Proteins are the major

components of living organisms

and perform a wide range of
essential functions in cells.

Proteins regulate metabolic

activity, catalyze biochemical
reactions and maintain structural
integrity of cells and organisms.
 Classification of Proteins
According to biological function:
Type: Example:
DNA-polymerase
1.Enzymes- catalyze biological reactions Dehydrogenases
Ribonuclease
2.Hormones Insulin
3.Transport protein Hemoglobin
4.Movement proteins Actin and Myosin in muscles
Immunoglobulins
5.Immune Protection proteins
(antibodies)
Hormone receptors
6.Receptors
rhodopsin
7.Signalling proteins - Transcription & Translation
regulatory function within cells Factors
Collagen
8.Structural proteins
Keratin
Egg ovalbumin
9.Storage proteins
Proteins are the most abundant and diverse molecules
found in living cells.
How does one group of molecules perform such a diferent set of
functions? The answer is found in the wide variety of possible
structures for proteins.

Ribonuclease
Collagen Hemoglobin
 An incredible variety of

proteins can be formed using

20 different monomers called

amino acids.
The a-amino acids in peptides and proteins (excluding proline) consist of
a carboxylic (-COOH) and an amino (-NH2) functional group
attached to the same carbon atom.
This carbon is the alpha-carbon.
R-groups (radicals), that distinguish one amino acid from another,
also are attached to the alpha-carbon.
All peptides and polypeptides are polymers of
alpha-amino acids.

Each of these amino acid has a different

chemical structure and different properties.

Each protein has a unique amino acid sequence

that is genetically determined by the order of
nucleotide bases in the DNA, the genetic code.

Since each protein has different numbers and

kinds of the twenty available amino acids, each
protein has a unique chemical composition and
structure.
 Amino acids are
joined together in
proteins by peptide
bonds.
 Formation of peptide bond:
A peptide bond forms between the carboxyl group of one amino
acid and the amino group of the adjacent amino acid.
Properties of peptide bond:
• The peptide bond is a rigid covalent bond and has partial double-
bond character.
• The peptide bond is planar and unrotatable.
• The peptide bond is generally in the trans conformation.
• Each peptide bond is able to form maximum 2 hydrogen bonds with
other polar atoms.
Peptide bond formation is reversible.

A peptide bond can be broken by hydrolysis (the

adding of water).

Enzymes such as pepsin and trypsin easily hydrolyse

proteins to free amino acids during digestion. However, to
break peptide bonds non-enzymically requires harsh
conditions (e.g. boiling in 6M HCl for 24 hours).
 The products of polycondensation of
a different number of α-aminoacids, bound
by peptide bond are named peptides:
• a peptide containing two amino acid units
is called a dipeptide;
• one containing three amino acids, a
tripeptide;
• one containing a large number, but less
than 50 amino acids, a peptide;
• one containing 50-100 amino acids –
polypeptide;
• if the number of aminoacids is higher then
100, the polypeptide is called protein.
• The end of the peptide chain with the -NH2 group is
known as the N-terminal, and the end with the -COOH
group is the C-terminal. A protein chain (with the N-
terminal on the left) will therefore look like this:

• The "R" groups come from the 20 amino acids which

occur in proteins. The peptide chain is known as the
backbone, and the "R" groups are known as side
chains.
• Each protein has a specific sequence of amino acids
which are assembled under the direction and control of
nucleic acids.
Levels of Protein Structure
There are 4 levels of the protein structure:

primary
secondary
tertiary
quaternary
The sequence of amino acids in a protein is called :

primary structure of protein

 Secondary structure
is a regular arrangement of polypeptides into more
compact shapes, stabilized by hydrogen bonds.
The secondary structure describes the
relative orientations of amino acids close in
sequence. There are three predominant structure

alpha-helix
•

•collagen-helix
•beta- sheets.
sheets.
 The alpha-helix – is a helical structure, a spring-like coil of
polypeptide that forms a rigid cylinder of great regularity. The alpha helix was
found to be exclusively right handed. The formation of the alpha-helix is
spontaneous and is stabilized by hydrogen bonds. The hydrogen bond forms
between C=O groups of one amino acid in the backbone with N-H groups located
four amino acid residues further along the chain.
This orientation of H-bonds produces a helical coiling of the peptide backbone
such that the R-groups lie on the exterior of the helix and perpendicular to its axis.
Alpha helix has 3.6 amino acids per turn of the helix. The pitch – the distance
separating each turn of the helix is 0,54 nm.
 Collagen helix.
Another type of helix occurs in the
collagens, which are important
constituents of the connective tissue
matrix. The collagen helix is left-
handed, and with a pitch of 0.96 nm
and 3.3 residues per turn, it is steeper
than the alpha-helix. In contrast to the
alpha-helix, H bonds are not possible
within the collagen helix. However, the
conformation is stabilized by the
association of three helixes to form a
right-handed collagen triple helix.
Types of secondary structure of protein

A. Alpha-helix B.Collagen helix

 Types of secondary structure of protein

Pleated-sheet structures
results from the alignment of the polypeptide backbone aside one
another. Beta-sheets is stabilized by hydrogen bonds between C=O
and N-H groups in different regions of the polypeptide, or even
between two different polypeptides.
 If extended strands are lined up side by side, H-bonds bridge from
strand to strand. Identical or opposed strand alignments make up
parallel or antiparallel beta sheets.
In parallel sheets adjacent peptide chains proceed in the same direction
(the direction of N-terminal to C-terminal ends is the same), whereas, in
antiparallel sheets adjacent chains are aligned in opposite directions.

1. Antiparallel 2. Parallel
The side-chain groups (radicals) are not involved in alpha-
helix or beta-sheet structure stabilization.

An antiparallel beta-sheet.
 Tertiary protein structure
refers to the complete three-dimensional folding of the entire polypeptide
chain
In general, proteins fold into two broad classes of structure
termed. In dependence of the protein’s shape there are 2 main
types of proteins:
Globular proteins
Fibrous proteins
Globular proteins are compactly folded and coiled:
Fibrous proteins are
filamentous or elongated:

Keratin fiber
Collagen fiber
 Stabilization of the protein's tertiary structure
may involve interactions between the
radicals of amino acids located far apart
along the primary sequence.
These may include several types of bonds:

Non-covalent Covalent
- weak bonds - strong bonds
 Non-covalent, weak bonds:
•Hydrogen bonds between side-chain
groups (if the side groups contain
hydroxyl or amino groups).
•Ionic bonds between positively and
negatively charged amino acid side
chains.
•Hydrophobic interactions (Van der
Waals bonds), between the nonpolar
side groups
 Covalent, strong bonds:
•Disulphide bridges - covalent bonds between
two -SH groups of cystein to form an -S-S linkages:

Cys-SH + HS-Cys Cys-S-S-Cys

•Estheric bonds – between a side-chain

carboxylic group (of asp or glu) and a side-chain
hydroxyl group (of ser, tre or tir)
Glu-COOH + HO-Ser Glu-CO-O-Ser

•Pseudo peptide bonds - between a side-chain

carboxylic group (of asp or glu) and a side-chain
amino group (of lys):
Glu-COOH + H2N-Lys Glu-CO-HN-Lys
Bonds that stabilized tertiary structure

Hydrophobic interactions
Tertiary protein structure

The tertiary structure is determined

by the sequence of amino acids in
the chain and is the most
energetically convenient.
A lot of proteins at this level begin
to show their biological properties
and are capable of carrying out
their designated function.
 Quaternary protein structure
refers to the regular association of two or more
polypeptide chains to form a complex (olygomer).
A multi-subunit protein may be composed of two or more
identical polypeptides, or it may include different
polypeptides (protomers).

Primary Secondary Tertiary Quaternary

structure structure structure structure

Amino acid Alpha-Helix Polypeptide chain Assembled subunits

residues
Different kinds of oligomers:
 Protein folding
Protein folding is the physical process by which a
polypeptide folds into its characteristic and functional
three-dimensional structure.

Information about the biologically active (native) conformation

of proteins is already encoded in their amino acid sequences.
The native forms of many proteins arise spontaneously. Nevertheless,
there are special auxiliary proteins (chaperonines) that support the
folding of other proteins in the conditions present within the cell
 Denaturation
The native conformation of
proteins can be lost as the
result of denaturation: the
destruction of the secondary,
tertiary and quaternary
structures at extreme pH
values, at high temperatures,
and in the presence of organic
solvents, detergents, and other
denaturing substances.
Since denaturation reactions are not strong enough to
break the peptide bonds, the primary structure (sequence
of amino acids) remains the same after a denaturation
process.
Effects of Denaturation

•Loss of biological activity

•Decreased solubility
•Improved digestibility
Refolding or renaturation

The denatured protein

can spontaneously
return to its native
conformation, but only if
the denaturating agent
was not strong enough
and its action was of
short duration.
 Classification of proteins
by chemical composition
• Simple proteins - contain only amino acids
and no other chemical groups; yield only amino acids upon
hydrolysis

• Conjugated proteins - proteins that contain

at least one prosthetic group – a nonproteic structure
attached by covalent bonds or by weak interactions, required
for the activity of the protein, for example the hem of
hemoglobin. Yield, on hydrolysis, some other chemical
component in addition to amino acids.
 Simple proteins
Proteins that yield only alpha-amino acids
by hydrolysis: albumins, globulins, histones,
glutelins, prolamines, protamines.

Albumin Globulin Histones

 Conjugated proteins
proteins that contains a nonproteic structure
called its prosthetic group. Conjugated proteins
are classified on the basis of the chemical nature
of their prosthetic groups.

Some examples of conjugated proteins are:

glycoproteins, lipoproteins,
phosphoproteins, hemoproteins,
flavoproteins, metalloproteins.
Glycoproteins

are generally the largest and most

abundant group of conjugated
proteins. They range from
glycoproteins in cell surface
membranes that constitute the
glycocalyx, to important antibodies
produced by leukocytes.
Hemoproteins
 Lipoproteins

Most lipids are transported in the

blood as part of soluble
complexes called lipoproteins
Cell membrane
 Physical-chemical properties of
proteins
Proteins differ by there physical and chemical
properties:

 Molecular mass
 Total electrical charge
 Termolability
 Solubility
Molecular mass of the
proteins
 Proteins are high molecular
compounds with molecular mass from
5 000 to
1 000 000 Da in dependence of the
number of amino acid residues and of
the number of protomers.
 Total electrical charge of
proteins
- depends on the presence and correlation
of charged radicals of amino acids;
- depends of the pH of the medium
If the protein has more negatively charged amino
acids (Glu and Asp)– in aqueous medium its total
charge will be negative. If the protein has more
(Lys, Arg and His) positively charged amino acids
– its charge will be positive (like in histones).
In acid medium the concentration of H+ is high and
neutralizes the COO- - groups of amino acids - the
negative charge decreases;
In basic medium the concentration of OH- is high
and neutralizes the positive charge of amino
groups -NH3+ - the positive charge decreases.
 The state of the protein when its
total electrical charge is equal to
zero is called isoelectrical state.
 The value of pH when the protein is
in the isoelectrical state is called
isoelectrical point.
 The proteins in the isoelectrical state
have a low solubility in water
medium and can easy precipitate.
 Termolability
- - the property of protein to maintain the
biological activity in narrow limits of temperature
(from 10 to 40°C)
- If the temperature is higher then 50-60°C the
protein denatures – loses its native conformation.
The destruction of all the structural levels of
protein (except the primary) takes place.
- There are several exceptions – the termostable
proteins (tripsin, lyzozime) – stable at high
temperature.
- If the temperature is low - the protein structure
doesn’t change, but the protein becomes
biologically inactive.
 Solubility of proteins
 The most of proteins are hydrophilic
compounds and are soluble in water.
 Water interacts with the polar groups of
proteins and forms an aqueous
membrane -hydration shell - at the
surface of protein
 Solubility of proteins depends
on:
1. presence of the polar
groups and hydration
shell;

2. shape of the molecule –

the globular proteins are
more soluble then fibrous;
 Solubility of proteins depends
on:
3. Solvent – albumins are soluble
both in water and salt solution of
different concentration, but
globulins are not soluble in water
and soluble only in weak salt
solution.
4. pH of the medium – pH influence
on the charge of the protein; in the
isoelectrical point solubility
decreases.
 The protein solution are
colloidal solution
and have the following
properties:
 Optical properties – Tindal effect

 A low speed of diffusion

 Osmotic (oncotic) properties
 A high viscosity of the solutions
 A capacity to form gels – structural
grating with water inside
Methods of protein purification
and fractionation:
 Salt precipitation
 Dialysis

 Electrophoresis

 Gel-filtration
 Salt-precipitation
 The solubility of proteins is strongly
dependent on the salt concentration
(ionic strength) of the medium. Proteins
are usually poorly soluble in pure water.
Their solubility increases as the ionic
strength increases, because more and
more of the well-hydrated anorganic
ions are bound to the protein’s surface,
preventing aggregation of the
molecules (salting in).
 Salt-precipitation
 At very high ionic strengths, the
salt withdraws the hydrate water
from the proteins and thus leads
to aggregation and precipitation
of the molecules (salting out).
For this reason, adding salts such
as ammonium sulfate (NH4)2SO4
makes it possible to separate
proteins from a mixture
according to their degree of
solubility (fractionation).
Dialysis
Dialysis is used to remove lower-molecular components
from protein solutions. Due to their size, protein
molecules are unable to pass through the pores of a
semipermeable membrane, while lower-molecular
substances are able.
Electrophoresis is a technique
used to separate different elements
(fractions) of a blood sample into
individual components. Serum
protein electrophoresis is a test that
measures the major blood proteins
by separating them into five distinct
fractions: albumin, alpha1, alpha2,
beta, and gamma proteins.