METHODS OF DETERMINING PROTEIN STRUCTURE

1. Topic: protein structure
determination methods
PRESENTED BY
KHIZAR ABBAS
ULFAT RANA
SABAHAT ALI

2. CONTENTS:
 INTRODUCTION
 EDMAN DEGRADATION
 X-RAY CRYSTALLOGRAPHY
 WESTERN BLOTTING
 SDS-PAGE
 2D-GEL ELECTROPHORESIS
 ISOELECTRIC FOCUSING

3. Introduction
Proteins, also known as polypeptides,
are organic compounds made up of
amino acids.
They are arranged in a linear chain and
folded into a globular form.
Proteins are essential parts of organisms
and they participate in virtually every
process within cells.
Many proteins are enzymes that
catalyze biochemical reactions and are
vital to metabolism.

4. The Structure of Proteins
Most proteins fold into unique
three-dimensional structures. The
shape that a protein folds into
naturally is known as its native
conformation. There are four distinct
aspects of a protein’s structure:

5. Continued..
 Primary structure: the amino acid sequence
 Secondary structure: regularly repeating local
structures stabilized by hydrogen bonds
 Tertiary structure: the overall shape of a single
protein molecule; the spatial relationship of the
secondary structures to one another
 Quaternary structure: the structure formed by
several protein molecules which function as a
single protein

6. Protein Analysis Techniques
1)Edman Degradation
 Introduction
 The strategy of Sanger for the sequencing of
insulin was to characterize series of small
overlapping peptides produced by cleavage
of the parent molecule.
 Determination of the overall amino acid
content and the identity of the amino- (N-
)terminal residue for each peptide allowed
deduction of the sequence of the whole
molecule.

7. Contd..
An alternative approach was that
described by Edman (1950). This allowed
determination of extended sequences of
peptides or whole protein.

8. Continued:
The method employs chemical reactions to
remove and identify the amino acid residue
that is at the N- terminus of the polypeptide
chain, i.e. the residue with a Free a amino
group.
Repeate this process,we can get the
sequence of the polypeptide.

9. The Basis of the
Method
Peptide
sequencing by
Edman chemistry
may be divided
into steps as given

10. Coupling: •Phenyl isothiocyanate
(PITC) reacts with an a-
amino group (or in the
case of prolyl residue
with an imino group) at
the N-terminal end of
the polypeptide chain,
to form a
phenylthiocarbamyl
derivative of the terminal
residue.

11. Contd..
Basic conditions are required for this reaction.
Edman originally used pyridine to generate a pH of
8.6.
 Clearly, a free a-amino group is required for this
reaction to occur.
If the amino group has become modified and will
no longer react with PITC, the polypeptide is said to
be ‘blocked’

12. Cleavage:
In the presence of strong acid, cleavage
occurs at the first peptide bond, giving the
peptide (minus the first residue) and the
liberated first residue as the
anilinothiazolinone (ATZ) form.

13. Cleavage conti…
Once other reactants and products
have been washed away, the shortened
polypeptide can be taken through
another round of coupling and
cleavage to release the second residue,
and so on in a cyclical fashion

14. 3-Conversion:
The ATZ residue is separated from the peptide
by extraction in organic solvent (ethyl acetate
or chlorobu- tane), and is then converted to a
more stable form to allow better analysis.
Conversion to the more stable phenylthio-
hydantoin (PTH) form is done in aqueous acid
(25% TFA, v/v in water).
.

15. Analysis of PTH residues:
The PTH residue generated by each
cycle of Edman chemistry is typically
identified by chromatography, originally
thin-layer chromatography and latterly
re- versed-phase high-performance
liquid chromatography

23. 3)Western blotting
 Blotting is a technique used to transfer DNA , RNA, Protein into a carrier so they can be
separated, often follows the use of gel electrophoresis.
 Three blotting techniques are used:
 Southern Blot
 Northern Blot
 Western Blot

24. Western blotting
 So we can define western blotting as
“An analytical method that involves the protein
immobilization on membranes before detection using monoclonal
or polyclonal antibodies”

25. Principle
• Immunoblotting technique which rely on the
specificity of binding between a molecule of
interest and a probe to allow detection of
molecule of interest In a mixture of molecule.

27. Principle…
• The molecule of interest is a
protein and the probe is an
antibody raised against that
particular protein.
• The SDS PAGE is prerequisite for
western blotting.

28. Procedure
Tissue preparation (Sample can be taken from
whole tissue or cell culture Detergents, salt and
buffers are employed to encourage lysis of cell)
Gel electrophoresis( Protein of sample separated
by using gel electrophoresis on the basis of
molecular weight, electric charge )

29. Procedure…
Transfer( Proteins are moved from the gel onto
membrane made of nitrocellulose .Electro
blotting can also be used.)
Blocking (Blocking is achieved by placing the
membrane in a dilute solution of protein with a
minute amount of detergent)

30. Procedure….
Detection(A modified antibody is used which is
linked to reporter enzyme , when exposed to
substrate it produces color)
Analysis (Size approximation are taken)

31. Advantages
It is specific test for detection of HIV as compare to
other tests.
It tells us how much protein has accumulated inside
the cell.
It gives the information about the size of protein.
It can detect one protein in a mixture of proteins.

32. Disadvantages
If a protein degraded , western blotting
detect it well.
This test takes longer than other existing
tests.
It might also be more costly.

33. Applications
It can identify the nature of the
protein
it can be applicated as a tool of
quantitative analysis of the micro
molecule antigen in cooperation with
Immunoprecipitation.

34. Applications…
The western blot is extensively used in biochemistry
for the qualitative detection of single proteins
protein-modifications (such as post-translational
modifications) are also determined.

35. Medical diagnostic applications
• The virus is enveloped with different proteins
• The detection of presence of these proteins
is useful in the detection of presence of virus.
• Some forms of Lyme disease testing employ
western blotting.

37. SDS-PAGE
SDS (also called lauryl sulfate) is an anionic
detergent, meaning that when dissolved its
molecules have a net negative charge within a
wide pH range.

38. SDS-PAGE
 A polypeptide chain binds amounts of SDS in
proportion to its relative molecular mass.
The negative charges on SDS destroy most of the
complex structure of proteins, and are strongly
attracted toward an anode (positively-charged
electrode) in an electric field.

39. 2D Gel Electrophoresis
2D gel electrophoresis (2DE) is a key
technique for purifying individual proteins
from complex samples based on their
Isoelectric points and molecular weights.

40. Isoelectric Focusing
 Isoelectric focusing (IEF), also known as
electrofocusing, is a technique for separating
different molecules by differences in their Isoelectric
point (pI.

41. Isoelectric Focusing
 Type of zone electrophoresis
 Electrophoresis is the separation of charged molecules,
particles and ions in a liquid medium under the influence of
an electric field.
 Is ideal for separation of amphoteric compounds.
 Separation is achieved by applying a potential difference
across the gel that contains a pH gradient.

42. Required for Isoelectric focusing
 Sample
 Ampholytes
 Buffer
 Voltage
 Supporting media
 Gel