3/3/2021

General facts:

• Proteins are the most abundant biological

macromolecules, occurring in all cells and in all
AMINO ACIDS, PEPTIDES & parts of cells

PROTEINS • Proteins also occur in great variety

• Proteins exhibit enormous diversity of biological

function
• The word protein is derived from the Greek
protos, meaning “first” or “foremost”
Nigani Willie, MSc (CWRU), BSc (Monash)
Discipline of Biochemistry and Molecular Biology
Division of Basic Medical Sciences
School Medicine and Health Sciences
University of Papua New Guinea
Taurama Campus
National Capital District, PNG

• Some examples of different type of proteins:

• All proteins, from all forms of life, are constructed
 Enzymes
from the same ubiquitous set of 20 amino acids
 Hormones
 Antibodies
• These amino acids are covalently linked in
 Transporters (transport proteins) characteristic linear sequences
 Muscle
 The lens protein of the eye • Cells can produce proteins with strikingly different
 Milk proteins properties and activities by joining the same 20
 Antibiotics amino acids in many different combinations and
 and many other substances having distinct sequences
biological activities

1
 3/3/2021

Amino Acid Structure and Composition

Amino Acids
• Proteins and peptides (polymers) are made up of • Amino acids have a
amino acids (monomers) carboxyl group and an
COO- amino group bonded to
the same carbon atom
• Proteins can be hydrolyzed (split) into their + (the -carbon)
constituent amino acids H3N C H

• They differ from each

• All amino acids have common names, in some cases R
other in their side chains
derived from the source from which they were first
(R groups)
isolated General Structure of Amino Acids

• Two conventions are used to identify carbons in
amino acids:
1) additional carbons in the R group are designated
, , , and  and so forth proceeding out from
the -carbon
2) the additional carbons in the R group are can
also be named using the numbering system i.e.
carboxylic carbon as C-1, α-carbon as C-2 and
followed by carbons in the R group

2
 3/3/2021

Standard Amino Acids • Two systems of abbreviations are used to shorten the
names of the 20 standard amino acids
• Cells use 20 standard (or common) amino Amino Acid 3 Letter 1 Letter Amino Acid 3 Letter 1 Letter
acids to synthesise polypeptides and proteins Threonine Thr T Alanine Ala A
Isoleucine Ile I Aspartate Asp D
Methionine Met M Asparagine Asn N
• The process of synthesizing peptides or
Histidine His H Cysteine Cys C
proteins is called Translation
Arginine Arg R Glutamate Glu E
Leucine Leu L Glutamine Gln Q
Lysine Lys K Glycine Gly G
Phenylalanine Phe F Proline Pro P
Tryptophan Trp W Serine Ser S
Valine Val V Tyrosine Tyr Y

ESSENTIAL AMINO ACIDS Memorizing the essential amino acids

• Essential amino acids are those that cannot be
synthesized by the body and thus must be obtained
from diet
8 of the 20 standard amino acids are essential for
adults
10 of the 20 standard amino acids are essential for
infants
• Using the first letter of standard amino acids (not to
be confused with the 1-letter abbreviation that is
used internationally)
• 8 essential amino acids for adults
–T V T I L L P M
• 10 essential amino acids for children
–P T V T I M H A L L

3
 3/3/2021

Amino Acids are Stereoisomers

• For all common amino acids except glycine, the -
carbon is bonded to four different groups and is
therefore a chiral center

• Tetrahedral arrangement of the bonding orbitals

occur around the -carbon atom

• All molecules with a chiral center are optically active

– they can rotate plane polarized light
• This results in the four different groups occupying
different spatial arrangements that are non-super-
imposable mirror images of each other
• All natural proteins consist of L-amino acids

Non-Protein Amino Acids Non-Protein Amino Acids

• Some non-protein amino acids are

• There are about some 300 additional amino acids intermediate metabolites in the biosynthesis
found in cells are not constituents of proteins
of standard amino acids
• These non-protein, (non-standard) amino acids have
e.g. homocysteine, an intermediate product
a variety of functions
in the biosynthesis of cysteine from
Examples: methionine
 Ornithine and citrulline
Both of which are key intermediates in the biosynthesis of the
amino acid arginine methionine homocysteine cysteine

4
 3/3/2021

Non-Standard Amino Acids R Group Classification of Amino Acids

• Non-standard amino acids are those that are
modified on a peptide or protein after the protein is • Nonpolar, Aliphatic R Groups
synthesized
• Aromatic R Groups
Amino acid Origin • Polar, Uncharged R Groups
5-hydroxylysine a derivative of lysine (found in collagen) • Positively Charged R Groups

6-N-Methyllysine found in myosin, contractile protein of muscle • Negatively Charged R Groups

found in blood clotting protein prothrombin and
-Carboxyglutamate
in other proteins that bind Ca2+
derived from four lysine residues, found in
Desmosine
fibrous protein elastin

5
 3/3/2021

• Cysteine can be readily oxidized to form a covalently

linked dimeric amino acid called Cystine

6
 3/3/2021

Amino Acids Can Act As Acids and Bases Amino Acids Can Act As Acids and Bases

COOH
• When an amino acid is • A zwitterion can gain a
+
dissolved in water, it exists H3N C H proton and have a positive
COO- in solution as a zwitterion, charge
a dipolar ion
+ R
H3N C H
• A zwitterion can act as COO-
R either acid (proton donor)
• A zwitterion can lose a
or base (proton acceptor)
H2N C H proton and have a
negative charge

Amino Acids Can Act As Acids and Bases

Amino Acids and Titration Curves
• Depending on the pH of the solution amino acids can exist in
solutions as either of the following forms: • Acid-base titration involves the gradual
addition or removal of protons
protonated zwitterion deprotonated
cationic form neutral anionic form
• Example: Glycine in acid pH is a fully
COOH COO- COO- protonated amino acid, diprotic acid
(i.e. one molecule of Glycine can donate two
+ +
H3 N C H H3 N C H H2 N C H

R R R
protons)

pH 1 pH 7 pH 13
Net charge: +1 Net charge: 0 Net charge: -1

7
 3/3/2021

An acid-base titration curve of Glycine

• Shows two stages and they correspond to the de- COOH COO- COO-
protonation of the two functional groups on Gly +
H3 N C H
+
H3 N C H H2 N C H

• Each stage of this acid-base titration curve R R R

resembles that of a monoprotic acid (such as

acetic acid)
• Remember that at the midpoint of any titration, a
point of inflection is reached where the pH is
equal to the pKa of the protonated group being
titrated

An acid-base titration curve of Glycine

• Remember that at the midpoint of any
= 9.60
titration, a point of inflection is reached where
the pH is equal to the pKa of the protonated
group being titrated

• pKa is a measure of the tendency of a group to

lose a proton.
= 2.34
• When the pKa increases by 1 unit, this
tendency decreases tenfold

8
 3/3/2021

1st stage
• At low pH (acidic conditions) in Isoelectric Point (pI)
the acid-base titration, the • As the titration
predominant ionic species of proceeds, another
Gly is the fully protonated important point is
form +H3N-CH2-COOH reached at pH 5.97
• the carboxyl group (-COOH) on • Here is another point
the Gly starts to lose its of inflection is reached
electrons as base (OH-) is at which the removal
added of the first proton (of
the carboxyl group is
complete
Midpoint of 1st stage
• When this reaches the midpoint there should be equal • At this point, the removal of a second proton, this time from
concentrations of proton-donor (+H3N-CH2-COOH) and proton the amino (+H3N-) group has just began
acceptor (+H3N-CH2-COO-) • This is also the pH at which the Gly is in zwitterion form (+H3N-
• For Gly, the pH at the midpoint is 2.34, thus the –COOH group of Gly CH2-COO-) i.e. the net electrical charge is zero
has a pKa (pK1) of 2.34 in the first stage of its acid-base titration curve

2nd stage Different Amino Acids have different

• Between the pI to the end
of the reaction Acid-Base Properties
• the removal of a second
proton from the amino • Amino acids that have a titration curve that
group resembles that of Glycine have
Midpoint of 2nd stage
• The pH at the midpoint at – a single -amino group
this stage is 9.60, which is
also the pKa (pK2) End of 2nd stage
• Completion of titration
– a single -carboxyl group
• At this point the zwitterion
• At about pH 12, all the protons
form (+H3N-CH2-COO-), which
is now the proton donor is
from the zwitterion form has been – an R group that does not dissociate
used up
equal in concentrations to
the proton acceptor (H2N- • The predominant form of Gly at
CH -COO ) - this pH is H2N- CH2-COO-
2

9
 3/3/2021

• More complex titration curves are seen in amino acids with:

– more than one amino group
– more than one carboxyl group
– an R group that dissociates
Titration of Glutamic acid
PEPTIDES AND PROTEINS
Titration of Lysine • Amino acids can be linked together by peptide
bonds to form peptides or proteins
• Two amino acids can be joined via a peptide
bond to form a dipeptide
• Three amino acids can be joined via two
peptide bonds to form a tripeptide

• All 20 standard amino acids found in proteins are -

amino acids

• In proteins and peptides, amino acids are joined to

each other by a covalent bond called the peptide
bond

• The peptide bond is formed from a dehydration

reaction between two amino acids • Peptide bond is formed by the removal of a hydroxide
from the carboxyl group of one amino acid and the
hydrogen from the amide group of another amino acid

10
 3/3/2021

• Four amino acids can be joined via three

peptide bonds to form a tetrapeptide peptide bond

• Five amino acids can be joined via four

peptide bonds to form a pentapeptide
peptide bond
• When few more amino acids are joined in this
fashion, the structure is called an oligopeptide
• When many amino acids are joined, the peptide bond
product is called a polypeptide

The Peptide Bond

• Covalent bonds have important constraints on the

conformation of a polypeptide

• A resonance or partial sharing of two pairs of

electrons occurs between the carbonyl oxygen and
the amide nitrogen

The terminal that ends with the amide group is called the amino-
terminal end (N-terminal) and the terminal that ends with the
carboxyl group is called the carboxyl-terminal end (C-terminal)

11
 3/3/2021

The Peptide Bond

• The oxygen has a partial negative charge and the
nitrogen has a partial positive charge setting up a
small electric dipole
• As a result the C-N peptide bonds are unable to
rotate freely
• rotation only happens for N-Cα and Cα –C

Acid-Base Behaviour of Proteins, Peptides Acid-Base Behaviour of Proteins, Peptides

• Peptides contain only one free -amino group and • Non-terminal amino acids do not contribute to acid-
one free -carboxyl group base behaviour of peptides

• These groups ionize as they do in free amino acids • However, the R groups of some non-terminal amino
but ionization constants are different acids in a peptide can ionize

• Non-terminal amino acids in a peptide chain are • Such R groups contribute to the acid-base behaviour
involved in peptide bonds and do not ionize of the peptide

12
 3/3/2021

• Acid-base properties of a pI values of some proteins

peptide can be predicted
from its: Protein pI
Pepsin ~1
1) free -amino group
Human serum albumin 5.9
2) free -carboxyl group 1-Lipoprotein 5.5
3) R group that can be Fibrinogen 5.8
ionized Haemoglobin A 7.1
Ribonuclease 7.8
• Therefore, just like free amino acids, peptides have: Cytochrome C 10.0
1. characteristic Titration Curves Thymohistone 10.6
2. characteristic Isoelectric Points (pI)

Electrophoresis equipment:

• This property (pI) of peptides/proteins is used

to separate different types of proteins in a
mixture
E.g. separating proteins in serum or plasma Gel

• The electrophoretic separation of plasma

proteins are commonly carried out for
disease diagnosis

13
 3/3/2021

Electrophoresis pattern for plasma proteins at pH 8.6

• Electrophoresis is usually done at a pH which
is higher than the pI of major proteins
• Order of migration is
(e.g. pH 8.6) + - along the horizontal
axis
• Proteins with highest
• At such a pH, the proteins are negatively
mobility are closest
charged and will move toward the anode to the anode
(positive electrode) in an electric field

• The rate at which the proteins migrate • Height of band

shows along vertical
(mobility) in an electrophoretic gel will axis reflects protein
depend on their mass (or charge) concentration
+ -

Length and Sizes of Peptides and Proteins

• The length of a peptide or protein alone
• Naturally occurring peptides range in length does not determine the function of the
from two to many thousands of residues peptide or protein

• The terms “protein” and “polypeptide” are

• Even the smallest peptides have biologically
sometimes used interchangeably
and physiological important effects
• In general the term protein is used for
molecules that contain over 50 amino acids • Many small peptides exert their effects are
and peptide is used for those that contain less very low concentrations
than 50 amino acids

14
 3/3/2021

Some examples of small but active peptides

No. of
Peptide Function
Residues
Stimulates uterine
Oxytocin 9
contractions
Inhibits inflammation of
Bradykinin 9
tissues
Thyrotrophic Stimulates release of
3
releasing factor thyrotrophic

Lehninger 2017

• Proteins can consist of a single polypeptide chain or

Amino acid sequence of bovine insulin may have two or more chains
• Complex proteins have two or more polypeptides
associated non-covalently. These are multi-subunit
proteins

Lehninger 2017

15
 3/3/2021

Human proinsulin Human insulin + C peptide

Slightly larger peptides: polypeptides

No. of No. of
Polypeptide Function
residues chains

glucose uptake
Insulin 30; 21 2
of cells
opposes action
Glucagon 29 1
of insulin
Stimulates
Corticotrophin 39 1
adrenal cortex

Modified Peptides and Proteins

• Example: Haemoglobin has four subunits:
• Simple proteins are those that contain only
2  chains amino acids and no other chemical groups
2  chains
• Examples:
 All four are held together by non-covalent oRibonuclease
interactions oChymotrypsinogen
oInsulin

16
 3/3/2021

Conjugated Proteins
• Some proteins contain permanently associated Class Prosthetic Groups Example
chemical components in addition to amino acids
Lipoproteins Lipids 1-Lipoprotein of blood
Glycoproteins Carbohydrates Immunoglobulin G
• These are called conjugated proteins Phosphoproteins Phosphate groups Casein of milk
Hemoproteins Heme (iron porphyrin) Haemoglobin
• The non-amino acid group that forms part of the Flavoproteins Flavin nucleotides Succinate dehydrogenase
conjugated protein is called the prosthetic group
Metalloproteins Iron Ferritin
Zinc Alcohol dehydrogenase
• Prosthetic groups are classified on the basis of the
Calcium Calmodulin
chemical nature of the prosthetic group
Molybdenum Dinitrogenase
Copper Plastocyanin
Lehninger 2017

An example of a hemoprotein: Myoglobin

3D structure model of Myoglobin

Lehninger 2017 Lehninger 2000

17
 3/3/2021

Structure of Proteins Levels of Protein Structure

• The covalent backbone of a typical protein contains
hundreds of individual bonds • Four levels of protein structure are commonly
• Each protein has a specific chemical or structural defined
function, strongly suggesting that each has a unique 1. Primary structure
three-dimensional structure
2. Secondary structure
• The spatial arrangement of atoms in a protein is
called its conformation 3. Tertiary structure
• Proteins in any of their functional, folded
conformations are called native proteins 4. Quaternary structure

Primary Structure
• The primary structure of a protein is the
amino acid sequence of the protein and all the
covalent bonds (peptide bonds and disulphide
bonds) linking the amino acid residues in that
protein or polypeptide chain

Lehninger 2017

18
 3/3/2021

Secondary Structure

• Stable arrangements of amino acid residues

Primary
structure of • Recurring structural patterns
(bovine) insulin

• the local conformation of some part of a

polypeptide

Secondary Structure Secondary Structure

• The most prominent secondary structures
that are stable and occur widely in proteins  helix
are:
– is a helical structure
1.  helix
– stabilised by internal hydrogen bonds
2.  conformations
– H bonds form between the hydrogen and the
nitrogen atoms and the carbon atoms

19
 3/3/2021

β conformations

– More extended conformation of

polypeptide chains

– Backbone of polypeptide chains is extended

into a zigzag rather than helical structure

β Conformations

– The zigzag polypeptide chain can be

arranged side by side to form a series of
pleats called a  sheet

– Hydrogen bonds are formed between

adjacent segments of polypeptide chain

20
 3/3/2021

β Conformations

– In proteins which have a compact folded

structure, nearly one-third of the amino
acid residues are in turns or loops where
the polypeptide reverses direction

– These turns link successive runs of α helix

and β conformation

β Conformations
Primary Structure
– The most
common are the • The primary structure of a peptide or protein is
β turns; the its amino acid sequence
structure is a 180
turn and involves
four amino acid
residues

– Gly and Pro often

occur in β turns
β turns

21
 3/3/2021

Tertiary Structure
Secondary Structure
• Repetitive structural patterns • Describes all aspects of the three-
• The most prominent secondary structures that are stable dimensional folding of a polypeptide
and occur widely in proteins are:
(1) -helix (2) -sheet

• Amino acids far apart in a polypeptide

sequence and those that are part of different
secondary structures may interact with each
other

Tertiary Structure Quaternary Structure

• Interacting segments
of polypeptide chains • When a protein has two or more polypeptide
are held in their subunits, their arrangement in space is
characteristic tertiary referred to as quaternary structure
positions by different
kinds of weak bonds,
such as disulphide • The interaction of these subunits in a
linkages, between quaternary structure is taken into
segments consideration

22
 3/3/2021

Quaternary Structure Fibrous Proteins

• Have polypeptide chains arranged in long
• In considering high levels of protein structure, strands or sheets
proteins are classified into two major groups
• Consist largely of a single type of secondary
1) Fibrous proteins structure

2) Globular proteins • Structures that provide support, shape and

external protection to vertebrates

• Examples: α-keratin, collagen, silk fibroin

23
 3/3/2021

Secondary Structures and Properties of Fibrous proteins

Structure of hair Cross section of hair
Structure Characteristics Examples of
occurrence
α helix, cross- Tough, insoluble α-keratin of hair,
linked by protective feathers and nails
disulfide bonds structures of
varying hardness
and flexibility
β conformation Soft, flexible Silk fibroin
filaments
Collagen triple High tensile Collagen
helix strength without tendons, bone
stretch matrix

Globular Proteins

Structure of • Have polypeptide chains folded into spherical

collagen fibrils or globular shape

• Often contains more than one type of

secondary structure

• Most enzymes and regulatory proteins are

globular proteins

• Examples: albumin, haemoglobin, amylase

24
 3/3/2021

More examples of Globular Proteins

Quaternary structure
of deoxyhaemoglobin,
a globular protein

(a) Ribbon diagram

(b) Space-filling model

Loss of Protein Structure results in Loss of Function • A denatured protein may regain its native structure thus
biological function if returned to conditions in which its
• A loss of three-dimensional structure sufficient to cause native conformation is stable
loss of function is called denaturation
• This process of a denatured protein returning to its
• Proteins don’t have to be completely unfolded to be native conformation is called renaturation
denatured
• Renaturation proves that tertiary structures are
• Proteins can be denatured by conditions such as heat, determined by amino acid sequences
extreme pH or certain miscible organic solvents such as
alcohol or acetone, by certain solutes such as urea or by
detergents

25
 3/3/2021

Study Questions 1
• What are amino acids?
• What is the difference between a peptide and protein?
• What are the common (or standard) Amino Acids?
• What do we mean by Essential and Non-essential amino
acids?
Denaturation Folding • What do the terms non-standard and non-protein amino
acids mean?
• How can an amino acid be amphoteric?
• Describe the classification of amino acids by R groups

Study Questions 2 REFERENCES

• Devlin, TM (editor). Textbook of Biochemistry with Clinical Correlations 6th edition, 2006, John
• Describe stereoisomerism in amino acids? Wiley & Sons Inc.

• What is a peptide bond? How is a peptide bond • Nelson DL and Cox MM. Lehninger Principles of Biochemistry 6th Edition, 2017, Worth
Publishers, New York.
formed?
• What are the different levels of organisation of • Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL and Darnell J.
Molecular Cell Biology 5th Edition, 2004, W.H. Freeman and Company, New York.
protein structure?
• Use myoglobin or haemoglobin as an example to
explain different levels of protein structure and their
relationship to protein function
• List the two major groups of proteins according to
three-dimensional structure and give at least two
examples of each

26