Protein protein interaction

2. INTRODUCTION
• Proteins are the workhorses that facilitate most
biological processes in a cell, including
• gene expression
• cell growth
• proliferation
• nutrient uptake
• morphology
• motility
• intercellular communication and apoptosis.

3. • Until the late 1990's, protein function analyses mainly
focused on single proteins. But because the majority of
proteins interact with other proteins for proper function,
they should be studied in the context of their interacting
partners to fully understand their function.
• And with the publication of the human genome and the
development of the field of proteomics, understanding
how proteins interact with each other and identifying
biological networks is vital to understanding how proteins
function within the cell.

4. • Proteins that are composed of more than one subunit and
purified as multi subunit complexes like hemoglobin, and
core RNA polymerase their protein-protein interactions
were self-evident.
• PPI are intrinsic to virtually every cellular process for
example DNA replication, transcription, translation,
splicing, secretion, cell cycle control, signal transduction,
and intermediary metabolism.

5. Critical aspects required to understand
the function of a protein include :
• Protein sequence and structure–used to discover motifs that
predict protein function
• Evolutionary history and conserved sequences–identifies
key regulatory residues
• Expression profile–reveals cell-type specificity and how
expression is regulated
• Post-translational modifications (e.g., phosphorylation,
acylation, glycosylation, ubiquitination)–suggests
localization, activation and/or function
• Intracellular localization–may allude to the function of the
protein
• Interactions with other proteins–function may be
extrapolated by knowing the function of binding partners.

6. Why are protein-protein
interactions so important?
The binding of one signaling protein to another can have a
number of consequences:
• Such binding can serve to recruit a signaling protein to a
location where it is activated and/or where it is needed to
carry out its function.
• The binding of one protein to another can induce
conformational changes that affect activity or
accessibility of additional binding domains, permitting
additional protein interactions

7. The types of protein
interactions
• Metabolic and signaling (genetic)pathways
• Morphogenic pathways in which groups of proteins
participate in the same cellular function during a
developmental process
• Structural complexes and molecular machines in which
numerous macromolecules are brought together.

8. Methods for Detection and
Analysis
• PHYSICAL METHODS
• LIBRARY-BASED METHODS
• GENETIC METHODS

9. Protein affinity chromatography
• This method was first used 20 years ago to detect phage
and host proteins that interacted with different forms of
E. coli RNA polymerase.
• The interactions were substantiated in two ways. First, the
interaction of T7 0.3 protein with RNA polymerase
second the interaction of T4 proteins with RNA
polymerase was shown to depend on the form of RNA
polymerase on the column.

10. Protein affinity chromatography

11. Advantages
1. Incredibly sensitive. With appropriate use (high
concentrations of immobilized test protein),it can detect
interactions with a binding constant as weak as 10-5 M.
2. Testing all proteins in an extract equally
3. Easy to examine both the domains of a protein and the
critical residues within it that are responsible for a
specific interaction, by preparing mutant derivatives.

12. Disadvantages
• Purity of the coupled protein and use of protein fusions.
An essential requirement for a successful protein affinity
chromatography experiment is pure protein; otherwise, any
interacting protein that is detected might be binding to a
contaminant in the preparation.
• Amount of extract applied. The amount of extract applied
to the column can be critical for two opposing reasons. If too
little extract is applied and the protein that binds is present at
low concentration, too little protein will be retained to be
detected, even if it binds with high affinity and is labelled
with 35S. Conversely, if too much protein is applied,
competition among potential ligands may result in failure to
detect minor species.

13. Affinity blotting
In a procedure 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 ligands.

14. Affinity blotting

15. Immunoprecipitation
• Co-immunoprecipitation is a classical method of
detecting protein-protein interactions.

16. DIRECT VS. INDIRECT
• Direct capture method:
Antibodies are immobilized on a solid-phase
substrate such as super paramagnetic micro beads or on
microscopic agarose(non-magnetic) beads. The beads with
bound antibodies are then added to the protein mixture and
the proteins that are targeted by the antibodies are captured
onto the beads via the antibodies.
• Indirect method:
After mixing of antibodies with proteins, the beads
coated in protein A/G are added.

17. DIRECT VS. INDIRECT
An indirect approach is sometimes
preferred when the concentration of the protein
target is low or when the specific affinity of the
antibody for the protein is weak.

18. Cross-Linking
• Used in two ways to deduce protein-protein interactions.
• Used to deduce the architecture of proteins or assemblies
that are readily isolated intact from the cell.
• Used to detect proteins that interact with a given test
protein ligand by probing extracts, whole cells, or
partially purified preparations.

19. Cross-Linking

20. Phage display
• E.coli filamentous phage can express a fusion protein
bearing a foreign peptide on its surface.
• These foreign amino acids were accessible to antibody,
such that the ‘‘fusion phage’’ could be enriched over
ordinary phage by immunoaffinity purification.

21. Phage display

22. Advantages
• Construction of large phage libraries (up to 10 9 to 1010
complexity) and affinity purification step can be carried
out at very high concentrations of phage (10 13 phage per
ml).
• High selectivity
• direct coupling of the fusion protein to its gene in a single
phage allows the immediate availability of sequence data
to generate one or more consensus sequences of bound
peptides.

23. Two-hybrid system
• uses transcriptional activity as a measure of
protein-protein interaction.
• consist of a DNA-binding domain(yeast Gal4 or the E.
coli LexA )and a transcriptional activation domain(herpes
simplex virus VP16).
• a DNA-binding domain fused to some protein, X, and a
transcription activation domain fused to some protein, Y.
• These two hybrids are expressed in a cell containing one
or more reporter genes(E. coli lacZ gene and selectable
yeast genes such as HIS3 and LEU2), If the X and Y
proteins interact.

24. Two-hybrid system

25. Advantages
• Highly sensitive
• interactions are detected within the native
environment of the cell and hence that no biochemical
purification is required.
• deletions can be made in the DNA encoding one of the
interacting proteins to identify a minimal domain for
interaction.

26. Disadvantages
• limited to proteins that can be localized to the nucleus,
able to fold and exist stably in yeast cells and to retain
activity as fusion proteins.
• Many proteins, including those not normally involved in
transcription ,will activate transcription when fused to a
DNA-binding domain.

27. Genetic methods
Classical genetics can be used to investigate
protein interactions by combining different
mutations in the same cell or organism and
observing the resulting phenotype.
Suppressor mutation: A secondary mutation that
can correct the phenotype of a primary mutation.

28. Suppressor mutation

29. Synthetic Lethal Effects
• Mutations in two genes can cause death (or another observable
defect) while mutation in either alone does not. This
phenomenon is called a synthetic effect and can result from
physical interactions between two proteins required for the
same essential function.

30. Synthetic lethal effect

31. Disadvantage
• Applied only to simple organisms such as phages,
bacteria, yeasts, nematodes, and Drosophila species.
• Requires not only the gene of interest but also a useful
mutant to initiate the analysis. Thus, identification of the
suppressors of interest against a background of these
other mutations can be a time-consuming process.

32. “Here one should remember that any protein
fails to execute its function unless it interacts
with other biomolecules”
- Takashi et al. (2001) Proc. Natl. Acad. Sci.
USA 98, 4569.
11-6
protein-protein interactions

33. References
• ERIC M. PHIZICKY1* AND STANLEY FIELDS” Protein-Protein
Interactions: Methods for Detection and Analysis”.
• Gary D. Bader Nucleic Acids Research, 2001, Vol. 29, No. 1 242-245
BIND—The Biomolecular Interaction Network Database
• http://bind.ca/
• http://nar.oupjournals.org/cgi/content/full/29/1/242
• http://dip.doe-mbi.ucla.edu/dip/Search.cgi
• (http://dip.doe-mbi.ucla.edu/dip/DLRP.cgi)
• http://dip.doe-mbi.ucla.edu/ldipc/tmpl/livedip.cgi
• http://www-cryst.bioc.cam.ac.uk/cgi-
bin/cgiwrap/homstrad/showpage.cgi?family=GroEL&disp=str
• http://www.nature.com/nsb/web_specials/movies/saibil_side.html

34. Thanks for your attention….