PROTEIN-PROTEIN INTERACTIONS

(PPI)

Dr. Sharda Bharti

Protein Cleavage
 Practice question 1
A given polypeptide chain is cut with trypsin followed by cyanogen bromide and generated different fragments.
The fragments are finally overlapped and a correct sequence is predicted.
• Trypsin cut generated four fragments:
T1 – GASMALIK, T2 – EGAAYHDFEPIDPR T3 - DCVHSD
T4 – YLIACGPMTK
• Cyanogen bromide cut generated three fragments:
C1 – EGAAYHDFEPIDPRGASM C2- TKDCVHSD
C3 – ALIKYLIACGPM
The correct sequence would be:
A. C3-T4-C2-T3-T2-C1-T1
B. T2-C1-T1-C3-T4-C2-T3
C. T1-C3-T4-C2-T3-T2-C1
D. C1-T2-T3-C2-T4-C3-T1
 Practice question (5 marks)

Question: A given polypeptide chain is cut with trypsin followed by cyanogen bromide and generated different
fragments. The fragments are finally overlapped and a correct sequence is predicted.

• Trypsin cut generated four fragments:

T1 – GASMALIK, T2 – EGAAYHDFEPIDPR T3 - DCVHSD T4 – YLIACGPMTK
• Cyanogen bromide cut generated three fragments:
C1 – EGAAYHDFEPIDPRGASM C2- TKDCVHSD C3 – ALIKYLIACGPM
The correct sequence would be: Answer: B: T2-C1-T1-C3-T4-C2-T3

A. C3-T4-C2-T3-T2-C1-T1
B. T2-C1-T1-C3-T4-C2-T3
C. T1-C3-T4-C2-T3-T2-C1
D. C1-T2-T3-C2-T4-C3-T1
 Practice question

In the given polypeptide sequence

Where might bends or beta turns occur?

a) 10 and 17
b) 6 and 21
c) 13 and 24
d) 7 and 19
1) In the given polypeptide sequence
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Ile – Ala – His – Thr – Tyr – Gly – Pro – Phe – Glu – Ala – Ala – Met – Cys – Lys – Trp – Glu –
17 18 19 20 21 22 23 24 25 26 27 28
Ala – Gln – Pro – Asp – Gly – Met – Glu – Cys – Ala – Phe – His – Arg
• Where might bends or beta turns occur?
a) 10 and 17
b) 6 and 21
c) 13 and 24
d) 7 and 19
Answer: D
PROTEIN-PROTEIN INTERACTIONS
 Protein-protein interactions (PPIs)

• Protein–protein interactions (PPIs) are physical contacts of high specificity established

between two or more protein molecules as a result of biochemical events.
• are physical contacts with molecular associations between chains that occur in a cell or in a
living organism in a specific biomolecular context
• control variety of biological phenomena including development, cell to cell interactions and
metabolic processes and a blend of these three pairs may develop a protein-protein
interaction. For example, a permanent interaction of the protein may be able to form a stable
protein complex while on the other hand a transient interaction among the proteins may
form a signaling pathway
• Proteins rarely act alone as their functions tend to be regulated.
• Many molecular processes within a cell are carried out by molecular machines that are built
from numerous protein components organized by their PPIs.
• These physiological interactions make up the so-called interactomics of the organism, while
aberrant PPIs are the basis of multiple aggregation-related diseases, such as Creutzfeldt–Jakob
and Alzheimer's diseases.
 Interactomics

• In molecular biology, an interactome is the whole set of molecular interactions in a particular cell.
• The term specifically refers to physical interactions among molecules (such as those among proteins,
also known as protein–protein interactions, PPIs; or between small molecules and proteins but can also
describe sets of indirect interactions among genes (genetic interactions).
• Interactomics is a discipline at the intersection of bioinformatics and biology that deals with studying
both the interactions and the consequences of those interactions between and among proteins, and
other molecules within a cell.
• Interactomics thus aims to compare such networks of interactions (i.e., interactomes) between and
within species in order to find how the traits of such networks are either preserved or varied.
• Interactomics is an example of "top-down" systems biology, which takes an overhead, as well as overall,
view of a biosystem or organism. Large sets of genome-wide and proteomic data are collected, and
correlations between different molecules are inferred. From the data, new hypotheses are formulated
about feedbacks between these molecules. These hypotheses can then be tested by new experiments.
 PPI
• The study of interactomes is called interactomics. The basic unit of a protein network is the
protein–protein interaction (PPI).
• While there are numerous methods to study PPIs, there are relatively few that have been
used on a large scale to map whole interactomes.
• The yeast two hybrid system (Y2H) is suited to explore the binary interactions among two proteins at a
time.
• Affinity purification and subsequent mass spectrometry is suited to identify a protein complex.
• Both methods can be used in a high-throughput (HTP) fashion.Yeast two hybrid screens allow false
positive interactions between proteins that are never expressed in the same time and place; affinity
capture mass spectrometry does not have this drawback, and is the current gold standard.
• Yeast two-hybrid data better indicates non-specific tendencies towards sticky interactions rather while
affinity capture mass spectrometry better indicates functional in vivo protein–protein interactions.
• To perform the function in a living cell, proteins rarely act as isolated species instead over 80% of the
proteins perform their functions in groups.
 PPI

• The function of an unidentified protein can be suggested by its interactions with a

protein of known function.
• The thorough study of PPIs is also important to demonstrate the molecular
mechanism of cellular processes of proteins.
• The momentous properties of PPIs are (a) the kinetic properties of the enzymes can
be modified by PPIs; (b) PPIs can allow substrate channeling; (c) they can create a new
binding site for the small molecules; (d) PPIs can suppress or activate a protein; (e)
PPIs can perform regulatory role in downstream or upstream regulation of the
protein; and (f) they can also alter the specificity of binding of the protein for its
particular substrate by changing its interactions.
 Examples of PPI
• Electron transfer proteins: In many metabolic reactions, a protein that acts as an electron carrier binds to an
enzyme that acts its reductase. After it receives an electron, it dissociates and then binds to the next enzyme that
acts its oxidase (i.e. an acceptor of the electron). These interactions between proteins are dependent on highly
specific binding between proteins to ensure efficient electron transfer. Examples: mitochondrial oxidative
phosphorylation chain system components cytochrome c-reductase / cytochrome c / cytochrome c oxidase;
microsomal and mitochondrial P450 systems.
• Signal transduction: The activity of the cell is regulated by extracellular signals. Signal propagation inside and/or
along the interior of cells depends on PPIs between the various signaling molecules. The recruitment of signaling
pathways through PPIs is called signal transduction and plays a fundamental role in many biological processes and in
many diseases including Parkinson's disease and cancer.
• Membrane transport: for example, from cytoplasm to nucleus or vice versa in the case of the nuclear pore
importins.
• Cell metabolism: In many biosynthetic processes, enzymes interact with each other to produce products.
• Muscle contraction: Myosin filaments act as molecular motors and by binding to actin enables filament sliding.
Furthermore, members of the skeletal muscle lipid droplet-associated proteins family associate with other
proteins, as activator of adipose triglyceride lipase and its coactivator comparative gene identification-58, to
regulate lipolysis in skeletal muscle
 Stability of Interacting Complexes

• Based on Stability of Interacting Complexes

• If protomers cannot exist in free form and only stable in multimeric association they form obligate
oligomers (Homo-obligomers and/or heteroobligomers), like the Arc repressor dimer where
dimerization is essential for DNA binding.
• When protomers can exist in free form as well, they are non obligate partners like antigen-
antibody complex or intracellular signal transmission complex. Mostly non obligate partners do not
co-localize, thus in need of self-stability.
• Based on Lifetime of PPI
• When an association between protomers is highly stable and need help from molecular switches to
break them, they are permanent complexes. Hetero-trimeric G protein (Gα, Gβγ and GDP) forms
this type of PPI.
• In contrast to this, Sperm Lysin, a homo-dimer, and many other show a dynamic equilibrium of
association and dissociation of oligo-meric form. This type of PPI is named as transient complexes
 Based on Nature of the Interaction Interface

• When protomers use the same interacting interface to join each other, they are isologous
complex. Arc repressor and Lysine; show this type of PPI with a two-fold symmetry axis.
They can only interact with other partners using a different interacting surface.
• In Heterologous assembly protomers use different interfaces to form PPI without any
closed symmetry. Thus, they have chances to aggregate infinitely. Amyloid aggregation might
have this type of PPI, allowing them to form a long multimeric disposition.
• Also, another type of grouping has been reported as Intra-domain, PPI within one structural
domain of protein, and Domain-domain, PPI of different domains within a single chain or
different chains of proteins.
• Oligomeric proteins do have selective advantages for benefits like surface area reduction,
increased stability and novel functions via communication among subunits. The interaction
surface of protomers is generally mentioned as PPI interface.
 IMPORTANCE OF PPI NETWORK

• Useful for isolating groups of interacting proteins that participate in the same biological
process
• Helps to understand the mechanism of regulating cell life
• Useful to predict the biological functions of uncharacterized proteins
 Assays

• Biophysical methods are somewhere in the middle.

• They can provide quantitative information on protein interaction, such as affinity rate
constants or thermodynamic parameters from which equilibrium dissociation
constant and free energy of binding can be derived
• Proteomic approaches are used to assess molecular networks within cells or cellular
compartments.
• Two most often used are affinity purification followed by mass spectrometry (AP-MS)
and yeast two hybrid (Y2H) approaches, which can both assess thousands of
interactions in a single study. Analysis of these requires computational approaches and
genome-wide mapping.
 Protein-protein interaction assays

• Protein-protein interaction (PPI) assays can be classified into three broad categories,
• i.e., in vivo, in vitro, and in silico.
• In vivo techniques apply the whole procedure on the living cell or organism itself.
• In vitro methods require the whole procedure completed outside the cell in a
controlled environment in a laboratory, i.e., affinity chromatography, tandem affinity
purification (TAP), protein fragment complementation, X-ray crystallography, co-
immunoprecipitation, phage display, nuclear magnetic resonance, spectroscopy, and
protein arrays.
• The techniques that are performed by using computers or computer simulations are
called in silico techniques. The sequence and structure-based approaches, gene
fusion, chromosome proximity, gene expression-based approaches, domain pair-based
approach, in silico two hybrid approaches, phylogenetic profile, and phylogenetic tree
are some approaches which are based on in silico methods.
 In vitro

• Affinity chromatography
• Coimmunoprecipitation
• Protein microarray
• TAP-MS

In vivo
PPI ASSAY
• Yeast two hybrid (Y2H) system

In silico

• Domain-pairs based sequence approach

• Structure based approach
• Gene neighbourhood
PPI ASSAY
 Immunoprecipitation

Immunoprecipitation (IP)
• The technique of precipitating a protein antigen out of solution
using an antibody specifically binding to the protein
• Used to isolate and concentrate a particular protein from a
sample containing many thousands of different proteins
• Requires that the antibody should be coupled to a solid
substrate at some point in the procedure
• Types of immunoprecipitation
• Individual protein immunoprecipitation (IP)
• Protein complex immunoprecipitation (Co-IP)
• Chromatin immunoprecipitation (ChIP)
• RNA immunoprecipitation (RIP)
• Tagged proteins
 Immunoprecipitation
Immunoprecipitation (IP)
To isolate a particular protein out of a solution containing many different
proteins
 Immunoprecipitation
Co-Immunoprecipitation (Co-IP)
• Immunoprecipitation of intact protein complexes
• A powerful technique that is used regularly to analyze protein-protein
interactions
 Immunoprecipitation
Chromatin immunoprecipitation (ChIP)

• A method to determine the location of DNA binding sites on

the genome for a particular protein of interest
• Based on DNA-protein interaction
• Using an antibody specific to a putative DNA binding proteins
→ immunoprecipitate the protein-DNA complex out of cellular
lysates
• Determine identity and quantity of the DNA fragment by PCR
or cloning the DNA into a plasmid vector
 Immunoprecipitation
Chromatin immunoprecipitation (ChIP)
 Immunoprecipitation
RNA immunoprecipitation (RIP)

• Similar to chromatin immunoprecipitation

• Targets RNA binding proteins rather than DNA binding proteins
• Isolate RNA-protein complex using an antibody specific for a
protein of interest
• Determination of identity of RNA by RT-PCR and cDNA
sequencing
 Immunoprecipitation
RNA immunoprecipitation (RIP)
 Immunoprecipitation
Tagged proteins

• Technical difficulty of immunoprecipiation: generation of an

antibody specifically targeting a single known protein is the
great difficulty
• To get around this obstacle, tags onto either C- or N-
terminal end of the protein of interest (GFP, Flag, HA tags,
GST)
• Presence of a good antibody against these tags and
substances specifically binding to tags
(immunoprecipitation or pull-down)
 Co-immunoprecipitation (co-IP)

• Co-immunoprecipitation (co-IP) is a popular technique to identify

physiologically relevant protein–protein interactions by using target protein-
specific antibodies to indirectly capture proteins that are bound to a specific
target protein.
• These protein complexes can then be analyzed to identify new binding
partners, binding affinities, the kinetics of binding and the function of the target
protein
 Pull down assay

Common fusion tags and their affinity binding ligands

Fusion tag Affinity ligand

Glutathione S-transferase (GST) Glutathione

Poly-histidine (polyhis or 6xhis) Nickel or cobalt chelate complexes

Biotin (vitamin B7), Streptavidin (bacterial proteins), avidin

Pull down assay
 In vitro techniques for the prediction of protein -protein
interactions

• To learn PPIs in the inherent environment of the cell, a technique called TAP tagging
was developed.
• TAP tagging method was first used to analyze the yeast interactome in a high
throughput way.
• TAP tagging involves two steps,
• first is double tagging of the protein of interest and
• second is two-step process of purification.
• After the process, the proteins that remain attached with the target protein can be studied by using
SDS-PAGE and then mass spectrometry analysis is performed to confirm the PPI partner of the
protein of interest.
• TAP tagging used in combination with mass spectrometry which can identify both
protein complexes and protein interactions.
 Affinity chromatography

• Affinity chromatography is also used to study PPIs in vitro. It is very sensitive technique and
can identify even the weakest interactions among the proteins. Though, it generates many of
the false positive results because of the great specificity of the proteins.
• Affinity Chromatography is an essential method used to separate desired protein from
complex mixtures, however it can also be used for studying Protein-Protein interaction.
• It is based on the affinity interaction among various groups of compatible proteins for its
separation from complex mixtures.
• Though, it generates many of the false positive results because of the great specificity of the
proteins.
• Therefore, studies of protein interactions cannot depend only on affinity chromatography. So,
other techniques are needed in combination with affinity chromatography to further confirm
the results generated. The affinity chromatography is often combined with mass spectroscopy
and SDS-PAGE to produce more convincing results.
Care should be taken during the
preparation of chromatographic
column

• The coupling of protein with the matrix should

not inhibit its functional activity otherwise it
would become inactive and incapable of
interacting and retaining the desired sample.
• Pure protein sample is to be used for column
preparation in order to prevent unwanted
protein interaction.
• The amount of sample protein applied must
not be too low to be unnoticeable for
detection within column and also must not be
too high for many small proteins to get eluted
without binding.
 Affinity Chromatography

• Affinity chromatography involves the retention of the desired sample within the
column from the sample mixture applied.
• In this method one of the protein ( against which interaction is to be checked) is
coupled with suitable matrix in the chromatographic column.
• The sample mixture containing the protein of interest from suitable source is applied
against it. Most of the proteins pass down through the column however, some
proteins may get retained into it. The protein which comes into affinity bonding or
interaction with the immobilized protein within the column gets retained and is
further eluted by suitable treatment. The eluted sample is collected for further
analysis (Fig. 1)
 Tandem Affinity Purification (TAP)

Principle
• A technique for studying protein-protein interactions
• A fusion protein with the TAP tag on the end
• TAP tag consists of calmodulin binding peptide (CBP), tabacco etch virus
protease (TEV protease) cleavage sit and protein A
• TEV protease: a highly specific cysteine protease found in tabacco etch virus,
recognize Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser
• Protein A – cell wall protein of bacteria, binds to immunoglobulins (IgG, Fc
region of Ab) → disrupts opsonization and phagocytosis
Tandem Affinity Purification (TAP)
Principle
 TAP (tandem affinity purification)

PCR product Spacer CBP TEV site Protein A

Homologous
recombination

Chromosome

Fusion protein Protein Spacer CBP TEV site Protein A

Calmodulin binding
peptide
 TAP process

"Taptag simple" by Chandres - Own work.

Licensed under CC BY-SA 3.0 via Wikimedia Commons
 Tandem Affinity Purification (TAP)
Advantages
• Simple to execute and often provide high yield
• Decrease the contamination of the target protein (two successive affinity purification)
• It can also provide effective and highly specific means to purify a protein
• No prior knowledge of complex composition
• Two-step purification increases specificity of pull-down

Disadvantages
• Tag can obscure binding of interacting proteins to the target protein
• Tag also can affect protein expression levels
• Tag may not be sufficiently exposed to the affinity beads
• The target protein may have TEV protease cleavage sites
• Hard to detect transient interactions (due to successive steps of purification)
• Transient interactions may not survive 2 rounds of washing
• Works less efficiently in mammalian cells
 Mass spectrometry
• Need to purify protein or protein complexes
• Use a affinity-tag system
• Need efficient method of recovering fusion protein in low
concentration
 Mass spectroscopy

• Mass spectroscopy can also be used to determine protein-protein interactions. There

are two ways to identify PPIs by using mass spectroscopy shotgun proteomics and
peptide finger printing
• To analyze complicated mixtures, shotgun proteomics is the most suitable technique
while in the peptide finger printing, SDS-PAGE is used to separate the eluted complex.
• X-ray crystallography can also be used to determine PPIs in vitro. It is a type of
microscopy with very high resolution that is used for the identification of proteins at
atomic level and it is helpful for functional analysis of proteins
 Other tags

• HA, Flag and His

• Anti-tag antibodies can interfere with MS analysis
• Streptavidin binding peptide (SBP)
• High affinity for streptavidin beads
• 10-fold increase in efficiency of purification compared to conventional TAP tag
• Successfully used to identify components of complexes in the Wnt/b-catenin pathway
 Protein Microarray
• Also called a protein binding microarray
• Provide a multiplex approach to identify protein-protein interactions, to identify
the substrates of protein kinases, to identify transcription factor or to identify
the targets of biologically active small molecules

Principle
▪ A piece of glass on which different molecules of protein are affixed at separate
locations in an ordered manner → labeled samples are added → interacted
proteins remained after washing
 TYPES OF PROTEIN MICROARRAYS

• Immunoassays, the first form of protein microarrays, take advantage of highly specific antigen-
antibody recognition to build a protein detection system.
• The expansion of the capability of conventional immunoassays into antibody array applications
enabled a parallel and multiple detection system using a small amount of sample.
• high sensitivity and good reproducibility in quantitative assays when studying complex biological samples.
• another type of protein microarray was developed via the immobilization of purified proteins on glass slides

• To distinguish this type of array from antibody arrays, they are divided into two classes:
analytical and functional.

• Unlike antibody arrays (analytical microarrays), functional protein microarrays are made by
spotting all the proteins encoded by an organism and therefore are useful for the characterization
of protein functions,
• More recently, reverse phase array has been developed, providing an alternative format to
analytical microarrays in which tissue/cell are used to form an array.
 Three categories of protein
microarrays

• Analytical protein microarrays are mostly

represented by antibody arrays and focus on protein
detection. In this class of microarrays, targeted
proteins can be detected either by direct labeling or
using a reporter antibody in sandwich assay format.
• Functional protein microarrays have broad
applications in studying protein interactions,
including protein binding and enzyme-substrate
reactions.
• Reverse-phase protein microarrays provide a
different array format by immobilizing many different
lysate samples on the same chip.
Forward Vs Reverse Phase Protein Microarray
Protein Microarray
 In vivo techniques for the prediction of protein -protein
interactions: yeast two hybrid (Y2H) method

• Given by Fields and Song (1989)

• The in vivo technique used to study PPIs is yeast two hybrid (Y2H) method
• The two protein domains are involved in the Y2H assay.
• First domain is the DNA binding domain which helps in binding the DNA and
• the second one is activation domain that is involved in activation of the transcription of the specific DNA.
These two domains are required for the transcription of a particular reporter gene.
• The interacting proteins that are involved in the Y2H assays must be present in the close vicinity or
inside the nucleus because these proteins have the capability to activate reporter gene and the
proteins that are not present in nucleus do not have the ability to activate reporter genes.
 PRINCIPLES of Y2H assay

Y2H assay relies on the expression of a reporter gene

(such as lacZ or GFP), which is activated by the
binding of a particular transcription factor.

The transcription factor is comprised of a DNA-

binding domain (BD) and an activation domain
(AD).

The query protein of interest fused with the BD is

known as the Bait, and the protein library fused
with the AD is referred to as the Prey.

In order to activate the reporter gene expression, a

transcriptional unit must be present at the gene
locus, which is only possible if Bait and Prey interact.
 Y2H assay

• Since AD and DB domains are

functionally and structurally
independent, they can be fused to
two separate proteins. The protein
that is fused to the DB domain is
called the Bait, while the one fused to
the AD domain is referred to as the
Prey.
• In the absence of Bait-Prey
interaction, the AD domain is unable
to localize to the reporter gene to
drive gene expression (Figure 1B).
• However, when Bait and Prey interact,
the DB domain binds to the DNA
localizing the AD domain upstream of
the reporter gene, leading to the
expression of reporter gene (Figure
1C).
 Yeast two hybrid system
Gal4 protein comprises DNA
binding and activating domains
Binding domain Activating domain
interacts with interacts with
promoter polymerase

Measure reporter enzyme activity (e.g. blue colonies)

Expression of a reporter gene requires the binding of a transcription factor, which normally consists of two
functionally and structurally independent domains: DNA-binding (DB) and activation (AD) domains.
The DB domain binds to the particular DNA sequence upstream of the reporter gene, while the AD domain activates
reporter gene expression
 Yeast two hybrid system
•Gal4 protein: two domains do not need to be transcribed
in a single protein
•If they come into close enough proximity to interact,
they will activate the RNA polymerase

Two other protein domains (A & B) interact

Activating domain
Binding domain
B interacts with
interacts with A
polymerase
promoter

Measure reporter enzyme activity (e.g. blue colonies)

 Yeast two hybrid system
• This is achieved using gene fusion
• Plasmids carrying different constructs can be expressed in
yeast

Binding domain as a translational Activating domain as a

fusion with the gene encoding translational fusion with the gene
another protein in one plasmid. encoding a different protein in a
second plasmid.
A B

If the two proteins interact, then GAL4 is expressed and blue colonies form
 Yeast two hybrid

• Advantages
• Fairly simple, rapid and inexpensive
• Requires no protein purification
• No previous knowledge of proteins needed
• Scalable to high-throughput
• Is not limited to yeast proteins
• Limitations
• Works best with cytosolic proteins
• Tendency to produce false positives
 ADVANTAGES AND LIMITATIONS

• ADVANTAGES AND LIMITATIONS

• Yeast 2-hybrid is a powerful technique to identify protein interactions because of its straightforward methodology and
fast turnaround time. Therefore, the throughput of the technique can be scaled up significantly to screen the entire
proteome
• In addition, the technique has been adapted in other model organisms to study organism specific interactions.
It is important to recognize the limitations of yeast 2-hybrid assays:
1. The assay can produce a high level of false positive and negative interactions. This is an important reason to validate any
interactions using other techniques such as co-immunoprecipitation.
2. Interaction must occur in the nucleus of the cell in order for the reporter gene to be activated. Proteins that are localized
to other cellular compartments may not produce a positive interaction, even if they interact directly. This can be
overcome by using a split ubiquitin 2-hybrid system or by performing the assay in bacteria, which do not possess a
nucleus.
3. Overexpression of recombinant fusion protein, which happens in most yeast 2-hybrid experiments, could produce
spurious interaction data. In addition, the fusion of AD/DB domains to query proteins may affect query protein
function in vivo.
4. Query proteins may not be correctly expressed, folded, or modified when expressed in yeast. Therefore, it is important to
confirm that query proteins are functional before deriving interaction data from the assay.
 In silico techniques

• In silico techniques to predict protein-protein interactions

• Many of the in vivo and in vitro techniques generate a large amount of data that is
helpful in the development of software and tools for the identification of PPIs among
various proteins that are found in many different combinations. The computational
methods used for the in silico prediction of interactions among proteins may include
the tools:
• Coev2Net: http://groups.csail.mit.edu/cb/coev2net/
• TSEMA- http://tsema.bioinfo.cnio.es/
• InterPreTS- http://www.russell.embl.de/interprets
• Struct2Net- http://groups.csail.mit.edu/cb/struct2net/webserver/
• PoiNet- http://poinet.bioinformatics.tw/