2 - COOH

pH=7:
ppH=12:
Biochemistry 3070 Amino Acids &
Proteins

13

Acid-Base Properties of Amino Acids

Draw the following chemical structures for glycine:
(Non-existent form:)

H2N CH2 - COOH

pH=1:

+H

pH=7:

+H

3N

CH2 - COOH

CH

COO
3
2

ppH=12:
Biochemistry 3070 Amino Acids &
Proteins

14

Acid-Base Properties
p
off Amino Acids
Draw the following chemical structures for glycine:
(Non-existent form:)

H2N CH2 - COOH

pH=1:

+H

pH=7:

+H

pH=12:
pH
12:

3N

CH2 - COOH

CH

COO
3
2

H2N CH2 COO15

Low pH
High
g pH
p

Neutral pH

16

Amino acids: (Aliphatic)

17

 Amino acid Proline

(The only secondary (2) amino acid or (imino acid.)

18

 Amino acids (Aromatic)

19

 Amino acids (Alcohols)

20

 Amino acids (Sulfur)

( f )

21

 Amino acids (Acids and related amides)

22

 Amino acids (Basic)

23

 Histidine (Acid/Base Activity)

24

25

26

Essential Amino Acids:

Isoleucine
Leucine
Lysine
Methionine
Phenylalanine a
Threonine
Tryptophan a
Valine
Arginine b
Histidine b
a

Aromatic

Probably
y essential
27

Biochemical Importance of
A i Acids
Amino
A id
Amino
Acids

Systematic
name

Importance

Glycine

Aminoetha
Aminoethanoic acid

Helps
p trigger
gg the release of oxygen
yg to
the energy requiring cell-making
process
Important
p
in the manufacture of
hormones for strong immune system

Alanine

-amino
propanoic
i
acid

Important AA as it is an energy source

for the liver,, muscles,, and CNS
Strengthens the immune system by
producing antibodies
Helps
Helps in the metabolism of sugars and
organic acids

Valine

-amino 3methylbutanoic
acid

Essential AA
Promotes
P
t mental
t l vigor,
i
muscle coordination and
calm emotions

Leucine

-amino
4-methyl
pentanoic acid

Essential AA
Provides necessary
substances for energy
production
Stimulants to the upper
brain and helps to be more
alert

Isoleucine -amino 3Essential AA

methylpentanoic Same functions as leucine
acid

Phenylalanine

-amino 3phenylpropanoic
acid

Essential AA
Used by the brain to produce
norepinephrine,
i
hi
Reduces hunger pains
Functions as antidepressant
Helps
H l iimprove memory

Tyrosine

-amino 3-(4hydroxyphenyl)pro
panoic acid

Transmits nerve impulses to the brain;

Tryptophan

-amino 3-indole
propanoic acid

p overcome depression;
p
; improves
p
helps
memory; increases mental alertness;
promotes the healthy functioning of the
endocrine glands
Essential AA
A natural relaxant, helps alleviate insomia by
inducing normal sleep
Reduces
R d
anxiety
i t and
dd
depression
i
Helps in the treatment of migraines and
headaches
Helps
p stabilize the immune system
y
Helps reduce risk of artery and heart spasms
Works with lysine in reducing cholesterol levels

Methionine

-amino 4methyl thiol

butanoic acid

Essential AA
Principal supplier of sulfur which
prevents disorder of the hair, skin
and nails
Helps lower cholesterol levels by
increasing the livers production of
lecithin
A natural chelating agent for heavy
metals
Regulates the formation of
ammonia and creates ammonia
ammonia-free
free
urine which reduces bladder
irritation
Influences hair follicles and
promotes hair growth

Cysteine

2-amino 3mercaptopropanoi
c acid

Functions as an antioxidant and is a

powerful aid to the body in protecting
g
radiation and p
pollution
against
Helps slow down the aging process,
deactivate free radicals, neutralizes toxins
Aids in protein synthesis and promotes
cellular repair
Necessary for skin formation, in the
recovery from burns and surgical operations

Serine

2-amino 3 hydroxy
propanoic acid

A storage source of glucose by the liver

and muscles
Helps strengthen immune system by
providing antibodies
Synthesizes fatty acid sheath around
nerve fibers

2 amino 3 h
2-amino
hydroxy
dro
Essential
Essential AA
Th
Threonine
i
butanoic acid

Important constituent of collagen,

Assists metabolism and assimilation
Helps
H l th
the digestive
di
ti and
d iintestinal
t ti l ttracts
t
functions normally
Helps prevents fat build-up in the liver

Histidine

-amino 3 (1Himidazol-4-yl)
propanoic
i acid
id

Essential AA
Found abundantly in hemoglobin
Used in the treatment of rheumatoid
arthritis, allergic diseases, ulcers, anemia

Lysine

2,6 diamino
hexanoic acid

Essential AA
Insures adequate absorption of calcium
Helps
p form collagen
g ((which makes up
p
bone and cartilages)
Aids in the production of antibodies,
hormones and enzymes

Arginine

-amino 5guanidino
pentanoic acid

Helps improve immune responses to

bacteria, viruses and tumor cells
Promotes
Promotes wound healing and
regeneration of the liver
Causes the release of growth hormones
p
muscle g
growth and
Crucial for optimal
tissue repair

Aspartic Acid -amino

butanedioic acid

Most easily used as energy source

Aids in the expulsion of toxic ammonia
from the body
bod
Located most closely to the TCA cycle,
the site of energy production
Found in increased levels in people with
epilepsy and in decreased amounts in
some cases of depression

Glutamic
acid

amino
-amino
Considered
Considered to be natures
nature s brain
brain food
food by
pentanedioic acid improving mental capacities
Helps speed the healing of ulcers; gives
a lift from fatigue
Helps control alcoholism, schizophrenia
and the craving of sugar, Parkinsons
disease, mental retardation, and muscular
dystrophy

Asparagine

-amino 3
carbamoyl
propanoic acid

Found in the surfaces of proteins where

they can interact with water molecules

Glutamine

-amino 4
carbamoyl
butanoic acid

Found in the surfaces of proteins where

they can interact with water molecules
The polar amide groups can also form
hydrogen bonds with atoms in the side
chains of other polar amino acids

Proline

Pyrrolidine-2carboxylic acid

Non-essential AA
Important for the proper functioning of
joints and tendons
Helps maintain and strengthen heart
muscles
Helps
p repair
p p
processes after cell injury
j y or
for any type of wound healing

Hydroxy
l i
lysine

Hydroxy
hexanoic acid

It is a hydroxy derivative of lysine.

It is most widely known as a component
of collagen

Biologically Important Proteins

Protein

No. of
AA

Function

Insulin

51

Cytochrome C

104

Enzyme for sugar

metabolism
Enzyme for cell respiration

Growth hormone

191

Used as anti-aging treatment

Hemoglobin

574

Oxygen transport in blood

Hexokinase
Gamma globulin

730
1320

Myosin

6100

Enzyme for glycolysis

Part of immune system in
blood
Muscle action

Blood Proteins
Albumins
Immunoglobulins

Create osmotic pressure and

transport other molecules
Participate in immune system

Fibrinogens

Blood coagulation

Alpha-1-Antitrypsin Neutralize trypsin that has

leaked from the digestive
g
system
g
yp
proteins Regulation
g
of g
gene
Regulatory
expression

 Amino acids are p

polymerized
y
via amide or
peptide bonds:

38

 Copolymer
p y
of amino acids:
a polypeptide

Definition:
Amino acid polymers of 50 amino acids are called
polypeptides, peptides, oligopeptides,
etc.

39

40

 An example of a dipeptide
dipeptide is the sweetener Aspartame.
Aspartame
Other names include:

NutraSweet
Equal
Tri-Sweet
Sanecta

IUPAC Name:
N-L- Aspartyl-L-phenylalanine 1-methyl ester
Abbreviated Structure:

Asp Phe - OCH3

41

 Cross links between peptide chains:

Di
Disulfide
lfid linkages
li k
between
b
individual
i di id l cysteines
t i
are called cystines:

42

 Insulin is the smallest protein, with 51 amino

acids in two chains linked by cystine
(disulfide) cross links:

43

 Peptide bonds have partial double bond

character
h
due
d to resonance that
h limits
li i rotation
i
about this bond:

44

45

Levels of Protein Structure

Primary (1) Protein Structure
linear sequence of amino acids.

Secondary
y (2)
( ) Protein Structure
localized regional structures

Teritary (3)
(3 ) Protein Structure
overal shape of proteins

Quaternary
Q t
(4) Protein
P t i Structure
St t
interactions between proteins
46

47

Proteins 3d conformation
Proteins-3d

Protein Structure
Sequence
determines
structure, structure
determines function
Most proteins can
fold byy itself very
y
quickly
Folded structure:
lowest energy state

49

Protein Structure:
Twisting about various bonds in the
polypeptide backbone gives proteins a variety
of shapes.
Bond angles give rise to secondary structures.
Then, localized secondary structures help drive
the peptide folding that gives rise to tertiary
structure.
50

PRIMARY STRUCTURE
The Amino acid sequence. The numbers of amino
acids
id vary (e.g.
(
insulin
i li 51,
51 lysozyme
l
129,
129
haemoglobin 574, gamma globulin 1250)
The pprimaryy structure determines the foldingg of the
polypeptide to give a functional protein
Polar amino acids (acidic, basic and neutral) are
hydrophilic and tend to be placed on the outside of
the protein.
Non-polar (hydrophobic) amino acids tend to be
placed
l d on the
h inside
i id off the
h protein
i

A Ramachandran plot (also known as a Ramachandran

diagram or a [,]
[ ] plot),
plot) originally developed in 1963 by G.
G N.
N
Ramachandran, C. Ramakrishnan, and V. Sasisekharan,is a way
to visualize backbone dihedral angles against of amino acid
residues in protein structure

Infinite variety
The number of p
possible sequences
q
is infinite
An average protein has 300 amino acids,
At each position there could be one of 20
different amino acids
= 10390 possible combinations
Most are useless
Natural selection picks out the best

SECONDARY STRUCTURE

The folding of the N-C-C

backbone of the
polypeptide chain using
weak hydrogen bonds

Science Student

Secondaryy Structure in Proteins:

Pauling and Corey proposed two secondary
structures in proteins many years before they
were actually proven:
alpha
l h

helix
h li

b t
beta

- sheet
h t

Both of these secondary protein structures are

stabilized by hydrogen bonding between the
carbonyl oxygen atoms and the nitrogen atoms
of amino acids in the protein chain.
chain
55

 This produces the alpha helix and beta pleating

The length of the helix or pleat is determined by certain amino
acids that will not participate in these structures
(e g proline)
(e.g.

 The alpha () helix:

57

 beta sheet (antiparallel):

58

 beta sheet (artistic representations):

59

TERTIARY STRUCTURE
The folding of the polypeptide into domains
whose chemical properties are determined by
tthee amino
a
o acids
ac ds in the
t e chain
c a

MIL1 protein
p

Anne-Marie Ternes

TERTIARY STRUCTURE
This folding is sometimes held together by strong
covalent bonds
(e.g. cysteine-cysteine disulphide bridge)
Bendingg of the chain takes place at certain amino
acids
(e.g. proline)
Hydrophobic amino acids tend to arrange
themselves inside the molecule
Hydrophilic amino acids arrange themselves on the
outside
t id

 Tertiary (3) Structure of Protein

Water-soluble
Water
soluble proteins fold into compact structures with nonpolar cores.

62

Tertiary (3) Structure the Protein Myoglobin

Water soluble proteins fold into compact structures with non-polar
Water-soluble
non polar cores
cores.

63

64

 In the case of myoglobin and many other proteins, the

majority of hydrophobic amino acids (yellow
yellow) are
found inside in structure:

65

 The Cro protein of Lambda bacteriophage is a

dimer of identical subunits:

66

 Hemoglobin is a protein tetramer, containing

two identical pairs of subunits:

67

 The coat of rhinovirus contains 60 copies of

each of four subunits (240 total)!

68

QUATERNARY STRUCTURE
Q
Some proteins are made
of several polypeptide
subunits
(e.g. haemoglobin has
four)

Max Planck Institute for Molecular Genetics

QUATERNARY STRUCTURE
These subunits fit together to form the
functional protein
p
Therefore, the sequence of the amino acids in
the primary structure will influence the
protein's structure at two, three or more levels

 In 1961 Christian Anfinsen published a classic

landmark work that clearly showed tertiary
structure was determined by primary structure!
His experiment was a classic example of welldesigned
g
experiments
p
that did not require
q
expensive equipment or years of work.
It deserves our attention.

71

 Anfinson chose the enzyme, ribonuclease, for

his experiments.
experiments This enzyme hydrolyzes
RNA and is composed of a single polypeptide
chain with 124 amino acids.
acids
Four disulfide (cystine) linkages are observed
in the active enzyme that stabilize the 33-D
D (3)
(3 )
shape of the enzyme.
The enzyme functions only when its 33
structure is properly aligned.

72

 Amino acid sequence of ribonuclease:

73

Anfinson used two chemicals to disrupt

p the
enzymes 3 structure [DENATURATION ]
1. urea - disrupts hydrogen bonds
2. -mercaptoethanol reduces disulfide bonds

74

Anfinsons
f
Experiment:
p

75

He also used dialysis to separate these chemicals from the

enzyme in different orders.
orders

Biochemistry 3070 Amino Acids &

Proeins

76

 By adding either one of these two chemicals to

the surrounding medium, it is not removed
during dialysis
dialysis.
In essence, Anfinson could remove either the
urea or the -mercaptoethanol
mercaptoethanol in any order he
chose.
The
Th order
d made
d a big
bi difference
diff
in
i the
h
enzymes ability to recover from the treatment!
Biochemistry 3070 Amino Acids &
Proteins

77

Separation of Amino Acids

and Proteins
1.
2.
3.
4.

5.

Chromatography the method of separating amino acids

on the basis of differences in absorption,
absorption ionic charges,
charges size
and solubility of molecules
Electrophoresis effects separation in an electric field on
the basis of differences in charges carried by amino acids
and proteins under specific condition
Ultracentrifugation effects separation on the basis of
molecular weight
g when large
g ggravitational forces are applied
pp
in the ultracentrifuge.
Precipitation Methods salts as sodium sulfate, ammonium
sulfate,, cadmium nitrate,, silver nitrate and mercuric chloride
at specific conc. precipitate some proteins while others
remain in solution
y is for the removal of small, crystalloidal
y
molecules
Dialysis
from protein solution.

H pure should
How
h ld my protein
i be?
b ?
Application
Therapeutic use,
use in vivo
studies
Biochemical assays, X-ray
crystallography
N-terminal sequencing,
antigen for antibody
production,, NMR
p

Required Purity
Extremely high > 99%

High 95-99%

Moderately high < 95%

Separation of proteins based on physical and

chemical properties
Solubility
Bindingg interactions
Surface-exposed hydrophobic residues
Charged surface residues
Isoelectric Point
Size and shape

Basic scheme of protein purification

Fractional precipitation of proteins

Precipitate
contaminants

Add Precipitant,
Centrifugation
g

Precipitate
protein of
interest

Discard pellet

Add Precipitant,
Centrifugation, Discard
supernatant,
t t Resuspend
R
d
protein

Discard
supernatant,
Resuspend
protein

Chromatography

Precipitation of proteins
by salting
salting out
out
The ability of a salt to precipitate proteins is
described byy the Hofmeister series:
_

Anions: SCN < ClO4 <

_
NO3

< Br < Cl < acetate < SO4 < PO4

Cations: Na+ < K+ < NH4+

Precipitation
p
of p
proteins

Chromatography
Much of modern biochemistry depends on the use of
column chromatographic methods to separate
molecules.
Chromatographic methods involve passing a solution
(the mobile phase) through a medium (the immobile
phase) that shows selective solute components
components.
The important methods of chromatography are:
1. Ion-Exchange Chromatography
2. Antibody Affinity Chromatography
3. Gel Filtration Chromatography
4 HPLC (High Performance Liq
4.
Liquid
id Chromatography)
Chromatograph )

Liquid chromatography
Protein solution applied
pp
to a column
Column = solid porous matrix
(stationary phase) + liquid (mobile
phase)
h )
Proteins separated based on differing
interactions with stationary and mobile
phases
Mobile pphase conditions can be
adjusted to increase or decrease
affinity of protein for stationary phase
(gradient)

Types of liquid chromatography

Adsorption Chromatography
Proteins bind to stationary phase
Proteins eluted by altering mobile phase
Includes: affinity, hydrophobic interaction, ion exchange, and
chromatofocusing

S l ti Phase
Solution
Ph
Chromatography
Ch
t
h
Proteins do not bind to stationary phase
Progress of proteins through column impeded by matrix of stationary
phase
Includes: size exclusion chromatography (aka gel filtration)

HPLC

Gel Filtration Chromatography

g p y