The interface in a complex involves two structurally matched protein subunits, and the binding sites can be predicted by identifying structural matches at protein surfaces. Identification of Protein–Ligand Binding Sites by Sequence & Identifying protein–ligand binding sites is an important process in drug discovery and structure-based drug design. Detecting protein–ligand binding sites is expensive and time-consuming by traditional experimental methods. Hence, computational approaches provide many effective strategies to deal with this issue. Recently, lots of computational methods are based on structure information on proteins. However, these methods are limited in the common scenario, where both the sequence of protein target is known and sufficient 3D structure information is available. Studies indicate that sequence-based computational approaches for predicting protein–ligand binding sites are more practical. Different methods were used to determine protein binding sites fir instance, chromatin immuno preciptitation assay ( ChIP), Electrophoretic mobility shift assay (EMSA), Dnase footprinting assay etc.

1. IDENTIFICATION OF
PROTEIN BINDING SITE
By :- SNEHA AGRAWAL

2. INDEX
ABOUT DNA
HISTORY
TYPES OF DNA
PROTEIN BINDING SITES
IDENTIFICATION OF PROTEIN BINDING SITE
REFERENCE
CONTENTS

3. WHAT IS DNA
• DNA, abbreviation of Deoxyribonucleic acid .
• DNA is one of the most important molecules in all of
biology.
• DNA, or deoxyribonucleic acid, is the hereditary
material in humans and almost all other organisms.
• Deoxyribonucleic acid is a polymer composed of two
polynucleotide chains that coil around each other to
form a double helix.
• DNA is made of nucleotides, A nucleotide has two
components: a backbone, made from the
sugar deoxyribose and phosphate groups,
and nitrogenous bases, known
as cytosine, thymine, adenine, and guanine.

4. HISTORY
•The discovery of DNA’s
double-helix structure is
credited to the
researchers JAMES
WATSON & FRANCIS
CRICK
•Watson and Crick presented
a model for the DNA’s
double-helix structure.

5. TYPES OF DNA
• A-DNA: It is a right-handed double helix
similar to the B-DNA form. Dehydrated
DNA takes an A form that protects the
DNA during extreme condition such as
desiccation. Protein binding also
removes the solvent from DNA and the
DNA takes an A form.
• B-DNA: This is the most common DNA
conformation and is a right-handed
helix. Majority of DNA has a B type
conformation under normal
physiological conditions.
• Z-DNA: Z-DNA is a left-handed DNA
where the double helix winds to the left
in a zig-zag pattern. It was discovered by
Andres Wang and Alexander Rich. It is
found ahead of the start site of a gene
and hence, is believed to play some role
in gene regulation.

6. DNA BINDINGDOMAINS
• It has two basic domains a DNA binding domain
specifically recognizes the target sequence -an
activation domain contacts a basal transcription
factor.
• DNA-Binding proteins serve two principal
functions
i. To organize and compact the chromosomal
DNA.
ii. To regulate and effect the processes of
transcription, DNA replication, and DNA
recombination

7. • The transcription factors which modulate
process of transcription, various polymerases,
nucleases which cleave DNA molecules
involving in chromosome packaging.
• DNA binding proteins are :-
Non-Histone
Histone
• There are several motifs present which are
involved in DNA binding that facilitate binding
to nucleic acid such as :-
Helix turn helix
Zinc fingers
Leucine zippers
Helix loop helix

8. IDENTIFICATION OF PROTEIN BINDING
SITE
• It is a technique that predicts the interaction
between a macromolecules and a chemical
molecule.
• Its major application is to Identify the protein
ligand binding sites is an important process in
drug discovery and structure based drug design.
• Earlier, detecting protein ligand binding site is
expensive and time consuming by traditional
experimental method. Hence, computational
approches provide many effective strategies to
deal with this issue.

9. • It quantifies the interaction by calculating
binding energies.
• It provides 3-D information such as
orientation of binding.
• There are many vitro and vivo techniques
which are useful in detecting DNA protein
interactions such as :-
A. ELECTROPHORETIC MOBILITY SHIFT
ASSAY
B. CHROMATIN IMMUNOPRECIPITATION
C. DNase FOOTPRINTING ASSAY

10. A. ELECTROPHORETICMOBILITYSHIFTASSAY
• An Electrophoretic mobility shift
assay (EMSA) or mobility shift electrophoresis,
also referred as a gel shift assay, gel mobility
shift assay, band shift assay, or gel retardation
assay, is a common affinity
electrophoresis technique used to study protein
DNA or protein RNA interactions.
• It is discovered by GARNER AND REVZIN
AND FRIED AND CROTHERS.

11. PRINCIPLE
It is based on the observation that the
electrophoretic mobility of a protein-nucleic
acid complex is typically less than that of the
free nucleic acid.
• Smaller molecules of DNA migrates faster
• Larger molecules of DNA migrates slower.
• Binding of protein will reduce the mobility of
DNA.

12. STEPS
PREPARATION OF CELL PROTEIN EXTRACT
PREPARE RADIOACTIVELY LABELLED DNA.
BINDING REACTION
NON DENATURING GEL ELECTROPHORESIS
DETECTION OF OUTCOME.

13. FIG :- EMSA

14. ADVANTAGES
• It is a simple, rapid &
sensitive method.
• Low cost
• It proves useful in detection
of protein that bind to DNA.
• Quick and versatile.
DISADVANTAGES
• It doesn’t provide a straight
forward measure of the
weights and actual
sequences of the proteins.
• Dissociation is one of the
drawbacks of EMSA. It is not
a appropriate method for
kinetic studies.

15. B. CHROMATINIMMUNOPRECIPITATION
• Chromatin immunoprecipitation (ChIP)
is a type of immunoprecipitation
experimental technique used to
investigate the interaction between the
proteins and DNA.
• It is discovered by JOHN T. LIS &
DAVID GILMOUR IN 1984.

16. PRINCIPLE
The principle behind ChIP is relatively
straightforward and relies on the use of
an antibody to precipitate, a certain
protein, and its bound chromatin from a
protein mixture that was extracted from
cells or tissues. Hence, the name of the
technique: Chromatin Immunoprecipitati
on then DNA fragments are then able to
be identified and quantified using widely
available PCR.

17. STEPS
CROSSLINKING
CELL LYSIS
CHROMATIN PREPARATION
IMMUNOPRECIPITATATION
REVERSAL OF CROSSLINKING AND DNA
CLEAN UP AND DNA QUANTITATION.

18. FIG :- ChIP

19. • Efficient precipitation of
DNA and protein.
• High resolution (175
bp/monosomes).
• Good for non-histone
proteins binding weakly or
indirectly to DNA.
• Protein Selective nuclease
digestion may bias input
chromatin.
• Nucleosomes may
rearrange during digestion.
• May be inefficient antibody
binding due to epitope
disruption.
ADVANTAGES DISADVANTAGES

20. C. DNase FOOTPRINTINGASSAY
• It is a method of investigating DNA sequence
specificity bind proteins in vitro.
• It is discovered by DAVID GALAS AND
ALBERT SCHMITZ IN 1978.
• This phenomenon can be used to locate the
protein binding site on DNA molecule.
• Labels can be used are fluorescence &
radioactivity.

21. PRINCIPLE
In this technique, nucleases like DNAse I is
used which will degrade DNA molecule.
Nucleases cannot degrade DNA if it is
bounded by a protein. Thus that region is
protected from degradation by nucleases.
This protected DNA region is called the foot
print.

22. STEPS
PREPARE END LABELELD DNA.
BIND PROTEIN
MILD DIGESTION WITH DNAASE 1
SEPARATES DNA FRAGMENTS ON
GEL

23. FIG :- DNase FOOTPRINTING ASSAY

24. • Powerful enough to
differentiate many
fragments.
• It can exactly locate the
binding site of the particular
ligand.
• The enzyme does not cut
DNA randomly
• Its activity is affected by
local DNA structure and
sequence and therefore
results in an uneven ladder.
ADVANTAGES DISADVANTAGES

25. REFERENCE
 https://www.creativebiomart.net/resource/principle-
protocol-protein-interaction-4-yeast-one-hybrid-assay-
374.htm
 https://www.creativebiomart.net/resource/principle-
protocol-dnase-i-footprinting-
377.htm#:~:text=DNase%20I%20footprinting%20assay%20i
s,the%20target%20protein%20is%20bound
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757439/
 https://youtu.be/kWeg-5FRqlk
 https://en.m.wikipedia.org/wiki/Chromatin_immunoprecipi
tation

26. THANK
YOU