Protein and its various structures

PROJECT WORK

SUBMITTED FOR THE DEGREE OF

MASTER OF SCIENCE

in
CHEMISTRY

BY

MUKUL KUSHWAHA

Under the supervision of

Dr. Dileep Kumar Singh

Department of Chemistry

Bipin Bihari College, Jhansi – 284001 , U.P

2023-24
 CERTIFICATE

This is to certify that the project entitled “Protein Protein

interaction” submitted to “Bipin Bihari Degree College, Jhansi”

for the fulfilment of the requirement for the Master of Science in a

project work is carried out by Mukul Kushwaha student of M.Sc.

chemistry under the supervision Dr. Dileep Kumar Singh during the

session 2023-24.

Dr. Yogesh pandey Dr.Dileep Kumar Singh

(Project Supervisor)
(Head of Department)
Department of Chemistry Department of Chemistry
Bipin Bihari College, Jhansi
Bipin Bihari College, Jhansi
 DECLARATION

I hereby declare that this reported title “Protein Protein interaction”

is submitted to the Department of Chemistry “Bipin Bihari Degree

College, Jhansi” is a record of original work done by me under the

guidance of Dr. Dileep Kumar Singh. The information and data

given in this report is authentic to the best of my knowledge. This

report project is not submitted to any other university or institution for

the award of any degree, diploma or fellowship or published any time

before.

Submitted by

Mukul Kushwaha

ACKNOWLEDGEMENT
This project is a result of dedicated effort. It gives us immense pleasure to
prepare this report on “Protein Protein interaction”. I would like to
thank my guide, Dr. Dileep Kumar Singh for consultative help and
suggestion on matter in this project.

I am greatly indebted to Prof. T. K. Sharma, Principal, Bipin

Bihari College, Jhansi, for permitting me to avail the facilities for my
work and rendering every sort of encouragement at each stage during my
tenure.

I would like to extend my deep and sincere thanks to Prof. D. K.

Agarwal, Head, Department of Chemistry, Bipin Bihari College, Jhansi,
for giving their valuable suggestions and guidelines in selecting my project
work & for their endless support, care & for being a kind & helpful all the
time.

I am also very thankful to all the faculty member and associated staff of the
Department of Chemistry Bipin Bihari College, Jhansi.

My sincere thanks to my parents as well as my friends whose efforts and

suggestion helped me in the completion of this work.

Date: Mukul Kushwaha

M.Sc III Semester

 Protein and its various structures

Abstract:

Proteins are the most abundant organic molecule of the living system. In 1839,

Dutch chemist G. J. Mulder was first to describe about protein. The term protein is

derived from a Greek word proteious, meaning first place. The proteins are

nitrogenous macromolecules that are composed of many amino acids. We are

discussing about protein and their structure.

Introduction

Protein is a chain of amino acids joined by peptide bonds in a specific sequence.

Protein is an essential nutrient. There is no life without protein. Protein is

contained in every part of your body, the skin, muscles, hair, body organs, eyes,

even fingernails and bone. Next to water, protein is the most plentiful substance in

your body. The two General categories of Protein are Fibrous proteins and

Globular proteins.

Protein is vital in the maintenance of body tissue. Including:

 Development and repair

 Protein is the major source of energy

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  Protein is involved in the creation of some hormones, help control body

functions that involve the interaction of several organs and help regulate cell

growth.

 Protein produces enzymes that increase the rate of chemical reactions in the

body

 Protein transport small molecules through the organism. Hemoglobin is the

protein that transports oxygen to the cells and it is called as transport protein.

 Protein called antibodies helps rid the body of foreign protein and help

prevent infections, illness.

Structures of proteins:
Proteins are composed of small units. These units are the amino acids which are

called the building blocks of protein there are about 20 different amino acids which

are commonly known. Each different protein is composed of various amino acids

put together in varying order with almost limitless combinations. Most proteins are

large molecules that may contain several hundred amino acids arranged in

branches and chains. We can thus see that he peptide bond (CO-NH) is formed

between the amine group of one molecule and the carboxyl group of the adjacent

molecule followed by the elimination of a water molecule. This bond is otherwise

a water molecule.

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 Types of Protein-Protein Interactions

On the basis of their Composition

Homo-Oligomers: These are macromolecule complexes having one type of protein

subunits.

e.g.: PPIs in Muscle Contraction

➤ Hetero-Oligomers: These are macromolecule complexes having multiple types

protein subunits.

e.g.: PPI between Cytochrome Oxidase and TRPC3 (Transient receptor potential
cation channels

On the basis of duration

Stable Interactions: These comprise of interactions that last for a long duration.
These Interactions carry out Functional or Structural roles.

e.g.: Haemoglobin structure

Transient Interactions: Interactions that last a short period of time.

e.g.: Muscle Contraction.

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 Effects of protein interaction

• They can alter the kinetic properties of proteins

• Protein interactions are one common mechanism to allow for substrate

channeling.

• Can result in the formation of a new binding site

Can inactivate a protein

• Can change the specificity of a protein for its substrate

Identify the different interactions, understand the extent to which they take place in
the cell, and determine the consequences of the interaction.

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Another way of classification for methods for identification
of PPis

 The first is 'atomic observation' in which the protein interaction detected

using, for example, X-ray crystallography. These experiments can yield
specific information on the atoms or residues involved in the interaction.

 The second is a 'direct interaction observation' where protein interaction

between two partners can be detected as in a two-hybrid experiment.

 At a third level of observation, multi-protein complexes can be detected

using methods such as immuno-precipitation or mass- specific analysis. This
type of experiment does not reveal the chemical detail of the interactions or
even reveal which proteins are in direct contact but gives information as to
which proteins are found in a complex at a given time.

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 Yeast two-hybrid (Y2H) system

 Yeast two-hybrid (Y2H) system has variations involving different reagents

and has been adapted to high-throughput screening.

 The strategy interrogates two proteins, called bait and prey,

 Coupled to two halves of a transcription factor and expressed in yeast.

 If the proteins make contact, they reconstitute a transcription factor that

activates a reporter gene.

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 Used to detect interactions between candidate proteins whose genes are
available by constructing the appropriate hybrids and testing for reporter
gene activity

 Point mutations can be assayed to identify specific amino acid residues

critical for the interaction

 Can be used to screen libraries of activation domain hybrids to identify

proteins that bind to a protein of interest

 Transcriptional activation domains are commonly derived from the Gal4

protein or the herpes simplex virus VP16 protein

 Reporter genes include the E. coli lacz gene and selectable yeast genes such
as HIS3 and LEU2

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 Uses/ Advantages of Yeast Two Hybrid Method

 It is highly sensitive, detecting interactions that are not detected by other

methods

 Minimal binding constant required to detect an interaction in two hybrid

system is on the order of 1 µM

 Interactions are detected within the native environment of the cell and hence
that no biochemical purification is required

 Study of oncogenes and tumor suppressors and the related area of cell cycle
control

Limitations

 limited to proteins that can be localized to the nucleus, which may

prevent its use with certain extracellular proteins

 Proteins must be able to fold and exist stably in yeast cells and to retain
activity as fusion proteins

 Interactions dependent on a posttranslational modification that does not

occur in yeast cell will not be detected.

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 Yeast three Hybrid

 The three-hybrid system enables the detection of RNA-protein interactions

in yeast using simple phenotypic assays.

 It was developed in collaboration with Stan Fields laboratory (University of

Washington).

 The LexA DNA binding domain is fused to the MS2 coat protein to form
Hybrid Protein 1.

 Hybrid Protein 2 consists of the Gal4 activation domain linked to the RNA
binding domain, Y, you wish to test.

 The Hybrid RNA consists of two MS2 RNA binding sites and the RNA
sequence you wish to test, RNAX.

 Hybrid Protein 1 and the presence of MS2 sites in the Hybrid RNA are
fixed, as is the Gal4 Activation Domain. RNAX and RNA Binding Domain
Y vary.

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 This method consists in the expression in yeast cells of three chimerical
molecules, which assemble in order to activate two reporter genes.

 Thus, using the yeast three-hybrid system, in contrast to other methods,

RNA-protein interactions are detected in vivo.

 This system uses a transactivator protein in yeast, such as Gal4p, that is able
to recruit the transcriptional machinery and trigger transcription of a gene.

 It consists of a DNA binding domain (DB) and an activation domain (AD)

and, importantly, these two domains are functionally independent, meaning
that they can be inserted into other molecules.

 In this method, the three components of the system are expressed from two
plasmids allowing the use of any previously described yeast strains for two
hybrid system that provide the two reporter HIS3 and lacZ under the control
of GaI4 operator.

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 CO-IMMUNOPRECIPITATION (coIP)

 Co-immunoprecipitation (colP) is the most complex method.

 Co-immunoprecipitation (co-IP) is a popular technique for protein

interaction discovery. Co-IP is conducted in essentially the same manner as
an immunoprecipitation (IP) of a single protein, except that the target protein
precipitated by the antibody, also called the "bait", is used to co- precipitate
a binding partner/protein complex, or "prev". from a Ivsate.

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 Immunoprecipitation

 This is classical method of detecting protein-protein interactions

 Cell lysates are generated, antibody is added, the antigen is precipitated and
washed, and bound proteins are eluted and analyzed.

 Co-precipitated protein is precipitated by the antibody itself and not by a

contaminating antibody in the preparation

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 If the interaction is direct through another protein that contacts both the
antigen and the coprecipitated protein determining that the interaction takes
place in the cell.

 Adenovirus E1A protein interacts with Rb protein detects the interactions

present in a crude lysate

 Both the antigen and the interacting proteins are present in the same relative
concentrations as found in the cell.

Pathway Databases and Algorithms

 KEGG (Kyoto Encyclopedia of Genes and Genomes): Representation of

higher order functions in terms of the network of interaction molecules

 GENES database contains 240 943 entries from the published genomes,
including the bacteria, mouse and human.

 KEGG has 3 databases, GENES, PATHWAY and LIGAND databases.

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 By matching genes in the genome and gene products in the pathway, KEGG
can be used to predict protein interaction networks and associated cellular
function.

 KEGG is a network of gene products with three types of interactions or

relations: enzyme-enzyme relations which catalyzes the successive reaction
steps in the metabolic pathway, direct protein-protein interactions and gene
expression relations.

 PATHWAY database contain 5761 entries including 201 pathway diagram

with 14960 enzyme-enzyme relations.

Protein Affinity Chromatography

 Affinity Chromatography is essentially a sample purification technique, used

primarily for biological molecules such as proteins.

 It is a method of separating a mixture of proteins or nucleic acids

(molecules) by specific interactions of those molecules with a component
known as a ligand, which is immobilized on a support.

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 If a mixture of proteins is passed over (through) the column, one of the
proteins binds to the ligand on the basis of specificity and high affinity (they
fit together like a lock and key).

 The other proteins in the solution wash through the column because they
were not able to bind to the ligand.

 Protein fusions
- Glutathione S-transferase
-Staphylococcus protein A
-Maltose-binding protein.

Affinity Blotting

 Analogous to the use of affinity columns, proteins can be fractionated by

PAGE transferred to a nitrocellulose membrane, and identified by their
ability to bind a protein, peptide, or other ligand

 Complex mixtures of proteins, such as total-cell lysates, can be analyzed

without any purification

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 Biological activity of the proteins on the membrane, the preparation of the
protein probe, and the method of detection widely used in studies of the
regulatory subunit of the type II CAMP- dependent protein kinase with
numerous specific anchoring proteins

 Two-dimensional procedures of isoelectric focusing followed by SDS-

PAGE have been used to increase the separation of proteins.

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