1. Protein-Protein Interaction

2. Interaction in biological systems
Occurs when two or more proteins bind together
Biochemical interaction (Physical interaction of proteins)
Genetic interaction: the phenomenon where the effects of one gene are modified by one or several other genes. In the pathway, there is no biochemical interaction among gene products, but they are affected by one another.
Important for the majority of biological functions
Signals from the exterior of a cell are mediated to the inside of that cell by protein-protein interactions of the signaling molecules

3. Methods for Proteins-Protein Interaction
Important to understand biological processes
Multiple methods exist  its own strengths and weaknesses in terms of the sensitivity and specificity of the method
1. Yeast two hybrid
2. Immunoprecipiation (pull-down assay)
3. Phage display
4. Tandem Affinity Purification
5. Bimolecular fluorescence complementation (BiFC)
6. Fluorescence resonance energy transfer (FRET)
7. Protein microarray

4. Yeast two hybrid Yeast Two Hybrid (Y2H)
A molecular-biological technique is used to detect protein-protein interaction
A transcription factor is split into two separate fragments called the binding domain (BD) and activation domain (AD) one of a protein fragment complementation method
Usually, library is linked to activation domain and the target protein is fused to DNA binding domain of a transcription factor

5. Yeast two hybrid Protein-fragment complementation assay
A method for the identification of protein-protein interactions
The proteins of interest are linked to incomplete fragments of a third protein
Interaction brings the fragments of the reporter protein  close enough proximity to allow them to become the functional reporter protein

6. Yeast two hybrid Host Yeast (S. cerevisiae)
Ability to form tertiary protein structure
Neutral internal pH
Complete genome sequence is known
Tolerant of diverse culture conditions and harsh chemicals that could not be applied to mammalian tissue culture
E. coli
Higher transformation efficiency
Faster rate of growth
The absence of requirement for a nuclear localization signal
Study proteins which would be toxic to yeast

7. Yeast two hybrid Yeast Two hybrid - term Transcription factor
A protein that binds to specific DNA sequence and controls the flow of genetic information from DNA to mRNA
Activator: promotes the recruitment of RNA polymerase
Repressor: blocks the recruitment of RNA polymerase
 Lac operon: CAP (CRP), Repressor I
Binary structure: DNA binding domain and trans-activation domain
Binds to either enhancer or promoter region of DNA  up/down regulation
Mechanism: stabilize or block the binding of RNA polymerase to DNA, catalyze acetylation or deacetylation of histone or recruit coactivator or corepressor to the transcription factor DNA complex

8. Yeast two hybrid Yeast Two hybrid - terms
UAS (upstream activation sequence)
Regulatory sequence, distinct from the promoter and increase the expression of a neighboring gene, activator binding site
Reporter gene
A gene is attached to a regulatory sequence of another gene of interest (GFP, Luciferase, beta-galactosidase, auxotrophic genes)
Bait/Prey
Bait-the gene you have, Prey-the gene you catch with the bait
In yeast two hybrid, a bait protein is fused to the DNA binding domain and library (prey) is linked to the activation domain

9. Yeast two hybrid Yeast Two hybrid – transcriptional activator (Gal4)
Gal4 (Gal80, Gal3, Gal1): yeast transcription activator for a gene (Gal1-kinase) required for galactose metabolism
In the absence of galactose  Gal80 binds to and inhibits the transcriptional activation domain of Gal4  preventing Gal1 gene expression
In the presence of galactose  Gal80-Gal3 interaction  relieve Gal80 inhibition of Gal4  transcription of the Gal1 gene

11. Yeast two hybrid
Yeast Two hybrid – Mechanism

12. Yeast two hybrid
Yeast Two hybrid – Mechanism

13. Growth on the selective plate, color change of colonies
Yeast two hybrid
Yeast Two hybrid – Mechanism
Growth on the selective plate, color change of colonies

14. Yeast two hybrid Yeast Two hybrid – Strengths
Low-tech: carried out in any lab without sophisticated equipment
Cheap tech: not require expensive reagents
Provide an important first hint for the identification of interaction partners
Scalable: possible to screen for interactions among many proteins
Relatively high quality of interaction data generated (price vs data)
In vivo technique used to screen for interactions

15. Yeast two hybrid Yeast Two hybrid – weaknesses
Overexpression of fusion proteins cause unnatural protein concentration that lead to unspecific (false) positives
The hybrid proteins are fusion protein  inhibit certain interactions, especially an interaction takes place at the N-terminus of a test protein
Prokaryotic protein or mammalian protein  improper folding and modification
Test protein should be localized to the nucleus (other localization signals  problematic)
 High number of false positive and false negative identification

16. Yeast two hybrid
Yeast Two hybrid – strains

17. Yeast two hybrid
Yeast Two hybrid – strains

18. Yeast two hybrid Yeast Two hybrid-transformation/selection
Yeast transformation is similar to that of bacteria
It has the really thick cells wall  hard to transfer of naked DNA into the cells
LiAc method  loose the cell wall, make yeasts easily uptake DNA
Yeast vectors have autotrophic markers as selection markers in yeast  media lacking certain amino acids used to select transformants
Auxotrophy: the inability of an organism to synthesize a particular organic compound required for its growth –amino acids

19. Yeast two hybrid
Yeast Two-hybrid screen

20. Yeast two hybrid One hybrid
Designed to investigate Protein-DNA interactions
Use a single fusion protein: AD is linked directly to library which you want to test
Library can be selected against the desired target sequence which is inserted in the promoter region of the reporter gene

21. Yeast two hybrid One hybrid
Aureobasidin A: a cyclic depsipeptide antibiotic from Aureobasidium pullulans (yeast like fungus), toxic to fungi and yeast at low concentration

22. Yeast two hybrid Three hybrid Used to detect RNA-protein interactions
Two fusion proteins interact each other through intermediary RNA molecule
BD is fused to a known RNA binding protein (MS2-the bacteriophage coat protein) and MS2 binding RNA is linked to a target RNA of interest
Library is fused to activation domain
Hybrid RNA binds to a protein fused to activation domain (complex formation)  reporter gene expression

23. Yeast two hybrid
Three hybrid

24. 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

25. Immunoprecipitation Immunoprecipitation (IP)
To isolate a particular protein out of a solution containing many different proteins

26. Immunoprecipitation Co-Immunoprecipitation (Co-IP)
Immunoprecipitation of intact protein complexes
A powerful technique that is used regularly to analyze protein-protein interactions

27. 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

28. Immunoprecipitation
Chromatin immunoprecipitation (ChIP)

29. 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

30. Immunoprecipitation
RNA immunoprecipitation (RIP)

31. 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)

32. 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

33. Pull down assay

34. Phage display Phage display
A laboratory technique for the study of protein-protein, protein-peptide and protein-DNA interaction that uses bacteriphage to connect proteins with the gene encoding proteins/peptide
Bacteriophage: any one of a number of viruses that infect bacteria
Common phages in phage display: M13, T4, T7 and l phage

35. Phage display Principle
DNA encoding the protein or peptide of interest (or library, prey) is ligated into the pIII or pVIII gene encoding either the minor or major coat protein of the virus
Exogenous proteins are expressed at the virus surface ( on the coat protein of virus)
A target molecules (bait-protein, peptide or DNA) is immobilized on the surface of a plate
Phage displaying a protein/peptide specifically binding to the target molecules (protein, peptide or DNA) will remain after washing unbound phages off

36. Phage display
Filamentous bacteriophage (M13)

37. Phage display General protocol
Target (bait) proteins or DNA sequences are immobilized to the wells of a microtiter plate.
Many genetic sequences are expressed in a bacteriophage library in the form of fusions with the bacteriophage coat protein, so that they are displayed on the surface of the viral particle. The protein displayed corresponds to the genetic sequence within the phage.
This phage-display library is added to the dish and after allowing the phage time to bind, the dish is washed.

38. Phage display General protocol
Phage-displaying proteins that interact with the target molecules remain attached to the dish, while all others are washed away.
Attached phage may be eluted and used to create more phage by infection of suitable bacterial hosts. The new phage constitutes an enriched mixture, containing considerably less irrelevant phage (i.e. non-binding) than were present in the initial mixture.
Above steps are optionally repeated one or more times, further enriching the phage library in binding proteins.
Following further bacterial-based amplification, the DNA within in the interacting phage is sequenced to identify the interacting proteins or protein fragments.

39. Phage display

40. Phage display Advantages
A means of rapidly screening large number of proteins against potential binding partners  billions of clones can be screened within a week
Disadvantages
The possibility of sterically hindering access to the displayed protein
Difficulty to express a protein larger than a coat protein

41. 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

42. Tandem Affinity Purification (TAP)
Principle

43. 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
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)

44. Bimolecular fluorescence complementation (BiFC)
Principle
A technology used to validate protein interactions
Proteins to be tested are fused to unfolded complementary fragments of a fluorescent reporter protein
Interaction of proteins brings the fluorescent fragments within proximity and allows the reporter protein to reform its 3-D structure and emit its fluorescent signal
The intensity of fluorescent signal is proportional to the intensity of interaction
Biochemical complementation proteins: ribonuclease, b-glactosidase, GFP , YFP

45. Bimolecular fluorescence complementation (BiFC)
Principle

46. Bimolecular fluorescence complementation (BiFC)
Strengths
Direct visualization: direct visualization rather than secondary effects or staining by exogenous molecules
Sensitivity: can detect weak interactions and low expression of proteins due to the stable complementation of the reporter proteins, strength of interaction measured by fluorescent signal strength
Spatial resolution: measure spatial protein-protein interaction (for example: drug responses)
No specialized equipment: a inverted fluorescent microscope is good enough
Live cell image

47. Bimolecular fluorescence complementation (BiFC)
Limitations
Unable to detect real time detection of protein interactions (the signal is not produced right after interaction, takes hours)
Irreversible BiFC formation
Altering protein structure and steric hindrance
Fluorescence complementation may indicate either direct or indirect interaction
Obligate anaerobes: due to the requirement of oxygen for fluorophore formation, BiFC cannot be used in obligate anaerobes (used in aerobic organisms)

48. Fluorescence Resonance Energy Transfer (FRET)
Principle
Useful tool to quantify molecular dynamics in biophysics and biochemistry such as protein-protein interactions, protein-DNA interactions and protein conformational changes
Proteins are labeled with a donor and acceptor
Two proteins are separated  the donor emission is detected upon the donor excitation
The donor and acceptor are in proximity due to interaction  the acceptor emission is predominantly observed because of the intermolecular fret from the donor to the acceptor
In order to detect conformational changes of a protein  similar method, a target protein is labeled with both the donor and acceptor molecules

49. Fluorescence Resonance Energy Transfer (FRET)
Principle

50. BiFC VS FRET Similarity
Ability to detect and locate protein interaction sites within live cells
Differences
FRET need close spatial proximity (60-100A, BiFC: 7nm)
FRET has decreased sensitivity (background  difficult to detect weak interactions)
FRET has irreversible photo-bleaching (the fluorescence will be destroyed over time by the light needed to excite the fluorophores
FRET can detect instantaneous real-time protein interactions and dynamics of interaction due to reversible fluorophore interaction (advantage)

51. 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

52. Protein Microarray