The Major

Biomolecules of
Mammalian Cells II

Amino Acids & Proteins

1
Part A: Amino acids (AA)

1. Definitions & nomenclature

2. Structure & common features

3. Classification

2
 AMINO ACIDS
Definition:
- organic acids containing an amine (–NH3) & a
carboxylic (-COOH) group

- the molecular units that make up proteins

- 20 naturally occurring AA are used in protein

synthesis

Nomenclature:
- three letter abbreviation (e.g. Ala, Arg, Asp)

- one-letter symbol (e.g., A, R, D)

3
 Abbreviations for amino acids

Amino acid Three-letter abbreviation One-letter symbol

Alanine Ala A
Arginine Arg R
Asparagine Asn* N
Aspartic acid Asp D
Glutamine Gln* Q
Cysteine Cys G
Isoleucine Ile* I
- - -
- - -
- - -
Tryptophan Trp* W
Tyrosine Tyr Y
4
Valine Val V
 Structure & Common Features

 AA as stereoisomers:

 a central -carbon

 4 different groups (- -amino acid

COOH, -NH3, -H & -R)

 with 4 groups on -C, AA are

chiral with L and D mirror
images
5
L-ala D-ala
 molecules with mirror images are also
referred to as stereoisomers

 most AA are -amino acids & the most

common -AA are the L--AA.

The L- and D- isomers of amino acids 6

 As protein constituents:

 only L--AA are constituents of proteins

 D-amino acids are never found in proteins, although

they exist in nature; exceptions are:
 bacterial membrane proteins, (D-Ala & D-Glu)
 modified AA - e.g. (hydroxy-Lys) & (hydroxyl-Pro) in
collagen
 occasional incorporation of the rare AA, seleno-Cys into
few proteins

7
 Several common L--AA found in cells, such as
ornithine and citrulline, are not used to make proteins

8
How did this particular set of AA become the
building blocks of proteins?

1. They are diverse; endowing proteins with the versatility

to assume many functional roles

2. Many of these amino acids were probably available

from prebiotic reactions

3. Excessive intrinsic reactivity may have eliminated other

possible amino acids:

9
 Acid-base properties:

 The -COOH & -NH2 groups in AA are capable of

ionizing due to a change in pH.
--COOH  --COO- + H+ (3.1)
--NH3+  --NH2 + H+ (8.0)

 At low pH (~ 1.0) both –COOH and –NH2 are

protonated & the molecule has net charge of +1

 At pH (~ 7.4) the –COOH is deprotonated & the –NH2

is protonated (neutral at this pH)  zwitterion; & the net
charge is 0 (isoelectric point). 10
 Zwitterions are substances containing equal numbers of
positive & negative charge (dipolar ions)

 At higher pH both groups becomes deprotonated with a

net charge of -1

11
pH 1 6-7 14
Charge +1 0* -1

cation zwitterion anion

(dipole)
pK1 pK2

12
Titration curve for Ala, showing isoelectric point (pI)
13
Also read “ Biochem Pract. Handbook” pg 27)
What is the major application of pI?
 Electrophoresis: a method of separation of proteins
and other charged molecules using an electric field
(isoelectric focusing)

 Principle: a molecule with a charge will move in an

electric field

14
 Classification of AA Found in Proteins

A: Based on the R-Groups:

 AA can be distinguished by R-groups which differ in:

- size, shape
- electric charge
- H-bonding capacity
- hydropathy
- chemical reactivity
15
1. With aliphatic side chains:

 Gly is the simplest, followed by Ala, Val, Leu, Ile

 The side chain of Ile becomes more extended & more

hydrophobic -  cluster together rather than contact
water

 Proline is a cyclic but it shares many properties with

the aliphatic group

 Proline influences protein architecture because its

ring structure makes it more conformationally
restricted than the other AA 16
1.

17
18
2. Hydroxyl-containing side chains:

 Two AA, Ser & Thr, contain aliphatic hydroxyl

groups

 The hydroxyl groups on Ser and Thr make them

much more hydrophilic (water loving) and reactive than
alanine & valine

19
2.

20 **
3. Sulphur-containing side chains:

 Cys & Met have weak polar side chains and are more
hydrophilic than their aliphatic analogs

 Met contains an aliphatic side chain with S-atom in a

thio-ether linkage (-S-CH3)

 Cyst contains a sulfhydryl group (-SH). The

sulfhydryl group is much more reactive

 Pairs of sulfhydryl groups may come together to form

disulfide bonds, which are particularly important in
stabilizing some proteins 21
3.

22
23
4. Acidic side chains:

 Asp & Glu acid - the only AA that carry negative

charges at pH 7 i.e., the -ve charge in the side chain is
retained under physiological conditions

 Therefore often referred to as aspartate and

glutamate (i.e., the conjugate bases rather than the
acids)

 Uncharged derivatives Asn & Gln with a terminal

–CONH2 (carboxamide) in place of a –COOH
(carboxylic acid); they are both hydrophilic

24
4.

25
5. Basic side chains:

 Lys, Arg & His – relatively long side chains which

carry positive charge under physiological conditions (c.f.
the acidic group)

 Lys is capped by a primary amino group whereas Arg

has a guanidinium group

 His is the least basic of the three and contains an

imidazole group, an aromatic ring that also can be
positively charged. Found in many enzymes as proton
donor

 They are all strongly polar (hydrophilic) 26

5.

NH2

27
6. Aromatic side chains:
 Phe, contains a phenyl ring attached in place of one of
the hydrogen atoms of Ala. It is hydrophobic

 Tyr contains a -OH group; it is the most reactive,

amino acid

 Trp has an indole ring joined to a methylene (-

CH2-) group

 The aromatic AA, like most compounds carrying

conjugated rings, contain delocalized  electrons that
strongly absorb light in the near-ultraviolet region of the
spectrum at 280 nm
28
6.

29
B: Based on nutritional requirements:

30
31
1. Essential amino acids:

Definition & Facts

 These are AA that MUST be provided in the diet to

meet an animal’s metabolic needs are called essential
amino acids

 Generally, EAA include those with complex

structures, including some aromatic rings and
hydrocarbon side chains
32
 They require a large number of steps for their
synthesis and some of the enzymes for these steps have
been lost in the course of evolution

 Mammals require about HALF of the AA in their

diet for growth & maintenance of normal N-balance

33
2. Non-Essential amino acids:

Definition & Facts

 AA that need not be provided because they cay be

biosynthesized in adequate amounts are called
nonessential amino acids

 These include those that are readily synthesized from

abundant metabolites, such as intermediates in glycolysis
or the citric acid cycle

34
 Many bacteria & most plants can synthesize all of
their N metabolites starting from a single N source
such as NH3 or nitrate.

35
 Amino acids classification

Light polarization Nutritional requirement

-L - EAA
-D - NEAA

Side chain/R-group
- aliphatic
- OH group
- acidic/basis
- S content
- hydropathy
- etc **
36
End of
Part A 37
Part B: Proteins
1. Definition
2. General properties
3. The peptide bond & its formation
4. Protein structure (10 – 40)
5. Strategy to sequence a protein
6. Examples of different structural proteins

38
Definition:
 proteins are biopolymers (polypeptides) of L--
AA

 functionally they are "workhorses" of the cell

because of their many functions*

 in keeping with their functions, proteins are

extremely complex molecules

39
General Properties:

1. Most abundant biomolecules; accounts for 50%

of dry weight of most cells

2. Built by assembling long chains of amino acids

(monomers), followed by intricate folding

3. Final shape of protein is very specific. Unless

correctly folded, is not functional

40
4. Several 1000’s different types of proteins are
found in any cell

5. They are most structurally complex

macromolecules known

6. Each type of protein has its own unique

structure and function

41
The peptide bond (PB):

 AA are linked together by peptide bonds

(amide bond) to form polypeptide chains or a
‘protein’ molecule

 Proteins may consist of more than one

polypeptide chain

42
 PB form in the process of translation (protein
synthesis) when the -NH3 of one AA residue forms
a covalent bond with the -COOH of another

---COOH + -NH3--

 H2O is eliminated; the -C=O & the -N-H bonds

are nearly parallel & that the C, O, N, and H atoms
are usually coplanar

43
Monomer 1 Monomer 2

44
45
Chain size 46
 little twisting possible around the
C-N bond because the PB has
double-bond character i.e.
(metastable)

 the peptide bond can be

considered a resonance hybrid of
the two bonds

47
Formation the polypeptide bond:

 In aqueous environment PB formation requires energy

(G ~ +10 kJ/mol, RT); therefore not favored (c.f.
hydrolysis)

 AA has to be activated and then attached to a transfer

RNA (tRNA)

 ATP is hydrolyzed to AMP & PPi and the bond is

formed between –COOH & 3'-OH of the adenosine of
tRNA molecule  aminoacyl-tRNA
48
 Activated AA

Activation of amino acid ** 49

 Protein Structure & Organization

Protein molecules have four levels of structural

organization:

- Primary (or amino acid sequence)

- Secondary (or local regular folding)

- Tertiary (or overall folding)

- Quaternary (multi-chain association)

50
 The primary structure (1)

• Refers to the unique & defined sequence of AA in a

protein molecule

• Depending on the characteristics of AA, the 1

determines the properties and shape of the protein 
function

51
• Proteins evolve over time thru changes in their AA
sequences:

• Determined by the nucleotide base sequence of DNA

(genetic code); not just a random sequence

52
• The primary structure of bovine insulin

• A- chain 21 AA
• B- chain 30 AA
• Total 51 AA

53
The primary structure of bovine insulin
54
 Determination of primary structure
(protein sequencing)
- determine the number of polypeptide or subunits

- cleave the disulphide bonds

- separate subunits and purify

- determine component AA using Edman reaction

& ion exchange chromatography

55
Edman degradation reaction

Definition:
• A reaction in which AA residues are removed from the
polypeptide chain one by one at a time, starting from N-
terminus (automated systems)

The reaction:
• Phenyl-iso-thio-cyanate (PITC) reacts with the AA
residue at the N-terminus under basic conditions to form a
PITC-protein derivative

56
• Trifluoroacetic acid (TFA) then cleaves off the first
AA as its anilino-thiolinone derivative (ATZ-amino
acid) and leaves the new N-terminus for the next
degradation cycle

• Using N-butyl chloride and with 25% TFA/water, the

ATZ-amino acid is then converted to a phenyl-thio-
hydantoin derivative (PTH-amino acid)

57
• The PTH-amino acid is transferred to a reverse-phase
HPLC column for detection at 270nm; a std mixture of
AA is used

• Std retention times of the AA compared with each

Edman degradation cycle chromatogram

• The HPLC chromatograms are analyzed using a

computer data analysis system

58
•The procedure is repeated again and again
 The Secondary Structure (2)
• The spatial arrangement of AA residues that
are near one another in the linear sequence i.e.
local regular folding

• In the myoglobin molecule

the chain appears to be
locally coiled into regions of
helical structures

• Pauling: showed most 2

structures are the -helix
and the -strands

59
Helix repeat

60
Helix pitch

P = nh

P = pitch (nm/turn)
h = rise (nm/res)
n = number of residue per turn (res/turn)
61
1.  helix: PP
chain twists into a tightly
packed rod; the H- bonds
(NH—COO-) are within a
single PP chain parallel to
the helix axis

2. Beta sheet: PP
chain is nearly fully
extended; the H- bonds
are between the adjacent
chains (NH—COO-) &
nearly perpendicular to
the chains

62
-helix -sheet
63
64
 Proteins with 2 Structure

Fibrous proteins are specific

examples of proteins with 2
structure and they usually play
structural roles in the cell

i. The keratins -
-keratins they are coiled -
helical and found in skin, hair
& nails

-keratins found mainly in

birds and reptiles; they are -
sheet
65
ii. Fibroin -

- a -sheet protein spun by silkworm and spiders; half of

its AA residues are Gly with a few Ala & Ser

- this sequence allows the sheets to fit and pack on top of

one another

- resulting in a fiber that is strong and relatively

inextensible

66
iii. Collagen –
- most abundant in vertebrates;
~1/3; it is for strength &
toughness (bone & tendons, skin)

- basic unit tropo-collagen

molecule, a triple helix of three
polypeptide chains (rich in Gly
and Pro, each ~ 1000 AA

- Pro in the collagen fiber are

mostly modified to hydroxy-
proline** to stabilize the
structure

67
68
69
Collagen & vitamin C

- the enzyme catalyzing hydroxylation

of Proline requires ascorbic acid

- severe vitamin C deficiency leads to

scurvy

- the condition recovers quickly after

administration of the vitamin

70
iv. Elastin –

- has a random coil structure with little of 2

- required for highly elastic tissues e.g. arterial blood

vessels, & ligaments

- the PP chain is rich in Gly, Ala & Val

- to prevent the fibers from indefinite extension the

polypeptide chain has a few Lys residues for cross-
linkage
71
 The Tertiary Structure (3)
• The 2 themselves are in turn folded into a
specific compact structure for the entire
polypeptide chain

• Tertiary structures are

found in globular proteins;
they are responsible for most
of the work in the cell –
transport, metabolic,
biosynthetic, etc
72
Examples of 3o proteins:
i. Myoglobin -
- has 70% -helix with a prosthetic group (heme)

ii. Bovine pancreatic trypsin inhibitor (BPTI) -

- smallest globular protein (58 AA).

- mostly -sheet structure connected by bends in the

chain

- sole function is to bind to and inhibit proteolytic

enzyme trypsin to prevent autocatalysis of pancreas

- three disulphide bonds are also found

73
iii. Larger globular proteins -

- these are composed of multiple domains i.e., compact

folded 3 structures

- domains are connected by specific polypeptide strands

that run thru the molecule

- all GP have defined inside and outside (hydrophobic &

hydrophilic residues respectively)

74
75
 The Quaternary Structure (4)

• The association of PP chains to form specific multi-subunit

structures; as most proteins in the cell exist

Levels of 4 organization

1. Homotypic -

– identical or nearly identical PP chains e.g. hemoglobin

(Hb), a tetramer of myoglobin-like chains

76
2. Heterotypic –

- interaction between subunits of very different

structures e.g. Bovine pancreatic trypsin inhibitor BPTI
interaction with trypsin:

- the two protein surfaces fit one another closely

to form a specific complex

- some are very complex e.g. Pyruvate

dehydrogenase with 72 subunits

77
78
79
 Complex Protein Structures
• Proteins covalently conjugated with other molecules;
this occurs as post-translational modification

Examples of complex proteins:

i. Glycoproteins – associated with CH2O; found on the
surface of RBC are extremely important as they
determine blood group specificities

ii. Lipoproteins - proteins associated with lipids via non-

covalent interactions (transport & storage of lipid and
cholesterol)
80
81
 82
***
 Forces Controlling Protein Structure
1. Hydrogen Bonding -

• Interaction between a covalently bonded H atom on a

donor group (e.g. -OH) & a pair of non-bonded electrons
on an acceptor group e.g. O / N

• In proteins, H-bonding, occurs not only within and

between peptide chains but also with the surrounding
aqueous medium

83
84
2. Hydrophathic Forces -

• H-bonding between hydrophilic R-groups and the

aqueous environment

• Repulsion from the aqueous environment by the

hydrophobic R-groups (& vice versa)

• All these forces stabilizes the protein molecule

85
3. Electrostatic Forces -

• charge-charge – between oppositely charged R-groups

• charge dipole - the interaction of ionized R-groups of AA

with the dipole of the H2O

• dipole-dipole - the slight dipole moment that exists in the

polar R-groups of AA also influences their interaction
with H2O
86
87
4. van der Waaals Forces -

Very weak attractive & repulsive forces:

• Attractive – among induced dipoles that arise from

fluctuations in the charge densities that occur between
adjacent uncharged non-bonded atoms

• Repulsive - occur when uncharged non-bonded atoms

come very close together but do not induce dipoles (i.e.,
electron repulsion)

88
89
90
 Denaturation & Renaturation of Proteins

Denaturation

• Disruption and possible destruction of both the secondary

and tertiary structures leading to unfolding and loss of
function ( coagulation & precipitation)

• The 1 (sequence of AA) remains the same after a

denaturation process

• When the 2 and 3 structures are lost the polypeptide

becomes a random coil 91
92
Renaturation
• = reversible denaturation process

• Sometimes an unfolded protein can be restored to correct

folding and regain biological activity

• Irreversible as when egg white protein or albumin is

denatured by boiling

• Renaturation was first demonstrated by Christian

Anfinsen  “Anfinsen experiment”

93
 B-mercaptol-ethanol
and 8M urea
Denatured
Ribonuclease Ribonuclease
(active) (inactive)

B-mercaptol-ethanol
and 8M urea

Ribonuclease
(active)

Anfinsen experiment 94
Denaturation agents

• Heat - heat increases the kinetic energy and causes the

molecules to vibrate so rapidly and violently that the
bonds are disrupted

• Alcohol – it disrupts H-bonding. E.g. a 70% alcohol

solution for disinfection

• Acid and bases – they disrupt salt bridges (ionic bonds)

which result from the neutralization of an acid and
amine on side chains
95
• Heavy metal salts – usually contain Hg+2, Pb+2, Ag+1 Tl+1,
Cd+2 etc with high atomic weights  disrupt salt bridges

• Reducing agents – they act to denature proteins by

disrupting disulfide bonds. Reducing agents add
hydrogen atoms to make the thiol group, -SH

96
97
 End of
Part B

End of topic 98